Method for regulating progranulin expression using antisense oligonucleotides targeting regulatory RNA

Abstract

Methods are described herein for modulating GRN gene transcription using antisense oligonucleotides (ASOs) that target GRN regulatory RNAs such as promoter-associated RNAs and enhancer RNAs. These methods are useful for modulating the level of GRN gene products and, for example, increasing the expression of progranulin (PGRN), thereby being useful for treating diseases and disorders of interest.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 351,263, filed Jun. 10, 2022; U.S. Provisional Application No. 63 / 369,907, filed Jul. 29, 2022; and U.S. Provisional Application No. 63 / 381,910, filed Nov. 1, 2022, each of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in XML format and is hereby incorporated by reference in its entirety. The name of the XML file created on XX / XX / 20XX is CTC - 027WO_SL.xml, and its size is X,XXX,XXX bytes.

[0003] Field of the Invention The present invention relates to a method for up - regulating or down - regulating GRN gene transcription using antisense oligonucleotides (ASOs) that target GRN - regulatory RNAs such as promoter - associated RNAs and enhancer RNAs.

Background Art

[0004] Background Transcription factors bind to specific sequences in promoter and enhancer DNA elements to regulate gene transcription. It has recently been reported that active promoter and enhancer elements are themselves transcribed to generate non-coding regulatory RNAs (regRNAs) such as promoter-associated RNAs (paRNAs) and enhancer RNAs (eRNAs) (see Sartorelli and Lauberth, Nat. Struct. Mol. Biol. (2020) 27:521-28 (Non-Patent Document 1)). Unlike coding RNAs, regRNAs are transcribed bidirectionally. Various models for the function of regRNAs have been proposed, including nucleosome remodeling (see Mousavi et al., Mol. Cell (2013) 51(5):606-17 (Non-Patent Document 2)), regulation of enhancer-promoter loops (see Lai et al., Nature (2013) 494(7438):497-501 (Non-Patent Document 3)), and direct interaction with transcription regulatory factors (see Sigova et al., Science (2015) 350:978-81 (Non-Patent Document 4)).

[0005] Progranulin (PGRN) is encoded by the human GRN gene and is a precursor of granulin peptides. PGRN is a highly conserved secreted protein expressed in multiple cell types, including the central nervous system (CNS) and peripheral tissues. There is increasing evidence that PGRN and its proteolytic granulin peptide products are involved in lysosomal function and have trophic and neuroprotective effects.

[0006] Deficiency of PGRN and mutations in GRN can lead to various neurological diseases and disorders, including frontotemporal dementia. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease, and patients rapidly progress to severe dementia and death. Approximately 40% of FTD patients have familial FTD, and approximately 30% (10% overall) have heterozygous GRN mutations that result in haploinsufficiency. Generally, PGRN protein levels remain constant throughout a patient's life and are not prognostic factors at the time of initial onset (usually 55 - 65 years old). The disease of GRN - frontotemporal dementia (GRN - FTD, also known as FTD - GRN) is difficult to catch in terms of the onset of the disease in FTD patients. Behavioral and language symptoms begin several years before diagnosis. Within 3 - 4 years from diagnosis, patients progress to end - stage dementia and require sufficient support from caregivers. The life expectancy after diagnosis of GRN - FTD is typically about 7 years. However, there are currently only symptomatic treatments for FTD, and there are no available disease - restoring or disease - modulating agents.

[0007] Gene expression is generally known as an undruggable biological process. Despite ongoing efforts to understand gene transcription and the biology of regRNA, clinically appropriate methods for regulating gene expression are limited. There remains a need for new and useful methods for treating diseases associated with abnormal (e.g., decreased) expression of PGRN, such as FTD.

Prior Art Documents

Non - Patent Documents

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Non - Patent Document 1

Non - Patent Document 2

Non - Patent Document 3

Non-Patent Document 4

Summary of the Invention

[0009] Summary In one aspect, provided herein is an antisense oligonucleotide (ASO) complementary to at least 5 consecutive nucleotides of a regulatory RNA of progranulin (pGRN), and this regulatory RNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0010] In some embodiments, the ASO is complementary to a sequence in the regRNA that is 200 nucleotides or less from the 3' end of the regRNA.

[0011] In some embodiments, the ASO is complementary to a sequence in the regRNA that is 200 nucleotides or less from the 5' end of the regRNA.

[0012] In some embodiments, the ASO comprises a nucleotide sequence selected from Table 17, 18, or 19.

[0013] In some embodiments, the ASO comprises any one nucleotide sequence of SEQ ID NOs: 1369-4738.

[0014] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 1, and the ASO comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10-268, 691, 991-1368, or 4743-4915.

[0015] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 2, and the ASO comprises the nucleotide sequence of SEQ ID NOs: 269-279.

[0016] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 3, and the ASO comprises the nucleotide sequence of SEQ ID NOs: 280-291 or 336-359.

[0017] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 4, and the ASO comprises the nucleotide sequence of SEQ ID NOs: 292-313 or 360-380.

[0018] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 5, and the ASO comprises the nucleotide sequence of SEQ ID NOs: 314-335 or 381-416.

[0019] In some embodiments, the regulatory RNA has the nucleotide sequence of SEQ ID NO: 6, and the ASO comprises the nucleotide sequence of SEQ ID NOs: 417-442.

[0020] In some embodiments, the ASO is 50, 40, 30, 25, or 20 nucleotides in length or less.

[0021] In some embodiments, the ASO comprises an RNA polynucleotide containing one or more chemical modifications.

[0022] In some embodiments, each nucleotide of the ASO comprises a ribonucleotide having one or more chemical modifications.

[0023] In some embodiments, at least 3, 4, or 5 nucleotides at the 5' end of the ASO, and at least 3, 4, or 5 nucleotides at the 3' end of the ASO, comprise ribonucleotides having one or more chemical modifications.

[0024] In some embodiments, the one or more chemical modifications include 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), and phosphorothioate nucleotide linkages.

[0025] In some embodiments, the ASO does not contain 10 or more consecutive nucleotides of unmodified DNA.

[0026] In some embodiments, the ASO does not contain deoxyribonucleotides.

[0027] In some embodiments, the ASO does not contain unmodified ribonucleotides.

[0028] In some embodiments, each ribonucleotide of the ASO is modified by 2'-O-methoxyethyl.

[0029] In some embodiments, the length of the ASO is 3×n + 10 nucleotides (n is an integer of 4 or more), the nucleotides at the 3×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0030] In some embodiments, the length of the ASO is 2×n + 4 nucleotides (n is an integer of 8 or more), the nucleotides at the 2×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0031] In some embodiments, the length of the ASO is 3×n + 2 nucleotides (n is an integer of 6 or more), the nucleotides at the 3×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0032] In some embodiments, the length of the ASO is 4×n + 4 nucleotides (n is an integer of 4 or more), the nucleotides at the 4×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0033] In some embodiments, the length of the ASO is 5×n + 5 nucleotides (n is an integer of 3 or more), the nucleotides at the 5×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0034] In some embodiments, the length of the ASO is 2×n + 8 nucleotides (n is an integer of 8 or more), the nucleotides at the 2×m position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the 2×m + 1 position are ribonucleotides modified by 2'-O-methoxyethyl.

[0035] In some embodiments, the length of the ASO is 2×n + 8 nucleotides (n is an integer of 8 or more), the nucleotides at the 2×m + 1 position are ribonucleotides modified by LNA (m is an integer from 1 to n), and the nucleotides at the 2×m position are ribonucleotides modified by 2'-O-methoxyethyl.

[0036] In some embodiments, the ASO contains at least one phosphodiester bond.

[0037] In some embodiments, the ASO contains 10 or more consecutive nucleotides of unmodified DNA, with at least 3 nucleotides of modified ribonucleotides adjacent to each other at each of the 5' end and the 3' end.

[0038] In some embodiments, each cytidine in the ASO is modified by 5-methyl.

[0039] In some embodiments, the ASO contains 2 or more consecutive nucleotides of unmodified DNA, with at least 3 nucleotides of modified ribonucleotides adjacent to each other at each of the 5' end and the 3' end.

[0040] In some embodiments, the regRNA is eRNA.

[0041] In some embodiments, the regRNA is paRNA.

[0042] In one aspect, there is provided herein a pharmaceutical composition comprising an ASO disclosed herein and a pharmaceutically acceptable carrier.

[0043] In one aspect, there is provided herein a method of increasing the transcription of pGRN in a human cell, the method comprising contacting the cell with an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0044] In some embodiments, the cell is a neuron.

[0045] In some embodiments, the ASO increases the amount of regulatory RNA in the cell.

[0046] In some embodiments, the ASO increases the stability of regulatory RNA in the cell.

[0047] In some embodiments, the ASO increases the amount of pGRN mRNA in the cell.

[0048] In some embodiments, the ASO increases the amount of pGRN protein in the cell.

[0049] In one aspect, there is provided herein a method of treating frontotemporal dementia (FTD), the method comprising administering to a subject in need thereof an effective amount of an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0050] In some embodiments, the ASO increases the amount of regulatory RNA in the subject's cells.

[0051] In some embodiments, the ASO increases the stability of regulatory RNA in the subject's cells.

[0052] In some embodiments, the ASO increases the amount of pGRN mRNA in the cell.

[0053] In some embodiments, the ASO increases the amount of pGRN protein in the cell.

[0054] In some embodiments, the cell is a neuron. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0083] Detailed Description The present disclosure provides antisense oligonucleotides (ASOs) that target regulatory RNAs such as promoter-associated RNAs and enhancer RNAs, and methods of using these ASOs to regulate gene expression. These methods are useful for regulating the level of a gene product, for example, by regulating the expression level of progranulin or progranulin (PGRN, encoded by the GRN gene), thereby treating diseases associated with abnormal GRN gene expression in a subject, such as, but not limited to, frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathological frontotemporal lobar degeneration associated with the accumulation of TDP-43 inclusions (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerosis dementia, spinocerebellar ataxia type 3, chronic traumatic encephalopathy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Gaucher disease (GD), Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL) type 11 (CLN11), limbic-predominant age-related TDP-43 encephalopathy (LATE) Gaucher disease, autism, cerebral ischemia-reperfusion injury, lysosomal storage diseases (LSD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), ischemic heart disease, intervertebral disc degeneration, and acute kidney injury.

[0084] In some embodiments, the ASOs of the present disclosure may be used to restore the expression of progranulin or granulin in cells such as cells exhibiting progranulin haploinsufficiency, or to enhance the expression of progranulin in cells (e.g., neurons). In some embodiments, the ASOs of the present disclosure may be used to restore or increase the levels of secreted progranulin or granulin in a subject (e.g., a subject having progranulin haploinsufficiency).

[0085] Various aspects of the multispecific binding proteins described in this application are described in the following sections.

[0086] I. Definitions To facilitate understanding of this application, a number of terms and phrases are defined below.

[0087] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0088] As used herein, the term "granulin precursor", or "progranulin", or "PGRN" refers to the protein with UniProt accession number P28799 (human) when used with respect to the human version of the protein, the protein with UniProt accession number P28798 (mouse) when used with respect to the mouse version of the protein, as well as related isoforms and orthologs.

[0089] As used herein, the terms "regulatory RNA" and "regRNA" are used interchangeably to refer to non-coding RNAs transcribed from regulatory elements of a gene (e.g., a protein-coding gene), where the gene is not the non-coding RNA itself. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, and super-enhancers. Non-coding RNAs transcribed in the antisense direction from a promoter are also referred to as "promoter RNA" or "paRNA". Non-coding RNAs transcribed from an enhancer or super-enhancer, either in the sense or antisense direction, are also referred to as "enhancer RNA" or "eRNA". It is understood that natural antisense transcripts (NATs) that are at least partially complementary to a transcript of a gene are not regulatory RNAs as used herein.

[0090] As used herein, the term "nascent RNA" refers to RNA that is still being transcribed or has just been transcribed by RNA polymerase and remains attached to the transcribed DNA. RNA that has dissociated from the transcribed DNA is also referred to as "untethered RNA".

[0091] As used herein, the term "antisense oligonucleotide" or "ASO" refers to a single-stranded oligonucleotide having a nucleotide sequence that hybridizes to a target nucleic acid under appropriate conditions, or a conjugate comprising such a single-stranded oligonucleotide. In some embodiments, the disclosure encompasses pharmaceutically acceptable salts of any of the ASOs described herein. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium salts, potassium salts, calcium salts, and magnesium salts. In some embodiments, the ASOs provided herein are lyophilized and isolated as salts (e.g., sodium salts).

[0092] As used herein, in some embodiments, the stability of a regRNA is inversely correlated with the rate of degradation of the regRNA. In some embodiments, when an ASO increases the stability of a regRNA, it decreases the rate of degradation of the regRNA. In some embodiments, when an ASO decreases the stability of a regRNA, this increases the rate of degradation of the regRNA. In some embodiments, the rate of degradation of a regRNA can be measured by blocking the synthesis of new regRNA and assessing the half-life of existing regRNA.

[0093] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., rodents, primates, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0094] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present application) sufficient to produce a beneficial or desired result. The effective amount may be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration. As used herein, the terms "treating," "treatment" refer to any effect, e.g., reduction, decrease, modulation, amelioration, or elimination, of a condition, disease, disorder, etc., or an improvement in its symptoms.

[0095] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that renders the composition substantially suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0096] As used herein, the term "pharmaceutically acceptable carrier" refers to any of standard pharmaceutical carriers such as phosphate buffered saline, water, emulsions (e.g., oil-in-water or water-in-oil emulsions), and various types of wetting agents. The compositions may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington’s Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0097] Throughout this description, when a composition is described as having, containing, or comprising a particular component, or a process and method are described as having, containing, or comprising particular steps, it is contemplated that in addition there exist compositions of this application consisting essentially of or consisting of the recited components, and processes and methods according to this application consisting essentially of or consisting of the recited process steps.

[0098] In general, compositions specifying percentages are by weight unless otherwise specified. Further, where a variable element is not accompanied by a definition, the previous definition of that variable element prevails.

[0099] II. Antisense Oligonucleotides In some embodiments, the antisense oligonucleotides (ASOs) disclosed herein hybridize to a regulatory RNA (e.g., regulatory RNA or promoter-associated RNA, also referred to herein as "GRN regulatory RNA" or "GRN regRNA") transcribed from a regulatory element of the GRN gene. It is understood that both eRNA and paRNA are regulatory RNAs that regulate (e.g., promote or upregulate) gene expression (Figure 1). In some embodiments, the GRN regRNA is a mouse GRN regRNA. In some embodiments, the GRN regRNA is a human GRN regRNA. In certain embodiments, the target GRN regRNA is eRNA. In certain embodiments, the target GRN regRNA is paRNA. eRNA can be identified using methods known in the art, such as assays of transposase-accessible chromatin using sequencing (ATAC-seq), global run-on sequencing, precision run-on sequencing, cap analysis of gene expression, and histone modification analysis (see, e.g., Sartorelli & Lauberth, Nat. Struct. Mol. Biol. (2020) 27:521-28; PCT Application Publication No. WO2013 / 177248). PaRNA is RNA transcribed from the promoter of a target gene in the antisense orientation (the transcript in the sense orientation is the mRNA of the target gene). They can be identified by similar methods, taking into account their specific positions and orientations. Exemplary nucleotide sequences of regulatory RNAs are shown in Table 1 below. Any of these GRN regRNAs are contemplated as target GRN regRNAs for the ASOs disclosed herein.

[0100] (Table 1) Exemplary regulatory RNAs TIFF2025522380000002.tif182152TIFF2025522380000003.tif223152TIFF2025522380000004.tif223152TIFF2025522380000005.tif241152

[0101] The present disclosure describes ASOs that increase the amount or stability of a target GRN regRNA, thereby increasing the expression of the GRN gene. These ASOs are different from the previously described ASOs designed to inhibit eRNA (see, e.g., PCT Publication No. WO2013 / 177248 and PCT Publication No. WO2017 / 075406). Without wishing to be bound by theory, the ability of the ASOs to upregulate GRN regRNA is hypothesized to result from the selection of the target sequence in the regRNA and / or chemical modification of the ASO.

