# Patent

## Title
Compositions and methods for modulating Tau expression

## Abstract

Disclosed are methods for modulating splicing of Tau mRNA in an animal with Tau antisense compounds. Also disclosed herein are methods for reducing expression of Tau mRNA and protein in an animal with Tau antisense compounds. Such compounds and methods are useful to treat, prevent, or ameliorate neurodegenerative diseases in an individual in need thereof. Examples of neurodegenerative diseases that can be treated, prevented, and ameliorated with the administration Tau antisense oligonucleotides include Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy, Chronic Traumatic Encephalopathy, Epilepsy, and Dravet's Syndrome.

## Background
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 BIOL0220USASEQ—ST25.txt created Sep. 14, 2015, which is approximately 228 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND
1. Field
Disclosed herein are compositions and methods related to the fields of antisense compounds biochemistry, molecular biology, and medicine. Embodiments described herein relate to compounds, compositions, and methods for treating, preventing, or ameliorating 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 or modulating the splicing of Tau in a cell and/or in an animal.
2. Description
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 0N, 1N, 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 and thus may be amenable to a similar splicing strategy.
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 (Aβ)plaques.
In seizure disorders, the brain's electrical activity is periodically disturbed, resulting in some degree of temporary brain dysfunction. Normal brain function requires an orderly, organized, coordinated discharge of electrical impulses. Electrical impulses enable the brain to communicate with the spinal cord, nerves, and muscles as well as within itself. Seizures may result when the brain's electrical activity is disrupted. There are two basic types of seizures; epileptic and nonepileptic. Epileptic seizures have no apparent cause or trigger and occur repeatedly. Nonepileptic seizures are triggered orprovoked by a disorder or another condition that irritates the brain. Certain mental disorders can cause seizure symptoms referred to as psychogenic nonepileptic seizures.
Alzheimer's Disease(AD) is known to be a clinical risk factor for late onset seizures. Multiple AD mouse models recapitulate this increased seizure susceptibility. Within the last 5 years, many of these AD models have been studied in the setting of mouse tau knockout (tau−/−). Increased seizure susceptibility was ameliorated in these amyloid-depositing tau knockout lines. Further, tau−/− alone interestingly appeared to be protective against chemically induced seizures.
Anticonvulsants represent the common treatment regime for seizures. However, anticonvulsants are ineffective in a significant percent of people with a seizure disorder and for these individuals, surgery is the only option. Amidst the lack of available treatments for seizure disorders and neurodegenerative diseases, certain methods of the present embodiments provide methods for treating, preventing or ameliorating a seizure disorder and neurodegenerative diseases by inhibiting expression of Tau or modulating the splicing of Tau in an animal.
SUMMARY
Provided herein are methods for modulating splicing of Tau mRNA in cells, tissues, and animals. Also provided herein are methods for modulating the expression product of a Tau mRNA in cells, tissues, and animals.
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 human. In certain embodiments, Tau mRNA levels are reduced. In certain embodiments, Tau protein levels are reduced. In certain embodiments, splicing of Tau mRNA is modulated. In certain embodiments, the expression product of a Tau mRNA is modulated. In certain embodiments, exclusion of Tau exon 10 is promoted. In certain embodiments, expression of the 4R isoform of Tau RNA or protein is reduced. In certain embodiments, expression of the 3R isoform of Tau RNA or protein is increased. In certain embodiments, expression of the 4R isoform of Tau RNA or protein is reduced and expression of the 3R isoform of Tau RNA or protein is increased. In certain embodiments, hyperphosphorylated Tau is reduced. Such reduction and modulation can occur in a time-dependent manner or in a dose-dependent manner.
Several embodiments are drawn to methods of reducing or decreasing one or more symptoms of a tau-associated disorder. In certain embodiments, the symptom is a seizure. In certain embodiments, the tau-associated disorder or neurodegenerative disorder is Alzheimer's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal ganglionic degeneration, dementia pugilistica, parkinsonism linked to chromosome, LyticoBodig 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. Certain embodiments are drawn to a method of decreasing seizures in a subject with a high 4R:3R tau isoform ratio. In certain embodiments, the methods comprise administering an antisense agent to the subject, wherein the agent decreases expression of tau or decreases the 4R:3R tau ratio in the central nervous system of the subject.
Also provided are methods useful for preventing, treating, and ameliorating diseases, disorders, and conditions associated with Tau. In certain embodiments, such diseases, disorders, and conditions associated with Tau are neurodegenerative diseases. In certain embodiments, the neurodegenerative disease is any of Alzheimer's Disease, Fronto-temporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy, Chronic Traumatic Encephalopathy, Epilepsy, or Dravet's Syndrome. In certain embodiments, one or more symptoms of a neurodegenerative disease is ameliorated, prevented, or delayed (progression slowed). In certain embodiments, the symptom is memory loss, anxiety, or loss of motor function. In certain embodiments, neurodegenerative function is improved. In certain embodiments, neurofibrillary inclusions are reduced.
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 a neurodegenerative disease include genetic predisposition and older age.
In certain embodiments, methods of treatment include administering a Tau antisense compound to an individual in need thereof. The antisense compound may inhibit expression of Tau or modulate splicing of Tau. In certain embodiments, the antisense compound is a single-stranded antisense oligonucleotide. In certain embodiments, the single-stranded antisense oligonucleotide is complementary to a Tau nucleic acid.
In certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides are complementary to a Tau transcript. In certain such embodiments, oligonucleotides are complementary to a target region of the Tau transcript comprising exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the Tau transcript comprising an intron adjacent to exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the Tau transcript comprising an intron adjacent to exon 10 and downstream of exon 10. In certain such embodiments, oligonucleotides are complementary to a target region of the Tau transcript comprising an intron adjacent to exon 10 and upstream of exon 10. In certain embodiments, the Tau transcript comprises an exonic splice silencer for exon 10. In certain embodiments, oligonucleotides inhibit inclusion of exon 10. In certain embodiments, oligonucleotides promote skipping of exon 10. In certain such embodiments, 3R Tau mRNA is increased. In certain such embodiments, Tau mRNA with exon 10 mRNA is decreased. In certain embodiments, the 4R isoform of the Tau protein is decreased. In certain embodiments, the 3R isoform of the Tau protein is increased.
In certain embodiments, including, but not limited to any of the above numbered embodiments, the Tau transcript is in a human. In certain embodiments, including, but not limited to any of the above numbered embodiments, the Tau transcript is in a mouse.
The present disclosure provides the following non-limiting numbered embodiments:

    Embodiment 1. A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a Tau transcript.
    Embodiment 2. The compound of embodiment 1, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
    Embodiment 3. The compound of embodiment 1 or 2, wherein the complementary region of the nucleobase sequence of the modified oligonucleotide comprises at least 10 contiguous nucleobases.
    Embodiment 4. The compound of embodiment 1 or 2, wherein the complementary region of the nucleobase sequence of the modified oligonucleotide comprises at least 15 contiguous nucleobases.
    Embodiment 5. The compound of embodiment 1 or 2, wherein the complementary region of the nucleobase sequence of the modified oligonucleotide comprises at least 18 contiguous nucleobases.
    Embodiment 6. The compound of any of embodiments 1-5, wherein the nucleobase sequence of the modified oligonucleotide is at least 80% complementary to an equal-length region of the Tau transcript, as measured over the entire length of the oligonucleotide.
    Embodiment 7. The compound of any of embodiments 1-5, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to an equal-length region of the Tau transcript, as measured over the entire length of the oligonucleotide.
    Embodiment 8. The compound of any of embodiments 1-5, wherein the nucleobase sequence of the modified oligonucleotide is 100% complementary to an equal-length region of the Tau transcript, as measured over the entire length of the oligonucleotide.
    Embodiment 9. The compound of any of embodiments 1-8, wherein the target region is within exon 10 of the Tau transcript.
    Embodiment 10. The compound of any of embodiments 1-8, wherein the target region is within intron 9 of the Tau transcript.
    Embodiment 11. The compound of any of embodiments 1-8, wherein the target region is within intron 10 of the Tau transcript.
    Embodiment 12. The compound of any of embodiments 1-8, wherein the target region comprises a portion of intron 9 and a portion of exon 10.
    Embodiment 13. The compound of any of embodiments 1-8, wherein the target region comprises a portion of exon 10 and a portion of intron 10.
    Embodiment 14. The compound of any of embodiments 1-8, wherein the target region comprises a portion of exon 10 and a portion of the intron at the 5′-end of exon 10.
    Embodiment 15. The compound of any of embodiments 1-8, wherein the target region comprises a portion of exon 10 and a portion of the intron at the 3′-end of exon 10.
    Embodiment 16. The compound of any of embodiments 1-15, wherein the target region is within nucleobase 121708 and nucleobase 122044 of SEQ ID NO.: 1.
    Embodiment 17. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121796 and nucleobase 122044 of SEQ ID NO.: 1.
    Embodiment 18. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121796 and nucleobase 121885 of SEQ ID NO.: 1.
    Embodiment 19. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121873 and nucleobase 121898 of SEQ ID NO.: 1.
    Embodiment 20. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121869 and nucleobase 121943 of SEQ ID NO.: 1.
    Embodiment 21. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 122020 and nucleobase 122044 of SEQ ID NO.: 1.
    Embodiment 22. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121881 and nucleobase 121898 of SEQ ID NO.: 1.
    Embodiment 23. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121880 and nucleobase 121897 of SEQ ID NO.: 1.
    Embodiment 24. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121879 and nucleobase 121896 of SEQ ID NO.: 1.
    Embodiment 25. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121878 and nucleobase 121895 of SEQ ID NO.: 1.
    Embodiment 26. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121877 and nucleobase 121894 of SEQ ID NO.: 1.
    Embodiment 27. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121876 and nucleobase 121893 of SEQ ID NO.: 1.
    Embodiment 28. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121875 and nucleobase 121892 of SEQ ID NO.: 1.
    Embodiment 29. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121874 and nucleobase 121891 of SEQ ID NO.: 1.
    Embodiment 30. The compound of any of embodiments 1-9, wherein the target region is within nucleobase 121873 and nucleobase 121890 of SEQ ID NO.: 1.
    Embodiment 31. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of any of SEQ ID NOs: 8-200.
    Embodiment 32. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 8-200.
    Embodiment 33. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of any of SEQ ID NOs: 33, 34, 147, 148, 149, 150, 151, 152, or 153.
    Embodiment 34. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 33, 34, 147, 148, 149, 150, 151, 152, or 153.
    Embodiment 35. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of any of SEQ ID NOs: 31, 33, 34, 35, 36, 37, 45, 72, 79, 82, 83, 97, 106, 107, 112, 113, 130, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 157, 158, 160, or 200.
    Embodiment 36. The compound of any of embodiments 1-15, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of any of SEQ ID NOs: 31, 33, 34, 35, 36, 37, 45, 72, 79, 82, 83, 97, 106, 107, 112, 113, 130, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 157, 158, 160, or 200.
    Embodiment 37. The compound of any of embodiments 1-36, wherein the modified oligonucleotide comprises at least one modified nucleoside.
    Embodiment 38. The compound of embodiment 37, wherein at least one modified nucleoside comprises a modified sugar moiety.
    Embodiment 39. The compound of embodiment 37, wherein at least one modified sugar moiety is a 2′-substituted sugar moiety.
    Embodiment 40. The compound of embodiment 39, wherein the 2′-substitutent of at least one 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F, and 2′-MOE.
    Embodiment 41. The compound of embodiment 39, wherein the 2′-substituent of at least one 2′-substituted sugar moiety is a 2′-MOE.
    Embodiment 42. The compound of any of embodiments 37-38, wherein at least one modified sugar moiety is a bicyclic sugar moiety.
    Embodiment 43. The compound of embodiment 42, wherein at least one bicyclic sugar moiety is LNA or cEt.
    Embodiment 44. The compound of any of embodiments 37-43, wherein at least one sugar moiety is a sugar surrogate.
    Embodiment 45. The compound of embodiment 44, wherein at least one sugar surrogate is a morpholino.
    Embodiment 46. The compound of embodiment 44, wherein at least one sugar surrogate is a modified morpholino.
    Embodiment 47. The compound of any of embodiment 1-46, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.
    Embodiment 48. The compound of embodiment 47, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
    Embodiment 49. The compound of embodiment 47, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
    Embodiment 50. The compound of embodiment 47, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside and each independently comprises a modified sugar moiety, wherein the modified sugar moieties of each modified nucleosides are each the same as one another.
    Embodiment 51. The compound of embodiment 47, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety.
    Embodiment 52. The compound of any of embodiments 1-51, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
    Embodiment 53. The compound of any of embodiments 1-52, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.
    Embodiment 54. The compound of any of embodiments 1-53, wherein the modified oligonucleotide comprises a modified region comprising at least 5 contiguous modified nucleosides.
    Embodiment 55. The compound of embodiment 54, wherein the modified oligonucleotide comprises a modified region comprising at least 10 contiguous modified nucleosides.
    Embodiment 56. The compound of embodiment 54, wherein the modified oligonucleotide comprises a modified region comprising at least 15 contiguous modified nucleosides.
    Embodiment 57. The compound of embodiment 54, wherein the modified oligonucleotide comprises a modified region comprising at least 18 contiguous modified nucleosides.
    Embodiment 58. The compound of embodiment 54, wherein the modified oligonucleotide comprises a modified region comprising at least 20 contiguous modified nucleosides.
    Embodiment 59. The compound of any of embodiments 53-58, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA, morpholino, and modified morpholino.
    Embodiment 60. The compound of any of embodiments 54-59, wherein the modified nucleosides of the modified region each comprise the same modification as one another.
    Embodiment 61. The compound of embodiment 60, wherein the modified nucleosides of the modified region each comprise the same 2′-substituted sugar moiety.
    Embodiment 62. The compound of embodiment 61, wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2′-F, 2′-OMe, and 2′-MOE.
    Embodiment 63. The compound of embodiment 61, wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2′-MOE.
    Embodiment 64. The compound of embodiment 59, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.
    Embodiment 65. The compound of embodiment 64, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.
    Embodiment 66. The compound of embodiment 59, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
    Embodiment 67. The compound of embodiment 66, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.
    Embodiment 68. The compound of embodiment 66, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.
    Embodiment 69. The compound of any of embodiments 1-68, wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.
    Embodiment 70. The compound of any of embodiments 1-68, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.
    Embodiment 71. The compound of embodiment 70 wherein each modified nucleoside comprises a modified sugar moiety.
    Embodiment 72. The compound of embodiment 71, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
    Embodiment 73. The compound of embodiment 72, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2′-substituted sugar moiety.
    Embodiment 74. The compound of embodiment 73, wherein the 2′-substituted sugar moiety of the modified oligonucleotide is selected from 2′-F, 2′-OMe, and 2′-MOE.
    Embodiment 75. The compound of embodiment 74, wherein the 2′-substituted sugar moiety of the modified oligonucleotide is 2′-MOE.
    Embodiment 76. The compound of embodiment 72, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
    Embodiment 77. The compound of embodiment 76, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.
    Embodiment 78. The compound of embodiment 72, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.
    Embodiment 79. The compound of embodiment 78, wherein the sugar surrogate of the modified oligonucleotide is a morpholino.
    Embodiment 80. The compound of embodiment 78, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
    Embodiment 81. The compound of any of embodiments 1 to 36, wherein the modified oligonucleotide has an A-B2-A-B2-A-B2-A-B2-A-B2-A-B2-A2 motif, wherein each A comprises a bicyclic sugar moiety, and wherein each B is selected from a 2′-substituted sugar moiety and an unmodified 2′-deoxy sugar moiety.
    Embodiment 82. The compound of embodiment 81, wherein each A is a cEt sugar moiety.
    Embodiment 83. The compound of embodiment 81, wherein each A is an LNA sugar moiety.
    Embodiment 84. The compound of any of embodiments 81 to 83, wherein each B is an unmodified 2′-deoxy sugar moiety.
    Embodiment 85. The compound of any of embodiments 81 to 83, wherein each B is 2′-MOE sugar moiety.
    Embodiment 86. The compound of any of embodiments 1-85, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
    Embodiment 87. The compound of embodiment 86, wherein each internucleoside linkage is a modified internucleoside linkage.
    Embodiment 88. The compound of embodiment 86 or 87, comprising at least one phosphoro-thioate internucleoside linkage.
    Embodiment 89. The compound of any of embodiments 1-85, wherein each internucleoside linkage is a modified internucleoside linkage and wherein each internucleoside linkage comprises the same modification.
    Embodiment 90. The compound of embodiment 89, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
    Embodiment 91. The compound of embodiment 86, wherein the modified oligonucleotide comprises 4 phosphodiester internucleoside linkages.
    Embodiment 92. The compound of embodiment 86, wherein the modified oligonucleotide comprises 5 phosphodiester internucleoside linkages.
    Embodiment 93. The compound of embodiment 86, wherein the modified oligonucleotide comprises 6 phosphodiester internucleoside linkages.
    Embodiment 94. The compound of embodiment 86, wherein the modified oligonucleotide comprises 7 phosphodiester internucleoside linkages.
    Embodiment 95. The compound of any of embodiments 91 to 94, wherein each remaining internucleoside linkage is a phosphodiester internucleoside linkage.
    Embodiment 96. The compound of any of embodiments 1-85, wherein the compound has an NSNSNONSNONSNONSNONSNONSNONSNONSNSN internucleoside linkage motif, wherein each N represents a nucleoside, each S represents a phosphorothioate internucleoside linkage, and each 0 represents a phosphodiester internucleoside linkage.
    Embodiment 97. The compound of any of embodiments 1-85, wherein the compound has an NSNONONONSNSNSNSNSNSNSNSNONONONSNSN internucleoside linkage motif, wherein each N represents a nucleoside, each S represents a phosphorothioate internucleoside linkage, and each 0 represents a phosphodiester internucleoside linkage.
    Embodiment 98. The compound of any of embodiments 1-97 comprising at least one conjugate.
    Embodiment 99. The compound of any of embodiments 1-98 consisting of the modified oligonucleotide.
    Embodiment 100. The compound of any of embodiments 1-99, wherein the compound modulates splicing of the Tau transcript.
    Embodiment 101. The compound of any of embodiments 1-99, wherein the compound decreases expression of the 4R Tau isoform.
    Embodiment 102. The compound of any of embodiments 1-99, wherein the compound decreases expression Tau mRNA comprising exon 10.
    Embodiment 103. The compound of any of embodiments 1-99, wherein the compound decreases expression Tau protein comprising amino acids encoded from exon 10 mRNA.
    Embodiment 104. The compound of any of embodiments 1-8 or 30-103, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 8-200.
    Embodiment 105. The compound of any of embodiments 1-8 or 30-103, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 33, 34, 147, 148, 149, 150, 151, 152, or 153.
    Embodiment 106. The compound of any of embodiments 1-8 or 30-103, having a nucleobase sequence consisting of any of the sequences as set forth in SEQ ID NOs. 8-200.
    Embodiment 107. The compound of any of embodiments 1-8 or 30-103, having a nucleobase sequence consisting of any of the sequences as set forth in SEQ ID NOs. 33, 34, 147, 148, 149, 150, 151, 152, or 153.
    Embodiment 108. A double stranded compound comprising the compound of any of embodiments 1-107.
    Embodiment 109. A compound consisting of ISIS 670011.
    Embodiment 110. A compound consisting of ISIS 670012.
    Embodiment 111. A compound consisting of ISIS 670013.
    Embodiment 112. A compound consisting of ISIS 670014.
    Embodiment 113. A compound consisting of ISIS 670015.
    Embodiment 114. A compound consisting of ISIS 670016.
    Embodiment 115. A compound consisting of ISIS 670017.
    Embodiment 116. A compound consisting of ISIS 670018.
    Embodiment 117. A compound consisting of ISIS 670019.
    Embodiment 118. A compound consisting of ISIS 670020.
    Embodiment 119. A compound consisting of ISIS 670021.
    Embodiment 120. A compound consisting of ISIS 670022.
    Embodiment 121. A compound consisting of ISIS 670023.
    Embodiment 122. A compound consisting of ISIS 670024.
    Embodiment 123. A compound consisting of ISIS 670025.
    Embodiment 124. A compound consisting of ISIS 670026.
    Embodiment 125. A compound consisting of ISIS 670027.
    Embodiment 126. A compound consisting of ISIS 670028.
    Embodiment 127. A compound consisting of ISIS 678329.
    Embodiment 128. A compound consisting of ISIS 678330.
    Embodiment 129. A compound consisting of ISIS 678331.
    Embodiment 130. A compound consisting of ISIS 678332.
    Embodiment 131. A compound consisting of ISIS 678333.
    Embodiment 132. A compound consisting of ISIS 678334.
    Embodiment 133. A compound consisting of ISIS 693840.
    Embodiment 134. A compound consisting of ISIS 693841.
    Embodiment 135. A compound consisting of ISIS 693842.
    Embodiment 136. A compound consisting of ISIS 693843.
    Embodiment 137. A compound consisting of ISIS 693844.
    Embodiment 138. A compound consisting of ISIS 693845.
    Embodiment 139. A compound consisting of ISIS 693846.
    Embodiment 140. A compound consisting of ISIS 693847.
    Embodiment 141. A compound consisting of ISIS 693848.
    Embodiment 142. A compound consisting of ISIS 693849.
    Embodiment 143. A compound consisting of ISIS 549577.
    Embodiment 144. A compound consisting of ISIS 549580.
    Embodiment 145. A compound consisting of ISIS 549581.
    Embodiment 146. A compound consisting of ISIS 549582.
    Embodiment 147. A compound consisting of ISIS 549583.
    Embodiment 148. A compound consisting of ISIS 549584.
    Embodiment 149. A compound consisting of ISIS 549585.
    Embodiment 150. A compound consisting of ISIS 549586.
    Embodiment 151. A compound consisting of ISIS 617341.
    Embodiment 152. A compound consisting of ISIS 617351.
    Embodiment 153. A compound consisting of ISIS 617352.
    Embodiment 154. A compound consisting of ISIS 617353.
    Embodiment 155. A compound consisting of ISIS 617358.
    Embodiment 156. A compound consisting of ISIS 617360.
    Embodiment 157. A compound consisting of ISIS 617361.
    Embodiment 158. A compound consisting of ISIS 617362.
    Embodiment 159. A method of modulating splicing of a Tau transcript in a cell comprising contacting the cell with a compound according to any of embodiments 1-158.
    Embodiment 160. The method of embodiment 158, wherein the cell is in vitro.
    Embodiment 161. The method of embodiment 158, wherein the cell is in an animal.
    Embodiment 162. A method of modulating the expression of Tau protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-158.
    Embodiment 163. The method of embodiment 162, wherein expression of the 4R isoform of Tau protein is decreased.
    Embodiment 164. The method of embodiment 162, wherein expression of the 3R isoform of Tau protein is increased.
    Embodiment 165. The method of any of embodiments 162 to 164, wherein the cell is in vitro.
    Embodiment 166. The method of any of embodiments 162 to 164, wherein the cell is in an animal.
    Embodiment 167. A method of reducing or ameliorating one or more symptoms associated with a tau-associated disorder, comprising contacting a cell with a compound according to any of embodiments 1-158.
    Embodiment 168. The method of embodiment 167, wherein the symptom is seizure.
    Embodiment 169. The method of any of embodiments 167-168, wherein the cell is in an animal.
    Embodiment 170. A pharmaceutical composition comprising a compound according to any of embodiments 1-158 and a pharmaceutically acceptable carrier or diluent.
    Embodiment 171. The pharmaceutical composition of embodiment 170, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
    Embodiment 172. A method comprising administering the pharmaceutical composition of embodiments 170 or 171 to an animal.
    Embodiment 173. The method of embodiment 172, wherein the administration is by injection.
    Embodiment 174. The method of embodiment 172 or 173, wherein the administration is systemic.
    Embodiment 175. The method of embodiment 172 or 173, wherein the administration is to the CNS.
    Embodiment 176. The method of any of embodiments 172-175, wherein the animal has one or more symptoms associated with one or more tau-associated disorders.
    Embodiment 177. The method of embodiment 176, wherein the administration results in amelioration of one or more symptoms associated with one or more tau-associated disorders.
    Embodiment 178. The method of any of embodiments 172-175, wherein the animal has one or more symptoms associated with Alzheimer's disease.
    Embodiment 179. The method of embodiment 178, wherein the administration results in amelioration of one or more symptoms associated with Alzheimer's disease.
    Embodiment 180. The method of any of embodiments 172-179, wherein the animal is a mouse.
    Embodiment 181. The method of any of embodiments 172-179, wherein the animal is a human.
    Embodiment 182. Use of the compound of any of embodiments 1 to 158 or the composition of embodiments 170-171 for the preparation of a medicament for use in the treatment of a tau-associated disorder.
    Embodiment 183. Use of the compound of any of embodiments 1 to 158 or the composition of embodiments 170-171 for the preparation of a medicament for use in the amelioration of one or more symptoms associated a tau-associated disorder.
    Embodiment 184. The compound, composition, or method of any of embodiments 1-183, wherein the nucleobase sequence does not consist of the nucleobase sequence as set forth in SEQ ID NO.: 45, 66, 68, 69, or 200.
    Embodiment 185. The compound, composition, or method of any of embodiments 1-183, wherein the nucleobase sequence does not consist of the nucleobase sequence











CCAGCTTCTTATTAATTATC



or



 



TAAGATCCAGCTTCTTATTA.






    Embodiment 186. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121828 and nucleobase 121847 of SEQ ID NO.: 1.
    Embodiment 187. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121834 and nucleobase 121853 of SEQ ID NO.: 1.
    Embodiment 188. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121840 and nucleobase 121859 of SEQ ID NO.: 1.
    Embodiment 189. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121924 and nucleobase 121941 of SEQ ID NO.: 1.
    Embodiment 190. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121012 and nucleobase 121029 of SEQ ID NO.: 1.
    Embodiment 191. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121020 and nucleobase 121037 of SEQ ID NO.: 1.
    Embodiment 192. The compound, composition, or method of any of embodiments 1-183, wherein the target region is not within nucleobase 121024 and nucleobase 121041 of SEQ ID NO.: 1.





BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 depicts a graphical representation of Tau isoforms. The isoforms can differ from each other in the number of tubulin-binding domains (three or four repeats located in the C-terminal half of the protein) and are referred to as 3R or 4R Tau isoforms, respectively. They can also differ in the presence or absence of either one or two 29-amino-acid-long, highly acidic inserts at the N-terminal portion of the protein (the projection domain). Between the projection domain and the microtubule-binding domain lies a basic proline-rich region.



DETAILED DESCRIPTION
Unless specific definitions are provided, the nomenclature used 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. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21st edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
As used herein, “modified sugar moiety” means a substituted sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.
As used herein, “substituted sugar moiety” means a furanosyl comprising at least one substituent group that differs from that of a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position.
As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
As used herein, “MOE” means —OCH2CH2OCH3.
As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholino, modified morpholinos, cyclohexenyls and cyclohexitols.
As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
As used herein, “heterocyclic base” or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.
As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.
As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′bridge.
As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2—O-2′bridge.
As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.
As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.
As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.
As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.
As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
As used herein, “detectable and/or measureable activity” means a statistically significant activity that is not zero.
As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.
As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes.
As used herein, “mRNA” means an RNA molecule that encodes a protein.
As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
As used herein, “transcript” means an RNA molecule transcribed from DNA. Transcripts include, but are not limited to mRNA, pre-mRNA, and partially processed RNA.
As used herein, “Tau transcript” means a transcript transcribed from a Tau gene. In certain embodiments, a Tau transcript is represented by GENBANK Accession No NT_010783.15, truncated from nucleotides 9240000 to 9381000 (SEQ ID NO: 1), or a variant thereof.
As used herein, “Tau gene” means a gene that encodes a Tau protein and any Tau protein isoforms. In certain embodiments, Tau protein isoforms include the 3R isoform and the 4R isoform.
As used herein, “3R” means a Tau transcript that includes does not include exon 10.
As used herein, “3R Tau isoform” means a Tau protein isoform that does not include amino acids encoded from exon 10.
As used herein, “4R” means a Tau transcript that includes exon 10.
As used herein, “4R Tau isoform” means a Tau protein isoform that includes amino acids encoded from exon 10.
As used herein, “Tau-associated disease” means any neurological or neurodegenerative disease associated with Tau. Non-limiting examples of Tau-associated disorders include Alzheimer's disease, progressive supranuclear palsy, dementia pugilistica, frontotemporal dementia, 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, corticobasal ganglionic degeneration, argyrophilic grain disease, supranuclear palsy, corticobasal degeneration, frontotemporal dementia, or frontotemporal lobar degeneration.
As used herein, “Tauopathy” means disorders characterized by a build-up of Tau protein in the brain.
As used herein, “Tau-specific inhibitor” includes but is not limited to a “antisense compound” targeted to Tau.
As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means 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 means 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. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.
As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
As used herein, “motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof.
As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(Rbb)(Rcc)), imino(═NRbb), amido (—C(O)N(Rbb)(Rcc) or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)N(Rbb)(Rcc) or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)N(Rbb)(Rcc)), thioureido (—N(Rbb)C(S)N(Rbb)(Rcc)), guanidinyl (—N(Rbb)C(═NRbb)N(Rbb)(Rcc)), amidinyl (—C(═NRbb)N(Rbb)(Rcc) or —N(Rbb)C(═NRbb)(Raa)), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb) and sulfonamidyl (—S(O)2N(Rbb)(Rcc) or —N(Rbb)S(O)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.
As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines Aliphatic groups as used herein may optionally include further substituent groups.
As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
As used herein, “aminoalkyl” means an amino substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or poly-cyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
Oligomeric Compounds
In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
Certain Sugar Moieties
In certain embodiments, oligomeric compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, O—C1-C10 substituted alkyl; O—C1-C10 alkoxy; O—C1-C10 substituted alkoxy, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′,2′-bis substituted sugar moieties and nucleosides).
Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, O—C1-C10 alkoxy; O—C1-C10 substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(Rm)-alkyl; O-alkenyl, S-alkenyl, or N(Rm)-alkenyl; O-alkynyl, S-alkynyl, N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH2, N3, OCF3, O—CH3, O(CH2)3NH2, CH2—CH═CH2, O—CH2—CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O—CH2—C(═O)—N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.
In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF3, O—CH3, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(CH3)2, —O(CH2)2O(CH2)2N(CH3)2, and O—CH2—C(═O)—N(H)CH3.
In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH3, and OCH2CH2OCH3.
Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or, —C(RaRb)—O—N(R)—; 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (cEt) and 4′-CH(CH2OCH3)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)—O-2′-, wherein each R is, independently, H, a protecting group, or C1-C12 alkyl; 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).
In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)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-C12 alkenyl, substituted C2-C12 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, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
each J1 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(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA (also referred to as constrained ethyl or cEt), (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.







wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.

Additional bicyclic sugar moieties are known in the art, 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., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).
In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:




wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a nucleobase moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group; q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and
each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:




In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH2—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
Certain Nucleobases
In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.
In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Certain Internucleoside Linkages
In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), α or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C(═O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O-5′), and thioformacetal (3′-S—CH2—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
Certain Motifs
In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
Certain Sugar Motifs
In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or “wings” and an internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5′-wing differs from the sugar modification motif of the 3′-wing (asymmetric gapmer). In certain embodiments, oligonucleotides comprise 2′-MOE modified nucleosides in the wings and 2′-F modified nucleosides in the gap.
In certain embodiments, oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2′-MOE. In certain embodiments, oligonucleotides are uniform 2′-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt.
In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified. In certain embodiments, the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a 2′-MOE region. In certain embodiments, the uniform region is a 2′-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region.
In certain embodiments, the oligonucleotide does not comprise more than 4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances, antisesense oligonucleotides comprising more than 4 contiguous 2′-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA. In certain embodiments, such cleavage is avoided by not having more than 4 contiguous 2′-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired.
Certain Internucleoside Linkage Motifs
In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphoro-thioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
In certain embodiments, compounds and compositions, including modified oligonucleotides, are delivered to the CNS. In certain embodiments, compounds and compositions, including modified oligonucleotides, are delivered to the CNS via intracerebroventricular administration or intracerebroventricular bolus administration. In certain embodiments, one or more modified oligonucleotides may exhibit high potency and high selectivity toward a nucleic acid target, but may possess certain degrees of acute toxicity when delivered into the CNS via intracerebroventricular administration. In certain embodiments, introduction of one or more modifications to the internucleoside linkages of the modified oligonucleotide ameliorates or reduces acute toxicity associated with CNS delivery via intracerebroventricular administration. In certain embodiments, introduction of one or more phosphodiester internucleoside linkages into the modified oligonucleotide ameliorates or reduces acute toxicity associated with CNS delivery via intracerebroventricular administration.
In certain embodiments, an oligomeric compound has an internucleoside linkage motif selected from the table below, wherein each “N” represents a nucleoside, each subscript “s” represents a phosphorothioate internucleoside linkage, and each subscript “o” represents a phosphodiester internucleoside linkage:










Internucleoside Linkage Motifs

















NSNSNONSNONSNONSNONSNONSNONSNONSNSN



NSNONONONSNSNSNSNSNSNSNSNONONONSNSN










In certain embodiments, the inclusion of 3, 4, 5, 6, 7, 8, or 9 phosphodiester internucleoside linkages into the modified oligonucleotide ameliorates or reduces acute toxicity associated with CNS delivery via intracerebroventricular administration.
Certain Nucleobase Modification Motifs
In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.
In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.
In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
Certain Overall Lengths
In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≦Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths.
One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5′-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3′-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in “10-20” has no effect in that embodiment.
Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.
Certain Oligonucleotides
In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
Certain Conjugate Groups
In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′ end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group. In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
Antisense Compounds
In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
In certain embodiments antisense compounds and antisense oligonucleotides comprise single-strand compounds. In certain embodiments antisense compounds and antisense oligonucleotides comprise double-strand compounds.
Certain Modified Oligonucleotides
In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 31. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 33. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO.34. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO.35. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 36. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 37. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 45.
In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 72. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 79. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 82. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 83. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 97. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 106. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 107. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 112. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 113. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 130. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 140.
In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 141. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 142. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 143. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 144. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 145. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 146. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 147. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 148. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 149.
In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 150. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 151. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 152. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 153. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 157. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 158. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 160. In certain embodiments, a modified oligonucleotide has the sequence of SEQ ID NO. 200.
Certain Pathways and Mechanisms Associated with Neurodegenerative Disorders
In certain embodiments, a neurodegenerative syndrome or disorder may be any neurodegenerative syndrome or disorder associated with Tau. Non limiting examples of a neurodegenerative disorder associated with Tau may include Alzheimer's disease, progressive supranuclear palsy, dementia pugilistica, frontotemporal dementia, 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, corticobasal ganglionic degeneration, argyrophilic grain disease, supranuclear palsy, corticobasal degeneration, frontotemporal dementia, or frontotemporal lobar degeneration. In some embodiments, the method of the invention comprises modifying frontotemporal dementia (FTD). In other embodiments, the method of the invention comprises modifying Alzheimer's disease (AD). In yet other embodiments, the method of the invention comprises modifying progressive supranuclear palsy. In other embodiments, the method of the invention comprises modifying corticobasalganglionic degeneration.
In certain embodiments, described herein are compositions and methods of modifying a neurodegenerative syndrome by altering the splicing of a nucleic acid encoding Tau. Tau is a protein found in multiple tissues, but is particularly abundant in axons of neurons. 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. 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 0N, 1N, or 2N Tau respectively. Inclusion of exon 10 at the C-terminus leads to inclusion of the microtubule binding domain encoded by exon 10. Since there are 3 mictrotubule 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. (FIG. 1). Tau without exon 10 is termed 3R Tau. In healthy subjects, the ratio of 3R:4R Tau is developmentally regulated, with fetal tissues expressing almost 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 syndromes such as FTD Tauopathies.
In certain embodiments, the 4R:3R Tau ratio in the central nervous system of the subject may be normal, low or high. As used herein, a “normal 4R:3R Tau ratio” in the central nervous system signifies a 4R:3R Tau ratio in the central nervous system that is substantially the same as the 4R:3R Tau ratio in the central nervous system of a subject from the same species and of approximately the same age not suffering from a neurodegenerative disease. In certain embodiments, certain antisense oligonucleotide compositions and/or methods decrease the normal 4R:3R Tau ratio in the central nervous system of a subject. In other embodiments, certain antisense oligonucleotide compositions and/or methods decreases a low 4R:3R Tau ratio in the central nervous system of a subject.
In certain embodiments, certain antisense oligonucleotide compositions and/or methods decrease a high 4R:3R Tau ratio in the central nervous system of a subject. In certain embodiments, certain antisense oligonucleotide compositions and/or methods decreases a high 4R:3R Tau ratio caused by a defect in splicing of the nucleic acid encoding Tau in the subject. Defects in splicing of the nucleic acid encoding Tau in the subject may be caused, for instance, by genetic mutations altering the splicing of the nucleic acid encoding Tau and leading to a high 4R:3R Tau ratio. A mutation may be either a substitution mutation or a deletion mutation which creates a new, aberrant, splice element. Non-limiting examples of genetic mutations that may alter the splicing of the nucleic acid encoding Tau and lead to a high 4R:3R Tau ratio may include N279K, P301S, 280, L284L, N296H, N296N, 296N, P301S, G303V, E10+11, E10+12, E10+13, E+10+14 and E10+16, and E10+19.
In certain embodiments, administration of an antisense oligonucleotide decreases the 4R:3R Tau ratio in the central nervous system of a subject by altering the splicing of a nucleic acid encoding Tau.
In certain embodiments, increasing exclusion of exon 10 of a Tau transcript inhibits one or more tau-associated disorders. In certain embodiments, the tau-associated disorder may be any of Alzheimer's Disease, frontotemporal Dementia (FTD), FTDP-17, Progressive Supranuclear Palsy (PSP), Chronic Traumatic Encephalopathy (CTE), Corticobasal Ganglionic Degeneration (CBD), epilepsy, Dravet's Syndrome, 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, supranuclear palsy, corticobasal degeneration, or frontotemporal lobar degeneration.
Certain Target Nucleic Acids and Mechanisms
In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target nucleic acid is a tau transcript. In certain embodiments, the target RNA is a Tau pre-mRNA.
In certain embodiments, an antisense compound is complementary to a region of Tau pre-mRNA. In certain embodiments, an antisense compound is complementary within a region of Tau pre-mRNA comprising an exon encoding the 4R isoform. In certain embodiments, an antisense compound is complementary to a region of Tau pre-mRNA comprising an intron-exon splice junction. In certain embodiments, an antisense compound is complementary to a region of Tau pre-mRNA comprising the intron-exon splice junction adjacent to exon 10. In certain embodiments, an antisense compound is complementary to a region of Tau pre-mRNA comprising the intron-exon splice junction between intron 9 and exon 10. In certain embodiments, an antisense compound is complementary to a region of Tau pre-mRNA comprising the intron-exon splice junction between exon 10 and intron 10. In certain embodiments, an antisense compound is complementary within a region of Tau pre-mRNA consisting of exon 10.
In certain embodiments, an antisense compound is complementary within a region of Tau pre-mRNA comprising an exonic splicing silencer within exon 10. In certain embodiments, an antisense compound is complementary within a region of Tau pre-mRNA comprising an exonic splicing enhancer within exon 10.
In certain embodiments, an antisense compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a Tau transcript.
In certain embodiments, the target region is within nucleobase 121708 and nucleobase 122044 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121796 and nucleobase 122044 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121796 and nucleobase 121885 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121873 and nucleobase 121898 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121869 and nucleobase 121943 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 122020 and nucleobase 122044 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121881 and nucleobase 121898 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121880 and nucleobase 121897 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121879 and nucleobase 121896 of SEQ ID NO.: 1.
In certain embodiments, the target region is within nucleobase 121878 and nucleobase 121895 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121877 and nucleobase 121894 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121876 and nucleobase 121893 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121875 and nucleobase 121892 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121874 and nucleobase 121891 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 121873 and nucleobase 121890 of SEQ ID NO.: 1.
In certain embodiments, an antisense oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing a Tau pre-mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of Tau 3R mRNA. In certain embodiments, an antisense oligonucleotide increases the exclusion of exon 10 in Tau mRNA. In certain embodiments, an antisense oligonucleotide decreases the inclusion of exon 10 in Tau mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of 3R Tau mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of 4R Tau mRNA.
In certain embodiments it is desirable to alter the splicing of Tau pre-mRNA to exclude exon 10. By altering the splicing of Tau pre-mRNA to exclude exon 10, expression of 3R Tau will increase and expression of 4R Tau will decrease. In certain embodiments it is desirable to alter the splicing of Tau pre-mRNA to decrease expression of 4R Tau.
In certain embodiments, an antisense oligonucleotide decreases the amount of Tau 3R mRNA. In certain embodiments, an antisense oligonucleotide decreases the exclusion of exon 10 in Tau mRNA. In certain embodiments, an antisense oligonucleotide increases the inclusion of exon 10 in Tau mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of 3R Tau mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of 4R Tau mRNA. In certain embodiments it is desirable to alter the splicing of Tau pre-mRNA to include exon 10, for example to produce a phenotypic effect in a mouse or animal model.
Certain Pharmaceutical Compositions
In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.
In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more 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 oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80TM; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the eyes, ears).
In certain embodiments, a pharmaceutical composition is administered to an animal having at least one Tau-associated disorder. In certain embodiments, such administration results in reduction in expression of the 4R isoform. In certain embodiments, a pharmaceutical composition is administered to an animal having at least one symptom associated with a Tau-associated disorder. In certain embodiments, such administration results in amelioration of at least one symptom. In certain embodiments, administration of a pharmaceutical composition to an animal results in a decrease of 4R mRNA in a cell of the animal. In certain embodiments, such administration results in an increase in 3R mRNA. In certain embodiments, such administration results in a decrease in 4R protein and an increase 3R protein. In certain embodiments, a 3R protein is preferred over a 4R protein. In certain embodiments, the administration of certain antisense oligonucleotides delays the onset of one or more Tau-associated disorders. In certain embodiments, the administration of certain antisense oligonucleotides prevents or reduces seizures. In certain embodiments, the administration of certain antisense oligonucleotides causes the amount of 4R protein to decrease in the CNS. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular phenotype.
Nonlimiting 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, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) for natural uracil of RNA).
Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “ATmeCGAUCG,” wherein meC indicates a cytosine base comprising a methyl group at the 5-position.
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
Effects of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
A series of modified oligonucleotides were designed to target exon 10 of human Tau and were screened for their effects in reducing exon 10 inclusion in vitro. These modified oligonucleotides were designed by shifting 4 nucleotides upstream or downstream (i.e. microwalk) across the target site. They are 18 nucleosides in length, wherein each nucleoside has a 2′-MOE modification. Each internucleoside linkage throughout the modified oligonucleotide are phosphorothioate internucleoside linkages (P═S). All cytosine residues throughout the modified oligonucleotides are 5-methylcytosines.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
A172 cells were transfected using Lipofectamine2000® with 5 nM concentration of modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human primer probe set 9_10 or 10_11 was used to measure mRNA levels. Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent of Tau exon 10 mRNA expression, relative to untreated control levels and is denoted as “% UTC.”
Human primer probe set 9_10 (forward sequence CACTGAGAACCTGAAGCACC, designated herein as SEQ ID NO: 2; reverse sequence GGACTGGACGTTGCTAAGATC, designated herein as SEQ ID NO: 3; probe sequence TTAATTATCTGCACCTTCCCGCCTCC, designated herein as SEQ ID NO: 4).
Human primer probe set 10_11 (forward sequence GGATAATATCAAACACGTCCCG, designated herein as SEQ ID NO: 5; reverse sequence TGCCTAATGAGCCACACTTG, designated herein as SEQ ID NO: 6; probe sequence GTCTACAAACCAGTTGACCTGAGC, designated herein as SEQ ID NO: 7).
As illustrated in Tables 1 and 2, ISIS 549583, 549584, 549585, 549586, 549595, 549571, 549566, 549570, 549587, 549568, 549617, 549567, 549576, 549577, 549580, and 549581 show 70% or greater reduction in human Tau exon 10 inclusion comparing to untreated control.







TABLE 1







Effects of uniform 2′-MOE modified oligonucleo-


tides on human Tau exon 10 using Primer Probe


Set 9_10















SEQ ID
SEQ ID






NO:
NO:
SEQ


ISIS

%
1 Start
1 Stop
ID


NO
Sequence
UTC
Site
Site
NO















549543
CCCGCCTACTTGCTCGCA
130
121708
121725
8


 


549544
TGGACCCGCCTACTTGCT
119
121712
121729
9


 


549545
ACCCTGGACCCGCCTACT
106
121716
121733
10


 


549546
CATGCGCCACCCTGGACC
84
121724
121741
11


 


549547
GTGACATGCGCCACCCTG
78
121728
121745
12


 


549548
ATGAGTGACATGCGCCAC
63
121732
121749
13


 


549549
TTCGATGAGTGACATGCG
70.0
121736
121753
14


 


549550
CACTTTCGATGAGTGACA
69
121740
121757
15


 


549551
CCTCCACTTTCGATGAGT
103
121744
121761
16


 


549552
GACGCCTCCACTTTCGAT
148
121748
121765
17


 


549553
CAAGGACGCCTCCACTTT
116
121752
121769
18


 


549554
CTCGCAAGGACGCCTCCA
80
121756
121773
19


 


549555
CTTGCTCGCAAGGACGCC
102
121760
121777
20


 


549556
CCTGCTTGCTCGCAAGGA
109
121764
121781
21


 


549557
CCCGCCTGCTTGCTCGCA
100
121768
121785
22


 


549558
TGGACCCGCCTGCTTGCT
111
121772
121789
23


 


549559
ACCCTGGACCCGCCTGCT
108
121776
121793
24


 


549560
CGCCACCCTGGACCCGCC
88
121720
121737
25





121780
121797



 


549561
GACACGCCACCCTGGACC
83
121784
121801
26


 


549562
GAGTGACACGCCACCCTG
77
121788
121805
27


 


549563
GGATGAGTGACACGCCAC
58
121792
121809
28


 


549564
AAAAGGATGAGTGACACG
49
121796
121813
29


 


549565
AGAAAAAAGGATGAGTGA
35
121800
121817
30


 


549566
AGCCAGAAAAAAGGATGA
21
121804
121821
31


 


549567
TGGTAGCCAGAAAAAAGG
32
121808
121825
32


 


549583
TTATCCTTTGAGCCACAC
11
121876
121893
33


 


549584
GATATTATCCTTTGAGCC
11
121880
121897
34


 


549585
GTTTGATATTATCCTTTG
13
121884
121901
35


 


549586
ACGTGTTTGATATTATCC
17
121888
121905
36


 


549587
CGGGACGTGTTTGATATT
24
121892
121909
37


 


549588
CTCCCGGGACGTGTTTGA
52
121896
121913
38


 


549589
CCGCCTCCCGGGACGTGT
57
121900
121917
39


 


549590
ACTGCCGCCTCCCGGGAC
84
121904
121921
40


 


549591
TCACACTGCCGCCTCCCG
48
121908
121925
41


 


549592
GTACTCACACTGCCGCCT
30
121912
121929
42


 


549593
GAAGGTACTCACACTGCC
35
121916
121933
43


 


549594
GTGTGAAGGTACTCACAC
63
121920
121937
44


 


549595
GGACGTGTGAAGGTACTC
20
121924
121941
45


 


549596
CATGGGACGTGTGAAGGT
125
121928
121945
46


 


549597
GGCGCATGGGACGTGTGA
216
121932
121949
47


 


549598
GCACGGCGCATGGGACGT
242
121936
121953
48


 


549599
CACAGCACGGCGCATGGG
158
121940
121957
49


 


549600
AAGCCACAGCACGGCGCA
162
121944
121961
50


 


549601
ATTCAAGCCACAGCACGG
175
121948
121965
51


 


549602
AATAATTCAAGCCACAGC
164
121952
121969
52


 


549603
TCCTAATAATTCAAGCCA
200
121956
121973
53


 


549604
CACTTCCTAATAATTCAA
133
121960
121977
54


 


549605
ACACCACTTCCTAATAAT
113
121964
121981
55


 


549606
ACTCACACCACTTCCTAA
136
121968
121985
56


 


549608
GTGTACGCACTCACACCA
80.6
121976
121993
57


 


549609
GCAAGTGTACGCACTCAC
49.9
121980
121997
58


 


549610
TCTCGCAAGTGTACGCAC
86.5
121984
122001
59


 


549611
AGTGTCTCGCAAGTGTAC
62.4
121988
122005
60


 


549612
ATGCAGTGTCTCGCAAGT
46.4
121992
122009
61


 


549613
TTCTATGCAGTGTCTCGC
46.6
121996
122013
62


 


549614
TTTATTCTATGCAGTGTC
33.2
122000
122017
63


 


549615
AGGATTTATTCTATGCAG
34.3
122004
122021
64


 


549616
AAGAAGGATTTATTCTAT
46.5
122008
122025
65


 


549617
GCCCAAGAAGGATTTATT
29.6
122012
122029
66


 


549618
GAGAGCCCAAGAAGGATT
38.6
122016
122033
67


 


549619
TCCTGAGAGCCCAAGAAG
36.9
122020
122037
68


 


549620
CAGATCCTGAGAGCCCAA
38.3
122024
122041
69
















TABLE 2







Effects of uniform 2′-MOE modified oligonucleotides on human Tau exon 10 using Primer Probe


Set 10_11
















SEQ ID NO: 1
SEQ ID NO: 1

SEQ


ISIS No.
Sequence
% UTC
Start Site
Stop Site
Mismatches
ID No.
















