Methods of modulating granulin precursor expression using antisense oligonucleotides targeting and modulating RNA

Abstract

Described herein are methods of regulating GRN gene transcription using antisense oligonucleotides (ASOs) that target GRN regulating RNAs, such as promoter-related RNAs and enhancer RNAs. These methods may be used to modulate the levels of GRN gene products, such as to increase the expression of granulin precursors (PGRNs), thereby treating diseases and disorders in subjects.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 351,263, filed on June 10, 2022, U.S. Provisional Application No. 63 / 369,907, filed on July 29, 2022, and U.S. Provisional Application No. 63 / 381,910, filed on November 1, 2022, each of which is hereby incorporated by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file was created on XX / XX / 20XX, is named CTC-027WO_SL.xml, and is X,XXX,XXX bytes in size. Technical Field

[0005] The present invention relates to methods for upregulating or downregulating GRN gene transcription using antisense oligonucleotides (ASOs) targeting GRN regulatory RNAs, such as promoter-associated RNAs and enhancer RNAs. Background Art

[0006] Transcription factors bind to specific sequences in promoter and enhancer DNA elements to regulate gene transcription. Recently, it has been reported that active promoters and enhancer elements themselves are transcribed to produce non-coding regulatory RNA (regRNA), such as promoter-related RNA (paRNA) and enhancer RNA (eRNA) (see Sartorelli and Lauberth, Nat. Struct. Mol. Biol. (2020) 27: 521–28). Unlike coding RNA, regRNA is bidirectionally transcribed. Various models have been proposed for the function of regRNA, including nucleosome remodeling (see Mousavi et al., Mol. Cell (2013) 51 (5): 606-17), regulation of enhancer-promoter loops (see Lai et al., Nature (2013) 494 (7438): 497-501) and direct interaction with transcriptional regulators (see Sigova et al., Science (2015) 350: 978–81).

[0007] Progranulin (PGRN) is encoded by the human GRN gene and is the precursor of granulin peptides. PGRN is a highly conserved secretory protein expressed in a variety of cell types, including the central nervous system (CNS) and peripheral tissues. Increasing evidence suggests that PGRN and its proteolytic granulin peptide products are involved in lysosomal function and have nutritional and neuroprotective effects.

[0008] Defects in PGRN and mutations in GRN may lead to a variety of neurological diseases and conditions, including frontotemporal dementia. Frontotemporal dementia (FTD) is a progressive neurodegenerative disease in which patients rapidly progress to severe dementia and death. Approximately 40% of FTD patients have familial FTD, of which approximately 30% (10% overall) have heterozygous GRN mutations, resulting in haploinsufficiency. In general, PGRN protein levels remain constant throughout the patient's life, with a non-prognostic factor for the time of first onset (usually 55-65 years of age). GRN-frontotemporal dementia (GRN-FTD, also known as FTD-GRN) disease onset in FTD patients is milder; behavioral and language symptoms begin to appear several years before diagnosis. Within 3-4 years of diagnosis, patients have progressed to advanced dementia and require full support from caregivers. Life expectancy after diagnosis of GRN-FTD is typically about 7 years. However, currently only symptomatic treatments for FTD exist, and there are no disease-reversing or disease-modifying agents available.

[0009] Gene expression is generally considered to be a non-druggable biological process. Despite continuous efforts to understand the biology of gene transcription and regRNA, clinically suitable methods for regulating gene expression are still limited. There is still a need for new and useful methods to treat diseases associated with abnormal (e.g., reduced) PGRN (such as FTD) expression. Summary of the invention

[0010] In one aspect, provided herein is an antisense oligonucleotide (ASO) that is complementary to at least 5 consecutive nucleotides of a regulatory RNA of progranulin (pGRN), wherein the regulatory RNA has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-6.

[0011] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 3' end of the regRNA.

[0012] In some embodiments, the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides from the 5' end of the regRNA.

[0013] In some embodiments, the ASO comprises a nucleotide sequence selected from Table 17, 18, or 19.

[0014] In some embodiments, the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 1369-4738.

[0015] 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 NO: 10-268, 691, 991-1368, or 4743-4915.

[0016] 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 NO: 269-279.

[0017] 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 NO: 280-291 or 336-359.

[0018] 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 NO: 292-313 or 360-380.

[0019] 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 NO: 314-335 or 381-416.

[0020] 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 NO:417-442.

[0021] In some embodiments, the ASO is no more than 50, 40, 30, 25, or 20 nucleotides in length.