[0102] Sequence of the ASO In certain embodiments, the ASOs disclosed herein are complementary to sequences within the GRN regRNA that are 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5’ or 3’ end of the GRN regRNA. In certain embodiments, the ASOs disclosed herein are complementary to sequences within the GRN regRNA that are 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5’ end of the GRN regRNA (i.e., the most 5’ nucleotide of the regRNA sequence that forms a duplex with the ASO is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 5’ end of the GRN regRNA). In certain embodiments, the ASOs disclosed herein are complementary to sequences within the target GRN regRNA that are 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 3’ end of the GRN regRNA (i.e., the most 3’ nucleotide of the GRN regRNA sequence that forms a duplex with the ASO is 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides or less from the 3’ end of the GRN regRNA).

[0103] In certain embodiments, the ASO is 25, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides in length or less. In certain embodiments, the ASO is designed to lack stable secondary structures formed within itself or between each other, thereby increasing the amount of single-stranded ASO ready to hybridize with the GRN regRNA. Methods for predicting secondary structures are known in the art (see, for example, Seetin and Mathews, Methods Mol. Biol. (2012) 905:99-122; Zhao et al., PLoS Comput. Biol. (2021) 17(8):e1009291) and web-based programs (such as RNAfold) are available to the public user.

[0104] For example, the ASO is designed to target human GRN paRNA or GRN eRNA. Some nucleotide sequences of these hGRN ASOs are shown in Table 17 below. Additional ASOs are being designed to target mouse GRN paRNA or eRNA. Some nucleotide sequences of these mGRN ASOs are shown in FIG. 18. In some embodiments, the ASO provided herein comprises any one nucleotide sequence of SEQ ID NOs: 1369 to 4738. In some embodiments, the ASO provided herein comprises the nucleotide sequences presented in Table 19 below.

[0105] (Table 19) Exemplary ASO Sequences TIFF2025522380000006.tif188155TIFF2025522380000007.tif223155TIFF2025522380000008.tif223155TIFF2025522380000009.tif223155TIFF2025522380000010.tif223155TIFF2025522380000011.tif223155TIFF2025522380000012.tif223155TIFF2025522380000013.tif223155TIFF2025522380000014.tif223155TIFF2025522380000015.tif223155TIFF2025522380000016.tif223155TIFF2025522380000017.tif223155TIFF2025522380000018.tif223155TIFF2025522380000019.tif226155TIFF2025522380000020.tif223155TIFF2025522380000021.tif223155TIFF2025522380000022.tif13155

[0106] Hybridization and ΔG As used herein, the terms "hybridize" or "hybridizing" are to be understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. The affinity of the bond between two nucleic acid strands is the strength of hybridization. This is often described in terms of the melting temperature (T m ) at which half of the oligonucleotide forms a duplex with the target nucleic acid. Under physiological conditions T m , it is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of the binding affinity, and ΔG° = -RTIn(K d ) for the dissociation constant (K d) (wherein R is the gas constant and T is the absolute temperature). Thus, the very low ΔG° of the reaction between the oligonucleotide and the target nucleic acid reflects the strong hybridization between the oligonucleotide and the target nucleic acid. ΔG° is the free energy associated with a reaction in an aqueous solution at a concentration of 1 M, a pH of 7, and a temperature of 37°C. The hybridization of the oligonucleotide to the target nucleic acid is a spontaneous reaction, and the ΔG° of a spontaneous reaction is less than zero. ΔG° can be measured experimentally, for example, using the isothermal titration calorimetry (ITC) method described in Hansen et al., 1965, Chem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. It is known to those skilled in the art that commercially available devices are available for ΔG° measurement. ΔG° can be numerically estimated using the nearest neighbor model described in SantaLucia, 1998, Proc Natl Aced Sci USA 95:1460-1465, using appropriately derived thermodynamic parameters described in Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the potential to modulate its intended nucleic acid target by hybridization, the oligonucleotides of the present disclosure hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal / mol for oligonucleotides 10-30 nucleotides in length. In some embodiments, the degree or strength of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotide can hybridize to the target nucleic acid with an estimated ΔG° value lower than the range of -10 kcal / mol, such as less than -15 kcal / mol, for example less than -20 kcal / mol, for example less than -25 kcal / mol, for oligonucleotides 8-30 nucleotides in length.In some embodiments, the oligonucleotide hybridizes to the target nucleic acid with an estimated ΔG° value of -10 to -60 kcal / mol, such as -12 to -40 kcal / mol, -15 to -30 kcal / mol, -16 to -27 kcal / mol, or -18 to -25 kcal / mol.

[0107] double-stranded region The term "duplex region" refers to a region in two complementary or substantially complementary polynucleotides that form base pairs with each other by either Watson-Crick base pairing or any other mode that allows for a stabilized duplex between polynucleotide strands that are complementary or substantially complementary. For example, a polynucleotide strand having 21 nucleotide units can base pair with another polynucleotide of 21 nucleotide units where only 19 bases on each strand are complementary or sufficiently complementary such that the "duplex region" is 19 base pairs. The remaining bases can exist, for example, as 5' and / or 3' overhangs. Further, 100% complementarity within the duplex is not required and substantial complementarity is acceptable within the duplex. Substantial complementarity refers to 70% or more complementarity. For example, a mismatch in a duplex consisting of 19 base pairs results in 94.7% complementarity, making the duplex region sufficiently complementary. The duplex region can be formed by two separate oligonucleotide strands as well as by a single oligonucleotide strand that can form a hairpin structure containing the duplex region.

[0108] dsRNA contains two RNA strands that are complementary and hybridize under the conditions in which the dsRNA is used to form a double-stranded structure. One strand of the dsRNA (the antisense strand) contains a region of complementarity that is substantially complementary, and generally fully complementary, to the target sequence. The target sequence may be derived from the sequence of a GRN regRNA such as an eRNA or a paRNA. The other strand (the sense strand) contains a region complementary to the antisense strand, such that the two strands hybridize and form a double-stranded structure when combined under appropriate conditions. As described elsewhere herein and as is known in the art, the complementary sequences of the dsRNA may be included as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides. Generally, the double-stranded structure is from 5 to 50 base pairs in length, for example, 5 to 50, 5 to 49, 5 to 48, 5 to 47, 5 to 46, 5 to 45, 5 to 44, 5 to 43, 5 to 42, 5 to 41, 5 to 40, 5 to 39, 5 to 38, 5 to 37, 5 to 36, 5 to 35, 5 to 34, 5 to 33, 5 to 32, 5 to 31, 5 to 30, 5 to 29, 5 to 28, 5 to 27, 5 to 26, 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 50, 6 to 49, 6 to 48, 6 to 47, 6 to 46, 6 to 45, 6 to 44, 6 to 43, 6 to 42, 6 to 41, 6 to 40, 6 to 39, 6 to 38, 6 to 37, 6 to 36, 6 to 35, 6 to 34, 6 to 33, 6 to 32, 6 to 31, 6 to 30, 6 to 29, 6 to 28, 6 to 27, 6 to 26, 6 to 25, 6 to 24, 6 to 23, 6 to 22, 6 to 21, 6 to 20, 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 8 to 50, 8 to 49, 8 to 48, 8 to 47, 8 to 46, 8 to 45, 8 to 44, 8 to 43, 8 to 42, 8 to 41, 8 to 40, 8 to 39, 8 to 38, 8 to 37, 8 to 36, 8 to 35, 8 to 34, 8 to 33, 8 to 32, 8 to 31, 8 to 30, 8 to 29, 8 to 28, 8 to 27, 8 to 26, 8 to 25, 8 to 24, 8 to 23, 8 to 22, 8 to 21, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12,8~11、8~10、8~9、10~50、10~49、10~48、10~47、10~46、10~45、10~44、10~43、10~42、10~41、10~40、10~39、10~38、10~37、10~36、10~35、10~34、10~33、10~32、10~31、10~30、10~29、10~28、10~27、10~26、10~25、10~24、10~23、10~22、10~21、10~20、10~19、10~18、10~17、10~16、10~15、10~14、10~13、10~12、10~11、10~10、10~9、12~50、12~49、12~48、12~47、12~46、12~45、12~44、12~43、12~42、12~41、12~40、12~39、12~38、12~37、12~36、12~35、12~34、12~33、12~32、12~31、12~30、12~29、12~28、12~27、12~26、12~25、12~24、12~23、12~22、12~21、12~20、12~19、12~18、12~17、12~16、12~15、12~14、12~13、15~50、15~49、15~48、15~47、15~46、15~45、15~44、15~43、15~42、15~41、15~40、15~39、15~38、15~37、15~36、15~35、15~34、15~33、15~32、15~31、15~30、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、18~50、18~49、18~48、18~47、18~46、18~45、18~44、18~43、18~42、18~41、18~40、18~39、18~38、18~37、18~36、18~35、18~34、18~33、18~32、18~31、18~30、18~30、18~29、18~28、18~27、18~26、18~25、18~24、18~23、18~22、18~21、18~20、19~50、19~49、19~48、19~47、19~46、19~45、19~44、19~43、19~42、19~41、19~40、19~39、19~38、19~37、19~36、19~35、19~34、19~33、19 to 32, 19 to 31, 19 to 30, 19 to 30, 19 to 29, 19 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 50, 20 to 49, 20 to 48, 20 to 47, 20 to 46, 20 to 45, 20 to 44, 20 to 43, 20 to 42, 20 to 41, 20 to 40, 20 to 39, 20 to 38, 20 to 37, 20 to 36, 20 to 35, 20 to 34, 20 to 33, 20 to 32, 20 to 31, 20 to 30, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 50, 21 to 49, 21 to 48, 21 to 47, 21 to 46, 21 to 45, 21 to 44, 21 to 43, 21 to 42, 21 to 41, 21 to 40, 21 to 39, 21 to 38, 21 to 37, 21 to 36, 21 to 35, 21 to 34, 21 to 33, 21 to 32, 21 to 31, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 50, 22 to 49, 22 to 48, 22 to 47, 22 to 46, 22 to 45, 22 to 44, 22 to 43, 22 to 42, 22 to 41, 22 to 40, 22 to 39, 22 to 38, 22 to 37, 22 to 36, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29, 22 to 28, 22 to 27, 22 to 26, 22 to 25, 22 to 24, 22 to 23, 23 to 50, 23 to 49, 23 to 48, 23 to 47, 23 to 46, 23 to 45, 23 to 44, 23 to 43, 23 to 42, 23 to 41, 23 to 40, 23 to 39, 23 to 38, 23 to 37, 23 to 36, 23 to 35, 23 to 34, 23 to 33, 23 to 32, 23 to 31, 23 to 30, 23 to 29, 23 to 28, 23 to 27, 23 to 26, 23 to 25, or 23 to 24 base pairs in length. Intermediate ranges and lengths within the above ranges and lengths are also contemplated as part of the present disclosure.,

[0109] Similarly, the region of complementarity to the target sequence has a length of 15 to 50 nucleotides, for example, 5 to 50, 5 to 49, 5 to 48, 5 to 47, 5 to 46, 5 to 45, 5 to 44, 5 to 43, 5 to 42, 5 to 41, 5 to 40, 5 to 39, 5 to 38, 5 to 37, 5 to 36, 5 to 35, 5 to 34, 5 to 33, 5 to 32, 5 to 31, 5 to 30, 5 to 29, 5 to 28, 5 to 27, 5 to 26, 5 to 25, 5 to 24, 5 to 23, 5 to 22, 5 to 21, 5 to 20, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 5 to 9, 5 to 8, 5 to 7, 5 to 6, 6 to 50, 6 to 49, 6 to 48, 6 to 47, 6 to 46, 6 to 45, 6 to 44, 6 to 43, 6 to 42, 6 to 41, 6 to 40, 6 to 39, 6 to 38, 6 to 37, 6 to 36, 6 to 35, 6 to 34, 6 to 33, 6 to 32, 6 to 31, 6 to 30, 6 to 29, 6 to 28, 6 to 27, 6 to 26, 6 to 25, 6 to 24, 6 to 23, 6 to 22, 6 to 21, 6 to 20, 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 6 to 9, 6 to 8, 6 to 7, 8 to 50, 8 to 49, 8 to 48, 8 to 47, 8 to 46, 8 to 45, 8 to 44, 8 to 43, 8 to 42, 8 to 41, 8 to 40, 8 to 39, 8 to 38, 8 to 37, 8 to 36, 8 to 35, 8 to 34, 8 to 33, 8 to 32, 8 to 31, 8 to 30, 8 to 29, 8 to 28, 8 to 27, 8 to 26, 8 to 25, 8 to 24, 8 to 23, 8 to 22, 8 to 21, 8 to 20, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 10 to 50, 10 to 49, 10 to 48, 10 to 47, 10 to 46, 10 to 45, 10 to 44, 10 to 43, 10 to 42, 10 to 41, 10 to 40, 10 to 39, 10 to 38, 10 to 37, 10 to 36, 10 to 35, 10 to 34, 10 to 33, 10 to 32, 10 to 31, 10 to 30, 10 to 29, 10 to 28, 10 to 27, 10 to 26, 10 to 25, 10 to 24, 10 to 23, 10 to 22, 10 to 21, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, 10 to 11, 10 to 10, 10 to 9, 12 to 50, 12 to 49, 12 to 48, 12 to 47, 12 to 46, 12 to 45, 12 to 44, 12 to 43, 12 to 42, 12 to 41,12~40、12~39、12~38、12~37、12~36、12~35、12~34、12~33、12~32、12~31、12~30、12~29、12~28、12~27、12~26、12~25、12~24、12~23、12~22、12~21、12~20、12~19、12~18、12~17、12~16、12~15、12~14、12~13、15~50、15~49、15~48、15~47、15~46、15~45、15~44、15~43、15~42、15~41、15~40、15~39、15~38、15~37、15~36、15~35、15~34、15~33、15~32、15~31、15~30、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、18~50、18~49、18~48、18~47、18~46、18~45、18~44、18~43、18~42、18~41、18~40、18~39、18~38、18~37、18~36、18~35、18~34、18~33、18~32、18~31、18~30、18~30、18~29、18~28、18~27、18~26、18~25、18~24、18~23、18~22、18~21、18~20、19~50、19~49、19~48、19~47、19~46、19~45、19~44、19~43、19~42、19~41、19~40、19~39、19~38、19~37、19~36、19~35、19~34、19~33、19~32、19~31、19~30、19~30、19~29、19~28、19~27、19~26、19~25、19~24、19~23、19~22、19~21、19~20、20~50、20~49、20~48、20~47、20~46、20~45、20~44、20~43、20~42、20~41、20~40、20~39、20~38、20~37、20~36、20~35、20~34、20~33、20~32、20~31、20~30、20~30、20~29、20~28、20~27、20~26、20~25、20~24、20~23、20~22、20~21、21~50、21~49、21~48、21~47、21~46、21~45、21~44、21~43、21~42、It may also be a length of 21 to 41, 21 to 40, 21 to 39, 21 to 38, 21 to 37, 21 to 36, 21 to 35, 21 to 34, 21 to 33, 21 to 32, 21 to 31, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, 21 to 22, 22 to 50, 22 to 49, 22 to 48, 22 to 47, 22 to 46, 22 to 45, 22 to 44, 22 to 43, 22 to 42, 22 to 41, 22 to 40, 22 to 39, 22 to 38, 22 to 37, 22 to 36, 22 to 35, 22 to 34, 22 to 33, 22 to 32, 22 to 31, 22 to 30, 22 to 29, 22 to 28, 22 to 27, 22 to 26, 22 to 25, 22 to 24, 22 to 23, 23 to 50, 23 to 49, 23 to 48, 23 to 47, 23 to 46, 23 to 45, 23 to 44, 23 to 43, 23 to 42, 23 to 41, 23 to 40, 23 to 39, 23 to 38, 23 to 37, 23 to 36, 23 to 35, 23 to 34, 23 to 33, 23 to 32, 23 to 31, 23 to 30, 23 to 29, 23 to 28, 23 to 27, 23 to 26, 23 to 25, or 23 to 24 nucleotides. Ranges and lengths intermediate to the above ranges and lengths are also contemplated to be part of the present disclosure.,

[0110] Chemical modification of ASO In certain embodiments, the ASO does not consist of only DNA. In certain embodiments, the ASO comprises at least one chemical modification to a native nucleotide (e.g., a ribonucleotide (e.g., a 2'-deoxy-2'-ribonucleotide)). A variety of chemical modifications can be included in the ASOs of the present disclosure. The modifications can include one or more modifications in the sugar (e.g., ribose), one or more modifications in the phosphate group, one or more modifications in the nucleobase, one or more terminal modifications, or combinations thereof. In some embodiments, the exemplary ASO sequences targeting regRNA shown in FIG. 17, FIG. 18, or other sections of the present disclosure are chemically modified. Such modifications can be, but are not limited to, 2'-O-(2-methoxyethyl) (2'-MOE), locked nucleic acid (LNA), 5-methyl on cytidine, constrained ethyl (cET), phosphorothioate (PS) linkages, and / or phosphodiester (PO) linkages, or any combination thereof. Chemical modifications of RNA are known in the art and are described, for example, in PCT Application Publication No. WO2013 / 177248, which is incorporated herein by reference. In certain embodiments, each cytidine in the ASOs provided herein is modified by 5-methyl.

