Methods for Treating Neurodegenerative Disorders

Abstract

The disclosure features useful compositions and methods for treating neurodegenerative disorders, for example, in a subject in need thereof.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international application claims the benefit of priority to U.S. Provisional Application No. 63 / 326,475, filed April 1, 2022, which is incorporated herein by reference in its entirety.

[0002] Oligonucleotide molecules, such as single-stranded oligonucleotides (e.g., antisense oligonucleotides (ASOs), mRNA), double-stranded oligonucleotides (e.g., siRNA molecules), are therapeutic agents that target messenger RNA (mRNA) to alter mRNA expression. Oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, have the potential to treat all genetic disorders, including neurodegenerative disorders, such as Huntington's disease, Alzheimer's disease, and Parkinson's disease.

[0003] Although the mechanisms underlying Alzheimer's disease are not fully understood, it is believed that certain genetic changes that disrupt brain proteins (e.g., tau protein) contribute to the development of the disease. Six isoforms of tau protein (encoded by the TAU gene) are useful in the internal support of neurons as well as in the transport system that carries nutrients and other essential substances. In Alzheimer's disease, tau protein changes shape and self-organizes into structures called neurofibrillary tangles that occur in neurons. In addition to the abundance of neurofibrillary tangles, neurofilament threads and senile plaque neurites contribute to the pathology of Alzheimer's disease. Abnormal hyperphosphorylation of tau protein that occurs in nerve cells can disrupt the neuronal transport system and lead to cell death. Many people with Down's syndrome develop Alzheimer's disease.

[0004] Similarly, the pathology of Parkinson's disease is believed to have a genetic component. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in Parkinson's disease. Furthermore, point mutations and gene amplifications in the α-synuclein gene (SNCA) play a central role in the development of Lewy bodies (LB) and the pathology of Lewy neuritis (LN). Lewy bodies are a pathological hallmark of Parkinson's disease and dementia, and constitute the second most common neuronal pathology after the neurofibrillary tangles of Alzheimer's disease.

[0005] Current therapies for neurodegenerative diseases focus on alleviating symptoms. There is an urgent need for effective treatments that can alter the expression of genes that contribute to the pathology of neurodegenerative diseases. [Brief description of the drawings]

[0006] [Figure 1] A shows the MSH3 mRNA response in the caudate nucleus of a non-human primate following intrathecal ("IT") administration of antisense oligo #1. B shows the MSH3 mRNA response in the putamen following IT administration of antisense oligo #1. C shows the MSH3 mRNA response in the cortex following IT administration of antisense oligo #1. D shows the MSH3 mRNA response in the lumbar region following IT administration of antisense oligo #1. [Diagram 2] A shows the MSH3 mRNA response in the caudate nucleus of a non-human primate following intracerebroventricular ("ICV") administration of antisense oligo #1. B shows the MSH3 mRNA response in the putamen following ICV administration of antisense oligo #1. C shows the MSH3 mRNA response in the cortex following IT administration of antisense oligo #1. D shows the MSH3 mRNA response in the lumbar region following IT administration of antisense oligo #1. [Diagram 3] 1 shows the relative expression of MSH3 obtained following IT, ICM, and intravenous ("IV") administration of antisense oligo #2 in non-human primates. [Figure 4A]MSH3 mRNA responses at t=0, t=15 days, and t=29 days following ICV administration of antisense oligo #1 in the ipsilateral caudate nucleus, ipsilateral frontal and temporal cortex, and ipsilateral nucleus accumbens are shown. [Figure 4B] Shown are MSH3 mRNA responses at t=0, t=15 days, and t=29 days following ICV administration of antisense oligo #1 in the contralateral caudate nucleus, contralateral frontal and temporal cortex, and contralateral nucleus accumbens. [Figure 5A] MSH3 mRNA responses after ICV administration of antisense oligo #1 in the ipsilateral and contralateral caudate nucleus at t=0, t=24 hrs, t=48 hrs, t=8 days, t=15 days, and t=29 days are shown. [Figure 5B] MSH3 mRNA responses after ICV administration of antisense oligo #1 in ipsilateral and contralateral putamen at t=0, t=24 hrs, t=48 hrs, t=8 days, t=15 days, and t=29 days are shown. [Figure 5C] MSH3 mRNA responses after ICV administration of antisense oligo #1 in the ipsilateral and contralateral nucleus accumbens at t=0, t=24 hrs, t=48 hrs, t=8 days, t=15 days, and t=29 days are shown. [Figure 5D] MSH3 mRNA responses after ICV administration of antisense oligo #1 in ipsilateral and contralateral frontal cortex at t=0, t=24 hrs, t=48 hrs, t=8 days, t=15 days, and t=29 days are shown. [Figure 5E] MSH3 mRNA responses after ICV administration of antisense oligo #1 in ipsilateral and contralateral temporal cortex at t=0, t=24 hrs, t=48 hrs, t=8 days, t=15 days, and t=29 days are shown. [Figure 6]Figure 1 shows MSH3 mRNA knockdown ("KD") in the frontal cortex following repeated intrathecal ("IT") dosing in non-human primates. The X-axis shows the antisense oligos tested. From left to right: artificial CSF control, antisense oligo #3, antisense oligo #4, antisense oligo #5, antisense oligo #1, and antisense oligo #2. The Y-axis shows the remaining MSH3 mRNA normalized to five housekeeping genes and compared to the aCSF group. [Figure 7] 1 shows MSH3 protein knockdown in the frontal cortex after repeated IT dosing in non-human primates. The X-axis shows the amount of MSH3 protein (normalized to β-tubulin) in the cortex of the aCSF-treated group versus the ASO-treated group (treated with antisense oligo #1) 15 days after repeated IT dosing using a proprietary antibody and Western blot. [Figure 8] 1 shows the QRT-PCR results of mouse MSH3 gene mRNA in the retina of the three experimental groups at doses of 50 μg and 100 μg compared to phosphate buffered saline (PBS). [Figure 9] A-D show MSH3 mRNA knockdown in the cortex up to 12 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows results achieved in the ipsilateral frontal cortex. B shows results achieved in the contralateral frontal cortex. C shows results achieved in the ipsilateral temporal cortex. D shows results achieved in the contralateral temporal cortex. [Figure 10] AB show MSH3 knockdown in the caudate nucleus up to 12 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows results achieved in the ipsilateral caudate nucleus. B shows results achieved in the contralateral caudate nucleus. [Figure 11] AB show MSH3 knockdown in the nucleus accumbens up to 12 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows results achieved in the ipsilateral nucleus accumbens. B shows results achieved in the contralateral nucleus accumbens. [Figure 12]A-B show MSH3 knockdown in the putamen 8 and 12 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows the results achieved in the ipsilateral putamen. B shows the results achieved in the contralateral putamen. [Figure 13] A-B show MSH3 knockdown in the cortex up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows results achieved in the ipsilateral and contralateral motor cortex. B shows results achieved in the ipsilateral and contralateral occipital cortex. [Figure 14A] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral caudate are shown. [Figure 14B] Shown is MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Shown are results achieved in the ipsilateral and contralateral body of globus pallidus. [Figure 14C] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral amygdala are shown. [Figure 14D] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral hypothalamus are shown. [Figure 14E] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral hypothalamus are shown. [Figure 14F] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral thalamus are shown. [Figure 14G]1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral substantia nigra are shown. [Figure 14H] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral pons are shown. [Figure 14I] 1 shows MSH3 knockdown in the ipsilateral caudate, amygdala, and thalamus up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. Results achieved in the ipsilateral and contralateral medulla oblongata are shown. [Figure 15] A-B show MSH3 knockdown in ipsilateral white matter surrounding the injection site up to 4 weeks after a single 10 mg ICV dose of antisense oligo #1. A shows results achieved in white matter at the level of the amygdala. B shows results achieved in white matter near the injection area. Summary of the Invention

[0007] Provided herein is a method of treating, preventing, or delaying the onset and / or progression of a neurodegenerative disorder in a subject in need thereof, comprising administering to the subject by intracerebroventricular ("ICV") injection a therapeutically effective amount of a pharma- cerebroventricular ("ICV") injection of one or more oligonucleotide molecules, or pharma- ceutical acceptable salts thereof, that are 15-30 linked nucleotides in length. In some embodiments, the neurodegenerative disorder is not a triplet repeat disorder. In some embodiments, the neurodegenerative disorder is not a nucleotide repeat disorder.

[0008] Provided herein is a method of reducing the amount of a target mRNA in a cell, the method comprising contacting the cell with one or more oligonucleotide molecules, 15-30 linked nucleotides in length, or a pharma- ceutically acceptable salt thereof, for a time sufficient to effect degradation of the target mRNA, wherein the target mRNA is derived from a target gene selected from the group consisting of MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43. In some embodiments, the target gene is MAPT. In some embodiments, the target gene is APP. In some embodiments, the target gene is LRRK2. In some embodiments, the target gene is SNCA.

[0009] In some embodiments, one or more oligonucleotide molecules hybridize to one or more of MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, or TDP43 genes. In some embodiments, one or more oligonucleotide molecules hybridize to one or more of MAPT, APP, LRRK2, SNCA, PSEN1, PSEN2, HSPA2, C4A, C4B, C9orf72, or TDP43 genes. In some embodiments, one or more oligonucleotide molecules hybridize to HTT gene. In some embodiments, one or more oligonucleotide molecules hybridize to ATXN1 gene. In some embodiments, one or more oligonucleotide molecules hybridize to ATXN2 gene. In some embodiments, one or more oligonucleotide molecules hybridize to ATXN3 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the MAPT gene. In some embodiments, one or more oligonucleotide molecules hybridize to the APP gene. In some embodiments, one or more oligonucleotide molecules hybridize to the LRRK2 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the SNCA gene. In some embodiments, one or more oligonucleotide molecules hybridize to the PSEN1 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the PSEN2 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the ATN1 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the HSPA2 gene. In some embodiments, one or more oligonucleotide molecules hybridize to the C4A gene. In some embodiments, one or more oligonucleotide molecules hybridize to the C4B gene. In some embodiments, one or more oligonucleotide molecules hybridize to the C9orf72 gene. In some embodiments, the one or more oligonucleotide molecules hybridize to the TDP43 gene.

[0010] In some aspects, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, Parkinson's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, cerebral amyloid angiopathy, schizophrenia, dentatorubral-pallidoluysian atrophy, amyotrophic lateral sclerosis, and Down's syndrome-associated Alzheimer's disease. In some aspects, the neurodegenerative disease is Alzheimer's disease. In some aspects, the neurodegenerative disease is Pick's disease. In some aspects, the neurodegenerative disease is Parkinson's disease. In some aspects, the neurodegenerative disease is frontotemporal dementia. In some aspects, the neurodegenerative disease is progressive supranuclear palsy. In some aspects, the neurodegenerative disease is corticobasal degeneration. In some aspects, the neurodegenerative disease is dementia with Lewy bodies. In some aspects, the neurodegenerative disease is multiple system atrophy. In some aspects, the neurodegenerative disease is cerebral amyloid angiopathy. In some embodiments, the neurodegenerative disease is schizophrenia. In some embodiments, the neurodegenerative disease is dentatorubral-pallidoluysian atrophy. In some embodiments, the neurodegenerative disease is Down's syndrome associated with Alzheimer's disease.

[0011] In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the temporal lobe. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the amygdala. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the hippocampus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the cerebral cortex. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the pons. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the basal ganglion. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the globus pallidus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the substantia nigra. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the ventral striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the nucleus accumbens. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the dorsal striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the caudate nucleus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the putamen. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the thalamus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the nucleus basalis of Meynert. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the brainstem. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the cerebellum.

[0012] In some embodiments, one or more oligonucleotide molecules or its pharmaceutically acceptable salts are single-stranded or double-stranded.In some embodiments, one or more oligonucleotide molecules or its pharmaceutically acceptable salts are single-stranded antisense oligonucleotides.In some embodiments, one or more oligonucleotide molecules or its pharmaceutically acceptable salts are siRNA oligonucleotides.In some embodiments, one or more oligonucleotide molecules or its pharmaceutically acceptable salts are mRNA oligonucleotides.In some embodiments, one or more oligonucleotide molecules or its pharmaceutically acceptable salts are CRISPR modalities.

[0013] In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are 17 to 27 linked nucleotides in length. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are 20 to 25 linked nucleotides in length.

[0014] In some embodiments, the one or more oligonucleotide molecules, or a pharma- ceutically acceptable salt thereof, comprises: (a) a DNA core sequence comprising linked deoxyribonucleosides; (b) a 5' flanking sequence comprising a linking nucleoside; and (c) a 3' flanking sequence containing a linked nucleoside Including, The DNA core comprises a region of at least 10 contiguous nucleobases located between the 5' flanking sequence and the 3' flanking sequence, the 5' flanking sequence and the 3' flanking sequence each comprising at least two linked nucleosides, and at least one nucleoside of each flanking sequence comprises an alternative nucleoside.

[0015] In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, comprise at least one alternative internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is a 2'-alkoxy internucleoside linkage. In some embodiments, at least one alternative internucleoside linkage is an alkylphosphate internucleoside linkage. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, comprise at least one alternative nucleobase. In some embodiments, the alternative nucleobase is 5'-methylcytosine, pseudouridine, or 5-methoxyuridine. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, comprise at least one alternative sugar moiety. In some embodiments, the alternative sugar moiety is 2'-OMe or a bicyclic nucleic acid. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, further comprise a ligand conjugated to the 5' or 3' end of the one or more oligonucleotide molecules via a monovalent or branched divalent or trivalent linker.

[0016] In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 50% at an oligonucleotide concentration of 10 nM. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 60% at an oligonucleotide concentration of 10 nM. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 70% at an oligonucleotide concentration of 10 nM. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 80% at an oligonucleotide concentration of 10 nM. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 50% at an oligonucleotide concentration of 1 nM. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, reduce target mRNA expression by at least 60% at an oligonucleotide concentration of 1 nM. In some embodiments, the one or more oligonucleotide molecules, or a pharma- ceutically acceptable salt thereof, reduce target mRNA expression by at least 70% at an oligonucleotide concentration of 1 nM.

[0017] In some embodiments, the target mRNA expression is assessed in vitro. In some embodiments, the target mRNA expression is assessed in a cell-based assay. In some embodiments, the target mRNA expression is assessed in HEK293 cells, LNCAP cells, primary neurons, and / or GABA neurons. In some embodiments, the target mRNA expression is determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). In some embodiments, the target mRNA expression is normalized relative to the mRNA expression of a reference gene. In some embodiments, the target mRNA expression is normalized relative to the mRNA expression of beta-glucuronidase (GUSB), TBP, GAPDH, HPRT, ACTB, NEFH, and / or ARL1. In some embodiments, the decrease in target mRNA expression is relative to a control. In some embodiments, the control is target mRNA expression in the absence of one or more oligonucleotide molecules, or a pharma- ceutically acceptable salt thereof. In some embodiments, the control is target mRNA expression in the absence of one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, but in the presence of a control oligonucleotide, or a salt thereof. In some embodiments, the control oligonucleotide, or a salt thereof, is a scrambled or luciferase-targeted oligonucleotide. In some embodiments, the reduction in target mRNA expression is calculated by the delta-delta Ct (ΔΔCT) method. In some embodiments, the delta-delta Ct (ΔΔCT) method includes normalizing target mRNA expression relative to the mRNA expression of a reference gene and relative to the target mRNA expression in the absence of one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, but in the presence of a control oligonucleotide, or a salt thereof. In some embodiments, the reference gene is beta-glucuronidase (GUSB), and / or the control oligonucleotide, or a salt thereof, is a scrambled or luciferase-targeted oligonucleotide.

[0018] In some embodiments, one or more of the oligonucleotide molecules are in free base form.

[0019] In some embodiments, one or more of the oligonucleotide molecules are in the form of a pharma- ceutically acceptable salt. In some embodiments, the pharma- ceutically acceptable salt of one or more of the oligonucleotide molecules is a sodium salt.

[0020] In some embodiments, the one or more oligonucleotide molecules are provided in a pharmaceutical composition further comprising a pharma- ceutical acceptable carrier or excipient. In some embodiments, the pharmaceutical composition further comprises artificial cerebrospinal fluid.

[0021] In some aspects, the subject is a primate. In some aspects, the primate is a human. In some aspects, the primate is a non-human primate.

[0022] In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 2 mg to about 300 mg. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 10 mg to about 250 mg. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 15 mg to about 200 mg. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 25 mg to about 200 mg. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 50 mg to about 200 mg. In some embodiments, the one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered in a dose of about 100 mg to about 150 mg.

[0023] In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once a week. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every two weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every three weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every four weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once a month. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every six weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every eight weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every two months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every ten weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 12 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 3 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 16 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 4 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 20 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 5 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 24 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 6 months.In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 28 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 7 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 32 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 8 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 36 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 9 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 40 weeks. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 10 months. In some embodiments, one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, are administered once every 44 weeks. In some embodiments, one or more oligonucleotide molecules or pharmaceutically acceptable salts thereof are administered once every 11 months. In some embodiments, one or more oligonucleotide molecules or pharmaceutically acceptable salts thereof are administered once every 48 weeks. In some embodiments, one or more oligonucleotide molecules or pharmaceutically acceptable salts thereof are administered once every 12 months. In some embodiments, one or more oligonucleotide molecules or pharmaceutically acceptable salts thereof are administered once a year.