549543
CCCGCCTACTTGCTCGCA
128
121708
121725
0
8





121768
121785
1



 


549544
TGGACCCGCCTACTTGCT
117
121712
121729
0
9





121772
121789
1



 


549545
ACCCTGGACCCGCCTACT
98
121716
121733
0
10





121776
121793
1



 


549546
CATGCGCCACCCTGGACC
78
121724
121741
0
11


 


549547
GTGACATGCGCCACCCTG
75
121728
121745
0
12


 


549548
ATGAGTGACATGCGCCAC
63
121732
121749
0
13


 


549549
TTCGATGAGTGACATGCG
74
121736
121753
0
14


 


549550
CACTTTCGATGAGTGACA
76
121740
121757
0
15


 


549551
CCTCCACTTTCGATGAGT
107
121744
121761
0
16


 


549552
GACGCCTCCACTTTCGAT
137
121748
121765
0
17


 


549553
CAAGGACGCCTCCACTTT
108
121692
121709
1
18





121752
121769
0



 


549554
CTCGCAAGGACGCCTCCA
71
121696
121713
1
19





121756
121773
0



 


549555
CTTGCTCGCAAGGACGCC
108
121700
121717
1
20





121760
121777
0



 


549556
CCTGCTTGCTCGCAAGGA
106
121704
121721
1
21





121764
121781
0



 


549557
CCCGCCTGCTTGCTCGCA
93
121708
121725
1
22





121768
121785
0



 


549558
TGGACCCGCCTGCTTGCT
111
121712
121729
1
23





121772
121789
0



 


549559
ACCCTGGACCCGCCTGCT
100
121716
121733
1
24





121776
121793
0



 


549560
CGCCACCCTGGACCCGCC
89
121720
121737
0
25





121780
121797
0



 


549561
GACACGCCACCCTGGACC
80
121784
121801
0
26


 


549562
GAGTGACACGCCACCCTG
81
121788
121805
0
27


 


549563
GGATGAGTGACACGCCAC
54
121792
121809
0
28


 


549564
AAAAGGATGAGTGACACG
45
121796
121813
0
29


 


549565
AGAAAAAAGGATGAGTGA
34
121800
121817
0
30


 


549566
AGCCAGAAAAAAGGATGA
19
121804
121821
0
31


 


549567
TGGTAGCCAGAAAAAAGG
31
121808
121825
0
32


 


549568
CCTTTGGTAGCCAGAAAA
23
121812
121829
0
70


 


549569
TGCACCTTTGGTAGCCAG
53
121816
121833
0
71


 


549570
TAATTATCTGCACCTTTG
26
121824
121841
0
72


 


549571
TTCTTAATTATCTGCACC
21
121828
121845
1
73


 


549572
CTTCTTCTTAATTATCTG
30
121832
121849
1
74


 


549573
CCAGCTTCTTCTTAATTA
34
121836
121853
1
75


 


549574
AGATCCAGCTTCTTCTTA
43
121840
121857
1
76


 


549575
GCTAAGATCCAGCTTCTT
25
121844
121861
0
77


 


549576
CGTTGCTAAGATCCAGCT
18
121848
121865
0
78


 


549577
TGGACGTTGCTAAGATCC
16
121852
121869
0
79


 


549578
GGACTGGACGTTGCTAAG
44
121856
121873
0
80


 


549579
ACTTGGACTGGACGTTGC
36
121860
121877
0
81


 


549580
CCACACTTGGACTGGACG
19
121864
121881
0
82


 


549581
TGAGCCACACTTGGACTG
16
121868
121885
0
83


 


549595
GGACGTGTGAAGGTACTC
20
121924
121941
0
45


 


549596
CATGGGACGTGTGAAGGT
128
121928
121945
0
46


 


549597
GGCGCATGGGACGTGTGA
199
121932
121949
0
47


 


549598
GCACGGCGCATGGGACGT
199
121936
121953
0
48


 


549599
CACAGCACGGCGCATGGG
149
121940
121957
0
49


 


549600
AAGCCACAGCACGGCGCA
156
121944
121961
0
50


 


549601
ATTCAAGCCACAGCACGG
166
121948
121965
0
51


 


549602
AATAATTCAAGCCACAGC
159
121952
121969
0
52


 


549603
TCCTAATAATTCAAGCCA
179
121956
121973
0
53


 


549604
CACTTCCTAATAATTCAA
123
121960
121977
0
54


 


549605
ACACCACTTCCTAATAAT
113
121964
121981
0
55


 


549606
ACTCACACCACTTCCTAA
119
121968
121985
0
56


 


549608
GTGTACGCACTCACACCA
77.2
121976
121993
0
57


 


549609
GCAAGTGTACGCACTCAC
53.0
121980
121997
0
58


 


549610
TCTCGCAAGTGTACGCAC
87.4
121984
122001
0
59


 


549611
AGTGTCTCGCAAGTGTAC
69.2
121988
122005
0
60


 


549612
ATGCAGTGTCTCGCAAGT
43.3
121992
122009
0
61


 


549613
TTCTATGCAGTGTCTCGC
41.4
121996
122013
0
62


 


549614
TTTATTCTATGCAGTGTC
29.2
122000
122017
0
63


 


549615
AGGATTTATTCTATGCAG
30.9
122004
122021
0
64


 


549616
AAGAAGGATTTATTCTAT
45.8
122008
122025
0
65


 


549617
GCCCAAGAAGGATTTATT
31.8
122012
122029
0
66


 


549618
GAGAGCCCAAGAAGGATT
41.8
122016
122033
0
67


 


549619
TCCTGAGAGCCCAAGAAG
41.7
122020
122037
0
68


 


549620
CAGATCCTGAGAGCCCAA
35.6
122024
122041
0
69









Example 2
Effects of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
A series of modified oligonucleotides were designed to target exon 10 of human Tau and were screened for their effects in reducing exon 10 inclusion in vitro. These modified oligonucleotides were designed by shifting 1 nucleotide upstream or downstream (i.e. microwalk) across the target site. They are 18 nucleosides in length, wherein each nucleoside has a 2′-MOE modification. Each internucleoside linkage throughout the modified oligonucleotide are phosphorothioate internucleoside linkages (P═S). All cytosine residues throughout the modified oligonucleotides are 5-methylcytosines.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
A172 cells were transfected using Lipofectamine2000® with 5 nM concentration of modified oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human Tau primer probe set 10_11 or 9_10 R5 was used to measure mRNA levels. Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent of Tau exon 10 mRNA expression, relative to untreated control levels and is denoted as “% UTC.”
Human primer probe set 10_11 (forward sequence GGATAATATCAAACACGTCCCG, designated herein as SEQ ID NO: 5; reverse sequence TGCCTAATGAGCCACACTTG, designated herein as SEQ ID NO: 6; probe sequence GTCTACAAACCAGTTGACCTGAGC, designated herein as SEQ ID NO: 7).
Human Tau primer probe set 9_10 R5 (forward sequence CACTGAGAACCTGAAGCACC, designated herein as SEQ ID NO: 2; reverse sequence GGACGTTGCTAAGATCCAGCT, designated herein as SEQ ID NO: 3; probe sequence TTAATTATCTGCACCTTCCCGCCTCC, designated herein as SEQ ID NO: 4).







TABLE 3







Effects of uniform 2′-MOE modified oligonucleo-


tides on human Tau exon 10 using Human Tau


primer probe set 10_11















SEQ ID
SEQ ID






NO: 1
NO:
SEQ


ISIS

%
Start
1 Stop
ID


NO
Sequence
UTC
Site
Site
NO















549564
AAAAGGATGAGTGACACG
43
121796
121813
29


 


617296
AAAAAGGATGAGTGACAC
38
121797
121814
84


 


617297
AAAAAAGGATGAGTGACA
40
121798
121815
85


 


617298
GAAAAAAGGATGAGTGAC
39
121799
121816
86


 


549565
AGAAAAAAGGATGAGTGA
45
121800
121817
30


 


617299
CAGAAAAAAGGATGAGTG
39
121801
121818
87


 


617300
CCAGAAAAAAGGATGAGT
34
121802
121819
88


 


617301
GCCAGAAAAAAGGATGAG
34
121803
121820
89


 


549566
AGCCAGAAAAAAGGATGA
21
121804
121821
31


 


617302
TAGCCAGAAAAAAGGATG
26
121805
121822
90


 


617303
GTAGCCAGAAAAAAGGAT
27
121806
121823
91


 


617304
GGTAGCCAGAAAAAAGGA
32
121807
121824
92


 


549567
TGGTAGCCAGAAAAAAGG
39
121808
121825
32


 


617305
TTGGTAGCCAGAAAAAAG
34
121809
121826
93


 


617306
TTTGGTAGCCAGAAAAAA
49
121810
121827
94


 


617307
CTTTGGTAGCCAGAAAAA
37
121811
121828
95


 


549568
CCTTTGGTAGCCAGAAAA
27
121812
121829
70


 


617308
ACCTTTGGTAGCCAGAAA
31
121813
121830
96


 


617309
CACCTTTGGTAGCCAGAA
23
121814
121831
97


 


617310
GCACCTTTGGTAGCCAGA
59
121815
121832
98


 


549569
TGCACCTTTGGTAGCCAG
59
121816
121833
71


 


617311
CTGCACCTTTGGTAGCCA
59
121817
121834
99


 


617312
TCTGCACCTTTGGTAGCC
58
121818
121835
100


 


617313
ATCTGCACCTTTGGTAGC
53
121819
121836
101


 


415866
TATCTGCACCTTTGGTAG
41
121820
121837
102


 


617314
TTATCTGCACCTTTGGTA
36
121821
121838
103


 


617315
ATTATCTGCACCTTTGGT
31
121822
121839
104


 


617316
AATTATCTGCACCTTTGG
38
121823
121840
105


 


549570
TAATTATCTGCACCTTTG
21
121824
121841
72


 


617317
TTAATTATCTGCACCTTT
23
121825
121842
106


 


617318
ATTAATTATCTGCACCTT
25
121826
121843
107


 


617319
TATTAATTATCTGCACCT
27
121827
121844
108


 


617320
TTATTAATTATCTGCACC
27
121828
121845
109


 


617321
CTTATTAATTATCTGCAC
26
121829
121846
110


 


617322
TCTTATTAATTATCTGCA
25
121830
121847
111


 


617323
TTCTTATTAATTATCTGC
25
121831
121848
112


 


617324
CTTCTTATTAATTATCTG
25
121832
121849
113


 


617325
GCTTCTTATTAATTATCT
26
121833
121850
114


 


617326
AGCTTCTTATTAATTATC
32
121834
121851
115


 


617327
CAGCTTCTTATTAATTAT
31
121835
121852
116


 


617328
CCAGCTTCTTATTAATTA
29
121836
121853
117


 


617329
TCCAGCTTCTTATTAATT
35
121837
121854
118


 


617330
ATCCAGCTTCTTATTAAT
29
121838
121855
119


 


617331
GATCCAGCTTCTTATTAA
33
121839
121856
120


 


617332
AGATCCAGCTTCTTATTA
33
121840
121857
121


 


617333
AAGATCCAGCTTCTTATT
37
121841
121858
122


 


617334
TAAGATCCAGCTTCTTAT
42
121842
121859
123


 


617335
CTAAGATCCAGCTTCTTA
34
121843
121860
124


 


549575
GCTAAGATCCAGCTTCTT
29
121844
121861
77


 


617336
TGCTAAGATCCAGCTTCT
42
121845
121862
125


 


617337
TTGCTAAGATCCAGCTTC
36
121846
121863
126


 


617338
GTTGCTAAGATCCAGCTT
35
121847
121864
127


 


549576
CGTTGCTAAGATCCAGCT
26
121848
121865
78


 


617339
ACGTTGCTAAGATCCAGC
25
121849
121866
128


 


617340
GACGTTGCTAAGATCCAG
30
121850
121867
129


 


617341
GGACGTTGCTAAGATCCA
24
121851
121868
130


 


549577
TGGACGTTGCTAAGATCC
25
121852
121869
79


 


617342
CTGGACGTTGCTAAGATC
27
121853
121870
131


 


617343
ACTGGACGTTGCTAAGAT
29
121854
121871
132


 


617344
GACTGGACGTTGCTAAGA
34
121855
121872
133


 


549578
GGACTGGACGTTGCTAAG
40
121856
121873
80


 


617345
TGGACTGGACGTTGCTAA
51
121857
121874
134


 


617346
TTGGACTGGACGTTGCTA
43
121858
121875
135


 


617347
CTTGGACTGGACGTTGCT
38
121859
121876
136


 


549579
ACTTGGACTGGACGTTGC
34
121860
121877
81


 


617348
CACTTGGACTGGACGTTG
39
121861
121878
137


 


617349
ACACTTGGACTGGACGTT
30
121862
121879
138


 


617350
CACACTTGGACTGGACGT
32
121863
121880
139


 


549580
CCACACTTGGACTGGACG
27
121864
121881
82


 


617351
GCCACACTTGGACTGGAC
23
121865
121882
140


 


617352
AGCCACACTTGGACTGGA
23
121866
121883
141


 


617353
GAGCCACACTTGGACTGG
26
121867
121884
142


 


549581
TGAGCCACACTTGGACTG
24
121868
121885
83
















TABLE 4







Effects of uniform 2′-MOE modified oligonucleo-


tides on human Tau exon 10 using Human Tau


primer probe set 9_10 R5















SEQ ID
SEQ ID






NO: 1
NO:
SEQ


ISIS

%
Start
1 Stop
ID


NO
Sequence
UTC
Site
Site
NO















617354
TTGAGCCACACTTGGACT
21
121869
121886
143


 


617355
TTTGAGCCACACTTGGAC
22
121870
121887
144


 


617356
CTTTGAGCCACACTTGGA
16
121871
121888
145


 


549582
CCTTTGAGCCACACTTGG
14
121872
121889
146


 


617357
TCCTTTGAGCCACACTTG
17
121873
121890
147


 


617358
ATCCTTTGAGCCACACTT
17
121874
121891
148


 


617359
TATCCTTTGAGCCACACT
19
121875
121892
149


 


549583
TTATCCTTTGAGCCACAC
20
121876
121893
33


 


617360
ATTATCCTTTGAGCCACA
21
121877
121894
150


 


617361
TATTATCCTTTGAGCCAC
15
121878
121895
151


 


617362
ATATTATCCTTTGAGCCA
16
121879
121896
152


 


549584
GATATTATCCTTTGAGCC
17
121880
121897
34


 


565989
TGATATTATCCTTTGAGC
17
121881
121898
153


 


565990
TTGATATTATCCTTTGAG
20
121882
121899
154


 


