WO2015010135A2 - Compositions for modulating tau expression - Google Patents

Compositions for modulating tau expression Download PDF

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Publication number
WO2015010135A2
WO2015010135A2 PCT/US2014/047486 US2014047486W WO2015010135A2 WO 2015010135 A2 WO2015010135 A2 WO 2015010135A2 US 2014047486 W US2014047486 W US 2014047486W WO 2015010135 A2 WO2015010135 A2 WO 2015010135A2
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Prior art keywords
compound
soooossssssssssooss
modified
seq
tau
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PCT/US2014/047486
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French (fr)
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WO2015010135A3 (en
Inventor
Holly Kordasiewicz
Eric E. Swayze
Susan M. Freier
Huynh-Hoa Bui
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Isis Pharmaceuticals, Inc.
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Priority to PL14826839T priority Critical patent/PL3022217T3/en
Priority to KR1020227025324A priority patent/KR102526525B1/en
Priority to DK14826839.4T priority patent/DK3022217T3/en
Priority to LTEP14826839.4T priority patent/LT3022217T/en
Priority to IL279572A priority patent/IL279572B2/en
Priority to SI201431848T priority patent/SI3022217T1/en
Priority to KR1020247016358A priority patent/KR20240093742A/en
Priority to KR1020217020564A priority patent/KR102424991B1/en
Priority to KR1020237013823A priority patent/KR20230062658A/en
Priority to RS20210805A priority patent/RS62054B1/en
Priority to RU2016104824A priority patent/RU2735551C2/en
Priority to IL302146A priority patent/IL302146A/en
Priority to KR1020167003896A priority patent/KR102273926B1/en
Priority to ES14826839T priority patent/ES2871533T3/en
Priority to EP21166766.2A priority patent/EP3910060A1/en
Priority to CN201480040726.6A priority patent/CN108064227B/en
Priority to AU2014290395A priority patent/AU2014290395B2/en
Application filed by Isis Pharmaceuticals, Inc. filed Critical Isis Pharmaceuticals, Inc.
Priority to BR112016000654-2A priority patent/BR112016000654B1/en
Priority to NZ715828A priority patent/NZ715828A/en
Priority to CA2918600A priority patent/CA2918600C/en
Priority to EP14826839.4A priority patent/EP3022217B1/en
Priority to JP2016527154A priority patent/JP6684707B2/en
Priority to US14/906,047 priority patent/US9683235B2/en
Priority to MX2016000782A priority patent/MX2016000782A/en
Publication of WO2015010135A2 publication Critical patent/WO2015010135A2/en
Publication of WO2015010135A3 publication Critical patent/WO2015010135A3/en
Priority to IL243609A priority patent/IL243609B/en
Priority to HK16111947.3A priority patent/HK1223626A1/en
Priority to US15/593,173 priority patent/US10793856B2/en
Priority to IL260378A priority patent/IL260378B/en
Priority to US16/986,770 priority patent/US11591595B2/en
Priority to AU2020250262A priority patent/AU2020250262B2/en
Priority to CY20211100565T priority patent/CY1124292T1/en
Priority to HRP20211051TT priority patent/HRP20211051T1/en
Priority to US18/155,945 priority patent/US12091662B2/en

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    • C12N2310/3525—MOE, methoxyethoxy

Definitions

  • Alzheimer's disease One of the early indicators that Tau may be important in neurodegenerative syndromes was the recognition that Tau is a key component of neurofibrillary inclusions in Alzheimer's disease.
  • neurofibrillary inclusions are aggregates of hyperphosphorylated Tau protein.
  • amyloid beta containing plaques neurofibrillary inclusions are a hallmark of Alzheimer's disease and correlate
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.
  • Certain embodiments provide methods for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with Tau in an individual in need thereof. Also contemplated are methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Tau.
  • Tau associated diseases, disorders, and conditions include neurodegenerative diseases.
  • tau associated diseases include Tauopathies,
  • Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135929 of SEQ ID NO: 1.
  • an antisense compound targeted to a Tau nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 12 to 25 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 15 to 25 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 18 to 22 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 19 to 21 subunits in length.
  • gapmers provided herein include, for example 20-mers having a motif of 5-
  • hybridization occurs between an antisense compound disclosed herein and a Tau nucleic acid.
  • the most common mechanism of hybridization involves hydrogen bonding (e.g., Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
  • an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity.
  • the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases.
  • the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
  • Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. Modified Internucleoside Linkages
  • Examples of chemically modified sugars include 2'-F-5 '-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein LNA is substituted with for example a 5'-methyl or a 5'-vinyl group).
  • the modified THP nucleosides of Formula VII are provided wherein q b q 2 , q3, q4, q 5 , q6 and q 7 are each H. In certain embodiments, at least one of qi, q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is other than H. In certain embodiments, at least one of qi, q 2 , q 3 , q 4 , q 5 , q 6 and q 7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R 2 is fluoro. In certain embodiments, Ri is fluoro and R 2 is H; R t is methoxy and R 2 is H, and Ri is H and R 2 is methoxyethoxy.
  • 2'-modified or “2 '-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH.
  • 2'-F refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.
  • compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • the effects of antisense compounds on the level, activity or expression of Tau nucleic acids can be tested in vitro in a variety of cell types.
  • Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, CA).
  • Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, and primary hepatocytes.
  • Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
  • Example 1 Antisense inhibition of human Tau in HepG2 cells by MOE gapmers
  • Example 2 Dose-dependent antisense inhibition of human tau in HepG2 cells by 5-10-5 MOE gapmers
  • Start site indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence.
  • “Stop site” indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence.
  • Each gapmer listed in the Tables below is targeted to either the human Tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK
  • Additional antisense oligonucleotides were designed targeting a Tau nucleic acid and were tested for their effects on Tau mRNA in vitro.
  • Cultured SH-SY5Y cells were plated at a density of 20,000 cells per well and were transfected using electroporation with 6,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.

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Abstract

Disclosed herein are antisense compounds and methods for decreasing Tau mRNA and protein expression. Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate Tau-associated diseases, disorders, and conditions.

Description

COMPOSITIONS FOR MODULATING TAU EXPRESSION
Sequence Listing
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0227WOSEQ_ST25.txt created July 17, 2014, which is 916 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
Field
Provided are compositions and methods for reducing expression of Tau mRNA and protein in an animal. Such methods are useful to treat, prevent, or ameliorate neurodegenerative diseases, including Tauopathies, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, and Dravet's Syndrome by inhibiting expression of Tau in an animal.
Background
The primary function of Tau is to bind to and stabilize microtubules, which are important structural components of the cytoskeleton involved in mitosis, cytokinesis, and vesicular transport. Tau is found in multiple tissues, but is particularly abundant in axons of neurons. In humans, there are six isoforms of Tau that are generated by alternative splicing of exons 2, 3, and 10. Splicing of exons 2 and 3 at the N-terminus of the protein leads to inclusion of zero, one, or two 29 amino acid acidic domains and is termed ON, IN, or 2N Tau respectively. The influence of these domains on Tau function is not fully clear, though may play a role in interactions with the plasma membrane. Inclusion of exon 10 at the C-terminus leads to inclusion of the microtubule binding domain encoded by exon 10. Since there are 3 microtubule binding domains elsewhere in Tau, this Tau isoform (with exon 10 included) is termed 4R Tau, where 'R' refers to the number of repeats of microtubule binding domains. Tau without exon 10 is termed 3R Tau. Since more microtubule binding domains (4R compared with 3R) increases the binding to microtubules, 4R Tau presumably significantly increases microtubule binding and assembly. The ratio of 3R''4R Tau is developmentally regulated, with fetal tissues expressing exclusively 3R Tau and adult human tissues expressing approximately equal levels of 3R/4R Tau. Deviations from the normal ratio of 3R/4R Tau are characteristic of neurodegenerative FTD Tauopathies. It is not known how changing the 3R/4R Tau ratio at a later stage in the adult animal will affect Tau pathogenesis. Serine -threonine directed phosphorylation regulates the microtubule binding ability of Tau.
Hyperphosphorylation promotes detachment of Tau from microtubules. Other post translational modifications of Tau have been described; however the significance of these is unclear. Phosphorylation of Tau is also developmentally regulated with higher phosphorylation in fetal tissues and much lower phosphorylation in the adult. One characteristic of neurodegenerative disorders is aberrantly increased Tau phosphorylation.
The microtubule network is involved in many important processes within the cell including structural integrity needed for maintaining morphology of cells and operating transport machinery. Since binding of Tau to microtubules stabilizes microtubules, Tau is likely to be a key mediator of some of these processes and disruption of normal Tau in neurodegenerative diseases may disrupt some of these key cellular processes.
One of the early indicators that Tau may be important in neurodegenerative syndromes was the recognition that Tau is a key component of neurofibrillary inclusions in Alzheimer's disease. In fact, neurofibrillary inclusions are aggregates of hyperphosphorylated Tau protein. Along with amyloid beta containing plaques, neurofibrillary inclusions are a hallmark of Alzheimer's disease and correlate
significantly with cognitive impairment. 95% of Tau accumulations in AD are found in neuronal processes and is termed neuritic dystrophy. The process(es) whereby this microtubule associated protein becomes disengaged from microtubules and forms accumulations of proteins and how this relates to neuronal toxicity is not well understood.
Neuronal Tau inclusions are a pathological characteristic of not only Alzheimer's disease, but also a subset of Frontotemporal dementia (FTD), PSP, and CBD. The link between Tau and neurodegeneration was solidified by the discovery that mutations in the Tau gene cause a subset of FTD. These genetic data have also highlighted the importance of the 3R:4R ratio of Tau. Many of the Tau mutations that cause FTD lead to a change in Tau splicing which leads to preferential inclusion of exon 10, and thus to increased 4R Tau. The overall Tau levels are normal. Whether the Tau isoform change or the amino acid change or both cause neurodegeneration remains unknown. Recent data suggest that PSP may also be associated with an increased 4R:3R Tau ratio.
To help understand the influence of Tau ratios on neurodegeneration, a mouse model based on one of the splicing Tau mutations (N279K) has been generated using a minigene that includes the Tau promoter and the flanking intronic sequences of exon 10. As in humans, these mice demonstrate increased levels of 4R Tau compared with transgenics expressing WT Tau and develop behavioral and motor abnormalities as well as accumulations of aggregated Tau in the brain and spinal cord.
The protein "Tau" has been associated with multiple diseases of the brain including Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal ganglionic degeneration, dementia pugilistica, parkinsonism linked to chromosome, Lytico-Bodig disease, tangle-predominant dementia, ganglioglioma, gangliocytoma, meningioangiomatosis, subacute sclerosing panencephalitis, lead encephalopathy, tuberous sclerosis, Hallervorden-Spatz disease, Pick's disease, argyrophilic grain disease, corticobasal degeneration or frontotemporal lobar degeneration and others. Tau-associated disorders such as AD are the most common cause of dementia in the elderly. AD affects an estimated 15 million people worldwide and 40% of the population above 85 years of age. AD is characterized by two pathological hallmarks: Tau neurofibrillary inclusions (NFT) and amyloid-β (Αβ) plaques.
There is currently a lack of acceptable options for treating such neurodegenerative diseases. It is therefore an object herein to provide methods for the treatment of such diseases.
Summary
Provided herein are methods, compounds, and compositions for modulating expression of Tau mRNA and protein. In certain embodiments, compounds useful for modulating expression of Tau mRNA and protein are antisense compounds. In certain embodiments, the antisense compounds are antisense oligonucleotides.
In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, Tau mRNA levels are reduced. In certain embodiments, Tau protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.
Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such Tau related diseases, disorders, and conditions are neurodegenerative diseases. In certain embodiments, such neurodegenerative diseases, disorders, and conditions include Tauopathies, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, and Dravet's Syndrome
Such diseases, disorders, and conditions can have one or more risk factors, causes, or outcomes in common. Certain risk factors and causes for development of neurodegenerative disorder include growing older, having a personal or family history, or genetic predisposition. Certain symptoms and outcomes associated with development of a neurodegenerative disorder include but are not limited to: presence of hyperphosphorylated Tau, presence of neurofibrillary inclusions, reduction of neurological function, reduced memory, reduced motor function, reduced motor coordination, and confusion.
In certain embodiments, methods of treatment include administering a Tau antisense compound to an individual in need thereof. In certain embodiments, methods of treatment include administering a Tau antisense oligonucleotide to an individual in need thereof.
The present disclosure provides the following non- limiting numbered embodiments: Embodiment 1 : A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2443 and SEQ ID NOs: 2478-2483.
Embodiment 2: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 2444-2477 and SEQ ID NOs: 2484-2565.
Embodiment 3: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2565.
Embodiment 4: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135980 of SEQ ID NO: 1.
Embodiment 5: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135853-135872 of SEQ ID NO: 1.
Embodiment 6: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135929 of SEQ ID NO: 1.
Embodiment 7: A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135914 of SEQ ID NO: 1. Embodiment 8: The compound of embodiments 4-7, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: l .
Embodiment 9: The compound of any preceding embodiment, consisting of a single-stranded modified oligonucleotide.
Embodiment 10: The compound of any preceding embodiment, wherein at least one internucleoside linkage is a modified internucleoside linkage.
Embodiment 11 : The compound of embodiment 10, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
Embodiment 12: The compound of embodiment 10, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.
Embodiment 13: The compound of any preceding embodiment, wherein at least one internucleoside linkage is a phosphodiester internucleoside linkage.
Embodiment 14: The compound of any preceding embodiment, wherein at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.
Embodiment 15: The compound of any preceding embodiment, wherein at least one nucleoside comprises a modified nucleobase.
Embodiment 16: The compound of embodiment 15, wherein the modified nucleobase is a 5- methylcytosine.
Embodiment 17: The compound of any preceding embodiment, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar.
Embodiment 18: The compound of embodiment 17, wherein the at least one modified sugar is a bicyclic sugar.
Embodiment 19: The compound of embodiment 18, wherein the bicyclic sugar comprises a chemical link between the 2' and 4' position of the sugar 4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, CI -CI 2 alkyl, or a protecting group.
Embodiment 20: The compound of embodiment 18, wherein the bicyclic sugar comprises a
4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, C1 -C12 alkyl, or a protecting group. Embodiment 21 : The compound of embodiment 17, wherein at least one modified sugar comprises a 2'-0-methoxyethyl group.
Embodiment 22: The compound of embodiment 17, wherein the modified sugar comprises a 2'-0(CH2)2-OCH3 group.
Embodiment 23 : The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 10 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 24: The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 9 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 25: The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 7 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 26: The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 27: The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 4 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 28: The compound of any preceding embodiment, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 6 linked nucleosides; and
a 3' wing segment consisting of 4 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
Embodiment 29: The compound of any preceding embodiment, wherein the modified oligonucleotide consists of 20 linked nucleosides.
Embodiment 30: The compound of any preceding embodiment, wherein the modified oligonucleotide consists of 19 linked nucleosides.
Embodiment 31 : The compound of any preceding embodiment, wherein the modified oligonucleotide consists of 18 linked nucleosides.
Embodiment 32: A composition comprising the compound of any preceding embodiment or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
Embodiment 33: A method comprising administering to an animal the compound or composition of any preceding embodiment.
Embodiment 34: The method of embodiment 33, wherein the animal is a human.
Embodiment 35: The method of embodiment 33, wherein administering the compound prevents, treats, ameliorates, or slows progression of a tau associated disease, disorder or condition. Embodiment 36: The method of embodiment 35, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
Embodiment 37: Use of the compound or composition of any preceding embodiment for the manufacture of a medicament for treating a neurodegenerative disorder.
Embodiment 38: A compound consisting of ISIS 613099. Embodiment 39: A compound consisting of ISIS 613361. Embodiment 40: A compound consisting of ISIS 613370.
Embodiment 41 : A compound consisting of ISIS 623782.
Embodiment 42: A compound consisting of ISIS 623996.
Embodiment 43: A composition comprising the compound of any of embodiments 38 - 42, or salt thereof, and at least one of a pharmaceutically acceptable carrier or diluent.
Embodiment 44: A method comprising administering to an animal the compound or composition of any of embodiments 38 - 43.
Embodiment 45: The method of embodiment 44, wherein the animal is a human.
Embodiment 46: The method of embodiment 44, wherein administering the compound prevents, treats, ameliorates, or slows progression of a tau associated disease, disorder or condition.
Embodiment 47: The method of embodiment 46, wherein the disease, disorder or condition is a
Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
Embodiment 48: Use of the compound or composition of any of embodiments 38 - 43 for the manufacture of a medicament for treating a neurodegenerative disorder.
Detailed Description
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Additionally, as used herein, the use of "and" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this disclosure, including, but not limited to, patents, patent applications, published patent applications, articles, books, treatises, and GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Definitions
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis.
Unless otherwise indicated, the following terms have the following meanings:
"2'-0-methoxyethyl" (also 2'-MOE and 2'-OCH2CH2-OCH3 and MOE) refers to an O-methoxy- ethyl modification of the 2' position of a furanose ring. A 2'-0-methoxyethyl modified sugar is a modified sugar.
"2'-MOE nucleoside" (also 2'-0-methoxyethyl nucleoside) means a nucleoside comprising a 2'- MOE modified sugar moiety.
"2 '-substituted nucleoside" means a nucleoside comprising a substituent at the 2 '-position of the furanose ring other than H or OH. In certain embodiments, 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.
"5-methylcytosine" means a cytosine modified with a methyl group attached to the 5 position. A 5- methylcytosine is a modified nucleobase.
"About" means within ±7% of a value. For example, if it is stated, "the compounds affected at least about 70% inhibition of Tau", it is implied that the Tau levels are inhibited within a range of 63% and 77%.
"Administered concomitantly" refers to the co-administration of two pharmaceutical agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time.
Concomitant administration does not require that both pharmaceutical agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both pharmaceutical agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive. "Administering" means providing a pharmaceutical agent to an animal, and includes, but is not limited to administering by a medical professional and self-administering.
"Amelioration" refers to a lessening, slowing, stopping, or reversing of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.
"Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
"Antibody" refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, Fab region, and Fc region.
"Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
"Antisense compound" means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single- stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
"Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels or in the absence of the antisense compound.
"Antisense mechanisms" are all those mechanisms involving hybridization of a compound with a target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
"Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid.
"Base complementarity" refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.
"Bicyclic sugar" means a furanose ring modified by the bridging of two atoms. A bicyclic sugar is a modified sugar.
"Bicyclic nucleoside" (also BNA) means a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4 '-carbon and the 2 '-carbon of the sugar ring. "Cap structure" or "terminal cap moiety" means chemical modifications, which have been incorporated at either terminus of an antisense compound.
"cEt" or "constrained ethyl" means a bicyclic nucleoside having a sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH3)-0-2'.
"Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge.
"Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'- O-methoxyethyl nucleosides is chemically distinct from a region having nucleosides without 2'-0- methoxyethyl modifications.
"Chimeric antisense compound" means an antisense compound that has at least two chemically distinct regions, each position having a plurality of subunits.
"Co-administration" means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration.
"Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
"Comprise," "comprises," and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
"Contiguous nucleobases" means nucleobases immediately adjacent to each other.
"Designing" or "designed to" refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
"Diluent" means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g. saline solution.
"Dose" means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In certain embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In certain embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month. "Effective amount" in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
"Efficacy" means the ability to produce a desired effect.
"Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
"Fully complementary" or "100% complementary" means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
"Gapmer" means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as a "gap" and the external regions may be referred to as the "wings."
"Gap-narrowed" means a chimeric antisense compound having a gap segment of 9 or fewer contiguous 2'-deoxyribonucleosides positioned between and immediately adjacent to 5' and 3' wing segments having from 1 to 6 nucleosides.
"Gap-widened" means a chimeric antisense compound having a gap segment of 12 or more contiguous 2'-deoxyribonucleosides positioned between and immediately adjacent to 5' and 3' wing segments having from 1 to 6 nucleosides.
"Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a target nucleic acid. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
"Identifying an animal having a Tau associated disease" means identifying an animal having been diagnosed with a Tau associated disease or predisposed to develop a Tau associated disease. Individuals predisposed to develop a Tau associated disease include those having one or more risk factors for developing a Tau associated disease, including, growing older, having a personal or family history, or genetic predisposition of one or more Tau associated diseases. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments, such as genetic testing. "Immediately adjacent" means there are no intervening elements between the immediately adjacent elements.
"Individual" means a human or non-human animal selected for treatment or therapy.
"Inhibiting Tau" means reducing the level or expression of a Tau mRNA and/or protein. In certain embodiments, Tau mRNA and/or protein levels are inhibited in the presence of an antisense compound targeting Tau, including an antisense oligonucleotide targeting Tau, as compared to expression of Tau mRNA and/or protein levels in the absence of a Tau antisense compound, such as an antisense oligonucleotide.
"Inhibiting the expression or activity" refers to a reduction or blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
"Internucleoside linkage" refers to the chemical bond between nucleosides.
"Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.
"Locked nucleic acid" or " LNA" or "LNA nucleosides" means nucleic acid monomers having a bridge connecting two carbon atoms between the 4' and 2 'position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) a-L- Methyleneoxy (4'-CH2-0-2') LNA , (B) β-D-Methyleneoxy (4'-CH2-0-2') LNA , (C) Ethyleneoxy (4'-
(CH2)2-0-2') LNA , (D) Aminooxy (4'-CH2-0-N(R)-2') LNA and (E) Oxyamino (4'-CH2-N(R)-0-2') LNA,
Figure imgf000014_0001
As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from -[C(Ri)(R2)]n-,
Figure imgf000014_0002
-C(Ri)=N- , -C(=NR -, -C(=0)-, -C(=S)-, -0-, -Si(Ri)2-, -S(=0)x- and -N(Ri)-; wherein: x is 0, 1, or 2; n is
1, 2, 3, or 4; each Ri and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted Cp C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-Ci2 alkynyl, substituted C2-Ci2 alkynyl, C5-C2o aryl, substituted C5-C2o aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)-H), substituted acyl, CN, sulfonyl
Figure imgf000014_0003
or sulfoxyl and each Ji and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group. Examples of 4'- 2' bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: -[C(R (R2)]n-, -[C(R1)(R2)]n-0-, -C(R1R2)-N(R1)-0- or -C(R1R2)-0-N(R1)-. Furthermore, other bridging groups encompassed with the definition of LNA are 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R1)-2' and 4'-CH2-N(R1)-0-2'- bridges, wherein each Ri and R2 is, independently, H, a protecting group or CrCi2 alkyl.
Also included within the definition of LNA according to the invention are LNAs in which the 2'- hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH2-0-2') bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'- CH2-0-2') LNA is used. Furthermore; in the case of the bicylic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4'-CH2CH2-0-2') LNA is used, a -L- methyleneoxy (4'-CH2- 0-2'), an isomer of methyleneoxy (4'-CH2-0-2') LNA is also encompassed within the definition of LNA, as used herein.
"Mismatch" or "non-complementary nucleobase" refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
"Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a phosphodiester internucleoside bond).
"Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
A "modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
"Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and/or modified nucleobase.
"Modified oligonucleotide" means an oligonucleotide comprising at least one modified
internucleoside linkage, modified sugar, and/or modified nucleobase.
"Modified sugar" means substitution and/or any change from a natural sugar moiety.
"Monomer" means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.
"Motif means the pattern of unmodified and modified nucleosides in an antisense compound.
"Natural sugar moiety" means a sugar moiety found in DNA (2'-H) or RNA (2' -OH).
"Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.
"Non-complementary nucleobase" refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization. "Nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
"Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid. "Nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
"Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
"Nucleoside" means a nucleobase linked to a sugar.
"Nucleoside mimetic" includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo, or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by -N(H)-C(=0)-0- or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
"Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
"Off-target effect" refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.
"Oligomeric compound" or "oligomer" means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
"Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another. "Parenteral administration" means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.
"Peptide" means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, peptide refers to polypeptides and proteins.
"Pharmaceutical agent" means a substance that provides a therapeutic benefit when administered to an individual. For example, in certain embodiments, an antisense oligonucleotide targeted to Tau is a pharmaceutical agent.
"Pharmaceutical composition" means a mixture of substances suitable for administering to a subject.
For example, a pharmaceutical composition may comprise an antisense oligonucleotide and a sterile aqueous solution.
"Pharmaceutically acceptable derivative" encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.
"Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
"Phosphorothioate linkage" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
"Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
"Prevent" or "preventing" refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to days, weeks to months, or indefinitely.
"Prodrug" means a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
"Prophylactically effective amount" refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.
"Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
"Ribonucleotide" means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents. "Salts" mean a physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.
"Segments" are defined as smaller or sub-portions of regions within a target nucleic acid.
"Shortened" or "truncated" versions of antisense oligonucleotides taught herein have one, two or more nucleosides deleted.
"Side effects" means physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.
"Single-stranded oligonucleotide" means an oligonucleotide which is not hybridized to a
complementary strand.
"Sites," as used herein, are defined as unique nucleobase positions within a target nucleic acid.
"Slows progression" means decrease in the development of the said disease.
"Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.
"Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
"Subject" means a human or non-human animal selected for treatment or therapy.
"Target" refers to a protein, the modulation of which is desired.
"Target gene" refers to a gene encoding a target.
"Targeting" or "targeted" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
"Target nucleic acid," "target RNA," and "target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by antisense compounds.
"Target region" means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
"Target segment" means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5 '-most nucleotide of a target segment. "3' target site" refers to the 3 '-most nucleotide of a target segment.
"Tau" means mammalian microtubule-associated protein tau (MAPT), including human microtubule - associated protein tau (MAPT).
"Tau associated disease" means any disease associated with any Tau nucleic acid or expression product thereof. Such diseases may include a neurodegenerative disease. Such neurodegenerative diseases may include Tauopathies, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Cortieobasal Ganglionic Degeneration (CBD), Epilepsy, and Dravet's Syndrome.
"Tau mRNA" means any messenger RNA expression product of a DNA sequence encoding Tau. "Tau nucleic acid" means any nucleic acid encoding Tau. For example, in certain embodiments, a
Tau nucleic acid includes a DNA sequence encoding Tau, an RNA sequence transcribed from DNA encoding Tau (including genomic DNA comprising introns and exons), and an mRNA sequence encoding Tau. "Tau mRNA" means an mRNA encoding a Tau protein.
"Tau protein" means the polypeptide expression product of a Tau nucleic acid.
"Therapeutically effective amount" means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
"Treat" or "treating" or "treatment" refers administering a composition to effect an alteration or improvement of the disease or condition.
"Unmodified nucleobases" mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
"Unmodified nucleotide" means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
"Wing segment" means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Certain Embodiments
Certain embodiments provide methods, compounds, and compositions for inhibiting Tau mRNA and protein expression. Certain embodiments provide methods, compounds, and composition for decreasing Tau mRNA and protein levels.
Certain embodiments provide antisense compounds targeted to a Tau nucleic acid. In certain embodiments, the Tau nucleic acid is the sequence set forth in GENBANK Accession No. GENBANK Accession No. NT O 10783.15 truncated from nucleotides 9240000 to 9381000 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NM 001123066.3 (incorporated herein as SEQ ID NO: 2),
GENBANK Accession No. NM 016841.4, a variant mRNA sequence which skips exons 3, 4, 6, 8, 10, and 12 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. NT 010783.14 truncated from nucleotides 2624000 to 2761000 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No.
DR002467.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. NM 001203251.1 (incorporated herein as SEQ ID NO: 6), and GENBANK Accession No. NM 016835.4 (incorporated herein as SEQ ID NO: 7). Certain embodiments provide methods for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with Tau in an individual in need thereof. Also contemplated are methods for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with Tau. Tau associated diseases, disorders, and conditions include neurodegenerative diseases. In certain embodiments, tau associated diseases include Tauopathies,
Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2443 and SEQ ID NOs: 2478-2483.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 2444-2477 and SEQ ID NOs: 2484-2565.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2565.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135980 of SEQ ID NO: 1.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135853-135872 of SEQ ID NO: 1.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135929 of SEQ ID NO: 1.
Certain embodiments provide compounds, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135914 of SEQ ID NO: 1.
In certain embodiments the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: 1.
In certain embodiments, the compound is a single-stranded modified oligonucleotide.
In certain embodiments, at least one internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.
In certain embodiments, at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, each modified internucleoside linkage is a phosphorothioate internucleoside linkage.
In certain embodiments, at least one internucleoside linkage is a phosphodiester internucleoside linkage.
In certain embodiments, at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.
In certain embodiments, at least one nucleoside comprises a modified nucleobase.
In certain embodiments, the modified nucleobase is a 5-methylcytosine.
In certain embodiments, at least one nucleoside of the modified oligonucleotide comprises a modified sugar.
In certain embodiments, at least one modified sugar is a bicyclic sugar.
In certain embodiments, the bicyclic sugar comprises a chemical link between the 2' and 4' position of the sugar 4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, C1-C12 alkyl, or a protecting group.
In certain embodiments, the bicyclic sugar comprises a 4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, CI -CI 2 alkyl, or a protecting group.
In certain embodiments, at least one modified sugar comprises a 2'-0-methoxyethyl group.
In certain embodiments, the modified sugar comprises a 2'-0(CH2)2-OCH3 group.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 10 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 9 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 7 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 4 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 6 linked nucleosides; and
a 3' wing segment consisting of 4 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides.
In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides.
In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides. Certain embodiments provide compositions comprising any compound described herein or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
Certain embodiments provide methods comprising administering to an animal any compound or composition described herein.
In certain embodiments, the animal is a human.
In certain embodiments, administering the compound prevents, treats, ameliorates, or slows progression of a tau associated disease, disorder or condition.
In certain embodiments, the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
Certain embodiments provide use of any of the compounds or compositions of described herein for the manufacture of a medicament for treating a neurodegenerative disorder.
Certain embodiments provide compounds according to the following formula (la):
Figure imgf000023_0001
, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided are
pharmaceutical compositions comprising the compound having the formula (la).
Certain embodiments provide compounds according to the following formula (Ila):
Figure imgf000024_0001
, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided are pharmaceutical compositions comprising the compound having the formula (Ila).
Certain embodiments provide compounds according to the following formula (Ilia):
Figure imgf000025_0001
(Ilia)
, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided are pharmaceutical compositions comprising the compound having the formula (Ilia).
Certain embodiments provide compounds according to the following formula (IVa):
Figure imgf000026_0001
(IVa)
, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided are pharmaceutical compositions comprising the compound having the formula (IVa).
Certain embodiments provide compounds according to the following formula (Va):
Figure imgf000027_0001
(Va)
, or a pharmaceutically acceptable salt thereof. In certain embodiments, provided are
pharmaceutical compositions comprising the compound having the formula (Va).
Antisense Compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 12 to 30 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 12 to 25 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 12 to 22 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 14 to 20 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 15 to 25 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 18 to 22 subunits in length. In certain embodiments, an antisense compound targeted to Tau nucleic acid is 19 to 21 subunits in length. In certain embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 30, 18 to 50, 19 to 30, 19 to 50, or 20 to 30 linked subunits in length.
In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 12 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 13 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 14 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 15 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 16 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 17 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 18 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 19 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 20 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 21 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 22 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 23 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 24 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 25 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 26 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 27 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 28 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 29 subunits in length. In certain embodiments, an antisense compound targeted to a Tau nucleic acid is 30 subunits in length. In certain embodiments, the antisense compound targeted to a Tau nucleic acid is 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In certain embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleosides. In certain embodiments antisense oligonucleotides targeted to a Tau nucleic acid may be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated antisense compound targeted to a Tau nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5' or 3' end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model.
Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this
oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358,1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a Tau nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such a enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may optionally serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2'- modified nucleosides (such 2'-modified nucleosides may include 2'-MOE, and 2'-0-CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a 4'- (CH2)n-0-2' bridge, where n=l or n=2 and 4'-CH2-0-CH2-2'). In certain embodiments, wings may include several modified sugar moieties, including, for example 2'-MOE. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2'-MOE nucleosides and 2'-deoxynucleosides.
Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as "X-Y-Z", where "X" represents the length of the 5' wing, "Y" represents the length of the gap, and "Z" represents the length of the 3' wing. "X" and "Z" may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, "X" and "Y" may include one or more 2'-deoxynucleosides. "Y" may comprise 2'-deoxynucleosides. As used herein, a gapmer described as "X-Y-Z" has a configuration such that the gap is positioned immediately adjacent to each of the 5' wing and the 3' wing. Thus, no intervening nucleotides exist between the 5' wing and gap, or the gap and the 3 ' wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same; in other embodiments they are different.
In certain embodiments, gapmers provided herein include, for example 20-mers having a motif of 5-
10-5.
In certain embodiments, gapmers provided herein include, for example 19-mers having a motif of 5-
9-5.
In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 5-
8-5. In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 4-
8-6.
In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 6-
8-4.
In certain embodiments, gapmers provided herein include, for example 18-mers having a motif of 5-
7-6.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode Tau include, without limitation, the following: GENBANK Accession No. NT O 10783.15 truncated from nucleotides 9240000 to 9381000 (incorporated herein as SEQ ID NO: 1), GENBANK Accession No. NM 001123066.3 (incorporated herein as SEQ ID NO: 2),
GENBANK Accession No. NM 016841.4, a variant mRNA sequence which skips exons 3, 4, 6, 8, 10, and 12 (incorporated herein as SEQ ID NO: 3), GENBANK Accession No. NT 010783.14 truncated from nucleotides 2624000 to 2761000 (incorporated herein as SEQ ID NO: 4), GENBANK Accession No.
DR002467.1 (incorporated herein as SEQ ID NO: 5), GENBANK Accession No. NM 001203251.1
(incorporated herein as SEQ ID NO: 6), and GENBANK Accession No. NM 016835.4 (incorporated herein as SEQ ID NO: 7).
It is understood that the sequence set forth in each SEQ ID NO in the Examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3' UTR, a 5' UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for Tau can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5' target site of one target segment within the target region to a 3' target site of another target segment within the same target region.
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. In certain embodiments, the desired effect is reduction of levels of protein encoded by the target nucleic acid or a phenotypic change associated with the target nucleic acid.
A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In certain embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In certain emodiments, target segments within a target region are separated by a number of nucleotides that is, is about, is no more than, is no more than about, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the preceeding values. In certain embodiments, target segments within a target region are separated by no more than, or no more than about, 5 nucleotides on the target nucleic acid. In certain embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5' target sites or 3' target sites listed herein.
Suitable target segments may be found within a 5' UTR, a coding region, a 3' UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon.
The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense compound sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).
There may be variation in activity (e.g., as defined by percent reduction of target nucleic acid levels) of the antisense compounds within an active target region. In certain embodiments, reductions in Tau mRNA levels are indicative of inhibition of Tau expression. Reductions in levels of a Tau protein are also indicative of inhibition of target mRNA expression. Reduction of percent of cells staining positive for hyperphosphorylated Tau are indicative of inhibition of Tau expression. Further, phenotypic changes are indicative of inhibition of Tau expression. Improvement in neurological function is indicative of inhibition of Tau expression. Improved memory and motor function are indicative of inhibition of Tau expression.
Reduction of neurofibrillary inclusions is indicative of inhibition of Tau expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and a Tau nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a Tau nucleic acid. Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a Tau nucleic acid).
Non-complementary nucleobases between an antisense compound and a Tau nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a Tau nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%o, 99%), or 100%> complementary to a Tau nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four)
noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or
complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary {i.e., 100%> complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a Tau nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Tau nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a Tau nucleic acid, or specified portion thereof.
The antisense compounds provided herein also include those which are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds, are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%,
85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid. Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3 Or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides. Modified Internucleoside Linkages
The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
Representative phosphorus containing internucleoside linkages include, but are not limited to,
phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a Tau nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are interspersed throughout the antisense compound. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(Ri)(R2) (R, Ri and R2 are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5 '-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on 8/21/08 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on June 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on 11/22/07 wherein LNA is substituted with for example a 5'-methyl or a 5'-vinyl group). Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, 2'-OCH2CH3, 2'-OCH2CH2F and - 0(CH2)20CH3 substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O-CrC10 alkyl, OCF3, OCH2F, 0(CH2)2SCH3, 0(CH2)2-0-N(Rm)(Rn), 0-CH2-C(=0)- N(Rm)(Rn), and 0-CH2-C(=0)-N(R1)-(CH2)2-N(Rm)(Rn), where each Rh Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl.
As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2' and 4'-CH(CH2OCH3)-0-2' (and analogs thereof see U.S. Patent 7,399,845, issued on July 15, 2008); 4'-C(CH3)(CH3)-0-2' (and analogs thereof see published International Application WO/2009/006478, published January 8, 2009); 4'-CH2-N(OCH3)-2' (and analogs thereof see published International Application WO/2008/150729, published December 11 , 2008); 4'-CH2-0-N(CH3)-2' (see published U.S. Patent Application US2004-0171570, published September 2, 2004 ); 4'-CH2-N(R)-0-2', wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Patent 7,427,672, issued on September 23, 2008); 4'-CH2-C(H)(CH3)-2' (see Chattopadhyaya et al, J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C- (=CH2)-2' (and analogs thereof see published International Application WO 2008/154401, published on December 8, 2008).
Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al, Chem. Commun., 1998, 4, 455-456; Koshkin et al, Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al, Proc. Natl Acad. Sci. U. S. A. , 2000, 97, 5633-5638; Kumar et al, Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al, J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al, J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al, Curr. Opinion Invest. Drugs, 2001, 2, 558-561 ; Braasch et al, Chem. Biol, 2001, 8, 1-7; and Orum et al, Curr. Opinion Mol Ther., 2001, 3, 239-243; U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461 ; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Serial Nos.
60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231 ; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181 ; WO 2008/150729; WO 2008/154401 ; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example a-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on March 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from - [C(Ra)(Rb)]n-, -C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=0)-, -C(=NRa)-, -C(=S)-, -0-, -Si(Ra)2-, -S(=0)x-, and -N(Ra)-; wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJi, NJiJ2, SJi, N3, COOJi, acyl (C(=0)- H), substituted acyl, CN, sulfonyl (S(=0)2-Ji), or sulfoxyl (S(=0)-Ji); and
each Ji and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=0)- H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-0-
, -C(RaRb)-N(R)-0- or -C(RaRb)-0-N(R)-. In certain embodiments, the bridge is 4'-CH2-2', 4'-(CH2)2-2', 4'- (CH2)3-2', 4'-CH2-0-2', 4'-(CH2)2-0-2', 4'-CH2-0-N(R)-2' and 4'-CH2-N(R)-0-2'- wherein each Ris, independently, H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4' -2' methylene-oxy bridge, may be in the a-L configuration or in the β- D configuration. Previously, a-L-methyleneoxy (4'-CH2-0-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et ah, Nucleic Acids Research, 2003, 21, 6365- 6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) a-L-methyleneoxy (4'-CH2-0-2') BNA , (B) β-D-methyleneoxy (4'-CH2-0-2') BNA , (C) ethyleneoxy (4'-(CH2)2-0-2') BNA , (D) aminooxy (4'-CH2-0-N(R)-2') BNA, (E) oxyamino (4'-CH2-N(R)-0-2') BNA, and (F)
methyl(methyleneoxy) (4'-CH(CH3)-0-2') BNA, (G) methylene-thio (4'-CH2-S-2') BNA, (H) methylene- amino (4'-CH2-N(R)-2') BNA, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA, and (J) propylene carbocyclic (4'-(CH2)3-2') BNA as depicted below.
Figure imgf000039_0001
Figure imgf000039_0002
wherein Bx is the base moiety and R is independently H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are provided having Formula I:
Figure imgf000039_0003
wherein:
Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is -CH2-N(RC)-CH2-, -C(=0)-N(Rc)-CH2-, -CH2-0-N(Rc)-, -CH2-N(Rc)-0- or -N(Rc)-0-
CH2
Rc is C1-C12 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleosides are provided having Formula II:
Figure imgf000040_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is Ci-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted Ci-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJC, N3, OC(=X)Jc, and NJeC(=X)NJcJd, wherein each Jc, Jd and Je is, independently, H, CrC6 alkyl, or substituted CrC6 alkyl and X is O or NJC.
In certain embodiments, bicyclic nucleosides are provided having Formula III:
Figure imgf000040_0002
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(=0)-).
In cert leosides are provided having Formula IV:
Figure imgf000040_0003
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Pvj is Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, Ci-C6 alkoxyl, substituted Cr C6 alkoxyl, acyl, substituted acyl, Ci-C6 aminoalkyl or substituted Ci-C6 aminoalkyl;
In certain embodiments, bicyclic nucleosides are provided having Formula V:
Figure imgf000041_0001
wherein:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
qa, qb, qe and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2- C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk,
N(H)C(=NH)NJjJk, N(H)C(=0)NJjJk or N(H)C(=S)NJjJk;
or qe and qf together are =C(qg)(qh);
qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4'-CH2-0-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4'-CH2-0-2') BNA and 2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226 ). Furthermore, synthesis of 2'-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
lic nucleosides are provided having Formula VI:
Figure imgf000042_0001
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
each q;, q,-, qk and ¾ is, independently, H, halogen, C1-C12 alkyl, substituted CrCi2 alkyl, C2-Ci2 alkenyl, substituted C2-Ci2 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, CrCi2 alkoxyl, substituted Cr C12 alkoxyl, OJj, SJj, SOJj, S02Jj, NJjJk, N3, CN, C(=0)OJj, C(=0)NJjJk, C(=0)Jj, 0-C(=0)NJjJk,
N(H)C(=NH)NJjJk, N(H)C(=0)NJjJk orN(H)C(=S)NJjJk; and
¾ and ¾ or ¾ and qk together are =C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4'-(CH2)3-2' bridge and the alkenyl analog bridge 4'- CH=CH-CH2-2' have been described (Freier et al, Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al, J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al, J. Am. Chem. Soc, 2007, 129(26), 8362-8379).
As used herein, "4 '-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.
As used herein, "monocytic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, "2 '-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to: 0[(CH2)nO]mCH3, 0(CH2)nNH2, 0(CH2)nCH3, 0(CH2)nF, 0(CH2)nONH2, OCH2C(=0)N(H)CH3, and 0(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other 2'- substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, CI, Br, CN, F, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modifed nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'- O- methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854), fluoro HNA (F-HNA) or those compounds having Formula VII:
Figure imgf000043_0001
VII wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of Ta and Tb is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of Ta and Tb is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;
qi, ¾2, ¾3, ¾4, ¾5, q6 ¾nd q7 are each independently, H, Ci-C6 alkyl, substituted Ci-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of Ri and R2 is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJiJ2, SJi, N3,
Figure imgf000043_0002
Figure imgf000044_0001
and CN, wherein X is O, S or NJi and each J J2 and J3 is, independently, H or CrC6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein qb q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of qi, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of Ri and R2 is fluoro. In certain embodiments, Ri is fluoro and R2 is H; Rt is methoxy and R2 is H, and Ri is H and R2 is methoxyethoxy.
As used herein, "2'-modified" or "2 '-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2'substituents, such as allyl, amino, azido, thio, O-allyl, O-C Ci0 alkyl, -OCF3, 0-(CH2)2-0-CH3, 2'-0(CH2)2SCH3, 0-(CH2)2-0- N(Rm)(Rn), or 0-CH2-C(=0)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted Ci-Cio alkyl. 2'-modifed nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position.
As used herein, "2'-OMe" or "2'-OCH3" or "2'-0-methyl" each refers to a nucleoside comprising a sugar comprising an -OCH3 group at the 2' position of the sugar ring.
As used herein, "MOE" or "2'-MOE" or "2'-OCH2CH2OCH3" or "2'-0-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a -OCH2CH2OCH3 group at the 2' position of the sugar ring.
As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides.
In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article:
Leumann, Bioorg. Med. Chem., 2002, 10, 841-854).
Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2' -MOE. In certain embodiments, the 2' -MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH3)-0-2') bridging group. In certain embodiments, the (4'- CH(CH3)-0-2') modified nucleosides are arranged throughout the wings of a gapmer motif. Compositions and Methods for Formulating Pharmaceutical Compositions
Antisense oligonucleotides may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
An antisense compound targeted to a Tau nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. A pharmaceutically acceptable diluent includes phosphate -buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to a Tau nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the antisense compound is an antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound. Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides.
Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3'- cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5 '-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on January 16, 2003. Cell culture and antisense compounds treatment
The effects of antisense compounds on the level, activity or expression of Tau nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and are cultured according to the vendor's instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, HepG2 cells, Hep3B cells, and primary hepatocytes.
In vitro testing of antisense oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, CA). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, CA) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes
LIPOFECTAMINE (Invitrogen, Carlsbad, CA). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, CA) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommended protocols.
Analysis of inhibition of target levels or expression
Inhibition of levels or expression of a Tau nucleic acid can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer's instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents may be obtained from Invitrogen (Carlsbad, CA). RT real-time -PCR reactions are carried out by methods well known to those skilled in the art.
Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A
CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.