[0024] In some embodiments, administration of one or more oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, delays the onset and / or progression of a neurodegenerative disorder by at least 120 days, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more as compared to expected onset and / or progression.

[0025] In some embodiments, the method further comprises administering an additional therapeutic agent.

[0026] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to help describe certain embodiments and are not intended to limit the claimed technology, since the scope of the technology is limited only by the claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0027] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," and (iii) the terms "including" and "comprising" may be understood to encompass the listed elements or steps, whether presented by themselves or with one or more additional elements or steps.

[0028] As used herein, the terms "about" and "approximately" refer to values ​​within 10% above and below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 to 5.5 nM.

[0029] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range. "At least" is also not limited to integers (e.g., "at least 5%" includes 5.0%, 5.1%, and 5.18% without considering the number of significant digits).

[0030] As used herein, "less than" or "less than" is understood as the value adjacent to the phrase and the logically lower value or integer to zero, as is logical from the context.For example, an oligonucleotide with "3 or less mismatches to target sequence" has 3, 2, 1 or 0 mismatches to target sequence.When "less than" is present before a series of numbers or a range, it is understood that "less than" can modify each of the numbers in the series or range.

[0031] As used herein, the term "administration" refers to administration of a composition (e.g., a compound or a preparation containing a compound as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) can be by any suitable route, such as those routes described herein.

[0032] As used herein, "combination therapy" or "administered in combination" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen specifies the dose and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or parallel, and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated, but are administered in a sequential manner as part of a given regimen. In some embodiments, the administration of two or more agents or treatments in combination is such that the reduction in symptoms, or other parameters associated with the disorder, is greater than that observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent may be by any suitable route, including, but not limited to, oral, intraocular, subcutaneous, intracisternal, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents may be administered by the same route or by different routes. For example, one therapeutic agent of the combination may be administered by intravenous injection, while an additional therapeutic agent of the combination may be administered orally.

[0033] As used herein, a "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of any of the genes described herein, i.e., MAPT, APP, LRRK2, or SNCA genes. When referring to a nucleotide sequence, the terms "target sequence" and "target gene" are used interchangeably herein. The term "target sequence" also refers to an mRNA that is a product of RNA processing of a primary transcript. In one aspect, the target portion of the sequence is at or near a portion of the nucleotide sequence of an mRNA molecule formed during transcription of the target gene, at least long enough to serve as a substrate for oligonucleotide-directed (e.g., antisense oligonucleotide (ASO)-directed) cleavage. The target sequence can be, for example, about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence may be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-25, 18-26, 18-27, 18-28, 18-29, 18-30, 18-30, 18-4 ... The length may be 22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the ranges and lengths listed above are also contemplated.

[0034] As used herein, an mRNA is "derived from" a target gene when it is the product of transcription of the target gene and proper processing of the resulting pre-mRNA transcript, which may include removal of one or more introns (if present) from the pre-mRNA transcript, depending on the target gene sequence. Those skilled in the relevant art will understand that the mRNAs described herein may be the product of alternative splicing of the pre-mRNA transcript.

[0035] "G", "C", "A", "T" and "U" each generally represent naturally occurring nucleotides containing guanine, cytosine, adenine, thymidine and uracil, respectively, as bases. However, it will be understood that the term "nucleotide" may refer to alternative nucleotides or surrogate replacement moieties, as further detailed below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil may be substituted with other moieties without substantially changing the base pairing properties of an oligonucleotide containing such a substituted nucleotide. For example, but not limited to, a nucleotide containing inosine as its base may base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine may be replaced in the nucleotide sequence of an oligonucleotide with, for example, a nucleotide containing inosine. In another example, adenine and cytosine anywhere in an oligonucleotide may be replaced with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured herein.

[0036] The terms "nucleobase" and "base" include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but function during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1.

[0037] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring as well as alternative nucleosides, such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0038] The term "alternative nucleoside" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein.

[0039] In some aspects, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, such as a substituted purine or substituted pyrimidine, such as an "alternate nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0040] The nucleobase moieties may be designated by the letter code of the corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may include alternative nucleobases with equivalent functions. In some embodiments, e.g., for gapmers, 5-methylcytosine LNA nucleosides may be used.

[0041] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," defined as structures that can replace the furanose ring of a nucleoside. In some embodiments, the sugar substitute is a non-furanose (or 4'-substituted furanose) ring or ring system or open system. Such structures can include simple changes compared to the natural furanose ring, such as a six-membered ring, or can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. The sugar substitute can include sugar surrogates in which the furanose ring is replaced with another ring system, such as, for example, a morpholino or hexitol ring system. Sugar moieties useful for preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis-substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose), bicyclic sugar surrogates (such as bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose) and sugar surrogates (such as where the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position varies and is not a determining factor of the motif. In most nucleosides with surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0042] "Nucleotide" as used herein refers to a monomeric unit of an oligonucleotide or polynucleotide that includes a nucleoside and an internucleoside linkage. The internucleoside linkage may include a phosphate linkage. Similarly, a "linked nucleoside" may be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs (e.g., A-LNA, 5mC L-NA, G-LNA, T-LNA)) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants on the phosphate backbone of natural nucleosides, including those described herein.

[0043] "Alternative nucleotide," as used herein, refers to a nucleotide having an alternative nucleoside or sugar and internucleoside linkage, which may include alternative nucleoside linkages.

[0044] The terms "oligonucleotide" and "polynucleotide" as used herein are defined as molecules that contain two or more covalently linked nucleosides as generally understood by those skilled in the art. Such covalently linked nucleosides may be referred to as nucleic acid molecules or oligomers. Oligonucleotides are usually produced in a laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, it refers to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides, or modifications thereof. An oligonucleotide may be artificial. For example, an oligonucleotide may be chemically synthesized and purified or isolated. Oligonucleotides are also intended to include compounds with (i) one or more furanose moieties replaced by furanose derivatives or any cyclic or acyclic structure that can be used as a covalent attachment point for the base moiety, (ii) one or more phosphodiester linkages that are either modified as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced with a suitable linkage moiety as in the case of formacetal or riboacetal linkages, and / or (iii) one or more linked furanose-phosphodiester linkages that are replaced by any cyclic or acyclic structure that can be used as a covalent attachment point for the base moiety. Oligonucleotides may include one or more alternative nucleosides or nucleotides (e.g., including those described herein). It is further understood that oligonucleotides include compositions that lack sugar moieties or nucleobases, but can still pair with or hybridize to a target sequence. "Oligonucleotide" refers to short polynucleotides (e.g., 100 or fewer linked nucleosides).

[0045] As used herein, the term "oligonucleotide containing a nucleobase sequence" refers to an oligonucleotide that contains a chain of nucleotides or nucleosides described by a sequence referenced using standard nucleotide nomenclature.

[0046] The term "contiguous nucleobase region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleobase sequence". In some embodiments, all nucleotides of an oligonucleotide are present within a contiguous nucleotide or nucleoside region. In some embodiments, an oligonucleotide comprises a contiguous nucleotide region and may further comprise a nucleotide linker region that may be used to attach a nucleotide(s) or nucleoside(s), e.g., a functional group, to the contiguous nucleotide sequence. The nucleotide linker region may be complementary to a target nucleic acid. In some embodiments, the internucleoside linkages present between the nucleotides of the contiguous nucleotide region are all phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

[0047] The term "gapmer," as used herein, refers to an oligonucleotide that includes a region of an RNase H recruiting oligonucleotide (the gap or DNA core) flanked on the 5' and 3' sides by regions that include one or more affinity-enhancing alternative nucleosides (wing or flanking sequences). Various gapmer designs are described herein. Headmers and tailmers are oligonucleotides capable of recruiting RNase H that lack one of the flanks, i.e., only one end of the oligonucleotide contains the affinity-enhancing alternative nucleosides. In the case of a headmer, the 3' flanking sequence is lacking (i.e., the 5' flanking sequence includes the affinity-enhancing alternative nucleosides), and in the case of a tailmer, the 5' flanking sequence is lacking (i.e., the 3' flanking sequence includes the affinity-enhancing alternative nucleosides). A "mixed flanking sequence gapmer" refers to a gapmer in which the flanking sequence comprises at least one alternative nucleoside, such as at least one DNA nucleoside or at least one 2'-substituted alternative nucleoside, such as 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-F-ANA nucleoside(s), or bicyclic nucleosides (e.g., locked or constrained ethyl (cEt) nucleosides). In some embodiments, a mixed flanking sequence gapmer has one flanking sequence (e.g., on the 5' or 3' side) that comprises an alternative nucleoside and the other flanking sequence (on the 3' or 5' side, respectively) comprises the 2'-substituted alternative nucleoside(s).

[0048] A "linker" or "linking group" is a bond between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest by one or more covalent bonds. The oligonucleotide molecules disclosed herein can include one or more linkers that can link one or more oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein, and / or to any other oligonucleotides, and / or to any conjugate moiety.

[0049] The linker may be susceptible to cleavage ("cleavable linker"), thereby facilitating the release of different oligonucleotides and / or different conjugate moieties disclosed herein. Such cleavable linkers may be susceptible under suitable conditions, for example, to nuclease-induced cleavage, acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Suitable cleavable linkers for use in cleavable linkers include linkers that are sufficiently stable outside a cell, but are cleaved upon entry into a target cell to release the two moieties held together by the linker.

[0050] Alternatively, the linker may be substantially resistant to cleavage ("non-cleavable linker"). Such non-cleavable linker may be any chemical moiety capable of linking one or more different oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein and / or any complex moiety that is stably covalently linked, and does not fall into the categories listed above for cleavable linkers. Thus, non-cleavable linkers are substantially resistant to acid-induced cleavage, nuclease-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage. Furthermore, "non-cleavable" refers to the ability of the chemical bond in or adjacent to the linker to resist cleavage induced by acid, nuclease, photocleavable cleavage agent, peptidase, esterase, or chemical or physiological compound that cleaves disulfide bonds, provided that the oligonucleotides disclosed herein do not lose their activity or intended purpose.

[0051] The conjugate moiety can be attached to the oligonucleotide directly or via a linking moiety (e.g., a linker or tether). The linker serves to covalently link the third region, e.g., the conjugate moiety, to the oligonucleotide (e.g., at the end of region A or C). In some embodiments, the conjugate or oligonucleotide conjugate can include a linker region disposed between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide is biochemically cleavable. Phosphodiester containing biochemically cleavable linkers are described in more detail in WO2014 / 076195 (incorporated herein by reference).

[0052] In some embodiments, two or more linkers may be linked in tandem. When multiple linkers connect one or more oligonucleotides disclosed herein to one or more other oligonucleotides disclosed herein and / or to any conjugate moiety, each of the linkers may be the same or different.

[0053] As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize to form a double-stranded structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as would be understood by one of skill in the art. Such conditions may be, for example, stringent conditions, which may include 400 mM NaCl, 40 mM PIPES (pH 6.4), 1 mM EDTA, at 50°C or 70°C for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as those that may occur inside an organism, such as physiologically relevant conditions, may be used. Those skilled in the art will be able to determine the optimal set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotide or nucleoside.

[0054] "Complementary" sequences, as used herein, may include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements regarding their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairing or Hoogsteen base pairing. Complementary sequences between an oligonucleotide and a target sequence as described herein include base pairing over the entire length of one or both nucleotide or nucleoside sequences of an oligonucleotide or polynucleotide containing a first nucleotide or nucleoside sequence and an oligonucleotide or polynucleotide containing a second nucleotide or nucleoside sequence. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to as "substantially complementary" to a second sequence herein, the two sequences may be completely complementary, or they may form one or more, but generally no more than five, four, three or two mismatched base pairs upon hybridization into a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate use, e.g., inhibition of gene expression by the RNase H-mediated pathway. "Substantially complementary" may refer to a polynucleotide that is substantially complementary to a contiguous portion of an mRNA of interest (e.g., an mRNA derived from a target gene). For example, a polynucleotide is complementary to at least a portion of a target mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding from a target gene.

[0055] As used herein, the term "region of complementarity" refers to a region on an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., a target sequence, e.g., an mRNA nucleotide sequence), or processed mRNA, so as to interfere with the expression of an endogenous gene (e.g., MAPT, APP, LRRK2, or SNCA). If the region of complementarity is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. In general, the most tolerated mismatches occur in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the oligonucleotide.

[0056] As used herein, "an agent that reduces the level and / or activity of a target gene" refers to any polynucleotide agent (e.g., an oligonucleotide, e.g., ASO) that reduces the level or inhibits the expression of a target gene in a cell or subject. As used herein, the phrase "inhibits expression of a target gene or mRNA" includes inhibition of expression of any target gene and any target gene variant or mutant that encodes a protein. Thus, a target gene can be a wild-type gene, a mutant gene, or a transgenic gene in the context of a genetically engineered cell, cell population, or organism.

[0057] "Reducing the activity of a target gene" refers to reducing the level of activity associated with a target gene (e.g., MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43). The reduction in the activity of a target gene can be characterized by one or more mechanisms, such as a reduction, suppression, or elimination of the amount of target mRNA derived from the target gene or the amount of protein synthesized by ribosomal translation of the target mRNA. The activity level of a target gene can be measured using any method known in the art (e.g., by quantifying the polypeptide product of the target mRNA or ribosomal translation of the target mRNA, quantifying the polypeptide product, or quantifying the activity of the polypeptide product).

[0058] "Reducing the level" of any of the target genes means, for example, by administering an oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, to a cell or a subject, thereby reducing the level of target mRNA in the cell or subject.The level of target mRNA can be measured using any method known in the art (for example, by measuring the level of mRNA or protein in a cell or a subject).

[0059] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein (e.g., MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43). Non-limiting examples of inhibitors include polynucleotides (e.g., oligonucleotides, e.g., ASOs). The term "inhibit" as used herein is used interchangeably with "reduce," "silence," "downregulate," "suppress," "knockdown," and other similar terms, and includes any level of inhibition.

[0060] The phrase "contacting a cell with an oligonucleotide molecule" such as an oligonucleotide molecule, as used herein, includes contacting a cell by any possible means.Contacting a cell with an oligonucleotide molecule includes contacting a cell with an oligonucleotide molecule in vitro or contacting a cell with an oligonucleotide molecule in vivo.Contacting can be performed directly or indirectly.Thus, for example, the oligonucleotide molecule can be physically contacted with the cell by the individual who performs the method, or alternatively, the oligonucleotide molecule can be in a situation that allows or will cause it to contact the cell later.

[0061] Contacting cells in vitro can be performed, for example, by incubating cells with oligonucleotide molecules.Contacting cells in vivo can be performed, for example, by injecting oligonucleotide molecules into or near the tissue where the cells are located, or by injecting oligonucleotide molecules into another area, for example, bloodstream or subcutaneous space, so that the molecules will then reach the tissue where the contacted cells are located.For example, the oligonucleotide molecules can contain and / or be bound to a ligand, for example, GalNAc3, that guides the oligonucleotide to the site of interest, for example, the liver.Combination of in vitro and in vivo contact methods is also possible.For example, cells can be contacted with oligonucleotide molecules in vitro and then transplanted into a subject.

[0062] In one embodiment, contacting a cell with an oligonucleotide molecule includes "introducing" or "delivering" an oligonucleotide molecule into a cell by promoting or causing uptake or absorption into the cell. Absorption or uptake of an oligonucleotide molecule can be through unassisted diffusive or active cellular processes, or by auxiliary agents or devices. Introduction of an oligonucleotide molecule into a cell can be in vitro and / or in vivo. For example, in in vivo introduction, one or more oligonucleotide molecules can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below and / or known in the art.

[0063] The term "antisense oligonucleotide," as used herein, refers to a nucleic acid comprising an oligonucleotide or polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA to interfere with the expression of an endogenous gene (e.g., MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43). A "complementary" polynucleotide is one that can base pair according to standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and either adenine paired with thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

[0064] The terms "antisense strand" and "guide strand" refer to the strand of a dsRNA that includes a region that is substantially complementary to a target sequence, eg, APP mRNA.

[0065] The terms "sense strand" and "passenger strand," as used herein, refer to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand, as that term is defined herein.

[0066] The term "dsRNA" refers to an agent that includes a sense strand and an antisense strand that contain linked nucleosides, as that term is defined herein. dsRNA includes, for example, siRNA and shRNA that mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. dsRNA directs sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). dsRNA reduces the expression of target mRNA in cells, such as cells in a subject, such as a mammalian subject. Generally, the majority of linked nucleosides in each strand of dsRNA are ribonucleosides, but each or both strands may include one or more non-ribonucleosides, such as deoxyribonucleosides and / or alternative nucleosides, as described in detail herein.