[0111] Various chemical modifications for use with the ASOs of the present disclosure include, but are not limited to, the following: 3'-terminal deoxythymidine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, higher order structure restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxyl modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, unnatural bases including nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphate, and nucleotides containing 5'-phosphate mimics.

[0112] In certain embodiments, the ASO comprises an RNA polynucleotide that is chemically modified to be resistant to one or more nucleases (e.g., a nuclear RNase such as an exosome complex or RNaseH). In some embodiments, all nucleotide bases are modified in the ASO. In certain embodiments, the chemical modifications include β-D-ribonucleotides, 2'-modified nucleotides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, or 2'-fluoro-arabino (FANA)), bicyclic sugar-modified nucleotides (e.g., having a constrained ethyl or locked nucleic acid (LNA)), and / or one or more modified internucleotide linkages (e.g., phosphorothioate internucleotide linkages). In certain embodiments, the chemical modifications include 2'-MOE and phosphorothioate internucleotide linkages. In certain embodiments, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive nucleotides of the ASO are modified by 2'-MOE. In certain embodiments, each nucleotide of the ASO is modified by 2'-MOE. In certain embodiments, at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutive internucleotide linkages of the ASO are phosphorothioate internucleotide linkages. In certain embodiments, each internucleotide linkage of the ASO is a phosphorothioate internucleotide linkage.

[0113] Internucleotide linkage modifications that can be used with the ASOs of the present disclosure include, but are not limited to, phosphorothioate "PS" (P(S)), phosphoramidate (P(NR1R2), e.g., dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2) n COOR), e.g., phosphonoacetate "PACE" (P(CH2COO - )), thiophosphonocarboxylate ((S)P(CH2) n COOR), e.g., thiophosphonoacetate, "thioPACE" ((S)P(CH2COO -), alkyl phosphonate (P(C 1~3 alkyl), for example, methylphosphonic acid - P(CH3), borane phosphonic acid (P(BH3)), and phosphorodithioate (P(S)2).

[0114] In some embodiments, the ASOs provided herein include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more PO linkages. In some embodiments, all nucleotide linkages of the ASOs provided herein are PO nucleotide linkages. In some embodiments, the ASOs provided herein do not include PO nucleotide linkages. In some embodiments, the ASOs provided herein include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more PS nucleotide linkages. In some embodiments, all nucleotide linkages of the ASOs provided herein are PS linkages. In some embodiments, the ASOs provided herein do not include PS nucleotide linkages.

[0115] In certain embodiments, the ASO comprises one or more chemical modifications at the 5' end, 3' end, or both. Without wishing to be bound by theory, chemical modifications at one or both ends of a polynucleotide (e.g., a polyribonucleotide) can stabilize the polynucleotide. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO. In certain embodiments, the ASO comprises one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO and one or more chemical modifications at at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO.

[0116] Chemical structures can also be described in letters. In such cases, "M" represents MOE, "d" represents DNA, "L" represents LNA, "m" represents 2'O-methyl, "=" represents a phosphorothioate (PS) bond, "-" represents a phosphodiester (PO) bond; "5C" represents 5-methylcytosine, "ag" represents GalNAc, "tg" represents Teg-GalNAc, "^" represents FANA, "BioTeg" represents biotin, "Palm" represents palmitic acid; "C18" represents a spacer 18 moiety.

[0117] To avoid ambiguity, this LNA has the following formula: TIFF2025522380000023.tif45128wherein B is a specific designated base.

[0118] Visual representations of exemplary ASOs with chemical modifications are shown in FIGS. 17 and 18. Additional exemplary ASOs with chemical modifications are shown in Tables 17 and 18. In some embodiments, the ASOs provided herein comprise the nucleotide sequences presented in Table 17 below. In some embodiments, the ASOs provided herein comprise the nucleotide sequences presented in Table 18 below. In some embodiments, the ASO comprises the nucleotide sequence and / or chemical modification of any one of the oligonucleotides presented in Tables 17 and 18 below. In some embodiments, the ASO comprises the nucleotide sequence and / or chemical modification of any one of SEQ ID NOs: 1-442, 691, 991-1368, or 4743-4915. In some embodiments, the ASO comprises the nucleotide sequence and / or chemical modification of any one of SEQ ID NOs: 443-690, 692-990, or 4916.

[0119] (Table 17) Exemplary ASOs targeting hGRN regRNA with chemical modifications TIFF2025522380000024.tif66159TIFF2025522380000025.tif223159TIFF2025522380000026.tif226159TIFF2025522380000027.tif223159TIFF2025522380000028.tif223159TIFF2025522380000029.tif226159TIFF2025522380000030.tif225159TIFF2025522380000031.tif225159TIFF2025522380000032.tif223159TIFF2025522380000033.tif220159TIFF2025522380000034.tif224159TIFF2025522380000035.tif223159TIFF2025522380000036.tif226159TIFF2025522380000037.tif223159TIFF2025522380000038.tif189159

[0120] (Table 18) Exemplary ASOs Targeting mGrn regRNA with Chemical Modifications TIFF2025522380000039.tif19153TIFF2025522380000040.tif226153TIFF2025522380000041.tif223153TIFF2025522380000042.tif226153TIFF2025522380000043.tif222153TIFF2025522380000044.tif224153TIFF2025522380000045.tif223153TIFF2025522380000046.tif223153TIFF2025522380000047.tif157153

[0121] In some embodiments, the ASO comprises a sequence selected from the sequences shown in any one of SEQ ID NOs: 10 to 4916 and / or chemical modifications. In some embodiments, the ASO comprises a sequence selected from the sequences shown in any one of SEQ ID NOs: 1369 to 4738. In some embodiments, the ASO comprises a sequence selected from the sequences shown in any one of SEQ ID NOs: 10 to 1368, or 4734 to 4916 and chemical modifications. In some embodiments, the ASO comprises a sequence selected from the sequences shown in any one of SEQ ID NOs: 10 to 442, 691, 991 to 1368 or 4743 to 4915 and / or chemical modifications. In some embodiments, the ASO comprises a sequence selected from the sequences shown in any one of SEQ ID NOs: 443 to 690, 692 to 990, or 4916 and / or chemical modifications.

[0122] High-affinity modified nucleotides High-affinity modified nucleotides, when incorporated into an oligonucleotide, for example, melting temperature (T m) When measured by, it is a modified nucleotide that enhances the affinity of the oligonucleotide for its complementary target. The high-affinity modified nucleotides of the present invention preferably result in an increase in melting temperature of +0.5 to +12 °C, for example, +1.5 to +10 °C or +3 to +8 °C per modified nucleotide. A number of high-affinity modified nucleotides are known in the art, including, for example, a number of 2'-substituted nucleotides as well as locked nucleic acids (LNAs) (see, for example, Freier & Altmann (1997) Nucl. Acid Res. 25:4429-43 and Uhlmann (2000) Curr. Opinion in Drug Development 3(2):203-213, which are each incorporated herein by reference).

[0123] Sugar modification The ASOs described herein may include one or more nucleotides having a modified sugar moiety, i.e., a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA. A number of nucleotides having a modified ribose sugar moiety have been made primarily for the purpose of improving certain properties of the oligonucleotide, such as affinity and / or nuclease resistance. Such modifications include modifications in which the ribose ring structure is modified, for example, by substitution with a hexose ring (HNA) or a bicyclic ring (which typically has a biradial bridge between the C2 and C4 carbons on the ribose ring (LNA)), or an unlinked ribose ring (which typically lacks a bond between the C2 and C3 carbons) (e.g., UNA). Other sugar-modified nucleotides include, for example, bicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798), both of which are incorporated herein by reference. Modified nucleotides also include, for example, nucleotides in which the sugar moiety is replaced by a non-sugar moiety in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0124] Sugar modifications also include modifications made by changing substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group naturally found in RNA nucleosides. The substituents may be introduced, for example, at the 2', 3', 4' or 5' positions.

[0125] In some embodiments, the oligonucleotide comprises a modified sugar moiety such as a 2'-O-methyl (2'OMe) moiety, a 2'-O-methoxyethyl moiety, a bicyclic sugar moiety, a PNA (e.g., an oligonucleotide comprising one or more N-(2-aminoethyl)-glycine units linked by amide bonds or carbonylmethylene bonds as repeating units instead of a sugar phosphate backbone), a locked nucleotide (LNA) (e.g., an oligonucleotide comprising one or more locked riboses and which can be a mixture of 2'-deoxynucleotides or 2'OMe nucleotides), a cET (e.g., an oligonucleotide comprising one or more cET sugars), a cMOE (e.g., an oligonucleotide comprising one or more cMOE sugars), a morpholino oligomer (e.g., an oligonucleotide comprising a backbone comprising one or more phosphorodiamidate morpholino oligomers), a 2'-deoxy-2'-fluoronucleotide (e.g., an oligonucleotide comprising one or more 2'-fluoro-β-D-arabinonucleotides), a tcDNA (e.g., an oligonucleotide comprising one or more tcDNA modified sugars), a constrained ethyl 2'-4' bridged nucleic acid (cEt), an S-cEt, an ethylene bridged nucleic acid (ENA) (e.g., an oligonucleotide comprising one or more ENA modified sugars), a hexitol nucleic acid (HNA) (e.g., an oligonucleotide comprising one or more HNA modified sugars), or a tricyclic analog (tcDNA) (e.g., an oligonucleotide comprising one or more tcDNA modified sugars).

[0126] In some embodiments, the oligonucleotide comprises a nucleobase modification selected from the group consisting of 2-thiouracil ("2-thio U"), 2-thiocytosine ("2-thio C"), 4-thiouracil ("4-thio U"), 6-thioguanine ("6-thio G"), 2-aminoadenine ("2-amino A"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methyl C"), 5-methyluracil ("5-methyl U"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dihydrouracil, 5-propynylcytosine, 5-propynyluracil, 5-ethynylcytosine, 5-ethynyluracil, 5-allyluracil ("5-allyl U"), 5-allylcytosine ("5-allyl C"), 5-aminoallyluracil ("5-aminoallyl U"), 5-aminoallyl-cytosine ("5-aminoallyl C"), abasic nucleotide, Z base, P base, unstructured nucleic acid ("UNA"), isoguanine ("iso G"), and isocytosine ("iso C"), glycerol nucleic acid (GNA), thiomorpholino (C4H9NS) or thiophosphoramidate morpholino (TMO). The synthesis of glycerol nucleic acid (GNA) (also known as glycol nucleic acid) is described in Zhang et al, Current Protocols in Nucleic Acid Chemistry 4.40.1-4.40.18, September 2010, which is incorporated herein by reference. The synthesis of thiophosphoramidate morpholino oligonucleotides is described in Langer et al. J. Am. Chem. Soc. 2020, 142(38):16240-253.

[0127] 2'-sugar modified nucleotide A 2'-sugar modified nucleotide is a nucleotide having a substituent other than H or -OH at the 2'-position (a 2'-substituted nucleotide), or includes a 2'-linked bicyclic ring capable of forming a bridge between the 2'-carbon and the second carbon in the ribose ring, such as an LNA (2'-4'-bicyclic bridge) nucleotide.

[0128] While not wishing to be bound by theory, 2'-modified sugars can provide enhanced binding affinity to oligonucleotides and / or increased nuclease resistance. Examples of 2'-substituted modified nucleotides are 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, and 2'-FANA nucleotides. For further examples, see, for example, Freier & Altmann 1997, (supra); Uhlmann 2000 (supra), and Deleavey and damha (2012) Chemistry and Biology 19:937 (each of which is incorporated herein by reference).

[0129] Locked nucleic acid nucleotides (LNA nucleotides) An "LNA nucleotide" is a 2'-sugar modified nucleotide that contains a biradical (also called a "2'-4' bridge") that links the C2' and C4' of the ribose sugar ring of the nucleotide, which restricts or locks the higher-order structure of the ribose ring. In other words, a locked nucleotide is a nucleotide that contains a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in the higher-order structure of the 3'-end conformation. The addition of locked nucleotides to oligonucleotides has been shown to enhance the stability of oligonucleotides in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). These nucleotides are also sometimes referred to as bridged nucleic acids or bicyclic nucleic acids (BNA). The locking of the higher-order structure of ribose is associated with an enhancement of hybridization affinity (duplex stabilization) when LNA is incorporated into an oligonucleotide that has complementarity to an RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex. Exemplary LNA nucleotides include beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET) and ENA.

[0130] Examples of bicyclic nucleotides for use with the polynucleotides of the present disclosure include, but are not limited to, nucleotides that include a bridge between the 4’ ribosyl ring atom and the 2’ ribosyl ring atom. In certain embodiments, the polynucleotide agents of the present disclosure include one or more bicyclic nucleotides that include a 4’ to 2’ bridge. Examples of such 4’ to 2’ bridged bicyclic nucleotides include, but are not limited to, 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(CH2OCH3)-O-2’ (and analogs thereof; see, e.g., U.S. Patent No. 7,399,845); 4’-C(CH3)(CH3)-O-2’ (and analogs thereof; see, e.g., U.S. Patent No. 8,278,283); 4’-CH2-N(OCH3)-2’ (and analogs thereof; see, e.g., U.S. Patent 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. Patent 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. Patent No. 8,278,426). The entirety of each of the foregoing is incorporated herein by reference.

[0131] Additional representative U.S. patents and U.S. patent application publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following, the entire contents of each of which are incorporated herein by reference: 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; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; U.S. Patent Application Publication No. 2008 / 0039618; and U.S. Patent Application Publication No. 2009 / 0012281.

[0132] For example, any of the aforementioned bicyclic nucleotides having one or more stereochemical sugar configurations such as α-L-ribofuranose and β-D-ribofuranose can be prepared (see PCT Application Publication No. WO99 / 14226, the contents of which are incorporated herein by reference).

[0133] The oligonucleotides of the present disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, "constrained ethyl nucleotide" or "cEt" is a locked nucleotide containing a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleotide is in the S conformation, referred to herein as "S-cEt".

[0134] The oligonucleotides of the present disclosure may also include one or more "conformationally restricted nucleotides" ("CRNs"). A CRN is a nucleotide analog having a linker that connects the C2' and C4' carbons of ribose, or the C3 and -C5' carbons of ribose. CRNs lock the ribose ring into a stable higher-order structure and enhance hybridization affinity to RNA (e.g., regRNA or mRNA). The linker is of sufficient length to position oxygen optimally for stability and affinity and reduces ribose ring packing.