Probes and primers are designed to hybridize to a Tau nucleic acid. Methods for designing real- time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, CA). Analysis of Protein Levels
Antisense inhibition of Tau nucleic acids can be assessed by measuring Tau protein levels. Protein levels of Tau can be evaluated or quantitated in a variety of ways well known in the art, such as
immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
In vivo testing of antisense compounds
Antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to inhibit expression of Tau and produce phenotypic changes, such as, improved cognition and motor function. In certain embodiments, cognition is measured by novel object recognition and nestlet building activity. In certain embodiments, motor function is measured by walking initiation analysis, rotarod, grip strength, pole climb, open field performance, balance beam, hindpaw footprint testing in the animal. In certain embodiments, antisense compounds, for example, antisense oligonucleotides, are tested in animals to assess their ability to reduce hyperphosphorylated tau and neurofibrillary tangles. In certain embodiments, antisense compounds, for example, antisense oligonucleotides, are tested to assess their ability to prevent, and/or reduce severity of, seizures in a pentylenetetrazol (PTZ) induced seizure model.
Testing may be performed in normal animals, or in experimental disease models. For
administration to animals, antisense oligonucleotides are formulated in a pharmaceutically acceptable diluent, such as phosphate -buffered saline. Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous. Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with antisense oligonucleotides, RNA is isolated from CNS tissue or CSF and changes in Tau nucleic acid expression are measured. Certain Indications
In certain embodiments, provided herein are methods, compounds, and compositions of treating an individual comprising administering one or more pharmaceutical compositions described herein. In certain embodiments, the individual has a neurodegenerative disease. In certain embodiments, the individual is at risk for developing a neurodegenerative disease, including, but not limited to, a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic
Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, and Dravet's Syndrome. In certain embodiments, the individual has been identified as having a Tau associated disease. In certain embodiments, provided herein are methods for prophylactically reducing Tau expression in an individual. Certain embodiments include treating an individual in need thereof by administering to an individual a therapeutically effective amount of an antisense compound targeted to a Tau nucleic acid.
In one embodiment, administration of a therapeutically effective amount of an antisense compound targeted to a Tau nucleic acid is accompanied by monitoring of Tau levels in an individual, to determine an individual's response to administration of the antisense compound. An individual's response to
administration of the antisense compound may be used by a physician to determine the amount and duration of therapeutic intervention.
In certain embodiments, administration of an antisense compound targeted to a Tau nucleic acid results in reduction of Tau expression by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values. In certain embodiments, administration of an antisense compound targeted to a Tau nucleic acid results in improved motor function in an animal. In certain embodiments, administration of a Tau antisense compound improves motor function by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or a range defined by any two of these values.
In certain embodiments, pharmaceutical compositions comprising an antisense compound targeted to Tau are used for the preparation of a medicament for treating a patient suffering or susceptible to a neurodegenerative disease including a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, and Dravet's Syndrome. Certain Hotspot Regions
1. Nucleobases 135783-135980 of SEQ ID NO: 1
In certain embodiments, antisense oligonucleotides are designed to target nucleobases 135783- 135980 of SEQ ID NO: 1 (GENBANK Accession No. GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000). In certain embodiments, nucleobases 135783-135980 are a hotspot region. In certain embodiments, nucleobases 135783-135980 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 5-9-5 MOE gapmers, 5-7-6 MOE gapmers, and 5-8-5 MOE gapmers. In certain
embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate
internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have "mixed backbones").
In certain embodiments, nucleobases 135783-135980 are targeted by the following ISIS numbers: 424879, 424880, 548937, 613114-613120, 622096-622150, 623988-623996, 664511-664542, and 664661- 664819.
In certain embodiments, nucleobases 135783-135980 are targeted by the following SEQ ID NOs: 56, 57, 248, 462-467, 1668-1698, 2025-2048, 2301-2309, 2331- 2443, and 2478-2483.
In certain embodiments, antisense oligonucleotides targeting nucleobases 135783-135980 achieve at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, 20%), at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, or at least 93% reduction of Tau m NA and/or protein levels in vitro and/or in vivo.
2. Nucleobases 135853-135872 of SEQ ID NO: 1
In certain embodiments, antisense oligonucleotides are designed to target nucleobases 135853- 135872 of SEQ ID NO: 1 (GENBANK Accession No. GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000). In certain embodiments, nucleobases 135853-135872 are a hotspot region. In certain embodiments, nucleobases 135853-135872 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 5-9-5 MOE gapmers, 5-7-6 MOE gapmers, or 5-8-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have "mixed backbones").
In certain embodiments, nucleobases 135853-135872 are targeted by the following ISIS numbers: 424879, 424880, 613117, 613118, 622114-622125, 623993-623996, 664522-664542, 664676-664713, 664729-664766, and 664783-664819. In certain embodiments, nucleobases 135853-135872 are targeted by the following SEQ ID NOs: 56, 57, 248, 464-465, 1668-1673, 2039-2048, 2306-2309, 2345-2443, and 2478-2483.
In certain embodiments, antisense oligonucleotides targeting nucleobases 135853-135872 achieve at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, or at least 87% reduction of Tau mRNA and/or protein levels in vitro and/or in vivo.
3. Nucleobases 135783-135929 of SEP ID NO: 1
In certain embodiments, antisense oligonucleotides are designed to target nucleobases 135783- 135929 of SEQ ID NO: 1 (GENBANK Accession No. GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000). In certain embodiments, nucleobases 135783-135929 are a hotspot region. In certain embodiments, nucleobases 135783-135929 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 5-9-5 MOE gapmers, 5-7-6 MOE gapmers, or 5-8-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have "mixed backbones").
In certain embodiments, nucleobases 135783-135929 are targeted by the following ISIS numbers:
424879, 424880, 548937, 613114-613119, 622096-622138, 623988-623996, 664511-664542, and 664661- 664819.
In certain embodiments, nucleobases 135783-135929 are targeted by the following SEQ ID NOs: 56, 57, 248, 462-466, 1668-1686, 2025-2048, 2301-2309, 2331-2443, and 2478-2483.
In certain embodiments, antisense oligonucleotides targeting nucleobases 135783-135929 achieve at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, 20%), at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, or at least 93% reduction of Tau mRNA and/or protein levels in vitro and/or in vivo.
4. Nucleobases 135783-135914 of SEQ ID NO: 1
In certain embodiments, antisense oligonucleotides are designed to target nucleobases 135783- 135914 of SEQ ID NO: 1 (GENBANK Accession No. GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000). In certain embodiments, nucleobases 135783-135914 are a hotspot region. In certain embodiments, nucleobases 135783-135914 are targeted by antisense oligonucleotides. In certain embodiments, the antisense oligonucleotides are 18, 19, or 20 nucleobases in length. In certain embodiments, the antisense oligonucleotides are gapmers. In certain embodiments, the gapmers are 5-10-5 MOE gapmers, 5-9-5 MOE gapmers, 5-7-6 MOE gapmers, or 5-8-5 MOE gapmers. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphodiester internucleoside linkages. In certain embodiments, the nucleosides of the antisense oligonucleotides are linked by phosphorothioate and phosphodiester internucleotide linkages (e.g., the antisense oligonucleotides have "mixed backbones").
In certain embodiments, nucleobases 135783-135914 are targeted by the following ISIS numbers: 424879, 424880, 548937, 613114-613119, 622096-622133, 623988-623996, 664511-664542, 664661- 664819.
In certain embodiments, nucleobases 135783-135914 are targeted by the following SEQ ID NOs: 56, 57, 248, 462-466, 1668-1681, 2025-2048, 2301-2309, 2331-2443, and 2478-2483.
In certain embodiments, nucleobases 135783-135914 are targeted by the following ISIS numbers: 424879, 424880, 548937, 613114-613119, 622096- 622133, and 623988- 623996.
In certain embodiments, nucleobases 135783-135914 are targeted by the following SEQ ID NOs: 56, 57, 248, 462-466, 1668-1681, 2025-2048, and 2301-2309.
In certain embodiments, antisense oligonucleotides targeting nucleobases 135783-135914 achieve at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, or at least 93% reduction of Tau mRNA and/or protein levels in vitro and/or in vivo.
EXAMPLES
Non-limiting disclosure and incorporation by reference
While certain compounds, compositions, and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1 : Antisense inhibition of human Tau in HepG2 cells by MOE gapmers
Antisense oligonucleotides were designed targeting a tau nucleic acid and were tested for their effects on tau mRNA in vitro. Cultured HepG2 cells were transfected using Lipofectin reagent with 100 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 (forward sequence AAGATTGGGTCCCTGGACAAT, designated herein as SEQ ID NO: 10; reverse sequence AGCTTGTGGGTTTCAATCTTTTTATT, designated herein as SEQ ID NO: 11 ; probe sequence CACCCACGTCCCTGGCGGA, designated herein as SEQ ID NO: 12) was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by
RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in Table 1 below is targeted to either the human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000) or to the human tau mRNA sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NM 001123066.3). 'n/a' indicates that the oligonucleotide does not target the gene sequence with 100% complementarity. The sequences listed in Table 2 do not target either SEQ ID NO: 1 or 2 with 100% complementarity, but instead target SEQ ID NO: 3 (GENBANK Accession No. NM_016841.4, a variant mRNA sequence which skips exons 3, 4, 6, 8, 10, and 12) or SEQ ID NO: 4 (GENBANK Accession No. NT 010783.14 truncated from nucleotides 2624000 to 2761000).
Table 1
Inhibition of Tau mRNA by 5-10-5 MOE gapmers targeting SEQ ID NOs: 1 and 2
Figure imgf000054_0001
433479 130188 130207 GGACCCAATCTTCGACTGGA 70 2376 2395 52
424926 135467 135486 GTGGGTTTCAATCTGCAAGA 39 n/a n/a 53
424869 135475 135494 GTCAGCTTGTGGGTTTCAAT 74 2438 2457 54
433480 135673 135692 GATCACAAACCCTGCTTGGC 60 2636 2655 55
424879 135848 135867 TGATTTTGAAGTCCCGAGCC 64 2811 2830 56
424880 135853 135872 ATCACTGATTTTGAAGTCCC 54 2816 2835 57
433481 136033 136052 CAGAAGCAGCTTTCAGAGCC 38 2996 3015 58
433482 136118 136137 AAATCCTTTGTTGCTGCCAC 37 3081 3100 59
424882 136425 136444 CCCACAGGCTGCCCTGCAGA 57 3388 3407 60
433483 136491 136510 GGAGGTCATCCACGAAGTGC 67 3454 3473 61
433484 136577 136596 GGAAGCCCCTCAACTCAGGC 28 3540 3559 62
433485 136655 136674 GGTCTGCAAAGTGGCCAAAA 49 3618 3637 63
424883 136675 136694 TGGTTAGCCCTAAAGTCCCA 54 3638 3657 64
433486 136686 136705 ACAAAGAGAACTGGTTAGCC 39 3649 3668 65
424884 136703 136722 AAGAGGCACAAGTCCTTACA 40 3666 3685 66
433487 136748 136767 CAGAGATGCCAGTGGCCCAG 70 3711 3730 67
433488 137083 137102 GGAACCGAATCAGATCATGA 54 4046 4065 68
433489 137387 137406 TGGAGGGCTGATACTATGCA 45 4350 4369 69
433490 137430 137449 AAAAGAACCATTTCCAAGGG 41 4393 4412 70
433491 137540 137559 ATCCAACTACAACTCAACAG 17 4503 4522 71
424894 137579 137598 ATCATAGTCACTCTGGTGAA 56 4542 4561 72
433492 137694 137713 GCCACACGAGTCCCAGTGTG 45 4657 4676 73
433493 137731 137750 AAAACTTGGGAGGCCCCAGC 50 4694 4713 74
433494 138173 138192 GATCCAGGACAGGCAATTCA 58 5136 5155 75
433495 138205 138224 TCCTCAGGCAGGCAGCTTGG 49 5168 5187 76
433496 138303 138322 TTCTCATGGCAGCAGATGGA 51 5266 5285 77
433497 138338 138357 TTAGGCAGCAATGTTTTGCA 18 5301 5320 78
433498 138503 138522 AGAGTTCTGGGCCCAGAGAC 73 5466 5485 79
433499 138675 138694 AGGAAGAGGAACCGAGGTGC 59 5638 5657 80
433500 138774 138793 TCTTAGGCTGGCCCCAAGAG 36 5737 5756 81
433501 138812 138831 TCAATTTATCTGCCAGCACT 28 5775 5794 82
433502 138845 138864 TCCTCATTTAAGATCACAAG 36 5808 5827 83
433503 138983 139002 ATGGAACTATTGATAAAGTG 47 5946 5965 84
433504 139003 139022 CACCACTGAAGTCAATTTAA 23 5966 5985 85
433505 139112 139131 AGTTTAAGTGCTGCACCCCA 52 6075 6094 86
433506 139137 139156 GAAATCATGAAAAGGGTTAC 25 6100 6119 87
433507 139154 139173 CTCTAGCAAATGTGGTTGAA 42 6117 6136 88
433508 139229 139248 AGCCAGCTGCCTGGGAAAGC 70 6192 6211 89
433509 139359 139378 TAGAGGGAAGGATGCCAAGG 56 6322 6341 90
433510 139406 139425 GTGTGTCTGGAGCCAGTGTG 49 6369 6388 91
424910 139675 139694 GAAATCATGGGACTTGCAAG 52 6638 6657 92
433512 139729 139748 AAAGCTAAGCTAAGATGATT 31 6692 6711 93
424913 139797 139816 TTACAGCAACAGTCAGTGTA 42 6760 6779 94 Table 2
Inhibition of Tau mRNA by 5-10-5 MOE gapmers targeting SEQ ID NOs: 3 and 4
Figure imgf000056_0001
Example 2: Dose-dependent antisense inhibition of human tau in HepG2 cells by 5-10-5 MOE gapmers
Gapmers from studies described above exhibiting significant in vitro inhibition of Tau mRNA were selected and tested at various doses in HepG2 cells. Cells were plated at a density of 10,000 cells per well and transfected using Lipofectin reagent with 12.5 nM, 25.0 nM, 50.0 nM, 100.0 nM, or 200.0 nM concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 3
Figure imgf000056_0002
Example 3: Antisense inhibition of human Tau in SH-SY5Y cells by 5-10-5 MOE gapmers
Additional antisense oligonucleotides were designed targeting a Tau nucleic acid and were tested for their effects on Tau mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured SH-SY5Y cells were transfected using electroporation with 7,000 nM antisense
oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either the human Tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK
Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000) or to the human Tau mRNA sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NM 001123066.3). 'n/a' indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
Table 4
Figure imgf000057_0001
548793 73908 73927 TTCCTGTCCCCCAACCCGTA 58 374 393 105
548794 73910 73929 CTTTCCTGTCCCCCAACCCG 55 376 395 106
548795 73912 73931 ATCTTTCCTGTCCCCCAACC 14 378 397 107
548796 73914 73933 TGATCTTTCCTGTCCCCCAA 59 380 399 108
548797 73916 73935 CCTGATCTTTCCTGTCCCCC 67 382 401 109
548798 73918 73937 CCCCTGATCTTTCCTGTCCC 67 384 403 110
548799 73920 73939 CCCCCCTGATCTTTCCTGTC 33 386 405 111
548800 73922 73941 AGCCCCCCTGATCTTTCCTG 45 388 407 112
548801 73924 73943 GTAGCCCCCCTGATCTTTCC 62 390 409 113
548802 73926 73945 GTGTAGCCCCCCTGATCTTT 42 392 411 114
548803 73928 73947 TGGTGTAGCCCCCCTGATCT 58 394 413 115
548804 73930 73949 CATGGTGTAGCCCCCCTGAT 62 396 415 116
548805 73932 73951 TGCATGGTGTAGCCCCCCTG 82 398 417 117
548806 73934 73953 GGTGCATGGTGTAGCCCCCC 79 400 419 118
548807 73936 73955 TTGGTGCATGGTGTAGCCCC 58 402 421 119
548808 73938 73957 TCTTGGTGCATGGTGTAGCC 60 404 423 120
548809 73940 73959 GGTCTTGGTGCATGGTGTAG 67 406 425 121
548810 73945 73964 CTCTTGGTCTTGGTGCATGG 61 411 430 122
548811 73958 73977 CGTCCGTGTCACCCTCTTGG 73 424 443 123
548812 98559 98578 GCTTTTACTGACCATGCGAG 68 1449 1468 124
548813 98561 98580 TTGCTTTTACTGACCATGCG 64 1451 1470 125
548814 98563 98582 CTTTGCTTTTACTGACCATG 40 1453 1472 126
548815 98565 98584 GTCTTTGCTTTTACTGACCA 60 1455 1474 127
548816 98567 98586 CCGTCTTTGCTTTTACTGAC 50 1457 1476 128
548817 98569 98588 TCCCGTCTTTGCTTTTACTG 46 1459 1478 129
548818 98571 98590 AGTCCCGTCTTTGCTTTTAC 41 1461 1480 130
548819 98573 98592 CCAGTCCCGTCTTTGCTTTT 37 1463 1482 131
548820 98577 98596 GCTTCCAGTCCCGTCTTTGC 43 1467 1486 132
548821 98579 98598 TCGCTTCCAGTCCCGTCTTT 35 1469 1488 133
548822 98581 98600 CATCGCTTCCAGTCCCGTCT 73 1471 1490 134
548823 98583 98602 GTCATCGCTTCCAGTCCCGT 63 1473 1492 135
548824 98585 98604 TTGTCATCGCTTCCAGTCCC 50 1475 1494 136
548825 98587 98606 TTTTGTCATCGCTTCCAGTC 46 1477 1496 137
548826 103023 103042 GGCCCTTCTGGCCTGGAGGG 64 1747 1766 138
548827 103025 103044 CTGGCCCTTCTGGCCTGGAG 29 1749 1768 139
548828 103027 103046 GCCTGGCCCTTCTGGCCTGG 40 1751 1770 140
548829 103029 103048 TGGCCTGGCCCTTCTGGCCT 12 1753 1772 141
548830 103031 103050 GTTGGCCTGGCCCTTCTGGC 53 1755 1774 142
548831 103052 103071 TTTTGCTGGAATCCTGGTGG 20 1776 1795 143
548832 103054 103073 GTTTTTGCTGGAATCCTGGT 29 1778 1797 144
548833 103056 103075 GGGTTTTTGCTGGAATCCTG 51 1780 1799 145
548834 103075 103094 GGTGTCTTTGGAGCGGGCGG 44 1799 1818 146
548835 103077 103096 GTGGTGTCTTTGGAGCGGGC 30 1801 1820 147 548836 103079 103098 GGGTGGTGTCTTTGGAGCGG 38 1803 1822 148
548837 103081 103100 CTGGGTGGTGTCTTTGGAGC 29 1805 1824 149
548838 103083 103102 AGCTGGGTGGTGTCTTTGGA 37 1807 1826 150
548839 103085 103104 AGAGCTGGGTGGTGTCTTTG 0 1809 1828 151
548840 107921 107940 ATCCCCTGATTTTGGAGGTT 27 1887 1906 152
548841 107923 107942 CGATCCCCTGATTTTGGAGG 65 1889 1908 153
548842 107925 107944 TGCGATCCCCTGATTTTGGA 53 1891 1910 154
548843 107927 107946 GCTGCGATCCCCTGATTTTG 23 1893 1912 155
548844 107929 107948 CCGCTGCGATCCCCTGATTT 32 1895 1914 156
548845 107931 107950 AGCCGCTGCGATCCCCTGAT 51 1897 1916 157
548846 107933 107952 GTAGCCGCTGCGATCCCCTG 14 1899 1918 158
548847 107968 107987 CGGCTGCCGGGAGTGCCTGG 33 1934 1953 159
548848 107970 107989 AGCGGCTGCCGGGAGTGCCT 52 1936 1955 160
548849 107972 107991 GGAGCGGCTGCCGGGAGTGC 44 1938 1957 161
548850 108047 108066 CGACTTGGGTGGAGTACGGA 10 2013 2032 162
548851 108049 108068 GGCGACTTGGGTGGAGTACG 29 2015 2034 163
548852 108051 108070 ACGGCGACTTGGGTGGAGTA 39 2017 2036 164
548853 108053 108072 AGACGGCGACTTGGGTGGAG 6 2019 2038 165
548854 108055 108074 GAAGACGGCGACTTGGGTGG 0 2021 2040 166
548855 108057 108076 CGGAAGACGGCGACTTGGGT 40 2023 2042 167
548856 108059 108078 GGCGGAAGACGGCGACTTGG 38 2025 2044 168
548857 108061 108080 TTGGCGGAAGACGGCGACTT 41 2027 2046 169
548858 108063 108082 TCTTGGCGGAAGACGGCGAC 35 2029 2048 170
548859 108065 108084 GCTCTTGGCGGAAGACGGCG 54 2031 2050 171
548860 108067 108086 CGGCTCTTGGCGGAAGACGG 58 2033 2052 172
548861 108069 108088 GGCGGCTCTTGGCGGAAGAC 55 2035 2054 173
433498 138503 138522 AGAGTTCTGGGCCCAGAGAC 53 5466 5485 79
Table 5
Figure imgf000059_0001
548888 85939 85958 TGCGCGGCAGCCTGCTTGCC 32 578 597 184
548889 94699 94718 ACCACCTTACCACTTTCAGG 35 698 717 185
548890 94775 94794 GGGAGCCCCAGGCATGCCGG 45 774 793 186
548891 95125 95144 TTGGAGAGGAAATCCACAGG 13 1124 1143 187
548892 95131 95150 GAAACTTTGGAGAGGAAATC 0 1130 1149 188
548896 98557 98576 TTTTACTGACCATGCGAGCT 40 1447 1466 189
548897 101425 101444 AAGGCAAGGCCTATTTTTCA 34 1533 1552 190
548898 101469 101488 TGGATCAGAGGGTCTGAGCT 40 1577 1596 191
548899 101501 101520 GTGGCTCTGGGCACACAGCA 63 1609 1628 192
548900 108037 108056 GGAGTACGGACCACTGCCAC 37 2003 2022 193
433477 108040 108059 GGTGGAGTACGGACCACTGC 21 2006 2025 40
548862 108071 108090 CAGGCGGCTCTTGGCGGAAG 24 2037 2056 194
548863 108073 108092 TGCAGGCGGCTCTTGGCGGA 34 2039 2058 195
548864 108075 108094 TCTGCAGGCGGCTCTTGGCG 40 2041 2060 196
548865 108129 108148 TGGAGCCGATCTTGGACTTG 23 2095 2114 197
548866 108131 108150 AGTGGAGCCGATCTTGGACT 4 2097 2116 198
548901 108134 108153 CTCAGTGGAGCCGATCTTGG 28 2100 2119 199
548902 108136 108155 TTCTCAGTGGAGCCGATCTT 9 2102 2121 200
548903 108155 108174 TCCCGGCTGGTGCTTCAGGT 33 2121 2140 201
548904 121675 121694 TTTCGGACTTGGCAGAGGCA 50 n/a n/a 202
548905 121728 121747 GAGTGACATGCGCCACCCTG 65 n/a n/a 203
548906 121736 121755 CTTTCGATGAGTGACATGCG 19 n/a n/a 204
548907 121794 121813 AAAAGGATGAGTGACACGCC 58 n/a n/a 205
548908 121916 121935 GTGAAGGTACTCACACTGCC 34 n/a n/a 206
548909 121917 121936 TGTGAAGGTACTCACACTGC 21 n/a n/a 207
548910 121992 122011 CTATGCAGTGTCTCGCAAGT 39 n/a n/a 208
548911 122001 122020 GGATTTATTCTATGCAGTGT 38 n/a n/a 209
548912 122002 122021 AGGATTTATTCTATGCAGTG 37 n/a n/a 210
548913 122015 122034 TGAGAGCCCAAGAAGGATTT 15 n/a n/a 211
548914 122022 122041 CAGATCCTGAGAGCCCAAGA 33 n/a n/a 212
548915 122025 122044 AGCCAGATCCTGAGAGCCCA 55 n/a n/a 213
548916 122137 122156 GGACCCAGAAATGCTGGGAC 18 n/a n/a 214
548917 125555 125574 AAATGTGTTGTCGAAATTCT 0 n/a n/a 215
548918 125568 125587 AGTCAGGGTGGAAAAATGTG 0 n/a n/a 216
548919 125578 125597 AGCACATCCTAGTCAGGGTG 39 n/a n/a 217
548920 125587 125606 GCCATGAGGAGCACATCCTA 45 n/a n/a 218
548921 125600 125619 GTGGTTCCCAGCTGCCATGA 38 n/a n/a 219
548922 125603 125622 ACAGTGGTTCCCAGCTGCCA 38 n/a n/a 220
548923 125619 125638 CCCAGGCCCTTATTGGACAG 19 n/a n/a 221
548867 125778 125797 CTTGCTCAGGTCAACTGGTT 45 2259 2278 222
548925 125812 125831 GGATGTTGCCTAATGAGCCA 35 2293 2312 223
548926 130194 130213 GTCCAGGGACCCAATCTTCG 87 2382 2401 224
548927 130196 130215 TTGTCCAGGGACCCAATCTT 48 2384 2403 225 548928 135478 135497 AAGGTCAGCTTGTGGGTTTC 52 2441 2460 226
548868 135518 135537 CTCCGCCCCGTGGTCTGTCT 37 2481 2500 227
548869 135520 135539 ATCTCCGCCCCGTGGTCTGT 25 2483 2502 228
548870 135522 135541 CGATCTCCGCCCCGTGGTCT 48 2485 2504 229
548871 135524 135543 CACGATCTCCGCCCCGTGGT 38 2487 2506 230
548872 135526 135545 TACACGATCTCCGCCCCGTG 33 2489 2508 231
548873 135528 135547 TGTACACGATCTCCGCCCCG 22 2491 2510 232
548874 135530 135549 CTTGTACACGATCTCCGCCC 38 2493 2512 233
548875 135532 135551 GACTTGTACACGATCTCCGC 13 2495 2514 234
548876 135534 135553 GCGACTTGTACACGATCTCC 40 2497 2516 235
548877 135536 135555 TGGCGACTTGTACACGATCT 33 2499 2518 236
548878 135538 135557 ACTGGCGACTTGTACACGAT 25 2501 2520 237
548879 135540 135559 CCACTGGCGACTTGTACACG 19 2503 2522 238
548929 135546 135565 CAGACACCACTGGCGACTTG 33 2509 2528 239
548930 135593 135612 GCTGCCGGTGGAGGAGACAT 29 2556 2575 240
548931 135594 135613 TGCTGCCGGTGGAGGAGACA 19 2557 2576 241
548932 135600 135619 TGTCGATGCTGCCGGTGGAG 20 2563 2582 242
548933 135601 135620 ATGTCGATGCTGCCGGTGGA 20 2564 2583 243
548934 135610 135629 GAGTCTACCATGTCGATGCT 23 2573 2592 244
548880 135654 135673 CCAGGGAGGCAGACACCTCG 28 2617 2636 245
548935 135662 135681 CTGCTTGGCCAGGGAGGCAG 14 2625 2644 246
548936 135674 135693 TGATCACAAACCCTGCTTGG 25 2637 2656 247
548937 135786 135805 CCGAACTGCGAGGAGCAGCT 66 2749 2768 248
433498 138503 138522 AGAGTTCTGGGCCCAGAGAC 34 5466 5485 79
Example 4: Dose-dependent antisense inhibition of human Tau in SH-SY5Y cells by 5-10-5 MOE gapmers
Gapmers from studies described above exhibiting significant in vitro inhibition of Tau mRNA were selected and tested at various doses in SH-SY-5Y cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 1.25 μΜ, 2.50 μΜ, 5.00 μΜ, 10.00 μΜ, and 20.00 μΜ concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Tau, relative to untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Table 6
Figure imgf000062_0001
Example 5: Antisense inhibition of human Tau in SH-SY5Y cells by 5-10-5 MOE gapmers
Additional antisense oligonucleotides were designed targeting a Tau nucleic acid and were tested for their effects on Tau mRNA in vitro. Cultured SH-SY5Y cells were plated at a density of 20,000 cells per well and were transfected using electroporation with 6,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to the human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000).
Table 7 SEQ ID SEQ ID
ISIS % SEQ
NO: 1 NO: 1 Sequence
NO inhibition ID NO Start Site Stop Site
433474 73879 73898 GTGATCTTCCATCACTTCGA 63 25
26760 26779
559166 TGCTTACACACCACACACAC 0 249
26944 26963
26761 26780
559167 ATGCTTACACACCACACACA 4 250
26945 26964
26762 26781
559168 CATGCTTACACACCACACAC 0 251
26946 26965
81022 81041
559169 TACAGATATATGATTGGATG 0 252
81125 81144
81023 81042
559170 GTACAGATATATGATTGGAT 0 253
81126 81145
81024 81043
559171 TGTACAGATATATGATTGGA 0 254
81127 81146
81025 81044
559172 ATGTACAGATATATGATTGG 0 255
81128 81147
81026 81045
559173 TATGTACAGATATATGATTG 0 256
81129 81148
81027 81046
559174 TTATGTACAGATATATGATT 0 257
81130 81149
81028 81047
559175 ATTATGTACAGATATATGAT 0 258
81131 81150
81029 81048
559176 GATTATGTACAGATATATGA 0 259
81132 81151
81030 81049
559177 GGATTATGTACAGATATATG 0 260
81133 81152
81031 81050
559178 TGGATTATGTACAGATATAT 0 261
81134 81153
81032 81051
559179 ATGGATTATGTACAGATATA 0 262
81135 81154
81033 81052
559180 AATGGATTATGTACAGATAT 0 263
81136 81155
81034 81053
559181 GAATGGATTATGTACAGATA 0 264
81137 81156
81035 81054
559182 AGAATGGATTATGTACAGAT 0 265
81138 81157
81036 81055
559183 AAGAATGGATTATGTACAGA 0 266
81139 81158
81037 81056
559184 GAAGAATGGATTATGTACAG 0 267
81140 81159
559185 81038 81057 GGAAGAATGGATTATGTACA 0 268 81141 81160
81039 81058
559186 GGGAAGAATGGATTATGTAC 0 269
81142 81161
81040 81059
559187 AGGGAAGAATGGATTATGTA 0 270
81143 81162
81041 81060
559188 GAGGGAAGAATGGATTATGT 0 271
81144 81163
81042 81061
559189 CGAGGGAAGAATGGATTATG 0 272
81145 81164
81043 81062
559190 CCGAGGGAAGAATGGATTAT 33 273
81146 81165
81044 81063
559191 ACCGAGGGAAGAATGGATTA 12 274
81147 81166
81045 81064
559192 AACCGAGGGAAGAATGGATT 1 275
81148 81167
81046 81065
559193 GAACCGAGGGAAGAATGGAT 0 276
81149 81168
81047 81066
559194 TGAACCGAGGGAAGAATGGA 0 277
81150 81169
81048 81067
559195 ATGAACCGAGGGAAGAATGG 0 278
81151 81170
81049 81068
559196 GATGAACCGAGGGAAGAATG 0 279
81152 81171
81050 81069
559197 GGATGAACCGAGGGAAGAAT 0 280
81153 81172
81051 81070
559198 TGGATGAACCGAGGGAAGAA 0 281
81154 81173
81052 81071
559199 ATGGATGAACCGAGGGAAGA 0 282
81155 81174
81053 81072
559200 GATGGATGAACCGAGGGAAG 0 283
81156 81175
81054 81073
559201 GGATGGATGAACCGAGGGAA 1 284
81157 81176
81055 81074
559202 TGGATGGATGAACCGAGGGA 0 285
81158 81177
81056 81075
559203 ATGGATGGATGAACCGAGGG 0 286
81159 81178
81057 81076
559204 GATGGATGGATGAACCGAGG 0 287
81160 81179
81058 81077
559205 GGATGGATGGATGAACCGAG 0 288
81161 81180
81096 81115
559206 AAGGATGAAGGAAGGGATGG 0 289
81199 81218 81097 81116
559207 GAAGGATGAAGGAAGGGATG 0 290
81200 81219
81098 81117
559208 GGAAGGATGAAGGAAGGGAT 0 291
81201 81220
81100 81119
559209 TAGGAAGGATGAAGGAAGGG 0 292
81203 81222
81101 81120
559210 ATAGGAAGGATGAAGGAAGG 0 293
81204 81223
81102 81121
559211 GATAGGAAGGATGAAGGAAG 0 294
81205 81224
81103 81122
559212 TGATAGGAAGGATGAAGGAA 0 295
81206 81225
81104 81123
559213 ATGATAGGAAGGATGAAGGA 0 296
81207 81226
81105 81124
559214 GATGATAGGAAGGATGAAGG 0 297
81208 81227
81106 81125
559215 GGATGATAGGAAGGATGAAG 0 298
81209 81228
81107 81126
559216 TGGATGATAGGAAGGATGAA 0 299
81210 81229
81108 81127
559217 ATGGATGATAGGAAGGATGA 0 300
81211 81230
81109 81128
559218 GATGGATGATAGGAAGGATG 0 301
81212 81231
81110 81129
559219 GGATGGATGATAGGAAGGAT 0 302
81213 81232
81111 81130
559220 TGGATGGATGATAGGAAGGA 0 303
81214 81233
81112 81131
559221 TTGGATGGATGATAGGAAGG 0 304
81215 81234
81113 81132
559222 ATTGGATGGATGATAGGAAG 0 305
81216 81235
81114 81133
559223 GATTGGATGGATGATAGGAA 0 306
81217 81236
81115 81134
559224 TGATTGGATGGATGATAGGA 0 307
81218 81237
81116 81135
559225 ATGATTGGATGGATGATAGG 0 308
81219 81238
81117 81136
559226 TATGATTGGATGGATGATAG 0 309
81220 81239
80890 80909
559227 AGATATGGGTGGATGGATGG 0 310
81472 81491
559228 80891 80910 AAGATATGGGTGGATGGATG 0 311 81473 81492
80892 80911
559229 GAAGATATGGGTGGATGGAT 0 312
81474 81493
98881 98900
559230 ACACACCTTCATTTACTGTC 79 313
98918 98937
98882 98901
559231 AACACACCTTCATTTACTGT 56 314
98919 98938
98883 98902
559232 AAACACACCTTCATTTACTG 59 315
98920 98939
98884 98903
559233 CAAACACACCTTCATTTACT 51 316
98921 98940
98885 98904
559234 TCAAACACACCTTCATTTAC 25 317
98922 98941
98887 98906
559235 TTTCAAACACACCTTCATTT 25 318
98924 98943
98888 98907
559236 TTTTCAAACACACCTTCATT 14 319
98925 98944
98889 98908
559237 GTTTTCAAACACACCTTCAT 42 320
98926 98945
98890 98909
559238 GGTTTTCAAACACACCTTCA 79 321
98927 98946
98891 98910
559239 TGGTTTTCAAACACACCTTC 87 322
98928 98947
111795 111814
559240 GAAAACAGAGAAAAGCACAA 12 323
111819 111838
119594 119613
559241 AGAAGTGATATCATATCCTA 63 324
119905 119924
119595 119614
559242 AAGAAGTGATATCATATCCT 74 325
119906 119925
Example 6: Dose-dependent antisense inhibition of human tau in SH-SY5Y cells by 5-10-5 MOE gapmers Gapmers from studies described above exhibiting significant in vitro inhibition of Tau mRNA were selected and tested at various doses in SH-SY-5Y cells. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.625 μΜ, 1.25 μΜ, 2.500 μΜ,5.00 μΜ, 10.00 μΜ, and 20.00 μΜ concentrations of antisense oligonucleotide, as specified in the Table below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mR A levels. Tau mR A levels were adjusted according to total R A content, as measured by RIBOGREEN®. Results are presented as percent inhibition of Tau, relative to untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells. Table 8
Figure imgf000067_0001
Example 7: Antisense inhibition of human Tau in SH-SY5Y cells by MOE gapmers with
Phosphorothioate and Phosphodiester Internucleoside Linkages
Antisense oligonucleotides were designed targeting a tau nucleic acid and were tested for their effects on tau mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured SH-SY5Y cells were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by
RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification. The internucleoside linkages throughout each gapmer are either phosphorothioate linkages or phosphodiester linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. The 'Chemistry' column describes the internucleoside linkages of each oligonucleotide, 's' indicates phosphorothioate linkage and Ό' indicates phosphodiester linkage. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3' -most nucleoside to which the gapmer is targeted human gene sequence.