[0067] The terms "siRNA" and "small interfering RNA" (also known as "small interfering RNA") refer to an RNA agent, preferably a double-stranded agent, of about 10-30 nucleotides in length, whose strands optionally have overhanging ends that contain, for example, one, two, or three overhang-linked nucleosides capable of directing or mediating RNA interference. Naturally occurring siRNAs are generated from longer dsRNA molecules (e.g., greater than 25 linked nucleosides in length) by the cellular RNAi machinery (e.g., Dicer or its homologs).

[0068] As used herein, the terms "shRNA" and "short hairpin RNA" refer to an RNA agent having a stem-loop structure comprising first and second regions of complementary sequence, the degree and orientation of complementarity of which is sufficient to allow base pairing between the regions, and the first and second regions are connected by a loop region, where the loop results from the lack of base pairing between nucleobases in the loop region.

[0069] In the context of the present invention, a "chimeric" dsRNA or "chimera" is a dsRNA that contains two or more chemically distinct regions, each of which is composed of at least one monomeric unit, i.e., a nucleoside, or, in the case of dsRNA, a nucleotide.

[0070] As used herein, the terms "effective amount," "therapeutically effective amount," and "sufficient amount" of an agent that reduces the level and / or activity of a target mRNA described herein (e.g., in a cell or subject) refer to an amount sufficient to produce a beneficial or desired result, including a clinical result, when administered to a subject, including a human, and thus the "effective amount" or its synonyms will depend on the context in which it is applied. For example, in the context of treating a neurodegenerative disorder, the amount is the amount of agent that reduces the level and / or activity of a target gene sufficient to achieve a therapeutic response when compared to the response obtained without administering the agent that reduces the level and / or activity of the target mRNA. The amount of a given agent that reduces the level and / or activity of a target mRNA described herein that would correspond to such an amount will vary depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity of the subject or host being treated (e.g., age, sex, and / or weight), etc., but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, the "therapeutically effective amount" of an agent that reduces the level and / or activity of a target mRNA of the present disclosure is an amount that produces a beneficial or desired result in a subject when compared to a control. As defined herein, the therapeutically effective amount of an agent that reduces the level and / or activity of a target mRNA of the present disclosure can be easily determined by those skilled in the art by routine methods known in the art. The administration regimen can be adjusted to provide an optimal therapeutic response.

[0071] A "prophylactically effective amount," as used herein, is intended to include an amount of an oligonucleotide molecule sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject having or predisposed to having a neurodegenerative disorder. Ameliorating a disease includes slowing the course of the disease or reducing the severity of a disease that subsequently develops. A "prophylactically effective amount" may vary depending on the oligonucleotide, the method of administration of the agent, the degree of disease risk, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment, if any, of the patient being treated, and other individual characteristics. A prophylactically effective amount may refer, for example, to an amount of an agent that reduces the level and / or activity of a target gene (e.g., in a cell or subject) described herein, or may refer to an amount sufficient, when administered to a subject, including a human, to delay the onset and / or progression of one or more of the neurodegenerative disorders described herein by at least 120 days, e.g., at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, or more, when compared to the expected onset and / or progression.

[0072] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of oligonucleotide (either administered in single or multiple doses) that produces some desired local or systemic effect, at a reasonable benefit / risk ratio applicable to any treatment. The oligonucleotide molecules used in the methods herein can be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0073] As used herein, the term "region of complementarity" refers to a region on an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., target sequence), or processed mRNA so as to interfere with the expression of an endogenous gene (e.g., APP). If the region of complementarity is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. In general, the most tolerated mismatches occur in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the oligonucleotide.

[0074] As used herein, the term "neurodegenerative disease" refers to a group of disorders characterized by neurodegeneration. Neurodegeneration is the gradual loss of neuronal structure or function, or the death of neurons. Neurodegenerative disease can refer to any of the diseases disclosed herein, including, but not limited to, Huntington's disease, Alzheimer's disease, and Parkinson's disease, all of which have a genetic component and are targets for antisense oligonucleotide therapy as disclosed herein.

[0075] As used herein, "trinucleotide repeat expansion disorder" refers to a classification of genetic diseases or disorders characterized by excess trinucleotide repeats (e.g., trinucleotide repeats such as CAG) in a target gene or intron that exceed the normal stable threshold of the gene or intron. Nucleotide repeats are common in the human genome and are not usually associated with disease. However, in some cases, expansion of the repeat number beyond a stable threshold leads to disease, and the severity of symptoms can generally be correlated with the number of repeats. Nucleotide repeat expansion disorders include "polyglutamine" disorders and "non-polyglutamine" disorders.

[0076] "Determining the level of a protein" refers to detecting the protein, or the mRNA encoding the protein, by methods known in the art, either directly or indirectly. "Directly determining" refers to performing a process to obtain a physical entity or value (e.g., performing an assay or test on a sample, or "analyzing a sample" as that term is defined herein). "Indirectly determining" refers to receiving a physical entity or value from another party or source (e.g., a third party laboratory that obtains the physical entity or value directly). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC) mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, as well as assays based on protein properties, including, but not limited to, enzyme activity or interaction with other protein partners.Methods for measuring mRNA levels are known in the art.

[0077] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps (DNA core sequences) if necessary to obtain the maximum percent sequence identity. Alignment to determine percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can be translated as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids that a sequence alignment program (e.g., BLAST) scores as identical matches in that program's alignment of A and B, and Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0078] By "level" is meant the level or activity of a protein or an mRNA encoding a protein (e.g., MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43), optionally compared to a reference. The reference may be any useful reference as defined herein. A "decreased level" or "increased level" of a protein refers to a decrease or increase in protein levels (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more decrease or increase, for example, a decrease or increase in protein levels (e.g., about 5%, about 10%, about 15%, about 20%, about 3 ... By "protein level" is meant a 10%, 15%, 20%, 50%, 75%, 100%, or more than 200% decrease or increase, less than 0.01-fold, 0.02-fold, 0.1-fold, 0.3-fold, 0.5-fold, 0.8-fold decrease or increase, or more than 1.2-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000-fold increase, or more, as compared to a control. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, or ng / mL) or as a percentage compared to the total protein or mRNA in the sample.

[0079] The term "pharmaceutical composition" as used herein refers to a composition containing a compound described herein formulated with a pharma- ceutical acceptable excipient, which may be manufactured or sold as part of a therapeutic regimen for the treatment of a disease in a mammal, subject to approval by a government regulatory agency. The pharmaceutical composition may be formulated, for example, for intracerebroventricular injection, or in any other pharma- ceutical acceptable formulation.

[0080] In some aspects, provided herein are pharmaceutical compositions formulated for intraventricular injection.

[0081] "Pharmaceutically acceptable excipient" as used herein refers to any ingredient other than the compounds described herein (e.g., a vehicle capable of suspending or dissolving an active compound) and has the properties of being substantially non-toxic and non-inflammatory in a patient. Excipients may include artificial cerebrospinal fluid (acsf).

[0082] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of a compound for any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are suitable for use in contact with human and animal tissues without causing undue toxicity, irritation, or allergic response within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977, and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting a free base group with a suitable organic acid.

[0083] The compounds described herein may have ionic groups so that they can be prepared as pharmaceutically acceptable salts.These salts may be acid addition salts, including inorganic or organic acids, or salts may be prepared from inorganic or organic bases in the case of the acidic form of the compounds described herein.In many cases, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.Suitable pharmaceutically acceptable acids and bases and methods for preparing suitable salts are well known in the art.Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0084] By "reference" is meant any useful reference used to compare protein or mRNA levels or activity. A reference can be any sample, standard, calibration curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, e.g., a predefined negative control value such as a "normal control" or a previous sample taken from the same subject, a sample from a normal healthy subject, e.g., a normal cell or normal tissue, a sample (e.g., cell or tissue) from a subject without a disease, a sample from a subject diagnosed with a disease but not yet treated with a compound described herein, a sample from a subject being treated with a compound described herein, or a sample of a known normal concentration of purified protein (e.g., any described herein). By "reference standard or level" is meant a value or number obtained from a reference sample. A "normal control value" is a predefined value indicative of a non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("X to Y"), a high threshold value ("below X"), or a low threshold value ("above X"). A subject having a measurement value within the normal control value for a particular biomarker is usually referred to as being "within the normal range" for that biomarker. The normal reference standard or level can be a value or value obtained from a normal subject without a disease or disorder (e.g., a neurodegenerative disorder) and a subject treated with a compound described herein. In some embodiments, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, stage of disease, and overall health. A standard curve of purified protein within the normal reference range, for example any of the levels described herein, can be used as a reference.

[0085] As used herein, the term "subject" refers to any organism to which a composition may be administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals, such as mice, rats, rabbits, non-human primates, and humans). A subject may be a human or animal that is seeking or in need of treatment, is in need of treatment, is undergoing treatment, will be undergoing treatment in the future, or is under the care of a professional trained in a particular disease or condition.

[0086] As used herein, the terms "treat", "treated" and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow (reduce) an undesirable physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of the condition, disorder or disease, stabilization (i.e., not worsening) of the condition, disorder or disease, delay in onset of the condition, disorder or disease or slowing of its progression, improvement or remission (whether partial or total) of the condition, disorder or disease state, whether detectable or undetectable, improvement in at least one measurable physical parameter, not necessarily discernible by the patient, or improvement or amelioration of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without causing an excessive level of side effects. Treatment also includes extending survival as compared to expected survival in the absence of treatment.

[0087] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original material.

[0088] The details of one or more embodiments are set forth in the description below. Other features, objects, and advantages will be apparent from the description, and from the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] The present inventors have found that suppressing, inhibiting, or depleting the level and / or activity of a protein in a cell is effective in treating neurodegenerative disorders (e.g., Huntington's disease, Alzheimer's disease, and Parkinson's disease). Accordingly, useful compositions and methods for treating neurodegenerative disorders, e.g., in a subject in need thereof, are provided herein.

[0090] The agents described herein that decrease the level and / or activity of a target protein in a cell can be, for example, a polynucleotide, e.g., an oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, which can be utilized in the methods described herein. These agents decrease the level of activity associated with a protein involved in a neurodegenerative disease, or associated downstream effects, or decrease the level of a target protein in a cell or subject.

[0091] In some embodiments, the agent that reduces the level and / or activity of a target protein is an oligonucleotide molecule. In some embodiments, the oligonucleotide molecule is a single-stranded oligonucleotide that acts, for example, by an RNase H-mediated pathway. Single-stranded oligonucleotides include DNA and DNA / RNA chimeric molecules that are typically about 15-30 nucleotides in length and recognize a target sequence or sequence portion by hydrogen bonding interactions with the nucleotide bases of the target sequence (e.g., APP). The oligonucleotide molecule can reduce the expression level (e.g., protein level or mRNA level) of a target gene. For example, the oligonucleotide molecule includes an oligonucleotide that targets full-length APP. In some embodiments, the oligonucleotide molecule recruits RNase H enzymes that result in target mRNA degradation.

[0092] In some aspects, the oligonucleotide molecule is an inhibitory RNA molecule, e.g., acting through the RNA interference (RNAi) pathway. The inhibitory RNA molecule can reduce the expression level (e.g., protein level or mRNA level) of a target mRNA. The inhibitory RNA molecule can be a double-stranded (dsRNA) molecule. For example, the dsRNA includes a small interfering RNA (siRNA) that targets a full-length target gene. The siRNA is a double-stranded RNA molecule that typically has a length of about 19-25 base pairs. In other embodiments, the dsRNA is a short hairpin RNA (shRNA) that targets a full-length target gene. The shRNA is a dsRNA molecule that contains a hairpin turn that reduces expression of the target gene via the RNAi pathway. In some aspects, the dsRNA molecule recruits an RNAse H enzyme. Degradation is triggered by the enzymatic RNA-induced silencing complex (RISC).

[0093] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, decreases the level and / or activity of a positive regulator of function. In other embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, increases the level and / or activity of an inhibitor of a positive regulator of function. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, increases the level and / or activity of a negative regulator of function.

[0094] In some embodiments, the oligonucleotide molecule, or a pharmaceutically acceptable salt thereof, reduces the level and / or activity or function of a target gene, such as MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 genes. In some embodiments, the oligonucleotide molecule, or a pharmaceutically acceptable salt thereof, reduces the level and / or activity or function of a target mRNA, such as APP. The oligonucleotide molecule, or a pharmaceutically acceptable salt thereof, can be chemically synthesized.

[0095] The oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, can be synthesized by standard methods known in the art, as further described below, for example, by use of an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0096] The oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, may be prepared using liquid-phase or solid-phase organic synthesis, or both. Organic synthesis offers the advantage that oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, that contain non-natural or alternative nucleotides may be easily prepared. The oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, may be prepared using liquid-phase or solid-phase organic synthesis, or both.

[0097] In some aspects, the oligonucleotide molecules or pharma- ceutically acceptable salts thereof described herein reduce target mRNA expression, e.g., MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression, by at least 50% at an oligonucleotide concentration of 10 nM. In some embodiments, the oligonucleotide molecules or pharma- ceutically acceptable salts thereof described herein reduce MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression by at least 60% at an oligonucleotide concentration of 10 nM. In some embodiments, the oligonucleotide molecules or pharma- ceutically acceptable salts thereof described herein reduce MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression by at least 70% at an oligonucleotide concentration of 10 nM. In some aspects, the oligonucleotide molecules or pharma- ceutically acceptable salts thereof described herein reduce MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression by at least 80% at an oligonucleotide concentration of 10 nM.

[0098] In some aspects, the oligonucleotides or pharma- ceutically acceptable salts thereof described herein reduce target mRNA expression, e.g., expression of MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43, by at least 50% at an oligonucleotide concentration of 1 nM. In some aspects, the oligonucleotides or pharma- ceutically acceptable salts thereof described herein reduce target mRNA expression, e.g., expression of MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression, by at least 60% at an oligonucleotide concentration of 1 nM. In some aspects, the oligonucleotides or pharma- ceutically acceptable salts thereof described herein reduce target mRNA expression, e.g., expression of MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 mRNA expression, by at least 70% at an oligonucleotide concentration of 1 nM.

[0099] The cellular assay may include transfecting mammalian cells, such as HEK293 cells, LNCAP cells, primary neurons, and / or GABA neurons, with a desired concentration of oligonucleotide (e.g., 1 nM or 10 nM) using Lipofectamine 2000 (Invitrogen), and comparing the levels of the transfected cells to those of control cells, e.g., comparing the target mRNA levels of the transfected cells to the mRNA levels of the control cells. The control cells may be transfected with an oligonucleotide that is not specific for the target gene, or may be mock transfected. The mRNA levels may be determined using RT-qPCR, and the mRNA levels may be normalized to GAPDH mRNA levels. The inhibition rate may be calculated as the ratio of the target mRNA concentration to the target mRNA concentration of the control cells.

[0100] In some embodiments, the mRNA expression is evaluated in vitro. By way of example, in some embodiments, the mRNA expression is evaluated in vitro. In some embodiments, the mRNA expression is evaluated in a cell-based assay. In some embodiments, the mRNA expression is evaluated in HEK293 cells, LNCAP cells, primary neurons, and / or GABA neurons. In some embodiments, the mRNA expression is determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR). In some embodiments, the mRNA expression is normalized relative to the mRNA expression of a reference gene. In some embodiments, the mRNA expression is normalized relative to the mRNA expression of beta-glucuronidase (GUSB), TBP, GAPDH, HPRT, ACTB, NEFH, and / or ARL1. In some embodiments, the decrease in mRNA expression is relative to a control. In some embodiments, the control is the mRNA expression in the absence of the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof. In some embodiments, the control is mRNA expression in the absence of the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, but in the presence of a control oligonucleotide, or a salt thereof. In some embodiments, the control oligonucleotide, or a salt thereof, is a scrambled or luciferase-targeted oligonucleotide. In some embodiments, the decrease in mRNA expression is calculated by the delta-delta Ct (ΔΔCT) method. In some embodiments, the delta-delta Ct (ΔΔCT) method includes normalizing the mRNA expression relative to the mRNA expression of a reference gene and relative to the mRNA expression in the absence of the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, but in the presence of a control oligonucleotide, or a salt thereof. In some embodiments, the reference gene is beta-glucuronidase (GUSB), and / or the control oligonucleotide, or a salt thereof, is a scrambled or luciferase-targeted oligonucleotide.

[0101] In some embodiments, the oligonucleotide molecule, or its adjacent nucleotide regions, has a gapmer design or structure, also referred to herein simply as a "gapmer." In a gapmer structure, the oligonucleotide comprises at least three distinct structural regions in a "5->3" orientation: a 5' flanking sequence (also known as a 5' wing), a DNA core sequence (also known as a gap), and a 3' flanking sequence (also known as a 3' wing). In this design, the 5' and 3' flanking sequences comprise at least one alternative nucleoside adjacent to the DNA core sequence, and in some embodiments may comprise a continuous stretch of 2-7 alternative nucleosides, or a continuous stretch of alternative and DNA nucleosides (mixed flanking sequences containing both alternative and DNA nucleosides).