[0135] Representative publications teaching the preparation of the specific CRNs described above include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and PCT Application Publication WO2013 / 036868, the entire contents of each of which are incorporated herein by reference.

[0136] In some embodiments, the oligonucleotides of the present disclosure include one or more monomers that are unlocked nucleotide (UNA) nucleotides. UNA is an unlocked acyclic nucleotide in which the sugar linkage is removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) is removed. In another example, the C2'-C3' bond of the sugar (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) is 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).

[0137] Representative U.S. patent publications teaching the preparation of UNA include, but are not limited to, U.S. Patent No. 8,314,227 and U.S. Patent Application Publications 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of each of which are incorporated herein by reference.

[0138] Ribose can also be modified with a cyclopropane ring to produce tricyclodeoxyribonucleic acid (tricycloDNA). The ribose moiety may be replaced with another sugar, such as 1,5-anhydrohexitol, replaced with threose to produce threose nucleotides (TNA), or replaced with arabinose to produce arabinonucleotides. The ribose molecule may be replaced with a non-sugar, such as cyclohexene, to produce cyclohexene nucleotides, or replaced with glycol to produce glycol nucleotides.

[0139] Potentially stabilizing modifications to the ends of nucleotide molecules may include N-(acetylaminocaproyl)-4-hydroxyproline (Hyp-C6-NHAc), N-(caproyl-4-hydroxyproline (Hyp-C6), N-(acetyl-4-hydroxyproline (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyproline (Hyp-C6-amino), 2-docosanoyl-uridine-3'-phosphate, inverted base dT (idT), and the like. The disclosure of this modification can be found in PCT Publication No. WO2011 / 005861.

[0140] Other alternative chemistries of the oligonucleotides of the present disclosure may include 5'-phosphate or 5'-phosphate mimics of oligonucleotides, such as 5'-terminal phosphate or phosphate mimics. Suitable phosphate mimics are disclosed, for example, in U.S. Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0141] Additional non-limiting exemplary LNA nucleotides are disclosed in WO99 / 014226, WO00 / 66604, WO98 / 039352, WO2004 / 046160, WO00 / 047599, WO2007 / 134181, WO2010 / 077578, WO2010 / 036698, WO2007 / 090071, WO2009 / 006478, WO2011 / 156202, WO2008 / 154401, WO2009 / 067647, WO2008 / 150729, Morita et al., Bioorganic & Med.Chem.Lett.12,73-76, Seth et al.J.Org.Chem.2010,Vol 75(5)pp.1569-81, Mitsuoka et al., Nucleic Acids Research 2009,37(4),1225-1238, and Wan and Seth, J.Medical Chemistry 2016,59,9645-9667, each incorporated herein by reference.

[0142] In some embodiments, the length of the ASO is 5×n + 5 nucleotides (n is an integer of 3 or more), the nucleotide at the 5×m position is a ribonucleotide modified by LNA (m is an integer from 1 to n), and the nucleotides at the remaining positions are ribonucleotides modified by 2'-O-methoxyethyl.

[0143] In some embodiments, the nucleotide sugar modification is 2'-O-C1-4 alkyl, such as 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-C1-3 alkyl-O-C1-3 alkyl, such as 2'-methoxyethyl ("2'-MOE" or "MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotide, 2'-F-arabinosyl ("2'-F-arabino") nucleotide, 2'-locked nucleic acid ("LNA") nucleotide, 2'-amide-bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotide, L-type sugar ("L-sugar"), or 4'-thiothribosyl nucleotide.

[0144] Mixmer and Gapmer The ASO can have a mixmer and / or gapmer structure in a pattern disclosed by, for example, the ASO of FIG. 17 or FIG. 18.

[0145] In certain embodiments, the ASO is a mixmer. As used herein, the term "mixmer" refers to an oligonucleotide comprising an alternating composition of DNA monomers and nucleotide analog monomers over at least a portion of the oligonucleotide sequence. In certain embodiments, the ASO is a mixmer based on a gapmer structure, containing a mixture of DNA nucleotides and 2'-MOE nucleotides in the gap, with RNA sequences (e.g., 2'-modified RNA sequences) in the wings adjacent. The mixmer can be designed to include a mixture of affinity-enhancing nucleotide analogs, for example, in non-limiting examples, 2'-O-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, arabinonucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers, 2'-MOE-RNA (2'-O-methoxyethyl-RNA), 2'-fluoro-DNA, and LNA. In some embodiments, the mixmer cannot recruit RNase H. In some embodiments, this mixmer contains one type of affinity-enhancing nucleotide analog together with DNA and / or RNA.

[0146] Multiple different modifications may be spaced within the mixmer. For example, an ASO may include LNA modifications at multiple nucleotides and may include different modifications at some or all of the remaining nucleotides. In some embodiments, any two adjacent LNA-modified nucleotides are separated by at least 1, 2, 3, 4, or 5 nucleotides. Throughout the ASO, the distance between adjacent LNA-modified nucleotides may be constant (e.g., any two adjacent LNA-modified nucleotides are separated by 1, 2, 3, 4, or 5 nucleotides) or variable. In some embodiments, the length of the ASO is 3×n, 3×n−1, or 3×n−2 nucleotides (n is an integer of 6 or greater), where (a) (i) the nucleotide at position 3×m−2 (m is an integer from 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA), (ii) the nucleotide at position 3×m−1 (m is an integer from 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA), or (iii) the nucleotide at position 3×m (m is an integer from 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions contain a second different modification (e.g., 2′-O-methoxyethyl). In some embodiments, the length of the ASO is 2×n or 2×n−1 nucleotides (n is an integer of 9 or greater), where (a) (i) the nucleotide at position 2×m−1 (m is an integer from 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA), or (ii) the nucleotide at position 2×m (m is an integer from 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) containing a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions contain a second different modification (e.g., 2′-O-methoxyethyl). Similar modification patterns, e.g., patterns where the first modification is repeated every 4, 5, or more nucleotides, are also contemplated.

[0147] In certain embodiments, the ASO comprises a DNA sequence (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous nucleotides of unmodified DNA) flanked on both sides by an RNA sequence (e.g., a 2'-modified RNA sequence or 2'-modified ribonucleotide). Such a structure is known as a "gapmer," with the DNA region called the "gap" and the RNA region called the "wing" (see, e.g., PCT Publication No. WO2013 / 177248). Gapmers are known to promote degradation of target RNA by recruiting nucleases (e.g., nuclear RNAse such as RNase H). Surprisingly, in some embodiments of the present disclosure, it has been discovered that a gapmer that binds to a regRNA (having the same sequence as the parental ASO but different chemical modifications) can also increase target gene expression.

[0148] In some embodiments, the ASO gapmer comprises an internal DNA region flanked by two external RNA “wings”. For example, the internal DNA gap can comprise at least 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide(s), while each of the external RNA wing(s) can independently comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides. Exemplary gapmer structures include, but are not limited to, 1-10-9, 2-10-8, 3-10-7, 4-10-6, 6-10-4, 7-10-3, 8-10-2, 9-10-1, 1-18-1, 2-16-2, 3-14-3, 4-12-4, 5-10-5, 6-8-6, 7-6-7, 8-5-7, 7-5-8, 8-4-8, or 9-2-9 structures, where the first and third numbers indicate the number of external RNA nucleotides and the second number indicates the number of internal DNA nucleotides.

[0149] This ASO may also be a mixmer comprising one DNA region linked to one RNA region. In some embodiments, the mixmer comprises at least 10 DNA nucleotides linked to at least 10 RNA nucleotides, and the DNA nucleotides are at the 5' end or the 3' end of the mixmer. In some embodiments, the mixmer comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 DNA nucleotides (s) linked to at least 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 RNA nucleotide (s), wherein the DNA nucleotides are at the 5' end or the 3' end of the mixmer. In some embodiments, the RNA region of the gapmer or mixmer may comprise any additional chemical modifications disclosed herein.

[0150] In certain embodiments, the ASO (e.g., a gapmer or mixmer) is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides in length. In certain embodiments, the gap is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more nucleotides in length. In certain embodiments, one or both wings are about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more nucleotides in length. In certain embodiments, one or both RNA regions or wings contain RNA modifications such as β-D-ribonucleotides, 2'-modified nucleotides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3, or 2'-fluoro-arabino (FANA)), and bicyclic sugar-modified nucleotides (e.g., having locked ethyl or locked nucleic acid (LNA)). In certain embodiments, each ribonucleotide in the mixmer or gapmer is modified by 2'-MOE. In certain embodiments, the mixmer or gapmer contains one or more modified internucleotide linkages, such as phosphorothioate (PS) internucleotide linkages. In certain embodiments, each two adjacent nucleotides in the mixmer or gapmer are linked by a phosphorothioate internucleotide linkage.

[0151] In certain embodiments, the ASO does not contain one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, thirty-five or more, forty or more, or forty-five or more consecutive nucleotides of unmodified DNA. In some embodiments, such DNA sequences are interrupted by modified (e.g., 2'-MOE modified) ribonucleotides every 2, 3, 4, 5, or more nucleotides. In some embodiments, the ASO contains only ribonucleotides and no deoxyribonucleotides.

[0152] The structural features of mixmers and gapmers may be combined. In certain embodiments, the ASO has a structure similar to that of the mixmers disclosed herein (e.g., a structure having intervening modifications), except that in the gap a second modification is changed to a third modification (e.g., a deoxyribonucleotide). In certain embodiments, the ASO has a structure similar to that of the gapmers disclosed herein, except that in the gap the nucleotides are modified in a mixmer pattern.

[0153] In certain embodiments, the ASO further comprises a ligand moiety, e.g., a ligand moiety that specifically targets a tissue or organ of interest. For example, N-acetylgalactosamine (GalNAc) specifically targets the liver. In certain embodiments, the ligand moiety comprises GalNAc. In certain embodiments, the ligand moiety comprises a 3-cluster GalNAc moiety (commonly referred to as GalNAc3). Other types of GalNAc moieties are 1-cluster, 2-cluster, or 4-cluster GalNAc, designated GalNAc1, GalNAc2, or GalNAc4. In certain embodiments, the ligand moiety comprises GalNAc1, GalNAc2, GalNAc3, or GalNAc4.

[0154] In certain embodiments, the ligand moiety comprises biotin. In certain embodiments, the ligand moiety comprises palmitic acid. In certain embodiments, the ligand moiety comprises a spacer 18 moiety (C18).

[0155] III. Pharmaceutical Compositions In certain embodiments, the ASOs disclosed herein may be present in a pharmaceutical composition. The pharmaceutical composition can be formulated for use in various drug delivery systems. One or more pharmaceutically acceptable excipients or carriers may also be included in the composition for proper formulation. In some embodiments, the pharmaceutically acceptable carrier includes sterile saline, sterile water, phosphate-buffered saline (PBS), or aCSF. Formulations suitable for use in the present disclosure can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249:1527-1533, 1990).

[0156] Exemplary carriers and pharmaceutical formulations suitable for delivering nucleic acids are described in Darymanov and Reineke (2018) Front. Pharmacol. 9:971; Barba et al. (2019) Pharmaceutics 11(8):360; Ni et al. (2019) Life (Basel) 9(3):59 (each incorporated herein by reference). It is understood that the presence of the ligand moiety conjugated to the ASO can obviate the need for a carrier for delivery to the tissue or organ targeted by the ligand moiety.

[0157] Delivery of the oligonucleotides of the present disclosure to cells, such as cells within a subject, such as a human subject, such as a subject in need thereof, such as a subject having a GRN-related disorder, can be accomplished in many different ways. For example, delivery may be effected by contacting the cells with the oligonucleotides of the present disclosure, either in vitro or in vivo. In vivo delivery can also be effected directly by administering to the subject a composition comprising the oligonucleotides. These alternatives are considered further below.

[0158] In general, any method for delivering nucleic acid molecules (in vitro or in vivo) may be adapted for use with the oligonucleotides of the present disclosure (see, for example, Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94 / 02595, which are incorporated herein by reference in their entirety). In the case of in vivo delivery, factors to consider for delivering oligonucleotide molecules include, for example, the biological stability of the molecule being delivered, prevention of non-specific effects, and accumulation of the molecule being delivered in the target tissue. Non-specific effects of the oligonucleotides may be minimized by local administration, such as by direct injection or implantation into the tissue, or by administering the preparation locally. Local administration to the site of treatment maximizes the local concentration of the agent and limits exposure of the agent to systemic tissues that may otherwise be harmed by the agent or may degrade the agent, and allows for administration of a lower total dose of oligonucleotide molecules.

[0159] For systemic administration of oligonucleotides for the treatment of diseases, the oligonucleotides may contain alternative nucleobases, alternative sugar moieties, and / or alternative internucleotide linkages, or alternatively may be delivered using a drug delivery system; both methods act to prevent rapid degradation of the oligonucleotides by endo- and exonucleases in vivo. Modification of the oligonucleotides or pharmaceutical carriers can also enable targeting of the oligonucleotide composition to the target tissue and avoid unwanted off-target effects. Oligonucleotide molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to facilitate cellular uptake and prevent degradation. In alternative embodiments, the oligonucleotides can be delivered using a drug delivery system such as nanoparticles, lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of the oligonucleotide molecule (negatively charged) and also enhance interaction with the negatively charged cell membrane to enable efficient uptake of the oligonucleotide by the cell. Cationic lipids, dendrimers, or polymers can be induced to bind to the oligonucleotide or form vesicles or micelles that encapsulate the oligonucleotide. Formation of vesicles or micelles further prevents degradation of the oligonucleotides when administered systemically. In general, any method of nucleic acid delivery known in the art may be adaptable for delivery of the oligonucleotides of the present disclosure. Methods for making and administering cationic oligonucleotide complexes are within the capabilities of those skilled in the art (see, e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety).Some non-limiting examples of drug delivery systems useful for systemic delivery of oligonucleotides include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptide (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamine (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, the oligonucleotide forms a complex with cyclodextrin for systemic administration. The methods of administration and pharmaceutical compositions of the oligonucleotide and cyclodextrin can be found in U.S. Patent No. 7,427,605, which is hereby incorporated by reference in its entirety. In some embodiments, the oligonucleotides of the present disclosure are delivered by polyplexes or lipoplex nanoparticles.Methods of administration and pharmaceutical compositions of oligonucleotides and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Application Nos. 2017 / 0121454; 2016 / 0369269; 2016 / 0279256; 2016 / 0251478; 2016 / 0230189; 2015 / 0335764; 2015 / 0307554; 2015 / 0174549; 2014 / 0342003; 2014 / 0135376; and 2013 / 0317086, which are hereby incorporated by reference in their entirety.

[0160] In some embodiments, the compounds described herein can be administered in combination with additional therapeutic agents. Examples of additional therapies include standard therapeutic anti-seizure medications such as quinidine and / or sodium channel blockers. Further, the compounds described herein may be administered in combination with recommended lifestyle modifications such as a ketogenic diet.