Each gapmer listed in the Tables below is targeted to either the human Tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000), human Tau mRNA sequences, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NM 001123066.3) or SEQ ID NO: 3 (GENBANK Accession No. NM_016841.4). Several oligonucleotides, presented in Tables 10, 12, and 16, target variant mRNA sequences, designated herein as SEQ ID NO: 5 (GENBANK Accession No. DR002467.1), SEQ ID NO: 6 (GENBANK Accession No. NM 001203251.1) or SEQ ID NO: 7 (GENBANK Accession No. NM_016835.4). The oligonucleotides are
10 presented in the various tables according to the main gene sequence that they target with 100%
complementarity, 'n/a' indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
Table 9
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester
15 inter ucleoside linkages targetin; g SEQ ID NOs: 1 and 3
Figure imgf000068_0001
613183 138116 138135 AGGCTGCGCTGCCCCTCCAG 68 3807 3826 soooossssssssssooss 342
613184 138149 138168 AATCCCTGCTGTGGTCGCAG 63 3840 3859 soooossssssssssooss 343
613185 138182 138201 TCTAGAGCAGATCCAGGACA 53 3873 3892 soooossssssssssooss 344
613186 138215 138234 AAGTCATCCTTCCTCAGGCA 63 3906 3925 soooossssssssssooss 345
613187 138248 138267 C AAGGCTTT GGGAAC AGT GT 78 3939 3958 soooossssssssssooss 346
613188 138250 138269 GTCAAGGCTTTGGGAACAGT 84 3941 3960 soooossssssssssooss 347
613189 138281 138300 TTGTGCAAGGTCAGCGGGCT 75 3972 3991 soooossssssssssooss 348
613190 138314 138333 CGGCTTCCCTTTTCTCATGG 75 4005 4024 soooossssssssssooss 349
613191 138347 138366 TGAGTTTCTTTAGGCAGCAA 82 4038 4057 soooossssssssssooss 350
613192 138380 138399 AACCAGAAGTGGCAGAATTG 47 4071 4090 soooossssssssssooss 351
613193 138413 138432 CAAGTCCCTCAGGGTTGCCT 78 4104 4123 soooossssssssssooss 352
613194 138454 138473 CGAAGCTGCCAGCCCCAGGG 70 4145 4164 soooossssssssssooss 353
613195 138487 138506 AGACTT CCTTT C AGGT AAAG 77 4178 4197 soooossssssssssooss 354
613196 138503 138522 AGAGTTCTGGGCCCAGAGAC 58 4194 4213 soooossssssssssooss 79
613197 138520 138539 AGGGAGGCTCTTGGTGGAGA 35 4211 4230 soooossssssssssooss 355
613198 138553 138572 ACTTAGGAGAATTGCTGGGA 75 4244 4263 soooossssssssssooss 356
613199 138586 138605 CCCACATTTCCTTCTCCTTC 72 4277 4296 soooossssssssssooss 357
613200 138636 138655 ACTGTTGGCAGTAATGAGGG 71 4327 4346 soooossssssssssooss 358
613201 138674 138693 GGAAGAGGAACCGAGGTGCG 61 4365 4384 soooossssssssssooss 359
613202 138707 138726 CATGGTGCTGAAGAGCAGGG 71 4398 4417 soooossssssssssooss 360
613203 138740 138759 GGGAGATCCCAGAGCCTTCC 53 4431 4450 soooossssssssssooss 361
613204 138773 138792 CTTAGGCTGGCCCCAAGAGC 64 4464 4483 soooossssssssssooss 362
613205 138806 138825 TATCTGCCAGCACTGATCAC 56 4497 4516 soooossssssssssooss 363
613206 138839 138858 TTTAAGATCACAAGCCAGCG 62 4530 4549 soooossssssssssooss 364
613207 138872 138891 GAGGAGTGCCCAGCCCTGGG 54 4563 4582 soooossssssssssooss 365
613208 138905 138924 CTGGCTCTGCAGGTGGGAGA 44 4596 4615 soooossssssssssooss 366
613209 138938 138957 T AC AGT AT ATC CT AT CT AGC 57 4629 4648 soooossssssssssooss 367
613210 138971 138990 ATAAAGTGAGTCAGCAGCTT 71 4662 4681 soooossssssssssooss 368
613211 139004 139023 TCACCACTGAAGTCAATTTA 72 4695 4714 soooossssssssssooss 369
613212 139037 139056 AGCACAACAAGCAATAGCAA 80 4728 4747 soooossssssssssooss 370
613213 139070 139089 ACTATCTTACACATTCCTCC 48 4761 4780 soooossssssssssooss 371
613214 139103 139122 GCTGCACCCCAAGATCTCCC 79 4794 4813 soooossssssssssooss 372
613215 139136 139155 AAATCATGAAAAGGGTTACG 61 4827 4846 soooossssssssssooss 373
613216 139169 139188 GTGGCTGCTCCCTCCCTCTA 60 4860 4879 soooossssssssssooss 374
613217 139188 139207 CCCAAGGGCCTCTAACTCCG 64 4879 4898 soooossssssssssooss 375
613218 139202 139221 TGGAAAAGAGAAACCCCAAG 56 4893 4912 soooossssssssssooss 376
613219 139229 139248 AGCCAGCTGCCTGGGAAAGC 63 4920 4939 soooossssssssssooss 89
613220 139235 139254 TGAACTAGCCAGCTGCCTGG 46 4926 4945 soooossssssssssooss 377
613221 139268 139287 TATTCCTACGCCTGCACCTG 54 4959 4978 soooossssssssssooss 378
613222 139301 139320 AGAGGGCAGCAGGCCAAAGC 48 4992 5011 soooossssssssssooss 379
613223 139337 139356 TTAGGGAGGCATGATTGTGG 55 5028 5047 soooossssssssssooss 380
613224 139368 139387 CCAACGGCTTAGAGGGAAGG 61 5059 5078 soooossssssssssooss 381
613225 139370 139389 TGCCAACGGCTTAGAGGGAA 65 5061 5080 soooossssssssssooss 382 613226 139403 139422 TGTCTGGAGCCAGTGTGAGA 56 5094 5113 soooossssssssssooss 383
613227 139436 139455 CGAGTGATCTCAGCTCCAAA 79 5127 5146 soooossssssssssooss 384
613228 139502 139521 TGATCACCTCTGCCCTCGCC 72 5193 5212 soooossssssssssooss 385
613229 139535 139554 TGAAGCTGCAGGTCTGTAGA 57 5226 5245 soooossssssssssooss 386
613230 139568 139587 CTTTTC AAAGCT GAAGAGAA 57 5259 5278 soooossssssssssooss 387
613231 139601 139620 AGGTGAGGCTCTAGGCCAGT 76 5292 5311 soooossssssssssooss 388
613232 139636 139655 CAACATGGCAAACTCATGGG 76 5327 5346 soooossssssssssooss 389
613233 139669 139688 ATGGGACTTGCAAGTGCCAG 83 5360 5379 soooossssssssssooss 390
613234 139702 139721 CCTCCCCCCACCCTCAGAAT 15 5393 5412 soooossssssssssooss 391
613235 139735 139754 AGACAGAAAGCTAAGCTAAG 50 5426 5445 soooossssssssssooss 392
613236 139768 139787 TTAAAACACACAATACACTA 46 5459 5478 soooossssssssssooss 393
613237 139801 139820 ACTTTTACAGCAACAGTCAG 54 5492 5511 soooossssssssssooss 394
613238 139834 139853 TTTAATCAGAGTAATAACTT 12 5525 5544 soooossssssssssooss 395
Table 10
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages targeting SEQ ID NOs: 5 and 6
5
Figure imgf000070_0001
Table 11
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages targeting SEQ ID NOs: 1 and 3
Figure imgf000070_0002
613036 73869 73888 ATCACTTCGAACTCCTGGCG 72 335 354 soooossssssssssooss 99
613037 73871 73890 CCATCACTTCGAACTCCTGG 69 337 356 soooossssssssssooss 100
613412 73879 73898 GTGATCTTCCATCACTTCGA 72 345 364 soooossssssssssooss 25
613038 73906 73925 CCTGTCCCCCAACCCGTACG 44 372 391 soooossssssssssooss 104
613039 73932 73951 TGCATGGTGTAGCCCCCCTG 80 398 417 soooossssssssssooss 117
613040 73934 73953 GGTGCATGGTGTAGCCCCCC 73 400 419 soooossssssssssooss 118
613041 73939 73958 GTCTTGGTGCATGGTGTAGC 77 405 424 soooossssssssssooss 407
613042 73958 73977 CGTCCGTGTCACCCTCTTGG 79 424 443 soooossssssssssooss 123
613044 83397 83416 CCTCAGATCCGTCCTCAGTG 67 n/a n/a soooossssssssssooss 408
613045 83430 83449 TGCTCTTAGCATCAGAGGTT 89 n/a n/a soooossssssssssooss 409
613046 85902 85921 CTAAGGGTGCTGTCACATCT 69 n/a n/a soooossssssssssooss 410
613047 85934 85953 GGCAGCCTGCTTGCCGGGAG 51 n/a n/a soooossssssssssooss 29
613048 85935 85954 CGGCAGCCTGCTTGCCGGGA 51 n/a n/a soooossssssssssooss 411
613049 85968 85987 TGGTTCCTTCTGGGATCTCC 76 n/a n a soooossssssssssooss 412
613051 94719 94738 CTCGGAGGAAGCCTTCCTGG 53 n/a n/a soooossssssssssooss 413
613052 94752 94771 TGAGCTGGTGGCTCAGACCT 41 n a n a soooossssssssssooss 414
613053 94825 94844 CCTGTCCCCGAAGGTTGGCG 61 n a n a soooossssssssssooss 415
613054 94875 94894 GGTGCTTGAGCAGCTCAGGG 70 n/a n/a soooossssssssssooss 416
613055 94908 94927 GCGGCCCCTCCTGGTGCAGG 47 n/a n a soooossssssssssooss 417
613056 94941 94960 CCGGCCTCTCTTTGCCCCCT 31 n/a n/a soooossssssssssooss 418
613057 94974 94993 CGTCGCGGTCTTCATCCACC 42 n/a n/a soooossssssssssooss 419
613058 95038 95057 GGCCGCCCATCTTGGGCTGG 0 n/a n/a soooossssssssssooss 420
613059 95071 95090 ATGCTGGTGGCTTCTCTGGC 53 n/a n a soooossssssssssooss 421
613060 95098 95117 ATGGCACCCTCCGCTGGGAA 39 n/a n a soooossssssssssooss 422
613061 95121 95140 AGAGGAAATCCACAGGGAGG 30 n/a n/a soooossssssssssooss 33
613062 95154 95173 GCTCTGAGGCTGGGATCTCT 70 n/a n/a soooossssssssssooss 423
613063 95187 95206 GCCCTTTGGCCCGCCCTACA 67 n/a n a soooossssssssssooss 424
613064 95235 95254 CGTTGGGTGTGATTTCCACG 61 n/a n a soooossssssssssooss 425
613065 95268 95287 AATGCTCCTCCGAGTGCGCC 85 n/a n a soooossssssssssooss 426
613066 95313 95332 CCTCTGGCCCCTCTCCAGGG 43 n/a n/a soooossssssssssooss 427
613067 95346 95365 CTTTTGTGTCCTCTCCCAAA 42 n/a n a soooossssssssssooss 428
613068 95379 95398 GCTGCTTTTCAGAGGGCTCT 51 n/a n/a soooossssssssssooss 429
613069 95412 95431 GGCTGACGGGCTTCCCCCGC 61 n/a n a soooossssssssssooss 430
613070 95429 95448 TTTGAGTTGAGGGACCCGGC 33 n/a n/a soooossssssssssooss 431
613073 98559 98578 GCTTTTACTGACCATGCGAG 79 522 541 soooossssssssssooss 124
613074 98581 98600 CATCGCTTCCAGTCCCGTCT 78 544 563 soooossssssssssooss 134
613075 98587 98606 TTTTGTCATCGCTTCCAGTC 35 550 569 soooossssssssssooss 137
613076 101406 101425 AAGGTTTTAGCAGAGGAACG 63 n/a n a soooossssssssssooss 37
613077 101457 101476 TCTGAGCTACCAGGAGTGGG 65 n/a n a soooossssssssssooss 432
613078 101491 101510 GCACACAGCAGGGCTGGAGG 68 n/a n a soooossssssssssooss 433
613079 101501 101520 GTGGCTCTGGGCACACAGCA 77 n a n a soooossssssssssooss 192
613080 101507 101526 AGGAAGGTGGCTCTGGGCAC 69 n/a n a soooossssssssssooss 38
613081 101537 101556 GGGAAGTGACAGAAGAGACG 46 n/a n a soooossssssssssooss 434 613082 101570 101589 GTTTCATCTCCTTTGCTCCA 57 n/a n a soooossssssssssooss 435
613083 102995 103014 CCGCGGTGTGGCGATCTTCG 9 594 613 soooossssssssssooss 436
613084 103028 103047 GGCCTGGCCCTTCTGGCCTG 14 627 646 soooossssssssssooss 437
613085 103075 103094 GGTGTCTTTGGAGCGGGCGG 37 674 693 soooossssssssssooss 146
613086 105445 105464 CTT CTCT GGACTT GCTT AGT 45 n/a n/a soooossssssssssooss 438
613087 105475 105494 CTCTCAGATCTGGGCCCTGC 41 n/a n/a soooossssssssssooss 439
613088 105478 105497 CCTCTCTCAGATCTGGGCCC 49 n/a n a soooossssssssssooss 440
613089 107932 107951 TAGCCGCTGCGATCCCCTGA 19 719 738 soooossssssssssooss 441
613090 107968 107987 CGGCTGCCGGGAGTGCCTGG 31 755 774 soooossssssssssooss 159
613091 108021 108040 CCACCTTCTTGGGCTCCCGG 17 808 827 soooossssssssssooss 442
613092 108040 108059 GGTGGAGTACGGACCACTGC 20 827 846 soooossssssssssooss 40
613093 108054 108073 AAGACGGCGACTTGGGTGGA 0 841 860 soooossssssssssooss 41
613094 108101 108120 CAGGTCTGGCATGGGCACGG 67 888 907 soooossssssssssooss 443
613095 108134 108153 CTCAGTGGAGCCGATCTTGG 31 921 940 soooossssssssssooss 199
613096 108167 108186 CACCTTCCCGCCTCCCGGCT 47 954 973 soooossssssssssooss 444
613097 121845 121864 GTTGCTAAGATCCAGCTTCT 49 n/a n/a soooossssssssssooss 445
613098 121865 121884 GAGCCACACTTGGACTGGAC 85 n/a n/a soooossssssssssooss 47
613099 121878 121897 GATATTATCCTTTGAGCCAC 88 n/a n/a soooossssssssssooss 446
613101 125784 125803 GGTCACCTTGCTCAGGTCAA 68 993 1012 soooossssssssssooss 447
613102 125817 125836 ATGATGGATGTTGCCTAATG 14 1026 1045 soooossssssssssooss 448
613103 130141 130160 ATTTTACTTCCACCTGGCCA 22 1057 1076 soooossssssssssooss 51
Table 12
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages targeting SEQ ID NOs: 6 and 7
5
Figure imgf000072_0001
Table 13
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages targeting SEQ ID NOs: 1 and 3
10
SEQ SEQ
ID ID
% NO: NO:
Sequence Linkage chemistry
inhibition 3 3
Start Stop
Figure imgf000072_0002
Site Site 433475 n a n/a TCTTCAGCTTTCAGGCCAGC 23 443 462 sssssssssssssssssss 96
613412 73879 73898 GTGATCTTCCATCACTTCGA 76 345 364 soooossssssssssooss 25
613104 130143 130162 AGATTTTACTTCCACCTGGC 59 1059 1078 soooossssssssssooss 453
613105 130176 130195 CGACTGGACTCTGTCCTTGA 47 1092 1111 soooossssssssssooss 454
613106 130225 130244 TATTTCCTCCGCCAGGGACG 90 1141 1160 soooossssssssssooss 455
613107 135483 135502 CGCGGAAGGTCAGCTTGTGG 43 1174 1193 soooossssssssssooss 456
613108 135516 135535 CCGCCCCGTGGTCTGTCTTG 44 1207 1226 soooossssssssssooss 457
613109 135549 135568 CCCCAGACACCACTGGCGAC 36 1240 1259 soooossssssssssooss 458
613110 135590 135609 GCCGGTGGAGGAGACATTGC 51 1281 1300 soooossssssssssooss 459
613111 135654 135673 CCAGGGAGGCAGACACCTCG 61 1345 1364 soooossssssssssooss 245
613112 135697 135716 AATTATTGACCGCCCCAGGG 33 1388 1407 soooossssssssssooss 460
613113 135730 135749 TTTTTCCACACTCTCTCATT 41 1421 1440 soooossssssssssooss 461
613114 135783 135802 AACTGCGAGGAGCAGCTGGG 51 1474 1493 soooossssssssssooss 462
613115 135786 135805 CCGAACTGCGAGGAGCAGCT 78 1477 1496 soooossssssssssooss 248
613116 135816 135835 CAAAAGCAGGTTAAGTGATT 28 1507 1526 soooossssssssssooss 463
613117 135849 135868 CTGATTTTGAAGTCCCGAGC 69 1540 1559 soooossssssssssooss 464
613118 135869 135888 ATTTGCTCTTACTCCCATCA 80 1560 1579 soooossssssssssooss 465
613119 135882 135901 TTGGAAAGATGAAATTTGCT 52 1573 1592 soooossssssssssooss 466
613120 135948 135967 TGTTGGATGTGGCCATGTTT 63 1639 1658 soooossssssssssooss 467
613121 136049 136068 GTCCCTTGAAATCCCCCAGA 45 1740 1759 soooossssssssssooss 468
613122 136082 136101 CCACAACAGGGCCAGAGGTG 55 1773 1792 soooossssssssssooss 469
613123 1361 15 136134 TCCTTTGTTGCTGCCACTGC 59 1806 1825 soooossssssssssooss 470
613124 136148 136167 GCCTGTGGCTCCACGAACAC 72 1839 1858 soooossssssssssooss 471
613125 136181 136200 CCCCGTCACACTCACACAAG 43 1872 1891 soooossssssssssooss 472
613126 136214 136233 GGCCTCCCCCGTGGCCTCCC 37 1905 1924 soooossssssssssooss 473
613127 136247 136266 TGCTTCCTCTCCCCTCTGCC 41 1938 1957 soooossssssssssooss 474
613128 136295 136314 GATGTCTACTCTCCAGCACG 32 1986 2005 soooossssssssssooss 475
613129 136328 136347 ATAGGCCTTGGCTCTCCCAG 73 2019 2038 soooossssssssssooss 476
613130 136361 136380 AGGACAGGCGGCCGCTCAGA 46 2052 2071 soooossssssssssooss 477
613131 136394 136413 TGACCCACAGCAGGCCCCCA 14 2085 2104 soooossssssssssooss 478
613132 136427 136446 CTCCCACAGGCTGCCCTGCA 46 2118 2137 soooossssssssssooss 479
613133 136460 136479 CAGCTTGCCTTCTCTTTTTA 58 2151 2170 soooossssssssssooss 480
613134 136491 136510 GGAGGTCATCCACGAAGTGC 45 2182 2201 soooossssssssssooss 61
613135 136493 136512 AAGGAGGTCATCCACGAAGT 41 2184 2203 soooossssssssssooss 481
613136 136515 136534 GACATCAAGGTCAGTCTTTT 80 2206 2225 soooossssssssssooss 482
613137 136541 136560 AGGGAGGAAGAGGCCAGCGC 60 2232 2251 soooossssssssssooss 483
613138 136574 136593 AGCCCCTCAACTCAGGCCCC 53 2265 2284 soooossssssssssooss 484
613139 136607 136626 TCAATAAAACAGGGTTTCTG 38 2298 2317 soooossssssssssooss 485
613140 136640 136659 CAAAATCATGGCAGCAGTTC 26 2331 2350 soooossssssssssooss 486
613141 136673 136692 GTTAGCCCTAAAGTCCCAGG 76 2364 2383 soooossssssssssooss 487
613142 136706 136725 CCCAAGAGGCACAAGTCCTT 57 2397 2416 soooossssssssssooss 488
613143 136739 136758 CAGTGGCCCAGGCTTGGAAA 63 2430 2449 soooossssssssssooss 489
613144 136748 136767 CAGAGATGCCAGTGGCCCAG 43 2439 2458 soooossssssssssooss 67 613145 136772 136791 TGCCTCCCAGACCCCCACAC 23 2463 2482 soooossssssssssooss 490
613146 136805 136824 AGTGGCCGTGGGAAGGACAG 17 2496 2515 soooossssssssssooss 491
613147 136838 136857 ACAACAGCACAGCGGCGCAG 61 2529 2548 soooossssssssssooss 492
613148 136845 136864 AC GGC AG AC AAC AGC AC AGC 59 2536 2555 soooossssssssssooss 493
613149 136899 136918 GAATTCGGGACATTGTGACG 42 2590 2609 soooossssssssssooss 494
613150 136933 136952 CAGGGTCATTACTGAGAAGG 39 2624 2643 soooossssssssssooss 495
613151 136966 136985 CTCAGTATGGAGTAGGTACC 52 2657 2676 soooossssssssssooss 496
613152 136999 137018 TGTGCCTGGACTTTGCCTTC 61 2690 2709 soooossssssssssooss 497
613153 137032 137051 GGAACTGAGAGTGAGAGGCT 62 2723 2742 soooossssssssssooss 498
613154 137077 137096 GAATCAGATCATGAGATTCG 30 2768 2787 soooossssssssssooss 499
613155 1371 10 137129 CACATCTGTGACGGGAGGAG 26 2801 2820 soooossssssssssooss 500
613156 137143 137162 CACCTAGGGTCACAGCTGAG 33 2834 2853 soooossssssssssooss 501
613157 137176 137195 GAAAGGGCTCTCTCCATGTC 54 2867 2886 soooossssssssssooss 502
613158 137214 137233 GTGGGCTCAGCACAGGAAGG 23 2905 2924 soooossssssssssooss 503
613159 137229 137248 ACCCAGCCTGCTGCTGTGGG 36 2920 2939 soooossssssssssooss 504
613160 137247 137266 ACCACTGACAACCAAGACAC 22 2938 2957 soooossssssssssooss 505
613161 137280 137299 GCCCTGGGTGCCTTGCCCTT 53 2971 2990 soooossssssssssooss 506
613162 137322 137341 ACAAGCTAGGGTGCAAGTGG 47 3013 3032 soooossssssssssooss 507
613163 137364 137383 GGAGCTGAGCAGCGGGCTGG 45 3055 3074 soooossssssssssooss 508
613164 137401 137420 CCCTTTGTCGGGTGTGGAGG 8 3092 3111 soooossssssssssooss 509
613165 137453 137472 TGGCTTCCAGCTGGGACTGG 65 3144 3163 soooossssssssssooss 510
613166 137486 137505 ATATGTTCAGCTGCTCCAGC 58 3177 3196 soooossssssssssooss 511
613167 137529 137548 ACTCAACAGGGTGCAGATGG 54 3220 3239 soooossssssssssooss 512
613168 137562 137581 GAATCCAAGCATAAACAGAC 55 3253 3272 soooossssssssssooss 513
613169 137595 137614 TTTTTTTCTTTTCACTATCA 34 3286 3305 soooossssssssssooss 514
613170 137629 137648 AGCATTTCAAGATACATGCG 59 3320 3339 soooossssssssssooss 515
613171 137692 137711 CACACGAGTCCCAGTGTGGG 11 3383 3402 soooossssssssssooss 516
613172 137761 137780 TTGGGTCCCAGGTGCTGAGG 40 3452 3471 soooossssssssssooss 517
613173 137794 137813 ACGGCCTCCTTAGCTGCTAG 73 3485 3504 soooossssssssssooss 518
613174 137827 137846 TAATCCTGTGCTTCAGGCCT 32 3518 3537 soooossssssssssooss 519
613175 137881 137900 TGACACAGGGAGCCCCAAGG 40 3572 3591 soooossssssssssooss 520
613176 137914 137933 AGCCAGACCAGCCACAAGAC 16 3605 3624 soooossssssssssooss 521
613177 137947 137966 GCTATGACCAGAGAGAACCA 29 3638 3657 soooossssssssssooss 522
613178 137967 137986 GGACTGCCATGAGACTTCGG 26 3658 3677 soooossssssssssooss 523
613179 137980 137999 TAAGCCTCCTTTGGGACTGC 64 3671 3690 soooossssssssssooss 524
613180 138013 138032 AGTGGCTTCCTTTTTCTTGT 69 3704 3723 soooossssssssssooss 525
Table 14
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester
internucleoside linkages targeting SEQ ID NOs: 1 and 3
5
SEQ SEQ SEQ SEQ
SEQ
ISIS ID ID % ID ID
Sequence Linkage chemistry ID NO NO: 1 NO: 1 inhibition NO: NO:
NO
Start Stop 3 3 Site Site Start Stop
Site Site
433475 n/a n/a TCTTCAGCTTTCAGGCCAGC 23 433 452 sssssssssssssssssss 96
613258 18371 18390 GGCAATGCCTGGAAGAAACT 75 n/a n a soooossssssssssooss 526
613259 18411 18430 CGCTGGTGAGAGTGTGACTG 69 n/a n a soooossssssssssooss 527
613260 18449 18468 ATCCGAGGGTGAGATGATAA 75 n/a n/a soooossssssssssooss 528
613261 19611 19630 AATAATTTCTCCAGGCTCAT 62 n/a n a soooossssssssssooss 529
613262 20955 20974 GCTGACTTCGATTTGTTATT 84 n/a n/a soooossssssssssooss 530
613263 22139 22158 TATCATATGGCACAAATTCT 32 n/a n a soooossssssssssooss 531
613264 23662 23681 CTACCTGGGACCACACCTTA 61 n/a n/a soooossssssssssooss 532
613265 25046 25065 GGGTGGAGAGCCTGATGGTC 47 n/a n/a soooossssssssssooss 533
613266 25418 25437 CTGTGAGCATCATGAAAGCC 56 n/a n/a soooossssssssssooss 534
613267 25606 25625 TTTTATTATGAGGGAGATCA 48 n/a n/a soooossssssssssooss 535
613268 26186 26205 AATAGTAGAGAAATGGAAAT 39 n/a n/a soooossssssssssooss 536
613269 26690 26709 TAACATGACAGGGTTAGGCC 67 n/a n/a soooossssssssssooss 537
613270 26917 26936 GCC C AC AC AC AT GCT C ATGC 57 n/a n/a soooossssssssssooss 538
613271 27160 27179 TGTGTTCAGTTACATGCCTT 82 n/a n/a soooossssssssssooss 539
613272 27569 27588 GACCACCATGGTGGGCTGAC 39 n/a n/a soooossssssssssooss 540
613273 28227 28246 GGCACCTCTCTGCAGACTTT 85 n/a n/a soooossssssssssooss 541
613274 30102 30121 TGCTTCACACACCACAATGG 60 n/a n/a soooossssssssssooss 542
613275 31769 31788 AACATTGCCGAGCACCAGCC 63 n/a n/a soooossssssssssooss 543
613276 31832 31851 GACTCAGGAACATGTTAGCT 70 n/a n a soooossssssssssooss 544
613277 32045 32064 CACGGCTCAGCAGAGGAGAC 23 n/a n/a soooossssssssssooss 545
613278 32324 32343 GCTGCTATCCTTCCTGGGCC 57 n/a n/a soooossssssssssooss 546
613279 32760 32779 GGAAATGAAAGCTTCCCACG 61 n/a n/a soooossssssssssooss 547
613280 33359 33378 AACAGTCAGAATACCCCCAA 72 n/a n/a soooossssssssssooss 548
613281 34056 34075 TATAGGTAGAATATAAAACT 17 n/a n a soooossssssssssooss 549
613282 35266 35285 TATGAACAAAACTGCAGGAC 45 n/a n/a soooossssssssssooss 550
613283 35839 35858 AGCCTCCAGAGTGACAGGTG 63 n/a n/a soooossssssssssooss 551
613284 36223 36242 CCATAAGCCACTCCTGGTTG 0 n/a n/a soooossssssssssooss 552
613285 36410 36429 CAGCTTGGAAGATACAGGAG 63 n/a n/a soooossssssssssooss 553
613286 38647 38666 CAAACTTAAGAAAAGTTGCA 67 n/a n/a soooossssssssssooss 554
613287 39104 39123 TGCTGGCAAAGAAGACAGGA 69 n/a n/a soooossssssssssooss 555
613288 40506 40525 CCACTGATGGCCGGGTGCAG 27 n/a n/a soooossssssssssooss 556
613289 42727 42746 TGGGTGTCAAATGGCTGGCT 55 n/a n/a soooossssssssssooss 557
613290 42955 42974 TAGAAAGCTATTTAATAAAT 0 n/a n/a soooossssssssssooss 558
613291 43109 43128 AGCCGTACATCAATGGTACA 71 n/a n/a soooossssssssssooss 559
613292 46627 46646 GGCCCTACCATGGTCGATTT 57 n/a n/a soooossssssssssooss 560
613293 47399 47418 ATCAGTGCCAGAGACAGAGG 5 n/a n/a soooossssssssssooss 561
613294 48115 48134 TATTATTAACGAACAAAAAA 0 n/a n/a soooossssssssssooss 562
613295 48223 48242 AGAGCAATACTTTGTGCTTA 51 n/a n/a soooossssssssssooss 563
613296 48608 48627 AGACTCCTCAAATGTGCGCC 53 n/a n/a soooossssssssssooss 564 613297 49204 49223 GAATGACGGCCGCCTCCTCT 39 n/a n a soooossssssssssooss 565
613298 49696 49715 TATGGAGGGCTTCTGGCCTC 18 n/a n/a soooossssssssssooss 566
613299 51 161 51180 GAGAAAGAGCGAGTAATTAG 44 n/a n/a soooossssssssssooss 567
613300 52250 52269 TAGCTAGAAACTGTGTCCCT 41 n/a n a soooossssssssssooss 568
613301 53908 53927 CTGTGAGATCATCCCCTGGT 51 n/a n/a soooossssssssssooss 569
613302 54457 54476 CAGCGCAGTGCAGGGAGTCA 41 n/a n/a soooossssssssssooss 570
613303 55975 55994 GGGTACACAGAGAGGCCACT 53 n/a n/a soooossssssssssooss 571
613304 56155 56174 GAACGATGCACTGCCTTGGC 54 n/a n/a soooossssssssssooss 572
613305 57402 57421 CACCATATTACACCAGTTGT 60 n/a n/a soooossssssssssooss 573
613306 57696 57715 CTGAAATGAATCCCCTGAAC 0 n/a n/a soooossssssssssooss 574
613307 57733 57752 GTGTCCACGAATGCCCCTGG 40 n/a n/a soooossssssssssooss 575
613308 58392 58411 ACGGATGGTTGCTGCTGATG 47 n/a n/a soooossssssssssooss 576
613309 61917 61936 GTCCCTTTTGCTCCCAAAGA 18 n/a n/a soooossssssssssooss 577
613310 62489 62508 CAGTCTTTTAATAAGTGAAA 50 n/a n/a soooossssssssssooss 578
613311 63219 63238 TTAGCTAAATTGAGGCCCGG 31 n/a n/a soooossssssssssooss 579
613312 63257 63276 GGAAAGGTATTGCCTCCTGA 42 n/a n/a soooossssssssssooss 580
613313 63627 63646 GGTGACTACGCAGCCTGGAC 57 n/a n/a soooossssssssssooss 581
613314 64559 64578 TTCAGGGCAATCCTTGAGGT 41 n/a n/a soooossssssssssooss 582
613315 64872 64891 TGCTAATGCTTTGGGACCTA 44 n/a n/a soooossssssssssooss 583
613316 66922 66941 TGGGTAGAGAGCAACTGTTC 32 n/a n/a soooossssssssssooss 584
613317 67104 67123 ACACCCCTTCTCTGCACATC 25 n/a n/a soooossssssssssooss 585
613318 68893 68912 CTGCTAGAGTTCGCCAACAG 59 n/a n/a soooossssssssssooss 586
613319 69457 69476 TCTTATTCTCTAGGTGGGCT 70 n/a n/a soooossssssssssooss 587
613320 70922 70941 CAGTGCAAGTGGGTATGAAG 30 n/a n/a soooossssssssssooss 588
613321 70928 70947 CAGATGCAGTGCAAGTGGGT 44 n/a n/a soooossssssssssooss 589
613322 71310 71329 GAAAAACTTTGTCCATAATT 41 n/a n/a soooossssssssssooss 590
613323 71665 71684 TAATGAATGTATAACAGAAA 0 n/a n/a soooossssssssssooss 591
613324 71702 71721 AAAAGGCAGATACTTTGTGG 56 n/a n/a soooossssssssssooss 592
613325 72456 72475 CAGAGGTGTGAAAGTAAAAG 0 n/a n/a soooossssssssssooss 593
613326 72566 72585 CAGCTAAAGCTTTGTGAACA 31 n/a n/a soooossssssssssooss 594
613327 72612 72631 TGCAGCCCAGGTGAGCCAGC 49 n/a n/a soooossssssssssooss 595
613328 73748 73767 GCCTCTCCTCCCAGTTCATG 12 n/a n/a soooossssssssssooss 596
613329 75140 75159 GTTTTCTTATTTTTAGCATT 87 n/a n/a soooossssssssssooss 597
613330 76340 76359 GGAGGGACGAGAAACAGCAG 0 n/a n/a soooossssssssssooss 598
613331 79041 79060 AGCTTCCAGCATGAACATAG 77 n/a n/a soooossssssssssooss 599
613332 79971 79990 GAGCCCTGATATGATAGAGG 57 n/a n/a soooossssssssssooss 600
613333 80360 80379 TGTACCATGCCTGGCAGATG 78 n/a n/a soooossssssssssooss 601
613334 81325 81344 GTTAGATGTATGTATGATGG 0 n/a n/a soooossssssssssooss 602
613412 73879 73898 GTGATCTTCCATCACTTCGA 80 345 364 soooossssssssssooss 25
Table 15
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester
internucleoside linkages targeting SEQ ID NOs: 1 and 3
5 SEQ SEQ
SEQ SEQ ID ID ID ID SEQ
ISIS % NO: NO:
NO: 1 NO: 1 Sequence Linkage chemistry ID NO inhibition 3 3
Start Stop NO
Start Stop
Site Site
Site Site
433475 n/a n/a TCTTCAGCTTTCAGGCCAGC 31 443 462 sssssssssssssssssss 96
613335 82136 82155 GCCTCCTCCTAGAGCTCCTG 81 n/a n a soooossssssssssooss 603
613336 83231 83250 CGGCCTCGCAACAAACCCAC 55 n/a n a soooossssssssssooss 604
613337 83820 83839 TCCACTGACCTGCCCCTTCC 66 n/a n a soooossssssssssooss 605
613338 84269 84288 TTTGCAACTTGTTCCTGCTT 84 n/a n a soooossssssssssooss 606
613339 85073 85092 TCTTGGGTGCAGGCGGAGCA 53 n/a n a soooossssssssssooss 607
613340 86117 86136 ACCCAAGATTCCCAGGAGCC 77 n/a n a soooossssssssssooss 608
613341 86191 86210 AGGCCCTAAATGCTCTGAGA 75 n/a n a soooossssssssssooss 609
613342 86251 86270 AGAAGTCTCTGGAACTGACG 70 n/a n a soooossssssssssooss 610
613343 86714 86733 TCACCAAACCAATGGCTGGC 67 n/a n a soooossssssssssooss 611
613344 86774 86793 GGTGAAATCCTGGCTAGGCC 89 n/a n a soooossssssssssooss 612
613345 88487 88506 TGCAGATTCGGCCTGAGTTT 72 n/a n a soooossssssssssooss 613
613346 89131 89150 CCAAACAAGAAAGACTGAGA 55 n/a n a soooossssssssssooss 614
613347 89994 90013 CCATGTCCCTCCCCCCAACC 12 n/a n a soooossssssssssooss 615
613348 90126 90145 ATTAGAAAAAAACCACTAGG 37 n/a n a soooossssssssssooss 616
613349 92246 92265 AGCTT ACT AGAGT GCTCTT G 81 n/a n a soooossssssssssooss 617
613350 92915 92934 ATTTTTTTCT GGTTC ATT C A 71 n/a n a soooossssssssssooss 618
613351 94316 94335 CGCTTAAGAGAGGGAGCATC 72 n/a n a soooossssssssssooss 619
613352 94673 94692 GTGGAGATACGCAGTGGTGG 68 n/a n a soooossssssssssooss 620
613353 95790 95809 ACACCATGAGGGCACCCGTC 82 n/a n a soooossssssssssooss 35
613354 95918 95937 CCCAAACCTGGCTCTGTCAC 65 n/a n a soooossssssssssooss 621
613355 97224 97243 ATCAACCAGCACCATCCACG 65 n/a n a soooossssssssssooss 622
613356 97314 97333 GGGTCTGTAAGCCTAAAGTG 53 n/a n a soooossssssssssooss 623
613357 97315 97334 TGGGTCTGTAAGCCTAAAGT 56 n/a n a soooossssssssssooss 624
613358 97368 97387 TATACTATGTGGCAGAATCA 48 n/a n a soooossssssssssooss 625
613359 97478 97497 CACATTCCCTCTACCCCATG 57 n/a n a soooossssssssssooss 626
613360 97507 97526 TC AT AGATTTT CTTCTTT GG 75 n/a n a soooossssssssssooss 627
98881 98900 n/a n a soooossssssssssooss
613361 ACACACCTTCATTTACTGTC 95 313
98918 98937 n/a n a soooossssssssssooss
98882 98901 n/a n a soooossssssssssooss
613362 AAC AC AC CTTC ATTT ACT GT 83 314
98919 98938 n/a n a soooossssssssssooss
98883 98902 n/a n a soooossssssssssooss
613363 AAACACACCTTCATTTACTG 79 315
98920 98939 n/a n a soooossssssssssooss
98884 98903 n/a n a soooossssssssssooss
613364 CAAACACACCTTCATTTACT 63 316
98921 98940 n/a n a soooossssssssssooss
98885 98904 n/a n a soooossssssssssooss
613365 TCAAACACACCTTCATTTAC 59 317
98922 98941 n/a n a soooossssssssssooss
613366 98886 98905 TT C AAAC AC ACCTT C ATTT A 67 n/a n a soooossssssssssooss 628 98923 98942 n/a n a soooossssssssssooss
98887 98906 n/a n a soooossssssssssooss
613367 TTTCAAACACACCTTCATTT 72 318
98924 98943 n/a n a soooossssssssssooss
98888 98907 n/a n a soooossssssssssooss
613368 TTTTCAAACACACCTTCATT 59 319
98925 98944 n/a n a soooossssssssssooss
98889 98908 n/a n a soooossssssssssooss
613369 GTTTTCAAACACACCTTCAT 90 320
98926 98945 n/a n a soooossssssssssooss
98890 98909 n/a n a soooossssssssssooss
613370 GGTTTTCAAACACACCTTCA 96 321
98927 98946 n/a n a soooossssssssssooss
98891 98910 n/a n a soooossssssssssooss
613371 1 LrO 1 1 1 1 LAAALALALL 1 1 95 322
98928 98947 n/a n a soooossssssssssooss
613372 99560 99579 CCCCAGCTGGCCGAGGCCCA 81 n/a n a soooossssssssssooss 629
613373 99816 99835 GAGAAGGGTCCTGTCCCAGA 76 n/a n a soooossssssssssooss 630
613374 100743 100762 AGTAAGTTGATGGAGATCAT 83 n/a n a soooossssssssssooss 631
613375 102894 102913 CACCAGCCCCACTCCGCCAC 61 n/a n a soooossssssssssooss 632
613376 103875 103894 TGCTCTGTCTCCAGAGACAT 70 n/a n a soooossssssssssooss 633
613377 104615 104634 ATCCAAGATCCAGGCCAGGC 63 n/a n a soooossssssssssooss 634
613378 105870 105889 AGAGACCCAGGGTGACCAGT 76 n/a n a soooossssssssssooss 635
613379 106909 106928 CAAATTTCTCGATCCCCTTC 18 n/a n a soooossssssssssooss 636
613380 108411 108430 CCTGAGGAGGGCACTCACAG 61 n/a n a soooossssssssssooss 637
613381 108891 108910 GTGGCTACCCACGGCCACAG 1 n/a n a soooossssssssssooss 638
613382 108939 108958 CTACCAGGAGCACACACAGA 65 n/a n a soooossssssssssooss 639
613383 109303 109322 CCCTAAGCCATGGTGGGTTT 72 n/a n a soooossssssssssooss 640
613384 109995 110014 CCCTTGTCCCCACAGCCACA 72 n/a n a soooossssssssssooss 641
613385 110198 110217 GGGTCACCTGTGAGTTATTT 62 n/a n a soooossssssssssooss 642
613386 113567 113586 AGCTGCCTCCAGTTGTTTAT 85 n/a n a soooossssssssssooss 643
613387 114056 114075 ACTTTAGCTCCATCTTCTCA 49 n/a n a soooossssssssssooss 644
613388 114755 114774 CTATAATGGTCAGTGGTGTA 67 n/a n a soooossssssssssooss 645
613389 115899 115918 TGAAGGAGAGGCTGTGGTGA 41 n/a n a soooossssssssssooss 646
613390 117387 117406 TGTGACAACAAAGTTGTCCT 46 n/a n a soooossssssssssooss 647
613391 117654 117673 CCCTGCAATCAACACAGGAG 65 n/a n a soooossssssssssooss 648
613392 119050 119069 TCTAACATGCACATATTTAC 60 n/a n a soooossssssssssooss 649
613393 119190 119209 AGCACTGGGTGTTTACAACA 84 n/a n a soooossssssssssooss 650
119594 119613 n/a n a soooossssssssssooss
613394 AGAAGTGATATCATATCCTA 85 324
119905 119924 n/a n a soooossssssssssooss
119595 119614 n/a n a soooossssssssssooss
613395 AAGAAGTGATATCATATCCT 68 325
119906 119925 n/a n a soooossssssssssooss
613396 120782 120801 AC CC GGTGAC CTTTCCTCTC 67 n/a n a soooossssssssssooss 651
613397 121728 121747 GAGTGACATGCGCCACCCTG 87 n/a n a soooossssssssssooss 203
613398 121794 121813 AAAAGGATGAGTGACACGCC 66 n/a n a soooossssssssssooss 205
613399 121940 121959 GCCACAGCACGGCGCATGGG 89 n/a n a soooossssssssssooss 652
613400 124954 124973 GCACCGCTCCCTCTGGGAAT 49 n/a n a soooossssssssssooss 653
613401 125298 125317 AAATCTTGCTGGAAAGGCAG 64 n/a n a soooossssssssssooss 654 613402 125428 125447 GACCCAGACATTTGCTCAGC 86 n/a n a soooossssssssssooss 655
613403 125982 126001 GGGCTCCCGCAAGTTTCACA 71 n/a n/a soooossssssssssooss 656
613404 127460 127479 GAGAGGCCCTGTGGACAGCT 75 n/a n/a soooossssssssssooss 657
613405 130381 130400 CCCACTGGATGCTGCTGAGG 34 n/a n/a soooossssssssssooss 658
613406 131662 131681 GTGCCTAGTAACCCATTTAG 54 n/a n/a soooossssssssssooss 659
613407 131796 131815 CACCCACAGCAAGACCTAGA 49 n a n/a soooossssssssssooss 660
613408 133056 133075 ACACAGAGAAGCTTCCAGAG 70 n/a n/a soooossssssssssooss 661
613409 133304 133323 AGCTATCACATGGACGAGTT 67 n/a n/a soooossssssssssooss 662
613410 133548 133567 CATTTAATACAGGCTTTGAT 38 n/a n/a soooossssssssssooss 663
613411 134599 134618 AGCCACTGATGCCCAGACAT 69 n/a n/a soooossssssssssooss 664
613412 73879 73898 GTGATCTTCCATCACTTCGA 82 345 364 soooossssssssssooss 25
Table 1
Inhibition of Tau mRNA by 5-10-5 MOE gapmers with phosphorothioate and phosphodiester
internucleoside linkages targeting SEQ ID NOs: 5 and 6
5
Figure imgf000079_0001
Example 8: Dose-dependent antisense inhibition of human tau in SH-SY5Y cells by 5-10-5
MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages
Gapmers from studies described above exhibiting significant in vitro inhibition of tau mRNA were selected and tested at various doses in SH-SY-5Y cells. Cells were plated at a density of
10 20,000 cells per well and transfected using electroporation with 1.25 μΜ, 2.500 μΜ,5.00 μΜ, 10.00 μΜ, and 20.00 μΜ concentrations of antisense oligonucleotide, as specified in the Table below.
After a treatment period of approximately 16 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as
15 measured by RIBOGREEN®. Results are presented as percent inhibition of Tau, relative to
untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Table 17
Figure imgf000079_0002
613136 36 70 83 88 92
613115 32 49 82 86 94
613329 73 80 92 94 92
613273 46 24 48 60 85
613262 31 72 76 87 94
613271 44 66 84 89 94
613370 92 92 95 98 98
613361 88 86 89 94 96
613371 80 88 91 95 97
613369 58 85 89 95 96
613399 61 77 90 94 96
613344 80 79 93 95 95
613397 64 74 79 93 95
613412 45 66 70 81 96
Table 18
Figure imgf000080_0001
Example 9: Antisense inhibition of human Tau in SH-SY5Y cells by 5-10-5 MOE, 5-8-5 MOE, 4-8-6 MOE, or 6-8-4 MOE gapmers
Antisense oligonucleotides were designed targeting a tau nucleic acid and were tested for their effects on Tau mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. ISIS 613412 was also included in the assays. The results for each experiment are presented in separate tables shown below. Cultured SH-SY5Y cells at a density of 20,000 cells per well were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and Tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-8-5
MOE, 4-8-6 MOE, or 6-8-4 MOE gapmers. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2' -deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. The 4-8-6 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising four and six nucleosides respectively. The 6-8-4 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising six and four nucleosides respectively. Each nucleoside in the 5' wing segment and each nucleoside in the 3 ' wing segment has a 2' -MOE modification. The internucleoside linkage motif throughout for each gapmer in the tables below, except for ISIS 613412, is 5'-sooosssssssssooss-3', wherein each "s" represents a phosphorothioate internucleoside linkage and wherein each "o" represents a phosphodiester internucleoside linkage. The internucleoside linkage motif for ISIS 613412 is 5'-soooossssssssssooss-3', wherein each "s" represents a phosphorothioate internucleoside linkage and wherein each "o" represents a phosphodiester internucleoside linkage. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to either SEQ ID NO: 1 (GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000), SEQ ID NO: 4 (GENBANK Accession No.