565991
TTTGATATTATCCTTTGA
28
121883
121900
155


 


549585
GTTTGATATTATCCTTTG
22
121884
121901
35


 


617363
TGTTTGATATTATCCTTT
25
121885
121902
156


 


617364
GTGTTTGATATTATCCTT
22
121886
121903
157


 


617365
CGTGTTTGATATTATCCT
20
121887
121904
158


 


549586
ACGTGTTTGATATTATCC
21
121888
121905
36


 


617366
GACGTGTTTGATATTATC
24
121889
121906
159


 


617367
GGACGTGTTTGATATTAT
16
121890
121907
160


 


617368
GGGACGTGTTTGATATTA
33
121891
121908
161


 


549587
CGGGACGTGTTTGATATT
20
121892
121909
37


 


617369
CCGGGACGTGTTTGATAT
25
121893
121910
162


 


617370
CCCGGGACGTGTTTGATA
43
121894
121911
163


 


617371
TCCCGGGACGTGTTTGAT
52
121895
121912
164


 


549588
CTCCCGGGACGTGTTTGA
53
121896
121913
38


 


549590
ACTGCCGCCTCCCGGGAC
48
121904
121921
40


 


617372
CACTGCCGCCTCCCGGGA
71
121905
121922
165


 


617373
ACACTGCCGCCTCCCGGG
60
121906
121923
166


 


617374
CACACTGCCGCCTCCCGG
40
121907
121924
167


 


549591
TCACACTGCCGCCTCCCG
47
121908
121925
41


 


617375
CTCACACTGCCGCCTCCC
33
121909
121926
168


 


617376
ACTCACACTGCCGCCTCC
34
121910
121927
169


 


617377
TACTCACACTGCCGCCTC
34
121911
121928
170


 


549592
GTACTCACACTGCCGCCT
31
121912
121929
42


 


617378
GGTACTCACACTGCCGCC
29
121913
121930
171


 


617379
AGGTACTCACACTGCCGC
34
121914
121931
172


 


617380
AAGGTACTCACACTGCCG
43
121915
121932
173


 


549593
GAAGGTACTCACACTGCC
33
121916
121933
43


 


617381
TGAAGGTACTCACACTGC
28
121917
121934
174


 


617382
GTGAAGGTACTCACACTG
41
121918
121935
175


 


617383
TGTGAAGGTACTCACACT
43
121919
121936
176


 


549594
GTGTGAAGGTACTCACAC
68
121920
121937
44


 


617384
CGTGTGAAGGTACTCACA
35
121921
121938
177


 


617385
ACGTGTGAAGGTACTCAC
34
121922
121939
178


 


617386
GACGTGTGAAGGTACTCA
29
121923
121940
179


 


549595
GGACGTGTGAAGGTACTC
26
121924
121941
45


 


617387
GGGACGTGTGAAGGTACT
36
121925
121942
180


 


617388
TGGGACGTGTGAAGGTAC
36
121926
121943
181


 


617389
ATGGGACGTGTGAAGGTA
94
121927
121944
182


 


549596
CATGGGACGTGTGAAGGT
141
121928
121945
46


 


617390
GCATGGGACGTGTGAAGG
297
121929
121946
183


 


510184
CGCATGGGACGTGTGAAG
295
121930
121947
184


 


617391
GCGCATGGGACGTGTGAA
274
121931
121948
185


 


549597
GGCGCATGGGACGTGTGA
284
121932
121949
47


 


510185
CGGCGCATGGGACGTGTG
276
121933
121950
186


 


617392
ACGGCGCATGGGACGTGT
304
121934
121951
187


 


510186
CACGGCGCATGGGACGTG
268
121935
121952
188


 


549598
GCACGGCGCATGGGACGT
305
121936
121953
48


 


617393
AGCACGGCGCATGGGACG
237
121937
121954
189


 


510187
CAGCACGGCGCATGGGAC
144
121938
121955
190


 


617394
ACAGCACGGCGCATGGGA
170
121939
121956
191


 


549599
CACAGCACGGCGCATGGG
183
121940
121957
49


 


549619
TCCTGAGAGCCCAAGAAG
42
122020
122037
68


 


617395
ATCCTGAGAGCCCAAGAA
38
122021
122038
192


 


617396
GATCCTGAGAGCCCAAGA
44
122022
122039
193


 


617397
AGATCCTGAGAGCCCAAG
35
122023
122040
194


 


549620
CAGATCCTGAGAGCCCAA
35
122024
122041
69


 


617398
CCAGATCCTGAGAGCCCA
39
122025
122042
195


 


617399
GCCAGATCCTGAGAGCCC
47
122026
122043
196


 


617400
AGCCAGATCCTGAGAGCC
38
122027
122044
197









Example 3
Inhibitory Effect of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
Several modified oligonucleotides from Tables 3 and 4 were selected and further evaluated for their effects on inhibiting human Tau exon 10 expression in vitro. ISIS 549595 was included in the study for comparison.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
A172 cells were transfected using Lipofectamine2000® with 0, 0.1, 0.3, 1, 3, 10, or 30 nM concentration of modified oligonucleotide as specified in the table below. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human Tau primer probe set 9_10 R5 was used to measure mRNA levels, except for ISIS 549581. Human Tau primer probe set 10_11 was used for this oligonucleotide. Human Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is presented in the table below and was calculated by plotting the concentrations of oligonucleotides used versus the percent inhibition of human Tau exon 10 mRNA expression achieved at each concentration, and noting the concentration of oligonucleotide at which 50% inhibition of human Tau exon 10 mRNA expression was achieved compared to the control. Results are presented below.







TABLE 5







Inhibitory effect of uniform 2′-MOE modified


oligonucleotides on human Tau exon 10 expression















SEQ ID
SEQ ID






NO:
NO:
SEQ


ISIS

IC50
1 Start
1 Stop
ID


NO
Sequence
(nM)
Site
Site
NO















549581
TGAGCCACACTTGGACTG
1.20
121868
121885
83


 


617354
TTGAGCCACACTTGGACT
1.39
121869
121886
143


 


617355
TTTGAGCCACACTTGGAC
1.08
121870
121887
144


 


617356
CTTTGAGCCACACTTGGA
0.74
121871
121888
145


 


549582
CCTTTGAGCCACACTTGG
0.49
121872
121889
146


 


617357
TCCTTTGAGCCACACTTG
0.64
121873
121890
147


 


617358
ATCCTTTGAGCCACACTT
0.44
121874
121891
148


 


617359
TATCCTTTGAGCCACACT
0.54
121875
121892
149


 


549583
TTATCCTTTGAGCCACAC
0.37
121876
121893
33


 


617360
ATTATCCTTTGAGCCACA
0.39
121877
121894
150


 


617361
TATTATCCTTTGAGCCAC
0.38
121878
121895
151


 


549595
GGACGTGTGAAGGTACTC
0.97
121924
121941
45









Example 4
Inhibitory Effect of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
Several modified oligonucleotides from Table 4 were selected and further evaluated for their effects on inhibiting human Tau exon 10 expression in vitro. ISIS 549595 was included in the study for comparison.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
A172 cells were transfected using Lipofectamine2000® with 0, 0.1, 0.3, 1, 3, 10, or 30 nM concentration of modified oligonucleotide as specified in the table below. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human Tau primer probe set 9_10 R5 was used to measure mRNA levels. Human Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
The IC50 of each oligonucleotide was calculated in the same manner as illustrated in Example 3 and the results are presented below.







TABLE 6







Inhibitory effect of uniform 2′-MOE modified


oligonucleotides on human Tau exon 10 expression















SEQ ID
SEQ ID






NO:
NO:
SEQ


ISIS

IC50
1 Start
1 Stop
ID


NO
Sequence
(nM)
Site
Site
NO















617362
ATATTATCCTTTGAGCCA
0.27
121879
121896
152


 


549584
GATATTATCCTTTGAGCC
0.40
121880
121897
34


 


565989
TGATATTATCCTTTGAGC
0.58
121881
121898
153


 


565990
TTGATATTATCCTTTGAG
1.23
121882
121899
154


 


549585
GTTTGATATTATCCTTTG
0.71
121884
121901
35


 


617364
GTGTTTGATATTATCCTT
0.61
121886
121903
157


 


617365
CGTGTTTGATATTATCCT
0.74
121887
121904
158


 


549586
ACGTGTTTGATATTATCC
0.93
121888
121905
36


 


617367
GGACGTGTTTGATATTAT
0.64
121890
121907
160


 


549587
CGGGACGTGTTTGATATT
0.97
121892
121909
37


 


549620
CAGATCCTGAGAGCCCAA
3.46
122024
122041
69


 


549595
GGACGTGTGAAGGTACTC
1.25
121924
121941
45









Example 5
Inhibitory Effect of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
Several modified oligonucleotides from Tables 3 and 4 were selected and further evaluated for their effects on inhibiting human Tau exon 10 expression in vitro. ISIS 549595 was included in the study for comparison.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
A172 cells were transfected using Lipofectamine2000® with 0, 0.1, 0.3, 1, 3, 10, or 30 nM concentrations of modified oligonucleotide as specified in the table below. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human Tau primer probe set 10_11 was used to measure mRNA levels, except for ISIS 549595. Human Tau primer probe set 9_10 R5 was used for this oligonucleotide. Human Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
The IC50 of each oligonucleotide was calculated in the same manner as illustrated in Example 3 and the results are presented below.







TABLE 7







Inhibitory effect of uniform 2′-MOE modified


oligonucleotides on human Tau exon 10 expression















SEQ ID
SEQ ID






NO:
NO:
SEQ


ISIS

IC50
1 Start
1 Stop
ID


NO
Sequence
(nM)
Site
Site
NO















549566
AGCCAGAAAAAAGGATGA
1.00
121804
121821
31


 


617309
CACCTTTGGTAGCCAGAA
1.11
121814
121831
97


 


549570
TAATTATCTGCACCTTTG
1.39
121824
121841
72


 


617317
TTAATTATCTGCACCTTT
1.26
121825
121842
106


 


617318
ATTAATTATCTGCACCTT
1.21
121826
121843
107


 


617323
TTCTTATTAATTATCTGC
0.96
121831
121848
112


 


617324
CTTCTTATTAATTATCTG
1.06
121832
121849
113


 


617341
GGACGTTGCTAAGATCCA
0.82
121851
121868
130


 


549577
TGGACGTTGCTAAGATCC
0.87
121852
121869
79


 


617351
GCCACACTTGGACTGGAC
0.91
121865
121882
140


 


617352
AGCCACACTTGGACTGGA
0.79
121866
121883
141


 


549595
GGACGTGTGAAGGTACTC
0.73
121924
121941
45









Example 6
Inhibitory Effect of Uniform 2′-MOE Modified Oligonucleotides on Human Tau Exon 10
Several modified oligonucleotides from Tables 1 and 4 were selected and further evaluated for their effect on inhibiting human Tau exon 10 expression in vitro. ISIS 617782, 617781, and 415833 were included in the study for comparison.
“Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
ISIS 617782 is 21 nucleosides in length, wherein each nucleoside has a 2′-OCH3 modification and is denoted as the subscript “m”. Each internucleoside linkage throughout the modified oligonucleotide are phosphorothioate internucleoside linkages (P═S).
ISIS 617781 is 18 nucleosides in length, wherein each nucleoside has a 2′-OCH3 modification and is denoted as the subscript “m”. Each internucleoside linkage throughout the modified oligonucleotide are phosphorothioate internucleoside linkages (P═S).
ISIS 415833 is 20 nucleosides in length, wherein each nucleoside has a 2′-MOE modification. Each internucleoside linkage throughout the modified oligonucleotide are phosphorothioate internucleoside linkages (P═S). All cytosine residues throughout the modified oligonucleotides are 5-methylcytosines.
A172 cells were transfected using Lipofectamine2000® with 0, 0.1, 0.3, 1, 3, 10, or 30 nM concentration of modified oligonucleotide as specified in the table below. After a treatment period of approximately 24 hours, RNA was isolated from the cells and the mRNA levels of Tau transcripts containing exon 10 were measured by quantitative real-time PCR. Human Tau primer probe set 9_10 R5 was used to measure mRNA levels, except for ISIS 617781 and 415883. Human Tau primer probe set 10_11 was used for these two oligonucleotides. Human Tau exon 10 mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®.
The IC50 of each oligonucleotide was calculated in the same manner as illustrated in Example 3 and the results are presented below.