[0161] Methods of Delivery of Membrane Molecular Assemblies The oligonucleotides of the present disclosure may also be delivered using various membrane molecular assembly delivery methods including polymers, biodegradable microparticles, or microcapsule delivery devices known in the art. For example, colloidal dispersions may be used for the targeted delivery of the oligonucleotide agents described herein. Colloidal dispersions include polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems including water-in-oil emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles useful as delivery vehicles in vitro and in vivo. Large unilamellar vesicles (LUVs) in the size range of 0.2 - 4.0 μm have been shown to be able to encapsulate a significant proportion of an aqueous buffer containing large polymers. Liposomes are useful for the introduction and delivery of active ingredients to the site of action. Since the liposome membrane is structurally similar to the biological membrane, when liposomes are applied to tissues, the liposome bilayer fuses with the bilayer of the cell membrane. As the fusion of liposomes and cells progresses, the internal aqueous contents containing the oligonucleotide are delivered to the cells, where the oligonucleotide can specifically bind to the target RNA. In some cases, liposomes are also specifically targeted, for example, to direct the oligonucleotide to a specific cell type. The composition of liposomes is usually a combination of phospholipids, usually in combination with steroids, particularly cholesterol. Other phospholipids or other lipids may also be used. The physical properties of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0162] Liposomes containing oligonucleotides can be prepared in various ways. In one example, the lipid component of the liposome is dissolved in a surfactant to form a lipid component and micelles. For example, the lipid component may be an amphiphilic cationic lipid or a lipid conjugate. The surfactant may have a high critical micelle concentration and may be nonionic. Exemplary surfactants include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. Next, an oligonucleotide formulation is added to the micelles containing the lipid component. The cationic groups on the lipid interact with the oligonucleotide and condense around the oligonucleotide to form liposomes. After condensation, the surfactant is removed, for example, by dialysis, to obtain a liposome formulation of the oligonucleotide.

[0163] Optionally, during the condensation reaction, a carrier compound that aids in condensation may be added, for example, by controlled addition. For example, the carrier compound may be a polymer other than nucleic acid (e.g., spermine or spermidine). The pH may be adjusted to promote condensation.

[0164] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic lipid complexes as a structural component of the delivery vehicle are further described, for example, in WO96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation may also include one or more aspects of the exemplary methods described in Feigner, P.L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Patent No. 4,897,355; U.S. Patent No. 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75:4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Techniques commonly used to prepare lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, for example, Mayer et al., (1986) Biochim. Biophys. Acta 858:161). Microfluidization can be used when consistently small (50-200 nm), relatively uniform aggregates are required (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adaptable to packaging oligonucleotide formulations into liposomes.

[0165] Liposomes are broadly classified into two major classes. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized into endosomes. Due to the acidic pH within endosomes, the liposomes rupture and release their contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0166] pH-sensitive or negatively charged liposomes do not form complexes with nucleic acids but rather entrap them. Since both nucleic acids and lipids are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acids are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0167] One major type of liposome composition contains phospholipids other than naturally derived phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are mainly formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed from phosphatidylcholine (PC), such as soy PC and egg PC, etc. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0168] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Patent No. 5,283,185; U.S. Patent No. 5,171,678; WO94 / 00569; WO93 / 24640; WO91 / 16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

[0169] Nonionic liposome systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in drug delivery to the skin. Nonionic liposome formulations containing NOVASOME™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A to the dermis of mouse skin. The results showed that such nonionic liposome systems were effective in promoting the deposition of cyclosporin A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

[0170] Liposomes can also be sterically stabilized liposomes and contain one or more special lipids that result in an extended circulation lifetime compared to liposomes lacking such special lipids. Examples of sterically stabilized liposomes are those in which a portion of the vesicle-forming lipid moiety of the liposome is (A) monosialoganglioside G M1comprising one or more glycolipids such as, or (B) liposomes derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. Without wishing to be bound by any particular theory, for at least sterically stabilized liposomes comprising gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes is thought in the art to result from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0171] A variety of liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G M1 , galactosylcerebroside sulfate, and phosphatidylinositol to improve the blood half-life of liposomes. These findings were elaborated by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). Both U.S. Patent No. 4,837,028 and WO88 / 04924 to Allen et al. disclose (1) sphingomyelin and (2) liposomes containing ganglioside G M1 or galactosylcerebroside sulfate ester. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO97 / 13499 (Lim et al).

[0172] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Non-cationic liposomes cannot fuse efficiently with the plasma membrane, but can be taken up by macrophages in vivo and used to deliver oligonucleotides to macrophages.

[0173] Further advantages of liposomes include that liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; liposomes can protect oligonucleotides encapsulated in internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the surface charge of the lipid, the size of the vesicles, and the water content of the liposomes.

[0174] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids and fuse with the negatively charged lipids of the cell membranes of tissue culture cells to form lipid-nucleic acid complexes that can deliver oligonucleotides (e.g., Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and for an explanation of DOTMA and its use with DNA, see U.S. Patent No. 4,897,355).

[0175] When 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane (DOTAP), an analog of DOTMA, is used in combination with a phospholipid, a DNA complexing vesicle can be formed. LIPOFECTIN (trademark, Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for delivering highly anionic nucleic acids to cultured cells of living tissues, which contains positively charged DOTMA liposomes that interact spontaneously with negatively charged polynucleotides to form complexes. When sufficient positively charged liposomes are used, the net charge of the resulting complex is also positive. The positively charged complex thus prepared adheres spontaneously to the negatively charged cell surface, fuses with the plasma membrane, and efficiently delivers functional nucleic acids, for example, to cultured cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonio)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.), differs from DOTMA in that the oleoyl moieties are linked by esters rather than ether bonds.

[0176] Other reported cationic lipid compounds include, for example, compounds conjugated to various moieties including carboxyspermine conjugated to either of two lipids, for example, 5-carboxyspermill glycine dioctaoleylamide (“DOGS”) (TRANSFECTAM (trademark), Promega, Madison, Wis.), and dipalmitoylphosphatidylethanolamine 5-carboxyspermill-amide (“DPPES”) (see, for example, U.S. Patent No. 5,171,678), and the like.

[0177] Another cationic lipid conjugate involves derivatization of a lipid with cholesterol (“DC-Chol”) formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolyllysine made by conjugating polylysine to DOPE has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). In certain cell lines, these liposomes containing conjugated cationic lipids are said to be less toxic and provide more efficient transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO98 / 39359 and WO96 / 37194.

[0178] Liposomal formulations are particularly suitable for topical administration, and liposomes exhibit several advantages compared to other formulations. Such advantages include a reduction in side effects associated with high systemic absorption of the administered drug, an increase in the accumulation of the administered drug at the desired target, and the ability to administer oligonucleotides to the skin. In some embodiments, liposomes are used to deliver oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into skin tissue, such as the skin. For example, liposomes may be applied topically. Topical delivery of drugs formulated as liposomes has been documented (see, for example, Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R.J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R.M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C.Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0179] Nonionic liposomal systems, particularly those containing nonionic surfactants and cholesterol, have also been investigated to determine their usefulness in drug delivery to the skin. Nonionic liposomal formulations containing NOVASOME I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin. Such formulations containing oligonucleotides are useful for treating skin disorders.

[0180] Targeting of liposomes is also possible, for example, based on organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of a liposome targeting delivery system, a lipid group may be incorporated into the lipid bilayer of the liposome to maintain the targeting ligand in stable association with the liposome bilayer. Various linking groups may be used to attach the lipid chain to the target ligand. Additional methods are known in the art, for example, as described in U.S. Patent Application Publication No. 20060058255, the contents of which are incorporated herein by reference.

[0181] Liposomes containing oligonucleotides can be made highly deformable. Such deformability can enable the liposomes to penetrate pores smaller than the average radius of the liposomes. For example, transferosomes are yet another type of liposome and are highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transferosomes can be described as fat droplets that are very easily deformable and can readily penetrate pores smaller than droplets. Transferosomes may be made by adding a surface edge activator, which is usually a surfactant, to a standard liposome composition. Transferosomes containing oligonucleotides may be delivered subcutaneously, for example, by infection, in order to deliver the oligonucleotides to keratinocytes of the skin. In order to pass through the intact mammalian skin, the lipid vesicles must each pass through a series of fine pores each less than 50 nm in diameter under the influence of an appropriate transdermal gradient. Further, due to the properties of the lipids, these transferosomes can self-optimize (e.g., in the skin, adapt to the shape of the pores), self-repair, and often reach the target by self-loading without fragmentation. Transferosomes have been used to deliver serum albumin to the skin. Delivery of serum albumin via transferosomes has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0182] Other formulations suitable for the present disclosure are described in PCT Publications WO2009 / 088891, WO2009 / 132131, and WO2008 / 042973, which are incorporated herein by reference in their entirety.

[0183] Surfactants are widely used in formulations such as emulsions (including microemulsions) and liposomes. The most common method for classifying and ranking the properties of many different types of surfactants, both natural and synthetic, is by use of the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for classifying the various surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0184] When the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants are widely used in pharmaceuticals and cosmetics and can be used over a wide range of pH values. Generally, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this class. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0185] When surfactant molecules carry a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Examples of anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0186] When surfactant molecules carry a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. Quaternary ammonium salts are the most widely used members of this class.

[0187] When surfactant molecules have the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Examples of amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phosphatides.

[0188] The use of surfactants in pharmaceuticals, formulations, and emulsions has been considered (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0189] Oligonucleotides for use in the methods of the present disclosure can also be provided as micelle formulations. A micelle is a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all hydrophobic portions of the molecules are directed inward and the hydrophilic portions remain in contact with the surrounding aqueous phase. When the environment is hydrophobic, the reverse arrangement exists.

[0190] Lipid nanoparticle-based delivery methods The oligonucleotides in the present disclosure can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs), or other nucleic acid-lipid particles. LNPs are useful for systemic administration because they have an extended circulation lifetime after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the administration site). Examples of LNPs include "pSPLP", which includes encapsulated condensing agent-nucleic acid complexes described in PCT Publication No. WO00 / 03683. The particles of the present disclosure typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm and are substantially non-toxic. Further, the nucleic acid is resistant to nuclease degradation in aqueous solution when present in the nucleic acid-lipid particles of the present disclosure. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010 / 0324120 and PCT Publication No. WO96 / 40964.

[0191] Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.(Cl), 1,2-dilinoleoyl-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyl-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or an analog thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can constitute, for example, about 20 mol% to about 50 mol%, or about 40 mol% of the total lipids present in the particles.

[0192] The ionizable / non-cationic lipid may be an anionic lipid or a neutral lipid, including but not limited to distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid can be, for example, about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipids present in the particles when cholesterol is included.

[0193] The complex lipid that inhibits particle aggregation can be a polyethylene glycol (PEG)-lipid including but not limited to PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG dilauryl oxypropyl (C 12 ), PEG dimyristyl oxypropyl (C 14 ), PEG-dipalmityl oxypropyl (C 16 ), or PEG-distearyl oxypropyl (C 18 ). The complex lipid that prevents particle aggregation can be, for example, 0 mol% to about 20 mol%, or about 2 mol% of the total lipids present in the particles.

[0194] In some embodiments, the nucleic acid-lipid particles further comprise, for example, from about 10 mole % to about 60 mole %, or about 50 mole % cholesterol, of the total lipids present in the particles.

[0195] The ASO can also be delivered with a lipidoid. The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suitable for the delivery of modified nucleic acid molecules or ASOs (see Mahon et al, Bioconjug Chem. 2010 21:1448-1454; Schroeder et al, J Intern Med. 2010 267:9-21; Akinc et al, Nat Biotechnol. 2008:26:561-569; Love et al, Proc Natl Acad Sci U A. 2010 107:1864-1869; Siegwart et al, Proc Natl Acad Sci U S A. 2011 108:12996-3001, all of which are incorporated herein by reference in their entirety).

[0196] Lipid compositions for RNA delivery are disclosed in WO2012170930A1, WO2013149141A1, and WO2014152211A1, each of which is incorporated herein by reference.

[0197] IV. Therapeutic uses The present disclosure provides methods for treating diseases and disorders associated with decreased GRN gene expression and other diseases and disorders. In some embodiments, the method uses an ASO or a pharmaceutical composition comprising an ASO that hybridizes to a GRN regRNA transcribed from a regulatory element of the GRN gene. The oligonucleotide compositions described herein are useful in the methods of the present disclosure and, without being bound by theory, in cells of a subject (e.g., a mammal, mouse, hamster, non-human primate (e.g., monkey), or human), the levels of PGRN protein (and its proteolytic GRN peptide products) and / or GRN mRNA, the status and / or activity of GRN are thought to exert their desired effects (e.g., by increasing the level of PGRN protein, through the ability to regulate).

[0198] One aspect of the present disclosure is a method of treating a disorder associated with GRN (e.g., a GRN-related disorder) in a subject in need thereof, the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing the expression of GRN in the cells of the subject. In some embodiments, the GRN-related disorder is frontotemporal dementia (FTD) (e.g., GRN-FTD, also known as FTD-GRN) or frontotemporal lobar degeneration (e.g., GRN-related frontotemporal lobar degeneration). In some embodiments, the subject comprises progranulin haploinsufficiency.

[0199] Another aspect of the present disclosure is a method of treating a disease or disorder (e.g., a disease or disorder provided herein) in a subject in need thereof, the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby treating the disease or disorder in the subject. In some embodiments, the disease or disorder is frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathological frontotemporal lobar degeneration associated with the accumulation of TDP-43 inclusions (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerosis dementia, spinocerebellar ataxia type 3, chronic traumatic encephalopathy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Gaucher disease (GD), and Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL) type 11 (CLN11), limbic-predominant age-related TDP-43 encephalopathy (LATE), autism, cerebral ischemia-reperfusion injury, lysosomal storage disease (LSD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), ischemic heart disease, intervertebral disc degeneration, and acute kidney injury.In some embodiments, the disease or disorder is a sphingolipidosis disorder (e.g., GM2 gangliosidosis type A (also known as Tay-Sachs disease); GM2 gangliosidosis type O (also known as Sandhoff disease); GM2 gangliosidosis type AB (also known as GM2 activator deficiency); Niemann-Pick disease (e.g., Niemann-Pick disease type A; Niemann-Pick disease type B; Niemann-Pick disease type C; Niemann-Pick disease type D; Niemann-Pick disease type E; and Niemann-Pick disease type F); Gaucher disease (e.g., Gaucher disease type 1; Gaucher disease type 2; and Gaucher disease type 3); Fabry disease (also known as Anderson-Fabry disease) (e.g., classical Fabry disease and late-onset Fabry disease); metachromatic leukodystrophy; globoid cell leukodystrophy (also known as Krabbe disease), GM1 gangliosidosis (e.g., GM1 gangliosidosis type 1, GM1 gangliosidosis type 2, and GM1 gangliosidosis type 3); and multiple sulfatase deficiency); an oligosaccharidosis disorder (e.g., alpha-mannosidosis, Schindler disease, aspartylglucosaminuria, and fucosidosis); a mucopolysaccharidosis (MPS) (e.g., Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome (e.g., Sanfilippo syndrome type A, Sanfilippo syndrome type B, Sanfilippo syndrome type C, and Sanfilippo syndrome type D), Morquio syndrome (e.g., Morquio syndrome type A and Morquio syndrome type B), Maroteaux-Lamy syndrome, and Sly syndrome; neuronal ceroid lipofuscinosis (NCL; also known as Batten disease) (e.g., CLN1, CLN2, CLN3, CLN4, CLN5, CLN6, CLN7, CLN8, CLN9, CLN10, CLN11, CLN12, CLN13, and CLN14), a sialic acid disorder (e.g., galactosialidosis, infantile sialic acid storage disease, Salla disease, and sialuria), a mucolipidosis (e.g., sialidosis I, sialidosis II, I-cell disease, pseudo-Hurler-polydystrophy, and mucolipidosis IV), lysosomal acid lipase deficiency, Pompe disease, Danon disease, and cystinosis selected LSD.Lysosomal storage diseases are generally described in Rajjumur and Dumpa, “Lysosomal Storage Disease,” In: StatPearls [internet] Treasure Island (FL): StatPearls Publishing; 2023 Jan - (available at: ncbi.nlm.nih.gov / books / NBK563270 / ) (incorporated herein by reference in its entirety).

[0200] Another aspect of the present disclosure is a method of increasing the level of PGRN protein or GRN mRNA in the cells of a subject (e.g., a subject identified as having a GRN - related disorder or any other disease or disorder provided herein), the method comprising contacting the cells of the subject with an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing the level of PGRN protein or GRN mRNA in the cells of the subject.

[0201] Another aspect of the present disclosure is a method of increasing the expression of the GRN gene in the cells of a subject (e.g., a subject having a disease or disorder described herein), the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing the expression of the GRN gene in the cells of the subject.