NT_010783.14 truncated from nucleotides 2624000 to 2761000), SEQ ID NO: 5 (GENBANK Accession No. DR002467.1), or SEQ ID NO: 6 (GENBANK Accession No. NM_001203251.1). 'n a' indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
Table 19
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1 and SEQ
ID NO: 4
Figure imgf000081_0001
621196 67898 67915 TGAAGGAGCCTGGGAAGT 15 63891 63908 5-8-5 667
621198 69079 69096 AGCAGAGATGTGGCTGGG 50 65044 65061 5-8-5 668
621199 69622 69639 GTGAGGTTGAAAAGTTTC 34 65587 65604 5-8-5 669
621200 69802 69819 TGGTTTCTGATGGTTTTT 43 65767 65784 5-8-5 670
621201 70023 70040 GTTGGAACTCCTGGGATC 27 65988 66005 5-8-5 671
621202 70259 70276 TCAGCATTTTTCTCCTCT 47 66224 66241 5-8-5 672
621203 70531 70548 CTGGAATGGTACGATCTC 52 66496 66513 5-8-5 673
621204 70750 70767 CCACTCTGGTGCAGGACG 29 66715 66732 5-8-5 674
621205 70930 70947 CAGATGCAGTGCAAGTGG 43 66895 66912 5-8-5 675
621206 71110 71127 GGGTTTCACCAGACCTCA 29 67075 67092 5-8-5 676
621207 71306 71323 CTTTGTCCATAATTTTTT 34 67271 67288 5-8-5 677
621208 71487 71504 TATAGCATGAGCATTTGT 42 67452 67469 5-8-5 678
621209 71667 71684 TAATGAATGTATAACAGA 2 67632 67649 5-8-5 679
621210 71850 71867 TCCTCCTGGCTGCCCTGT 33 67815 67832 5-8-5 680
621211 72030 72047 CATCCTGCCATACCAGAC 32 67995 68012 5-8-5 681
621212 72224 72241 TATCCGCCTCTCAAAGTG 0 68189 68206 5-8-5 682
621213 72410 72427 CACATTTGCATATTTTCA 65 68373 68390 5-8-5 683
621214 72590 72607 TCGGGTTGCCCCATTCAA 27 68553 68570 5-8-5 684
621215 72770 72787 AACCACCATTTACTCACA 35 68733 68750 5-8-5 685
621216 72950 72967 ACCGGCACCCACCAGGTC 22 68913 68930 5-8-5 686
621217 73130 73147 AGGGACAGCATCAGCAGA 22 69093 69110 5-8-5 687
621218 73437 73454 AGGCATGTGGCAGGTGCC 12 69400 69417 5-8-5 688
621219 73617 73634 ACCAACACCGCAGCAGTT 35 69580 69597 5-8-5 689
621220 73797 73814 GTTCTGAGGAGTGTTGGG 0 69760 69777 5-8-5 690
621221 73978 73995 ACTAACCTTTCAGGCCAG 33 69941 69958 5-8-5 691
621222 74273 74290 TAAATTATTTTAGAGACG 0 70236 70253 5-8-5 692
621223 74467 74484 CAAGGTAAATTTCTTTCT 69 70430 70447 5-8-5 693
621224 74647 74664 TCAGGCCTGATCTAAGTA 14 70610 70627 5-8-5 694
621225 74827 74844 TCCCCAACCATGTGGTCT 40 70790 70807 5-8-5 695
621226 75007 75024 TGCCAGCCACACCCATGG 52 70970 70987 5-8-5 696
621227 75187 75204 AACTCCACGGCTTCCATG 50 71150 71167 5-8-5 697
621228 75371 75388 AGATAGAAATCTGAAACG 34 71334 71351 5-8-5 698
621229 75551 75568 GCTGGGCCGAGCTGCATT 66 71514 71531 5-8-5 699
621230 75891 75908 GGATTCAAAGGAGAAAAC 35 71854 71871 5-8-5 700
621231 76192 76209 ATTATTATTTGACATGGG 75 72155 72172 5-8-5 701
621232 76373 76390 AGGGTAGGCAGTGTTGTG 4 72336 72353 5-8-5 702
621233 76553 76570 CTCTCTTTGTCAGGAAAA 58 72516 72533 5-8-5 703
621234 76942 76959 CCTTTTTTTTTAGGACGG 0 72905 72922 5-8-5 704
621235 77222 77239 GAGTTGACTGGGCACGGT 33 73185 73202 5-8-5 705
621236 77414 77431 TCATCTGTGAAGCGGACG 81 73377 73394 5-8-5 706
621237 77594 77611 GGCACAATCCATATGAGG 56 73557 73574 5-8-5 707
621238 77781 77798 GTTATTTTGGAACAGTTT 87 73744 73761 5-8-5 708
621239 78117 78134 CTGGACTTTATTTCATTT 77 74080 74097 5-8-5 709 621240 78319 78336 CAGTATATATAGTGCATA 76 74282 74299 5-8-5 710
621241 78499 78516 CAAAACAGCTCCTTGTAA 14 74462 74479 5-8-5 711
621242 78679 78696 ACTTTCATCTACTTTTCA 32 74642 74659 5-8-5 712
621243 78859 78876 CCAATCTGTCCCCAGCTT 42 74822 74839 5-8-5 713
621244 79039 79056 TCCAGCATGAACATAGCT 65 75002 75019 5-8-5 714
621245 79219 79236 TAGAGTCAGTTTCAGGAT 75 75182 75199 5-8-5 715
621246 79399 79416 GGTGGTATCACCAACAGC 35 75362 75379 5-8-5 716
621247 79589 79606 TTAAGGAATGGCTCTGGG 42 75552 75569 5-8-5 717
621248 79769 79786 GAACATGAGACATCTTGA 8 75732 75749 5-8-5 718
621249 79949 79966 AGCTGTGCCCAGTTAAAA 53 75912 75929 5-8-5 719
621250 80129 80146 TCTGGTCATATGAGGAAA 55 76092 76109 5-8-5 720
621251 80309 80326 AGTCATTATCATGTCACC 89 76272 76289 5-8-5 721
621252 80489 80506 GCTGAGCGAATTACCTAA 75 76452 76469 5-8-5 722
621253 80669 80686 CCTCTGTATGACAGAAAT 48 76632 76649 5-8-5 723
621254 80849 80866 GGTAACATGTAAAGCTTC 79 76812 76829 5-8-5 724
81033 81050 76996 77013
621255 TGGATTATGTACAGATAT 28 5-8-5 725
81136 81153 77099 77116
81110 81127 77073 77090
621256 ATGGATGATAGGAAGGAT 20 5-8-5 726
81213 81230 77176 77193
621257 81393 81410 GATGGAGGAAGGAATGAT 5 77356 77373 5-8-5 727
621258 81629 81646 CTGGATGGTTACATGGAT 31 77592 77609 5-8-5 728
621259 81812 81829 ATAATTGATGTATAATTA 0 77775 77792 5-8-5 729
621260 81992 82009 GTTCTGCCCCACCTGGGC 56 77955 77972 5-8-5 730
621261 82198 82215 GCCTCTCAGGGCCTCCGT 76 78161 78178 5-8-5 731
621262 82378 82395 GAAGGATGGCCACACAGA 39 78341 78358 5-8-5 732
621263 82558 82575 ATGGGCTTATCAATGCAT 85 78521 78538 5-8-5 733
621264 82738 82755 GAGGCCCAAATGATCACA 56 78701 78718 5-8-5 734
621265 82918 82935 GCTCAGGGCAGACACGGT 66 78881 78898 5-8-5 735
621266 83098 83115 CTTAACCAGCTAGTGGTG 38 79061 79078 5-8-5 736
621267 83278 83295 GTCCTGTGGAGCTGAAAA 54 79241 79258 5-8-5 737
621268 83486 83503 GGCTGGAGGCATGGAGGG 7 79449 79466 5-8-5 738
621269 83666 83683 TGCCTCCAGAGCACACAC 55 79629 79646 5-8-5 739
621270 83846 83863 AGGATACTAAACCAAGAT 24 79809 79826 5-8-5 740
621271 84026 84043 CACAGATGGGAAGCAAGA 20 79989 80006 5-8-5 741
621272 84206 84223 CATGAGGCAACAATCCAA 70 80169 80186 5-8-5 742
Table 20
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO:
SEQ SEQ ID ID SEQ
ISIS %
NO: 1 NO: 1 Sequence Motif ID NO inhibition
Start Stop NO Site Site
613412 73879 73898 GTGATCTTCCATCACTTCGA 80 5-10-5 25
620887 98891 98908 GTTTTCAAACACACCTTC 92 5-8-5 665 98928 98945
621273 84386 84403 GCCGTAAGACCCAGCTCT 58 5-8-5 743
621274 84566 84583 GAAGAAGTACTGAGAAAG 14 5-8-5 744
621275 84746 84763 TAAGAACCTCAGCGGCAA 56 5-8-5 745
621276 84926 84943 TGAGGGAGCACTGAGAGT 21 5-8-5 746
621277 85106 85123 AGTTCAATGAAGGACATT 69 5-8-5 747
621278 85287 85304 ACTGTTGGATGGGTCCAC 70 5-8-5 748
621279 85467 85484 TTGGTCCATTTTGATTGG 0 5-8-5 749
621280 85647 85664 CGGCAGCCCTGTGTAAAC 48 5-8-5 750
621281 85828 85845 CCTTTGCTTCTTGCGCAG 66 5-8-5 751
621282 86014 86031 AGGGCCTTGACTGCCTGG 15 5-8-5 752
621283 86194 86211 AAGGCCCTAAATGCTCTG 57 5-8-5 753
621284 86384 86401 CGCTCAGGTGATCTTGGG 76 5-8-5 754
621285 86564 86581 CTGATTGATTCCCCATCA 64 5-8-5 755
621286 86746 86763 TTCTTGTTGGGCAACTGG 59 5-8-5 756
621287 86928 86945 GAAATGCACTCAGAAGGG 52 5-8-5 757
621288 87108 87125 CCTCAGTGAAATAACTGT 55 5-8-5 758
621289 87288 87305 TGGCTGCAACTTTGAATG 0 5-8-5 759
621290 87470 87487 CTGGAGATGGCAGGCTGG 39 5-8-5 760
621291 87653 87670 CCCGTGGGCCCCATGTGG 1 5-8-5 761
621292 87834 87851 ATTGAGGACACCTGGTGT 6 5-8-5 762
621293 88014 88031 AAGGTCTGCATTGTCAGT 61 5-8-5 763
621294 88194 88211 CTGCTGGCCTCTCTGTAC 56 5-8-5 764
621295 88374 88391 TCGGACACGGTCACTGCC 60 5-8-5 765
621296 88554 88571 GTGCCTTGTCATGTGACA 79 5-8-5 766
621297 88734 88751 GGAAGCCATGTGGTAGCC 50 5-8-5 767
621298 88918 88935 CAGAGGCCACTGCCTCTG 0 5-8-5 768
621299 89098 89115 TTAATAGTACCAAAATCA 25 5-8-5 769
621300 89278 89295 TACATTTCCTTCCTCCCA 10 5-8-5 770
621301 89470 89487 CGGAGTCAGGCAGATGGG 43 5-8-5 771
621302 89650 89667 GCTATGACCTAGTAGGAA 84 5-8-5 772
621303 89830 89847 ATTGTTTACTAGAAACCA 39 5-8-5 773
621304 90010 90027 GGGCAGAGCCCACCCCAT 40 5-8-5 774
621305 90190 90207 AGGCTGGACAAGGCTAGC 63 5-8-5 775
621306 90531 90548 CATATTTGAGTTTCTTTT 62 5-8-5 776
621307 90711 90728 AATGATTACACAAAGCTG 61 5-8-5 777
621308 90891 90908 GAACAGTGTCTTTCCAGC 73 5-8-5 778
621309 91071 91088 ATGTGCCTTTCTGTGCCA 89 5-8-5 779
621310 91251 91268 AATGATTTCTAGAGGTCA 54 5-8-5 780
621311 91431 91448 CCATGATTCCAGGCTGCT 80 5-8-5 781
621312 91816 91833 CAGCCAGGCTAGTCTTGC 80 5-8-5 782
621313 92037 92054 AGGAGAATGTTCTTTTTT 53 5-8-5 783
621314 92219 92236 TTACTGTACTCTGATATA 41 5-8-5 784 621315 92399 92416 CCTTATGGGAGCCCTTTG 54 5-8-5 785
621316 92579 92596 CAGAGCGCAGCAGCACCT 64 5-8-5 786
621317 92759 92776 CTCACCAGCTCTGCTTTA 53 5-8-5 787
621318 92939 92956 CAGCTGATCAAGGGCACA 84 5-8-5 788
621319 93123 93140 AGACCTGCCATAAGACTC 39 5-8-5 789
621320 93380 93397 CTGACTAAAATGTTATTT 34 5-8-5 790
621321 93570 93587 ACAGGCATGAGTTTTTTT 61 5-8-5 791
621322 93840 93857 CCCCTTTTTTTTGGTTTG 2 5-8-5 792
621323 94020 94037 TCTGGAGCCGCCCTGGGC 36 5-8-5 793
621324 94200 94217 ATGACAGCTCTCCTGGTC 38 5-8-5 794
621325 94380 94397 ACTCTCAACTCCTCTGGT 49 5-8-5 795
621326 94576 94593 TCCTGTTGGAGGAGGCGC 7 5-8-5 796
621327 95433 95450 CCTTTGAGTTGAGGGACC 60 5-8-5 797
621328 95613 95630 GCCTGAGCACGGGAGGAG 41 5-8-5 798
621329 95793 95810 GACACCATGAGGGCACCC 69 5-8-5 799
621330 95986 96003 AGCACCTCCTGGGAGGCG 18 5-8-5 800
621331 96166 96183 GGTGGCATGTGGACCAGG 50 5-8-5 801
621332 96346 96363 TGTGGCTCTGAGGTCTCC 72 5-8-5 802
621333 96526 96543 GCCTCTGTCTTAACTTTT 50 5-8-5 803
621334 96778 96795 TTAGGGTACAGTGGTGTG 18 5-8-5 804
621335 96958 96975 CCTCTAGTAGGCCAGTAT 62 5-8-5 805
621336 97141 97158 AATCAAGTAAGTTTGGGA 57 5-8-5 806
621337 97321 97338 CAACTGGGTCTGTAAGCC 47 5-8-5 807
621338 97506 97523 TAGATTTTCTTCTTTGGT 45 5-8-5 808
621339 97686 97703 GGGCACGAATTCTCACTG 53 5-8-5 809
621340 97866 97883 TCCAGAATAGGAGAGGCT 9 5-8-5 810
621341 98046 98063 ACACTGCAGCCAGGCTTG 63 5-8-5 811
621342 98226 98243 TGCAGTCTCCCTAACCCA 59 5-8-5 812
621343 98406 98423 CTCTTTTCCCTTGAATCT 33 5-8-5 813
621344 98607 98624 GTCAGCTTACCTTGGCTT 44 5-8-5 814
621345 98802 98819 CAACTGCTCTTCCCTGGG 68 5-8-5 815
621346 98982 98999 TCTGGGTGCAGTTTATGC 80 5-8-5 816
621347 99162 99179 AGAATTTATGCTGGAAAT 38 5-8-5 817
621348 99342 99359 GGCCAGCTCTCCAAATCC 56 5-8-5 818
621349 99522 99539 CCCTAGACTCTGGGCAAC 66 5-8-5 819
Table 21
Inhibition of Tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ SEQ ID ID
ISIS % SEQ NO: 1 NO: 1 Sequence Motif NO inhibition ID NO Start Stop
Site Site
613412 73879 73898 GTGATCTTCCATCACTTCGA 91 5-10-5 25
620887 98891 98908 GTTTTCAAACACACCTTC 96 5-8-5 665 98928 98945
620965 20187 20204 TCTAAGTAATTCGATATG 27 5-8-5 820
620966 20375 20392 AAACAGAAACTAGGGTGG 63 5-8-5 821
620967 20571 20588 TCCCGCTGGGATCCATGG 40 5-8-5 822
620968 20751 20768 TGTTGCCATTGGGCAGCC 0 5-8-5 823
620969 20931 20948 GACCTTCTAATTAGTCCA 58 5-8-5 824
620970 21291 21308 GCGATTCTCTCCTGTCTC 53 5-8-5 825
620971 21567 21584 ACTCAGATTTATTATTTT 87 5-8-5 826
620972 21747 21764 AAAGTACTATCCACTTGT 77 5-8-5 827
620973 21927 21944 CAAATCCTGGGTTCGAAT 39 5-8-5 828
620974 22107 22124 AGGTCTCTGCCCATGAAA 59 5-8-5 829
620975 22287 22304 AGATTTCTCCCTTCACCC 46 5-8-5 830
620976 22467 22484 TTAACATGATGGTGGTTT 86 5-8-5 831
620977 22652 22669 CATTGTGGAAATTAAACA 50 5-8-5 832
620978 22837 22854 TGTAGTCAATATTGAACT 86 5-8-5 833
620979 23269 23286 TTTTGAGAGCAGGTCTCG 61 5-8-5 834
620980 23449 23466 ATGGCAAACAGTACATGT 80 5-8-5 835
620981 23631 23648 AGGATTTAAGTGATTTTC 66 5-8-5 836
620982 23826 23843 TACAAAGACCCCTTTTCC 51 5-8-5 837
620983 24016 24033 AGCTGCCAGCAGTCCTTG 81 5-8-5 838
620984 24196 24213 CTGCTGTAGGAAATGACC 77 5-8-5 839
620985 24377 24394 AGCTCCAACCAGCTTTCT 58 5-8-5 840
620986 24557 24574 GTCCTGATTAACAGCTAA 69 5-8-5 841
620987 24744 24761 GTCTGTCTGCTGCCATCC 81 5-8-5 842
620988 24924 24941 GTAACTGATATGGTAACA 85 5-8-5 843
620989 25286 25303 AGCCCTCTTTGTACAGGA 75 5-8-5 844
620990 25466 25483 CTGTTCCAGTTGATCCAG 53 5-8-5 845
620991 25668 25685 GCAGTCTTTTTAAATTAA 80 5-8-5 846
620992 25848 25865 CCTCTGCTCACATAGAAA 64 5-8-5 847
620993 26028 26045 AGTACCTGCCTTGTTCCT 52 5-8-5 848
620994 26208 26225 AAGCATGGTAATACAAAA 78 5-8-5 849
620995 26404 26421 TTGATTAAAAAAAATAGC 4 5-8-5 850
620996 26584 26601 TTCATCCTTTCCAAGTGT 58 5-8-5 851
26764 26781
620997 CATGCTTACACACCACAC 23 5-8-5 852
26948 26965
26762 26779
620998 TGCTTACACACCACACAC 38 5-8-5 853
26946 26963
620999 27140 27157 ATATGCTGAACACACACA 75 5-8-5 854
621000 27320 27337 CCACCGAGGTCTCATTGG 52 5-8-5 855
621001 27500 27517 GGGAGACCTCCCTTTCAA 0 5-8-5 856
621002 27680 27697 TCTCTGGGTGTAGAGACG 54 5-8-5 857
621003 27861 27878 GCAGGCTCGGGCTCCACG 61 5-8-5 858
621004 28041 28058 CCTTGTCAGATGGTTGAT 55 5-8-5 859 621005 28221 28238 TCTGCAGACTTTTCTCAA 22 54 i-5 860
621006 28426 28443 CTGGAGGGAGGTGATGTG 7 54 i-5 861
621007 28616 28633 GAAAGCTGCTGCTGAGGG 63 54 -5 862
621008 28981 28998 GGGCAAAGATAATTCGAA 61 54 -5 863
621009 29165 29182 CCTCACCCTGGGCAAGAA 68 54 -5 864
621010 29409 29426 TTGCTGCTCGGGAGGCCG 14 54 i-5 865
621011 29589 29606 AAATGAAAGATGCTGGCT 23 54 i-5 866
621012 29953 29970 CAGAGAGGTCCAGCTACT 58 54 i-5 867
621013 30133 30150 AGCTCACTACAGCAGGCA 93 54 -5 868
621014 30607 30624 TATTTCTTGCAATTCTTT 78 54 i-5 869
621015 30787 30804 GGAGGACTGTCTCTAGAG 59 54 i-5 870
621016 30967 30984 TCCGGCCTGGCGAACTGA 40 54 i-5 871
621017 31278 31295 CCTGCAAAGCTAATTTTA 18 54 i-5 872
621018 31458 31475 GAGCCCCCTTTAAGCCTT 65 54 i-5 873
621019 31654 31671 GTACAAAGACTGTGTACG 47 54 i-5 874
621020 31834 31851 GACTCAGGAACATGTTAG 81 54 i-5 875
621021 32023 32040 TGAGGCAGCAATGCTGGG 34 54 i-5 876
621022 32203 32220 CACTGGGAGGTGTATAGA 0 54 i-5 877
621023 32383 32400 GCGAGGTGGGTGAAGGTT 25 54 i-5 878
621024 32563 32580 AAAAACTTTGGCAGTCAG 80 54 i-5 879
621025 32743 32760 GGTCTGCCCTGCACCAGG 59 54 i-5 880
621026 32923 32940 CTGAGCTCTCCGAGCTGC 61 54 i-5 881
621027 33103 33120 GTGAGGTGGTATCATTGG 51 54 i-5 882
621028 33283 33300 CTAGCTTGAATTCCTCCA 81 54 i-5 883
621029 33463 33480 AATCTGTGACTCAAGAAC 47 54 i-5 884
621030 33708 33725 GGGCCGTCTCTATTAAAA 56 54 i-5 885
621031 33888 33905 GGTATAATTTGTTTGGAC 85 54 i-5 886
621032 34068 34085 CTTTATTGCATATAGGTA 86 54 i-5 887
621033 34248 34265 ATCCAGTCCCAACATTGG 30 54 i-5 888
621034 34428 34445 CGGTCTCTCTGACTTGCC 78 54 i-5 889
621035 34620 34637 GAGGGCATTCCTCAAAGG 10 54 i-5 890
621036 34800 34817 AGTTCTCTGATTCTCAAA 63 54 i-5 891
621037 34980 34997 CCCCCAGTGCCCACAAGT 48 54 i-5 892
621038 35160 35177 CTAAAAGCTAAAGTGGGT 49 54 i-5 893
621039 35340 35357 AGGACCTGGCAGAGCTGC 73 54 i-5 894
621040 35520 35537 AAATCGAACACTTACATA 20 54 i-5 895
621041 35721 35738 TTGCCATCTTGGACAGGG 89 54 i-5 896 Table 22
Figure imgf000088_0001
620918 10961 10978 ACCTGCTACATAGCGGAA 87 5-8-5 927
620919 11141 11158 TTATAGATCATATCTGGG 82 5-8-5 928
620920 11321 11338 AAACAGAGCACCAATGTA 36 5-8-5 929
620921 11501 11518 ACCTCAGAGACTTGACCT 54 5-8-5 930
620922 11681 11698 CCCAGGAAGTGAGAAAAG 20 5-8-5 931
620923 11864 11881 CAGTTGGGAGCCATCTGG 51 5-8-5 932
620924 12044 12061 ATTGGGCTCAGCATGGAG 30 5-8-5 933
620925 12224 12241 TCCTTAACCATTAGGCCA 70 5-8-5 934
620926 12404 12421 TGGCCCTAGTGAGTACCG 75 5-8-5 935
620927 12597 12614 GCCCAGTTTGGTGCAGGG 79 5-8-5 936
620928 12777 12794 GGCGGGAAGGCAGGTCAG 51 5-8-5 937
620929 12957 12974 CAGTTTACCAATGTCAGC 77 5-8-5 938
620930 13137 13154 ATATATTGTTACTTAGTC 83 5-8-5 939
620931 13317 13334 CATAGGCAAAACAGACAA 68 5-8-5 940
620932 13504 13521 TAGTTGAATGTTTTGGAA 73 5-8-5 941
620933 13702 13719 ACGGCGGAGGCTGAGGCG 10 5-8-5 942
620934 13882 13899 AGGTGCAAGCTGGCCGGG 44 5-8-5 943
620935 14062 14079 TGATACCCTGTAAGAATA 82 5-8-5 944
620936 14243 14260 AATATAGCAAAGGGAATT 54 5-8-5 945
620937 14423 14440 TAGAGATAATTATGTCCC 73 5-8-5 946
620938 14609 14626 GCAATTACAGAGCCAGGG 76 5-8-5 947
620939 14789 14806 GCAGAACTGGATCCGATC 69 5-8-5 948
620940 15035 15052 CAAAGCCAGGATGGTCTC 92 5-8-5 949
620941 15246 15263 CCAAAGAGATTCCTTTTT 79 5-8-5 950
620942 15924 15941 GGCCTCATTTATATATAT 20 5-8-5 951
620943 16156 16173 TATGGGACAATCATAGCT 56 5-8-5 952
620944 16336 16353 AATTCTGATACATTGTCA 81 5-8-5 953
620945 16516 16533 GATGACTCTTCCAAATGG 70 5-8-5 954
620946 16696 16713 TCACTTCATTATTCAAGC 84 5-8-5 955
620947 16876 16893 GTGCAGCCGCCACTGGCC 86 5-8-5 956
620948 17085 17102 CGATAAGATCTTTTTAAA 25 5-8-5 957
620949 17266 17283 GACAGTATAATCTCCATT 69 5-8-5 958
620950 17446 17463 AGGCTAAAGTCAGCCCAC 29 5-8-5 959
620951 17639 17656 CTTGCACCCTGGTTTGGG 40 5-8-5 960
620952 17819 17836 TCCCTGTCTGGTTAGGAA 80 5-8-5 961
620953 17999 18016 ACGCGCCTGCCCCGTGCC 50 5-8-5 962
620954 18179 18196 CTCAAACCTGGCCACGCT 64 5-8-5 963
620955 18359 18376 GAAACTGGCGGGTCTGTT 64 5-8-5 964
620956 18539 18556 GGGAGGAATTTGGCAATG 37 5-8-5 965
620957 18719 18736 AAAGAGCACGGCATCTAT 76 5-8-5 966
620958 18905 18922 AAACTGTGAGGCACTGGG 87 5-8-5 967
620959 19088 19105 CATTTGACATTGGCCTGT 72 5-8-5 968
620960 19283 19300 ATTATTATCAGCATCTTC 77 5-8-5 969 620961 19467 19484 TTGCAGAGGCAGGAAGGC 67 5-8-5 970
620962 19647 19664 AGCAGCCTGGTTTAGAGG 31 5-8-5 971
620963 19827 19844 TCCACATCTTCCAGCCTC 74 5-8-5 972
620964 20007 20024 ATACAGACTCAGTCTCCT 79 5-8-5 973
Table 23
Inhibition of Tau mRNA by 5-10-5 and 5-8-5 MOE gapmers targeting SEQ ID ] VO: 1
SEQ SEQ ID ID
ISIS % SEQ NO: 1 NO: 1 Sequence Motif NO inhibition ID NO Start Stop
Site Site
613412 73879 73898 GTGATCTTCCATCACTTCGA 78 5-10-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 93 5-8-5 665
98928 98945
621042 35901 35918 AGTCGACCGCACTCCTGC 42 5-8-5 974
621043 36106 36123 GCCCTGAGGGTCCACAGG 20 5-8-5 975
621044 36286 36303 AGGGCAGTGTCCTACCTC 50 5-8-5 976
621045 36466 36483 GTCCTCCCTTCCCCACAA 29 5-8-5 977
621046 36653 36670 CTGAATTCCTATCATGCG 51 5-8-5 978
621047 36833 36850 GAGAAAGAAGAGTGTGGT 23 5-8-5 979
621048 37024 37041 CTTAGGACAGATTCCTAG 29 5-8-5 980
621049 37204 37221 GTCAGAAGTGTTTTCCCA 80 5-8-5 981
621050 37413 37430 TCCAATTTTAAACTTAAA 28 5-8-5 982
621051 37750 37767 CTACTGTTTAAAAAGGCT 66 5-8-5 983
621052 38013 38030 GTAAACACCATCTCTAAA 19 5-8-5 984
621053 38193 38210 CTCTGATACTATTATAAG 31 5-8-5 985
621054 38374 38391 CTGAGCTATTTGAGAATT 21 5-8-5 986
621055 38557 38574 TCTGCATATATAATTATA 57 5-8-5 987
621056 38737 38754 CACATTGTTTAGATTTTC 79 5-8-5 988
621057 38922 38939 TTAGCACACCTGAAACGC 51 5-8-5 989
621058 39102 39119 GGCAAAGAAGACAGGAGA 68 5-8-5 990
621059 39541 39558 TGCTGAGGATGCTTTTTT 72 5-8-5 991
621060 39721 39738 GCTGGAAGATGAGGTGGT 47 5-8-5 992
621061 39901 39918 TGGGTGTTGGAGAAAACT 24 5-8-5 993
621062 40100 40117 GCTTTAAAAGCTCCTCAG 65 5-8-5 994
621063 40347 40364 CGAGCACTTGTAGTTCCA 35 5-8-5 995
621064 40528 40545 GCAGTGCTTTAAAAATAT 22 5-8-5 996
621065 40709 40726 GAAATTTCATGAAATATG 37 5-8-5 997
621066 40889 40906 TTACTTGCAATTGAAAGA 6 5-8-5 998
621067 41069 41086 GGAAAGACTACTCAGAGC 57 5-8-5 999
621068 41249 41266 GATGAAAGAGAATAACAA 20 5-8-5 1000
621069 41429 41446 ACCCATTATCTGCTCCCC 66 5-8-5 1001
621070 41611 41628 TTTTCAGTAACAACATAA 51 5-8-5 1002
621071 41944 41961 CCTTTAGCTTTCCTTTTC 47 5-8-5 1003 621072 42124 42141 CAAATTCTGTTTCTGTAA 47 5-8-5 1004
621073 42304 42321 GCAAAAATAAGTGAACTG 38 5-8-5 1005
621074 42492 42509 TTGCAGTGACTTCTTGGG 71 5-8-5 1006
621075 42672 42689 AAACCCCTTCAACATCAG 45 5-8-5 1007
621076 42853 42870 ACAAGAGAAACATTTTAC 53 5-8-5 1008
621077 43033 43050 CACTTAGTAACCAAGCAA 53 5-8-5 1009
621078 43213 43230 CATAGATGATATATTTTG 78 5-8-5 1010
621079 43393 43410 AAATCTTGAACTCCTGAA 58 5-8-5 1011
621080 43620 43637 GGAGTTTTTGTGTTTCTT 77 5-8-5 1012
621081 43804 43821 AAATTCTTAATGGTTCAG 75 5-8-5 1013
621082 44051 44068 AGATAGATCTCGGCTCAC 81 5-8-5 1014
621083 44231 44248 AACCTTTAATAAACTTTT 43 5-8-5 1015
621084 44617 44634 GTCCAAGAGGTTTTTTTT 41 5-8-5 1016
621085 44809 44826 GCAGTCACTGCATTCCAG 27 5-8-5 1017
621086 45050 45067 CAAAAACAGGGCTAGGCA 21 5-8-5 1018
621087 45230 45247 TTCCCTGAACAAATGGCA 59 5-8-5 1019
621088 45412 45429 CTCAAATGTAACATTTTA 84 5-8-5 1020
621089 45592 45609 TACACCTGGCCCATGACC 41 5-8-5 1021
621090 45772 45789 AAGTTCTGGCTCAAGCAA 21 5-8-5 1022
621091 45955 45972 ACAACTCTCTGGAAAAAA 30 5-8-5 1023
621092 46135 46152 AGTCCACGCCTGCATCTG 63 5-8-5 1024
621093 46315 46332 CAAGGCAACAGAGAGGCA 42 5-8-5 1025
621094 46495 46512 AACATGCATATAATTACC 40 5-8-5 1026
621095 46675 46692 TTGGAGGGACAATTCTCA 46 5-8-5 1027
621096 46855 46872 CGAAGAGGCCAGGACTGC 34 5-8-5 1028
621097 47036 47053 TTTGGCTCTGGTGATGGT 45 5-8-5 1029
621098 47216 47233 ACCAGGCAAAGGGACACC 68 5-8-5 1030
621099 47396 47413 TGCCAGAGACAGAGGTTG 68 5-8-5 1031
621100 47576 47593 CACACGCATGGCTTCATG 55 5-8-5 1032
621101 47756 47773 GAGGAAGGCATGAACTAA 60 5-8-5 1033
621102 47936 47953 TCCCATGCCCACGCATGT 26 5-8-5 1034
621103 48116 48133 ATTATTAACGAACAAAAA 2 5-8-5 1035
621104 48302 48319 ATGTCCAGATGTGGATCG 52 5-8-5 1036
621105 48482 48499 ATCAGACAGAAGAGCCAT 55 5-8-5 1037
621106 48678 48695 GCACAGTGCAACAGTGGG 78 5-8-5 1038
621107 49171 49188 GGCTTTCCCTTCCCTTCT 33 5-8-5 1039
621108 49361 49378 AGCTGGGAAAGTGGCAGG 23 5-8-5 1040
621109 49561 49578 ATGACAACTTGATTTGGG 65 5-8-5 1041
621110 49741 49758 GACCGCAACCTTGCCAAA 43 5-8-5 1042
621111 50111 50128 CTTTCAGAGACAGGCTCG 41 5-8-5 1043
621112 50298 50315 GGGCTCACCCCTGTAGTT 9 5-8-5 1044
621113 50766 50783 CTCTTAAGGCCGAGTGCA 39 5-8-5 1045
621114 50970 50987 TGCTAGGCAAAAAAACAA 35 5-8-5 1046 621115 51150 51167 TAATTAGAAACCTCCTCG 10 5-8-5 1047
621116 51330 51347 CAGGTCATATATGAAATC 33 5-8-5 1048
621117 51521 51538 TGTGCTTCCGAAAGTAGT 63 5-8-5 1049
621118 51702 51719 GCATCAAATCCATGCATT 38 5-8-5 1050
Table 24
Inhibition of tau mRNA by 5-10-5 and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ SEQ ID ID
ISIS % SEQ NO: 1 NO: 1 Sequence Motif NO inhibition ID NO Start Stop
Site Site
613412 73879 73898 GTGATCTTCCATCACTTCGA 71 5-10-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 84 5-8-5 665
98928 98945
621119 51894 51911 ACTCTGAAGCACAGACAG 21 5-8-5 1051
621120 52081 52098 CTTACCTGGGCTGGATGG 5 5-8-5 1052
621121 52261 52278 TTCTGTCTATAGCTAGAA 32 5-8-5 1053
621122 52441 52458 AGATGGCTTCCTATTGTT 32 5-8-5 1054
621123 52621 52638 TGGTTTGGTCAGGCCTCA 51 5-8-5 1055
621124 52803 52820 ATTGTAACTCAAAGTGGG 50 5-8-5 1056
621125 53004 53021 AAACTCTACACCCCTGGG 17 5-8-5 1057
621126 53264 53281 CATACCAGGAAGGAAGGA 17 5-8-5 1058
621127 53444 53461 TGGACATATCAGAATTTG 64 5-8-5 1059
621128 53624 53641 CCCTTATTAGTGTCTACA 68 5-8-5 1060
621129 53805 53822 ACACTCTAGAGAGGTCCA 68 5-8-5 1061
621130 53985 54002 GACAGTACCCACGACACG 32 5-8-5 1062
621131 54165 54182 CCCCATTCTGGATCCTGG 40 5-8-5 1063
621132 54348 54365 ATTGTCTGTAAAATGTGG 57 5-8-5 1064
621133 54528 54545 ATTCTGTCTGACAGAGAC 40 5-8-5 1065
621134 54711 54728 CGATGGTGCACATCTATA 45 5-8-5 1066
621135 54891 54908 CAGTGTCCGCTGAACACA 37 5-8-5 1067
621136 55073 55090 AGGAGGATTAGGAAGAAA 14 5-8-5 1068
621137 55261 55278 CAAGGAAGGCGATCTGGG 17 5-8-5 1069
621138 55623 55640 GGTGGCACTTGAACAAGC 61 5-8-5 1070
621139 55803 55820 GGTGGAAAGGGAACCCGG 43 5-8-5 1071
621140 55983 56000 CCCCACGGGTACACAGAG 20 5-8-5 1072
621141 56163 56180 CGCTGCGAACGATGCACT 63 5-8-5 1073
621142 56343 56360 GAATGGTCCTTCTTACAG 13 5-8-5 1074
621143 56526 56543 CTGGGAAAACCAAGGCAA 57 5-8-5 1075
621144 56706 56723 CCCAGGCTGAGGAAGGAC 40 5-8-5 1076
621145 56886 56903 GATTGGCTTTTACATCTT 48 5-8-5 1077
621146 57289 57306 CGGTTTGAGTACAGTGGT 50 5-8-5 1078
621147 57470 57487 CTAACATCTTAGGGCACA 73 5-8-5 1079
621148 57650 57667 TTCCTCTGATGTCAGAAT 51 5-8-5 1080 621 149 58090 58107 CCCCCTCGGTCGCCCAGG 37 54 i-5 1081
621 150 58271 58288 TGTTTTAACTAAAAGCTT 24 54 i-5 1082
621 151 58451 58468 GTATGAGGGCATAGATGA 26 54 i-5 1083
621 152 58631 58648 ATCACACCACAGAATGTT 29 54 1-5 1084
621 153 58812 58829 TACTCTTCTGTGGCAGCT 66 54 i-5 1085
621 154 59198 59215 CAACAGCAAGGCCAGGCG 44 54 i-5 1086
621 155 59380 59397 TAGATAATTTGAAATTTA 0 54 i-5 1087
621 156 59786 59803 ACCTTAATCCCAGTATTT 12 54 i-5 1088
621 157 59966 59983 TGCTTATGGTCCAGCTGT 40 54 i-5 1089
621 158 60146 60163 GAGCCAGGCTTCAAAACC 26 54 i-5 1090
621 159 60326 60343 TCCAGCACATTTAGGCGG 32 54 i-5 1091
621 160 60522 60539 ATCTCAATAAAGCTATCG 70 54 i-5 1092
621 161 60702 60719 CGCCCGCGGTGACTCAGC 32 54 i-5 1093
621 162 60906 60923 GGAGTCAAGAGTCCAGGG 44 54 i-5 1094
621 163 61109 61 126 AGAAGGAATGATGAGGAA 24 54 i-5 1095
621 164 61290 61307 GCAGTACAGTTTGGTAAT 32 54 i-5 1096
621 165 61476 61493 TGGCTACTCTCTCAGGAG 9 54 i-5 1097
621 166 61656 61673 GACCCTAGGTGGGTAAAG 21 54 i-5 1098
621 167 61836 61853 GTCCCCTGAGGTGTTGTG 0 54 i-5 1099
621 168 62016 62033 CCCAGACAACCTTCACCT 34 54 i-5 1100
621 169 62208 62225 TGGGTTAAGACTCTGTCT 11 54 i-5 1101
621 170 62467 62484 TGGAGCCGAGTGCGGTGG 12 54 i-5 1102
621 171 62657 62674 TCTCTTCTGGATATTTCG 49 54 i-5 1103
621 172 62843 62860 CGATTTAAAATGAATGAT 20 54 i-5 1104
621 173 63023 63040 AGAACAAAGATAACAGTT 0 54 i-5 1105
621 174 63218 63235 GCTAAATTGAGGCCCGGG 42 54 i-5 1106
621 175 63398 63415 CTTTCTCCTTTCCTCAAT 26 54 i-5 1107
621 176 63578 63595 TCTAAGCGAGACACACAG 26 54 i-5 1108
621 177 63758 63775 TGATTTCTCTGGAAGAAC 27 54 i-5 1109
621 178 63938 63955 GCCTGTGTCTTCTAACTC 32 54 i-5 1110
621 179 64184 64201 GGCTGAGAAGCTGGGACC 40 54 i-5 1111
621 180 64365 64382 TTTTTACTTGAGATGTCA 38 54 i-5 1112
621 181 64543 64560 GTAGGTGTCATCATCATC 83 54 i-5 1113
621 182 64723 64740 CAACACTGACTGAGCACA 56 54 i-5 1114
621 183 64903 64920 GCATTGGCTGGGCTAGGT 73 54 i-5 1115
621 184 65092 65109 TATTTACTATGCAAAATA 18 54 i-5 1116
621 185 65273 65290 AGCACCACAGAGCAGATG 19 54 i-5 1117
621 186 65453 65470 CTCAGTGTGGAGTTCTGC 44 54 i-5 1118
621 187 65634 65651 CATTTTTCAACCACTTAA 44 54 i-5 1119
621 188 65815 65832 GTAAGGCTTTGTGGGCCA 32 54 i-5 1120
621 189 66148 66165 TACTTTGCATTATTTATT 44 54 i-5 1121
621 190 66535 66552 TTGGCTTTCATTATAATT 13 54 i-5 1122
621 191 66736 66753 GGACGGTTGGGAAATAGG 18 54 i-5 1123 621192 66916 66933 GAGCAACTGTTCATAGGG 49 5-8-5 1124
621193 67096 67113 TCTGCACATCGACACATC 34 5-8-5 1125
621194 67538 67555 AGGCTAGGCCCCATGGCT 21 5-8-5 1126
621195 67718 67735 GCACCCCCATCCTTCAGC 30 5-8-5 1127
Table 25
Inhibition of Tau mRNA by 5-10-5 and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
Figure imgf000094_0001
621381 106545 106562 CGGAGTCTTATTCTGATG 39 5-8-5 1159
621382 106725 106742 CCGCAGCAATGCCCCTGC 65 5-8-5 1160
621383 106905 106922 TCTCGATCCCCTTCAAGA 23 5-8-5 1161
621384 107085 107102 CTGGGTGTCCTTTACCCT 5 5-8-5 1162
621385 107265 107282 CCTGCTCCACGCCTGCCT 82 5-8-5 1163
621386 107445 107462 TCAGGGCCTTTATCCTAA 51 5-8-5 1164
621387 107625 107642 TCGGCTCCACAGTCTTTG 60 5-8-5 1165
621388 107805 107822 CACCACTGGGTTAGGCAG 38 5-8-5 1166
621389 108170 108187 TCACCTTCCCGCCTCCCG 16 5-8-5 1167
621390 108359 108376 TTGAAGAGGGTCCAGAGG 3 5-8-5 1168
621391 108548 108565 TGCCCAGAAGGCAGGTGG 38 5-8-5 1169
621392 108728 108745 GGCCCCCGAAGTCTGTGC 62 5-8-5 1170
621393 108908 108925 CGGTGCAGGACAGAGGTG 57 5-8-5 1171
621394 109135 109152 CGCGCACCACCACCACGC 78 5-8-5 1172
621395 109315 109332 TCAGGCTGGGCCCTAAGC 50 5-8-5 1173
621396 109495 109512 TCACCCTTCCCCAGCTCC 45 5-8-5 1174
621397 109675 109692 TCAGACCCAAGTGATAAG 64 5-8-5 1175
621398 109855 109872 GCAGGGTCTACACATGCG 76 5-8-5 1176
621399 110045 110062 CTGAGATGTTCTCTTCCT 73 5-8-5 1177
621400 110225 110242 AGCTGCGGTACAGGACAG 68 5-8-5 1178
621401 110405 110422 CCAGTGAGGGCCCCTCTG 20 5-8-5 1179
621402 110585 110602 GCCCCCAGAGGTGCATGG 58 5-8-5 1180
621403 110769 110786 GGAGCAGCAGACACATGC 79 5-8-5 1181
621404 110949 110966 GGAAGCTACTTCCCATGC 43 5-8-5 1182
621406 111495 111512 GGAGGAGGCCTGACACCC 58 5-8-5 1183
621407 111675 111692 TTTCCCTTTGGTGTTAGC 91 5-8-5 1184
621408 111856 111873 CCCCAAAGCAATCTATGT 47 5-8-5 1185
621409 112036 112053 TCTGGAGGGAACACTGCC 60 5-8-5 1186
621410 112216 112233 GGTTGAGGTTGAGGGTAG 37 5-8-5 1187
621411 112396 112413 ACTCCCGCAGGCCAAACA 36 5-8-5 1188
621412 112603 112620 GAGTGCCAACAGGCCCAG 75 5-8-5 1189
621413 112784 112801 TTCTAAGTACAATTTGGG 67 5-8-5 1190
621414 112984 113001 TTACTGGTTGTGTTTTCT 94 5-8-5 1191
621415 113164 113181 GAGTCTCAGTCTCACTGT 76 5-8-5 1192
621416 113347 113364 CATTCCTCCTGCTGCTGT 83 5-8-5 1193
621417 113540 113557 GCTCTGAAGAGCTCCACG 83 5-8-5 1194
621418 113720 113737 CTCTCTTCAGGGCCACCG 82 5-8-5 1195
621419 113900 113917 CTCTCTCGCCCTGCATGG 48 5-8-5 1196
621420 114080 114097 CTGGACATCCTCCGAGAA 27 5-8-5 1197
621421 114260 114277 CACCCAGGCTGCGGCCCA 64 5-8-5 1198
621422 114440 114457 CTGTGGTGCAAGCCTGTG 42 5-8-5 1199
621423 114639 114656 AGACGAGAGTGCGCCCAC 78 5-8-5 1200
621424 115155 115172 CCACATAAATGTTCTACA 94 5-8-5 1201 621425 115335 115352 GGTACAGGAAAAGATGCC 88 5-8-5 1202
621426 115515 115532 ATCAGCTTAGGAACTGAC 85 5-8-5 1203
Table 26
Inhibition of tau mRNA by 5-10-5 and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ ID SEQ ID
ISIS % SEQ ID NO: 1 start NO: 1 stop Sequence Motif
NO inhibition NO:
site site
613412 73879 73898 GTGATCTTCCATCACTTCGA 86 5-10-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 97 5-8-5 665
98928 98945
621427 115695 115712 GTGCCTCACAGTTGGCTC 39 5-8-5 1204
621428 115896 115913 GAGAGGCTGTGGTGAGGT 25 5-8-5 1205
621429 116077 116094 GAAATCAGAATCCTGAAA 66 5-8-5 1206
621430 116259 116276 ATGTGGCTTTTTCTTGTT 64 5-8-5 1207
621431 116595 116612 GTTTTTTTCTGGCCGGGC 90 5-8-5 1208
621432 116955 116972 TTTTCAGTGGATAAGGCT 78 5-8-5 1209
621433 117135 117152 ACATAAACTCCTTCTTTG 53 5-8-5 1210
621434 117315 117332 TGGTGAATGTTGAAATTC 78 5-8-5 1211
621435 117495 117512 AGGACAAGGAGGCCATGT 56 5-8-5 1212
621436 117787 117804 TCAAATGACAGATTCTCA 78 5-8-5 1213
621437 117967 117984 CATCCCTACTTTCTCCCT 24 5-8-5 1214
621438 118420 118437 TAGAATCTTGCCTTGTCG 34 5-8-5 1215
621439 118600 118617 GATACAGACATACATTGT 68 5-8-5 1216
621440 118831 118848 AATTCTTTGTGATGATGG 82 5-8-5 1217
621441 119044 119061 GCACATATTTACATTTTA 94 5-8-5 1218
621442 119224 119241 GGTGAGAGAGCCAGCCTC 59 5-8-5 1219
621443 119404 119421 CAAACAGCCTCCCATGAA 38 5-8-5 1220
621444 119584 119601 ATATCCTAAGCATTGTCT 71 5-8-5 1221
621445 119880 119897 TACAGTGGCTCCTGTAAT 18 5-8-5 1222
621446 120060 120077 GCTCATCAAAGCAAAAAC 84 5-8-5 1223
621447 120248 120265 CTGGGCTGTCGGATCTGG 69 5-8-5 1224
621448 120439 120456 AAGCCACCATGCCTGTAA 89 5-8-5 1225
621449 120660 120677 ACACCATCACGGCTCAGT 56 5-8-5 1226
621450 120840 120857 AGGGAGTTGGAAAAACTG 46 5-8-5 1227
621451 121209 121226 CAGGTTTGAGAAGCCCTG 0 5-8-5 1228
621452 121389 121406 AGACCCACCGGCACATTC 56 5-8-5 1229
621453 121569 121586 ATTTCCAGCGGGCTTTAC 62 5-8-5 1230
621454 121761 121778 GCTTGCTCGCAAGGACGC 93 5-8-5 1231
621455 121941 121958 CCACAGCACGGCGCATGG 65 5-8-5 1232
621456 122121 122138 ACACCCCCTCCTAGAATA 0 5-8-5 1233
621457 122301 122318 ACCACCTTCAGCCCAACT 22 5-8-5 1234
621458 122481 122498 TAAGCTGGAGGCTTAGGA 40 5-8-5 1235
621459 122678 122695 GAACCTCCCGCCTTAGTC 19 5-8-5 1236 621460 122913 122930 TATATGATTGTTTTTTGA 0 5-8-5 1237