[0102] The length of the 5' flanking sequence region can be at least two nucleosides long (e.g., at least two, at least three, at least four, at least five, at least six, or more nucleosides long). The length of the 3' flanking sequence region can be at least two nucleosides long (e.g., at least two, at least three, at least four, at least five, at least six, or more nucleosides long). The 5' and 3' flanking sequences can be symmetric or asymmetric with respect to the number of nucleosides they contain. In some embodiments, the DNA core sequence comprises about 10 nucleosides adjacent to the 5' and 3' flanking sequences, each of which comprises about five nucleosides. In some embodiments, the DNA core sequence comprises about 11 nucleosides adjacent to the 5' and 3' flanking sequences, each of which comprises about five or about six nucleosides. In some embodiments, the DNA core sequence comprises about 12 nucleosides flanked by a 5' sequence comprising about 5 nucleosides and a 3' flanking sequence comprising about 6 nucleosides. In some embodiments, the DNA core sequence comprises about 12 nucleosides flanked by a 5' sequence comprising about 6 nucleosides and a 3' flanking sequence comprising about 5 nucleosides. In some embodiments, the DNA core sequence comprises about 12 nucleosides flanked by 5' and 3' flanking sequences each comprising about 6 nucleosides.

[0103] Thus, the nucleosides of the 5' and 3' flanking sequences adjacent to the DNA core sequence are alternative nucleosides, such as 2' alternative nucleosides. The DNA core sequence comprises a contiguous stretch of nucleotides that can recruit RNase H when the oligonucleotide is duplexed with a target nucleic acid, such as an MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43 target nucleic acid. In some embodiments, the DNA core sequence comprises a contiguous stretch of 5-16 DNA nucleosides. In other embodiments, the DNA core sequence comprises a region of at least 10 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementarity to the target gene. In some embodiments, the gapmer comprises a region that is complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, or 19 contiguous nucleotides of the target gene. The gapmer can be a contiguous nucleoside region of an oligonucleotide, as it is complementary to a target nucleic acid, such as a MAPT target nucleic acid. In some embodiments, the gapmer comprises a region that is complementary to at least 21 contiguous nucleotides, 20-25 contiguous nucleotides, or 23 contiguous nucleotides of a target gene, such as a target gene. The gapmer can be a contiguous nucleoside region of an oligonucleotide, as it is complementary to a target nucleic acid, such as a MAPT target nucleic acid.

[0104] The 5' and 3' flanking sequences adjacent to the 5' and 3' ends of the DNA core sequence may comprise one or more affinity enhancing surrogate nucleosides. In some embodiments, the 5' and / or 3' flanking sequences comprise at least one 2'-O-methoxyethyl (MOE) nucleoside. In some embodiments, the 5' and / or 3' flanking sequences contain at least two MOE nucleosides. In some embodiments, the 5' flanking sequences comprise at least one, at least two, at least three, at least four, at least five, or at least six or more MOE nucleosides. In some embodiments, the 5' flanking sequences comprise at least one, at least two, at least three, at least four, at least five, or at least six or more MOE nucleosides. In some embodiments, both the 5' and 3' flanking sequences comprise MOE nucleosides. In some embodiments, all nucleosides in the flanking sequences are MOE nucleosides. In other embodiments, the flanking sequences can include both MOE nucleosides and other nucleosides (mixed flanking sequences), such as DNA nucleosides and / or non-MOE surrogate nucleosides, such as bicyclic nucleosides (BNAs) (e.g., LNA nucleosides (e.g., A-LNA, 5mCL-NA, G-LNA, T-LNA) or cET nucleosides), or other 2'-substituted nucleosides. In this case, the DNA core sequence is defined as a flanking sequence of at least five RNase H recruiting nucleosides (e.g., 5-16 DNA nucleosides) flanked on the 5' and 3' ends by affinity enhancing surrogate nucleosides, such as MOE nucleosides.

[0105] In other embodiments, the 5' and / or 3' flanking sequences comprise at least one BNA (e.g., at least one LNA nucleoside (e.g., A-LNA, 5mCL-NA, G-LNA, T-LNA) or a cET nucleoside). In some embodiments, the 5' and / or 3' flanking sequences comprise at least two bicyclic nucleosides. In some embodiments, the 5' flanking sequence comprises at least one BNA. In some embodiments, both the 5' and 3' flanking sequences comprise BNAs. In some embodiments, all nucleosides in a flanking sequence are BNAs. In other embodiments, a flanking sequence may comprise both BNAs and other nucleosides (mixed flanking sequences), such as DNA nucleosides and / or non-BNA alternative nucleosides, such as 2' substituted nucleosides. In this case, the DNA core sequence is defined as a flanking sequence of at least five RNase H recruiting nucleosides (e.g., 5-16 DNA nucleosides) flanked at the 5' and 3' ends by affinity enhancing surrogate nucleosides such as BNAs, e.g., LNAs, e.g., beta-D-oxy-LNAs.

[0106] The 5' flank attached to the 5' end of the DNA core sequence comprises, contains or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some embodiments, the flanking sequence comprises or consists of 1-7 alternative nucleobases, such as 2-6 alternative nucleobases, such as 2-5 alternative nucleobases, such as 2-4 alternative nucleobases, such as 1-3 alternative nucleobases, such as 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

[0107] The 3' flank attached to the 3' end of the DNA core sequence comprises, contains or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some embodiments, the flanking sequence comprises or consists of 1-7 alternative nucleobases, such as 2-6 alternative nucleobases, such as 2-5 alternative nucleobases, such as 2-4 alternative nucleobases, such as 1-3 alternative nucleobases, such as 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the flanking sequence comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

[0108] In one aspect, one or more or all of the alternative sugar moieties in the flanking sequences are 2' alternative sugar moieties.

[0109] In a further aspect, one or more of the 2' alternative sugar moieties in the wing region are selected from a 2'-O-alkyl-sugar moiety, a 2'-O-methyl-sugar moiety, a 2'-amino-sugar moiety, a 2'-fluoro-sugar moiety, a 2'-alkoxy-sugar moiety, an MOE sugar moiety, an LNA sugar moiety, an arabinonucleic acid (ANA) sugar moiety, and a 2'-fluoro-ANA sugar moiety.

[0110] In one embodiment, all alternative nucleosides in the flanking sequences are bicyclic nucleosides. In a further embodiment, the bicyclic nucleosides in the flanking sequences are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and / or ENA, or combinations thereof, in either the beta-D or alpha-L configuration.

[0111] In some embodiments, one or more alternative internucleoside linkages in the flanking sequences are phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate linkages are stereochemically pure phosphorothioate linkages. In some embodiments, the phosphorothioate linkages are Sp phosphorothioate linkages. In other embodiments, the phosphorothioate linkages are Rp phosphorothioate linkages. In some embodiments, the alternative internucleoside linkages are 2'-alkoxy internucleoside linkages. In other embodiments, the alternative internucleoside linkages are alkylphosphate internucleoside linkages.

[0112] The DNA core sequence may comprise, contain, or consist of at least 5-16 consecutive DNA nucleosides capable of recruiting RNase H. In some embodiments, all of the nucleosides of the DNA core sequence are DNA units. In further embodiments, the DNA core region may consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. In some embodiments, at least 50% of the nucleosides of the DNA core sequence are DNA, such as at least 60%, at least 70%, or at least 80%, or at least 90% are DNA. In some embodiments, all of the nucleosides of the DNA core sequence are RNA units.

[0113] The oligonucleotide molecule comprises a flanking region that is complementary to the target nucleic acid. In some embodiments, the oligonucleotide may further comprise additional linked nucleosides located 5' and / or 3' to either the 5' and 3' flanking sequences. These additional linked nucleosides may be attached to the 5' end of the 5' flanking sequence or the 3' end of the 3' flanking sequence, respectively. The additional nucleosides may form part of the flanking sequence that is complementary to the target nucleic acid in some embodiments, or may be non-complementary to the target nucleic acid in other embodiments.

[0114] The inclusion of additional nucleosides in either or both of the 5' and 3' flanking sequences can independently include 1, 2, 3, 4, or 5 additional nucleotides, which can be complementary or non-complementary to the target nucleic acid. In this regard, the oligonucleotide can, in some embodiments, include adjacent sequences that can modulate the target adjacent to the additional nucleotides at the 5' and / or 3' ends. Such additional nucleosides can function as biochemically cleavable linkers that are sensitive to nucleases, and can be used to attach functional groups, such as conjugate moieties, to the oligonucleotide. In some embodiments, the additional nucleosides at the 5' and / or 3' ends are linked with phosphodiester bonds and can be DNA or RNA. In other embodiments, the additional nucleosides at the 5' and / or 3' ends are alternative nucleosides that can be included, for example, to enhance nuclease stability or to facilitate synthesis.

[0115] In other embodiments, the oligonucleotide molecules utilize an "altimer" design, which contains alternating 2'-fluoro-ANA and DNA regions that are arranged every three nucleosides. Altimer oligonucleotides are described in more detail in Min, et al., Bioorganic & Medicinal Chemistry Letters, 2002, 12(18):2651-2654 and Kalota, et al., Nuc. Acid Res. 2006, 34(2):451-61, which are incorporated herein by reference.

[0116] In other embodiments, the oligonucleotide molecules utilize a "hemimer" design, which includes a single 2' modified flanking sequence adjacent to (either 5' or 3' of) a DNA core sequence. Hemimer oligonucleotides are described in detail in Geary et al., 2001, J. Pharm. Exp. Therap., 296:898-904, incorporated herein by reference.

[0117] The construction of vectors for expressing polynucleotides can be accomplished using conventional techniques that do not require detailed explanation to those skilled in the art. The creation of an efficient expression vector requires the presence of regulatory sequences that control the expression of the polynucleotide. These regulatory sequences include promoter and enhancer sequences, are influenced by specific cellular factors that interact with these sequences, and are well known in the art.

[0118] In one embodiment, one or more of the linking nucleosides or internucleoside bonds of the oligonucleotide molecule are naturally occurring and do not include, for example, chemical modifications and / or conjugation, as known in the art and described herein.In another embodiment, one or more of the linking nucleosides or internucleoside bonds of the oligonucleotide molecule are chemically modified to enhance stability or other beneficial properties.Without being bound by theory, it is believed that certain modifications can increase nuclease resistance and / or serum stability or reduce immunogenicity.For example, the oligonucleotide molecule can contain nucleotides (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) found to be naturally occurring in DNA or RNA, or can contain alternative nucleosides or internucleoside bonds that have one or more chemical modifications to one or more components of the nucleotide (e.g., nucleobase, sugar, or phospholinker moiety). The oligonucleotide molecules may be linked to each other via naturally occurring phosphodiester bonds or may contain alternative linkages (e.g., phosphorothioate (e.g., Sp phosphorothioate or Rp phosphorothioate), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoamidate, 2'-5' phosphodiester, guanidinium, S-methylthiourea, 2'-alkoxy, alkyl phosphate, or covalently linked via peptide bonds).

[0119] In some embodiments, substantially all of the nucleosides or internucleotide bonds of the oligonucleotide molecule are alternative nucleosides.In other embodiments, substantially all of the nucleosides or internucleotide bonds of the oligonucleotide molecule are alternative nucleosides.The oligonucleotide molecule in which "substantially all of the nucleosides are alternative nucleosides" can be mostly, but not completely, modified and can contain 5, 4, 3, 2, or 1 or less naturally occurring nucleosides.In still other embodiments, the oligonucleotide molecule can contain 5, 4, 3, 2, or 1 or less alternative nucleosides.

[0120] Nucleic acids can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SLet al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Alternative nucleotides and nucleosides include those that include modifications, such as terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, DNA nucleotides, reverse linkage, etc.), base modifications, such as replacement with stabilizing bases, destabilizing bases, or bases that base pair with a broad repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and / or backbone modifications, including modification or substitution of phosphodiester linkages. The nucleobase may be an isonucleoside in which the nucleobase is moved from the C1 position of the sugar moiety to a different position (e.g., C2, C3, C4, or C5). Specific examples of oligonucleotide compounds useful in the embodiments described herein include, but are not limited to, alternative nucleosides containing modified backbones or non-natural internucleoside linkages. Nucleotides and nucleosides with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referred to in the art, alternative RNAs that do not have a phosphorus atom in the internucleoside backbone of RNA can be considered to be oligonucleosides. In some embodiments, the oligonucleotide will have a phosphorus atom in its internucleoside backbone.

[0121] Alternative internucleoside linkages include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boronophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with inverted polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0122] Representative United States patents which teach the preparation of the above phosphorus-containing linkages include U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,133, and 5,476,201. No. 1, No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476,925, No. 5,5 19,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,799, 5,587,361, No. 5,625,050, No. 6,028,188, No. 6,124,445, No. 6,160,109, No. 6,169,170, No. 6,172,209, No. 6,239,2 No. 65, No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639, No. 6,6 Nos. 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Patent No. RE39464, the contents of each of which are incorporated herein by reference in their entirety.

[0123] Alternative internucleoside linkages that do not include a phosphorus atom in the linkage have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH2 constituent moieties.

[0124] Representative U.S. patents which teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, and 5,489,677. Nos. 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the contents of each of which are incorporated herein by reference in their entirety.

[0125] In other aspects, suitable oligonucleotide molecules include those in which both the sugar and the internucleoside linkage of the nucleotide unit, i.e., the backbone, are replaced. The base unit is maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, a mimetic that has been shown to have excellent hybridization properties, is called peptide nucleic acid (PNA). In PNA compounds, the sugar of the nucleoside is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the contents of each of which are incorporated herein by reference in their entirety. Additional PNA compounds suitable for use in oligonucleotides are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0126] Some embodiments include oligonucleotide molecules with phosphorothioate backbones and oligonucleotides with heteroatom backbones, and in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, -CH2-N(CH3)-N(CH3)-CH2- and -N(CH3)-CH2-CH2- [wherein the natural phosphodiester backbone is represented as -OPO-CH2-] of the aforementioned U.S. Patent No. 5,489,677, as well as the amide backbones of the aforementioned U.S. Patent No. 5,602,240. In some embodiments, the oligonucleotide molecules featured herein have the morpholino backbone structure of the aforementioned U.S. Patent No. 5,034,506. In other aspects, the oligonucleotide molecules described herein comprise phosphorodiamidate morpholino oligomers (PMOs), in which the deoxyribose moieties are replaced by morpholine rings and the charged phosphodiester intersubunit linkages are replaced by uncharged phosphorodiamidate linkages, as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70.

[0127] Alternative nucleosides and nucleotides can contain one or more substituted sugar moieties. Oligonucleotides, such as those featured herein, can include one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 (Alkenyl and alkynyl). Exemplary and preferred modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2, -O(CH2) n CH3, -O(CH2) n-ONH2 and -O(CH2) n -ON[(CH2) n CH3]2, where n and m are from 1 to about 10. In other embodiments, the oligonucleotide includes one of the following at the 2' position: C1 to C 10 Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification comprises 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. MOE nucleosides confer several beneficial properties to oligonucleotides, including, but not limited to, increased nuclease resistance, improved pharmacokinetic properties, reduced non-specific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity compared to unmodified oligonucleotides.

[0128] Another exemplary alternative contains the group -O(CH2)2ON(CH3)2, also known as 2'-dimethylaminooxyethoxy, i.e. 2'-DMAOE, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e. 2'-O-(CH2)2-O-(CH2)2-N(CH3)2, as described in the examples herein below. Further exemplary alternatives include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families), 2'-alkoxyalkyl, and 2'-NMA (N-methylacetamide).

[0129] Other alternatives include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can be made at other positions on the nucleosides and nucleotides of the oligonucleotide molecule, particularly the 3' position of the sugar in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotide molecules can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents which teach the preparation of such modified sugar structures include U.S. Pat. Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,520,136, and 5,530,782. Nos. 5,576,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920, several of which are commonly owned with the present application. The entire contents of each of the aforementioned patents are incorporated herein by reference.

[0130] Oligonucleotide molecules may contain nucleobase (often simply referred to in the art as "base") alternatives (e.g., modifications or substitutions). Unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Alternative nucleobases include other synthetic and natural nucleobases, such as 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine, 5-methoxyuridine, 5-hydroxydeoxyuridine, dihydrouridine, 4-thiouridine, pseudouridine, 1-methyl-pseudouridine, deoxyuridine, 5-hydroxyuridine, 5-hydroxy-1,1-trimethyl ... Sibutinyl-2'-deoxyuridine, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-methylguanosine, 7-deazaguanosine, 6-aminomethyl-7-deazaguanosine, 8-aminoguanine, 2,2,7-trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-deazaadenine, 3-deazaadenosine purine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluridine and cytidine, 6-azouridine, cytidine and thymine, 4-thiouridine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uridines and cytidines, 8-azaguanine and 8-azaadenine, and 3-deazaguanine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30: 613, and those disclosed by Sanghvi, Y S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of oligonucleotides. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including, for example, 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278), and are an exemplary base substitution, especially when combined with a 2′-O-methoxyethyl sugar modification.

[0131] Representative United States patents that teach the preparation of other alternative nucleobases, in addition to some of the alternative nucleobases mentioned above, include the above-mentioned U.S. Patent Nos. 3,687,808, 4,845,205, 5,130,30, 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, 5,587,469, 5,594,121, 5,595,121, 5,596,121, 5,597,121, 5,598,121, 5,599,122, 5,600, 5,601, 5,610, 5,611, 5,612, 5,613, 5,614, 5,615, 5,616, 5,617, 5,618, 5,619, 5,620, 5,621, 5,625, 5,630, 5,631, 5,632, 5,635, 5,640, 5,641, 5,642, 5,643, 5,645, 5,650, 5,651, 5,652, 5,655, 5,656, 5,657, 5,658, 5,65 Nos. 5,596,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the contents of each of which are incorporated herein by reference in their entirety.