[0202] Another aspect of the present disclosure relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby treating the disease or disorder of the subject.

[0203] Yet another aspect of the present disclosure is a method of increasing the expression of GRN in the cells of a subject, the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby treating the disease or disorder of the subject.

[0204] In yet another aspect, the present disclosure provides the ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) for use as a medicament. Further, the present disclosure provides the ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) for use in therapy.

[0205] Yet another aspect of the present disclosure includes a method of modulating (e.g., increasing or decreasing) the expression of the GRN gene in a cell (e.g., in vivo, ex vivo, or in vitro) by contacting the cell with the ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing the expression of the GRN gene in the cell. In some embodiments, the cell is a human cell or a mammalian cell. This method can include contacting the cell with the ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) in an amount effective to modulate (e.g., increase) the expression of GRN in the cell, thereby increasing the expression of PGRN protein or GRN mRNA in the cell. In some embodiments, contacting the cell with the ASO (or a pharmaceutical composition comprising the ASO) modulates (e.g., increases) the amount of GRN mRNA in the cell. In some embodiments, contacting the cell with the ASO (or a pharmaceutical composition comprising the ASO) modulates (e.g., increases) the amount of PGRN protein in the cell.

[0206] Based on the above methods, further aspects of the present disclosure include oligonucleotides of the present disclosure, or compositions comprising such oligonucleotides, for use in treatment, or for use as a medicament, or for use in treating a disease or disorder (e.g., a GRN-related disorder or FTD) in a subject in need thereof, or for use in increasing the level of PGRN in the cells of a subject (e.g., a subject identified as having a GRN-related disorder), or for use in increasing the expression of GRN in the cells of a subject. This use involves contacting the cell and the oligonucleotide in an amount effective to increase the expression of GRN in the cell, thereby increasing the expression of GRN in the cell. The embodiments described below with respect to the methods of the present disclosure are also applicable to these further aspects.

[0207] Contact between the cell and the ASO may occur in vitro, ex vivo, or in vivo. Contacting the cell with the ASO in vivo involves contacting a cell or cell population within a subject, e.g., a human subject, with the oligonucleotide. Combinations of methods for contacting the cell in vitro, ex vivo, and in vivo are also possible. As described above, the contact with the cell may be direct or indirect. Further, the contact with the cell can be achieved via a targeting ligand comprising any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc3 ligand, or another ligand that directs the oligonucleotide to the site of interest. The cell may be a CNS cell, e.g., a neuron or a brain cell, a microglia cell.

[0208] Administration of the ASO or pharmaceutical composition disclosed herein to a subject may be by intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, perfusion through a catheter, or direct lesion injection. In certain embodiments, the ASO or pharmaceutical composition is administered systemically. In certain embodiments, the ASO or pharmaceutical composition is administered by a parenteral route. For example, in certain embodiments, the ASO or pharmaceutical composition is administered intravenously (e.g., by intravenous infusion), e.g., using a prefilled bag, prefilled pen, or prefilled syringe. In other embodiments, the ASO or pharmaceutical composition is administered locally to an organ or tissue where an increase in target gene expression is desired (e.g., the liver or brain tissue (e.g., cortex, hypothalamus, hippocampus, cerebellum, and coronary brain tissue)).

[0209] In some embodiments, the oligonucleotide is administered to a subject such that the oligonucleotide is delivered to a specific site within the subject. Such targeted delivery can be achieved by either systemic or local administration. An increase in GRN expression can be evaluated using measurement of the level or change in level of GRN mRNA or progranulin protein in a sample (e.g., blood, tissue, or CNS sample) derived from a specific site within the subject. In certain embodiments, the method includes a clinically relevant increase in GRN expression, as indicated by a clinically relevant outcome, e.g., after treating the subject with an agent that decreases GRN expression.

[0210] In other embodiments, the oligonucleotide is administered in an amount and for a time effective to result in a reduction (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of one or more symptoms of a disease or disorder (e.g., a GRN-related disorder), e.g., deterioration of behavior or personality, language impairment, muscle or motor function impairment or change, memory loss, dysfunction, tremors, seizures, and dizziness.

[0211] Increase in GRN expression level In some embodiments, the therapeutic methods disclosed herein that use ASOs targeting GRN regRNA are designed to increase the GRN expression level in a subject. An increase in the expression of the GRN gene includes any level of increase in the GRN gene, such as at least a partial increase in the expression of the GRN gene. The increase in expression can be evaluated by an increase in the absolute or relative level of one or more of these variable elements compared to a control level. The control level can be any type of control that is used in the art, such as a baseline level before administration, or a level determined from a similar subject, cell, or a sample treated with an untreated or control (e.g., buffer only (vehicle) control or inactive agent control). In certain embodiments, the method results in a clinically relevant increase in the expression of GRN, as indicated by a clinically relevant outcome, for example, after treating the subject with an agent that increases the expression of GRN.

[0212] In certain embodiments, the methods disclosed herein increase GRN gene expression by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% relative to a baseline level prior to dosing, prior to administration, or prior to exposure. In certain embodiments, the methods disclosed herein increase GRN gene expression by at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more relative to a baseline level prior to dosing, prior to administration, or prior to exposure. In certain embodiments, the subject has a deficiency in GRN expression and the methods disclosed herein restore the GRN expression level or activity to at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the average GRN expression level or activity in subjects of the same age and sex species.

[0213] In some embodiments, the ASO of the present disclosure can enhance the production of GRN mRNA (e.g., in cells or in the cells, tissues or samples of a subject) to at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or more, relative to the baseline level before dosing, before administration, or before exposure. In some embodiments, the ASO of the present disclosure can enhance the production of GRN mRNA (e.g., in cells or in the cells, tissues or samples of a subject) to at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold or more, relative to the baseline level before dosing, before administration, or before exposure.

[0214] In some embodiments, the ASOs of the present disclosure enhance the production of PGRN protein (e.g., in cells or in the cells, tissues, or samples of a subject) to at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, or more, relative to the baseline level before dosing, before administration, or before exposure. In some embodiments, the ASOs of the present disclosure enhance the production of PGRN protein (e.g., in cells or in the cells, tissues, or samples of a subject) to at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold, or more, relative to the baseline level before dosing, before administration, or before exposure.

[0215] The expression of the GRN gene can be evaluated based on the level of any variable element related to GRN gene expression, e.g., the mRNA level of GRN or the protein level of PGRN. In certain embodiments, the expression level or activity of GRN herein refers to the average expression level or activity in neurons or the brain (e.g., brain cells of the brain regions described herein).

[0216] In certain embodiments, alternative markers may be used to detect an increase in GRN expression levels. For example, effective treatment of GRN-related disorders, as indicated by acceptable diagnostic and monitoring criteria for agents that increase GRN expression, may be understood to indicate a clinically relevant increase in GRN.

[0217] An increase in the expression of the GRN gene may be evidenced by an increase in the amount of GRN mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which the GRN gene is transcribed, processed, or being processed (e.g., by contacting the cell(s) with an oligonucleotide of the present disclosure or administering an oligonucleotide of the present disclosure to a subject in which the cell(s) are present or were present), such that the expression of the GRN gene is increased as compared to a second cell or group of cells (control cell(s) not so treated or not treated at all, i.e., not treated with an oligonucleotide or not treated with an oligonucleotide targeting the gene of interest) that is substantially identical to the first cell or group of cells but not so treated.

[0218] In other embodiments, an increase in the expression of the GRN gene may be evaluated with respect to a parameter that is functionally related to GRN gene expression, such as an increase in progranulin (PGRN) protein expression, granulin peptide levels, or PGRN activity. An increase in GRN expression may be determined by any assay known in the art in any cell expressing GRN, either endogenously or heterologously, from an expression construct.

[0219] An increase in GRN expression may be evidenced by an increase in the level of PGRN protein (or its proteolytic granulin peptide products) expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject) as compared to a control cell or control group of cells. An increase in GRN expression may also be evidenced by an increase in GRN mRNA levels in a treated cell or group of cells as compared to a control cell or control group of cells.

[0220] As control cells or cell populations that can be used to evaluate an increase in the expression of the GRN gene, cells or cell populations that have not yet been contacted with the oligonucleotides of the present disclosure can be mentioned. For example, the control cells or cell populations can be derived from individual subjects (e.g., human or animal subjects) before treating the subject with the oligonucleotide.

[0221] The level of GRN mRNA expressed by a cell or cell population can be determined using any method known in the art for evaluating mRNA expression. In one embodiment, the expression level of GRN in a sample is determined by detecting the transcribed polynucleotide, or a portion thereof, e.g., the mRNA of the GRN gene. RNA can be extracted from cells using RNA extraction techniques including, for example, acid phenol / guanidinium isothiocyanate extraction (RNAzol™ B; Biogenesis), RNeasy™ RNA preparation kits (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 GRN mRNA can be detected using the methods described in PCT Publication WO2012 / 177906, the entire content of which is incorporated herein by reference. In some embodiments, the level of expression of GRN is determined using a nucleic acid probe. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific GRN or PGRN sequence, e.g., to an mRNA or polypeptide. The probe may be synthesized by one of ordinary skill in the art or derived from a suitable biological preparation. The probe may be specially designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0222] In some embodiments, the disclosed methods of treatment using an ASO targeting a GRN regRNA are designed to reduce the level of immune response gene expression in a subject. Such immune response genes include, but are not limited to, cytokines and chemokines. Exemplary cytokines and chemokines are IL-8, IL-6, CCL4, and CCL2. In some embodiments, the ASO of the GRN reduces the expression of IL-8, IL-6, CCL4, and CCL2 in cells or in a subject.

[0223] 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 for determining the mRNA level involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of the GRN. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by electrophoresing 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. One of ordinary skill in the art can readily adapt known methods of mRNA detection for use in determining the level of mRNA of the GRN.

[0224] Alternative methods for determining the expression level of GRN in a sample include, for example, the process of nucleic acid amplification and / or reverse transcription (to prepare cDNA) of mRNA in the sample, such as, for example, RT-PCR (experimental embodiments described in Mullis, 1987, U.S. Patent 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 system (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. Patent 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 detecting such molecules when the nucleic acid molecules are present in very small numbers. In certain aspects of the present disclosure, the level of GRN expression is determined by quantitative fluorescence-generating RT-PCR (i.e., the TAQMAN (trademark) system) or the DUAL-GLO (registered trademark) luciferase assay.

[0225] The expression level of GRN mRNA can be monitored using a membrane blot (such as those used in hybridization analyses such as Northern, Southern, dot, etc.), or using a microwell, sample tube, gel, bead, or fiber (or any solid support containing the 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. Determination of GRN expression levels can also include using nucleic acid probes in solution.

[0226] In some embodiments, the level of GRN mRNA expression is evaluated using branched DNA (bDNA) assay or real-time PCR (qPCR). Such methods can also be used for the detection of GRN nucleic acids.

[0227] The level of PGRN protein expression and the level of granulin peptides can be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, fluid or gel precipitation reactions, absorption spectroscopy, colorimetric analysis, spectrophotometric analysis, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, and the like. Such assays can also be used for the detection of proteins that indicate the presence of PGRN protein and granulin peptides.

Example

[0228] Example 1: Synthesis and in vitro characterization of human GRN regRNA-targeted ASO Six GRN regRNAs (one paRNA and five eRNAs) were identified within the human genome. 177 steric ASOs targeting GRN regRNAs were designed and synthesized. 29 ASOs were selected for tiling after the first-pass screening. 52 ASOs were designed and synthesized for fine-tuning. Of these, 5 ASOs were gapmers, 27 had PO / PS linkages, 5 were mixmers, and 15 consisted of LNA.

[0229] To evaluate the expression of hGRN paRNA and mRNA in iPSC-derived neurons and iMGL cells, the following experiments were conducted. After exposing cells to the histone deacetylase (HDAC) inhibitor vorinostat (VOR) at either 1 μM or 3 μM for 24 hours (it is known to increase the expression of GRN mRNA), the expression of hGRN paRNA and mRNA in these cells was evaluated using real-time quantitative PCR (qPCR). As a control, cells were exposed to a DMSO vehicle control. In this analysis, the qPCR reference genes were the geometric mean of GAPDH and PPIA, normalized to iPSC-derived neurons treated with the DMSO sample. n = 3.

[0230] hGRN mRNA (Figure 2A) and paRNA (Figure 2B) were detected in iPSC-derived neurons and iMGL cells. hGRN mRNA and paRNA were 15 - 20-fold more abundant in microglia (iMGL cells) compared to iPSC-derived neurons. Vorinostat treatment induced both hGRN mRNA levels and paRNA levels.

[0231] To detect whether hGRN paRNA is also expressed in human cortical tissue, a similar qPCR analysis was performed using human cortical tissue. As shown in Figure 3, hGRN paRNA was detected in human cortical tissue.

[0232] To evaluate the ability of ASOs targeting hGRN paRNA to regulate the expression of human GRN mRNA, 110 ASOs targeting hGRN paRNA (-strand) were tested in the hepatocellular carcinoma cell line HepG2. Briefly, HepG2 cells were transfected with 100 nM of the ASOs shown in Table 2. Forty-eight hours after transfection, the cells were harvested for mRNA analysis via qPCR. The expression levels of hGRN mRNA in cells treated with each ASO are shown in Table 2.

[0233] (Table 2) TIFF2025522380000048.tif221165

[0234] For further characterization in dose - setting studies, the best ASOs were selected based on the increased fold - change of GRN mRNA. Briefly, human neuroblastoma SK - N - AS cells were transfected with ASOs CO - 3423, CO - 3431, CO - 3463, and CO - 3503, which are ASOs at 20 - 160 nM. A stereotactic non - targeting control ASO (sNTC) was used as a control. After 48 hours, cells were harvested for mRNA analysis and GRN mRNA was quantified by qPCR. Housekeeping genes for normalization were GAPDH and PPIA, and the mRNA fold - change (FC) was normalized to sNTC. CO - 3423 up - regulated GRN mRNA by approximately 2.5 - fold in a dose - dependent manner compared to sNTC at the same dose (Figure 4). CO - 3431 down - regulated GRN levels by approximately 50%.

[0235] Thirty - six additional ASOs were designed by base - walking around different modifications and selected ASOs. The effects of increasing doses of these ASOs were evaluated in the human neuroblastoma cell line SK - N - AS after transfection. Forty - eight hours after transfection, the relative GRN mRNA levels in treated SK - N - AS cells were evaluated, and data from exemplary ASOs are shown in Figures 5A and 5B.

[0236] Additional chemical modifications, including LNA modifications, were made to CO - 3423. SK - N - AS cells were transfected with increasing concentrations of LNA / PS - modified ASO and GRN mRNA quantified by qPCR. ASOs containing different LNA modifications of CO - 3423 had similar efficacy (i.e., similar increases in GRN mRNA expression). The fold - change of GRN mRNA induced by each ASO is shown in Table 3. GRN mRNA was normalized to the average fold - change of cells treated with two stereotactic non - targeting control ASOs (CO - 3772 and CO - 1589 (sNTC1)).

[0237] (Table 3) TIFF2025522380000049.tif87165

[0238] The effect of the length of the ASO on the regulation of GRN gene expression was also examined. SK-N-AS cells were transfected with increasing concentrations of longer ASO and GRN mRNA, which were quantified by qPCR. As the length of CO-3423 was increased, the potency of the ASO increased, as shown in Table 4. The mRNA was normalized against the average fold change in cells treated with two sterically non-targeting control ASOs (CO-3772 and CO-1589 (sNTC1)).

[0239] (Table 4) TIFF2025522380000050.tif58165

[0240] To determine whether variations in the linkage of the PO / PS nucleotide-to-nucleotide bond affect the ability of CO-3423 and CO-3431 to regulate gene expression, several modified ASOs based on these parental ASOs were prepared. SK-N-AS cells were transfected with increasing concentrations of the PO / PS mixed-bond ASOs described in Tables 5 and 6, and GRN mRNA was quantified by qPCR. The ASOs containing the mixed PO / PS nucleotide-to-nucleotide bond linkages did not affect the efficacy of CO-3423 or CO-3431.