621461 123093 123110 AGGAGCCTCGGGTGCCCA 30 5-8-5 1238
621462 123273 123290 TCAGGCCAGGCATTTTCT 58 5-8-5 1239
621463 123453 123470 CACTTAGCAGACACTGGT 77 5-8-5 1240
621464 123633 123650 TCCTCTGGCCGAGCTCAC 58 5-8-5 1241
621465 123824 123841 ACAGCGCGGGACACACGG 77 5-8-5 1242
621466 124004 124021 GCATCTCTTCTCACCAGG 13 5-8-5 1243
621467 124184 124201 GGATACCTGGAGGGCAGG 13 5-8-5 1244
621468 124379 124396 TGAGGCCAGCACTGAGGG 29 5-8-5 1245
621469 124559 124576 ACTGGAACCATCCCGAAT 37 5-8-5 1246
621470 124752 124769 GCAGTGGCCTTGTGTGGG 19 5-8-5 1247
621471 124932 124949 ACCCTGAGCTGCCAGCTG 53 5-8-5 1248
621472 125112 125129 CTGCACACTCAGTGTCCT 78 5-8-5 1249
621473 125292 125309 CTGGAAAGGCAGGAGTGG 33 5-8-5 1250
621474 125472 125489 AAGAGCCTCTGGGAAAAA 50 5-8-5 1251
621475 125652 125669 GGTGTAACTCAATGAGAA 41 5-8-5 1252
621476 125832 125849 GGCTACCTGGTTTATGAT 32 5-8-5 1253
621477 126012 126029 GGAGCGAGCTGGAGCCAC 75 5-8-5 1254
621478 126332 126349 GCTCACTCCGCTCACTGC 74 5-8-5 1255
621479 126527 126544 CTAGGTCTTATTCTATTT 38 5-8-5 1256
621480 127208 127225 AGGTTTTTGTTGTGTGTT 54 5-8-5 1257
621481 127396 127413 CCTCTTCACCTTAAAAAA 24 5-8-5 1258
621482 127591 127608 AAACAAAGTCACAGAGGG 67 5-8-5 1259
621483 127934 127951 AGGATCACTTTCTTTTCT 31 5-8-5 1260
621484 128271 128288 TTGCTGAGTCTCACTCTG 87 5-8-5 1261
621485 128451 128468 CAGAATCTATAGCTGTGT 51 5-8-5 1262
621486 128631 128648 ATACTTTCCACAGGGAGA 45 5-8-5 1263
621487 128811 128828 GAAGAAGGGTCCCTCTCT 38 5-8-5 1264
621488 128993 129010 ATGAGAGTACAACTCCAT 44 5-8-5 1265
621489 129269 129286 ACCCTTTCAGGCCGGGCG 44 5-8-5 1266
621490 129678 129695 TAAGTAAAGTCTGTTTTT 47 5-8-5 1267
621491 129861 129878 TAGAATGGTGTTTGGATA 42 5-8-5 1268
621492 130040 130057 GCAGTCTACAGGGCAAAG 79 5-8-5 1269
621493 130234 130251 ACCTTTTTATTTCCTCCG 14 5-8-5 1270
621494 130414 130431 AGCTCCAGGTGATTGAGA 68 5-8-5 1271
621495 130691 130708 GTCGCCATGTAAGAAATG 46 5-8-5 1272
621496 130902 130919 AGGGAGACTGATATGGTT 31 5-8-5 1273
621497 131113 131130 ATATTGCTTGACCTCAGG 18 5-8-5 1274
621498 131543 131560 AGTGGTTCTCGCTTTTTT 75 5-8-5 1275
621499 131725 131742 CAAGACAGGCAGGCAGAG 19 5-8-5 1276
621500 131905 131922 GTGCTTGGCTGCGCTTCT 45 5-8-5 1277
621501 132085 132102 CAGACCTCTATCTTTTAG 24 5-8-5 1278
621502 132266 132283 AGGAGGGTCTTTCCCGTG 57 5-8-5 1279 1503 132446 132463 AACTGGGCCACCATGAGA 30 5-8-5 1280
Table 27
Inhibition of Tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NO: 1
Figure imgf000098_0001
621556 6057 6074 GCGAGGGCGCGCGCCGGC 15 5-8-5 1318
621557 6060 6077 ACTGCGAGGGCGCGCGCC 30 5-8-5 1319
621558 6063 6080 GTGACTGCGAGGGCGCGC 38 5-8-5 1320
621559 6066 6083 GCGGTGACTGCGAGGGCG 30 5-8-5 1321
621560 6069 6086 GTGGCGGTGACTGCGAGG 37 5-8-5 1322
621561 6072 6089 TGGGTGGCGGTGACTGCG 48 5-8-5 1323
621562 6075 6092 TGGTGGGTGGCGGTGACT 34 5-8-5 1324
621563 6078 6095 AGCTGGTGGGTGGCGGTG 54 5-8-5 1325
621564 6081 6098 CGGAGCTGGTGGGTGGCG 36 5-8-5 1326
621565 6084 6101 TGCCGGAGCTGGTGGGTG 52 5-8-5 1327
621566 6087 6104 TGGTGCCGGAGCTGGTGG 64 5-8-5 1328
621567 6090 6107 TGTTGGTGCCGGAGCTGG 74 5-8-5 1329
621568 6093 6110 TGCTGTTGGTGCCGGAGC 50 5-8-5 1330
621569 6096 6113 CGCTGCTGTTGGTGCCGG 47 5-8-5 1331
621570 6111 6128 GGGCGGTGGCAGCGGCGC 86 5-8-5 1332
621571 6114 6131 GGTGGGCGGTGGCAGCGG 61 5-8-5 1333
621572 6117 6134 GAAGGTGGGCGGTGGCAG 19 5-8-5 1334
621573 6120 6137 GCAGAAGGTGGGCGGTGG 22 5-8-5 1335
621574 6123 6140 GCGGCAGAAGGTGGGCGG 18 5-8-5 1336
621575 6126 6143 GCGGCGGCAGAAGGTGGG 62 5-8-5 1337
621576 6129 6146 GTGGCGGCGGCAGAAGGT 79 5-8-5 1338
621577 6132 6149 GTGGTGGCGGCGGCAGAA 70 5-8-5 1339
621578 6135 6152 GCTGTGGTGGCGGCGGCA 78 5-8-5 1340
621579 6138 6155 GTGGCTGTGGTGGCGGCG 78 5-8-5 1341
621580 6141 6158 AAGGTGGCTGTGGTGGCG 72 5-8-5 1342
613412 73879 73898 GTGATCTTCCATCACTTCGA 78 5-8-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 94 5-8-5 665
98928 98945
621504 132626 132643 AAACCCTTCTCACAAAAC 17 5-8-5 1343
621505 132812 132829 CCTCCTGGTCCCTCCTCG 12 5-8-5 1344
621506 132992 133009 CCTATCCAGACCCACTCA 10 5-8-5 1345
621507 133179 133196 CACAACCACCCCGGGACG 8 5-8-5 1346
621508 133359 133376 GGACCAGGATGGTGTTTT 44 5-8-5 1347
621509 133539 133556 GGCTTTGATAAAAATATT 8 5-8-5 1348
621510 133788 133805 CGCCTCCGGGATCAAGCA 47 5-8-5 1349
621511 133968 133985 GGTTCGCATTTAAGCTAG 28 5-8-5 1350
621512 134148 134165 ACGGTCATTCTCAGCCCT 59 5-8-5 1351
621513 134333 134350 TAACCTCCAGAGCAACTG 26 5-8-5 1352
621514 134513 134530 GGCAGGTGGGCCACAAAA 17 5-8-5 1353
621515 134696 134713 TCAAAAGGCATGCACATT 44 5-8-5 1354
621516 134876 134893 TAGTATTCCGTCGCCACG 40 5-8-5 1355
621517 135057 135074 GAGCTGTTGTTTTTATTG 7 5-8-5 1356
621518 135369 135386 GCCAGGGACTGTTTTTTG 62 5-8-5 1357 Table 28
Inhibition of tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NOs: 1 and 2
Figure imgf000100_0001
621610 73882 73899 CGTGATCTTCCATCACTT 13 348 365 5-8-5 1394
621611 73906 73923 TGTCCCCCAACCCGTACG 46 372 389 5-8-5 1395
621612 73909 73926 TCCTGTCCCCCAACCCGT 54 375 392 5-8-5 1396
621613 73912 73929 CTTTCCTGTCCCCCAACC 43 378 395 5-8-5 1397
621614 73915 73932 GATCTTT CCT GT CC CC C A 71 381 398 5-8-5 1398
621615 73918 73935 CCTGATCTTTCCTGTCCC 71 384 401 5-8-5 1399
621616 73921 73938 CCCCCTGATCTTTCCTGT 54 387 404 5-8-5 1400
621617 73924 73941 AGCCCCCCTGATCTTTCC 40 390 407 5-8-5 1401
621618 73927 73944 TGTAGCCCCCCTGATCTT 35 393 410 5-8-5 1402
621619 73930 73947 TGGTGTAGCCCCCCTGAT 31 396 413 5-8-5 1403
621620 73933 73950 GCATGGTGTAGCCCCCCT 71 399 416 5-8-5 1404
621621 73936 73953 GGTGCATGGTGTAGCCCC 62 402 419 5-8-5 1405
621622 73939 73956 CTTGGTGCATGGTGTAGC 54 405 422 5-8-5 1406
621623 73942 73959 GGTCTTGGTGCATGGTGT 65 408 425 5-8-5 1407
621624 73945 73962 CTTGGTCTTGGTGCATGG 63 411 428 5-8-5 1408
621625 73948 73965 CCTCTTGGTCTTGGTGCA 49 414 431 5-8-5 1409
621626 73951 73968 C ACC CT CTTGGT CTTGGT 65 417 434 5-8-5 1410
621627 73956 73973 CGTGTCACCCTCTTGGTC 49 422 439 5-8-5 1411
621628 73959 73976 GTCCGTGTCACCCTCTTG 70 425 442 5-8-5 1412
621631 83395 83412 AGATCCGTCCTCAGTGGG 48 473 490 5-8-5 1413
621632 83398 83415 CTCAGATCCGTCCTCAGT 38 476 493 5-8-5 1414
621633 83401 83418 TTCCTCAGATCCGTCCTC 11 479 496 5-8-5 1415
621634 83404 83421 CGGTTCCTCAGATCCGTC 57 482 499 5-8-5 1416
621635 83407 83424 GCCCGGTTCCTCAGATCC 38 485 502 5-8-5 1417
621636 83410 83427 AGAGCCCGGTTCCTCAGA 63 488 505 5-8-5 1418
621637 83413 83430 TTCAGAGCCCGGTTCCTC 50 491 508 5-8-5 1419
621638 83416 83433 GGTTTCAGAGCCCGGTTC 48 494 511 5-8-5 1420
621639 83419 83436 AGAGGTTTCAGAGCCCGG 41 497 514 5-8-5 1421
621640 83422 83439 ATCAGAGGTTTCAGAGCC 40 500 517 5-8-5 1422
621641 83425 83442 AGCATCAGAGGTTTCAGA 23 503 520 5-8-5 1423
621642 83428 83445 CTTAGCATCAGAGGTTTC 50 506 523 5-8-5 1424
621643 83431 83448 GCTCTTAGCATCAGAGGT 69 509 526 5-8-5 1425
621644 83434 83451 AGTGCTCTTAGCATCAGA 80 512 529 5-8-5 1426
621645 83437 83454 TGGAGTGCTCTTAGCATC 50 515 532 5-8-5 1427
621646 83440 83457 TGTTGGAGTGCTCTTAGC 59 518 535 5-8-5 1428
621647 83443 83460 CGCTGTTGGAGTGCTCTT 70 521 538 5-8-5 1429
621648 83446 83463 TTCCGCTGTTGGAGTGCT 52 524 541 5-8-5 1430
621654 85904 85921 CTAAGGGTGCTGTCACAT 64 543 560 5-8-5 1431
621655 85907 85924 CCACTAAGGGTGCTGTCA 44 546 563 5-8-5 1432
621656 85910 85927 CATCCACTAAGGGTGCTG 55 549 566 5-8-5 1433
621657 85913 85930 CCTCATCCACTAAGGGTG 41 552 569 5-8-5 1434
98891 98908 n/a n/a
620887 GTTTTCAAACACACCTTC 81 5-8-5 665
98928 98945 n/a n/a Table 29
Inhibition of Tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NOs: 1 and 2
SEQ ID SEQ ID SEQ ID SEQ ID SEQ
%
ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO: 2 Motif ID inhibition
start site stop site start site stop site NO
621672 n/a n/a GCTGTGGTTCCTTCTGGG 51 613 630 5-8-5 1435
621676 n/a n/a TCAGGCTCTTGGGTCACG 0 685 702 5-8-5 1436
621677 n/a n/a CTTTCAGGCTCTTGGGTC 0 688 705 5-8-5 1437
621678 n/a n/a CCACTTTCAGGCTCTTGG 51 691 708 5-8-5 1438
613412 73879 73898 GTGATCTTCCATCACTTCGA 79 345 364 5-10-5 25
621658 85916 85933 CTCCCTCATCCACTAAGG 27 555 572 5-8-5 1439
621659 85919 85936 GAGCTCCCTCATCCACTA 35 558 575 5-8-5 1440
621660 85922 85939 CGGGAGCTCCCTCATCCA 25 561 578 5-8-5 1441
621661 85925 85942 TGCCGGGAGCTCCCTCAT 40 564 581 5-8-5 1442
621662 85928 85945 GCTTGCCGGGAGCTCCCT 47 567 584 5-8-5 1443
621663 85931 85948 CCTGCTTGCCGGGAGCTC 46 570 587 5-8-5 1444
621664 85934 85951 CAGCCTGCTTGCCGGGAG 45 573 590 5-8-5 1445
621665 85937 85954 CGGCAGCCTGCTTGCCGG 1 576 593 5-8-5 1446
621666 85940 85957 GCGCGGCAGCCTGCTTGC 31 579 596 5-8-5 1447
621667 85943 85960 GCTGCGCGGCAGCCTGCT 50 582 599 5-8-5 1448
621668 85962 85979 TCTGGGATCTCCGTGTGG 39 601 618 5-8-5 1449
621669 85965 85982 CCTTCTGGGATCTCCGTG 56 604 621 5-8-5 1450
621670 85968 85985 GTTCCTTCTGGGATCTCC 79 607 624 5-8-5 1451
621671 85971 85988 GTGGTTCCTTCTGGGATC 49 610 627 5-8-5 1452
621673 89894 89911 CAATGCCTGCTTCTTCAG 31 630 647 5-8-5 1453
621674 89899 89916 GTCTCCAATGCCTGCTTC 43 635 652 5-8-5 1454
621675 89902 89919 GGTGTCTCCAATGCCTGC 74 638 655 5-8-5 1455
621679 94695 94712 TTACCACTTTCAGGCTCT 63 694 711 5-8-5 1456
621680 94700 94717 CCACCTTACCACTTTCAG 28 699 716 5-8-5 1457
621681 94703 94720 GGACCACCTTACCACTTT 62 702 719 5-8-5 1458
621682 94706 94723 CCTGGACCACCTTACCAC 33 705 722 5-8-5 1459
621683 94709 94726 CTTCCTGGACCACCTTAC 18 708 725 5-8-5 1460
621684 94712 94729 AGCCTTCCTGGACCACCT 61 711 728 5-8-5 1461
621685 94715 94732 GGAAGCCTTCCTGGACCA 54 714 731 5-8-5 1462
621686 94718 94735 GGAGGAAGCCTTCCTGGA 24 717 734 5-8-5 1463
621687 94721 94738 CTCGGAGGAAGCCTTCCT 48 720 737 5-8-5 1464
621688 94724 94741 GCTCTCGGAGGAAGCCTT 27 723 740 5-8-5 1465
621689 94727 94744 CTGGCTCTCGGAGGAAGC 19 726 743 5-8-5 1466
621690 94730 94747 GGCCTGGCTCTCGGAGGA 46 729 746 5-8-5 1467
621691 94749 94766 TGGTGGCTCAGACCTGGG 49 748 765 5-8-5 1468
621692 94752 94769 AGCTGGTGGCTCAGACCT 50 751 768 5-8-5 1469
621693 94755 94772 ATGAGCTGGTGGCTCAGA 17 754 771 5-8-5 1470
621694 94758 94775 GACATGAGCTGGTGGCTC 54 757 774 5-8-5 1471
621695 94761 94778 CCGGACATGAGCTGGTGG 26 760 111 5-8-5 1472 621696 94764 94781 ATGCCGGACATGAGCTGG 12 763 780 543-5 1473
621697 94767 94784 GGCATGCCGGACATGAGC 1 1 766 783 54 3-5 1474
621698 94770 94787 CCAGGCATGCCGGACATG 41 769 786 54 3-5 1475
621699 94773 94790 GCCCCAGGCATGCCGGAC 57 772 789 54 3-5 1476
621700 94776 94793 GGAGCCCCAGGCATGCCG 47 775 792 54 1-5 1477
621701 94793 94810 GGCCCTCAGGCAGGAGGG 0 792 809 54 ]-5 1478
621702 94825 94842 TGTCCCCGAAGGTTGGCG 50 824 841 54 1-5 1479
621703 94828 94845 TCCTGTCCCCGAAGGTTG 38 827 844 54 3-5 1480
621704 94831 94848 AGGTCCTGTCCCCGAAGG 41 830 847 54 1-5 1481
621705 94834 94851 CTCAGGTCCTGTCCCCGA 60 833 850 54 -5 1482
621706 94837 94854 GTCCTCAGGTCCTGTCCC 0 836 853 54 3-5 1483
621707 94840 94857 TGTGTCCTCAGGTCCTGT 23 839 856 54 3-5 1484
621708 94843 94860 CTCTGTGTCCTCAGGTCC 55 842 859 54 3-5 1485
621709 94846 94863 GCCCTCTGTGTCCTCAGG 35 845 862 54 3-5 1486
621710 94849 94866 GCCGCCCTCTGTGTCCTC 45 848 865 54 3-5 1487
62171 1 94852 94869 GCGGCCGCCCTCTGTGTC 0 851 868 54 3-5 1488
621712 94855 94872 GTGGCGGCCGCCCTCTGT 0 854 871 54 3-5 1489
621713 94875 94892 TGCTTGAGCAGCTCAGGG 67 874 891 54 3-5 1490
621714 94878 94895 TGGTGCTTGAGCAGCTCA 51 877 894 54 3-5 1491
621715 94882 94899 AAGCTGGTGCTTGAGCAG 27 881 898 54 3-5 1492
621716 94885 94902 TAGAAGCTGGTGCTTGAG 14 884 901 54 3-5 1493
621717 94888 94905 TCCTAGAAGCTGGTGCTT 0 887 904 54 3-5 1494
621718 94891 94908 GTCTCCTAGAAGCTGGTG 45 890 907 54 3-5 1495
621719 94894 94911 CAGGTCTCCTAGAAGCTG 47 893 910 54 3-5 1496
621720 94897 94914 GTGCAGGTCTCCTAGAAG 41 896 913 54 3-5 1497
621721 94900 94917 CTGGTGCAGGTCTCCTAG 36 899 916 54 3-5 1498
621722 94903 94920 CTCCTGGTGCAGGTCTCC 41 902 919 54 3-5 1499
621723 94906 94923 CCCCTCCTGGTGCAGGTC 39 905 922 54 3-5 1500
621724 94909 94926 CGGCCCCTCCTGGTGCAG 19 908 925 54 3-5 1501
621725 94912 94929 CGGCGGCCCCTCCTGGTG 22 911 928 54 3-5 1502
621726 94915 94932 CAGCGGCGGCCCCTCCTG 35 914 931 54 3-5 1503
621727 94918 94935 CTTCAGCGGCGGCCCCTC 46 917 934 54 3-5 1504
621728 94921 94938 CCCCTTCAGCGGCGGCCC 36 920 937 54 3-5 1505
621729 94924 94941 TGCCCCCTTCAGCGGCGG 17 923 940 54 3-5 1506
621730 94927 94944 CCCTGCCCCCTTCAGCGG 15 926 943 54 3-5 1507
621731 94930 94947 GCCCCCTGCCCCCTTCAG 27 929 946 54 3-5 1508
621732 94933 94950 TTTGCCCCCTGCCCCCTT 49 932 949 54 3-5 1509
621733 94936 94953 CTCTTTGCCCCCTGCCCC 22 935 952 54 3-5 1510
621734 94939 94956 CCTCTCTTTGCCCCCTGC 28 938 955 54 3-5 1511
98891 98908 n/a n/a
620887 GTTTTCAAACACACCTTC 90 54 1-5 665
98928 98945 n/a n/a Table 30
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ ID SEQ ID
% SEQ
ISIS NO NO: 1 start NO: 1 stop Sequence Motif
inhibition ID NO: site site
613412 73879 73898 GTGATCTTCCATCACTTCGA 82 5-10-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 92 5-8-5 665
98928 98945
621735 94942 94959 CGGCCTCTCTTTGCCCCC 34 5-8-5 1512
621736 94945 94962 CCCCGGCCTCTCTTTGCC 27 5-8-5 1513
621737 94948 94965 GCTCCCCGGCCTCTCTTT 44 5-8-5 1514
621738 94951 94968 CTTGCTCCCCGGCCTCTC 38 5-8-5 1515
621739 94954 94971 CTCCTTGCTCCCCGGCCT 37 5-8-5 1516
621740 94957 94974 CTCCTCCTTGCTCCCCGG 33 5-8-5 1517
621741 94960 94977 CACCTCCTCCTTGCTCCC 37 5-8-5 1518
621742 94963 94980 ATCCACCTCCTCCTTGCT 32 5-8-5 1519
621743 94967 94984 CTTCATCCACCTCCTCCT 51 5-8-5 1520
621744 94970 94987 GGTCTTCATCCACCTCCT 61 5-8-5 1521
621745 94973 94990 CGCGGTCTTCATCCACCT 71 5-8-5 1522
621746 94976 94993 CGTCGCGGTCTTCATCCA 48 5-8-5 1523
621747 95038 95055 CCGCCCATCTTGGGCTGG 12 5-8-5 1524
621748 95041 95058 AGGCCGCCCATCTTGGGC 18 5-8-5 1525
621749 95044 95061 GGGAGGCCGCCCATCTTG 21 5-8-5 1526
621750 95060 95077 CTCTGGCGGCTGTCTGGG 47 5-8-5 1527
621751 95063 95080 CTTCTCTGGCGGCTGTCT 41 5-8-5 1528
621752 95066 95083 TGGCTTCTCTGGCGGCTG 53 5-8-5 1529
621753 95069 95086 TGGTGGCTTCTCTGGCGG 26 5-8-5 1530
621754 95072 95089 TGCTGGTGGCTTCTCTGG 57 5-8-5 1531
621755 95075 95092 GGATGCTGGTGGCTTCTC 65 5-8-5 1532
621756 95078 95095 CTGGGATGCTGGTGGCTT 66 5-8-5 1533
621757 95081 95098 AGCCTGGGATGCTGGTGG 51 5-8-5 1534
621758 95084 95101 GGAAGCCTGGGATGCTGG 57 5-8-5 1535
621759 95092 95109 CTCCGCTGGGAAGCCTGG 48 5-8-5 1536
621760 95095 95112 ACCCTCCGCTGGGAAGCC 38 5-8-5 1537
621761 95098 95115 GGCACCCTCCGCTGGGAA 54 5-8-5 1538
621762 95101 95118 GATGGCACCCTCCGCTGG 27 5-8-5 1539
621763 95121 95138 AGGAAATCCACAGGGAGG 15 5-8-5 1540
621764 95124 95141 GAGAGGAAATCCACAGGG 29 5-8-5 1541
621765 95127 95144 TTGGAGAGGAAATCCACA 45 5-8-5 1542
621766 95130 95147 ACTTTGGAGAGGAAATCC 38 5-8-5 1543
621767 95134 95151 GGAAACTTTGGAGAGGAA 56 5-8-5 1544
621768 95137 95154 TGTGGAAACTTTGGAGAG 26 5-8-5 1545
621769 95140 95157 CTCTGTGGAAACTTTGGA 69 5-8-5 1546
621770 95143 95160 GATCTCTGTGGAAACTTT 62 5-8-5 1547 621771 95146 95163 TGGGATCTCTGTGGAAAC 61 5-8-5 1548
621772 95149 95166 GGCTGGGATCTCTGTGGA 47 5-8-5 1549
621773 95152 95169 TGAGGCTGGGATCTCTGT 32 5-8-5 1550
621774 95155 95172 CTCTGAGGCTGGGATCTC 64 5-8-5 1551
621775 95161 95178 GTCGGGCTCTGAGGCTGG 42 5-8-5 1552
621776 95164 95181 CCCGTCGGGCTCTGAGGC 34 5-8-5 1553
621777 95174 95191 CTACACTGGGCCCGTCGG 31 5-8-5 1554
621778 95177 95194 GCCCTACACTGGGCCCGT 48 5-8-5 1555
621779 95180 95197 CCCGCCCTACACTGGGCC 35 5-8-5 1556
621780 95183 95200 TGGCCCGCCCTACACTGG 51 5-8-5 1557
621781 95186 95203 CTTTGGCCCGCCCTACAC 4 5-8-5 1558
621782 95189 95206 GCCCTTTGGCCCGCCCTA 52 5-8-5 1559
621783 95192 95209 CCTGCCCTTTGGCCCGCC 42 5-8-5 1560
621784 95195 95212 CATCCTGCCCTTTGGCCC 49 5-8-5 1561
621785 95198 95215 GGGCATCCTGCCCTTTGG 15 5-8-5 1562
621786 95235 95252 TTGGGTGTGATTTCCACG 52 5-8-5 1563
621787 95253 95270 GCCTGCTCCTTCTGCACG 44 5-8-5 1564
621788 95256 95273 TGCGCCTGCTCCTTCTGC 64 5-8-5 1565
621789 95259 95276 GAGTGCGCCTGCTCCTTC 70 5-8-5 1566
621790 95262 95279 TCCGAGTGCGCCTGCTCC 53 5-8-5 1567
621791 95265 95282 TCCTCCGAGTGCGCCTGC 54 5-8-5 1568
621792 95268 95285 TGCTCCTCCGAGTGCGCC 52 5-8-5 1569
621793 95271 95288 AAATGCTCCTCCGAGTGC 72 5-8-5 1570
621794 95274 95291 CCCAAATGCTCCTCCGAG 75 5-8-5 1571
621795 95277 95294 CTTCCCAAATGCTCCTCC 47 5-8-5 1572
621796 95280 95297 GCCCTTCCCAAATGCTCC 58 5-8-5 1573
621797 95283 95300 GCAGCCCTTCCCAAATGC 39 5-8-5 1574
621798 95286 95303 AATGCAGCCCTTCCCAAA 61 5-8-5 1575
621799 95291 95308 CTGGAAATGCAGCCCTTC 64 5-8-5 1576
621800 95294 95311 CCCCTGGAAATGCAGCCC 49 5-8-5 1577
621801 95297 95314 GGGCCCCTGGAAATGCAG 29 5-8-5 1578
621802 95313 95330 TCTGGCCCCTCTCCAGGG 41 5-8-5 1579
621803 95316 95333 GCCTCTGGCCCCTCTCCA 42 5-8-5 1580
621804 95319 95336 CGGGCCTCTGGCCCCTCT 11 5-8-5 1581
621805 95322 95339 CCCCGGGCCTCTGGCCCC 13 5-8-5 1582
621806 95341 95358 GTCCTCTCCCAAAGAGGG 25 5-8-5 1583
621807 95344 95361 TGTGTCCTCTCCCAAAGA 20 5-8-5 1584
621808 95347 95364 TTTTGTGTCCTCTCCCAA 66 5-8-5 1585
621809 95365 95382 CTCTGGAAGGTCAGCCTC 53 5-8-5 1586
621810 95368 95385 GGGCTCTGGAAGGTCAGC 72 5-8-5 1587
621811 95371 95388 AGAGGGCTCTGGAAGGTC 57 5-8-5 1588 Table 31
Inhibition of tau mRNA by 5-10-5 and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ ID SEQ ID
ISIS % SEQ ID NO: 1 start NO: 1 stop Sequence Motif
NO inhibition NO:
site site
613412 73879 73898 GTGATCTTCCATCACTTCGA 88 5-10-5 25
98891 98908
620887 GTTTTCAAACACACCTTC 96 5-8-5 665
98928 98945
623737 111219 111236 CCTCCACCCAGCATGGTG 56 5-8-5 1589
623738 111267 111284 TCAAGTCACCCTTTCTCC 78 5-8-5 1590
623739 111368 111385 TTCAGACAATTTTTCTAG 66 5-8-5 1591
623740 111629 111646 TCAGACTCCGCCAGCTTT 79 5-8-5 1592
623741 111678 111695 TCTTTTCCCTTTGGTGTT 34 5-8-5 1593
623742 111753 111770 TTTCCCCCAATGATTTGC 77 5-8-5 1594
623743 112016 112033 CCACGACTCCCACAAGAT 32 5-8-5 1595
623744 112213 112230 TGAGGTTGAGGGTAGGTG 50 5-8-5 1596
623745 112219 112236 AGTGGTTGAGGTTGAGGG 31 5-8-5 1597
623746 112304 112321 CAGGCACTTGGAAACTGC 90 5-8-5 1598
623747 112877 112894 TTGTACTCTTTTTCCCCT 65 5-8-5 1599
623748 112949 112966 TTAGGAGTGCAAGGTTGT 49 5-8-5 1600
623749 113352 113369 TCCTACATTCCTCCTGCT 67 5-8-5 1601
623750 113523 113540 GTTGCAGTGTTCCACTAT 90 5-8-5 1602
623751 113783 113800 CCAGCAGATGCCGACAGC 74 5-8-5 1603
623752 113809 113826 GGGCCCTCACCCCTGCTT 14 5-8-5 1604
623753 113830 113847 CCAAGAAGGGCTGCTGAG 8 5-8-5 1605
623754 114267 114284 AAGCAGCCACCCAGGCTG 17 5-8-5 1606
623755 114739 114756 TATGAGGAGGGAGGAAAG 13 5-8-5 1607
623756 115242 115259 CTGACATCTCAGCCCAAG 93 5-8-5 1608
621425 115335 115352 GGTACAGGAAAAGATGCC 77 5-8-5 1202
623757 115522 115539 CAAGGTGATCAGCTTAGG 82 5-8-5 1609
623758 115526 115543 AGTCCAAGGTGATCAGCT 89 5-8-5 1610
623759 115547 115564 CCCACACAAGCCTCCTCT 66 5-8-5 1611
623760 115791 115808 TTTGTTTGGGTTCAGTTC 59 5-8-5 1612
623761 115803 115820 CCTTGATCTGGTTTTGTT 77 5-8-5 1613
623762 115993 116010 ATCAAGGGAGAGAACGAT 52 5-8-5 1614
623763 116229 116246 AAAAGGCAACAAGCCTTT 0 5-8-5 1615
623764 116769 116786 CGAGGCAAAAAGGGAAAT 45 5-8-5 1616
623765 116791 116808 TAACTTTGAGCTTCCTGG 65 5-8-5 1617
623766 116944 116961 TAAGGCTTAGAAGATTTG 45 5-8-5 1618
623767 116978 116995 GACGATCATTTTTTATTC 72 5-8-5 1619
623768 116983 117000 GGCTTGACGATCATTTTT 77 5-8-5 1620
623769 117421 117438 AGATCTCAGAGTTTGTAG 68 5-8-5 1621
623770 117472 117489 TCGCTGAGGTTCCGCAAC 37 5-8-5 1622
623771 117489 117506 AGGAGGCCATGTGAGGCT 61 5-8-5 1623 623772 117547 117564 TGGATAAATCTCTAGTGC 54 5-8-5 1624
623773 117564 117581 CACTCCTCCTTTAGAGAT 38 5-8-5 1625
623774 117599 117616 TTGTAATTCCTTCTCTGG 65 5-8-5 1626
623775 117971 117988 TAACCATCCCTACTTTCT 42 5-8-5 1627
623776 119098 119115 ACTGAAATCGCTCCTTTA 58 5-8-5 1628
623777 119107 119124 CCAATTATGACTGAAATC 32 5-8-5 1629
623778 119313 119330 GGTCAAAGTTATAAATAT 57 5-8-5 1630
623779 119431 119448 GGATTTGATGCAAAGAAA 70 5-8-5 1631
623780 119740 119757 GCGCAACCCAGCTACTCG 7 5-8-5 1632
621445 119880 119897 TACAGTGGCTCCTGTAAT 28 5-8-5 1222
623781 119964 119981 CTGTATACCTATACTTGG 34 5-8-5 1633
623782 120043 120060 CCGTTTTCTTACCACCCT 92 5-8-5 1634
623783 120177 120194 TTACTTGTCTGAATCTTC 83 5-8-5 1635
623784 120204 120221 TTCATCCCGTTTTTTTTC 54 5-8-5 1636
623785 120309 120326 GACCTCAGTGGCTCTTTT 68 5-8-5 1637
623786 120513 120530 TGTCCAGGATAGTTTGAA 44 5-8-5 1638
623787 121033 121050 GCTGAGCAAATGCTCCCG 54 5-8-5 1639
623788 121194 121211 CTGACCCCGTAGGCAGGA 29 5-8-5 1640
623789 121478 121495 ACCAGCCTGAGGTCAAGT 73 5-8-5 1641
623790 121538 121555 CAGGTCCTCACTTCACAA 42 5-8-5 1642
623791 121767 121784 CCGCCTGCTTGCTCGCAA 92 5-8-5 1643
623792 121787 121804 AGTGACACGCCACCCTGG 80 5-8-5 1644
623793 121797 121814 AAAAAGGATGAGTGACAC 28 5-8-5 1645
623794 121807 121824 GGTAGCCAGAAAAAAGGA 49 5-8-5 1646
623795 121812 121829 CCTTTGGTAGCCAGAAAA 63 5-8-5 1647
623796 121817 121834 CTGCACCTTTGGTAGCCA 87 5-8-5 1648
623797 121822 121839 ATTATCTGCACCTTTGGT 67 5-8-5 1649
623798 121824 121841 TAATTATCTGCACCTTTG 49 5-8-5 1650
623799 121905 121922 CACTGCCGCCTCCCGGGA 65 5-8-5 1651
623800 121909 121926 CTCACACTGCCGCCTCCC 75 5-8-5 1652
623801 121911 121928 TACTCACACTGCCGCCTC 54 5-8-5 1653
623679 121913 121930 GGTACTCACACTGCCGCC 81 5-8-5 1654
623802 121915 121932 AAGGTACTCACACTGCCG 69 5-8-5 1655
623803 121920 121937 GTGTGAAGGTACTCACAC 18 5-8-5 1656
623804 121942 121959 GCCACAGCACGGCGCATG 80 5-8-5 1657
623805 121952 121969 AATAATTCAAGCCACAGC 88 5-8-5 1658
623806 121962 121979 ACCACTTCCTAATAATTC 39 5-8-5 1659
623807 121972 121989 ACGCACTCACACCACTTC 83 5-8-5 1660
623808 121982 121999 TCGCAAGTGTACGCACTC 94 5-8-5 1661
623809 121992 122009 ATGCAGTGTCTCGCAAGT 93 5-8-5 1662
623810 122227 122244 TGATGGGTATTCTCAGCT 77 5-8-5 1663 Table 32
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ ID SEQ ID % SEQ
ISIS NO: 1 start NO: 1 stop Sequence inhibitio Motif ID NO
site site n NO:
GTGATCTTCCATCACTTCG
613412 73879 73898 87 5-10-5 25
A
98891 98908
620887 GTTTTCAAACACACCTTC 83 5-8-5 665
98928 98945
622186 5897 5914 CCGCTCGGCCGTCCGGCG 6 5-8-5 1664
622187 5900 5917 CTGCCGCTCGGCCGTCCG 6 5-8-5 1665
622174 103089 103106 CCAGAGCTGGGTGGTGTC 12 5-8-5 1666
622182 125760 125777 TGTAGACTATTTGCACCT 74 5-8-5 1667
622120 135855 135872 ATCACTGATTTTGAAGTC 36 5-8-5 1668
622121 135858 135875 CCCATCACTGATTTTGAA 59 5-8-5 1669
622122 135861 135878 ACTCCCATCACTGATTTT 70 5-8-5 1670
622123 135864 135881 CTTACTCCCATCACTGAT 41 5-8-5 1671
622124 135867 135884 GCTCTTACTCCCATCACT 82 5-8-5 1672
622125 135870 135887 TTTGCTCTTACTCCCATC 87 5-8-5 1673
622126 135873 135890 AAATTTGCTCTTACTCCC 62 5-8-5 1674
622127 135876 135893 ATGAAATTTGCTCTTACT 70 5-8-5 1675
622128 135879 135896 AAGATGAAATTTGCTCTT 55 5-8-5 1676
622129 135882 135899 GGAAAGATGAAATTTGCT 84 5-8-5 1677
622130 135885 135902 TTTGGAAAGATGAAATTT 12 5-8-5 1678
622131 135891 135908 CATCAATTTGGAAAGATG 43 5-8-5 1679
622132 135894 135911 ACCCATCAATTTGGAAAG 18 5-8-5 1680
622133 135897 135914 CCCACCCATCAATTTGGA 34 5-8-5 1681
622134 135900 135917 TAGCCCACCCATCAATTT 17 5-8-5 1682
622135 135903 135920 TACT AGCC C AC CC ATC AA 14 5-8-5 1683
622136 135906 135923 TATTACTAGCCCACCCAT 24 5-8-5 1684
622137 135909 135926 TTTTATTACTAGCCCACC 24 5-8-5 1685
622138 135912 135929 ATATTTTATTACTAGCCC 33 5-8-5 1686
622139 135915 135932 TAAATATTTTATTACTAG 0 5-8-5 1687
622140 135918 135935 TTTTAAATATTTTATTAC 0 5-8-5 1688
622141 135926 135943 ATGTTTTTTTTTAAATAT 0 5-8-5 1689
622142 135939 135956 GCCATGTTTTTGAATGTT 60 5-8-5 1690
622143 135942 135959 GTGGCCATGTTTTTGAAT 28 5-8-5 1691
622144 135945 135962 GATGTGGCCATGTTTTTG 63 5-8-5 1692
622145 135948 135965 TTGGATGTGGCCATGTTT 78 5-8-5 1693
622146 135951 135968 ATGTTGGATGTGGCCATG 71 5-8-5 1694
622147 135954 135971 GAAATGTTGGATGTGGCC 72 5-8-5 1695
622148 135957 135974 GAGGAAATGTTGGATGTG 55 5-8-5 1696
622149 135960 135977 CCTGAGGAAATGTTGGAT 77 5-8-5 1697
622150 135963 135980 TTGC CTGAGGAAATGTTG 55 5-8-5 1698 622151 13981 1 139828 CCAAATTCACTTTTACAG 54 5-8-5 1699
622152 139813 139830 TTCCAAATTCACTTTTAC 67 5-8-5 1700
622153 139815 139832 ATTTCCAAATTCACTTTT 52 5-8-5 1701
622154 139817 139834 TTATTTCCAAATTCACTT 52 5-8-5 1702
622155 139819 139836 CTTTATTTCCAAATTCAC 61 5-8-5 1703
622156 139821 139838 AACTTTATTTCCAAATTC 26 5-8-5 1704
622157 139823 139840 ATAACTTTATTTCCAAAT 23 5-8-5 1705
622158 139826 139843 GTAATAACTTTATTTCCA 65 5-8-5 1706
622159 139828 139845 GAGTAATAACTTTATTTC 25 5-8-5 1707
622160 139830 139847 CAGAGTAATAACTTTATT 9 5-8-5 1708
622161 139832 139849 ATCAGAGTAATAACTTTA 23 5-8-5 1709
622162 139834 139851 TAATCAGAGTAATAACTT 14 5-8-5 1710
622163 139836 139853 TTTAATCAGAGTAATAAC 4 5-8-5 1711
Table 33
Inhibition of Tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NO: 5 and 6
Figure imgf000109_0001
622193 442 459 GCCCCCTTGATCTTTCCT 7 n/a n/a 5-8-5 1734
622194 445 462 GTAGCCCCCTTGATCTTT 3 n/a n/a 5-8-5 1735
622195 448 465 GGTGTAGCCCCCTTGATC 8 n/a n/a 5-8-5 1736
622196 451 468 CATGGTGTAGCCCCCTTG 83 n/a n/a 5-8-5 1737
622168 564 581 ATGCGAGCTTGGGTCACG 65 598 615 5-8-5 1738
622169 567 584 ACCATGCGAGCTTGGGTC 62 601 618 5-8-5 1739
622170 570 587 CTGACCATGCGAGCTTGG 91 604 621 5-8-5 1740
Table 34
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1 and 2
Figure imgf000110_0001
621838 98562 98579 TGCTTTTACTGACCATGC 95 1452 1469 5-8-5 1769
621839 98565 98582 CTTTGCTTTTACTGACCA 85 1455 1472 5-8-5 1770
621840 98568 98585 CGTCTTTGCTTTTACTGA 66 1458 1475 5-8-5 1771
621841 98571 98588 TCCCGTCTTTGCTTTTAC 79 1461 1478 5-8-5 1772
621842 98575 98592 CCAGTCCCGTCTTTGCTT 87 1465 1482 5-8-5 1773
621843 98578 98595 CTTCCAGTCCCGTCTTTG 64 1468 1485 5-8-5 1774
621844 98581 98598 TCGCTTCCAGTCCCGTCT 88 1471 1488 5-8-5 1775
621845 98584 98601 TCATCGCTTCCAGTCCCG 90 1474 1491 5-8-5 1776
621846 98587 98604 TTGTCATCGCTTCCAGTC 88 1477 1494 5-8-5 1777
621847 98590 98607 TTTTTGTCATCGCTTCCA 80 1480 1497 5-8-5 1778
621850 101406 101423 GGTTTTAGCAGAGGAACG 75 1514 1531 5-8-5 1779
621851 101409 101426 CAAGGTTTTAGCAGAGGA 83 1517 1534 5-8-5 1780
621852 101412 101429 TTTCAAGGTTTTAGCAGA 35 1520 1537 5-8-5 1781
621853 101415 101432 ATTTTTCAAGGTTTTAGC 79 1523 1540 5-8-5 1782
621854 101418 101435 CCTATTTTTCAAGGTTTT 71 1526 1543 5-8-5 1783
621855 101425 101442 GGCAAGGCCTATTTTTCA 83 1533 1550 5-8-5 1784
621856 101428 101445 TAAGGCAAGGCCTATTTT 23 1536 1553 5-8-5 1785
621857 101431 101448 GGCTAAGGCAAGGCCTAT 69 1539 1556 5-8-5 1786
621858 101457 101474 TGAGCTACCAGGAGTGGG 75 1565 1582 5-8-5 1787
621859 101460 101477 GTCTGAGCTACCAGGAGT 78 1568 1585 5-8-5 1788
621860 101463 101480 AGGGTCTGAGCTACCAGG 82 1571 1588 5-8-5 1789
621861 101466 101483 CAGAGGGTCTGAGCTACC 88 1574 1591 5-8-5 1790
621862 101469 101486 GATCAGAGGGTCTGAGCT 72 1577 1594 5-8-5 1791
621863 101472 101489 TTGGATCAGAGGGTCTGA 83 1580 1597 5-8-5 1792
621864 101478 101495 GGAGGGTTGGATCAGAGG 0 1586 1603 5-8-5 1793
621865 101481 101498 GCTGGAGGGTTGGATCAG 16 1589 1606 5-8-5 1794
621866 101491 101508 ACACAGCAGGGCTGGAGG 34 1599 1616 5-8-5 1795
621867 101494 10151 1 GGCACACAGCAGGGCTGG 67 1602 1619 5-8-5 1796
621868 101499 101516 CTCTGGGCACACAGCAGG 75 1607 1624 5-8-5 1797
621869 101502 101519 TGGCTCTGGGCACACAGC 90 1610 1627 5-8-5 1798
621870 101505 101522 AGGTGGCTCTGGGCACAC 92 1613 1630 5-8-5 1799
621871 101508 101525 GGAAGGTGGCTCTGGGCA 67 1616 1633 5-8-5 1800
621872 101511 101528 AGAGGAAGGTGGCTCTGG 43 1619 1636 5-8-5 1801
621873 101514 101531 AGGAGAGGAAGGTGGCTC 65 1622 1639 5-8-5 1802
621874 101517 101534 TTTAGGAGAGGAAGGTGG 26 1625 1642 5-8-5 1803
621875 101520 101537 GTATTTAGGAGAGGAAGG 0 1628 1645 5-8-5 1804
621876 101537 101554 GAAGTGACAGAAGAGACG 37 1645 1662 5-8-5 1805
621877 101540 101557 CGGGAAGTGACAGAAGAG 59 1648 1665 5-8-5 1806
621878 101543 101560 GTTCGGGAAGTGACAGAA 65 1651 1668 5-8-5 1807
621879 101546 101563 CCAGTTCGGGAAGTGACA 35 1654 1671 5-8-5 1808
621880 101549 101566 CTGCC AGTTC GGGAAGT G 69 1657 1674 5-8-5 1809
621881 101552 101569 GAACTGCCAGTTCGGGAA 48 1660 1677 5-8-5 1810
621882 101555 101572 CCAGAACTGCCAGTTCGG 66 1663 1680 5-8-5 181 1 621883 101558 101575 GCTCCAGAACTGCCAGTT 69 1666 1683 5-8-5 1812
621884 101561 101578 TTTGCTCCAGAACTGCCA 74 1669 1686 5-8-5 1813
621885 101564 101581 TCCTTTGCTCCAGAACTG 61 1672 1689 5-8-5 1814
621886 101567 101584 ATCTCCTTTGCTCCAGAA 64 1675 1692 5-8-5 1815
621887 101570 101587 TTCATCTCCTTTGCTCCA 36 1678 1695 5-8-5 1816
621888 101573 101590 AGTTTCATCTCCTTTGCT 32 1681 1698 5-8-5 1817
Table 35
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1 and 2
SEQ ID SEQ ID SEQ ID SEQ ID SEQ
%
ISIS NO NO: 1 NO: 1 Sequence NO: 2 NO: 2 Motif ID inhibition
start site stop site start site stop site NO:
613412 73879 73898 GTGATCTTCCATCACTTCGA 87 345 364 5-10-5 25
98891 98908 n/a n/a
620887 GTTTTCAAACACACCTTC 95 5-8-5 665
98928 98945 n/a n/a
621890 n/a n a CCCTTGAGTTTCATCTCC 30 1687 1704 5-8-5 1818
621891 n/a n a GCCCCCTTGAGTTTCATC 8 1690 1707 5-8-5 1819
621892 n/a n a TCAGCCCCCTTGAGTTTC 0 1693 1710 5-8-5 1820
621893 n/a n a CCATCAGCCCCCTTGAGT 4 1696 1713 5-8-5 1821
621894 n/a n a TT AC CAT C AGC CC CCTT G 75 1699 1716 5-8-5 1822
621912 n/a n a CGCAGAGCTGGGTGGTGT 0 1814 1831 5-8-5 1823
621913 n/a n a AGTCGCAGAGCTGGGTGG 0 1817 1834 5-8-5 1824
621914 n/a n/a CTTAGTCGCAGAGCTGGG 21 1820 1837 5-8-5 1825
621915 n/a n a TTGCTTAGTCGCAGAGCT 12 1823 1840 5-8-5 1826
621916 n/a n/a GACTTGCTTAGTCGCAGA 29 1826 1843 5-8-5 1827
621925 n/a n a TTCACCTCTCTCAGATCT 4 1871 1888 5-8-5 1828
621926 n/a n a AGGTTCACCTCTCTCAGA 9 1874 1891 5-8-5 1829
621927 n/a n/a TGGAGGTTCACCTCTCTC 7 1877 1894 5-8-5 1830
621928 n/a n a TTTTGGAGGTTCACCTCT 1 1 1880 1897 5-8-5 1831
621889 101576 101593 TTGAGTTTCATCTCCTTT 23 1684 1701 5-8-5 1832
621895 102978 102995 GTTTTACCATCAGCCCCC 88 1702 1719 5-8-5 1833
621896 102995 103012 GCGGTGTGGCGATCTTCG 42 1719 1736 5-8-5 1834
621897 102998 103015 CCCGCGGTGTGGCGATCT 33 1722 1739 5-8-5 1835
621898 103001 103018 CTCCCCGCGGTGTGGCGA 37 1725 1742 5-8-5 1836
621899 103004 103021 CTGCTCCCCGCGGTGTGG 50 1728 1745 5-8-5 1837
621900 103007 103024 GGGCTGCTCCCCGCGGTG 43 1731 1748 5-8-5 1838
621901 103025 103042 GGCCCTTCTGGCCTGGAG 29 1749 1766 5-8-5 1839
621902 103029 103046 GCCTGGCCCTTCTGGCCT 34 1753 1770 5-8-5 1840
621903 103032 103049 TTGGCCTGGCCCTTCTGG 49 1756 1773 5-8-5 1841