[0022] In some embodiments, the ASO comprises an RNA polynucleotide comprising one or more chemical modifications.

[0023] In some embodiments, each nucleotide in the ASO comprises a ribonucleotide having one or more chemical modifications.

[0024] In some embodiments, at least 3, 4, or 5 nucleotides at the 5' end and at least 3, 4, or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

[0025] In some embodiments, the one or more chemical modifications comprise 2'-O-methoxyethyl, 5-methyl on cytidine, locked nucleic acid (LNA), and phosphorothioate internucleotide linkages.

[0026] In some embodiments, the ASO does not comprise 10 or more consecutive nucleotides of unmodified DNA.

[0027] In some embodiments, the ASO does not comprise deoxyribonucleotides.

[0028] In some embodiments, the ASO does not comprise unmodified ribonucleotides.

[0029] In some embodiments, each ribonucleotide of the ASO is modified with a 2'-O-methoxyethyl group.

[0030] In some embodiments, the length of the ASO is 3×n+10 nucleotides (n is an integer of 4 or greater), wherein the nucleotides at positions 3×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0031] In some embodiments, the length of the ASO is 2×n+4 nucleotides (n is an integer of 8 or greater), wherein the nucleotides at positions 2×m are LNA-modified ribonucleotides (m is an integer from 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0032] In some embodiments, the length of the ASO is 3×n+2 nucleotides (n is an integer of 6 or greater), wherein the nucleotides at positions 3×m are LNA-modified ribonucleotides (m is an integer from 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0033] In some embodiments, the length of the ASO is 4×n+4 nucleotides (n is an integer of 4 or greater), wherein the nucleotides at positions 4×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0034] In some embodiments, the length of the ASO is 5×n+5 nucleotides (n is an integer of 3 or greater), wherein the nucleotides at positions 5×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

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

[0036] In some embodiments, the length of the ASO is 2×n+8 nucleotides (n is an integer of 8 or greater), wherein the nucleotide at position 2×m+1 is a ribonucleotide modified with LNA (m is an integer from 1 to n), and the nucleotide at position 2×m is a ribonucleotide modified with 2'-O-methoxyethyl.

[0037] In some embodiments, the ASO comprises at least one phosphodiester bond.

[0038] In some embodiments, the ASO comprises 10 or more consecutive nucleotides of unmodified DNA flanked on each of the 5' and 3' ends by at least 3 nucleotides of modified ribonucleotides.

[0039] In some embodiments, each cytidine in the ASO is modified with a 5-methyl group.

[0040] In some embodiments, the ASO comprises 2 or more consecutive nucleotides of unmodified DNA flanked at each of the 5' and 3' ends by at least 3 nucleotides of modified ribonucleotides.

[0041] In some embodiments, the regRNA is an eRNA.

[0042] In some embodiments, the regRNA is a paRNA.

[0043] In one aspect, provided herein is a pharmaceutical composition comprising an ASO disclosed herein and a pharmaceutically acceptable carrier.

[0044] In one aspect, provided herein is a method of increasing transcription of pGRN in a human cell, the method comprising contacting the cell with an ASO disclosed herein or a pharmaceutical composition disclosed herein.

[0045] In some embodiments, the cell is a neuron.

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

[0047] In some embodiments, the ASO increases the stability of a regulatory RNA in a cell.

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

[0049] In some embodiments, the ASO increases the amount of pGRN protein in a cell.

[0050] In one aspect, provided herein is 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.

[0051] In some embodiments, the ASO increases the amount of a regulatory RNA in cells of a subject.

[0052] In some embodiments, the ASO increases the stability of a regulatory RNA in cells of a subject.

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

[0054] In some embodiments, the ASO increases the amount of pGRN protein in a cell.

[0055] In some embodiments, the cell is a neuron. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1A Illustrative schematic diagram showing eRNA, paRNA, mRNA and natural antisense transcripts (NAT) of genes on chromosomes. eRNA, paRNA and NAT are all non-coding RNAs. eRNA is bidirectionally transcribed from the enhancer of a gene. paRNA is transcribed from the promoter of a gene, identical to mRNA, but transcribed in the antisense direction. NAT is transcribed in the antisense direction from its own downstream promoter, so that the transcript overlaps at least partially with the mRNA. Typically, eRNA and paRNA upregulate gene expression, while NAT downregulates gene expression. Figure 1B Schematic diagram showing the interaction of regRNA with enhancer and promoter regions to recruit transcription factors and regulators that control gene expression.