TABLE 8







Inhibitory effect of uniform 2′-MOE modified


oligonucleotides on human Tau exon 10 expression















SEQ ID
SEQ ID






NO:
NO:
SEQ


ISIS

IC50
1 Start
1 Stop
ID


NO
Sequence
(nM)
Site
Site
NO















617357
TCCTTTGAGCCACACTTG
0.48
121873
121890
147


 


617358
ATCCTTTGAGCCACACTT
0.34
121874
121891
148


 


617359
TATCCTTTGAGCCACACT
0.41
121875
121892
149


 


549583
TTATCCTTTGAGCCACAC
0.27
121876
121893
33


 


617360
ATTATCCTTTGAGCCACA
0.33
121877
121894
150


 


617361
TATTATCCTTTGAGCCAC
0.24
121878
121895
151


 


617362
ATATTATCCTTTGAGCCA
0.27
121879
121896
152


 


549584
GATATTATCCTTTGAGCC
0.19
121880
121897
34


 


565989
TGATATTATCCTTTGAGC
0.39
121881
121898
153


 


617782
UmGmAmAmGmGmUmAmCm
4.33
121914
121934
198



UmCmAmCmAmCmUmGmCm







CmGmCm






 


617781
UmAmUmCmUmGmCmAmCm
20.25
121820
121837
199



CmUmUmUmGmGmUmAmGm






 


415883
TCTTATTAATTATCTGCA
0.65
121828
121847
200



CC









Example 7
Modified Oligonucleotides Targeting Exon 10 of Human Tau
A series of modified oligonucleotides were designed to target exon 10 of human Tau. “Start site” indicates the 5′-most nucleoside to which the modified oligonucleotide is targeted. “Stop site” indicates the 3′-most nucleoside to which the modified oligonucleotide is targeted. Each modified oligonucleotide listed in the tables below is targeted to SEQ ID NO: 1 (GENBANK Accession No NT_010783.15 truncated from nucleotides 9240000 to 9381000).
In the table below, “mC” indicates 5-methylcytosine, subscript “e” indicates 2′-O-methoxyethyl, subscript “f” indicates 2′-deoxyfluoro, subscript “d” indicates 2′-deoxy, subscript “p” indicates a peptide nucleic acid monomer, subscript “s” indicates a phosphorothioate internucleoside linkage, subscript “o” indicates a phosphodiester internucleoside linkage, “P-” indicates an O-linker at the 5′-end (PNA Bio, Thousand Oaks, Calif.), “-N” indicates a 3′-end carboxy amide.







TABLE 9







Modified oligonucleotides targeting human Tau











ISIS

SEQ ID NO:
SEQ ID NO:
SEQ


NO
Sequence
1 Start Site
1 Stop Site
ID NO














549607
AesmCesGesmCesAesmCesTesmCesAesmCesAesmCesmCes
121972
121989
201



AesmCesTesTesmCe





 


565985
mCesGesmCesmCesTesAesCesTesTesGesmCesTesCesGes
121706
121723
202



mCesAesAesGe





 


565986
GesAesmCesmCesmCesGesmCesmCesTesAesmCesTesTesGes
121710
121727
203



mCesTesmCesGe





 


565987
GesAesmCesmCesmCessGesmCesmCesTesGesmCesTesTesGes
121770
121787
204



mCesTesmCesGe





 


565988
mCesmCesTesGesGesAesmCesmCesmCesGesmCesmCesTes
121774
121791
205



GesmCesTesTesGe





 


566007
mCesGesCfsCfsUfsAfsCfsUfsUfsGfsCfsUfsCfsGfsCfsAfs
121706
121723
206



AesGe





 


566012
mCesmCesUfsGfsGfsAfsCfsCfsCfsGfsCfsCfsUfsGfsCfsUfs
121774
121791
207



TesGe





 


566013
TesGesAfsUfsAfsUfsUfsAfsmUfsCfsCfsUfsUfsUfsGfsAfsGes
121881
121898
208



mCe





 


566014
TesTesGfsAfsUfsAfsUfsUfsAfsUfsCfsCfsUfsUfsUfsGfsAes
121882
121899
209



Ge





 


566015
Tes Tes Ufs Gfs Afs Ufs Afs Ufs Ufs Afs Ufs Cfs Cfs Ufs
121883
121900
210



Ufs Ufs Ges Ae





 


566016
mCes Aes Cfs Ufs Ufs Cfs Cfs Ufs Afs Afs Ufs Afs Afs Ufs
121960
121977
211



Ufs Cfs Aes Ae





 


566017
Aes mCes Afs Cfs Cfs Afs Cfs Ufs Ufs Cfs Cfs Ufs Afs Afs
121964
121981
212



Ufs Afs Aes Te





 


566018
Aes mCes Ufs Cfs Afs Cfs Afs Cfs Cfs Afs Cfs Ufs Ufs Cfs
121968
121985
213



Cfs Ufs Aes Ae





 


568409
mCes mCes Cfs Gfs Cfs Cfs Ufs Afs Cfs Ufs Ufs Gfs Cfs Ufs
121708
121725
214



Cfs Gfs mCes Ae





 


568410
Ges Aes Cfs Cfs Cfs Gfs Cfs Cfs Ufs Afs Cfs Ufs Ufs Gfs
121710
121727
215



Cfs Ufs mCes Ge





 


568411
Ges Aes Cfs Cfs Cfs Gfs Cfs Cfs Ufs Gfs Cfs Ufs Ufs Gfs
121770
121787
216



Cfs Ufs mCes Ge





 


568412
Tes Ges Gfs Afs Cfs Cfs Cfs Gfs Cfs Cfs Ufs Gfs Cfs Ufs
121772
121789
217



Ufs Gfs mCes Te





 


624464
Ums Cms Ums Ums Ams Ums Ums Ams Ams Ums Ums Ams
121828
121847
218



Ums Cms Ums Gms Cms Ams Cms Cm





 


624465
Tks mCds Tds Tks Ads Tds Tks Ads Ads Tks Tds Ads Tks
121828
121847
200



mCds Tds Gks mCds Ads mCks mCk





 


624466
Tks mCes Tes Tks Aes Tes Tks Aes Aes Tks Tes Aes Tks
121828
121847
200



mCes Tes Gks mCes Aes mCks mCk





 


624467
Tks mCes Tes Tko Aes Tes Tko Aes Aes Tko Tes Aes Tko
121828
121847
200



mCes Tes Gko mCes Aes mCks mCk





 


624468
Tks mCds Tds Tko Ads Tds Tko Ads Ads Tko Tds Ads Tko
121828
121847
200



mcds Tds Gko mCds Ads mCks mCk





 


626881
P-Tp Cp Tp Tp Ap Tp Tp Ap Ap Tp Tp Ap Tp Cp Tp Gp
121828
121847
200



Cp Ap Cp Cp-N





 


670011
Ges Ges Aeo mCes Geo Tes Teo Ges mCeo Tes Aeo Aes Geo
121851
121868
130



Aes Teo mCes mCes Ae





 


670012
mCes mCes Teo Tes Teo Ges Aeo Ges mCeo mCes Aeo mCes 
121872
121889
146



Aeo mCes Teo Tes Ges Ge





 


670013
Aes Tes mCeo mCes Teo Tes Teo Ges Aeo Ges mCeo mCes
121874
121891
148



Aeo mCes Aeo mCes Tes Te





 


670014
Tes Tes Aeo Tes mCeo mCes Teo Tes Teo Ges Aeo Ges mCeo
121876
121893
33



mCes Aeo mCes Aes mCe





 


670015
Aes Tes Teo Aes Teo mCes mCeo Tes Teo Tes Geo Aes Geo
121877
121894
150



mCes mCeo Aes mCes Ae





 


670016
Tes Aes Teo Tes Aeo Tes mCeo mCes Teo Tes Teo Ges Aeo
121878
121895
151



Ges mCeo mCes Aes mCe





 


670017
Aes Tes Aeo Tes Teo Aes Teo mCes mCeo Tes Teo Tes Geo
121879
121896
152



Aes Geo mCes mCes Ae





 


670018
Ges Aes Teo Aes Teo Tes Aeo Tes mCeo mCes Teo Tes Teo
121880
121897
34



Ges Aeo Ges mCes mCe





 


670019
Ges Tes Teo Tes Geo Aes Teo Aes Teo Tes Aeo Tes mCeo
121884
121901
35



mCes Teo Tes Tes Ge





 


670020
Ges Geo Aeo mCeo Ges Tes Tes Ges mCes Tes Aes Aes Geo
121851
121868
130



Aeo Teo mCes mCes Ae





 


670021
mCes mCeo Teo Teo Tes Ges Aes Ges mCes mCes Aes mCes
121872
121889
146



Aeo mCeo Teo Tes Ges Ge





 


670022
Aes Teo mCeo mCeo Tes Tes Tes Ges Aes Ges mCes mCes
121874
121891
148



Aeo mCeo Aeo mCes Tes Te





 


670023
Tes Teo Aeo Teo mCes mCes Tes Tes Tes Ges Aes Ges mCeo
121876
121893
33



mCeo Aeo mCes Aes mCe





 


670024
Aes Teo Teo Aeo Tes mCes mCes Tes Tes Tes Ges Aes Geo
121877
121894
150



mCeo mCeo Aes mCes Ae





 


670025
Tes Aeo Teo Teo Aes Tes mCes mCes Tes Tes Tes Ges Aeo
121878
121895
151



Geo mCeo mCes Aes mCe





 


670026
Aes Teo Aeo Teo Tes Aes Tes mCes mCes Tes Tes Tes Geo
121879
121896
152



Aeo Geo mCes mCes Ae





 


670027
Ges Aeo Teo Aeo Tes Tes Aes Tes mCes mCes Tes Tes Teo
121880
121897
34



Geo Aeo Ges mCes mCe





 


670028
Ges Teo Teo Teo Ges Aes Tes Aes Tes Tes Aes Tes mCeo
121884
121901
35



mCeo Teo Tes Tes Ge





 


678329
Ges mCes mCeo Aes mCeo Aes mCeo Tes Teo Ges Geo Aes
121865
121882
140



mCeo Tes Geo Ges Aes mCe





 


678330
Aes Ges mCeo mCes Aeo mCes Aeo mCes Teo Tes Geo Ges
121866
121883
141



Aeo mCes Teo Ges Ges Ae





 


678331
Tes Ges Aeo Ges mCeo mCes Aeo mCes Teo Tes
121868
121885
83



Geo Ges Aeo mCes Tes Ge





 


678332
Ges mCeo mCeo Aeo mCes Aes mCes Tes Tes Ges Ges Aes
121865
121882
140



mCeo Teo Geo Ges Aes mCe





 


678333
Aes Geo mCeo mCeo Aes mCes Aes mCes Tes Tes Ges Ges
121866
121883
141



Aeo mCeo Teo Ges Ges Ae





 


678334
Tes Geo Aeo Geo mCes mCes Aes mCes Aes mCes Tes Tes
121868
121885
83



Geo Geo Aeo mCes Tes Ge





 


693840
Tes Ges Geo Aes mCeo Ges Teo Tes Geo mCes Teo Aes Aeo
121852
121869
79



Ges Aeo Tes mCes mCe





 


693841
mCes mCes Aeo mCes Aeo mCes Teo Tes Geo Ges Aeo mCes
121864
121881
82



Teo Ges Geo Aes mCes Ge





 


693842
Aes mCes Geo Tes Geo Tes Teo Tes Geo Aes Teo Aes Teo
121888
121905
36



Tes Aeo Tes mCes mCe





 


693843
Ges Ges Aeo mCes Geo Tes Geo Tes Geo Aes Aeo Ges Geo
121924
121941
45



Tes Aeo mCes Tes mCe





 


693844
Ges Aes Geo mCes mCeo Aes mCeo Aes mCeo Tes Teo Ges
121867
121884
142



Geo Aes mCeo Tes Ges Ge





 


693845
Tes Geo Geo Aeo mCes Ges Tes Tes Ges mCes Tes Aes Aeo
121852
121869
79



Geo Aeo Tes mCes mCe





 


693846
mCes mCeo Aeo mCeo Aes Mces Tes Tes Ges Ges Aes mCes
121864
121881
82



Teo Geo Geo Aes mCes Ge





 


693847
Aes mCeo Geo Teo Ges Tes Tes Tes Ges Aes Tes Aes Teo Teo
121888
121905
36



Aeo Tes mCes mCe





 


693848
Ges Geo Aeo mCeo Ges Tes Ges Tes Ges Aes Aes Ges Geo
121924
121941
45



Teo Aeo mCes Tes mCe





 


693849
Ges Aeo Geo mCeo mCes Aes mCes Aes mCes Tes Tes Ges
121867
121884
142



Geo Aeo mCeo Tes Ges Ge









Example 8
In Vivo Analysis of Oligonucleotides Targeting Human Tau Exon 10
Sprague Dawley rats were separated into groups of 3 rats for the experiment presented in Table 11 and into groups of 2 or 4 rats for the experiment presented in Table 12. Each rat in each group of rats was administered a single 3 mg intrathecal (IT) dose of an oligonucleotide selected from Table 3, 4, or 9. At various time points after injection from 3 hours to 8 weeks, 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 Tables 11 and 12.







TABLE 11







3 mg IT bolus in vivo study











ISIS
Score 3 hours after
Score 2 weeks
Score 8 weeks
SEQ ID


No.
injection
after injection
after injection
NO.