[0132] In other embodiments, the sugar moiety in a nucleotide can be a ribose molecule, optionally having a 2'-O-methyl, 2'-O-MOE, 2'-F, 2'-amino, 2'-O-propyl, 2'-aminopropyl, or 2'-OH modification.

[0133] An oligonucleotide molecule may contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a bridge of two atoms. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, an oligonucleotide may contain one or more locked nucleosides. A locked nucleoside is a nucleoside having a modified ribose moiety, where the ribose moiety includes an additional bridge connecting the 2'-carbon and the 4'-carbon. In other words, a locked nucleoside is a nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo structural conformation. The addition of locked nucleosides to oligonucleotides has been shown to increase oligonucleotide stability in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in polynucleotides include, but are not limited to, nucleosides that contain a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the polynucleotide agent includes one or more bicyclic nucleosides that contain a 4' to 2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH2)-O-2' (LNA), 4'-(CH2)-S-2', 4'-(CH2)2-O-2' (ENA), 4'-CH(CH3)-O-2' (also referred to as "constrained ethyl" or "cEt") and 4'-CH(CHOCH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 7,399,845), 4'-C (CH3)(CH3)-O-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,283), 4'-CH2-N(OCH3)-2' (and analogs thereof, see, e.g., U.S. Pat. No. 8,278,425), 4'-CH2-ON(CH3)2-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570), 4'-CH2-N(R)-O-2' (wherein R is H, C1-C. 12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672), 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, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing patents are incorporated herein by reference.

[0134] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include the following: U.S. Patent Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,420,117, and 7,525,191. Nos. 7,427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, US2008 / 0039618, and US2009 / 0012281, the contents of each of which are incorporated herein by reference in their entirety.

[0135] Any of the foregoing bicyclic nucleosides may be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0136] The oligonucleotide molecule may be modified to include one or more constrained ethyl nucleosides. As used herein, a "constrained ethyl nucleoside" or "cEt" is a locked nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleoside is in the S configuration, referred to herein as "S-cEt."

[0137] An oligonucleotide molecule may contain one or more "conformationally restricted nucleosides" ("CRNs"). CRNs are nucleoside analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and --C5' carbons of ribose. The CRNs lock the ribose ring into a stable conformation and increase hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, resulting in reduced puckering of the ribose ring.

[0138] Representative publications that teach the preparation of some of the above-mentioned CRNs include, but are not limited to, U.S. Patent Publication No. 2013 / 0190383, and PCT Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0139] In some embodiments, the oligonucleotide molecule comprises one or more monomers that are UNA (unlocked nucleosides) nucleosides. UNA is an unlocked acyclic nucleoside, in which any of the sugar bonds have been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0140] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the contents of each of which are incorporated by reference in their entirety herein.

[0141] The ribose molecule may be modified with a cyclopropane ring to produce tricyclodeoxynucleic acid (tricycloDNA). The ribose moiety may be replaced with another sugar, such as 1,5-anhydrohexitol, threose to produce threose nucleosides (TNAs), or arabinose to produce arabinonucleosides. The ribose molecule may be replaced with a non-sugar, such as cyclohexene to produce cyclohexene nucleosides, or glycol to produce glycol nucleosides.

[0142] Stabilizing modifications to the termini of the nucleoside molecules can potentially include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0143] Other alternative chemistries for oligonucleotide molecules include 5' phosphates or 5' phosphate mimetics of oligonucleotides, such as 5' terminal phosphates or phosphate mimetics.Suitable phosphate mimetics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0144] Exemplary oligonucleotide molecules include nucleosides with alternative sugar moieties and may include DNA or RNA nucleosides. In some embodiments, oligonucleotide molecules include nucleosides with alternative sugar moieties and DNA nucleosides. The incorporation of alternative nucleosides into oligonucleotide molecules may enhance the affinity of the oligonucleotide to a target nucleic acid. In this case, the alternative nucleoside may be referred to as an affinity enhancing alternative nucleotide.

[0145] In some embodiments, the oligonucleotide molecule comprises at least one alternative nucleoside, such as at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 alternative nucleosides. In other embodiments, the oligonucleotide molecule comprises 1-10 alternative nucleosides, such as 2-9 alternative nucleosides, such as 3-8 alternative nucleosides, such as 4-7 alternative nucleosides, such as 6 or 7 alternative nucleosides. In one embodiment, the oligonucleotide molecule can comprise alternatives, which are independently selected from these three types of alternatives (alternative sugar moieties, alternative nucleobases, and alternative internucleoside linkages), or combinations thereof. In one embodiment, the oligonucleotide molecule comprises one or more nucleosides comprising a sugar substitute moiety, e.g., a 2' sugar substitute nucleoside. In some embodiments, the oligonucleotide molecule comprises one or more 2' sugar substitute nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, and BNA (e.g., LNA) nucleosides.

[0146] An exemplary structure of an LNA is as follows:

[0147] [ka]

[0148] In some embodiments, one or more of the substituted nucleosides is a BNA.

[0149] In some embodiments, at least one of the alternative nucleosides is a BNA (e.g., an LNA (e.g., A-LNA, 5mC L-NA, G-LNA, T-LNA)), such as at least two of the alternative nucleosides, such as at least three, at least four, at least five, at least six, at least seven, or at least eight, etc. In even further embodiments, all of the alternative nucleosides are BNAs.

[0150] In further embodiments, the oligonucleotide molecule comprises at least one alternative internucleoside bond.In some embodiments, the internucleoside bond in adjacent nucleotide sequence is phosphorothioate or boronophosphate internucleoside bond.In some embodiments, all internucleoside bonds in adjacent sequences of oligonucleotide molecule are phosphorothioate bond.In some embodiments, phosphorothioate bond is stereochemically pure phosphorothioate bond.In some embodiments, phosphorothioate bond is Sp phosphorothioate bond.In other embodiments, phosphorothioate bond is Rp phosphorothioate bond.

[0151] In some embodiments, the oligonucleotide molecule comprises at least one alternative nucleoside that is 2'-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE-RNA nucleoside units. In some embodiments, the 2'-MOE-RNA nucleoside units are linked by phosphorothioate bonds. In some embodiments, at least one of the alternative nucleosides is 2'-fluoro-DNA, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleoside units. In some embodiments, the oligonucleotide molecule comprises at least one BNA unit and at least one 2'-substituted modified nucleoside. In some embodiments, the oligonucleotide molecule comprises both 2' sugar modified nucleosides and DNA units. In some embodiments, the oligonucleotide molecule or its adjacent nucleotide region is a gapmer oligonucleotide molecule.

[0152] Oligonucleotide molecules conjugated to ligands Oligonucleotide molecules can be chemically linked to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include lipid moieties, such as cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), thioethers, such as beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids, 1:106-1070), and cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060). Res., 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids, 75:49-54). Res., 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moieties (Mishra et al., (1995) Biochim. Biophys.Acta 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0153] In one aspect, a ligand alters the distribution, targeting, or lifetime of an oligonucleotide agent into which it is incorporated. In some aspects, a ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a body site, e.g., when compared to a species in which such ligand is not present.

[0154] Ligands can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulins), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid), or lipids. Ligands can be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of polyamine, or alpha-helical peptide.

[0155] The ligand may comprise a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a particular cell type, such as a kidney cell. The targeting group may be thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0156] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-( oleoyl), cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0157] The ligand may be a protein, e.g., a glycoprotein, or a peptide, e.g., a molecule with specific affinity for a co-ligand, or an antibody, e.g., an antibody that binds to a particular cell type, such as a hepatocyte. Ligands may include hormones and hormone receptors. They may include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.

[0158] A ligand can be a substance, e.g., a drug, that can increase uptake of an oligonucleotide agent into a cell, e.g., by disrupting the cytoskeleton of the cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0159] In some embodiments, the ligand attached to the oligonucleotide molecule as described herein functions as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing multiple phosphorothioate bonds are also known to bind to serum proteins, and therefore short oligonucleotides, for example, about 5-base, 10-base, 15-base, or 20-base oligonucleotides, containing multiple phosphorothioate bonds in the backbone, are also suitable as ligands (e.g., as PK-modulating ligands). In addition, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0160] Ligand-conjugated oligonucleotide molecules can be synthesized by using oligonucleotides with pendant reactive functional groups, such as those resulting from the attachment of a linking molecule onto an oligonucleotide (described below). The reactive oligonucleotide molecules can be reacted directly with commercially available ligands, with synthesized ligands having any of a variety of protecting groups, or with ligands having a linking moiety attached to the ligand.

[0161] The oligonucleotide molecules used in the conjugates can be conveniently and routinely produced by the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art can additionally or alternatively be used. It is also known to use similar techniques to prepare other oligonucleotides, such as phosphorothioates and alkylated derivatives.

[0162] For ligand-conjugated oligonucleotide molecules, such as ligand molecules having sequence-specific linked nucleosides, oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer using standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-bearing components.

[0163] When using a conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed and then the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotide or linked nucleoside is synthesized by an automated synthesizer using phosphoramidites derived from the ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0164] i. Lipid complex In one embodiment, the ligand or complex is lipid or lipid-based molecule.Such lipid or lipid-based molecule can bind to serum protein, for example, human serum albumin (HSA).HSA binding ligand allows the distribution of complex to target tissue, for example, non-renal target tissue in the body.Lipid or lipid-based ligand can (a) increase the degradation resistance of complex, (b) increase targeting or transport to target cell or cell membrane, and / or (c) be used to adjust the binding to serum protein, for example, HSA.

[0165] In another embodiment, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferative cell. Exemplary vitamins include vitamins A, E, and K.

[0166] ii. Cell-penetrating agents In another embodiment, the ligand is a cell-penetrating agent, such as a helical cell-penetrating agent. In one embodiment, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennapedia. If the agent is a peptide, it can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide bonds, and the use of D-amino acids. In one embodiment, the helical agent is an alpha-helical agent, which can have a lipophilic and lipophobic phase.

[0167] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of the oligonucleotide, such as by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0168] The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) can be targeting moieties. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, it has been found that sequences derived from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK) can function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library, or a one bead one compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic tethered to an oligonucleotide agent by a monomer unit incorporated for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. The peptide portion can range in length from about 5 amino acids to about 40 amino acids. The peptide portion can have structural modifications, such as to increase stability or induce conformational properties. Any of the structural modifications described below can be utilized.

[0169] RGD peptides for use in the compositions and methods can be linear or cyclic, and can be modified, for example, glycosylated or methylated, to facilitate targeting to a particular tissue(s).RGD-containing peptides and peptidomimetics can include synthetic RGD mimetics in addition to D-amino acids.In addition to RGD, other moieties can be used that target integrin ligands.Some complexes of this ligand target PECAM-1 or VEGF.

[0170] The cell-penetrating peptide can penetrate cells, such as microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. The microbial cell-penetrating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or cecropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin, or bactenecin), or a peptide containing only one or two key amino acids (e.g., PR-39 or indolicidin). The cell-penetrating peptide can include a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

[0171] iii. Carbohydrate Complex In some embodiments of the compositions and methods described herein, the oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated oligonucleotides are advantageous in compositions suitable for in vivo therapeutic applications in addition to in vivo delivery of nucleic acids, as described herein. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate itself, composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom, or a compound that has as part of it a carbohydrate moiety, each of which is composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units), as well as polysaccharides, such as starch, glycogen, cellulose and polysaccharide gums. Particular monosaccharides include sugars of C5 or higher (e.g., C5, C6, C7, or C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0172] In one aspect, a complex carbohydrate for use in the compositions and methods described herein is a monosaccharide.

[0173] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell penetrating peptide.

[0174] Additional carbohydrate conjugates (and linkers) suitable for use include those described in PCT Publication Nos. WO2014 / 179620 and WO2014 / 179627, the contents of each of which are incorporated herein by reference in their entirety.

[0175] iv. Linker In some aspects, the conjugates or ligands described herein may be attached to the oligonucleotide using a variety of linkers, which may be cleavable or non-cleavable.

[0176] The linker is typically a direct bond or an atom, such as oxygen or sulfur, or a unit, such as NR 8 , C(O), C(O)NH, SO, SO2, SO2NH, etc., or chains of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl ... and the like, wherein one or more methylenes are selected from the group consisting of O, S, S(O), SO, N(R), S ... 8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic (wherein R 8 is hydrogen, acyl, aliphatic or substituted aliphatic). In one embodiment, the linker is about 1-24, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, 8-16 or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 21, 22, 23, or 24 atoms.

[0177] A cleavable linking group is one that is sufficiently stable outside a cell, but that upon entry into a target cell is cleaved to release the two moieties that the linker is holding together. In some embodiments, the cleavable linking group is cleaved at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or more, or at least 100 times faster in the target cell or under a first reference condition (which may be selected, for example, to mimic or represent intracellular conditions) than in the subject's blood or under a second reference condition (which may be selected, for example, to mimic or represent conditions found in blood or serum).

[0178] Cleavable linking groups are susceptible to cleaving agents, such as pH, redox potential, or the presence of degradable molecules. Generally, cleaving agents are more prevalent or found at higher levels or activity inside cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a particular substrate or have no substrate specificity at all, such as oxidizing or reducing enzymes, or reducing agents, such as mercaptans present in cells, that can degrade redox-cleavable linking groups by reduction, esterases, endosomes or agents that can cause an acidic environment, such as those that cause a pH of 5 or less, general acids, peptidases (which can be substrate specific), and enzymes that can function as phosphatases to hydrolyze or degrade acid-cleavable linking groups.

[0179] Cleavable linking groups, such as disulfide bonds, may be sensitive to pH. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of approximately 5.0. Some linkers have cleavable linking groups that are cleaved at a preferred pH, releasing the cationic lipid from the ligand inside the cell or into a desired compartment of the cell.

[0180] The linker may include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. For example, a ligand that targets the liver may be linked to a cationic lipid via a linker that includes an ester group. Because liver cells are rich in esterase, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterase. Other cell types that are rich in esterase include lung, renal cortex, and testis cells.

[0181] Linkers containing peptide bonds may be used when targeting cell types that are rich in peptidases, such as liver cells and synovial cells.

[0182] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It may also be desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between at least two conditions, where at least one condition is selected to indicate cleavage in target cells and another condition is selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluation in a cell-free or culture conditions and confirm by further evaluation in whole animals. In some embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) when compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0183] a. Redox-cleavable linking group In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductively cleavable linking group is a disulfide linking group (--S--S--). The methods described herein can be used to determine whether a candidate cleavable linking group is a suitable "reductively cleavable linking group" or is suitable for use with, for example, a particular oligonucleotide moiety and a particular targeting agent. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that mimic the cleavage rate that would be observed in cells, for example, target cells. The candidate can be evaluated under conditions selected to mimic blood or serum conditions. In one embodiment, the candidate compound is cleaved at most about 10% in blood. In other embodiments, useful candidate compounds are degraded at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) when compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetics assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0184] b. Phosphate-based cleavable linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linking group. The phosphate-based cleavable linking group is cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group in a cell is an enzyme such as a phosphatase in the cell. An example of a phosphate-based linking group is OP(O)(OR k )-O-, -OP(S)(OR k )-O-, -OP(S)(SR k )-O-, -SP(O)(OR k )-O-, -OP(O)(OR k )-S-, -SP(O)(OR k )-S-, -OP(S)(ORk )-S-, -SP(S)(OR k )-O-, -OP(O)(R k )-O-, -OP(S)(R k )-O-, -SP(O)(R k )-O-, -SP(S)(R k )-O-, -SP(O)(R k )-S-, -OP(S)(R k )-S-. These candidates can be evaluated using methods similar to those described above.

[0185] c. acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid-cleavable linking group. An acid-cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments, the acid-cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent such as an enzyme that can function as a general acid. In cells, certain low pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for the acid-cleavable linking group. Examples of acid-cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN--, C(O)O, or -OC(O). In one embodiment, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0186] d. Ester-based linking groups In another embodiment, the cleavable linker comprises an ester-based cleavable linking group. The ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula --C(O)O--, or --OC(O)--. These candidates can be evaluated using methods similar to those described above.

[0187] e. Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable linking group. The peptide-based cleavable linking group is cleaved by enzymes, such as peptidases and proteases, in cells. A peptide-based cleavable linking group is a peptide bond formed between amino acids to generate oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. A peptide-based cleavable group does not include amide groups (-C(O)NH-). An amide group can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to generate peptides and proteins. A peptide-based cleaving group is generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to generate peptides and proteins, and does not include the entire amide functionality. A peptide-based cleavable linking group has the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0188] In one embodiment, the oligonucleotide is conjugated to the carbohydrate via a linker. The linker includes bivalent and trivalent branched linker groups. The linker of the oligonucleotide carbohydrate conjugate includes, but is not limited to, those described in formulas 24-35 of PCT Publication No. WO2018 / 195165.