621904 103050 103067 GCTGGAATCCTGGTGGCG 58 1774 1791 5-8-5 1842
621905 103053 103070 TTTGCTGGAATCCTGGTG 50 1777 1794 5-8-5 1843
621906 103056 103073 GTTTTTGCTGGAATCCTG 62 1780 1797 5-8-5 1844
621907 103075 103092 TGTCTTTGGAGCGGGCGG 21 1799 1816 5-8-5 1845
621908 103078 103095 TGGTGTCTTTGGAGCGGG 57 1802 1819 5-8-5 1846 621909 103081 103098 GGGTGGTGTCTTTGGAGC 17 1805 1822 5-8-5 1847
621910 103084 103101 GCTGGGTGGTGTCTTTGG 44 1808 1825 5-8-5 1848
621911 103087 103104 AGAGCTGGGTGGTGTCTT 47 1811 1828 5-8-5 1849
621917 105442 105459 CTGGACTTGCTTAGTCGC 27 1829 1846 5-8-5 1850
621918 105445 105462 TCTCTGGACTTGCTTAGT 0 1832 1849 5-8-5 1851
621919 105448 105465 TCTTCTCTGGACTTGCTT 48 1835 1852 5-8-5 1852
621920 105451 105468 TGGTCTTCTCTGGACTTG 49 1838 1855 5-8-5 1853
621921 105454 105471 GGGT GGTCTT CTCT GGAC 33 1841 1858 5-8-5 1854
621922 105473 105490 CAGATCTGGGCCCTGCAG 36 1860 1877 5-8-5 1855
621 23 105476 105493 TCTCAGATCTGGGCCCTG 70 1863 1880 5-8-5 1856
621924 105479 105496 CTCTCTCAGATCTGGGCC 32 1866 1883 5-8-5 1857
621929 107917 107934 TGATTTTGGAGGTTCACC 44 1883 1900 5-8-5 1858
621930 107920 107937 CCCTGATTTTGGAGGTTC 74 1886 1903 5-8-5 1859
621931 107923 107940 AT CC CCTGATTTTGGAGG 26 1889 1906 5-8-5 1860
621932 107926 107943 GCGATCCCCTGATTTTGG 45 1892 1909 5-8-5 1861
621933 107929 107946 GCTGCGATCCCCTGATTT 38 1895 1912 5-8-5 1862
621934 107932 107949 GCCGCTGCGATCCCCTGA 42 1898 1915 5-8-5 1863
621935 107935 107952 GTAGCCGCTGCGATCCCC 31 1901 1918 5-8-5 1864
621936 107938 107955 GCTGTAGCCGCTGCGATC 67 1904 1921 5-8-5 1865
621937 107941 107958 GCTGCTGTAGCCGCTGCG 23 1907 1924 5-8-5 1866
621938 107971 107988 GCGGCTGCCGGGAGTGCC 40 1937 1954 5-8-5 1867
621939 107974 107991 GGAGCGGCTGCCGGGAGT 1 1940 1957 5-8-5 1868
621 40 107978 107995 TGCGGGAGCGGCTGCCGG 48 1944 1961 5-8-5 1869
621941 108021 108038 ACCTTCTTGGGCTCCCGG 24 1987 2004 5-8-5 1870
621942 108024 108041 GCCACCTTCTTGGGCTCC 28 1990 2007 5-8-5 1871
621943 108027 108044 ACTGCCACCTTCTTGGGC 19 1993 2010 5-8-5 1872
621944 108030 108047 ACCACTGCCACCTTCTTG 28 1996 2013 5-8-5 1873
621945 108033 108050 CGGACCACTGCCACCTTC 72 1999 2016 5-8-5 1874
621 46 108036 108053 GTACGGACCACTGCCACC 57 2002 2019 5-8-5 1875
621947 108039 108056 GGAGTACGGACCACTGCC 25 2005 2022 5-8-5 1876
621948 108042 108059 GGTGGAGTACGGACCACT 30 2008 2025 5-8-5 1877
621949 108045 108062 TTGGGTGGAGTACGGACC 24 2011 2028 5-8-5 1878
621950 108052 108069 CGGCGACTTGGGTGGAGT 1 2018 2035 5-8-5 1879
621951 108055 108072 AGACGGCGACTTGGGTGG 4 2021 2038 5-8-5 1880
621 52 108058 108075 GGAAGACGGCGACTTGGG 50 2024 2041 5-8-5 1881
621 53 108061 108078 GGCGGAAGACGGCGACTT 66 2027 2044 5-8-5 1882
621954 108064 108081 CTTGGCGGAAGACGGCGA 38 2030 2047 5-8-5 1883
621955 108067 108084 GCT CTTGGC GGAAGACGG 24 2033 2050 5-8-5 1884
621956 108070 108087 GCGGCTCTTGGCGGAAGA 20 2036 2053 5-8-5 1885
621957 108073 108090 CAGGCGGCTCTTGGCGGA 46 2039 2056 5-8-5 1886
621958 108076 108093 CT GCAGGCGGCT CTT GGC 72 2042 2059 5-8-5 1887
621 59 108079 108096 TGTCTGCAGGCGGCTCTT 52 2045 2062 5-8-5 1888
621 60 108082 108099 GGCTGTCTGCAGGCGGCT 47 2048 2065 5-8-5 1889 621961 108101 108118 GGTCTGGCATGGGCACGG 73 2067 2084 5-8-5 1890
621962 108137 108154 TCTCAGTGGAGCCGATCT 39 2103 2120 5-8-5 1891
621963 108140 108157 GGTTCTCAGTGGAGCCGA 66 2106 2123 5-8-5 1892
621964 108143 108160 TCAGGTTCTCAGTGGAGC 65 2109 2126 5-8-5 1893
621965 108146 108163 GCTTCAGGTTCTCAGTGG 44 2112 2129 5-8-5 1894
Table 36
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1 and 2
Figure imgf000114_0001
621987 121873 121890 TCCTTTGAGCCACACTTG 66 2194 2211 5-8-5 1925
621988 121876 121893 TTATCCTTTGAGCCACAC 77 2197 2214 5-8-5 1926
621989 121879 121896 ATATTATCCTTTGAGCCA 54 2200 2217 5-8-5 1927
621990 121882 121899 TTGAT ATT AT CCTTT GAG 69 2203 2220 5-8-5 1928
621991 121885 121902 TGTTTGATATTATCCTTT 92 2206 2223 5-8-5 1929
621992 121903 121920 CTGCCGCCTCCCGGGACG 51 2224 2241 5-8-5 1930
621993 121906 121923 ACACTGCCGCCTCCCGGG 70 2227 2244 5-8-5 1931
621998 125764 125781 GGTTTGTAGACTATTTGC 76 2245 2262 5-8-5 1932
621999 125767 125784 ACTGGTTTGTAGACTATT 62 2248 2265 5-8-5 1933
622000 125770 125787 TCAACTGGTTTGTAGACT 39 2251 2268 5-8-5 1934
622001 125777 125794 GCTCAGGTCAACTGGTTT 87 2258 2275 5-8-5 1935
622002 125780 125797 CTTGCTCAGGTCAACTGG 53 2261 2278 5-8-5 1936
622003 125783 125800 CACCTTGCTCAGGTCAAC 68 2264 2281 5-8-5 1937
622004 125805 125822 CTAATGAGCCACACTTGG 51 2286 2303 5-8-5 1938
622005 125808 125825 TGCCTAATGAGCCACACT 65 2289 2306 5-8-5 1939
622006 125811 125828 T GTT GCCT AATGAGCC AC 69 2292 2309 5-8-5 1940
622007 125814 125831 GGATGTTGCCTAATGAGC 70 2295 2312 5-8-5 1941
622008 125817 125834 GATGGATGTTGCCTAATG 61 2298 2315 5-8-5 1942
622009 125820 125837 TATGATGGATGTTGCCTA 44 2301 2318 5-8-5 1943
622010 125823 125840 GTTTATGATGGATGTTGC 57 2304 2321 5-8-5 1944
622011 125826 125843 CTGGTTTATGATGGATGT 26 2307 2324 5-8-5 1945
622015 130141 130158 TTTACTTCCACCTGGCCA 36 2329 2346 5-8-5 1946
622016 130144 130161 GATTTTACTTCCACCTGG 49 2332 2349 5-8-5 1947
622017 130147 130164 TCAGATTTTACTTCCACC 76 2335 2352 5-8-5 1948
622018 130150 130167 TTCTCAGATTTTACTTCC 24 2338 2355 5-8-5 1949
62201 130153 130170 AGCTTCTCAGATTTTACT 30 2341 2358 5-8-5 1950
622020 130156 130173 TCAAGCTTCTCAGATTTT 39 2344 2361 5-8-5 1951
622021 130159 130176 AAGTCAAGCTTCTCAGAT 2 2347 2364 5-8-5 1952
622022 130162 130179 TTGAAGTCAAGCTTCTCA 53 2350 2367 5-8-5 1953
622023 130165 130182 TCCTTGAAGTCAAGCTTC 39 2353 2370 5-8-5 1954
622024 130168 130185 CTGTCCTTGAAGTCAAGC 19 2356 2373 5-8-5 1955
622025 130171 130188 ACTCTGTCCTTGAAGTCA 39 2359 2376 5-8-5 1956
622026 130174 130191 TGGACTCTGTCCTTGAAG 36 2362 2379 5-8-5 1957
622027 130177 130194 GACTGGACTCTGTCCTTG 75 2365 2382 5-8-5 1958
622028 130180 130197 TTCGACTGGACTCTGTCC 29 2368 2385 5-8-5 1959
622029 130183 130200 ATCTTCGACTGGACTCTG 33 2371 2388 5-8-5 1960
622030 130186 130203 CCAATCTTCGACTGGACT 49 2374 2391 5-8-5 1961
622031 130189 130206 GACCCAATCTTCGACTGG 74 2377 2394 5-8-5 1962
622032 130192 130209 AGGGACCCAATCTTCGAC 79 2380 2397 5-8-5 1963
622033 130195 130212 TCCAGGGACCCAATCTTC 77 2383 2400 5-8-5 1964
622034 130198 130215 TTGTCCAGGGACCCAATC 68 2386 2403 5-8-5 1965
622035 130201 130218 ATATTGTCCAGGGACCCA 6 2389 2406 5-8-5 1966
622036 130204 130221 GTGATATTGTCCAGGGAC 86 2392 2409 5-8-5 1967 622037 130207 130224 TGGGTGATATTGTCCAGG 68 2395 2412 5-8-5 1968
622038 130225 130242 TTTCCTCCGCCAGGGACG 77 2413 2430 5-8-5 1969
622039 130228 130245 TTATTTCCTCCGCCAGGG 52 2416 2433 5-8-5 1970
622040 130231 130248 TTTTTATTTCCTCCGCCA 61 2419 2436 5-8-5 1971
Table 37
Inhibition of tau mRNA by 5-10-5 MOE and 5-8-5 MOE gapmers targeting SEQ ID NO: 1 and 2
SEQ ID SEQ ID SEQ ID SEQ ID
ISIS % SEQ NO: 1 NO: 1 Sequence NO: 2 NO: 2 Motif NO inhibition ID NO: start site stop site start site stop site
613412 73879 73898 GTGATCTTCCATCACTTCGA 89 345 364 5-10-5 25
98891 98908 n/a n a
620887 GTTTTCAAACACACCTTC 98 5-8-5 665
98928 98945 n/a n/a
622043 n a n/a GGGTTTCAATCTTTTTAT 65 2430 2447 5-8-5 1972
622044 135477 135494 GTC AGCTT GT GGGTTTC A 86 2440 2457 5-8-5 1973
622045 135480 135497 AAGGTCAGCTTGTGGGTT 49 2443 2460 5-8-5 1974
622046 135484 135501 GCGGAAGGTCAGCTTGTG 26 2447 2464 5-8-5 1975
622047 135488 135505 TCTCGCGGAAGGTCAGCT 54 2451 2468 5-8-5 1976
622048 135491 135508 CGTTCTCGCGGAAGGTCA 63 2454 2471 5-8-5 1977
622049 135507 135524 TCT GT CTTGGCTTTGGC G 30 2470 2487 5-8-5 1978
622050 135510 135527 TGGTCTGTCTTGGCTTTG 72 2473 2490 5-8-5 1979
622051 135513 135530 CCGTGGTCTGTCTTGGCT 75 2476 2493 5-8-5 1980
622052 135516 135533 GCCCCGTGGTCTGTCTTG 39 2479 2496 5-8-5 1981
622053 135527 135544 ACACGATCTCCGCCCCGT 80 2490 2507 5-8-5 1982
622054 135530 135547 TGTACACGATCTCCGCCC 72 2493 2510 5-8-5 1983
622055 135533 135550 ACTTGTACACGATCTCCG 30 2496 2513 5-8-5 1984
622056 135536 135553 GCGACTTGTACACGATCT 72 2499 2516 5-8-5 1985
622057 135539 135556 CTGGCGACTTGTACACGA 65 2502 2519 5-8-5 1986
622058 135542 135559 CCACTGGCGACTTGTACA 35 2505 2522 5-8-5 1987
622059 135545 135562 ACACCACTGGCGACTTGT 46 2508 2525 5-8-5 1988
622060 135548 135565 CAGACACCACTGGCGACT 61 251 1 2528 5-8-5 1989
622061 135551 135568 CCCCAGACACCACTGGCG 74 2514 2531 5-8-5 1990
622062 135554 135571 TGTCCCCAGACACCACTG 38 2517 2534 5-8-5 1991
622063 135572 135589 TGAGATGCCGTGGAGACG 36 2535 2552 5-8-5 1992
622064 135575 135592 TGCTGAGATGCCGTGGAG 34 2538 2555 5-8-5 1993
622065 135599 135616 C GATGCTGC CGGTGGAGG 43 2562 2579 5-8-5 1994
622066 135602 135619 TGTCGATGCTGCCGGTGG 51 2565 2582 5-8-5 1995
622067 135605 135622 CCATGTCGATGCTGCCGG 66 2568 2585 5-8-5 1996
622068 135608 135625 CTACCATGTCGATGCTGC 68 2571 2588 5-8-5 1997
622069 135611 135628 AGTCTACCATGTCGATGC 56 2574 2591 5-8-5 1998
622070 135614 135631 GCGAGTCTACCATGTCGA 68 2577 2594 5-8-5 1999
622071 135654 135671 AGGGAGGCAGACACCTCG 24 2617 2634 5-8-5 2000
622072 135657 135674 GCCAGGGAGGCAGACACC 73 2620 2637 5-8-5 2001
622073 135661 135678 CTTGGCCAGGGAGGCAGA 50 2624 2641 5-8-5 2002 622074 135664 135681 CTGCTTGGCCAGGGAGGC 44 2627 2644 5-8-5 2003
622075 135667 135684 ACCCTGCTTGGCCAGGGA 18 2630 2647 5-8-5 2004
622076 135678 135695 CCTGATCACAAACCCTGC 65 2641 2658 5-8-5 2005
622077 135681 135698 GGGCCTGATCACAAACCC 82 2644 2661 5-8-5 2006
622078 135697 135714 TTATTGACCGCCCCAGGG 42 2660 2677 5-8-5 2007
622079 135700 135717 CAATTATTGACCGCCCCA 54 2663 2680 5-8-5 2008
622080 135703 135720 CCACAATTATTGACCGCC 95 2666 2683 5-8-5 2009
622081 135706 135723 TCTCCACAATTATTGACC 63 2669 2686 5-8-5 2010
622082 135709 135726 TCCTCTCCACAATTATTG 41 2672 2689 5-8-5 2011
622083 135712 135729 CTCTCCTCTCCACAATTA 76 2675 2692 5-8-5 2012
622084 135715 135732 ATTCTCTCCTCTCCACAA 57 2678 2695 5-8-5 2013
622085 135718 135735 CTCATTCTCTCCTCTCCA 72 2681 2698 5-8-5 2014
622086 135721 135738 TCTCTCATTCTCTCCTCT 69 2684 2701 5-8-5 2015
622087 135724 135741 CACTCTCTCATTCTCTCC 75 2687 2704 5-8-5 2016
622088 135727 135744 CCACACTCTCTCATTCTC 90 2690 2707 5-8-5 2017
622089 135730 135747 TTTCCACACTCTCTCATT 70 2693 2710 5-8-5 2018
622090 135733 135750 TTTTTTCCACACTCTCTC 76 2696 2713 5-8-5 2019
622091 135737 135754 CTTTTTTTTTCCACACTC 84 2700 2717 5-8-5 2020
622092 135740 135757 ATTCTTTTTTTTTCCACA 67 2703 2720 5-8-5 2021
622093 135746 135763 GTCATTATTCTTTTTTTT 54 2709 2726 5-8-5 2022
622094 135749 135766 CGGGTCATTATTCTTTTT 82 2712 2729 5-8-5 2023
622095 135752 135769 GGCCGGGTCATTATTCTT 72 2715 2732 5-8-5 2024
622096 135783 135800 CTGCGAGGAGCAGCTGGG 52 2746 2763 5-8-5 2025
622097 135786 135803 GAACTGCGAGGAGCAGCT 59 2749 2766 5-8-5 2026
622098 135789 135806 ACCGAACTGCGAGGAGCA 77 2752 2769 5-8-5 2027
622099 135792 135809 TTAACCGAACTGCGAGGA 62 2755 2772 5-8-5 2028
622100 135795 135812 CAATTAACCGAACTGCGA 55 2758 2775 5-8-5 2029
622101 135798 135815 AACCAATTAACCGAACTG 59 2761 2778 5-8-5 2030
622102 135801 135818 ATTAACCAATTAACCGAA 71 2764 2781 5-8-5 2031
622103 135804 135821 GTGATTAACCAATTAACC 70 2767 2784 5-8-5 2032
622104 135807 135824 TAAGTGATTAACCAATTA 42 2770 2787 5-8-5 2033
622105 135810 135827 GGTTAAGTGATTAACCAA 42 2773 2790 5-8-5 2034
622106 135813 135830 GCAGGTTAAGTGATTAAC 78 2776 2793 5-8-5 2035
622107 135816 135833 AAAGCAGGTTAAGTGATT 38 2779 2796 5-8-5 2036
622108 135819 135836 ACAAAAGCAGGTTAAGTG 55 2782 2799 5-8-5 2037
622109 135822 135839 GTGACAAAAGCAGGTTAA 82 2785 2802 5-8-5 2038
622110 135825 135842 CGAGTGACAAAAGCAGGT 86 2788 2805 5-8-5 2039
622111 135828 135845 AGCCGAGTGACAAAAGCA 93 2791 2808 5-8-5 2040
622112 135831 135848 CAAAGCCGAGTGACAAAA 73 2794 2811 5-8-5 2041
622113 135834 135851 AGCCAAAGCCGAGTGACA 82 2797 2814 5-8-5 2042
622114 135837 135854 CCGAGCCAAAGCCGAGTG 82 2800 2817 5-8-5 2043
622115 135840 135857 GTCCCGAGCCAAAGCCGA 63 2803 2820 5-8-5 2044
622116 135843 135860 GAAGTCCCGAGCCAAAGC 59 2806 2823 5-8-5 2045 622117 135846 135863 TTTGAAGTCCCGAGCCAA 76 2809 2826 5-8-5 2046
622118 135849 135866 GATTTTGAAGTCCCGAGC 72 2812 2829 5-8-5 2047
622119 135852 135869 ACTGATTTTGAAGTCCCG 73 2815 2832 5-8-5 2048
Table 38
Inhibition of tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NO: 1
Figure imgf000118_0001
623886 20943 20960 GTTATTGGGACTGACCTT 69 5-8-5 2082
623887 20948 20965 GATTTGTTATTGGGACTG 78 5-8-5 2083
613412 73879 73898 GTGATCTTCCATCACTTCGA 89 5-8-5 25
623811 122370 122387 CAGTGGAGCCACTCAACG 41 5-8-5 2084
623812 122380 122397 CACCTGTCCACAGTGGAG 58 5-8-5 2085
623813 122391 122408 AAC AAAC GGGTC AC CTGT 63 5-8-5 2086
623814 122445 122462 CGTGTAGGAGCAGCAGCT 81 5-8-5 2087
623815 122538 122555 CTTTGGTTTGGCTCTTTG 64 5-8-5 2088
623816 123104 123121 GCTGGTGGGAGAGGAGCC 9 5-8-5 2089
623817 123288 123305 ATGCGGGTGGCTGCCTCA 65 5-8-5 2090
623818 123293 123310 GCTGGATGCGGGTGGCTG 24 5-8-5 2091
623819 123341 123358 GTGCTCAGGGCAGGAAGC 45 5-8-5 2092
623820 123590 123607 CAAAGCTCAAACAGCTGA 56 5-8-5 2093
623821 123601 123618 AGAACCAGGATCAAAGCT 41 5-8-5 2094
623822 123704 123721 GCTCCGCCTCAGCAGCAC 71 5-8-5 2095
623823 123722 123739 GTGTGCACTCTCTCCCCA 45 5-8-5 2096
623824 123877 123894 GGCTAGCCCGCAGACGAG 31 5-8-5 2097
623825 123976 123993 CCTGTGAAGGTGCTCAGA 58 5-8-5 2098
623826 124033 124050 GATCCAGTGCCCCCAGAT 54 5-8-5 2099
623827 124057 124074 GGAGAGGCTAGGGCTCAG 68 5-8-5 2100
623828 124223 124240 AGAGGAGTGGATGGCAGT 37 5-8-5 2101
623829 124293 124310 TGGCCTGGGTGAGGTAAC 23 5-8-5 2102
623830 124296 124313 CTCTGGCCTGGGTGAGGT 28 5-8-5 2103
623831 124596 124613 AGGGCAGGTGGTGGTTTC 8 5-8-5 2104
623832 124780 124797 GACATTCCTGGAGTCCCC 83 5-8-5 2105
623833 124859 124876 TGGCAGACAGACAGGTCC 87 5-8-5 2106
623834 124909 124926 AGTCAAAGAACCAGCTCC 68 5-8-5 2107
623835 124949 124966 TCCCTCTGGGAATGATGA 41 5-8-5 2108
623836 124965 124982 GCCTCCAGGGCACCGCTC 76 5-8-5 2109
623837 124972 124989 GCCTGTGGCCTCCAGGGC 43 5-8-5 2110
623838 124977 124994 AGGAGGCCTGTGGCCTCC 11 5-8-5 2111
623839 125075 125092 AATAGGAACAAAGCAACA 25 5-8-5 2112
623840 125086 125103 CTGTCTTTAGCAATAGGA 84 5-8-5 2113
623841 125100 125117 TGTCCTGGACATTCCTGT 66 5-8-5 2114
623842 125291 125308 TGGAAAGGCAGGAGTGGG 0 5-8-5 2115
623843 125306 125323 TCTGAAAAATCTTGCTGG 51 5-8-5 2116
623844 125310 125327 AGCATCTGAAAAATCTTG 82 5-8-5 2117
623845 125322 125339 ATGAGTATGCACAGCATC 68 5-8-5 2118
623846 125337 125354 AAAGTGATCAATATGATG 33 5-8-5 2119
623847 125390 125407 GTCACTCCCTTTCCTGAC 68 5-8-5 2120
623848 125408 125425 ACGCTTAAGTGTAAAAAT 38 5-8-5 2121
623849 125557 125574 AAATGTGTTGTCGAAATT 19 5-8-5 2122
623850 125567 125584 CAGGGTGGAAAAATGTGT 33 5-8-5 2123 623851 125598 125615 TTCCCAGCTGCCATGAGG 33 5-8-5 2124
623852 125743 125760 TGGAGATGAGAGAGGAGG 0 5-8-5 2125
Table 39
Inhibition of Tau mRNA by 5-8-5 MOE gapmers targeting SEQ ID NO: 1
SEQ ID SEQ ID SEQ
%
ISIS NO NO: 1 start NO: 1 stop Sequence Motif ID inhibition
site site NO:
623661 6196 6213 GCGCTTACCTGATAGTCG 36 5-8-5 2126
613412 73879 73898 GTGATCTTCCATCACTTCGA 87 5-8-5 25
623662 73981 73998 TCCACTAACCTTTCAGGC 30 5-8-5 2127
623663 83368 83385 GGGAGATTCTGGAACACA 72 5-8-5 2128
623664 83455 83472 GGGCCCACCTTCCGCTGT 22 5-8-5 2129
623665 85895 85912 CTGTCACATCTAGAAACC 21 5-8-5 2130
623666 85982 85999 TACCCTCACCTGTGGTTC 0 5-8-5 2131
623667 89885 89902 CTTCTTCAGCTGGTGTAT 22 5-8-5 2132
623668 89951 89968 TTCACTGACCTTGGGTCA 0 5-8-5 2133
623669 94688 94705 TTTCAGGCTCTGTGTGGA 61 5-8-5 2134
623670 95441 95458 GACACAGACCTTTGAGTT 53 5-8-5 2135
623671 98550 98567 CCATGCGAGCTGATAAAA 39 5-8-5 2136
621344 98607 98624 GTCAGCTTACCTTGGCTT 34 5-8-5 814
98891 98908
620887 GTTTTCAAACACACCTTC 94 5-8-5 665
98928 98945
623672 101387 101404 GTGGATGTCTTAAACATA 89 5-8-5 2137
623673 101586 101603 TTTCCTTACCTTGAGTTT 0 5-8-5 2138
623674 102970 102987 ATCAGCCCCCTGTAAATG 7 5-8-5 2139
623675 103097 103114 TCTTCTTACCAGAGCTGG 0 5-8-5 2140
623676 107909 107926 GAGGTTCACCTGGGAAGG 0 5-8-5 2141
623677 108175 108192 CACTCTCACCTTCCCGCC 6 5-8-5 2142
623712 108255 108272 CCTCCAGGCGCAGCCCTA 92 5-8-5 2143
623713 108413 108430 CCTGAGGAGGGCACTCAC 28 5-8-5 2144
623714 108530 108547 GGAACCGCTGTGGGTGCC 60 5-8-5 2145
623715 108565 108582 TGGGTGGCAGTGTATTCT 63 5-8-5 2146
623716 108690 108707 GGAGAGCTCGCGAGCACC 69 5-8-5 2147
623717 108896 108913 GAGGTGGCTACCCACGGC 59 5-8-5 2148
623718 109561 109578 CCAGAAGGCCCAGCACAT 51 5-8-5 2149
623719 109574 109591 CGAGGCCCAGTGCCCAGA 59 5-8-5 2150
623720 109607 109624 AGGCCCAGGGTTCCAGAA 55 5-8-5 2151
623721 109623 109640 AGGCAAGCTGACACGCAG 73 5-8-5 2152
623722 110101 110118 CAGGAAAAGGCCGGACCT 37 5-8-5 2153
623723 110103 110120 AGCAGGAAAAGGCCGGAC 78 5-8-5 2154
623724 110182 110199 TTCCCCAAGGTCTCTAAC 14 5-8-5 2155
623725 110413 110430 AGGAAAGGCCAGTGAGGG 8 5-8-5 2156
623726 110500 110517 TTGTTATGTGACTTGAGG 74 5-8-5 2157 623727 110630 110647 ATTCCCCACCATGGGACA 17 5-8-5 2158
623728 110635 110652 AGGACATTCCCCACCATG 9 5-8-5 2159
623729 110648 110665 AGATAAGGAGAGAAGGAC 15 5-8-5 2160
623730 110755 110772 ATGCTCAGTGTGGTCAGA 73 5-8-5 2161
623731 110852 110869 AGGGCCGGCCACCTGCAC 56 5-8-5 2162
623732 110919 110936 TCTGTCTCTGGCAACCTG 85 5-8-5 2163
623733 110954 110971 AACAGGGAAGCTACTTCC 6 5-8-5 2164
623734 111077 111094 GGGCCTTCAATGGAAAGT 21 5-8-5 2165
623735 111153 111170 GGTCGCCTGACTCTCACC 66 5-8-5 2166
623736 111174 111191 CCCTTTCTACACTTGGCA 81 5-8-5 2167
623678 121820 121837 TATCTGCACCTTTGGTAG 30 5-8-5 2168
623685 121830 121847 TCTTATTAATTATCTGCA 81 5-8-5 2169
623686 121831 121848 TTCTTATTAATTATCTGC 74 5-8-5 2170
623687 121834 121851 AGCTTCTTATTAATTATC 46 5-8-5 2171
623688 121835 121852 CAGCTTCTTATTAATTAT 6 5-8-5 2172
623689 121837 121854 TCCAGCTTCTTATTAATT 4 5-8-5 2173
623690 121838 121855 ATCCAGCTTCTTATTAAT 2 5-8-5 2174
623691 121840 121857 AGATCCAGCTTCTTATTA 2 5-8-5 2175
623692 121841 121858 AAGATCCAGCTTCTTATT 17 5-8-5 2176
623693 121843 121860 CTAAGATCCAGCTTCTTA 41 5-8-5 2177
623694 121844 121861 GCTAAGATCCAGCTTCTT 75 5-8-5 2178
623695 121846 121863 TTGCTAAGATCCAGCTTC 50 5-8-5 2179
623696 121847 121864 GTTGCTAAGATCCAGCTT 52 5-8-5 2180
623697 121865 121882 GCCACACTTGGACTGGAC 88 5-8-5 2181
623698 121866 121883 AGCCACACTTGGACTGGA 89 5-8-5 2182
623699 121868 121885 TGAGCCACACTTGGACTG 59 5-8-5 2183
623700 121869 121886 TTGAGCCACACTTGGACT 62 5-8-5 2184
623701 121872 121889 CCTTTGAGCCACACTTGG 69 5-8-5 2185
623702 121874 121891 ATCCTTTGAGCCACACTT 64 5-8-5 2186
623703 121875 121892 TATCCTTTGAGCCACACT 69 5-8-5 2187
623704 121877 121894 ATTATCCTTTGAGCCACA 76 5-8-5 2188
623705 121878 121895 TATTATCCTTTGAGCCAC 76 5-8-5 2189
623706 121880 121897 GATATTATCCTTTGAGCC 54 5-8-5 2190
623707 121881 121898 TGATATTATCCTTTGAGC 65 5-8-5 2191
623708 121884 121901 GTTTGATATTATCCTTTG 94 5-8-5 2192
623709 121886 121903 GTGTTTGATATTATCCTT 85 5-8-5 2193
623710 121887 121904 CGTGTTTGATATTATCCT 78 5-8-5 2194
623711 121904 121921 ACTGCCGCCTCCCGGGAC 50 5-8-5 2195
623679 121913 121930 GGTACTCACACTGCCGCC 72 5-8-5 1654
623680 125753 125770 TATTTGCACCTGGAGATG 0 5-8-5 2196
623681 125835 125852 CAGGGCTACCTGGTTTAT 26 5-8-5 2197
623682 130128 130145 GGCCACCTCCTAGAACAC 8 5-8-5 2198
623683 130241 130258 CCCCTTTACCTTTTTATT 0 5-8-5 2199 623684 135466 135483 GGTTTCAATCTGCAAGAA 39 5-8-5 2200
Example 11: Dose-dependent antisense inhibition of human Tau in SH-SY5Y cells
Gapmers from studies described above exhibiting significant in vitro inhibition of tau mRNA were selected and tested at various doses in SH-SY5Y cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.938 μΜ, 0.1.875 μΜ, 3.750 μΜ, 7.500 μΜ, and 15.00 μΜ concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense
oligonucleotide treated cells.
Figure imgf000122_0001
Figure imgf000123_0001
620887 60 79 92 95 95
623746 19 43 54 81 91
623750 50 63 75 83 90
623758 37 54 72 80 82
623805 25 33 48 74 86
623807 15 29 44 75 88
623833 16 60 65 86 89
623860 51 71 80 85 85
623867 9 51 48 74 86
623874 24 28 72 84 94
623875 38 62 80 93 95
623876 40 63 83 93 95
623877 59 76 92 97 96
623878 50 73 90 94 95
Figure imgf000124_0001
621448 46 60 85 89 93
621454 63 70 81 89 94
621484 60 65 75 88 93
621570 26 55 81 79 90
621576 23 34 71 75 80
621578 30 45 67 81 90
621579 14 28 49 69 88
621598 38 55 73 88 94
621670 37 57 79 82 92
621675 31 35 51 79 87
Table 46
Figure imgf000125_0001
Table 47
Figure imgf000125_0002
623782 50 72 83 90 93
623791 58 71 87 93 95
623796 44 57 74 79 85
623808 30 55 73 84 91
623809 31 42 66 82 93
Example 12: Antisense inhibition of human Tau in HepG2 cells by 5-10-5 MOE, 5-8-5 MOE, 4-8-6 MOE, or 6-8-4 MOE gapmers
Antisense oligonucleotides were designed targeting a tau nucleic acid and were tested for their effects on tau mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. ISIS 613412 was also included in the assays. The results for each experiment are presented in separate tables shown below. Cultured HepG2 cells at a density of 20,000 cells per well were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-8-5 MOE, 4-8-6 MOE, or 6-8-4 MOE gapmers. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2' -deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. The 4-8-6 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising four and six nucleosides respectively. The 6-8-4 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising six and four nucleosides respectively. Each nucleoside in the 5' wing segment and each nucleoside in the 3 ' wing segment has a 2' -MOE modification. The internucleoside linkage motif throughout for each gapmer in the tables below, except for ISIS 613412, is 5'-sooosssssssssooss-3', wherein each "s" represents a phosphorothioate internucleoside linkage and wherein each "o" represents a phosphodiester internucleoside linkage. The internucleoside linkage motif for ISIS 613412 is 5'-soooossssssssssooss-3', wherein each "s" represents a phosphorothioate internucleoside linkage and wherein each "o" represents a phosphodiester internucleoside linkage.All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in the Tables below is targeted to human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT_010783.15 truncated from nucleotides 9240000 to 9381000).
Table 48
Inhibition of tau mRNA by 5-10-5 MOE gapmers targeting SEQ ID NO: 1
Figure imgf000127_0001
623920 86767 86784 CTGGCTAGGCCTCTTCTG 5-10-5 54 2233
623921 86770 86787 ATCCTGGCTAGGCCTCTT 5-10-5 69 2234
623922 86772 86789 AAATCCTGGCTAGGCCTC 5-10-5 67 2235
623923 86773 86790 GAAATCCTGGCTAGGCCT 5-10-5 55 2236
623924 86774 86791 TGAAATCCTGGCTAGGCC 5-10-5 64 2237
623925 86775 86792 GTGAAATCCTGGCTAGGC 5-10-5 77 2238
623926 86776 86793 GGTGAAATCCTGGCTAGG 5-10-5 57 2239
623927 86777 86794 TGGTGAAATCCTGGCTAG 5-10-5 8 2240
623928 86778 86795 CTGGTGAAATCCTGGCTA 5-10-5 45 2241
623929 86780 86797 TGCTGGTGAAATCCTGGC 5-10-5 63 2242
623930 86783 86800 CACTGCTGGTGAAATCCT 5-10-5 70 2243
623931 121716 121733 ACCCTGGACCCGCCTACT 5-10-5 49 2244
623932 121721 121738 GCGCCACCCTGGACCCGC 5-10-5 84 2245
623933 121724 121741 CATGCGCCACCCTGGACC 5-10-5 52 2246
623934 121726 121743 GACATGCGCCACCCTGGA 5-10-5 80 2247
623935 121727 121744 TGACATGCGCCACCCTGG 5-10-5 74 2248
623936 121728 121745 GTGACATGCGCCACCCTG 5-10-5 90 2249
623937 121729 121746 AGTGACATGCGCCACCCT 5-10-5 84 2250
623938 121730 121747 GAGTGACATGCGCCACCC 5-10-5 92 2251
623939 121731 121748 TGAGTGACATGCGCCACC 5-10-5 86 2252
623940 121732 121749 ATGAGTGACATGCGCCAC 5-10-5 64 2253
623941 121734 121751 CGATGAGTGACATGCGCC 5-10-5 76 2254
623942 121737 121754 TTTCGATGAGTGACATGC 5-10-5 60 2255
623943 121742 121759 TCCACTTTCGATGAGTGA 5-10-5 34 2256
623944 121938 121955 CAGCACGGCGCATGGGAC 5-10-5 26 2257
623945 121939 121956 ACAGCACGGCGCATGGGA 5-10-5 62 2258
623946 121940 121957 CACAGCACGGCGCATGGG 5-10-5 57 2259
621455 121941 121958 CCACAGCACGGCGCATGG 5-10-5 44 1232
623804 121942 121959 GCCACAGCACGGCGCATG 5-10-5 85 1657
623947 121943 121960 AGCCACAGCACGGCGCAT 5-10-5 83 2260
623948 121944 121961 AAGCCACAGCACGGCGCA 5-10-5 56 2261
623949 121946 121963 TCAAGCCACAGCACGGCG 5-10-5 58 2262
623950 121949 121966 AATTCAAGCCACAGCACG 5-10-5 52 2263
623951 121954 121971 CTAATAATTCAAGCCACA 5-10-5 56 2264
623952 125424 125441 GACATTTGCTCAGCAAAC 5-10-5 74 2265
623953 125426 125443 CAGACATTTGCTCAGCAA 5-10-5 62 2266
623954 125427 125444 CCAGACATTTGCTCAGCA 5-10-5 75 2267
623955 125428 125445 CCCAGACATTTGCTCAGC 5-10-5 65 2268
623956 125429 125446 ACCCAGACATTTGCTCAG 5-10-5 30 2269
623957 125430 125447 GACCCAGACATTTGCTCA 5-10-5 30 2270
623958 125431 125448 AGACCCAGACATTTGCTC 5-10-5 41 2271
623959 125432 125449 AAGACCCAGACATTTGCT 5-10-5 69 2272
623960 125434 125451 GCAAGACCCAGACATTTG 5-10-5 73 2273 623961 125437 125454 TGTGCAAGACCCAGACAT 5-10-5 62 2274
623962 125442 125459 GTCATTGTGCAAGACCCA 5-10-5 87 2275
Table 49
Inhibition of tau mRNA by 5-10-5 MOE, 5-8-5 MOE, 4-8-6 MOE, and 6-8-4 gapmers targeting SEQ
ID NO: 1
SEQ ID
SEQ ID SEQ NO: 1 %
ISIS NO NO: 1 Sequence Motif ID
Start inhibition
Stop Site NO Site
613412 73879 73898 GTGATCTTCCATCACTTCGA 5-10-5 87 25
98891 98908
620887 GTTTTCAAACACACCTTC 5-8-5 96 665
98928 98945
98881 98898
620888 ACACCTTCATTTACTGTC 5-8-5 96 897
98918 98935
98890 98907
620889 TTTTCAAACACACCTTCA 5-8-5 85 898
98927 98944
98892 98909
620890 GGTTTTCAAACACACCTT 5-8-5 95 899
98929 98946
98893 98910
620891 TGGTTTTCAAACACACCT 5-8-5 96 900
98930 98947
623963 73877 73894 TCTTCCATCACTTCGAAC 5-8-5 58 2276
623964 73878 73895 ATCTTCCATCACTTCGAA 5-8-5 44 2277
623965 73880 73897 TGATCTTCCATCACTTCG 5-8-5 51 2278
623966 73881 73898 GTGATCTTCCATCACTTC 5-8-5 45 2279
623967 73931 73948 ATGGTGTAGCCCCCCTGA 5-8-5 66 2280
623968 73932 73949 CATGGTGTAGCCCCCCTG 5-8-5 87 2281
623969 73934 73951 TGCATGGTGTAGCCCCCC 5-8-5 94 2282
623970 73935 73952 GTGCATGGTGTAGCCCCC 5-8-5 90 2283
623971 73957 73974 CCGTGTCACCCTCTTGGT 5-8-5 83 2284
623972 73958 73975 TCCGTGTCACCCTCTTGG 5-8-5 79 2285
623973 73960 73977 CGTCCGTGTCACCCTCTT 5-8-5 92 2286
623974 83429 83446 TCTTAGCATCAGAGGTTT 5-8-5 70 2287
623975 83430 83447 CTCTTAGCATCAGAGGTT 5-8-5 76 2288
623976 83432 83449 TGCTCTTAGCATCAGAGG 5-8-5 72 2289
623977 83433 83450 GTGCTCTTAGCATCAGAG 5-8-5 86 2290
623978 95266 95283 CTCCTCCGAGTGCGCCTG 5-8-5 24 2291
623979 95267 95284 GCTCCTCCGAGTGCGCCT 5-8-5 77 2292
623980 95269 95286 ATGCTCCTCCGAGTGCGC 5-8-5 58 2293
623981 95270 95287 AATGCTCCTCCGAGTGCG 5-8-5 79 2294
623982 95272 95289 CAAATGCTCCTCCGAGTG 5-8-5 34 2295 623983 98557 98574 TTACTGACCATGCGAGCT 5-8-5 79 2296
623984 98558 98575 TTTACTGACCATGCGAGC 5-8-5 73 2297
623985 98560 98577 CTTTTACTGACCATGCGA 5-8-5 85 2298
623986 98561 98578 GCTTTTACTGACCATGCG 5-8-5 91 2299
623987 98563 98580 TTGCTTTTACTGACCATG 5-8-5 96 2300
623988 135784 135801 ACTGCGAGGAGCAGCTGG 5-8-5 43 2301
623989 135785 135802 AACTGCGAGGAGCAGCTG 5-8-5 31 2302
623990 135787 135804 CGAACTGCGAGGAGCAGC 5-8-5 62 2303
623991 135788 135805 CCGAACTGCGAGGAGCAG 5-8-5 73 2304
623992 135790 135807 AACCGAACTGCGAGGAGC 5-8-5 73 2305
623993 135868 135885 TGCTCTTACTCCCATCAC 5-8-5 59 2306
623994 135869 135886 TTGCTCTTACTCCCATCA 5-8-5 85 2307
623995 135871 135888 ATTTGCTCTTACTCCCAT 5-8-5 77 2308
623996 135872 135889 AATTTGCTCTTACTCCCA 5-8-5 80 2309
625423 98879 98896 ACCTTCATTTACTGTCAG 5-8-5 90 2310
625424 98880 98897 CACCTTCATTTACTGTCA 5-8-5 94 2311
98882 98899 5-8-5
625425 CACACCTTCATTTACTGT 89 2312
98919 98936 5-8-5
98883 98900
625426 ACACACCTTCATTTACTG 5-8-5 87 2313
98920 98937
98884 98901
625427 AACACACCTTCATTTACT 5-8-5 86 2314
98921 98938
98885 98902
625428 AAACACACCTTCATTTAC 5-8-5 49 2315
98922 98939
98886 98903
625429 CAAACACACCTTCATTTA 5-8-5 66 2316
98923 98940
98887 98904
625430 TCAAACACACCTTCATTT 5-8-5 70 2317
98924 98941
98888 98905
625431 TTCAAACACACCTTCATT 5-8-5 74 2318
98925 98942
98889 98906
625432 TTTCAAACACACCTTCAT 5-8-5 81 2319
98926 98943
625433 98894 98911 TTGGTTTTCAAACACACC 5-8-5 92 2320
625434 75128 75145 AGCATTAAAAGAGAAAAG 5-8-5 13 2321
625435 75136 75153 TTATTTTTAGCATTAAAA 5-8-5 0 2322
625436 75138 75155 TCTTATTTTTAGCATTAA 5-8-5 52 2323
625437 75139 75156 TTCTTATTTTTAGCATTA 5-8-5 86 2324
625438 75140 75157 TTTCTTATTTTTAGCATT 5-8-5 40 2325 625439 75141 75158 TTTTCTTATTTTTAGCAT 541-5 0 2326
625440 75142 75159 GTTTTCTTATTTTTAGCA 54 1-5 82 2327
625441 75143 75160 TGTTTTCTTATTTTTAGC 54 1-5 68 2328
625442 75146 75163 AGGTGTTTTCTTATTTTT 54 -5 83 2329
625443 75154 75171 GGGCTGCAAGGTGTTTTC 54 1-5 69 2330
98881 98898
625444 ACACCTTCATTTACTGTC 44 1-6 90 897
98918 98935
98882 98899
625445 CACACCTTCATTTACTGT 44 i-6 82 2312
98919 98936
98883 98900
625446 ACACACCTTCATTTACTG 44 1-6 84 2313
98920 98937
98884 98901
625447 AACACACCTTCATTTACT 44 i-6 40 2314
98921 98938
98889 98906
625448 TTTCAAACACACCTTCAT 44 1-6 83 2319
98926 98943
98890 98907
625449 TTTTCAAACACACCTTCA 44 i-6 0 898
98927 98944
98891 98908
625450 GTTTTCAAACACACCTTC 44 -6 94 665
98928 98945
98892 98909
625451 GGTTTTCAAACACACCTT 44 1-6 94 899
98929 98946
98893 98910
625452 TGGTTTTCAAACACACCT 44 i-6 94 900
98930 98947
98881 98898 897
625453 ACACCTTCATTTACTGTC 1-4 96
98918 98935
98882 98899 2312
625454 CACACCTTCATTTACTGT 64 i-4 96
98919 98936
98883 98900 2313
625455 ACACACCTTCATTTACTG 64 i-4 93
98920 98937
98884 98901 2314
625456 AACACACCTTCATTTACT i-4
98921 98938
98889 98906 2319
625457 TTTCAAACACACCTTCAT 64 1-4 85
98926 98943
98890 98907 898
625458 TTTTCAAACACACCTTCA 64 1-4 90
98927 98944
98891 98908 665
625459 GTTTTCAAACACACCTTC 1-4 97
98928 98945
625460 98892 98909 GGTTTTCAAACACACCTT 64 1-4 97 899 98929 98946
98893 98910
625461 TGGTTTTCAAACACACCT 6-8-4 96 900
98930 98947
Example 13: Dose-dependent antisense inhibition of human tau in SH-SY5Y cells by MOE gapmers
Gapmers from studies described above exhibiting significant in vitro inhibition of tau mRNA were selected and tested at various doses in SH-SY5Y cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.938 μΜ, 0.1.875 μΜ, 3.750 μΜ, 7.500 μΜ, and 15.00 μΜ concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells. Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense
oligonucleotide treated cells.