670020
1.0
2.7
2.7
130


670011
2.3
2.3
2.3
130


617341
3.0
0.0
0.0
130


678329
2.3
0.0
0.0
140


678332
3.0
0.0
1.3
140


617351
7.0
0.0
4.0
140


678330
2.7
2.3
3.0
141


678333
2.7
2.3
2.3
141


617352
7.0
0.0
2.0
141


678331
3.0
0.0
0.0
83


678334
4.3
2.3
2.3
83


549581
7.0
0.0
0.3
83


670012
2.7
0.0
0.0
146


670021
3.0
0.0
1.0
146


549582
4.3
0.0
0.0
146


670013
0.3
2.3
2.3
148


670022
2.0
2.3
3.0
148


617358
3.7
0.0
1.3
148


670014
0.0
0.0
0.0
33


670023
2.0
1.0
0.3
33


549583
2.3
0.0
0.0
33


670015
1.0
2.0
0.7
150


670024
1.0
0.7
0.0
150


617360
5.3
0.0
1.0
150


670025
0.3
5.3
5.3
151


670016
2.3
1.3
1.7
151


617361
3.0
0.0
0.3
151


670017
0.0
1.7
1.3
152


670026
1.0
3.7
3.3
152


617362
4.7
3.7
2.7
152


670018
0.3
0.3
0.3
34


670027
1.0
0.7
0.7
34


549584
5.0
2.7
3.7
34


670028
2.0
0.0
0.0
35


670019
3.0
0.0
0.0
35


549585
4.7
5.3
7.0
35
















TABLE 12







3 mg IT bolus in vivo study













No. of rats in
Score 3 hours




ISIS No.
treatment group
after injection
SEQ ID NO.
















549577
4
3.3
79



693840
4
1.8
79



693845
4
0.0
79



549580
4
7.0
82



693841
4
1.8
82



693846
4
2.5
82



617353
4
7.0
142



693844
2
3.5
142



693849
4
5.0
142



549582
4
4.3
146



549586
4
5.0
36



693847
2
1.0
36



549595
4
6.5
45



693843
2
2.0
45



693848
2
7.0
45










Example 9
Inhibitory Effect In Vivo of a Modified Oligonucleotide Targeting Human Tau Exon 10
B6.Cg-Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J mice (The Jackson Laboratory) express the 3R and 4R isoforms of human Tau. The effect of oligonucleotides selected from Table 3, 4, or 8 on shifting of the 4R isoform to 3R isoform was assessed in this mouse model.
Groups of four B6.Cg-Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J mice (3-4 months of age) were administered 500 μg of oligonucleotide via an intracerebroventricular (ICV) bolus injection. A control group of four mice was similarly treated with PBS. Two weeks after the dose, animals were sacrificed and RNA was extracted from the cerebral cortex, hippocampus, and/or spinal cord. mRNA expression of the 4R isoform and total human Tau were analyzed by qRT-PCR. The 4R isoform was analyzed using primer probe set hTau 9_10 junction (forward sequence 5′-CACTGAGAACCTGAAGCACC-3′, SEQ ID NO: 219; reverse sequence 5′-GTTGCTAAGATCCAGCTTCTT-3′, SEQ ID NO: 220; probe sequence 5′-TTAATTATCTGCACCTTCCCGCCTCC-3′, SEQ ID NO: 221) or primer probe set hTau 10_11 junction (forward sequence 5′-AATATCAAACACGTCCCGGGAG-3′, SEQ ID NO: 222; reverse sequence 5′-TGCCTAATGAGCCACACTTG-3′, SEQ ID NO: 223; probe sequence 5′-GTCTACAAACCAGTTGACCTGAGC-3′, SEQ ID NO: 224). Total Tau was analyzed using primer probe set RTS3104 (forward sequence 5′-AAGATTGGGTCCCTGGACAAT-3′, SEQ ID NO: 225; reverse sequence 5′-AGCTTGTGGGTTTCAATCTTTTTATT-3′, SEQ ID NO: 226; probe sequence 5′-CACCCACGTCCCTGGCGGA-3′, SEQ ID NO: 227). The ratio of 4R isoform to total Tau mRNA levels was normalized to mouse GAPDH levels, which were analyzed using primer probe set mGapdh_LTS00102 (forward sequence 5′-GGCAAATTCAACGGCACAGT-3′, SEQ ID NO: 228; reverse sequence 5′-GGGTCTCGCTCCTGGAAGAT-3′, SEQ ID NO: 229; probe sequence 5′-AAGGCCGAGAATGGGAAGCTTGTCATC-3′, SEQ ID NO: 230). The results are presented in Tables 13-15 as the average ratio of 4R isoform to total Tau mRNA normalized to GAPDH levels for each treatment group divided by the average ratio for the PBS group. “ND” indicates no data.
Inflammation was also assessed by performing RT-PCT to determine the expression levels of AIF1 in the cortex, hippocampus, and/or spinal cord. After normalization of all samples to GADPH, the average AIF1 value for each treatment group was divided by the average AIF1 value for the PBS control group. The results are presented in Tables 13-15.







TABLE 13







Effect of modified oligonucleotides on Tau exon 10 skipping in transgenic


mice




















AIF1



ISIS

4R
4R spinal
AIF1
AIF1
spinal
SEQ


NO.
4R cortex
hippo
cord
cortex
hippo
cord
ID NO.

















415883
0.50
0.46
0.56
1.35
1.27
1.44
200


549566
0.54
0.54
0.66
1.09
1.13
1.46
31


549570
0.58
0.60
0.64
1.17
1.16
1.39
72


549577
0.25
0.30
0.38
2.92
3.27
4.39
79


549581
0.25
ND
ND
1.04
1.07
1.40
83


549582
0.07
0.10
0.12
0.89
0.95
1.15
146


549583
0.25
0.26
0.25
0.97
0.94
1.09
33


617309
0.36
0.36
0.51
1.06
1.06
1.09
97
















TABLE 14







Effect of modified oligonucleotides on Tau exon 10


skipping in transgenic mice












ISIS



AIF1
SEQ


NO.
4R cortex
4R hippo
4R spinal cord
spinal cord
ID NO.















549582
0.21
0.21
0.23
0.97
146


549584
0.32
0.28
0.28
1.12
34


549585
0.30
0.27
0.30
1.05
35


565989
0.36
0.32
0.36
1.03
153


565990
0.50
0.46
0.53
0.97
154


617341
0.25
0.28
0.28
1.16
130


617351
0.13
0.18
0.14
1.01
140


617352
0.09
0.13
0.09
1.06
141


617354
0.31
ND
ND
1.21
143


617355
0.43
0.50
0.37
1.18
144


617356
0.30
0.40
0.49
0.98
145


617357
0.33
0.33
0.27
1.31
147


617358
0.27
0.27
0.34
1.12
148


617359
0.32
0.32
0.37
0.95
149


617360
0.21
0.19
0.29
1.19
150


617361
0.26
0.21
0.37
1.04
151


617362
0.26
0.27
0.34
0.90
152


617364
0.39
0.33
0.43
1.09
157
















TABLE 15







Effect of modified oligonucleotides on Tau exon 10 skipping in


transgenic mice













ISIS

4R
4R spinal
AIF1
AIF1 spinal
SEQ


NO.
4R cortex
hippo
cord
cortex
cord
ID NO.
















549582
0.26
0.34
0.29
0.74
1.03
146


549586
0.25
0.43
0.38
0.97
0.98
36


549587
0.39
0.47
0.46
0.96
1.17
37


549595
0.27
0.39
0.39
0.84
0.92
45


617317
0.62
0.83
0.53
0.88
1.05
106


617318
0.57
0.69
0.49
0.94
1.06
107


617323
0.60
0.82
0.49
0.92
1.10
112


617324
0.72
0.88
0.49
0.70
1.03
113


617365
0.31
0.42
0.27
0.79
0.87
158


617367
0.39
0.46
0.35
0.90
1.29
160









Example 10
Effect In Vivo of Mixed Backbone Oligonucleotides on Human Tau Exon 10 Skipping
Groups of four B6.Cg-Mapttm1(EGFP)Klt Tg(MAPT)8cPdav/J mice (see Example 9) were administered 125 μg of an oligonucleotide selected from Table 3, 4, or 9 or PBS via ICV bolus injection. Two weeks after the dose, animals were sacrificed and RNA was extracted from the cerebral cortex and/or hippocampus and analyzed as described in Example 9. The results are presented in Tables 16 and 17 as the average ratio of the 4R isoform to total Tau mRNA normalized to PBS for each treatment group.
Expression of allograft inflammatory factor (AIF1) was tested as a measure of inflammation by performing RT-PCT to determine the expression levels of AIF1 in the cortex and spinal cord. After normalization of all samples to GADPH, the average AIF1 value for each treatment group was divided by the average AIF1 value for the PBS control group. The results are presented in Tables 16 and 17.







TABLE 16







Effect of modified oligonucleotides on Tau exon 10


skipping in transgenic mice















SEQ


ISIS NO.
4R Cortex
AIF1 cortex
AIF1 spinal cord
ID NO.














549582
0.42
1.0
0.9
146


549583
0.71
0.9
1.2
33


617341
0.64
1.0
0.9
130


617361
0.63
1.1
1.1
151


670011
0.78
1.0
1.0
130


670012
0.47
0.9
1.1
146


670013
0.63
1.0
0.9
148


670014
0.90
0.9
1.0
33


670015
0.70
0.9
0.9
150


670018
0.75
1.0
1.0
34


670019
0.58
1.1
1.0
35


670020
0.67
0.9
0.9
130


670023
0.87
1.0
1.0
33


670024
0.60
1.0
1.1
150


670025
0.72
1.1
0.9
151


670027
0.79
1.0
0.9
34


670028
0.66
1.1
1.0
35


678329
0.13
1.0
1.0
140


678330
0.51
0.9
0.9
141


678331
1.12
0.9
1.0
83


678332
0.27
0.9
1.0
140


678333
0.56
0.9
0.9
141


678334
1.10
0.9
1.2
83
















TABLE 17







Effect of modified oligonucleotides on Tau exon 10


skipping in transgenic mice















SEQ ID


ISIS No.
4R Cortex
AIF1 cortex
AIF1 spinal cord
NO.














549577
0.56
1.4
1.6
79


549580
0.52
0.9
1.2
82


549582
0.27
0.9
1.0
146


549586
0.90
1.1
1.3
36


549595
0.90
1.0
1.4
45


617353
1.35
1.0
1.3
142


693840
0.80
0.9
1.2
79


693841
0.91
1.1
1.1
82


693842
1.20
1.0
0.8
36


693843
1.31
1.0
1.6
45


693844
2.22
1.1
1.6
142


693845
0.86
1.0
1.4
79


693846
0.84
1.0
1.4
82


693847
0.89
1.0
1.8
36


693848
0.76
0.9
1.9
45


693849
2.08
0.8
1.8
142

## Claims

1. A compound comprising a modified oligonucleotide consisting of 15 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 15 contiguous nucleobases complementary to an equal length portion of nucleobases 121708 to 122044 of SEQ ID NO.: 1, wherein each nucleoside of the modified oligonucleotide comprises a 2′-substituted sugar moiety and wherein each 2′-substituted sugar moiety is 2′-MOE.

2. The compound of claim 1, wherein the modified oligonucleotide consists of 16 to 22 linked nucleosides.

3. The compound of claim 2, wherein at least one nucleobase of the modified oligonucleotide is a modified nucleobase.

4. The compound of claim 3, wherein the modified nucleobase is a 5-methylcytosine.

5. The compound of claim 4, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

6. The compound of claim 5, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

7. The compound of claim 4, wherein each internucleoside linkage is a modified internucleoside linkage.

8. The compound of claim 7, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.

9. A compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 15 contiguous nucleobases complementary to an equal length portion of nucleobases 121873 to 121898 of SEQ ID NO: 1, wherein each nucleoside of the modified oligonucleotide comprises a modified sugar moiety.

10. The compound of claim 9, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary to SEQ ID NO: 1.

11. The compound of claim 10, wherein the modified oligonucleotide consists of 16 to 22 linked nucleosides.

12. The compound of claim 11, wherein at least one nucleobase of the modified oligonucleotide is a modified nucleobase.

13. The compound of claim 12, wherein the modified nucleobase is a 5-methylcytosine.

14. The compound of claim 13, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

15. The compound of claim 14, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

16. The compound of claim 13, wherein each internucleoside linkage is a modified internucleoside linkage.

17. The compound of claim 16, wherein each modified internucleoside linkage is a phosphorothioate internucleoside linkage.

18. The compound of claim 17, wherein the modified sugar moiety is a 2′-substituted sugar moiety.

19. The compound of claim 18, wherein the 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F, and 2′-MOE.

20. The compound of claim 17, wherein the modified sugar moiety is a bicyclic sugar moiety.

21. The compound of claim 20, wherein the bicyclic sugar moiety is LNA or cEt.

22. The compound of claim 17, wherein the modified sugar moiety is a sugar surrogate.

23. The compound of claim 22, wherein the sugar surrogate is a morpholino.

24. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier or diluent.

25. A method of reducing or ameliorating one or more symptoms associated with a tau-associated disorder, comprising contacting a cell with a compound according to claim 1.

26. The method of claim 25, wherein the cell is in an animal.