[0189] Representative U.S. patents which teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603, No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4,605,735, No. 4,667,025, No. 4,762,779, No. 4,789,737, No. 4,824,941 , No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5,0 No. 82,830, No. 5,112,963, No. 5,214,136, No. 5,082,830, No. 5,112,963 No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5, No. 262,536, No. 5,272,250, No. 5,292,873, No. 5,317,098, No. 5,371,24 No. 1, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. 5 ,512,667, 5,514,785, 5,565,552, 5,567,810, 5,574,1 42, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, and 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022, the contents of each of which are incorporated herein by reference in their entirety.

[0190] Not all positions in a given compound need be uniformly modified, and in fact more than one of the above modifications may be incorporated into a single compound, or even into a single nucleoside within an oligonucleotide. Oligonucleotide compounds that are chimeric compounds are also contemplated. Chimeric oligonucleotides usually contain at least one region in which the RNA is modified to confer increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target nucleic acid to the oligonucleotide. Additional regions of the oligonucleotide may serve as substrates for enzymes capable of cleaving RNA:DNA. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly improving the efficiency of oligonucleotide inhibition of gene expression. As a result, comparable results can often be obtained with shorter oligonucleotides when chimeric oligonucleotides are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, optionally, associated nucleic acid hybridization techniques known in the art.

[0191] In certain cases, the nucleotides of the oligonucleotide may be modified by non-ligand groups. Numerous non-ligand molecules have been conjugated to oligonucleotides to enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide, and procedures for carrying out such conjugation are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids, 1997, 1:131-132), and the like. Res., 1992, 20:533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), palmityl moieties (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such oligonucleotide conjugates are listed above. A typical conjugation protocol involves the synthesis of an oligonucleotide bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule being conjugated using an appropriate coupling or activating reagent. The conjugation reaction can be carried out either with the oligonucleotide still attached to the solid support or in solution phase after oligonucleotide cleavage. Purification of the oligonucleotide conjugate by HPLC usually provides a pure conjugate.

[0192] Medical Use The oligonucleotide molecules, or pharma- ceutically acceptable salts thereof, compositions described herein are useful in the methods described herein and, without being bound by theory, are believed to exert their desired effects by their ability to modulate the level, status, and / or activity of target proteins.

[0193] One embodiment relates to a method of treating a disorder associated with a neurodegenerative disorder in a subject in need thereof. Another embodiment includes reducing the level of a target mRNA in a cell of a subject identified as having a neurodegenerative disorder. Yet another embodiment includes a method of inhibiting expression of a target gene, e.g., MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43, in a cell of the subject. The method includes contacting a cell with an oligonucleotide molecule or a pharma- ceutically acceptable salt thereof in an amount effective to inhibit expression of the target mRNA in the cell, thereby inhibiting expression of the target mRNA in the cell.

[0194] In some embodiments, the neurodegenerative disease is not a triplet repeat disorder. In some embodiments, the neurodegenerative disease is not a nucleotide repeat disorder. Nucleotide repeat expansion disorders (e.g., trinucleotide repeat expansion disorders) are a group of genetic disorders characterized by pathogenic expansion of repeat regions within genomic regions. In such disorders, the number of repeats exceeds the normal stable threshold number of genes and expands to the disease range. Exemplary trinucleotide repeat expansion disorders and the trinucleotide repeats of genes commonly associated with them are included in Table 1.

[0195] [Table 1-1]

[0196] [Table 1-2]

[0197] Based on the above method, the oligonucleotide molecule, or its pharma- ceutically acceptable salt, or a composition comprising such an oligonucleotide molecule or its pharma- ceutically acceptable salt, is for use in therapy, or for use as a medicine, or for use in treating a neurodegenerative disorder (e.g., Alzheimer's disease or Parkinson's disease) in a subject in need thereof, or for use in reducing the level of a target mRNA in a cell of a subject identified as having a neurodegenerative disorder, or for use in inhibiting the expression of a target mRNA in a cell of a subject. These uses include contacting a cell with the oligonucleotide molecule, or its pharma- ceutically acceptable salt, in an amount effective to inhibit the expression of the target mRNA in the cell, thereby inhibiting the expression of the target mRNA in the cell. The embodiments described below that relate to the methods described herein are also applicable to these further embodiments.

[0198] In some aspects, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, Parkinson's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, dementia with Lewy bodies, multiple system atrophy, cerebral amyloid angiopathy, schizophrenia, dentatorubral-pallidoluysian atrophy, and Down's syndrome-associated Alzheimer's disease. In some aspects, the neurodegenerative disease is Alzheimer's disease. In some aspects, the neurodegenerative disease is Pick's disease. In some aspects, the neurodegenerative disease is Parkinson's disease. In some aspects, the neurodegenerative disease is frontotemporal dementia. In some aspects, the neurodegenerative disease is progressive supranuclear palsy. In some aspects, the neurodegenerative disease is corticobasal degeneration. In some aspects, the neurodegenerative disease is dementia with Lewy bodies. In some aspects, the neurodegenerative disease is multiple system atrophy. In some aspects, the neurodegenerative disease is cerebral amyloid angiopathy. In some embodiments, the neurodegenerative disease is schizophrenia. In some embodiments, the neurodegenerative disease is dentatorubral-pallidoluysian atrophy. In some embodiments, the neurodegenerative disease is Down's syndrome-associated Alzheimer's disease.

[0199] Inhibition of expression of a target gene includes any level of inhibition of a target gene, such as MSH3, MLH3, MLH1, OGG1, MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43, at least partial suppression of expression of the target gene, for example, at least 20% inhibition. In some embodiments, inhibition is at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0200] Expression of a target gene can be assessed based on the level of any variable associated with gene expression, for example, mRNA levels or protein levels.

[0201] Inhibition can be assessed by a decrease in the absolute or relative level of one or more of these variables compared to a control level, which can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar subject, cell, or sample that is untreated or treated with a control, such as a buffer-only control or an inactive agent control.

[0202] The control cell or cell group that can be used to evaluate the inhibition of expression of the target gene includes a cell or cell group that has not yet been contacted with the oligonucleotide molecule.For example, the control cell or cell group can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the oligonucleotide.

[0203] The level of mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of a target gene in a sample is determined by detecting a transcribed polynucleotide, or a portion thereof, such as mRNA. RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B, Biogenesis), RNEASY™ RNA preparation kit (Qiagen) or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA can be detected using methods described in PCT Publication No. WO2012 / 177906, the entire contents of which are incorporated herein by reference. In some embodiments, the expression level of a target gene is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule that can selectively bind to a specific sequence, for example, to an mRNA or a polypeptide. Probes can be synthesized by those skilled in the art or obtained from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0204] The isolated mRNA can be used in hybridization or amplification assays, including, but not limited to, Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method of determining mRNA levels includes contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an AFFYMETRIX gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in determining mRNA levels.

[0205] Alternative methods for determining the expression level of a target protein in a sample include nucleic acid amplification of, e.g., mRNA and / or reverse transcriptase (to prepare cDNA) in a sample, e.g., RT-PCR (experimental aspects described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are particularly useful for detection of nucleic acid molecules when such molecules are present in very low numbers. In some embodiments, the expression level of the target gene is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ system) or the DUAL-GLO® luciferase assay.

[0206] The expression level of mRNA can be monitored using membrane blots (such as those used in hybridization analysis such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acid).See U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934, which are incorporated herein by reference.Determining the mRNA expression level can include using a nucleic acid probe in solution.

[0207] In some embodiments, the level of mRNA expression is evaluated using branched DNA (bDNA) assay or real-time PCR (qPCR).The use of this PCR method is described and illustrated in the examples presented herein.Such methods can be used for detecting nucleic acids.

[0208] The level of protein expression can be determined by using any method known in the art for measuring protein level.Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc.Such assays can be used to detect protein, which indicates the presence or replication of protein.

[0209] In some embodiments of the method described herein, the oligonucleotide molecule, or its pharmaceutically acceptable salt, is administered to the subject so that the oligonucleotide molecule, or its pharmaceutically acceptable salt, is delivered to a specific site in the subject.The inhibition of expression of the target gene can be evaluated using the measurement of the level or change in the level of mRNA or protein in the sample from a specific site in the subject.For example, in some embodiments, the method includes the clinically relevant inhibition of the expression of the target gene, for example, as demonstrated by the clinically relevant results after treating the subject with an agent for reducing the expression of the target gene.

[0210] In other aspects, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered in an amount and for a time effective to result in one (or more) of the following: (a) a reduction in cell death (e.g., CNS cell death and / or muscle cell death); (b) a delay in the onset of the disorder; (c) an increase in the subject's survival time; and (d) an increase in the subject's progression-free survival time.

[0211] Treatment of neurodegenerative disease may increase the average survival time of an individual or a population of subjects treated with the oligonucleotide molecule or a pharma- ceutically acceptable salt thereof described herein, compared to an untreated population of subjects. For example, the individual survival time or the population average survival time is increased by more than 30 days (more than 60 days, 90 days, or 120 days). The increase in the individual survival time or the population average survival time may be measured by any reproducible means. The increase in the individual survival time may be measured, for example, by calculating the length of survival time for an individual after the start of treatment with a compound described herein. The increase in the population average survival time may be measured, for example, by calculating the average length of survival time for an individual after the start of treatment with a compound described herein. The increase in the individual survival time may be measured, for example, by calculating the length of survival time for an individual after the completion of a first round of treatment with a compound described herein or a pharma- ceutically acceptable salt of the compound. The increase in the population average survival time may be measured, for example, by calculating the average length of survival time for a population after the completion of a first round of treatment with a compound described herein or a pharma- ceutically acceptable salt of the compound.

[0212] Neurodegenerative disease can result in a reduction in the mortality rate of a treated subject population compared to an untreated population.For example, the mortality rate is reduced by more than 2% (for example, more than 5%, 10%, or 25%).The reduction in the mortality rate of a treated subject population can be measured by any reproducible means, for example, by calculating the average number of disease-related deaths per unit time for a population after starting treatment with a compound or a pharma-ceutically acceptable salt of a compound described herein.The reduction in the mortality rate of a population can be measured by calculating the average number of disease-related deaths per unit time for a population after completing a first round of treatment with a compound or a pharma-ceutically acceptable salt of a compound described herein.

[0213] A. Delivery of Oligonucleotide Molecules Delivery of oligonucleotide molecules to cells, such as cells in a subject, such as a human subject, e.g., a subject in need thereof, e.g., a subject having a neurodegenerative disorder, can be achieved via intracerebroventricular (ICV) administration.

[0214] B. Combination Therapy The oligonucleotide molecule, or its pharmaceutically acceptable salt, can be used alone or in combination with at least one additional therapeutic agent, for example, other agents for neurodegenerative disorders or symptoms associated therewith, or in combination with other types of therapies for treating neurodegenerative disorders. In combination therapy, the dosage of one or more of the therapeutic compounds can be reduced from the standard dosage when administered alone. For example, dosage can be empirically determined from drug combinations and permutations, or estimated by isobologram analysis (e.g., Black et al., Neurology 65:S3-S6(2005)). In this case, the dosage of the compounds when combined should provide therapeutic effect.

[0215] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, that is the agent described herein can be used in combination with at least one additional therapeutic agent to treat a neurodegenerative disorder (e.g., Alzheimer's disease or Parkinson's disease). In some embodiments, at least one of the additional therapeutic agents can be an oligonucleotide (e.g., ASO) that hybridizes with the mRNA of a gene associated with a neurodegenerative disease.

[0216] In some embodiments, at least one of the additional therapeutic agents can be a therapeutic agent that is a non-drug treatment, for example, at least one of the additional therapeutic agents is physical therapy.

[0217] In any of the combination embodiments described herein, two or more therapeutic agents are administered simultaneously or sequentially in any order. For example, a first therapeutic agent can be administered immediately before or after one or more of the additional therapeutic agents, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7 days, 1-14 days, 1-21 days, or 1-30 days.

[0218] Pharmaceutical Compositions The oligonucleotide molecules described herein, or pharma- ceutically acceptable salts thereof, are formulated into pharmaceutical compositions for administration to human subjects in a biocompatible form suitable for administration in vivo.

[0219] The compounds described herein can be used in the form of free base, salt, solvate, and prodrug.All forms are within the scope of the methods described herein.As will be understood by those skilled in the art, according to the methods described herein, the described oligonucleotide molecules or their pharma-ceutically acceptable salts, solvates, or prodrugs can be administered to patients in various forms according to the route of administration selected.The compounds described herein can be administered, for example, by intracerebroventricular administration, and pharmaceutical compositions are formulated accordingly.

[0220] Solutions of the compounds described herein for intraventricular administration can be prepared in artificial cerebrospinal fluid or water, suitably mixed with suitable buffers and / or osmolarity agents, such as one or more of sodium chloride, potassium chloride, potassium phosphate, sodium carbonate, glucose, calcium chloride, sodium bicarbonate, and / or magnesium chloride. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. Suitable pharmaceutical forms for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that it can be easily administered by syringe.

[0221] The compounds described herein may be administered to animals, e.g., humans, alone or in combination with pharma- ceutically acceptable carriers, the proportions of which will be determined by the solubility and chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice, as described herein.

[0222] Dosage The dosage of the compositions described herein (e.g., compositions comprising oligonucleotide molecules, or pharmaceutically acceptable salts thereof) may vary depending on many factors, such as the pharmacodynamic properties of the compound, the method of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, the frequency of treatment, and the type of concomitant treatment, if any, as well as the clearance rate of the compound in the treated animal. The compositions described herein may be initially administered at a suitable dosage, which may be adjusted accordingly depending on the clinical response. In some aspects, the dosage of the compositions (e.g., compositions comprising oligonucleotide molecules, or pharmaceutically acceptable salts thereof) is a prophylactically or therapeutically effective amount.

[0223] In some aspects, provided herein are pharmaceutical compositions formulated for intraventricular injection.

[0224] In some embodiments, intraventricular administration of the pharmaceutical composition comprising oligonucleotide molecule or its pharmaceutical acceptable salt results in the administration of oligonucleotide to deep brain region.In some embodiments, the administration of oligonucleotide to deep brain means that intraventricular administration of one or more oligonucleotide molecules or its pharmaceutical acceptable salt results in the knockdown of target gene or the suppression of target mRNA in deep brain region.

[0225] In some embodiments, the deep brain region for knocking down target genes or suppressing target mRNA varies according to the disease. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the temporal lobe. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the amygdala. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the hippocampus. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the cerebral cortex. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the pons. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the basal ganglion. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the globus pallidus. In some embodiments, one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, are delivered to the substantia nigra. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the ventral striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the nucleus accumbens. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the dorsal striatum. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the caudate nucleus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the putamen. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the thalamus. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the nucleus basalis of Meynert. In some embodiments, one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, are delivered to the brainstem.In some embodiments, the one or more oligonucleotide molecules, or a pharma- ceutically acceptable salt thereof, are delivered to the cerebellum.

[0226] The compositions described herein can be initially administered intracerebroventricularly at a suitable dosage, which can be adjusted accordingly according to clinical response. In some embodiments, the composition (e.g., the composition comprising the oligonucleotide molecule, or its pharmaceutically acceptable salt) is administered at a prophylactically or therapeutically effective amount.

[0227] In some embodiments, provided herein is a method of treating, preventing, or delaying the onset and / or progression of a neurodegenerative disorder in a subject in need thereof, comprising intracerebroventricularly administering an oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, as described herein. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 2 mg to about 300 mg. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 10 mg to about 250 mg. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 15 mg to about 200 mg. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 25 mg to about 200 mg. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a dose of about 50 mg to about 200 mg. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered in a dose of about 100 mg to about 150 mg.

[0228] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered at a concentration of about 2 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 550 mg, about 600 mg, mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, about 200 mg, about 205 mg, about 210 mg, about 220 mg, about 225 mg, about 230 mg, about 235 mg, about 240 mg, about 245 mg, about 250 mg, about 255 mg, about 260 mg, about 265 mg, about 270 mg, about 275 mg, about 280 mg, about 285 mg, about 290 mg, about 295 mg, or about 300 mg.

[0229] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once a week. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every two weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every three weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every four weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every eight weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every sixteen weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every twenty weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every twenty-four weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every twenty-eight weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every thirty-two weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 36 weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 36 weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 40 weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 44 weeks. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 48 weeks.

[0230] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once a month. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every two months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every three months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every four months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every five months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every six months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every seven months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every eight months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every nine months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every eight months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 10 months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 8 months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 11 months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 8 months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every 12 months.

[0231] In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every eight months. In some embodiments, the oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, is administered once every year.

[0232] In some aspects, the methods described herein delay the onset and / or progression of a neurodegenerative disorder by at least 120 days, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more as compared to expected onset and / or progression.