[0241] The GRN mRNA fold change after treatment with the mixed PO / PS bond version of CO-3423 is shown in Table 5. The mRNA was normalized against the average fold change in cells treated with the sterically non-targeting controls (CO-3772, CO-1589 (sNTC1), and CO-1929 (sNTC3)).

[0242] (Table 5) TIFF2025522380000051.tif47165

[0243] As shown in Table 5, the PO / PS mixed version of CO-3423 had the same efficacy as the parental ASO CO-3423.

[0244] Table 6 shows the fold change of GRN mRNA after treatment with the mixed PO / PS conjugate version of CO-3431. The mRNA was normalized as described above.

[0245] (Table 6) TIFF2025522380000052.tif46165

[0246] Similarly, the PO / PS mixed version of CO-3431 had similar efficacy to CO-3431.

[0247] To further evaluate the effect of additional modifications on the ability of the parental ASO of CO-3423 to regulate GRN gene expression, additional LNA tiling and PO conjugation modifications were incorporated into CO-3423. SK-N-AS cells were transfected with increasing concentrations of modified ASO and GRN mRNA quantified by qPCR. CO-3423 was used for comparison. GRN mRNA was normalized to the average fold change in cells treated with the stereoisomeric non-targeting control ASO, CO-1589 (sNTC1). Table 7 shows the fold change of GRN mRNA induced by each ASO.

[0248] (Table 7) TIFF2025522380000053.tif80165

[0249] The ASOs with additional modifications had similar efficacy to the ASOs with LNA residues. CO-4452, CO-5268, and CO-5269 had similar or higher efficacy compared to CO-4113.

[0250] To further evaluate the effect of modifications on the ability of the parental ASO of CO-3463 to regulate GRN gene expression, an ASO based on this ASO incorporating additional LNA modifications was prepared and compared to CO-3462. SK-N-AS cells were transfected with increasing concentrations of ASO and GRN mRNA quantified by qPCR. GRN mRNA was normalized to the mean fold change in cells treated with the stereoisomeric non-targeting control ASO, CO-1589 (sNTC1). The fold changes in GRN mRNA induced by each ASO are shown in Table 8.

[0251] (Table 8) TIFF2025522380000054.tif53165

[0252] The ASOs of CO-3462, CO-5288, and CO-5289 upregulated GRN mRNA 2-fold compared to the control ASO-1589.

[0253] The PGRN protein levels after ASO treatment were also evaluated. To assess the effect of ASO treatment on PGRN protein expression, the following experiment was performed. Various ASOs (CO-3423, CO-3431, CO-4113) at 120 nM were transfected into SK-N-AS cells. As a control, cells were treated with the stereoisomeric non-targeting control ASO, CO-1589 (sNTC1). Forty-eight hours after transfection, proteins were extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific), and PGRN protein levels were determined using a GRN ELISA (PGRN ELISA kit, AdipoGen® Life Sciences; catalog number AG-45A-0018). Values were normalized to lysates from cells treated with sNTC1. As shown in Figure 6, CO-3423 and CO-4113 increased PGRN protein 1.5 - to 3-fold.

[0254] The ASOs of CO-3462, CO-3463, CO-41113, CO-4359, and CO-5269 also upregulate GRN mRNA in iMGL cells. To test the efficacy of the ability of GRN pRNA-targeting ASOs to regulate GRN gene expression in iMGL cells, the following experiments were conducted. Briefly, iMGL cells were nucleofected with ASO CO-3462, CO-3463, CO-4113, CO-4359, CO-5269, in addition to two steric non-targeting control ASOs (CO-3772 and CO-1589 (sNTC1)), and a negative control ASO, CO-5075 (described in Laudisi et al. (2019) Mol. Oncol. 13(10):2142-59). iMGL cells were collected 72 hours after nucleofection for quantification of mRNA using qPCR. Cell supernatants were also collected to quantify secreted PGRN protein using the GRN ELISA described above. mRNA was normalized to cells treated with CO-1589. As shown in Figure 7A, all tested ASOs (i.e., CO-3462, CO-3463, CO-4113, CO-4359, and CO-5269) upregulated GRN mRNA in iMGL cells compared to control CO-1589. The ASOs of CO-4113, CO-4359, and CO-5269 also increased secreted PGRN protein in iMGL cells (Figure 7B).

[0255] The ASOs of CO-3431, CO-3463, and CO-4113, CO-4359, and CO-5269 also upregulate GRN mRNA in iPSC wild-type or GRN M1L neurons. To test the efficacy of the ability of GRN paRNA-targeting ASOs to regulate GRN gene expression in iPSC-derived neurons, including wild-type iPSC-derived neurons and GRN M1L missense mutant (GRN M1L ) neurons, the following experiments were conducted. Briefly, iPSC-derived neuron cells and GRN M1LNeurons were nucleofected with the indicated ASO as a negative control or a scrambled non-targeting control (sNTC1; CO-1589). Cells were collected 120 hours after nucleofection for quantification of mRNA using qPCR, and the mRNA was normalized to cells treated with sNTC1. As shown in Figures 8A and 8B, the ASOs tested upregulated GRN mRNA in wild-type iPSC neurons and GRN M1L neurons.

[0256] CO-4113 rescued staurosporine-induced toxicity in neurons derived from GRN-FTD patients. Neurons derived from GRN-FTD patients were treated with 10 μM of CO-4113 or sNTC (CO-1589) ASO. As a positive control, cells were treated with either recombinant human PGRN protein (2.5 nM) or brain-derived neurotrophic factor (BDNF) protein (1 nM). One week after treatment, neurons were treated with DMSO, 10 nM, or 100 nM of staurosporine. The culture medium was collected 24 hours later, and cytotoxicity was measured using the LDH-Glo™ Cytotoxicity Assay (Promega Corp.) according to the manufacturer's instructions. Cells treated with Triton™ X-100 were used as a positive control. All values were normalized to positive control cells to calculate the percentage of cytotoxicity. The timeline of the assay is shown in Figure 9A. As shown in Figure 9B, treatment with the ASO CO-4113 reduced the cytotoxicity of staurosporine-induced GRN-FTD patient-derived neuron cells at 10 nM or 100 nM.

[0257] Sixty-eight additional ASOs were designed to target intergenic enhancers (eRNAs). SK-N-AS cells were transfected with 120 nM of the ASOs shown in Table 9. Forty-eight hours after transfection, the cells were harvested for GRN mRNA analysis using qPCR. The scrambled non-targeting control ASO, CO-1589, was used as a control. GRN mRNA was normalized to cells treated with CO-1589. The fold change in GRN mRNA induced by each ASO is shown in Table 9 below.

[0258] (Table 9) TIFF2025522380000055.tif234165

[0259] The best hits from these ASOs were tested in a dose escalation study. Briefly, SK-N-AS cells were transfected with escalating concentrations of the ASOs shown in Table 10 at 3.75 nM to 120 nM. The cells were incubated for 48 hours. Thereafter, the cells were harvested for quantification of mRNA via RT-qPCR. The GRN pRNA-targeting ASOs, CO-3431, CO-4113, and CO-4124, were used as positive controls. GRN mRNA was normalized to cells treated with CO-1589. The fold change in GRN mRNA induced by each ASO is shown in Table 10 below.

[0260] (Table 10) TIFF2025522380000056.tif92165

[0261] The ASOs of CO-4611, CO-4613, CO-4619, CO-4622, and CO-4631, CO-4637, CO-4649 were identified as initial hits. The ASOs of CO-4606 and CO-4619 showed upregulation of GRN mRNA exceeding 1.3-fold.

[0262] Twenty-six additional ASOs were designed to target enhancer RNAs (eRNAs) within the gene. SK-N-AS cells were treated with 120 nM of the ASOs shown in Table 11 for 48 hours. Cells were harvested for GRN mRNA analysis using qPCR. CO-1589 was used as a control. GRN mRNA was normalized to cells treated with CO-1589. The fold change in GRN mRNA induced by each ASO is shown in Table 11 below.

[0263] (Table 11) TIFF2025522380000057.tif98165

[0264] ASOs CO-6411, CO-6445, and CO-6452 showed upregulation of GRN mRNA by more than 1.3-fold.

[0265] Additional ASOs were designed based on the parental ASO CO-3462. SK-N-AS cells were transfected with increasing concentrations of ASOs based on CO-3462 designed to include chemical modifications and additional antisense nucleotide sequences. GRN mRNA was quantified using qPCR. CO-3462 and CO-4113 were used as controls. GRN mRNA was normalized to cells treated with CO-1589. The fold change in GRN mRNA induced by each ASO is shown in Table 12.

[0266] (Table 12) TIFF2025522380000058.tif162165

[0267] Additional ASOs were designed based on the parental ASOs CO-4363 and CO-4364. SK-N-AS cells were transfected with increasing concentrations of ASOs based on CO-4363 and CO-4364 designed to include additional chemical modifications and GRN mRNA was quantified using qPCR. GRN mRNA was normalized to cells treated with CO-1589. The fold change in GRN mRNA induced by each ASO is shown in Table 13.

[0268] (Table 13) TIFF2025522380000059.tif93165

[0269] ASO CO-6416, CO-6417, CO-6420, CO-6423, and CO-6224 showed upregulation of GRN mRNA more than two-fold.

[0270] The selected ASOs were evaluated for their immunosuppressive activity induced by IFNγ. To evaluate the ability of GRN regRNA-targeted ASOs to reduce immunosuppression induced by IFNγ, the following experiments were conducted. Briefly, iMGL cells were nucleofected with CO-4113, CO-4359, or CO-5269. A scrambled non-targeting control ASO was used as a control. After ASO nucleofection, IFNγ was added to the culture medium of the ASO-treated iMGL cells to induce an immune response. As an additional control, cells treated with the scrambled non-targeting control ASO were treated with phosphate-buffered saline (PBS) alone. Cells and supernatants were collected for qPCR and ELISA quantification of secreted proteins (PGRN, IL-8, and CCL4) and gene expression (IL-6 mRNA, CCL4 mRNA, and CCL2 mRNA).

[0271] As shown in Figure 19, treatment of iMGL cells with GRN regRNA-targeted ASOs upregulated the secreted PGRN protein level, as indicated by a decrease in the secretion of IL-8 and CCL4 proteins, and significantly reduced the IFNγ-mediated expression of the immune response genes IL-8 and CCL4. Treatment of iMGL cells with GRN regRNA-targeted ASOs also significantly reduced the IFNγ-mediated expression of the immune response genes IL-6, CCL4, and CCL2, as indicated by a reduction in their respective mRNA levels (Figure 19).

[0272] Example 2: Synthesis and In Vitro Characterization of Mouse GRN regRNA-Targeted ASOs The expression of mGRN in various mouse tissues was identified. To determine whether GRN pRNA and mRNA were expressed in mouse CNS tissues, CNS tissues from C57 / BL6 mice were lysed, RNA was extracted using TRIzol™ reagent (Thermo Fisher Scientific), and analyzed using qPCR. cDNA was synthesized using the presence or absence of reverse transcriptase (RT) to ensure that the amplification products were derived from the RNA in the samples. As shown in Figure 10, GRN mRNA and paRNA were detected and were expressed in the mouse cortex, hippocampus, striatum, cerebellum, and spinal cord.

[0273] To evaluate the levels of Pgrn protein in mouse samples, the following experiments were conducted. Briefly, mouse plasma, CSF, and brain tissues from C57 / BL6 mice were obtained. Brain tissues (cortex or mixed brain regions) were homogenized in RIPA buffer containing protease inhibitors. Mouse Pgrn protein levels were quantified by ELISA (Mouse Pgrn ELISA Kit, AdipoGen® Life Sciences; catalog number AG-45A-0019) as instructed by the manufacturer. Serum and brain samples were evaluated at various dilutions and the results for all samples were normalized against total protein. As shown in Figures 11A, 11B, and 11C, mouse Pgrn was detected in this assay in serum, CSF, and cortical and mixed brain region lysates.

[0274] Mouse Grn paRNA is also expressed in mouse neuroblastoma Neuro2a cells. To evaluate whether mouse Grn paRNA is expressed in the immortalized neuroblastoma cell line Neuro2a, the cells were exposed to vorinostat (VOR) at either 0.3 μM, 1 μM, or 3 μM or DMSO control for 24 hours, total RNA was extracted from Neuro2a cells using the Qiagen RNeasy Kit, and cDNA was synthesized using random hexamers. Mouse Grn pRNA was detected by real-time quantitative PCR (qPCR) assay using two different primer sets (1F / 1R or 3F / 3R). As shown in Figure 12, mouse Grn paRNA levels increase upon vorinostat treatment.

[0275] Ninety-one steric ASOs targeting mGrn regRNA were designed and synthesized. Thirty-three ASOs were selected for tiling after the initial pass screening. Eighty-eight ASOs were designed and synthesized for fine-tuning. Of these, 5 ASOs were gapmers, 11 were steric, 28 had PO / PS linkages, and 44 were mixmers.

[0276] Fifty-seven ASOs targeting the Grn promoter (-strand) were screened in Neuro2a cells. Neuro2a cells were transfected with each of the ASOs listed in Table 14 at 100 nM, and the cells were harvested after 48 hours for quantification of Grn mRNA using qPCR. The housekeeping genes for normalization were Gapdh and Ppia. The relative Grn mRNA levels were normalized to the mRNA extracted from cells treated with sNTC3 (steric non-targeting control). As shown in Figure 13, the Grn regRNA-targeting ASOs CO-3544 and CO-3595 upregulated mGrn mRNA in a dose-dependent manner, upregulating mGrn mRNA by 1.5-fold and 1.8-fold, respectively.

[0277] The fold change (FC) of mGrn mRNA induced by 100 nM of each ASO is shown in Table 14.

[0278] (Table 14) TIFF2025522380000060.tif204165

[0279] Next, 26 ASOs based on the parental ASOs CO-3544 and CO-3595 were designed using different modifications and base walking. Neuro2a cells were transfected with either 80, 120, or 160 nM of each of the ASOs listed in Table 15, and the cells were harvested 48 hours later for quantification of mGrn mRNA using qPCR. As a control, the cells were treated with sNTC3 or without ASO. The housekeeping genes for normalization were Gapdh and Ppia. The relative Grn mRNA levels were normalized to the mRNA extracted from cells treated with sNTC3. Four new ASOs (CO-4082, CO-4083, CO-4084, CO-4085) upregulated mGRN mRNA 1.5 - 2.5-fold in Neuro2a cells (Figure 14). Similar upregulation was observed with these ASOs when used to treat primary mouse neuron cells (1.5 - 6 μM in free uptake, Figure 15).

[0280] CO-3544 was modified to contain PO / PS linkages to determine its effectiveness in upregulating mGrn mRNA. As shown in Table 15 below, CO-3544 with up to nine PO - PS nucleotide - to - nucleotide bond substitutions was effective in upregulating mGrn mRNA after transfection in Neuro2a cells at 40, 80, and 160 nM after transfection.

[0281] (Table 15) TIFF2025522380000061.tif115165

[0282] Similarly, CO-3595 was modified to determine the effectiveness of these ASOs in upregulating mGrn mRNA in Neuro2A cells, including various PO / PS nucleotide - nucleotide linkages. As shown in Table 16 below, ASOs based on CO-3595 containing up to 13 PO / PS nucleotide - nucleotide linkage substitutions were effective in upregulating mGrn mRNA at 40, 80, and 160 nM after transfection.