Figure imgf000132_0001
Figure imgf000133_0001
620890 82 90 94 95 96
623969 41 62 84 94 n.d.
623987 46 71 89 96 97
625424 50 73 88 94 96
625433 64 83 90 95 95
625450 69 84 91 96 97
625451 85 91 95 93 95
625452 67 83 91 93 95
625453 72 85 91 94 96
625454 73 86 93 95 96
625455 44 68 86 92 94
625461 75 84 94 95 97
Figure imgf000134_0001
Example 14: Design of 5-7-6 MOE, 5-8-5 MOE, 5-9-5 MOE, and 5-10-5 MOE gapmers with phosphorothioate and phosphodiester internucleoside linkages at a hot spot region of human Tau
Antisense oligonucleotides were designed targeting a tau nucleic acid at a region identified as a 5 'hotspot' in the studies above.
The newly designed chimeric antisense oligonucleotides in the Table below were designed as 5-7-6 MOE, 5-8-5 MOE, 5-9-5 MOE, or 5-10-5 MOE gapmers. The 5-7-6 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of seven 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five and six nucleosides respectively. The 5-8-5 0 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment comprises of nine 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein 5 the central gap segment comprises often 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside linkages throughout each gapmer are either phosphorothioate linkages or phosphodiester linkages. The 'Chemistry' column describes the internucleoside linkages of each oligonucleotide. V indicates phosphorothioate linkage and Ό' 0 indicates phosphodiester linkage. All cytosine residues throughout each gapmer are 5-methylcytosines.
"Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in the Table below is targeted to either the human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT 010783.15 truncated from nucleotides 5 9240000 to 9381000) or to the human tau mRNA sequence, designated herein as SEQ ID NO : 2 (GENBANK Accession No. NM 001123066.3). 'n/a' indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.
Table 56
MOE gapmers targeting SEQ ID NOs: 1 and 2
Figure imgf000135_0001
664661 135820 135839 GTGACAAAAGCAGGTTAAGT 2783 2802 sooosssssssssssooss 5-10-5 2333
664767 135820 135837 GACAAAAGCAGGTTAAGT 2783 2800 sooossssssssoooss 5-7-6 2331
664512 135821 135838 TGACAAAAGCAGGTTAAG 2784 2801 sooosssssssssooss 5-8-5 2334
664715 135821 135839 GTGACAAAAGCAGGTTAAG 2784 2802 sooossssssssssooss 5-9-5 2335
664662 135821 135840 AGTGACAAAAGCAGGTTAAG 2784 2803 sooosssssssssssooss 5-10-5 2336
664768 135821 135838 TGACAAAAGCAGGTTAAG 2784 2801 sooossssssssoooss 5-7-6 2334
622109 135822 135839 GTGACAAAAGCAGGTTAA 2785 2802 sooosssssssssooss 5-8-5 2038
664716 135822 135840 AGTGACAAAAGCAGGTTAA 2785 2803 sooossssssssssooss 5-9-5 2337
664663 135822 135841 GAGTGACAAAAGCAGGTTAA 2785 2804 sooosssssssssssooss 5-10-5 2338
664769 135822 135839 GTGACAAAAGCAGGTTAA 2785 2802 sooossssssssoooss 5-7-6 2038
664513 135823 135840 AGTGACAAAAGCAGGTTA 2786 2803 sooosssssssssooss 5-8-5 2339
664717 135823 135841 GAGTGACAAAAGCAGGTTA 2786 2804 sooossssssssssooss 5-9-5 2340
664664 135823 135842 CGAGTGACAAAAGCAGGTTA 2786 2805 sooosssssssssssooss 5-10-5 2341
664770 135823 135840 AGTGACAAAAGCAGGTTA 2786 2803 sooossssssssoooss 5-7-6 2339
664514 135824 135841 GAGTGACAAAAGCAGGTT 2787 2804 sooosssssssssooss 5-8-5 2342
664718 135824 135842 CGAGTGACAAAAGCAGGTT 2787 2805 sooossssssssssooss 5-9-5 2343
664665 135824 135843 CCGAGTGACAAAAGCAGGTT 2787 2806 sooosssssssssssooss 5-10-5 2344
664771 135824 135841 GAGTGACAAAAGCAGGTT 2787 2804 sooossssssssoooss 5-7-6 2342
622110 135825 135842 CGAGTGACAAAAGCAGGT 2788 2805 sooosssssssssooss 5-8-5 2039
664719 135825 135843 CCGAGTGACAAAAGCAGGT 2788 2806 sooossssssssssooss 5-9-5 2345
664666 135825 135844 GCCGAGTGACAAAAGCAGGT 2788 2807 sooosssssssssssooss 5-10-5 2346
664772 135825 135842 CGAGTGACAAAAGCAGGT 2788 2805 sooossssssssoooss 5-7-6 2039
664515 135826 135843 CCGAGTGACAAAAGCAGG 2789 2806 sooosssssssssooss 5-8-5 2347
664720 135826 135844 GCCGAGTGACAAAAGCAGG 2789 2807 sooossssssssssooss 5-9-5 2348
664667 135826 135845 AGCCGAGTGACAAAAGCAGG 2789 2808 sooosssssssssssooss 5-10-5 2349
664773 135826 135843 CCGAGTGACAAAAGCAGG 2789 2806 sooossssssssoooss 5-7-6 2347
664516 135827 135844 GCCGAGTGACAAAAGCAG 2790 2807 sooosssssssssooss 5-8-5 2350
664721 135827 135845 AGCCGAGTGACAAAAGCAG 2790 2808 sooossssssssssooss 5-9-5 2351
664668 135827 135846 AAGCCGAGTGACAAAAGCAG 2790 2809 sooosssssssssssooss 5-10-5 2352
664774 135827 135844 GCCGAGTGACAAAAGCAG 2790 2807 sooossssssssoooss 5-7-6 2350
622111 135828 135845 AGCCGAGTGACAAAAGCA 2791 2808 sooosssssssssooss 5-8-5 2040
664722 135828 135846 AAGCCGAGTGACAAAAGCA 2791 2809 sooossssssssssooss 5-9-5 2353
664669 135828 135847 AAAGCCGAGTGACAAAAGCA 2791 2810 sooosssssssssssooss 5-10-5 2354
664775 135828 135845 AGCCGAGTGACAAAAGCA 2791 2808 sooossssssssoooss 5-7-6 2040
664517 135829 135846 AAGCCGAGTGACAAAAGC 2792 2809 sooosssssssssooss 5-8-5 2355
664723 135829 135847 AAAGCCGAGTGACAAAAGC 2792 2810 sooossssssssssooss 5-9-5 2356
664670 135829 135848 CAAAGCCGAGTGACAAAAGC 2792 2811 sooosssssssssssooss 5-10-5 2357 664776 135829 135846 AAGCCGAGTGACAAAAGC 2792 2809 sooossssssssoooss 5-7-6 2355
664518 135830 135847 AAAGCCGAGTGACAAAAG 2793 2810 sooosssssssssooss 5-8-5 2358
664724 135830 135848 CAAAGCCGAGTGACAAAAG 2793 2811 sooossssssssssooss 5-9-5 2359
664671 135830 135849 CCAAAGCCGAGTGACAAAAG 2793 2812 sooosssssssssssooss 5-10-5 2360
664777 135830 135847 AAAGCCGAGTGACAAAAG 2793 2810 sooossssssssoooss 5-7-6 2358
622112 135831 135848 CAAAGCCGAGTGACAAAA 2794 2811 sooosssssssssooss 5-8-5 2041
664725 135831 135849 CCAAAGCCGAGTGACAAAA 2794 2812 sooossssssssssooss 5-9-5 2361
664672 135831 135850 GCCAAAGCCGAGTGACAAAA 2794 2813 sooosssssssssssooss 5-10-5 2362
664778 135831 135848 CAAAGCCGAGTGACAAAA 2794 2811 sooossssssssoooss 5-7-6 2041
664519 135832 135849 CCAAAGCCGAGTGACAAA 2795 2812 sooosssssssssooss 5-8-5 2363
664726 135832 135850 GCCAAAGCCGAGTGACAAA 2795 2813 sooossssssssssooss 5-9-5 2364
664673 135832 135851 AGCCAAAGCCGAGTGACAAA 2795 2814 sooosssssssssssooss 5-10-5 2365
664779 135832 135849 CCAAAGCCGAGTGACAAA 2795 2812 sooossssssssoooss 5-7-6 2363
664520 135833 135850 GCCAAAGCCGAGTGACAA 2796 2813 sooosssssssssooss 5-8-5 2366
664727 135833 135851 AGC C AAAGCC GAGTG AC AA 2796 2814 sooossssssssssooss 5-9-5 2367
664674 135833 135852 GAGCCAAAGCCGAGTGACAA 2796 2815 sooosssssssssssooss 5-10-5 2368
664780 135833 135850 GCCAAAGCCGAGTGACAA 2796 2813 sooossssssssoooss 5-7-6 2366
622113 135834 135851 AGCCAAAGCCGAGTGACA 2797 2814 sooosssssssssooss 5-8-5 2042
664728 135834 135852 GAGCCAAAGCCGAGTGACA 2797 2815 sooossssssssssooss 5-9-5 2369
664675 135834 135853 CGAGCCAAAGCCGAGTGACA 2797 2816 sooosssssssssssooss 5-10-5 2419
664781 135834 135851 AGCCAAAGCCGAGTGACA 2797 2814 sooossssssssoooss 5-7-6 2042
664521 135835 135852 GAGCCAAAGCCGAGTGAC 2798 2815 sooosssssssssooss 5-8-5 2420
664729 135835 135853 CGAGCCAAAGCCGAGTGAC 2798 2816 sooossssssssssooss 5-9-5 2421
664676 135835 135854 CCGAGCCAAAGCCGAGTGAC 2798 2817 sooosssssssssssooss 5-10-5 2422
664782 135835 135852 GAGCCAAAGCCGAGTGAC 2798 2815 sooossssssssoooss 5-7-6 2420
664522 135836 135853 CGAGCCAAAGCCGAGTGA 2799 2816 sooosssssssssooss 5-8-5 2423
664730 135836 135854 CCGAGCCAAAGCCGAGTGA 2799 2817 sooossssssssssooss 5-9-5 2424
664677 135836 135855 CCCGAGCCAAAGCCGAGTGA 2799 2818 sooosssssssssssooss 5-10-5 2425
664783 135836 135853 CGAGCCAAAGCCGAGTGA 2799 2816 sooossssssssoooss 5-7-6 2423
622114 135837 135854 CCGAGCCAAAGCCGAGTG 2800 2817 sooosssssssssooss 5-8-5 2043
664731 135837 135855 CCCGAGCCAAAGCCGAGTG 2800 2818 sooossssssssssooss 5-9-5 2426
664678 135837 135856 TCCCGAGCCAAAGCCGAGTG 2800 2819 sooosssssssssssooss 5-10-5 2427
664784 135837 135854 CCGAGCCAAAGCCGAGTG 2800 2817 sooossssssssoooss 5-7-6 2043
664523 135838 135855 CCCGAGCCAAAGCCGAGT 2801 2818 sooosssssssssooss 5-8-5 2428
664732 135838 135856 TCCCGAGCCAAAGCCGAGT 2801 2819 sooossssssssssooss 5-9-5 2429
664679 135838 135857 GTCCCGAGCCAAAGCCGAGT 2801 2820 sooosssssssssssooss 5-10-5 2430
664785 135838 135855 CCCGAGCCAAAGCCGAGT 2801 2818 sooossssssssoooss 5-7-6 2428 664524 135839 135856 TCCCGAGCCAAAGCCGAG 2802 2819 sooosssssssssooss 5-8-5 2431
664733 135839 135857 GTCCCGAGCCAAAGCCGAG 2802 2820 sooossssssssssooss 5-9-5 2432
664680 135839 135858 AGTCCCGAGCCAAAGCCGAG 2802 2821 sooosssssssssssooss 5-10-5 2433
664786 135839 135856 TCCCGAGCCAAAGCCGAG 2802 2819 sooossssssssoooss 5-7-6 2431
622115 135840 135857 GTCCCGAGCCAAAGCCGA 2803 2820 sooosssssssssooss 5-8-5 2044
664734 135840 135858 AGTCCCGAGCCAAAGCCGA 2803 2821 sooossssssssssooss 5-9-5 2434
664681 135840 135859 AAGTCCCGAGCCAAAGCCGA 2803 2822 sooosssssssssssooss 5-10-5 2435
664787 135840 135857 GTCCCGAGCCAAAGCCGA 2803 2820 sooossssssssoooss 5-7-6 2044
664525 135841 135858 AGTCCCGAGCCAAAGCCG 2804 2821 sooosssssssssooss 5-8-5 2436
664735 135841 135859 AAGTCCCGAGCCAAAGCCG 2804 2822 sooossssssssssooss 5-9-5 2437
664682 135841 135860 GAAGTCCCGAGCCAAAGCCG 2804 2823 sooosssssssssssooss 5-10-5 2438
664788 135841 135858 AGTCCCGAGCCAAAGCCG 2804 2821 sooossssssssoooss 5-7-6 2436
664526 135842 135859 AAGTCCCGAGCCAAAGCC 2805 2822 sooosssssssssooss 5-8-5 2439
664736 135842 135860 GAAGTCCCGAGCCAAAGCC 2805 2823 sooossssssssssooss 5-9-5 2440
664683 135842 135861 TGAAGTCCCGAGCCAAAGCC 2805 2824 sooosssssssssssooss 5-10-5 2441
664789 135842 135859 AAGTCCCGAGCCAAAGCC 2805 2822 sooossssssssoooss 5-7-6 2439
622116 135843 135860 GAAGTCCCGAGCCAAAGC 2806 2823 sooosssssssssooss 5-8-5 2045
664737 135843 135861 TGAAGTCCCGAGCCAAAGC 2806 2824 sooossssssssssooss 5-9-5 2442
664684 135843 135862 TTGAAGTCCCGAGCCAAAGC 2806 2825 sooosssssssssssooss 5-10-5 2443
664790 135843 135860 GAAGTCCCGAGCCAAAGC 2806 2823 sooossssssssoooss 5-7-6 2045
664527 135844 135861 TGAAGTCCCGAGCCAAAG 2807 2824 sooosssssssssooss 5-8-5 2478
664738 135844 135862 TTGAAGTCCCGAGCCAAAG 2807 2825 sooossssssssssooss 5-9-5 2532
664685 135844 135863 TTTGAAGTCCCGAGCCAAAG 2807 2826 sooosssssssssssooss 5-10-5 2533
664791 135844 135861 TGAAGTCCCGAGCCAAAG 2807 2824 sooossssssssoooss 5-7-6 2478
664528 135845 135862 TTGAAGTCCCGAGCCAAA 2808 2825 sooosssssssssooss 5-8-5 2479
664739 135845 135863 TTTGAAGTCCCGAGCCAAA 2808 2826 sooossssssssssooss 5-9-5 2534
664686 135845 135864 TTTTGAAGTCCCGAGCCAAA 2808 2827 sooosssssssssssooss 5-10-5 2535
664792 135845 135862 TTGAAGTCCCGAGCCAAA 2808 2825 sooossssssssoooss 5-7-6 2479
622117 135846 135863 TTTGAAGTCCCGAGCCAA 2809 2826 sooosssssssssooss 5-8-5 2046
664740 135846 135864 TTTTGAAGTCCCGAGCCAA 2809 2827 sooossssssssssooss 5-9-5 2536
664687 135846 135865 ATTTTGAAGTCCCGAGCCAA 2809 2828 sooosssssssssssooss 5-10-5 2537
664793 135846 135863 TTTGAAGTCCCGAGCCAA 2809 2826 sooossssssssoooss 5-7-6 2538
664529 135847 135864 TTTTGAAGTCCCGAGCCA 2810 2827 sooosssssssssooss 5-8-5 2480
664741 135847 135865 ATTTTGAAGTCCCGAGCCA 2810 2828 sooossssssssssooss 5-9-5 2539
664688 135847 135866 GATTTTGAAGTCCCGAGCCA 2810 2829 sooosssssssssssooss 5-10-5 2540
664794 135847 135864 TTTTGAAGTCCCGAGCCA 2810 2827 sooossssssssoooss 5-7-6 2480
664530 135848 135865 ATTTTGAAGTCCCGAGCC 2811 2828 sooosssssssssooss 5-8-5 2481 664742 135848 135866 GATTTTGAAGTCCCGAGCC 2811 2829 sooossssssssssooss 5-9-5 2541
664689 135848 135867 TGATTTTGAAGTCCCGAGCC 1644 1663 sooosssssssssssooss 5-10-5 56
664795 135848 135865 ATTTTGAAGTCCCGAGCC 2811 2828 sooossssssssoooss 5-7-6 2481
622118 135849 135866 GATTTT GAAGTC CC GAGC 2812 2829 sooosssssssssooss 5-8-5 2047
664743 135849 135867 TGATTTTGAAGTCCCGAGC 2812 2830 sooossssssssssooss 5-9-5 2542
664690 135849 135868 CTGATTTTGAAGTCCCGAGC 2812 2831 sooosssssssssssooss 5-10-5 464
664796 135849 135866 GATTTTGAAGTCCCGAGC 2812 2829 sooossssssssoooss 5-7-6 2047
664531 135850 135867 TGATTTTGAAGTCCCGAG 2813 2830 sooosssssssssooss 5-8-5 2482
664744 135850 135868 CT GATTTTGAAGTC CC GAG 2813 2831 sooossssssssssooss 5-9-5 2543
664691 135850 135869 ACTGATTTTGAAGTCCCGAG 2813 2832 sooosssssssssssooss 5-10-5 2544
664797 135850 135867 TGATTTTGAAGTCCCGAG 2813 2830 sooossssssssoooss 5-7-6 2482
664532 135851 135868 CTGATTTTGAAGTC CC GA 2814 2831 sooosssssssssooss 5-8-5 2483
664745 135851 135869 ACTGATTTTGAAGTCCCGA 2814 2832 sooossssssssssooss 5-9-5 2545
664692 135851 135870 CACTGATTTTGAAGTCCCGA 2814 2833 sooosssssssssssooss 5-10-5 2546
664798 135851 135868 CTGATTTTGAAGTC CC GA 2814 2831 sooossssssssoooss 5-7-6 2483
622119 135852 135869 ACTGATTTTGAAGTCCCG 2815 2832 sooosssssssssooss 5-8-5 2048
664746 135852 135870 CACTGATTTTGAAGTCCCG 2815 2833 sooossssssssssooss 5-9-5 2547
664693 135852 135871 TC ACTGATTTTGAAGTC CC G 2815 2834 sooosssssssssssooss 5-10-5 2548
664799 135852 135869 ACTGATTTTGAAGTCCCG 2815 2832 sooossssssssoooss 5-7-6 2549
664533 135853 135870 CACTGATTTTGAAGTCCC 2816 2833 sooosssssssssooss 5-8-5 2370
664747 135853 135871 TCACTGATTTTGAAGTCCC 2816 2834 sooossssssssssooss 5-9-5 2371
664694 135853 135872 ATCACTGATTTTGAAGTCCC 1649 1668 sooosssssssssssooss 5-10-5 57
664800 135853 135870 CACTGATTTTGAAGTCCC 2816 2833 sooossssssssoooss 5-7-6 2370
664534 135854 135871 TCACTGATTTTGAAGTCC 2817 2834 sooosssssssssooss 5-8-5 2372
664748 135854 135872 ATCACTGATTTTGAAGTCC 2817 2835 sooossssssssssooss 5-9-5 2373
664695 135854 135873 CATCACTGATTTTGAAGTCC 2817 2836 sooosssssssssssooss 5-10-5 2374
664801 135854 135871 TCACTGATTTTGAAGTCC 2817 2834 sooossssssssoooss 5-7-6 2372
622120 135855 135872 ATCACTGATTTTGAAGTC 2818 2835 sooosssssssssooss 5-8-5 1668
664749 135855 135873 CATCACTGATTTTGAAGTC 2818 2836 sooossssssssssooss 5-9-5 2375
664696 135855 135874 CCATCACTGATTTTGAAGTC 2818 2837 sooosssssssssssooss 5-10-5 2376
664802 135855 135872 ATCACTGATTTTGAAGTC 2818 2835 sooossssssssoooss 5-7-6 1668
664535 135856 135873 CATCACTGATTTTGAAGT 2819 2836 sooosssssssssooss 5-8-5 2377
664750 135856 135874 CC ATC ACT GATTTT GAAGT 2819 2837 sooossssssssssooss 5-9-5 2378
664697 135856 135875 CCCATCACTGATTTTGAAGT 2819 2838 sooosssssssssssooss 5-10-5 2379
664803 135856 135873 CATCACTGATTTTGAAGT 2819 2836 sooossssssssoooss 5-7-6 2377
664536 135857 135874 CCATCACTGATTTTGAAG 2820 2837 sooosssssssssooss 5-8-5 2380
664751 135857 135875 CCCATCACTGATTTTGAAG 2820 2838 sooossssssssssooss 5-9-5 2381 664698 135857 135876 TCCCATCACTGATTTTGAAG 2820 2839 sooosssssssssssooss 5-10-5 2382
664804 135857 135874 CCATCACTGATTTTGAAG 2820 2837 sooossssssssoooss 5-7-6 2380
622121 135858 135875 CCCATCACTGATTTTGAA 2821 2838 sooosssssssssooss 5-8-5 1669
664752 135858 135876 TCCCATCACTGATTTTGAA 2821 2839 sooossssssssssooss 5-9-5 2383
664699 135858 135877 CTCCCATCACTGATTTTGAA 2821 2840 sooosssssssssssooss 5-10-5 2384
664805 135858 135875 CCCATCACTGATTTTGAA 2821 2838 sooossssssssoooss 5-7-6 1669
664537 135859 135876 TCCCATCACTGATTTTGA 2822 2839 sooosssssssssooss 5-8-5 2385
664753 135859 135877 CTCCCATCACTGATTTTGA 2822 2840 sooossssssssssooss 5-9-5 2386
664700 135859 135878 ACTCCCATCACTGATTTTGA 2822 2841 sooosssssssssssooss 5-10-5 2387
664806 135859 135876 TCCCATCACTGATTTTGA 2822 2839 sooossssssssoooss 5-7-6 2385
664538 135860 135877 CTCCCATCACTGATTTTG 2823 2840 sooosssssssssooss 5-8-5 2388
664754 135860 135878 ACTCCCATCACTGATTTTG 2823 2841 sooossssssssssooss 5-9-5 2389
664701 135860 135879 TACTCCCATCACTGATTTTG 2823 2842 sooosssssssssssooss 5-10-5 2390
664807 135860 135877 CTCCCATCACTGATTTTG 2823 2840 sooossssssssoooss 5-7-6 2388
622122 135861 135878 ACTCCCATCACTGATTTT 2824 2841 sooosssssssssooss 5-8-5 1670
664755 135861 135879 TACT CC CAT C ACT GATTTT 2824 2842 sooossssssssssooss 5-9-5 2391
664702 135861 135880 TT ACTCCCATCACTGATTTT 2824 2843 sooosssssssssssooss 5-10-5 2392
664808 135861 135878 ACTCCCATCACTGATTTT 2824 2841 sooossssssssoooss 5-7-6 1670
664539 135862 135879 TACTCCCATCACTGATTT 2825 2842 sooosssssssssooss 5-8-5 2393
664756 135862 135880 TTACTCCCATCACTGATTT 2825 2843 sooossssssssssooss 5-9-5 2394
664703 135862 135881 CTTACTCCCATCACTGATTT 2825 2844 sooosssssssssssooss 5-10-5 2395
664809 135862 135879 TACTCCCATCACTGATTT 2825 2842 sooossssssssoooss 5-7-6 2393
664540 135863 135880 TT ACT CC CAT C ACT GATT 2826 2843 sooosssssssssooss 5-8-5 2396
664757 135863 135881 CTTACTCCCATCACTGATT 2826 2844 sooossssssssssooss 5-9-5 2397
664704 135863 135882 TCTTACTCCCATCACTGATT 2826 2845 sooosssssssssssooss 5-10-5 2398
664810 135863 135880 TT ACT CC CAT C ACT GATT 2826 2843 sooossssssssoooss 5-7-6 2396
622123 135864 135881 CTTACTCCCATCACTGAT 2827 2844 sooosssssssssooss 5-8-5 1671
664758 135864 135882 TCTTACTCCCATCACTGAT 2827 2845 sooossssssssssooss 5-9-5 2399
664705 135864 135883 CTCTTACTCCCATCACTGAT 2827 2846 sooosssssssssssooss 5-10-5 2400
664811 135864 135881 CTTACTCCCATCACTGAT 2827 2844 sooossssssssoooss 5-7-6 1671
664541 135865 135882 TCTTACTCCCATCACTGA 2828 2845 sooosssssssssooss 5-8-5 2401
664759 135865 135883 CTCTTACTCCCATCACTGA 2828 2846 sooossssssssssooss 5-9-5 2402
664706 135865 135884 GCTCTTACTCCCATCACTGA 2828 2847 sooosssssssssssooss 5-10-5 2403
664812 135865 135882 TCTTACTCCCATCACTGA 2828 2845 sooossssssssoooss 5-7-6 2401
664542 135866 135883 CTCTTACTCCCATCACTG 2829 2846 sooosssssssssooss 5-8-5 2404
664760 135866 135884 GCTCTTACTCCCATCACTG 2829 2847 sooossssssssssooss 5-9-5 2405
664707 135866 135885 TGCTCTTACTCCCATCACTG 2829 2848 sooosssssssssssooss 5-10-5 2406 664813 135866 135883 CTCTTACTCCCATCACTG 2829 2846 sooossssssssoooss 5-7-6 2404
622124 135867 135884 GCTCTTACTCCCATCACT 2830 2847 sooosssssssssooss 5-8-5 1672
664761 135867 135885 TGCTCTTACTCCCATCACT 2830 2848 sooossssssssssooss 5-9-5 2407
664708 135867 135886 TTGCTCTTACTCCCATCACT 2830 2849 sooosssssssssssooss 5-10-5 2408
664814 135867 135884 GCTCTTACTCCCATCACT 2830 2847 sooossssssssoooss 5-7-6 1672
623993 135868 135885 TGCTCTTACTCCCATCAC 2831 2848 sooosssssssssooss 5-8-5 2306
664762 135868 135886 TTGCTCTTACTCCCATCAC 2831 2849 sooossssssssssooss 5-9-5 2409
664709 135868 135887 TTTGCTCTTACTCCCATCAC 2831 2850 sooosssssssssssooss 5-10-5 2410
664815 135868 135885 TGCTCTTACTCCCATCAC 2831 2848 sooossssssssoooss 5-7-6 2306
623994 135869 135886 TTGCTCTTACTCCCATCA 2832 2849 sooosssssssssooss 5-8-5 2307
664763 135869 135887 TTTGCTCTT ACT CC CATC A 2832 2850 sooossssssssssooss 5-9-5 2411
664710 135869 135888 ATTTGCTCTTACTCCCATCA 2832 2851 sooosssssssssssooss 5-10-5 2412
664816 135869 135886 TTGCTCTTACTCCCATCA 2832 2849 sooossssssssoooss 5-7-6 2307
622125 135870 135887 TTTGCTCTTACTCCCATC 2833 2850 sooosssssssssooss 5-8-5 1673
664764 135870 135888 ATTTGCTCTTACTCCCATC 2833 2851 sooossssssssssooss 5-9-5 2413
664711 135870 135889 AATTTGCTCTTACTCCCATC 2833 2852 sooosssssssssssooss 5-10-5 2414
664817 135870 135887 TTTGCTCTTACTCCCATC 2833 2850 sooossssssssoooss 5-7-6 1673
623995 135871 135888 ATTTGCTCTTACTCCCAT 2834 2851 sooosssssssssooss 5-8-5 2308
664765 135871 135889 AATTTGCTCTTACTCCCAT 2834 2852 sooossssssssssooss 5-9-5 2415
664712 135871 135890 AAATTTGCT CTT ACT CC CAT 2834 2853 sooosssssssssssooss 5-10-5 2416
664818 135871 135888 ATTTGCTCTTACTCCCAT 2834 2851 sooossssssssoooss 5-7-6 2308
623996 135872 135889 AATTTGCTCTTACTCCCA 2835 2852 sooosssssssssooss 5-8-5 2309
664766 135872 135890 AAATTTGCTCTTACTCCCA 2835 2853 sooossssssssssooss 5-9-5 2417
664713 135872 135891 GAAATTTGCTCTTACTCCCA 2835 2854 sooosssssssssssooss 5-10-5 2418
664819 135872 135889 AATTTGCTCTTACTCCCA 2835 2852 sooossssssssoooss 5-7-6 2309
Example 15: Intracerebroventricular administration of antisense oligonucleotides against human tau mR A in htau mice
5 Selected compounds were tested for efficacy by ICV administration in human tau transgenic mice
(Duff et al., Neurobiology of Disease 7:87-98, 2000).
Treatment and surgery
Groups of 4 mice each were administered ISIS 613255, ISIS 613329, ISIS 613344, ISIS 613361 ,0 ISIS 613369, ISIS 613370, ISIS 613397, ISIS 613045, ISIS 613099, ISIS 6131 18, ISIS 613136 with a 200 μg dose delivered by ICV bolus injection. A control group of 2 mice was similarly treated with ISIS 424880 and a control group of 4 mice was similarly treated with PBS. All procedures were performed under isoflourane anesthesia and in accordance with IACUC regulations. For mouse ICV bolus injections, the antisense oligonucleotide was injected into the right lateral ventricle of human tau transgenic mice. Ten microliters of solution containing 300 μg of oligonucleotide in PBS was injected over approximately 10 seconds. Tissue was collected 14 days after oligonucleotide administration.
RNA Analysis
On day 14 after the oligonucleotide administration, RNA was extracted from the hippocampus, spinal cord and cortex for real-time PCR analysis of tau iriRNA levels. Human tau mRNA levels were measured using the human primer probe set RTS3104. Results were calculated as percent inhibition of human tau mRNA expression compared to the control. All the antisense oligonucleotides effect significant inhibition of human tau mRNA levels.
Table 57
Percent reduction of human tau mRNA levels in hTau mice
Figure imgf000142_0001
Example 16: Antisense inhibition of human Tau in SH-SY5Y cells 5-7-6 MOE, 5-8-5 MOE, 5-9-5 MOE, and 5-10-5 MOE gapmers
The antisense oligonucleotides described in the Examples above, as well as newly designed antisense oligonucleotides targeting a human tau nucleic acid, were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured SH-SY5Y cells were transfected using electroporation with 8,000 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 5-7-6 MOE, 5-8-5 MOE, 5-9-5 MOE, or 5-10-5 MOE gapmers. The 5-7-6 MOE gapmers are 18 nucleosides in 5 length, wherein the central gap segment comprises of seven 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five and six nucleosides respectively. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises of eight - deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. The 5-9-5 MOE gapmers are 19 nucleosides in length, wherein the central gap segment 0 comprises of nine 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3'
direction comprising five nucleosides each. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises often 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside linkages throughout 5 each gapmer are either phosphorothioate linkages or phosphodiester linkages. The 'Linkage Chemistry' column describes the internucleoside linkages of each oligonucleotide, 's' indicates phosphorothioate linkage and Ό' indicates phosphodiester linkage. All cytosine residues throughout each gapmer are 5- methylcytosines.
"Start site" indicates the 5'-most nucleoside to which the gapmer is targeted in the human gene 0 sequence. "Stop site" indicates the 3'-most nucleoside to which the gapmer is targeted in the human gene sequence. Each gapmer listed in the Table below is targeted to either the human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000) or to the human tau mRNA sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NM 001123066.3). 'n a' indicates that the antisense oligonucleotide does not target that 5 particular gene sequence with 100% complementarity.