[0233] kit Kits are contemplated that include (a) a pharmaceutical composition comprising an oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, that reduces the level and / or activity of a target gene in a cell or subject as described herein, and (b) a package insert that includes instructions for carrying out any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising an oligonucleotide molecule, or a pharma- ceutically acceptable salt thereof, that is an agent that reduces the level and / or activity of a target gene in a cell or subject as described herein, (b) an additional therapeutic agent, and (c) a package insert that includes instructions for carrying out any of the methods described herein. EXAMPLES

[0234] Example 1. Antisense oligonucleotides

[0235] [Table 2]

[0236] In the following Table 3, the sequence number corresponds to the nucleobase sequence of antisense oligo number. However, certain antisense oligo number (for example, antisense oligo number 1) contains certain chemical modifications. It is well known in the art that all nucleobases in DNA core are deoxy. Therefore, in Table 3, when the nucleobase in DNA core of sequence is not declared by "o" to represent "moe", the nucleobase is DNA nucleobase (deoxy).

[0237] [Table 3]

[0238] Example 2. Pharmacokinetic profile and tolerability the purpose The aim of this study is to evaluate the pharmacokinetic profile of antisense oligo #1 administration at dose levels above 10mg, as well as its tolerability and efficacy in silencing MSH3 mRNA after a single intracerebroventricular (ICV) injection. The data obtained will also be used to perform pharmacokinetic and pharmacodynamic (PK / PD) modeling and simulation to predict dosing regimens that will achieve >40% and >50% MSH3 mRNA silencing in the caudate nucleus at trough levels within 12 weeks of dosing in a first-in-human (FIH) study.

[0239] Experimental design Sixteen female cynomolgus monkeys (NHPs) were randomized in this study. Necropsies were planned at 2 hours, 8 hours, 24 hours, 48 ​​hours, days 8, 15, and 29 after dosing (N=2 at each time point). Dose escalation was initiated with 15 mg as the starting dose level to monitor dose level tolerability. 2 mL of antisense oligo #1 was delivered via a single ICV injection at an infusion rate of 0.1 mL / min. Up to two sentinel animals were to be dosed at each dose level and monitored for clinical signs over 2-24 hours. If the 15 mg dose level was not well tolerated, monkeys were dosed with 12 mg via a single ICV injection and monitored for clinical signs over 2-24 hours.

[0240] Tissues from various central nervous system (CNS) and peripheral organs are collected at each necropsy time point. CSF and plasma samples are collected from live animals and at necropsy. Concentrations of antisense oligo #1 in CSF, plasma, brain tissue (cerebral cortex, caudate and putamen, nucleus accumbens), liver and kidney are measured using LC-MS / MS. Knockdown of MSH3 mRNA and protein in cerebral cortex, caudate, putamen and nucleus accumbens is measured using RT-qPCR and western blot or ELISA.

[0241] [Table 4]

[0242] Interim results of clinical observations Two female cynomolgus monkeys (day 29) were dosed with 15 mg via a single ICV injection. The first monkey became ataxic, began vomiting, and assumed a stooped posture approximately 3 hours after dosing. The subject stabilized after administration of an anti-nausea medication (Cerenia). The subject recovered after 24 hours and will remain on study until necropsy on day 29. The second monkey became ataxic approximately 4 hours after dosing, stooped, had significantly decreased activity, decreased pupillary reflexes, and assumed a lateral decubitus position. After consultation with a veterinarian, the animal was euthanized approximately 4-5 hours after dosing.

[0243] A third monkey received a single 12 mg injection ICV and dosing was well tolerated. Five additional NHPs received a single 12 mg injection ICV for PK / PD timepoints of 24 hours (n=2), 48 hours (n=2), day 8 (n=1), and day 15 (n=1). A total of eight NHPs were dosed (two at 15 mg and six at 12 mg). Two NHPs served as vehicle controls. Three of the five NHPs experienced ataxia, abnormal righting reflex, tremors, and were intermittently in a lateral decubitus position during the recovery period. However, all animals recovered to normal after 24-36 hours without intervention.

[0244] Example 3. Pharmacokinetic / pharmacodynamic (PK / PD) studies in non-human primates (NHPs) PK / PD investigation of antisense oligo no. 1 in NHPs using intrathecal (IT) injection route Experimental design Twelve male cynomolgus monkeys were administered 10 mg of antisense oligo #1 (2 mL) (in artificial CSF) by a single IT injection. Necropsies were performed at 2, 8, 24, 48, 168 (7 days) and 672 (28 days) hours after dosing (N=2 per time point). Cerebrospinal fluid (CSF) and tissues from various central nervous system (CNS) regions and peripheral organs were collected at each necropsy time point. Plasma samples were collected from live animals and at necropsy. Concentrations of antisense oligo #1 in CSF, plasma, brain tissues (cerebral cortex, caudate and putamen), liver and kidney were measured using liquid chromatography / tandem mass spectrometry (LC-MS / MS). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to measure mRNA levels of MSH3 in the cerebral cortex, caudate, putamen and lumbar spinal cord.

[0245] Experimental Results The concentration-time profile of antisense oligo #1 in CSF after a single IT injection was apparently multiphasic with a rapid initial distribution. A similar concentration-time profile was observed in plasma. Brain tissues (cerebral cortex, caudate and putamen) and peripheral tissues (liver and kidney) showed variable exposure levels of antisense oligo #1, with a slow apparent elimination phase. A multicompartment PK model was developed, which fit well to the concentration-time profiles observed in all tissues measured.

[0246] Compared to CNS tissue obtained from vehicle-dosed (artificial CSF) control animals in a separate study, or tissue collected at an earlier time point (2 hours) in this study, a significant decrease in MSH3 mRNA levels was observed only in the lumbar spinal cord, but not in the cerebral cortex, caudate nucleus, or putamen (Figures 1A-1D).

[0247] Investigation of PK / PD of antisense oligo no. 1 in NHPs using intracerebroventricular (ICV) injection route Experimental design Fifteen female cynomolgus monkeys were administered 10 mg of antisense oligo #1 (2 mL, in artificial CSF) by a single ICV injection. Antisense oligo #1 was injected into the dorsal horn of the lateral ventricle. Necropsies were performed at 2, 8, 24, 48, 168 (7 days), 336 (14 days), and 672 (28 days) hours after dosing (N=2 animals at each time point, except for N=3 at 336 (14 days)). Two naive animals that did not receive an injection were sacrificed and served as controls. The duration of action was further investigated up to day 84. Six female cynomolgus monkeys were administered 10 mg of antisense oligo #1 by a single ICV injection. Necropsies were performed on days 42, 56, and 84 (N=2 animals at each time point).

[0248] Tissues from various CNS regions and peripheral organs were collected at each necropsy time point. CSF and plasma samples were collected from selected survivors and at necropsy. Concentrations of antisense oligo #1 in CSF, plasma, brain tissue (cerebral cortex, caudate nucleus and putamen), liver and kidney were measured using LC-MS / MS. RT-qPCR was used to measure mRNA levels of MSH3 in the cerebral cortex, caudate nucleus, putamen and lumbar spinal cord.

[0249] Experimental Results The concentration-time profile of antisense oligo no. 1 in CSF after a single ICV injection was apparently multiphasic with a rapid initial distribution. A similar concentration-time profile was observed in plasma. Brain tissues (cerebral cortex, caudate and putamen) and peripheral tissues (liver and kidney) showed variable exposure levels of antisense oligo no. 1, with a slow apparent elimination phase. Significantly higher concentrations of antisense oligo no. 1 were observed in brain tissue than those observed after IT injection at the same dose (10 mg). A multicompartment PK model was developed, which provided a good fit to the concentration-time profiles observed in all tissues measured.

[0250] Compared to CNS tissues obtained from naive control animals in this study, a significant time-dependent decrease in MSH3 mRNA levels was observed in the cerebral cortex and caudate nucleus, with knockdown (KD) of 76% and 46%, respectively, at day 29 (28 days post-dosing, Figures 2A-2D). No significant KD of MSH3 mRNA was observed in the putamen.

[0251] Significant KD of MSH3 mRNA was observed in both ipsilateral and contralateral cortices (frontal and temporal cortices) and the nucleus accumbens (Figures 4A-4B). The KD rates on days 8, 15, and 29 are shown below, respectively. Frontal cortex: Same side: 53( ** ), 65( *** ) and 68% ( *** ) Contralateral: 63, 67 ( *** ) and 81% ( *** ) temporal cortex Same side: 23, 64 ( *** ) and 77% ( *** ) Contralateral: 36( * ), 51( *** ) and 66% ( *** ) Nucleus accumbens Same side: 43( ** ), 48( *** ) and 70% ( *** ) Contralateral: 20, 36 ( * ) and 48% ( ** )

[0252] In the ipsilateral caudate nucleus, approximately 40% or more of MSH3 mRNA was knockdown by day 15 (39% * ) and 29th day (46% * ) was observed in the contralateral caudate nucleus. No significant KD was detected in the contralateral caudate nucleus.

[0253] No significant KD of MSH3 mRNA was observed in the putamen.

[0254] MSH3 mRNA was increased by 47% ( *** ), and 39% on the 29th day ( ** ) and was significantly knocked down.

[0255] P values ​​from pairwise comparisons against naïve in a linear mixed-effects model: MSH3 fold change ∽(1|animal) + housekeeper + tissue * Time. Note: 2 h, 8 h, 24 h, and 48 h time points (no effect) were included in the analysis ( * P < 0.05, ** P < 0.01, *** P<0.001).

[0256] These results demonstrate that administration of antisense oligo #1 at a dose level of 10 mg results in significant and widespread knockdown of MSH3 mRNA in NHPs following a single ICV injection into the dorsal horn of the lateral ventricle. Similar mRNA knockdown effects were obtained in several brain regions, regardless of hemisphere.

[0257] An integrated PK / PD model was developed based on multicompartment tissue PK and indirect response PD (Sharma & Jusko, Br J Clin Pharmacol 45:229-239 (1998)). This PK / PD model was a good fit to the observed time-dependent MSH3 mRNA responses observed in the cerebral cortex, caudate, and putamen. Based on the current data on duration of action out to 84 days, the PK / PD model predicted S max =4.7(E max Approximately 18%), SC 50 = 8.6 μg / g, and a Hill coefficient γ = 1.8 were used to estimate the in vivo potency of antisense oligo no. 1 for stimulating MSH3 mRNA degradation in NHPs.

[0258] Conclusions regarding the PK / PD comparison following a single 10mg injection of antisense oligo #1 ICV vs IT in NHPs At the same dose (10 mg) in NHPs, administration of antisense oligo #1 by ICV route showed significantly higher concentrations in the cerebral cortex, caudate nucleus, and putamen than by IT route. Upon ICV administration, significant and time-dependent knockdown levels of MSH3 mRNA were observed in the cerebral cortex and caudate nucleus, but not in the putamen. Based on the current data, it may be possible to develop a PK / PD model that adequately describes the observed data and estimates the in vivo efficacy of antisense oligo #1 in stimulating MSH3 mRNA degradation.

[0259] Rationale and Design of a Dose-Escalating PK / PD Study of Antisense Oligo No. 1 in NHPs with a Single ICV Injection A dose escalation study in NHPs is ongoing to further validate the dose dependency of PK / PD responses and tolerability. 14 female cynomolgus monkeys will receive antisense oligo #1 (2 mL) (in artificial CSF) via a single ICV injection starting at 15 mg. Dose levels will be adjusted based on clinical findings of tolerability. Necropsies will be performed at 2, 8, 24, 48, 168 (7 days), 336 (14 days), and 672 (28 days) hours after dosing (N=2 animals per time point). Tissues from various CNS regions and peripheral organs will be collected at each necropsy time point. CSF and plasma samples will be collected from selected survivors and at necropsy. All relevant brain and peripheral tissues as well as CSF and plasma will be analyzed for antisense oligo #1 concentration and MSH3 mRNA levels as described above.

[0260] Example 4. Biodistribution study for three routes of administration (RoA) using SEQ ID NO:2 A pharmaceutical composition comprising the oligonucleotide of SEQ ID NO:2 was used in this study. The oligonucleotide of SEQ ID NO:2 was formulated in the CSF as a 7 mg bolus. This composition was used to evaluate brain biodistribution with IT, intracisternal magna ("ICM"), and ICV injections. The same dose was injected in all RoAs, and KD was evaluated after 15 days. As shown in Figure 3, ICV injection achieved the highest overall KD in all RoAs.

[0261] Example 5. Dose Escalation Study - ICV Administration of Antisense Oligo #1 The effect of antisense oligo no. 1 on reducing MSH3 mRNA after a single ICV injection in non-human primates was investigated.

[0262] An initial dose of 15 mg given to two animals caused adverse clinical signs, necessitating early euthanasia of one animal. Six animals were then dosed with a tolerated dose of 12 mg. Four of the six animals showed transient adverse clinical signs. Another group of six animals was then given a 10 mg ICV dose, and the duration of action of antisense oligo #1 was assessed by measuring MSH3 mRNA 6, 8, and 12 weeks after injection (two animals at each time point). MSH3 mRNA expression in various regions of the brain showed a dose-dependent decrease after ICV injection of a single dose (12 mg and 15 mg) (Figures 5A-5E).

[0263] The knockdown percentage of MSH3 mRNA after a single ICV injection of antisense oligo #1 on day 8 (10 mg and 12 mg doses) and day 29 (10 mg and 15 mg doses) is shown below in Tables 5 and 6. These results demonstrate that MSH3 mRNA expression is reduced after a single ICV dose of antisense oligo #1.

[0264] [Table 5]

[0265] [Table 6]

[0266] Example 6. Investigation of MSH3 mRNA and protein knockdown following IT administration of antisense oligo #1 Antisense oligo #1 was administered intrathecally to NHPs to investigate the results of MSH3 mRNA and protein knockdown in the frontal cortex. Antisense oligo #1 was injected twice on days 1 and 15. Three animals per group were sacrificed on day 30 (29 days after the first IT injection). Brains were removed and punches from regions of interest including the cortex, caudate and putamen were processed for RNA and protein assays. MSH3 mRNA in animals administered antisense oligo #1 was suppressed compared to animals administered other antisense oligos or control (aCSF) (Figure 6). Animals administered antisense oligo #1 produced approximately 67% of MSH3 protein compared to animals administered other antisense oligos (Figure 7).

[0267] Example 7: Dosing Policy - Safety Margin Assessment Predicted C of antisense oligo no. 1 in cerebrospinal fluid (CSF) while maintaining desired MSH3 mRNA knockdown (KD) in major brain tissues max We investigated various dosing strategies to maintain AF below preclinical thresholds.

[0268] Medication strategies are associated with the following clinical findings in NHPs: A single ICV dose of 10 mg achieved a CSF CO of 696 μg / mL. The medication was well tolerated. Assuming linear PK, a single 12 mg ICV dose extrapolated a CSF CO of 835 μg / mL. Adverse clinical findings at this dose were reversible.

[0269] For desired KDs of greater than 50% in the caudate and cortex in humans and approximately 30% KD in the putamen. A dosing regimen of 150 mg Q28Dx2 (2 x 150 mg ICV doses every 28 days) followed by 150 mg Q168Dxn (n maintenance ICV doses of 150 mg every 168 days) resulted in a CSF C of 610 μg / mL. max are expected to be 1.1- and 1.2-fold lower than CSF C0 values ​​from NHP studies. A dosing regimen of 75 mg Q14Dx2 (2 x 75 mg ICV doses every 14 days) followed by 75 mg Q84Dxn (n maintenance ICV doses of 75 mg every 84 days) provides a CSF C of 300 μg / mL. max are expected to be 2.3-fold and 2.8-fold lower than CSF C0 values ​​from NHP studies.

[0270] Example 8: Tolerability and Efficacy of a Single Intravitreal (IVT) Injection in the Mouse Retina The objective of this study was to evaluate antisense oligo number 2 at different doses for tolerability and efficacy in the retina of wild-type C57BL6 mice after a single intravitreal (IVT) injection. The protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and all animal welfare concerns were addressed and documented. Experimental Group: PBS 1μl:n=5 Antisense oligo number 2 50μg / 1μl:n=5 Antisense oligo number 2 100μg / 1μl:n=5

[0271] Study design Treatment solution: Antisense oligo number 2 stock solution (100 μl, 100 mg / ml) was received on dry ice and stored at -80°C. The stock solution was diluted 1:1 (e.g., 10 μl (stock): 10 μl (PBS)) with PBS (Gibco 10010-031) to make a final solution of 50 mg / ml for the 50 μg group. The stock solution was used for the 100 μg group. One microliter (1 μl) was given to each animal to give a final dose of 0 μg, 50 μg, or 100 μg.

[0272] Intravitreal injection (IVT) Animals were anesthetized with 2-3% isoflurane in 100% O2. After anesthesia, pupils were dilated using one drop of 0.5% tropicamide. Proparacaine was applied to the mouse eye as local anesthesia prior to IVT injection. Mice were positioned under a surgical scope to expose the superior temporal sclera region of the eye. A sterile 30-gauge needle was used to puncture the superior temporal sclera approximately at the level of the pars plana. Eye muscles and blood vessels were avoided. The tip of a prepared Hamilton syringe with a 32-gauge blunt needle containing the solution was inserted through the puncture hole in the sclera into the vitreous at an angle of approximately 45°. One microliter of solution was slowly injected into the vitreous humor and the needle was allowed to remain in place for a few seconds before being withdrawn. Special care was taken to avoid any contact with the lens and retina. After the needle was fully withdrawn, tobramycin ophthalmic antibiotic ointment was applied to the injection site to further prevent infection. This procedure was performed on both eyes. The animals were then placed on a heating pad in a recovery chamber to recover from anesthesia before being returned to their home cage.