[0283] (Table 16) TIFF2025522380000062.tif114165

[0284] Example 3: In Vivo Regulation of Mouse GRN Expression Using regRNA - Targeted ASO The ASO - mediated upregulation of progranulin in various brain regions in vivo was determined. ASO CO - 3544 (300 μg) in PBS was injected into the right lateral ventricle of 8 - week - old C57 / BL6 mice. A stereotactic non - targeted control ASO (CO - 1929), and a vehicle (PBS) control were used as controls. Mice were sacrificed on day 28 after injection. Tissue samples (cortex, hippocampus, and striatum) were collected and processed for total RNA and protein extraction as described above. Quantification of mouse Grn mRNA was performed using a real - time qPCR assay.

[0285] As shown in Figure 16, treatment with CO - 3544 increased Grn mRNA levels by more than 1.5 - fold in different brain regions. Thus, this ASO showed a therapeutic effect in vivo as indicated by its ability to upregulate Grn mRNA levels in mouse CNS tissues.

[0286] Example 4: hGRNT Tg In Vivo Regulation of PGRN in Mice To evaluate the ability of hGRN paRNA - targeted ASO to upregulate hGRN in vivo, an experiment using a human GRN transgenic mouse model was performed as follows.

[0287] Materials CO-8178 was obtained by modifying CO-4359 to remove the 3' and 5' terminal nucleotides. CO-8178 was characterized in the previously described SK-N-AS, HEK293T, and Vero-76 cell lines, as well as in NGN2 neurons and iMGL cells (data not shown).

[0288] B6.Cg-Grn tm1.2Blrl Hprt1 tm1(GRN)Blrl / J mice (hGRN Tg ; Jackson Laboratory, strain number 036240) were used to evaluate the efficacy of ASO CO-8178 in the upregulation of human GRN in vivo. These mice express the X-linked transgenic human GRN gene (Hprttm1(GRN)Blrl), and exons 3 and 4 of the mouse Grn gene are knocked out (Grntm1.2Blrl).

[0289] Mice were injected intracerebroventricularly (ICV) with 5 μL of aCSF (vehicle control) or 100 μg of CO-8178 at a rate of 1 μl / min. Animals were sacrificed 3 weeks after dosing for analysis. Mouse brain sections were processed to analyze the levels of PGRN protein and mRNA. GRN mRNA and intracellular and secreted PGRN were quantified using the methods described above.

[0290] hGRN Tg The in vivo assay using mice was repeated with the ASOs CO-4452, CO-8883, CO-8903, CO-8879, CO-8871, CO-8873, CO-8873, CO-3462, and CO-6424 as described above. aCSF and scrambled non-targeting (NTC) were used as controls. Mouse brain sections were processed to measure PGRN protein and mRNA. GRN mRNA and intracellular and secreted PGRN protein were quantified using the methods described above.

[0291] Results CO-8178 upregulated GRN mRNA in SK-N-AS, HEK293T, and Vero-76 cell lines, as well as in NGN2 neurons and iMGL cells (data not shown). CO-8178 also suppressed the immune response induced by IFNγ in iMGL cells (data not shown). Without wishing to be bound by theory, CO-8178, being 18mer, may distribute better throughout the tissue and has a high probability of escaping from endosomes.

[0292] As shown in FIGS. 20A and 20B, in vivo treatment with CO-8178 upregulated GRN mRNA and protein across all CNS tissues evaluated. FIG. 20A shows the quantification of hGRN mRNA, and FIG. 20B shows the quantification of hGRN T Tg protein in mouse. Samples from aCSF-treated control mice are shown in the bars on the left side of each tissue, and samples from CO-8178-treated mice are shown in the bars on the right side of each tissue. GRN mRNA and protein expression levels were correlated after treatment with CO-8178. r = 0.8158, 95% CI was 0.7079 - 0.8899, r 2 = 0.6699. P value < 0.0001. Additionally, the PGRN protein secreted in CSF was also upregulated after CO-8178 treatment (FIG. 20C). Therefore, CO-8178 upregulated GRN mRNA and PGRN protein in human GRN mice across all CNS tissues measured. CO-8178 upregulated the PGRN protein secreted in CSF. Furthermore, there was a significant correlation between the upregulation of GRN mRNA and PGRN protein.

[0293] As shown in FIGS. 21A and 21B, in vivo treatment with CO-4452, CO-8883, CO-8903, CO-8879, CO-8873 upregulated human GRN mRNA and PGRN protein in all tissues examined. FIG. 21A shows the quantification of hGRN mRNA, and FIG. 21B shows the quantification of hGRN T Tg Quantification of human PGRN protein in mouse brain tissue is shown.

[0294] Example 5: Grn + / - In vivo regulation of Grn mRNA in haploinsufficient mice To evaluate a mouse model analog of human GRN haploinsufficiency, the following experiments were conducted using Grn heterozygous knockout mice.

[0295] Materials and methods Grn tm1.1Far (B6.129S4(FVB)-Grn tm1.1Far / Mmjax; The Jackson Laboratory, catalog number MMRRC strain number 036771-JAX) heterozygous mice were used to evaluate the efficacy of CO-3544 and CO-10691, which upregulate mGrn in vivo. These mice lack exons 2-13 of the mouse Grn gene. CO-3544 and CO-10691 target mouse Grn paRNA.

[0296] Mice were injected intracerebroventricularly (ICV) with 5 μL of artificial cerebrospinal fluid (aCSF, vehicle control), 300 μg of ASO CO-3544, or 200 μg of ASO CO-10691 in aCSF at a rate of 1 μL / min. For ASO CO-3544, mice were sacrificed 4 weeks after dosing for analysis. For CO-10691, mice were sacrificed 2 and 4 weeks after dosing for analysis. Sections of mouse brains were processed and Pgrn protein and Grn mRNA were quantified as described above.

[0297] Results As shown in FIGS. 22A and 22B, in vivo treatment of heterozygous Grn tm1.1Far mice with the ASO CO-3544 resulted in a 1.5- to 2.0-fold increase in Pgrn protein expression in the hippocampus, striatum, and cerebellar regions compared to the aCSF vehicle control (FIG. 22B). Treatment with ASO CO-3544 resulted in a maximum 1.5-fold increase in Grn mRNA expression compared to the aCSF vehicle control (FIG. 22A).

[0298] ASO CO-10691 also upregulated the expression of mGrn mRNA (Figures 23A, 23B, 23C, and 23D) and mouse Pgrn protein (Figure 24) in the cortical, hippocampal, striatal, and cerebellar brain regions compared to the aCSF vehicle control.

[0299] Example 6: Synthesis and in vitro characterization of additional human GRN regRNA-targeting ASOs Additional hGRN regRNA-targeting ASOs were designed and synthesized using additional chemical modifications and characterized in vitro.

[0300] Materials and methods Secreted and intracellular GRN levels SK-N-AS cells were transfected with 90 nM of ASO CO-4359, CO-4452, CO-5268, CO-5269, CO-6424, and CO-8178. A stereotactic non-targeting control ASO (sNTC) was used as a control. As described in Example 1 above, cells were collected 48 hours after transfection and analyzed by ELISA. Protein levels were normalized to total protein and cells treated with sNTC.

[0301] mRNA expression assay SK-N-AS cells were transfected with selected ASOs at 3.75 - 90 nM. A scrambled ASO (sNTC) was used as a control. 48 hours after transfection, cells were collected for mRNA. Housekeeping genes were GAPDH and PPIA, and the fold change in mRNA was normalized to cells treated with sNTC.

[0302] iPSC-derived neurons were nucleofected with 20 μM of ASOs CO-8865, CO-8866, CO-8871, CO-8873, CO-8875, CO-8877, CO-8879, CO-8883, CO-8889, CO-8901, and CO-8903. Two non-targeting control ASOs (NTC-ASO-1 and NTC-ASO-2) were used as controls. Five days after nucleofection, cells were harvested for quantification of GRN mRNA. The qPCR reference genes were GAPDH and PPIA. The relative GRN mRNA levels were normalized to cells treated with either of the two non-targeting control ASOs.

[0303] Chemokine assay iMGL cells were nucleofected with 5 μM of CO-4452, CO-8865, CO-8866, and CO-8883. A non-targeting control ASO (NTC) was used as a control. After treatment, IFNγ was added to the ASO-treated iMGL cells to induce an immune response. Cells and supernatants were collected to quantify GRN, CCL3, and CCL4 gene expression by qPCR and ELISA.

[0304] Results As shown in Figures 25A and 25B, ASOs CO-4359, CO-4452, CO-5268, CO-5269, CO-6424, and CO-8178 upregulated both the secretion (Figure 25A) and intracellular (Figure 25B) levels of PGRN protein in SK-N-AS cells. A dose-dependent increase in GRN mRNA expression in SK-N-AS cells was also observed with CO-4113, CO-8877, CO-8879, CO-8883, CO-8889, CO-8901, and CO-8903 (Figures 26A and 26B).

[0305] Upregulation of GRN mRNA in iPSC-derived neuron cells was also observed with CO-8865, CO-8866, CO-8871, CO-8873, CO-8875, CO-8879, CO-8883, CO-8889, CO-8901, and CO-8903 (Figure 27).

[0306] As shown in FIGS. 28A, 28B, and 28C, the ASOs CO-4452, CO-8865, CO-8866, CO-8873, and CO-8883 also upregulate GRN mRNA expression (FIG. 28A) and decrease IFNγ-induced chemokine (CCL3 and CCL4) expression in iMGL cells (FIGS. 28B and 28C, respectively).

[0307] Incorporation by reference Unless otherwise indicated, all disclosures of each patent document and scientific paper referenced herein are incorporated by reference for all purposes.

[0308] Equivalents The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. An antisense oligonucleotide (ASO) that targets the regulatory RNA (regRNA) of progranulin (pGRN), comprising a nucleotide sequence complementary to at least five consecutive nucleotides of any one of sequence numbers 1 to 9.

2. The ASO according to claim 1, comprising any one nucleotide sequence from sequence numbers 1543, 1684-1688, 1902, 2292-2296, 2767, 3464, 3564, 1369-1542, 1544-1683, 1689-1901, 1903-2291, 2297-2766, 2768-3463, 3465-3563, or 3565-4738.

3. The ASO according to claim 1, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 1, and the ASO includes any one nucleotide sequence from SEQ ID NOs: 20, 59, 1085, 10-19, 21-58, 60-268, 691, 991-1084, 1086-1368, or 4743-4915.

4. (a) The regRNA has the nucleotide sequence of SEQ ID NO: 2, and the ASO includes any one nucleotide sequence from SEQ ID NOs: 269 to 279; (b) The regRNA has the nucleotide sequence of SEQ ID NO: 3, and the ASO includes one nucleotide sequence from SEQ ID NOs: 280-291 or 336-359; (c) The regRNA has the nucleotide sequence of SEQ ID NO: 4, and the ASO contains one of the nucleotide sequences of SEQ ID NOs: 292-313 or 360-380; (d) The regRNA has the nucleotide sequence of SEQ ID NO: 5, and the ASO contains any one of the nucleotide sequences of SEQ ID NOs: 314-335 or 381-416; or (e) The regRNA has the nucleotide sequence of SEQ ID NO: 6, and the ASO contains any one nucleotide sequence from SEQ ID NOs: 417 to 442; The ASO according to claim 1.

5. The ASO according to claim 1, comprising a nucleotide having one or more chemical modifications.

6. The ASO according to claim 5, wherein at least three, four, or five nucleotides at the 5' end and at least three, four, or five nucleotides at the 3' end of the ASO contain one or more ribonucleotides having one or more chemical modifications.

7. The one or more chemical modifications are 2'-O-C1-4 alkyl, for example, 2'-O-methyl (2'-OMe), 2'-deoxy (2'-H), 2'-O-C1-3 alkyl-O-C1-3 alkyl, for example, 2'-methoxyethyl ("2'-MOE" or "MOE"), 2'-fluoro ("2'-F"), 2'-amino ("2'-NH2"), 2'-arabinosyl ("2'-arabino") nucleotide, 2'-F-arabinosyl The ASO according to claim 5, comprising a nucleotide sugar modification comprising one or more of the following: 2'-F-arabino ("2'-F-arabino") nucleotide, 2'-locked nucleic acid ("LNA") nucleotide, 2'-amide crosslinked nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotide, L-type sugar ("L-sugar"), 4'-thioribosyl nucleotide, restricted ethyl (cET), 2'-fluoro-arabino (FANA), or thiomorpholino.

8. wherein the one or more chemical modifications are phosphorothioate ("PS" or (P(S))), phosphoramidate (P(NR ３ , ２ , １～３ R ２ )), for example, dimethylaminophosphoramidate (P(N(CH ３ ))), phosphonocarboxylate (P(CH ２ ))COOR), for example, phosphonoacetate "PACE" (P(CH ２ ))COO ｎ ), thiophosphonocarboxylate ((S)P(CH ２ ))COOR), for example, thiophosphonoacetate "thioPACE" ((S)P(CH － ))COO ２ ), alkylphosphonate (P(C ｎ alkyl)), for example, methylphosphonate - P(CH3), boranophosphonate (P(BH ２ ))), or phosphorodithioate (P(S)) － ))COO １～３ )) of one or more nucleotide - internucleotide linkage modifications, the ASO according to claim 5.​​​​

9. The one or more chemical modifications mentioned above are 2-thiouracil ("2-thioU"), 2-thiocytosine ("2-thioC"), 4-thiouracil ("4-thioU"), 6-thioguanine ("6-thioG"), 2-aminoadenine ("2-aminoA"), 2-aminopurine, pseudouracil, hypoxanthine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deazaadenine, 7-deaza-8-azaadenine, 5-methylcytosine ("5-methylC"), 5-methyluracil ("5-methylU"), 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 5-propynylcytosine, 5-propynyl The ASO according to claim 5, comprising nucleic acid base modification comprising one or more of the following: racil, 5-ethinylcytosine, 5-ethinyluracil, 5-allyluracil ("5-allyl U"), 5-allylcytosine ("5-allyl C"), 5-aminoallyluracil ("5-aminoallyl U"), 5-aminoallyl-cytosine ("5-aminoallyl C"), debasalized nucleotide, Z base, P base, unstructured nucleic acid ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), glycerol nucleic acid (GNA), glycerol nucleic acid (GNA), or thiophosphorumamide morpholino (TMO).

10. The ASO according to claim 5, wherein one or more of the chemical modifications include biotin, palmitic acid, or a C18 moiety linked to the 5' or 3' end of the ASO.

11. The ASO according to claim 5, wherein one or more of the chemical modifications include 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), phosphodiester (PO) nucleotide internucleotide bond, or phosphorothioate (PS) nucleotide internucleotide bond.

12. The ASO according to claim 5, which does not contain 10 or more consecutive nucleotides of unmodified DNA.

13. The ASO according to claim 12, which does not contain deoxyribonucleotides.

14. The ASO according to claim 5, which does not contain unmodified ribonucleotides.

15. The ASO according to claim 5, comprising at least one phosphodiester bond.

16. The ASO according to claim 5, wherein each cytidine in the ASO is modified with 5-methyl.

17. Two or more consecutive nucleotides of unmodified DNA, each with at least three nucleotides of modified ribonucleotides adjacent to each other at the 5' and 3' ends. The ASO according to claim 5, including the following:

18. A pharmaceutical composition comprising the ASO described in any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. A method for increasing GRN transcription in human cells, comprising contacting the cells with an ASO according to any one of claims 1 to 17.

20. A pharmaceutical composition comprising the ASO according to any one of claims 1 to 17 for treating a disease or disorder in a subject.

21. The aforementioned diseases or disorders include frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathological frontotemporal lobar degeneration associated with TDP-43 inclusion accumulation (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerotic dementia, spinocerebellar ataxia type 3, chronic traumatic encephalopathy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Gaucher disease (G D) and the pharmaceutical composition according to claim 20, selected from the group consisting of Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL) type 11 (CLN11), limbic-dominant age-related TDP-43 encephalopathy (LATE), autism, intracerebral ischemia-reperfusion injury, lysosomal storage disorder (LSD), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), ischemic heart disease, intervertebral disc senelation, and acute kidney injury.

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