Table 58
Figure imgf000143_0001
664768 135821 135838 TGACAAAAGCAGGTTAAG 2784 2801 sooossssssssoooss 5-7-6 30 2334
622109 135822 135839 GTGACAAAAGCAGGTTAA 2785 2802 sooosssssssssooss 5-8-5 72 2038
664769 135822 135839 GTGACAAAAGCAGGTTAA 2785 2802 sooossssssssoooss 5-7-6 49 2038
664513 135823 135840 AGTGACAAAAGCAGGTTA 2786 2803 sooosssssssssooss 5-8-5 56 2339
664770 135823 135840 AGTGACAAAAGCAGGTTA 2786 2803 sooossssssssoooss 5-7-6 45 2339
664514 135824 135841 GAGTGACAAAAGCAGGTT 2787 2804 sooosssssssssooss 5-8-5 84 2342
664771 135824 135841 GAGTGACAAAAGCAGGTT 2787 2804 sooossssssssoooss 5-7-6 63 2342
622110 135825 135842 CGAGTGACAAAAGCAGGT 2788 2805 sooosssssssssooss 5-8-5 77 2039
664772 135825 135842 CGAGTGACAAAAGCAGGT 2788 2805 sooossssssssoooss 5-7-6 37 2039
664515 135826 135843 CCGAGTGACAAAAGCAGG 2789 2806 sooosssssssssooss 5-8-5 70 2347
664773 135826 135843 CCGAGTGACAAAAGCAGG 2789 2806 sooossssssssoooss 5-7-6 62 2347
664516 135827 135844 GCCGAGTGACAAAAGCAG 2790 2807 sooosssssssssooss 5-8-5 79 2350
664774 135827 135844 GCCGAGTGACAAAAGCAG 2790 2807 sooossssssssoooss 5-7-6 70 2350
622111 135828 135845 AGCCGAGTGACAAAAGCA 2791 2808 sooosssssssssooss 5-8-5 75 2040
664775 135828 135845 AGCCGAGTGACAAAAGCA 2791 2808 sooossssssssoooss 5-7-6 78 2040
664517 135829 135846 AAGCCGAGTGACAAAAGC 2792 2809 sooosssssssssooss 5-8-5 67 2355
664776 135829 135846 AAGCCGAGTGACAAAAGC 2792 2809 sooossssssssoooss 5-7-6 50 2355
664518 135830 135847 AAAGCCGAGTGACAAAAG 2793 2810 sooosssssssssooss 5-8-5 47 2358
664777 135830 135847 AAAGCCGAGTGACAAAAG 2793 2810 sooossssssssoooss 5-7-6 42 2358
622112 135831 135848 CAAAGCCGAGTGACAAAA 2794 2811 sooosssssssssooss 5-8-5 46 2041
664778 135831 135848 CAAAGCCGAGTGACAAAA 2794 2811 sooossssssssoooss 5-7-6 34 2041
664519 135832 135849 CCAAAGCCGAGTGACAAA 2795 2812 sooosssssssssooss 5-8-5 57 2363
664779 135832 135849 CCAAAGCCGAGTGACAAA 2795 2812 sooossssssssoooss 5-7-6 34 2363
664520 135833 135850 GCCAAAGCCGAGTGACAA 2796 2813 sooosssssssssooss 5-8-5 63 2366
664780 135833 135850 GCCAAAGCCGAGTGACAA 2796 2813 sooossssssssoooss 5-7-6 66 2366
622113 135834 135851 AGCCAAAGCCGAGTGACA 2797 2814 sooosssssssssooss 5-8-5 70 2042
664781 135834 135851 AGCCAAAGCCGAGTGACA 2797 2814 sooossssssssoooss 5-7-6 74 2042
664521 135835 135852 GAGCCAAAGCCGAGTGAC 2798 2815 sooosssssssssooss 5-8-5 71 2420
664782 135835 135852 GAGCCAAAGCCGAGTGAC 2798 2815 sooossssssssoooss 5-7-6 31 2420
664522 135836 135853 CGAGCCAAAGCCGAGTGA 2799 2816 sooosssssssssooss 5-8-5 50 2423
664783 135836 135853 CGAGCCAAAGCCGAGTGA 2799 2816 sooossssssssoooss 5-7-6 30 2423
622114 135837 135854 CCGAGCCAAAGCCGAGTG 2800 2817 sooosssssssssooss 5-8-5 72 2043
664784 135837 135854 CCGAGCCAAAGCCGAGTG 2800 2817 sooossssssssoooss 5-7-6 76 2043
664523 135838 135855 CCCGAGCCAAAGCCGAGT 2801 2818 sooosssssssssooss 5-8-5 70 2428
664785 135838 135855 CCCGAGCCAAAGCCGAGT 2801 2818 sooossssssssoooss 5-7-6 56 2428
664524 135839 135856 TCCCGAGCCAAAGCCGAG 2802 2819 sooosssssssssooss 5-8-5 55 2431
664786 135839 135856 TCCCGAGCCAAAGCCGAG 2802 2819 sooossssssssoooss 5-7-6 36 2431
622115 135840 135857 GTCCCGAGCCAAAGCCGA 2803 2820 sooosssssssssooss 5-8-5 59 2044
664787 135840 135857 GTCCCGAGCCAAAGCCGA 2803 2820 sooossssssssoooss 5-7-6 54 2044
664525 135841 135858 AGTCCCGAGCCAAAGCCG 2804 2821 sooosssssssssooss 5-8-5 74 2436
664788 135841 135858 AGTCCCGAGCCAAAGCCG 2804 2821 sooossssssssoooss 5-7-6 77 2436
664789 135842 135859 AAGTCCCGAGCCAAAGCC 2805 2822 sooossssssssoooss 5-7-6 42 2439
664790 135843 135860 GAAGTCCCGAGCCAAAGC 2806 2823 sooossssssssoooss 5-7-6 38 2045
664791 135844 135861 TGAAGTCCCGAGCCAAAG 2807 2824 sooossssssssoooss 5-7-6 44 2478
664792 135845 135862 TTGAAGTCCCGAGCCAAA 2808 2825 sooossssssssoooss 5-7-6 44 2479
664793 135846 135863 TTTGAAGTCCCGAGCCAA 2809 2826 sooossssssssoooss 5-7-6 29 2046 664794 135847 135864 TTTTGAAGTCCCGAGCCA 2810 2827 sooossssssssoooss 5-7-6 32 2480
664795 135848 135865 ATTTTGAAGTCCCGAGCC 2811 2828 sooossssssssoooss 5-7-6 15 2481
664796 135849 135866 GATTTTGAAGTCCCGAGC 2812 2829 sooossssssssoooss 5-7-6 76 2047
664797 135850 135867 TGATTTTGAAGTCCCGAG 2813 2830 sooossssssssoooss 5-7-6 70 2482
664798 135851 135868 CTGATTTTGAAGTCCCGA 2814 2831 sooossssssssoooss 5-7-6 75 2483
664799 135852 135869 ACTGATTTTGAAGTCCCG 2815 2832 sooossssssssoooss 5-7-6 79 2048
664800 135853 135870 CACTGATTTTGAAGTCCC 2816 2833 sooossssssssoooss 5-7-6 82 2370
664534 135854 135871 TCACTGATTTTGAAGTCC 2817 2834 sooosssssssssooss 5-8-5 78 2372
664801 135854 135871 TCACTGATTTTGAAGTCC 2817 2834 sooossssssssoooss 5-7-6 29 2372
664802 135855 135872 ATCACTGATTTTGAAGTC 2818 2835 sooossssssssoooss 5-7-6 52 1668
664803 135856 135873 CATCACTGATTTTGAAGT 2819 2836 sooossssssssoooss 5-7-6 55 2377
664804 135857 135874 CCATCACTGATTTTGAAG 2820 2837 sooossssssssoooss 5-7-6 38 2380
664805 135858 135875 CCCATCACTGATTTTGAA 2821 2838 sooossssssssoooss 5-7-6 62 1669
664806 135859 135876 TCCCATCACTGATTTTGA 2822 2839 sooossssssssoooss 5-7-6 72 2385
664807 135860 135877 CTCCCATCACTGATTTTG 2823 2840 sooossssssssoooss 5-7-6 55 2388
664808 135861 135878 ACTCCCATCACTGATTTT 2824 2841 sooossssssssoooss 5-7-6 38 1670
664809 135862 135879 TACTCCCATCACTGATTT 2825 2842 sooossssssssoooss 5-7-6 54 2393
664810 135863 135880 TTACTCCCATCACTGATT 2826 2843 sooossssssssoooss 5-7-6 39 2396
664811 135864 135881 CTTACTCCCATCACTGAT 2827 2844 sooossssssssoooss 5-7-6 39 1671
664812 135865 135882 TCTTACTCCCATCACTGA 2828 2845 sooossssssssoooss 5-7-6 45 2401
664813 135866 135883 CTCTTACTCCCATCACTG 2829 2846 sooossssssssoooss 5-7-6 61 2404
664814 135867 135884 GCTCTTACTCCCATCACT 2830 2847 sooossssssssoooss 5-7-6 14 1672
664815 135868 135885 TGCTCTTACTCCCATCAC 2831 2848 sooossssssssoooss 5-7-6 70 2306
664816 135869 135886 TTGCTCTTACTCCCATCA 2832 2849 sooossssssssoooss 5-7-6 72 2307
664817 135870 135887 TTTGCTCTTACTCCCATC 2833 2850 sooossssssssoooss 5-7-6 62 1673
664818 135871 135888 ATTTGCTCTTACTCCCAT 2834 2851 sooossssssssoooss 5-7-6 69 2308
664819 135872 135889 AATTTGCTCTTACTCCCA 2835 2852 sooossssssssoooss 5-7-6 67 2309
Table 59
Figure imgf000145_0001
665054 33938 33955 CTGGCACAACAGGCATTG n/a n a sooosssssssssooss 5-8-5 25 2453
621041 35721 35738 TTGCCATCTTGGACAGGG n a n/a sooosssssssssooss 5-8-5 70 896
665055 35746 35763 GTGGTACAGAGGCTGTGT n a n a sooosssssssssooss 5-8-5 47 2454
665056 35771 35788 TTGCAGATTCTGAATACC n/a n a sooosssssssssooss 5-8-5 78 2455
665057 64499 64516 TATGATTCCCAGGAGTCT n/a n a sooosssssssssooss 5-8-5 0 2456
621181 64543 64560 GTAGGTGTCATCATCATC n/a n a sooosssssssssooss 5-8-5 77 1113
665058 64568 64585 GTGACCCTTCAGGGCAAT n/a n a sooosssssssssooss 5-8-5 35 2457
665059 64593 64610 ATGCAGGTGCCTTGCAGG n/a n a sooosssssssssooss 5-8-5 36 2458
623965 73880 73897 TGATCTTCCATCACTTCG n/a n a sooosssssssssooss 5-8-5 48 2278
665060 77364 77381 CCTGCTGCCAACAGTAGA n/a n a sooosssssssssooss 5-8-5 51 2459
665061 77389 77406 CCCTCACGGGCTGTTGTG n/a n a sooosssssssssooss 5-8-5 20 2460
621236 77414 77431 TCATCTGTGAAGCGGACG 73377 73394 sooosssssssssooss 5-8-5 79 706
665062 77439 77456 ACGGTCTGAGCATGAGGC n/a n a sooosssssssssooss 5-8-5 77 2461
665063 77464 77481 AGCCATGGACCTGCTCTG n/a n/a sooosssssssssooss 5-8-5 48 2462
665064 77731 77748 GACAGTATACCCCACATC n/a n a sooosssssssssooss 5-8-5 48 2463
621238 77781 77798 GTTATTTTGGAACAGTTT 73744 73761 sooosssssssssooss 5-8-5 89 708
665065 78142 78159 AAGAGTGGTAAAACCTAC n/a n a sooosssssssssooss 5-8-5 51 2464
665066 80259 80276 GCTCACCAGCAGGAACTT n/a n a sooosssssssssooss 5-8-5 56 2465
665067 80284 80301 AAGGGCCCAGTGTAGCAC n/a n a sooosssssssssooss 5-8-5 66 2466
621251 80309 80326 AGTCATTATCATGTCACC 76272 76289 sooosssssssssooss 5-8-5 73 721
665068 80334 80351 CCAATAAGTGGCAGTGAT n/a n a sooosssssssssooss 5-8-5 49 2467
665069 80359 80376 ACCATGCCTGGCAGATGA n/a n/a sooosssssssssooss 5-8-5 66 2468
665070 80799 80816 CTTAGTCATCTTCCCTCA n/a n/a sooosssssssssooss 5-8-5 64 2469
665071 80824 80841 ATTTCTGAATTCTTTGCC n/a n a sooosssssssssooss 5-8-5 69 2470
621254 80849 80866 GGTAACATGTAAAGCTTC 76812 76829 sooosssssssssooss 5-8-5 76 724
665072 82508 82525 CTTACACTCTGGAAGGTT n/a n a sooosssssssssooss 5-8-5 74 2471
665073 82533 82550 TATCCAGGGCTGAGCAGG n/a n a sooosssssssssooss 5-8-5 38 2472
621263 82558 82575 ATGGGCTTATCAATGCAT 78521 78538 sooosssssssssooss 5-8-5 80 733
665074 82583 82600 GGAGTCAATCTGCCCTGG n/a n a sooosssssssssooss 5-8-5 54 2473
665075 82608 82625 CTTCCCTGTGGCACTTTG n a n/a sooosssssssssooss 5-8-5 29 2474
665076 89600 89617 ATGCCTCACTCAACAAGG n/a n a sooosssssssssooss 5-8-5 26 2475
665077 89625 89642 CTCCACTTTGGGACCAGG n/a n a sooosssssssssooss 5-8-5 87 2476
621302 89650 89667 GCTATGACCTAGTAGGAA n/a n a sooosssssssssooss 5-8-5 68 772
665078 89675 89692 GGCACAGCACCCACATGC n/a n a sooosssssssssooss 5-8-5 66 2477
665079 89700 89717 GGTGATAAAAGTTCACTA n/a n a sooosssssssssooss 5-8-5 63 2531
98891 98908
620887 GTTTTCAAACACACCTTC n/a n a sooosssssssssooss 5-8-5 91 665
98928 98945
664526 135842 135859 AAGTCCCGAGCCAAAGCC 2805 2822 sooosssssssssooss 5-8-5 55 2439
622116 135843 135860 GAAGTCCCGAGCCAAAGC 2806 2823 sooosssssssssooss 5-8-5 63 2045
664527 135844 135861 TGAAGTCCCGAGCCAAAG 2807 2824 sooosssssssssooss 5-8-5 40 2478
664528 135845 135862 TTGAAGTCCCGAGCCAAA 2808 2825 sooosssssssssooss 5-8-5 61 2479
622117 135846 135863 TTTGAAGTCCCGAGCCAA 2809 2826 sooosssssssssooss 5-8-5 55 2046
664529 135847 135864 TTTTGAAGTCCCGAGCCA 2810 2827 sooosssssssssooss 5-8-5 25 2480 664530 135848 135865 ATTTTGAAGTCCCGAGCC 2811 2828 sooosssssssssooss 5-8-5 38 2481
622118 135849 135866 GATTTTGAAGTCCCGAGC 2812 2829 sooosssssssssooss 5-8-5 63 2047
664531 135850 135867 TGATTTTGAAGTCCCGAG 2813 2830 sooosssssssssooss 5-8-5 66 2482
664532 135851 135868 CTGATTTTGAAGTCCCGA 2814 2831 sooosssssssssooss 5-8-5 73 2483
622119 135852 135869 ACTGATTTTGAAGTCCCG 2815 2832 sooosssssssssooss 5-8-5 66 2048
664533 135853 135870 CACTGATTTTGAAGTCCC 2816 2833 sooosssssssssooss 5-8-5 72 2370
664534 135854 135871 TCACTGATTTTGAAGTCC 2817 2834 sooosssssssssooss 5-8-5 61 2372
622120 135855 135872 ATCACTGATTTTGAAGTC 2818 2835 sooosssssssssooss 5-8-5 47 1668
664535 135856 135873 CATCACTGATTTTGAAGT 2819 2836 sooosssssssssooss 5-8-5 59 2377
664536 135857 135874 CCATCACTGATTTTGAAG 2820 2837 sooosssssssssooss 5-8-5 34 2380
622121 135858 135875 CCCATCACTGATTTTGAA 2821 2838 sooosssssssssooss 5-8-5 49 1669
664537 135859 135876 TCCCATCACTGATTTTGA 2822 2839 sooosssssssssooss 5-8-5 61 2385
664538 135860 135877 CTCCCATCACTGATTTTG 2823 2840 sooosssssssssooss 5-8-5 76 2388
622122 135861 135878 ACTCCCATCACTGATTTT 2824 2841 sooosssssssssooss 5-8-5 38 1670
664539 135862 135879 TACTCCCATCACTGATTT 2825 2842 sooosssssssssooss 5-8-5 57 2393
664540 135863 135880 TTACTCCCATCACTGATT 2826 2843 sooosssssssssooss 5-8-5 32 2396
622123 135864 135881 CTTACTCCCATCACTGAT 2827 2844 sooosssssssssooss 5-8-5 33 1671
664541 135865 135882 TCTTACTCCCATCACTGA 2828 2845 sooosssssssssooss 5-8-5 50 2401
664542 135866 135883 CTCTTACTCCCATCACTG 2829 2846 sooosssssssssooss 5-8-5 58 2404
622124 135867 135884 GCTCTTACTCCCATCACT 2830 2847 sooosssssssssooss 5-8-5 64 1672
623993 135868 135885 TGCTCTTACTCCCATCAC n a n/a sooosssssssssooss 5-8-5 65 2306
623994 135869 135886 TTGCTCTTACTCCCATCA n a n/a sooosssssssssooss 5-8-5 63 2307
622125 135870 135887 TTTGCTCTTACTCCCATC 2833 2850 sooosssssssssooss 5-8-5 75 1673
623995 135871 135888 ATTTGCTCTTACTCCCAT n a n/a sooosssssssssooss 5-8-5 60 2308
623996 135872 135889 AATTTGCTCTTACTCCCA n/a n/a sooosssssssssooss 5-8-5 72 2309
Table 60
SEQ
SEQ ID
SEQ ID SEQ ID ID SEQ
ISIS NO: %
NO: 1 NO: 1 Sequence NO: Linkage Chemistry Motif ID NO 2 inhibition
start site stop site 2 stop NO start
site
site
665043 73879 73897 TGATCTTCCATCACTTCGA 345 364 sooossssssssssooss 5-9-5 67 2484
98890 98908
665044 GTTTTCAAACACACCTTCA n/a n/a sooossssssssssooss 5-9-5 90 2485
98927 98945
665081 120039 120057 TTTTCTTACCACCCTAACA n/a n/a sooossssssssssooss 5-9-5 28 2486
665082 120041 120059 CGTTTTCTTACCACCCTAA n/a n/a sooossssssssssooss 5-9-5 86 2487
665083 120045 120063 AAACCGTTTTCTTACCACC n/a n/a sooossssssssssooss 5-9-5 86 2488
665084 120047 120065 AAAAACCGTTTTCTTACCA n/a n/a sooossssssssssooss 5-9-5 65 2489
665085 120060 120078 AGCTCATCAAAGCAAAAAC n/a n/a sooossssssssssooss 5-9-5 65 2490
665086 1201 10 120128 TCAAAAGACTATGTATTTT n/a n/a sooossssssssssooss 5-9-5 54 2491
665087 120389 120407 TGTAAATAATTGCCAAGTG n/a n/a sooossssssssssooss 5-9-5 61 2492
665088 120439 120457 TAAGCCACCATGCCTGTAA n/a n/a sooossssssssssooss 5-9-5 61 2493
665089 12171 1 121729 TGGACCCGCCTACTTGCTC n/a n/a sooossssssssssooss 5-9-5 85 2494 665090 121736 121754 TTTCGATGAGTGACATGCG n/a n/a sooossssssssssooss 5-9-5 50 2495
665091 121761 121779 TGCTTGCTCGCAAGGACGC n/a n a sooossssssssssooss 5-9-5 80 2496
665092 121765 121783 CGCCTGCTTGCTCGCAAGG n/a n/a sooossssssssssooss 5-9-5 82 2497
665093 121767 121785 CCCGCCTGCTTGCTCGCAA n/a n/a sooossssssssssooss 5-9-5 92 2498
665094 121769 121787 GACCCGCCTGCTTGCTCGC n/a n/a sooossssssssssooss 5-9-5 91 2499
665095 121771 121789 TGGACCCGCCTGCTTGCTC n/a n/a sooossssssssssooss 5-9-5 83 2500
665096 121773 121791 CCTGGACCCGCCTGCTTGC n/a n/a sooossssssssssooss 5-9-5 88 2501
665097 121775 121793 ACCCTGGACCCGCCTGCTT n/a n/a sooossssssssssooss 5-9-5 60 2502
665098 121786 121804 AGTGACACGCCACCCTGGA n/a n/a sooossssssssssooss 5-9-5 62 2503
665099 12181 1 121829 CCTTTGGTAGCCAGAAAAA n/a n/a sooossssssssssooss 5-9-5 53 2504
665100 121817 121835 TCTGCACCTTTGGTAGCCA n/a n/a sooossssssssssooss 5-9-5 84 2505
665101 121938 121956 ACAGCACGGCGCATGGGAC n/a n/a sooossssssssssooss 5-9-5 54 2506
665102 121940 121958 CCACAGCACGGCGCATGGG n/a n/a sooossssssssssooss 5-9-5 84 2507
665103 121942 121960 AGCCACAGCACGGCGCATG n/a n/a sooossssssssssooss 5-9-5 85 2508
664714 135820 135838 TGACAAAAGCAGGTTAAGT 2783 2801 sooossssssssssooss 5-9-5 54 2332
664715 135821 135839 GTGACAAAAGCAGGTTAAG 2784 2802 sooossssssssssooss 5-9-5 65 2335
664716 135822 135840 AGTGACAAAAGCAGGTTAA 2785 2803 sooossssssssssooss 5-9-5 79 2337
664717 135823 135841 GAGTGACAAAAGCAGGTTA 2786 2804 sooossssssssssooss 5-9-5 70 2340
664718 135824 135842 CGAGTGACAAAAGCAGGTT 2787 2805 sooossssssssssooss 5-9-5 89 2343
664719 135825 135843 CCGAGTGACAAAAGCAGGT 2788 2806 sooossssssssssooss 5-9-5 84 2345
664720 135826 135844 GCCGAGTGACAAAAGCAGG 2789 2807 sooossssssssssooss 5-9-5 71 2348
664721 135827 135845 AGCCGAGTGACAAAAGCAG 2790 2808 sooossssssssssooss 5-9-5 81 2351
664722 135828 135846 AAGCCGAGTGACAAAAGCA 2791 2809 sooossssssssssooss 5-9-5 68 2353
664723 135829 135847 AAAGCCGAGTGACAAAAGC 2792 2810 sooossssssssssooss 5-9-5 51 2356
664724 135830 135848 CAAAGCCGAGTGACAAAAG 2793 281 1 sooossssssssssooss 5-9-5 47 2359
664725 135831 135849 CCAAAGCCGAGTGACAAAA 2794 2812 sooossssssssssooss 5-9-5 69 2361
664726 135832 135850 GCCAAAGCCGAGTGACAAA 2795 2813 sooossssssssssooss 5-9-5 76 2364
664727 135833 135851 AGCCAAAGCCGAGTGACAA 2796 2814 sooossssssssssooss 5-9-5 64 2367
664728 135834 135852 GAGCCAAAGCCGAGTGACA 2797 2815 sooossssssssssooss 5-9-5 78 2369
664729 135835 135853 CGAGCCAAAGCCGAGTGAC 2798 2816 sooossssssssssooss 5-9-5 74 2421
664730 135836 135854 CCGAGCCAAAGCCGAGTGA 2799 2817 sooossssssssssooss 5-9-5 76 2424
664731 135837 135855 CCCGAGCCAAAGCCGAGTG 2800 2818 sooossssssssssooss 5-9-5 75 2426
664732 135838 135856 TCCCGAGCCAAAGCCGAGT 2801 2819 sooossssssssssooss 5-9-5 80 2429
664733 135839 135857 GTCCCGAGCCAAAGCCGAG 2802 2820 sooossssssssssooss 5-9-5 70 2432
664734 135840 135858 AGTCCCGAGCCAAAGCCGA 2803 2821 sooossssssssssooss 5-9-5 59 2434
664735 135841 135859 AAGTCCCGAGCCAAAGCCG 2804 2822 sooossssssssssooss 5-9-5 58 2437
664736 135842 135860 GAAGTCCCGAGCCAAAGCC 2805 2823 sooossssssssssooss 5-9-5 76 2440
664737 135843 135861 TGAAGTCCCGAGCCAAAGC 2806 2824 sooossssssssssooss 5-9-5 46 2442
664738 135844 135862 TTGAAGTCCCGAGCCAAAG 2807 2825 sooossssssssssooss 5-9-5 46 2550
664739 135845 135863 TTTGAAGTCCCGAGCCAAA 2808 2826 sooossssssssssooss 5-9-5 56 2551
664740 135846 135864 TTTTGAAGTCCCGAGCCAA 2809 2827 sooossssssssssooss 5-9-5 44 2552
664741 135847 135865 ATTTTGAAGTCCCGAGCCA 2810 2828 sooossssssssssooss 5-9-5 61 2553
664742 135848 135866 GATTTTGAAGTCCCGAGCC 2811 2829 sooossssssssssooss 5-9-5 59 2554 664743 135849 135867 TGATTTTGAAGTCCCGAGC 2812 2830 sooossssssssssooss 5-9-5 64 2555
664744 135850 135868 CTGATTTTGAAGTCCCGAG 2813 2831 sooossssssssssooss 5-9-5 67 2556
664745 135851 135869 ACTGATTTTGAAGTCCCGA 2814 2832 sooossssssssssooss 5-9-5 73 2557
664746 135852 135870 CACTGATTTTGAAGTCCCG 2815 2833 sooossssssssssooss 5-9-5 86 2558
664747 135853 135871 TCACTGATTTTGAAGTCCC 2816 2834 sooossssssssssooss 5-9-5 76 2371
664748 135854 135872 ATCACTGATTTTGAAGTCC 2817 2835 sooossssssssssooss 5-9-5 74 2373
664749 135855 135873 CATCACTGATTTTGAAGTC 2818 2836 sooossssssssssooss 5-9-5 50 2375
664750 135856 135874 CCATCACTGATTTTGAAGT 2819 2837 sooossssssssssooss 5-9-5 57 2378
664751 135857 135875 CCCATCACTGATTTTGAAG 2820 2838 sooossssssssssooss 5-9-5 64 2381
664752 135858 135876 TCCCATCACTGATTTTGAA 2821 2839 sooossssssssssooss 5-9-5 59 2383
664753 135859 135877 CTCCCATCACTGATTTTGA 2822 2840 sooossssssssssooss 5-9-5 73 2386
664754 135860 135878 ACTCCCATCACTGATTTTG 2823 2841 sooossssssssssooss 5-9-5 75 2389
664755 135861 135879 TACTCCCATCACTGATTTT 2824 2842 sooossssssssssooss 5-9-5 40 2391
664756 135862 135880 TTACTCCCATCACTGATTT 2825 2843 sooossssssssssooss 5-9-5 43 2394
664757 135863 135881 CTTACTCCCATCACTGATT 2826 2844 sooossssssssssooss 5-9-5 70 2397
664758 135864 135882 TCTTACTCCCATCACTGAT 2827 2845 sooossssssssssooss 5-9-5 61 2399
664759 135865 135883 CTCTTACTCCCATCACTGA 2828 2846 sooossssssssssooss 5-9-5 68 2402
664760 135866 135884 GCTCTTACTCCCATCACTG 2829 2847 sooossssssssssooss 5-9-5 82 2405
664761 135867 135885 TGCTCTTACTCCCATCACT 2830 2848 sooossssssssssooss 5-9-5 76 2407
664762 135868 135886 TTGCTCTTACTCCCATCAC 2831 2849 sooossssssssssooss 5-9-5 77 2409
664763 135869 135887 TTTGCTCTTACTCCCATCA 2832 2850 sooossssssssssooss 5-9-5 83 241 1
664764 135870 135888 ATTTGCTCTTACTCCCATC 2833 2851 sooossssssssssooss 5-9-5 82 2413
664765 135871 135889 AATTTGCTCTTACTCCCAT 2834 2852 sooossssssssssooss 5-9-5 77 2415
664766 135872 135890 AAATTTGCTCTTACTCCCA 2835 2853 sooossssssssssooss 5-9-5 69 2417
Table 61
SEQ SEQ
SEQ SEQ ID ID ID ID SEQ
ISIS NO: NO: %
NO: 1 NO: 1 Sequence Linkage Chemistry Motif ID NO 2 2 inhibition start stop NO start stop
site site
site site
665041 73879 73898 GTGATCTTCCATCACTTCGA 345 364 sooosssssssssssooss 5-10-5 86 25
98890 98908
665044 GTTTTCAAACACACCTTCA n/a n/a sooossssssssssooss 5-9-5 90 2485
98927 98945
665201 102119 102138 GGTTT GTTTTTT AAACAATT n/a n/a sooosssssssssssooss 5-10-5 57 2509
665202 102144 102163 GAACCCAATGAGAGTAGCAA n/a n/a sooosssssssssssooss 5-10-5 86 2510
665203 102169 102188 TTGCCAAAATCAGGAATGGG n a n/a sooosssssssssssooss 5-10-5 87 2511
665204 102194 102213 TCAGGGCAATCTGGAAGCAT n/a n/a sooosssssssssssooss 5-10-5 79 2512
665205 102659 102678 ACTCTCCACTCCATGTCAAT n/a n/a sooosssssssssssooss 5-10-5 26 2513
665206 102684 102703 CAGAGCTCACAGCAATGATC n/a n/a sooosssssssssssooss 5-10-5 78 2514
665207 102709 102728 TCAGCATGAGTTGTGCCAAG n/a n/a sooosssssssssssooss 5-10-5 91 2515
665208 102734 102753 CCAGAGGAACTGTGTGCATT n/a n/a sooosssssssssssooss 5-10-5 79 2516
665209 102759 102778 CCAAGTTCCCTGAGGACATT n/a n/a sooosssssssssssooss 5-10-5 13 2517
665210 112934 112953 GTTGTGTTTTCTGGTTTATT n/a n/a sooosssssssssssooss 5-10-5 96 2518 665211 112959 112978 TTTTTTTTTAAGTTAGGAGT n/a n/a sooosssssssssssooss 5-10-5 20 2519
665212 112984 113003 TTTT ACTGGTT GT GTTTTCT n/a n/a sooosssssssssssooss 5-10-5 90 2520
665213 113009 113028 C AGCT CTT AAT GCT GTT AT A n a n/a sooosssssssssssooss 5-10-5 90 2521
665214 113498 113517 TTCAGCTCCTGCACCCAGCA n a n/a sooosssssssssssooss 5-10-5 54 2522
665215 113548 113567 TTTTGCTTTTGCTCTGAAGA n/a n/a sooosssssssssssooss 5-10-5 57 2523
665216 115105 115124 TTGCTATTAAATATAATGTA n/a n/a sooosssssssssssooss 5-10-5 35 2524
665217 115130 115149 GCTTTTTAAAGTGACAACTG n/a n/a sooosssssssssssooss 5-10-5 78 2525
665218 115155 115174 TTCCACATAAATGTTCTACA n/a n/a sooosssssssssssooss 5-10-5 93 2526
665219 115205 115224 CCCTGATTGAGGAGAGGCAA n/a n/a sooosssssssssssooss 5-10-5 77 2527
665220 116595 116614 ATGTTTTTTTCTGGCCGGGC n/a n/a sooosssssssssssooss 5-10-5 85 2528
665221 116620 116639 TTCAGTATCTGCCACATACT n/a n/a sooosssssssssssooss 5-10-5 64 2529
665222 116645 116664 TGAATCAAAGGACATTAAGC n/a n/a sooosssssssssssooss 5-10-5 69 2530
664661 135820 135839 GTGACAAAAGCAGGTTAAGT 2783 2802 sooosssssssssssooss 5-10-5 78 2333
664662 135821 135840 AGTGACAAAAGCAGGTTAAG 2784 2803 sooosssssssssssooss 5-10-5 18 2336
664663 135822 135841 GAGTGACAAAAGCAGGTTAA 2785 2804 sooosssssssssssooss 5-10-5 79 2338
664664 135823 135842 CGAGTGACAAAAGCAGGTTA 2786 2805 sooosssssssssssooss 5-10-5 66 2341
664665 135824 135843 CCGAGTGACAAAAGCAGGTT 2787 2806 sooosssssssssssooss 5-10-5 94 2344
664666 135825 135844 GCCGAGTGACAAAAGCAGGT 2788 2807 sooosssssssssssooss 5-10-5 89 2346
664667 135826 135845 AGCCGAGTGACAAAAGCAGG 2789 2808 sooosssssssssssooss 5-10-5 29 2349
664668 135827 135846 AAGCCGAGTGACAAAAGCAG 2790 2809 sooosssssssssssooss 5-10-5 72 2352
664669 135828 135847 AAAGCCGAGTGACAAAAGCA 2791 2810 sooosssssssssssooss 5-10-5 53 2354
664670 135829 135848 CAAAGCCGAGTGACAAAAGC 2792 2811 sooosssssssssssooss 5-10-5 55 2357
664671 135830 135849 CCAAAGCCGAGTGACAAAAG 2793 2812 sooosssssssssssooss 5-10-5 58 2360
664672 135831 135850 GCCAAAGCCGAGTGACAAAA 2794 2813 sooosssssssssssooss 5-10-5 80 2362
664673 135832 135851 AGCCAAAGCCGAGTGACAAA 2795 2814 sooosssssssssssooss 5-10-5 52 2365
664674 135833 135852 GAGCCAAAGCCGAGTGACAA 2796 2815 sooosssssssssssooss 5-10-5 71 2368
664675 135834 135853 CGAGCCAAAGCCGAGTGACA 2797 2816 sooosssssssssssooss 5-10-5 76 2419
664676 135835 135854 CCGAGCCAAAGCCGAGTGAC 2798 2817 sooosssssssssssooss 5-10-5 78 2422
664677 135836 135855 CCCGAGCCAAAGCCGAGTGA 2799 2818 sooosssssssssssooss 5-10-5 77 2425
664678 135837 135856 TCCCGAGCCAAAGCCGAGTG 2800 2819 sooosssssssssssooss 5-10-5 73 2427
664679 135838 135857 GTCCCGAGCCAAAGCCGAGT 2801 2820 sooosssssssssssooss 5-10-5 73 2430
664680 135839 135858 AGTCCCGAGCCAAAGCCGAG 2802 2821 sooosssssssssssooss 5-10-5 20 2433
664681 135840 135859 AAGTCCCGAGCCAAAGCCGA 2803 2822 sooosssssssssssooss 5-10-5 64 2435
664682 135841 135860 GAAGTCCCGAGCCAAAGCCG 2804 2823 sooosssssssssssooss 5-10-5 60 2438
664683 135842 135861 TGAAGTCCCGAGCCAAAGCC 2805 2824 sooosssssssssssooss 5-10-5 64 2441
664684 135843 135862 TTGAAGTCCCGAGCCAAAGC 2806 2825 sooosssssssssssooss 5-10-5 52 2443
664685 135844 135863 TTTGAAGTCCCGAGCCAAAG 2807 2826 sooosssssssssssooss 5-10-5 45 2559
664686 135845 135864 TTTTGAAGTCCCGAGCCAAA 2808 2827 sooosssssssssssooss 5-10-5 56 2560
664687 135846 135865 ATTTTGAAGTCCCGAGCCAA 2809 2828 sooosssssssssssooss 5-10-5 67 2561
664688 135847 135866 GATTTTGAAGTCCCGAGCCA 2810 2829 sooosssssssssssooss 5-10-5 73 2562
664689 135848 135867 TGATTTTGAAGTCCCGAGCC 1644 1663 sooosssssssssssooss 5-10-5 67 56
664690 135849 135868 CTGATTTTGAAGTCCCGAGC 2812 2831 sooosssssssssssooss 5-10-5 73 464
664691 135850 135869 ACTGATTTTGAAGTCCCGAG 2813 2832 sooosssssssssssooss 5-10-5 85 2563 664692 135851 135870 CACTGATTTTGAAGTCCCGA 2814 2833 sooosssssssssssooss 5-10-5 89 2564
664693 135852 135871 TCACTGATTTTGAAGTCCCG 2815 2834 sooosssssssssssooss 5-10-5 75 2565
424880 135853 135872 ATCACTGATTTTGAAGTCCC 2816 2835 sssssssssssssssssss 5-10-5 62 57
664694 135853 135872 ATCACTGATTTTGAAGTCCC 1649 1668 sooosssssssssssooss 5-10-5 85 57
664695 135854 135873 CATCACTGATTTTGAAGTCC 2817 2836 sooosssssssssssooss 5-10-5 83 2374
664696 135855 135874 CCATCACTGATTTTGAAGTC 2818 2837 sooosssssssssssooss 5-10-5 66 2376
664697 135856 135875 CCC AT C ACTGATTTT GAAGT 2819 2838 sooosssssssssssooss 5-10-5 58 2379
664698 135857 135876 TCCCATCACTGATTTTGAAG 2820 2839 sooosssssssssssooss 5-10-5 71 2382
664699 135858 135877 CTCCCATCACTGATTTTGAA 2821 2840 sooosssssssssssooss 5-10-5 32 2384
664700 135859 135878 ACTCCCATCACTGATTTTGA 2822 2841 sooosssssssssssooss 5-10-5 71 2387
664701 135860 135879 TACTCCCATCACTGATTTTG 2823 2842 sooosssssssssssooss 5-10-5 75 2390
664702 135861 135880 TTACTCCCATCACTGATTTT 2824 2843 sooosssssssssssooss 5-10-5 46 2392
664703 135862 135881 CTTACTCCCATCACTGATTT 2825 2844 sooosssssssssssooss 5-10-5 22 2395
664704 135863 135882 TCTTACTCCCATCACTGATT 2826 2845 sooosssssssssssooss 5-10-5 72 2398
664705 135864 135883 CTCTTACTCCCATCACTGAT 2827 2846 sooosssssssssssooss 5-10-5 70 2400
664706 135865 135884 GCTCTTACTCCCATCACTGA 2828 2847 sooosssssssssssooss 5-10-5 77 2403
664707 135866 135885 TGCTCTTACTCCCATCACTG 2829 2848 sooosssssssssssooss 5-10-5 88 2406
664708 135867 135886 TTGCTCTTACTCCCATCACT 2830 2849 sooosssssssssssooss 5-10-5 86 2408
664709 135868 135887 TTTGCTCTTACTCCCATCAC 2831 2850 sooosssssssssssooss 5-10-5 16 2410
664710 135869 135888 ATTTGCTCTTACTCCCATCA 2832 2851 sooosssssssssssooss 5-10-5 80 2412
664711 135870 135889 AATTTGCTCTTACTCCCATC 2833 2852 sooosssssssssssooss 5-10-5 65 2414
664712 135871 135890 AAATTTGCTCTTACTCCCAT 2834 2853 sooosssssssssssooss 5-10-5 60 2416
664713 135872 135891 GAAATTTGCTCTTACTCCCA 2835 2854 sooosssssssssssooss 5-10-5 85 2418
Example 17: Dose-dependent antisense inhibition of human Tau in SH-SY5Y cells
Gapmers from studies described above exhibiting significant in vitro inhibition of tau mRNA were selected and tested at various doses in SH-SY5Y cells. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with 0.247 μΜ, 0.741 μΜ, 2.22 μΜ, 6.67 μΜ, and 20.00 μΜ concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and tau mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3104 was used to measure mRNA levels. Tau mRNA levels were adjusted according to total RNA content, as measured by
RIBOGREEN®. Results are presented as percent inhibition of tau, relative to untreated control cells.
Tau mRNA levels were significantly reduced in a dose-dependent manner in antisense
oligonucleotide treated cells. Table 62
Figure imgf000152_0001
Table 63
Figure imgf000152_0002
Table 64
Figure imgf000152_0003
621041 26 55 70 89 92 0.7
621238 23 41 59 85 95 1.2
621251 33 56 74 88 92 0.5
664514 11 36 68 79 91 1.6
664516 16 36 64 82 94 1.5
664534 18 35 48 76 91 1.9
664775 24 30 59 79 92 1.6
664788 11 21 26 55 73 6.2
664799 26 37 63 79 90 1.3
664800 15 0 61 75 90 2.7
665047 43 65 82 95 98 0.2
665077 19 44 63 85 96 1.2
Example 18: Intracerebroventricular administration of antisense oligonucleotides against human tau mR A in htau mice
Selected compounds were tested for efficacy by ICV administration in human tau transgenic mice
(Duff et al., Neurobiology of Disease 7:87-98, 2000).
Treatment and surgery
Groups of 4 mice each were administered ISIS 613099, ISIS 613361, ISIS 613370, ISIS 623782, or ISIS 623996 with a 200 μg dose delivered by ICV bolus injection. A control group of 2 mice was similarly treated with ISIS 424880 and a control group of 4 mice was similarly treated with PBS. All procedures were performed under isoflourane anesthesia and in accordance with IACUC regulations. For mouse ICV bolus injections, the antisense oligonucleotide was injected into the right lateral ventricle of human tau transgenic mice. Ten microliters of solution containing 200 μg of oligonucleotide in PBS was injected over approximately 10 seconds. Tissue was collected 14 days after oligonucleotide administration.
RNA Analysis
On day 14 after the oligonucleotide administration, RNA was extracted from the hippocampus, spinal cord and cortex for real-time PCR analysis of tau mRNA levels. Human tau mRNA levels were measured using the human primer probe set RTS3104. Results were calculated as percent inhibition of human tau mRNA expression compared to the control. All the antisense oligonucleotides effect significant inhibition of human tau mRNA levels in several tissues. Table 65
Percent reduction of human tau mRNA levels in hTau mice
Figure imgf000154_0001
Example 19: Design of oligonucleotides targeting human Tau
ISIS No. 603054 was designed to target human Tau. The nucleobase sequence and linkage chemistry of of ISIS No. 603054 is given in table 66 below. ISIS No. 603054 is a 5-10-5 MOE gapmer. ISIS No. 603054 is 20 nucleosides in length, wherein the central gap segment comprises ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3 ' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2' -MOE modification. All cytosine residues throughout each gapmer are 5-methylcytosines. "Start site" indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence. "Stop site" indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence. Each gapmer listed in Table 1 below is targeted to either the human tau genomic sequence, designated herein as SEQ ID NO: 1 (GENBAN Accession No. NT 010783.15 truncated from nucleotides 9240000 to 9381000) or to the human tau mRNA sequence, designated herein as SEQ ID NO: 2 (GENBANK Accession No. NM_001 123066.3).
Table 66
ICV in vivo study in hTau mice and WT C57B16 mice
Figure imgf000154_0002
Example 20: In vivo analysis in mice of oligonucleotides targeting human Tau
Oligonucleotides, shown in the table below, were designed to target Tau. Mice, either human tau transgenic mice "hTau" (Duff et al., Neurobiology of Disease 7:87-98, 2000; Davies et al. J. Neurochem. (2003) 86, 582-590) or wild-type WT C57B16 mice were separated into groups of 3 or 4 mice. Each mouse in each group of mice was administered a single ICV dose of either 300ug or 200ug each of the oligonucleotides in the table below. At 3 hours post injection, each mouse was evaluated according to 7 different criteria. The 7 criteria are (1) the mouse was bright, alert, and responsive; (2) the mouse was standing or hunched without stimuli; (3) the mouse shows any movement without stimuli (4) the mouse demonstrates forward movement after its lifted; (5) the mouse demonstrates any movement after its lifted; (6) the mouse responds to a tail pinch; (7) regular breathing. For each of the 7 different criteria, each mouse was given a sub-score of 0 if it met the criteria or 1 if it did not. After all of the 7 criteria were evaluated, the sub-scores were summed for each mouse and then averaged for each group. For example, if a mouse was bright, alert, and responsive 3 hours after the 300 μg ICV dose, and met all other other criteria, it would get a summed score of 0. If another mouse was not bright, alert, and responsive 3 hours after the 300 μg ICV dose but met all other criteria, it would receive a score of 1. Saline treated mice generally receive a score of 0. Results are presented as the average score for each treatment group in Table 67 below. "ND" means no data. These results demonstrate that ISIS 613099, ISIS 613361, ISIS 613370, ISIS 623782, ISIS 623996, ISIS 424880, and ISIS 603054 were well tolerated.
Table 67
ICV in vivo study in hTau mice and WT C57B16 mice
Figure imgf000155_0001
Example 20: In vivo analysis in rats of oligonucleotides targeting human Tau
Sprague Dawley rats were separated into groups of 4 rats each. Each rat in each group of rats was administered a single 1 mg intrathecal (IT) dose or a single 3 mg intrathecal (IT) dose of ISIS 613099, ISIS 613361, ISIS 613370, ISIS 623782, ISIS 623996, ISIS 424880, or ISIS 603054. At 3 hours after injection, the movement of 7 different parts of the body was evaluated for each rat. The 7 body parts are (1) the rat's tail; (2) the rat's posterior posture; (3) the rat's hind limbs; (4) the rat's hind paws; (5) the rat's forepaws; (6) the rat's anterior posture; and (7) the rat's head. For each of the 7 different body parts, each rat was given a sub-score of 0 if the body part was moving or 1 if the body part was paralyzed. After each of the 7 body parts were evaluated, the sub-scores were summed for each rat and then averaged for each group. For example, if a rat's tail, head, and all other evaluated body parts were moving 3 hours after the 3 mg IT dose, it would get a summed score of 0. If another rat was not moving its tail 3 hours after the 3 mg IT dose but all other evaluated body parts were moving, it would receive a score of 1. Saline treated rats generally receive a score of 0. A score of at the top end of the range would be suggestive of toxicity. Results are presented as the average score for each treatment group in Table 68 below.
Table 68
and 3 mg IT bolus in vivo study
Figure imgf000156_0001

Claims

CLAIMS What is claimed is:
1. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2443 and SEQ ID NOs: 2478-2483.
2. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 446, 313, 321, 1634, and 2309.
3. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of SEQ ID NO: 446.
4. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of SEQ ID NO: 313.
5. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of SEQ ID NO: 321.
6. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of SEQ ID NO: 1634.
7. The compound of claim 1, wherein the modified oligonucleotide has a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of SEQ ID NO: 2309.
8. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 2444-2477 and SEQ ID NOs: 2484-2565.
9. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases of any of the nucleobase sequences of SEQ ID NOs: 20-2565.
10. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135980 of SEQ ID NO: 1.
11. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135853-135872 of SEQ ID NO: 1.
12. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135929 of SEQ ID NO: 1.
13. A compound, comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and comprising a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 consecutive nucleobases complementary to an equal length portion of nucleobases 135783-135914 of SEQ ID NO: 1.
14. The compound of claims 4-7, wherein the nucleobase sequence of the modified oligonucleotide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to SEQ ID NO: 1.
15. The compound of any preceding claim, consisting of a single-stranded modified oligonucleotide.
16. The compound of any preceding claim, wherein at least one internucleoside linkage is a modified internucleoside linkage.
17. The compound of claim 16, wherein at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage.
18. The compound of claim 16, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage or a phosphodiester internucleoside linkage.
19. The compound of any of claims 1 to 18, wherein at least one internucleoside linkage is a phosphodiester internucleoside linkage.
20. The compound of any of claims 1 to 18, wherein two internucleoside linkages are phosphodiester internucleoside linkages.
21. The compound of any of claims 1 to 18, wherein three internucleoside linkages are phosphodiester internucleoside linkages.
22. The compound of any of claims 1 to 18, wherein four internucleoside linkages are phosphodiester internucleoside linkages.
23. The compound of any of claims 1 to 18, wherein five internucleoside linkages are phosphodiester internucleoside linkages.
24. The compound of any of claims 1 to 18, wherein six internucleoside linkages are phosphodiester internucleoside linkages.
25. The compound of any of claims 1 to 18, wherein at least six internucleoside linkages are phosphodiester internucleoside linkages.
26. The compound of any preceding claim, wherein at least one internucleoside linkage is a phosphorothioate linkage and at least one internucleoside linkage is a phosphodiester linkage.
27. The compound of any of claims 1 to 17, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.
28. The compound of any preceding claim, wherein at least one nucleoside comprises a modified nucleobase.
29. The compound of claim 28, wherein the modified nucleobase is a 5-methylcytosine.
30. The compound of any preceding claim, wherein at least one nucleoside of the modified oligonucleotide comprises a modified sugar.
31. The compound of claim 30, wherein the at least one modified sugar is a bicyclic sugar.
32. The compound of claim 31 , wherein the bicyclic sugar comprises a chemical link between the 2' and 4' position of the sugar 4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, C1-C12 alkyl, or a protecting group.
33. The compound of claim 31, wherein the bicyclic sugar comprises a 4'-CH2-N(R)-0-2' bridge wherein R is, independently, H, CI -CI 2 alkyl, or a protecting group.
34. The compound of claim 31, wherein at least one modified sugar comprises a 2'-0- methoxyethyl group.
35. The compound of claim 31, wherein the modified sugar comprises a 2'-0(CH2)2-OCH3 group.
36. The compound of any preceding claim, wherein the modified oligonucleotide comprises: a gap segment consisting of 10 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3 ' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
37. The compound of any preceding claim, wherein the modified oligonucleotide comprises: a gap segment consisting of 9 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3 ' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
38. The compound of any preceding claim, wherein the modified oligonucleotide comprises:
a gap segment consisting of 7 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
39. The compound of any preceding claim, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 5 linked nucleosides; and
a 3' wing segment consisting of 5 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
40. The compound of any preceding claim, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 4 linked nucleosides; and
a 3' wing segment consisting of 6 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
41. The compound of any preceding claim, wherein the modified oligonucleotide comprises:
a gap segment consisting of 8 linked deoxynucleosides;
a 5' wing segment consisting of 6 linked nucleosides; and
a 3' wing segment consisting of 4 linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
42. The compound of any preceding claim, wherein the modified oligonucleotide consists of 20 linked nucleosides.
43. The compound of any preceding claim, wherein the modified oligonucleotide consists of 19 linked nucleosides.
44. The compound of any preceding claim, wherein the modified oligonucleotide consists of 18 linked nucleosides.
45. A composition comprising the compound of any preceding claim or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent.
46. A method comprising administering to an animal the compound or composition of any preceding claim.
47. The method of claim 46, wherein the animal is a human.
48. The method of claim 46, wherein administering the compound prevents, treats, ameliorates, or slows progression of a tau associated disease, disorder or condition.
49. The method of claim 48, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
50. The method of claim 48, wherein the disease, disorder or condition is Alzheimer's Disease.
51. Use of the compound or composition of any preceding claim for the manufacture of a medicament for treating a neurodegenerative disease, disorder or condition.
52. The use of claim 51, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
53. The use of claim 51, wherein the disease, disorder or condition is Alzheimer's Disease.
54. The compound or composition of any of claims 1 - 46 for use in treatment of a neurodegenerative disease, disorder, or condition.
55. The compound or composition of claim 54, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
56. The compound or composition of claim 54, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease.
57. A compound consisting of ISIS 613099.
58. A compound consisting of ISIS 613361.
59. A compound consisting of ISIS 613370.
60. A compound consisting of ISIS 623782.
61. A compound consisting of ISIS 623996.
62. A compound consisting of a modified oligonucleotide according to the following formula: Ges Aeo Teo Aeo Teo Tds Ads Tds mCds mCds Tds Tds Tds Gds Ads Geo mCeo mCes Aes mCe; wherein,
A = an adenine,
mC = a 5'-methylcytosine
G = a guanine,
T = a thymine,
e = a 2'-0-methoxyethyl modified nucleoside,
d = a 2'-deoxynucleoside, and
o = a phosphodiester internucleoside linkage, and s = a phosphorothioate internucleoside linkage.
63. A compound consisting of a modified oligonucleotide according to the following formula: Aes mCeo Aeo mCeo Aeo mCds mCds Tds Tds mCds Ads Tds Tds Tds Ads mCeo Teo Ges Tes mCe; wherein,
A = an adenine,
mC = a 5'-methylcytosine
G = a guanine,
T = a thymine,
e = a 2'-0-methoxyethyl modified nucleoside,
d = a 2'-deoxynucleoside, and
o = a phosphodiester internucleoside linkage, and
s = a phosphorothioate internucleoside linkage.
64. A compound consisting of a modified oligonucleotide according to the following formula: Ges Geo Teo Teo Teo Tds mCds Ads Ads Ads mCds Ads mCds Ads mCds mCeo Teo Tes mCes Ae; wherein,
A = an adenine,
mC = a 5'-methylcytosine
G = a guanine,
T = a thymine,
e = a 2'-0-methoxyethyl modified nucleoside,
d = a 2'-deoxynucleoside,
o = a phosphodiester internucleoside linkage, and
s = a phosphorothioate internucleoside linkage.
65. A compound consisting of a modified oligonucleotide according to the following formula: mCes mCeo Geo Teo Tes Tds Tds mCds Tds Tds Ads mCds mCds Aeo mCeo mCes mCes Te; wherein,
A = an adenine,
mC = a 5'-methylcytosine
G = a guanine, T = a thymine,
e = a 2'-0-methoxyethyl modified nucleoside,
d = a 2'-deoxynucleoside,
o = a phosphodiester internucleoside linkage, and
s = a phosphorothioate internucleoside linkage.
66. A compound consisting of a modified oligonucleotide according to the following formula: Aes Aeo Teo Teo Tes Gds mCds Tds mCds Tds Tds Ads mCds Teo mCeo mCes mCes Ae;
wherein,
A = an adenine,
mC = a 5'-methylcytosine
G = a guanine,
T = a thymine,
e = a 2'-0-methoxyethyl modified nucleoside,
d = a 2'-deoxynucleoside,
o = a phosphodiester internucleoside linkage, and
s = a phosphorothioate internucleoside linkage.
67. A composition comprising the compound of any of claims 57 - 66, or salt thereof, and at least one of a pharmaceutically acceptable carrier or diluent.
68. A method comprising administering to an animal the compound or composition of any of claims
57 - 66.
69. The method of claim 68, wherein the animal is a human.
70. The method of claim 67 or 68, wherein administering the compound prevents, treats, ameliorates, or slows progression of a tau associated disease, disorder or condition.
71. The method of claim 70, wherein the disease, disorder or condition is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
72. The method of claim 71, wherein the disease, disorder or condition is Alzheimer's Disease.
73. Use of the compound or composition of any of claims 57 - 66 for the manufacture of a medicament for treating a neurodegenerative disorder.
74. The use of claim 73, wherein the neurodegenerative disorder is a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
75. The use of claim 73, wherein the neurodegenerative disorder is Alzheimer's Disease
76. The compound or composition of any of claims 1 -45 or 57 - 66 for use in treatment of a neurodegenerative disorder.
77. The compound or composition of any of claims 1-45 or 57 - 66 for use in treatment of a neurodegenerative disorder selected from among a Tauopathy, Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), Epilepsy, or Dravet's Syndrome.
78. The compound or composition of any of claims 1-45 or 57 - 66 for use in treatment Alzheimer's
Disease.
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