[0273] Animal weights and observations On day 14 after IVT injection, mice were euthanized with CO2. Whole eyes were collected and briefly frozen on dry ice. Eyes were then partially thawed to remove the anterior chamber. For technical reasons, the lens was included in the collected vitreous humor samples to prevent loss of vitreous. Retinas (containing RPE (retinal pigment epithelium) cells) were collected and frozen separately.

[0274] QRT-PCR Due to small tissue size, left and right retinas including RPE were pooled for mRNA analysis. QRT-PCR was performed to evaluate the mRNA of mouse MSH3 gene in retinas from the three experimental groups. HPRT gene was used as a housekeeping gene for data normalization. The results are shown in Figure 8. As shown in Figure 8, there was significant MSH3 mRNA knockdown in the retinas of wild-type mice after a single IVT injection.

[0275] Example 9: Duration of action following a single ICV administration of antisense oligo #1 (10 mg) The duration of action of mRNA knockdown was investigated 6, 8, and 12 weeks after a single ICV administration of antisense oligo no. 1 in NHP. Two artificial CSF-treated NHPs and two naive NHPs were used as controls. Tissue samples from the PK / PD ICV modeling study at day 29 (Example 3 above, N=2) were used as comparisons at all different time points. In this study, 2mL of antisense oligo no. 1 in artificial cerebrospinal fluid (not equal volume) was administered into the dorsal horn of the lateral ventricle. Statistical analysis pairwise comparisons of this experiment were compared between treatment groups and artificial CSF-treated controls.

[0276] result A single 10 mg ICV dose of antisense oligo #1 resulted in sustained and significant MSH3 mRNA knockdown for up to 12 weeks. Notably, sustained and significant MSH3 mRNA knockdown for up to 12 weeks was achieved in the cortex (FIGS. 9A-9D), caudate nucleus (FIGS. 10A-10B), and nucleus accumbens (FIGS. 11A-11B). In the ipsilateral putamen, significant MSH3 mRNA knockdown was observed at 8 and 12 weeks (FIGS. 12A-12B). No significant knockdown was detected in the contralateral putamen.

[0277] Example 10: PK / PD modeling of a single ICV dose of antisense oligo #1 Single-dose ICV PK / PD modeling was used to predict dosing regimens that would achieve a KD of mRNA >50% in the caudate nucleus within 12 weeks. Study design: · ICV injection: dorsal horn (posterior lateral ventricle) Autopsy time points: 2 hours, 8 hours, 24 hours, 48 ​​hours, 8 days, 15 days, and 29 days.

[0278] Endpoint analysis was performed to determine the KD of mRNA in brain regions, PK in biofluids, brain regions, liver and kidneys, and collect results from simulation / prediction by PK / PD models.

[0279] result Significant mRNA KD was achieved in the ipsilateral and contralateral cortex for up to 4 weeks (FIGS. 13A and 13B).

[0280] Significant mRNA KD was achieved in the ipsilateral caudate head and body, amygdala, hypothalamus, hippocampus, thalamus, substantia nigra, pons, and medulla (Figures 14A-14I), as well as in the ipsilateral white matter proximal to the injection site for up to 4 weeks (Figures 15A-15B). Significant mRNA KD was also achieved in both the ipsilateral and contralateral pons.

[0281] These results demonstrate that a single ICV administration of antisense oligo no. 1 at well-tolerated dose levels achieves significant and widespread mRNA KD in NHPs.

[0282] Example 10: Effect of a 500 μg dose on intranuclear inclusions in Q111 mice Htt Q111 / + ICV administration of antisense oligo #2 at doses of 500 μg and 300 μg was investigated in mice. Q111 / + Mice received an initial ICV injection of either sterile PBS or antisense oligo #2 (300 μg or 500 μg dose levels) into the right ventricle (n=15 / group). All groups of mice were scheduled to receive three additional ICV injections, rotating between the left and right ventricles, once every 8 weeks (n=4 ICV injections) until the end of the study. Plasma and CSF were collected from each animal at necropsy. Whole brains were isolated and divided into hemispheres, and the striatum and cortex were snap frozen separately for either histological (e.g., intranuclear inclusions) or molecular (MSH3 mRNA and somatic proliferation) analysis.

[0283] In the 500 μg dose group, adverse clinical findings occurred in all animals after the third 500 μg ICV injection of antisense oligo no. 2. Elevated NfL and GFAP levels were observed in plasma, possibly suggesting persistent axonal damage and immune activation in the brain. However, a 55-70% MSH3 knockdown was observed in the striatum and cortex, which prevented further somatic proliferation. A mild increase in intranuclear inclusion size was observed in treated mice compared to controls (phosphate-buffered saline), but no statistical difference was observed between treated and control mice in the total number of intranuclear inclusions counted in the striatum.

[0284] No adverse clinical findings were observed throughout the study in the 300 μg dose group. However, Htt Q111 / + and wild-type mice took longer than control mice to cross the balance beam. The number and size of intranuclear inclusions were similar between treatment groups and controls.

[0285] Intranuclear inclusions (NIs) reflect the accumulation of mutant HTT protein, which can be detected by antibodies that recognize the mutant HTT protein. The appearance of NIs in neural tissues is a recognized part of HD neuropathology, but the formation, exact composition, and biological consequences of NIs are not fully understood. Genetic analysis in mice suggests that the formation of NIs is associated with somatic proliferation of neuronal tissue. Antisense oligo #2 failed to reduce NIs in this study, despite halting further somatic proliferation. There are several possible explanations.

[0286] Neuronal damage, as indicated by elevated NfL and GFAP, may have contributed to the lack of reduction.

[0287] Initiating therapeutic treatment at 5.5 months of age was too late, as somatic proliferation had already significantly intensified.

[0288] A greater than 55% reduction in MSH3 mRNA must be achieved in striatal tissue.

[0289] Example 11: Plasma levels of NfL and GFAP in mice treated with 500 μg of antisense oligo #2 Htt Q111 / + The effect of ICV administration of 500 μg of antisense oligo no. 2 in mice and wild type mice was investigated.

[0290] Briefly, NfL, GFAP, UCHL1, and tau were measured with a Quanterix 4-Plex human assay panel (tau was not detected due to poor preservation between humans and mice). All treated animals were euthanized approximately 6 weeks after the third 500 μg dose. Cerebrospinal fluid was not collected after euthanasia.

[0291] Increased plasma levels of NfL and GFAP were observed. These findings suggest that: Elevated plasma levels of NfL suggest persistent axonal damage. Increased plasma GFAP levels suggest activation of microglia in the brain. The increase in NfL is likely due to treatment of the brain with very high levels of antisense oligonucleotides.

[0292] Wild-type mice and Htt mice receiving ICV injection of PBS Q111 / + Mice did not show any elevation of either NfL or GFAP. To explore alternative tool compounds, additional ASOs were tested as described in Example 12.

[0293] Example 12: Screening of novel MSH3 ASOs for tolerability and activity in wild-type mice From the initial Axolab screen, four MSH3 ASOs were selected that yielded the greatest MSH3 KD. Antisense oligo number 2 was included as a control. C57BL6 mice (N=6 / group) were given a single ICV injection at two dose levels, 100ug and 300ug. The control group was a PBS-treated control. Mice were euthanized 4 weeks after dosing and the striatum, cortex, and remnants of the brain were collected. Knockdown of MSH3 mRNA was measured. Plasma was collected at baseline, 7 days, and 28 days after dosing, and CSF was collected at necropsy (day 28). Plasma and CSF were sent to Quanterix Corp. (Billerica, MA) for analysis of NfL, tau, GFAP, and UCHL1 using their Human Neuro 4-Plex assay. Note: Tau levels were not detected in all samples. This is likely due to poor preservation between humans and mice.

[0294] The four test ASOs were antisense oligo #9, antisense oligo #6, antisense oligo #7, and antisense oligo #8.

[0295] result Antisense oligo #6 was well tolerated after ICV administration, demonstrated strong MSH3 knockdown, and showed no clinical findings. Antisense oligo #6 showed similar MSH3 knockdown in the cortex compared to antisense oligo #2. However, administration of antisense oligo #9, antisense oligo #7, and antisense oligo #8 each caused negative clinical outcomes, including seizures and death.

[0296] These results show that: Tolerability of ASOs is not related to efficacy (i.e., MSH3 KD) Tolerability of ASOs was not associated with increased NfL. Increases in NfL appear to be ASO-specific and dose-dependent, Plasma NfL levels were persistently elevated, suggesting prolonged axonal damage.

[0297] None of the test ASOs tested increased CSF or plasma levels of GFAP after a single ICV dose of either 100 mg or 300 mg, suggesting that either higher and / or multiple doses may be necessary to increase GFAP and replicate the effects observed in the 500 mg antisense oligo #2 arm in the HTTQ111 / + study (Example 11).

[0298] Example 13: ICV Delivery Protocol of Antisense Oligo No. 1 to Determine Tolerability An alternative ICV delivery protocol for non-human primates that would allow for evaluation of the maximum tolerated dose was investigated. An initial ICV injection protocol was compared to a pilot dose escalation protocol. Findings are presented below.

[0299] The initial NHP dosing protocol was a 2 mL injection into the dorsal horn of the lateral ventricle (without removing CSF). At this dosing, antisense oligo #1 was well tolerated. The researchers observed significant knockdown in the caudate nucleus (>45%) and cortex (>75%) at day 29.

[0300] The pilot NHP dosing protocol was 2 mL injection into the dorsal horn of the lateral ventricle (without removing CSF). A 20% dose escalation caused transient ataxia in 4 of 6 NHPs, and a 50% dose escalation caused toxicity requiring euthanasia in 1 of 2 NHPs. The other NHP showed transient symptoms.

[0301] The planned NHP dosing protocol is a 1 mL injection into the anterior horn of the lateral ventricle (removing CSF).

[0302] Other Aspects All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. If any term in this application is found to be differently defined in a document incorporated herein by reference, the definition provided herein shall serve as the definition of that term.

[0303] While the invention has been described in relation to particular embodiments thereof, it will be understood that the invention can be further modified, and this application is intended to cover any variation, use, or adaptation of the invention generally in accordance with the principles of the invention, including such departures from the present disclosure as are within known or customary practice in the art to which the invention pertains and which may be applied to the essential features described above, subject to the scope of the appended claims.

Claims

1. An agent for the treatment, prevention, or delaying of the onset and / or progression of neurodegenerative disorders, comprising a therapeutically effective amount of a pharmaceutically acceptable composition containing one or more oligonucleotide molecules having a linked nucleotide length of 15 to 30, or a pharmaceutically acceptable salt thereof, and formulated for intraventricular ("ICV") injection.

2. The agent according to claim 1, wherein the neurodegenerative disease is not a triplet repeat disorder.

3. The agent according to claim 1, wherein the neurodegenerative disease is not a nucleotide repeat disorder.

4. The agent according to claim 1, wherein one or more oligonucleotide molecules hybridize to one or more of the following genes: MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, or TDP43.

5. The agent according to claim 1, wherein one or more oligonucleotide molecules hybridize to one or more of the following genes: MAPT, APP, LRRK2, SNCA, PSEN1, PSEN2, HSPA2, C4A, C4B, C9orf72, or TDP43.

6. The agent according to claim 1, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Pick's disease, Parkinson's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Lewy body dementia, multiple system atrophy, cerebral amyloid angiopathy, schizophrenia, dentatorubral-pallidoluysian atrophy, and Down syndrome-associated Alzheimer's disease.

7. The agent according to claim 1, wherein one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is delivered to the temporal lobe, amygdala, hippocampus, cerebral cortex, pons, brainstem ganglia, globus pallidus, substantia nigra, striatum, ventral striatum, nucleus accumbens, dorsal striatum, caudate nucleus, putamen, thalamus, nucleus basalis of Meynert, brainstem, or cerebellum.

8. The agent according to claim 1, wherein the one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is single-stranded or double-stranded.

9. The agent according to claim 1, wherein one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, (a) DNA core sequence containing linked deoxyribonucleosides, (b) A 5' flanking sequence containing a linked nucleoside, and (c) 3' flanking sequence containing linked nucleosides Includes, The agent wherein the DNA core comprises at least 10 adjacent nucleic acid base regions located between the 5' flanking sequence and the 3' flanking sequence, the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides, and at least one nucleoside in each flanking sequence comprises a substitute nucleoside.

10. The agent according to claim 9, wherein the one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, comprises at least one alternative nucleoside bond.

11. The agent according to claim 10, wherein the at least one alternative nucleoside bond is a phosphorothioate nucleoside bond.

12. The agent according to claim 10, wherein the at least one alternative nucleoside bond is a 2'-alkoxynucleoside bond.

13. The agent according to claim 10, wherein the at least one alternative nucleoside bond is an alkylphosphate nucleoside bond.

14. The agent according to claim 10, wherein the one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, comprises at least one alternative nucleic acid base.

15. The agent according to claim 14, wherein the substitute nucleic acid base is 5'-methylcytosine, pseudouridine, or 5-methoxyuridine.

16. The agent according to claim 1, wherein one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, comprises at least one alternative sugar moiety.

17. The agent according to claim 16, wherein the substitute sugar portion is 2'-OMe or a bicyclic nucleic acid.

18. The agent according to claim 1, further comprising a ligand in which one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is compounded to the 5' or 3' end of the one or more oligonucleotide molecules via a monovalent, branched divalent, or trivalent linker.

19. The agent according to claim 1, wherein the one or more oligonucleotide molecules are provided in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient.

20. An agent for reducing the amount of target mRNA in cells, comprising one or more oligonucleotide molecules having a linked nucleotide length of 15 to 30, or a pharmaceutically acceptable salt thereof, and being brought into contact with the cells for a time sufficient to achieve degradation of the target mRNA, wherein the target mRNA is derived from a target gene selected from the group consisting of MAPT, APP, LRRK2, SNCA, HTT, ATXN1, ATXN2, ATXN3, PSEN1, PSEN2, ATN1, HSPA2, C4A, C4B, C9orf72, and / or TDP43.

21. The agent according to claim 20, wherein the oligonucleotide is single-stranded or double-stranded.

22. The agent according to claim 20, wherein one or more oligonucleotide molecules, or pharmaceutically acceptable salts thereof, (a) DNA core sequence containing linked deoxyribonucleosides, (b) A 5' flanking sequence containing a linked nucleoside, and (c) 3' flanking sequence containing linked nucleosides Includes, The agent wherein the DNA core comprises at least 10 adjacent nucleic acid base regions located between the 5' flanking sequence and the 3' flanking sequence, the 5' flanking sequence and the 3' flanking sequence each comprise at least two linked nucleosides, and at least one nucleoside in each flanking sequence comprises a substitute nucleoside.

23. The agent according to claim 20, wherein the oligonucleotide or a pharmaceutically acceptable salt thereof comprises at least one alternative nucleoside bond.

24. The agent according to claim 23, wherein the at least one alternative nucleoside bond is a phosphorothioate nucleoside bond.

25. The agent according to claim 23, wherein the at least one alternative nucleoside bond is a 2'-alkoxynucleoside bond.

26. The agent according to claim 23, wherein the at least one alternative nucleoside bond is an alkylphosphate nucleoside bond.

27. The agent according to claim 20, wherein one or more oligonucleotides, or pharmaceutically acceptable salts thereof, comprises at least one alternative nucleic acid base.

28. The agent according to claim 27, wherein the substitute nucleic acid base is 5'-methylcytosine, pseudouridine, or 5-methoxyuridine.

29. The agent according to claim 20, wherein one or more oligonucleotides, or pharmaceutically acceptable salts thereof, comprises at least one alternative sugar moiety.

30. The agent according to claim 29, wherein the substitute sugar portion is 2'-OMe or a bicyclic nucleic acid.

31. The agent according to claim 20, further comprising a ligand in which one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is compounded to the 5' or 3' end of the oligonucleotide via a monovalent, branched divalent, or trivalent linker.

32. The agent according to claim 1 or 20, used in combination with an additional therapeutic agent.

33. The agent according to claim 1 or 20, wherein one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is administered in a dose of about 2 mg to about 300 mg.

34. The agent according to claim 1 or 20, wherein the one or more oligonucleotide molecules, or a pharmaceutically acceptable salt thereof, is administered once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every six weeks, once every eight weeks, once every two months, once every ten weeks, once every twelve weeks, once every three months, once every sixteen weeks, once every four months, once every twenty weeks, once every five months, once every twenty-four weeks, once every six months, once every twenty-eight months, once every seven months, once every thirty-two weeks, once every eight months, once every thirty-six weeks, once every nine months, once every forty weeks, once every ten months, once every forty-four weeks, once every eleven months, once every forty-eight weeks, once every twelve months, or once a year.

35. The agent according to claim 1 or 20, wherein administration of one or more oligonucleotide molecules or a pharmaceutically acceptable salt thereof delays the onset and / or progression of the neurodegenerative disorder by at least 120 days, at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years or more, compared to the predicted onset or progression.

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