[0057] Figure 2A and 2B Human GRN (hGRN) mRNA was detected in human induced pluripotent stem cell (iPSC)-derived neurons and induced microglia-like (iMGL) cells following exposure to 1 μM or 3 μM vorinostat or to DMSO alone (vehicle control), as determined using qPCR. Figure 2A ) and hGRN paRNA ( Figure 2B ).

[0058] Figure 3 It is shown that hGRN paRNA was detected in human cortical tissue from three donors (#1, #2, and #3) as determined using qPCR.

[0059] Figure 4 Shown are relative hGRN mRNA levels in human SK-N-AS cells as determined using qPCR following treatment with the indicated ASOs at the indicated concentrations.

[0060] Figure 5AShown are relative hGRN mRNA levels in human SK-N-AS cells following treatment with the indicated ASOs at the indicated concentrations, as determined using qPCR. Figure 5B Shown are relative hGRN mRNA levels in human SK-N-AS cells following treatment with the indicated ASOs at the indicated concentrations, as determined using qPCR.

[0061] Figure 6 Shown are relative human PGRN (hGRN) protein levels in SK-N-AS cells after treatment with the indicated ASOs as determined by ELISA.

[0062] Fig. 7A Shown are relative hGRN mRNA levels in iMGL cells following treatment with the indicated doses of the indicated ASOs, as determined using qPCR. Figure 7B Shown are the fold changes (FC) in secreted PGRN protein levels in iMGL cells following treatment with the indicated ASOs as determined using ELISA.

[0063] Fig. 8A and Figure 8B Shown are wild-type human iPSC-derived neurons ( Fig. 8A ) and FTD-GRN patient-derived GRN M1L Relative hGRN mRNA levels in neurons.

[0064] Fig. 9A Assay timeline for the staurosporine rescue assay is shown. Fig. 9B Shown are the percent levels of cytotoxicity in GRN-FTD patient-derived neurons following treatment with the indicated concentrations of the indicated ASOs, PGRN protein, or BDNF protein, and DMSO or staurosporine.

[0065] Fig.10 Shown is murine mGrn mRNA and paRNA expression in the indicated mouse brain tissue from C57 / BL6 as determined using qPCR. (RT = reverse transcriptase).

[0066] Fig.11A , 11B and 11C show that serum samples ( Fig.11A ), cerebrospinal fluid (CSF) samples ( Fig. 11B ) and brain tissue lysates derived from cortical or mixed brain regions ( Fig. 11C ) in the presence of PGRN protein.

[0067] Fig.12Shown are mouse GRN paRNA expression levels in Neuro2a cells following exposure to 0.3 μM, 1 μM, or 3 μM vorinostat or DMSO control as determined using qPCR with two different primer sets (1F / 1R and 3F / 3R).

[0068] Fig.13 Shown are relative mGrn mRNA levels in Neuro2a cells following treatment with the indicated concentrations of the indicated ASOs or a no-transfection control as determined using qPCR.

[0069] Fig.14 Shown are relative mGrn mRNA levels in Neuro2a cells after treatment with the indicated ASOs at the indicated concentrations or in untreated cells ("No ASO") as determined using qPCR.

[0070] Fig.15 Shown are relative mGrn mRNA levels in Neuro2a cells following treatment with the indicated ASOs at the indicated concentrations in mouse primary neurons as determined using qPCR.

[0071] Fig.16 Shown are relative mGrn mRNA levels in the cortex, hippocampus, and striatum mouse brain regions from C57 / BL6 mice following in vivo treatment with the indicated ASOs or PBS control.

[0072] Fig.17 Sequences and chemical modifications of human GRN ASOs are provided. Medium grey shading indicates MOE; * indicates 5Me-C; dark grey indicates LNA; light grey indicates 2′OMe (2'-O-methyl); black lines indicate phosphodiester (PO) bonds; white indicates DNA.

[0073] Fig.18 The sequences and chemical modifications of mouse GRN ASOs are provided. Medium grey shading indicates 2'-MOE; light grey indicates O-methyl; * indicates 5Me-C; dark grey indicates LNA; black lines indicate phosphodiester (PO); and white indicates DNA.

[0074] Fig.19 It was shown that in iMGL cells treated with PBS or IFNγ and the indicated ASOs, PGRN protein secretion was increased, as well as protein secretion of IL-8 and CCL4 was decreased, and gene expression of IL-6, CCL4, and CCL2 was reduced.

[0075] Fig. 20A hGRNT expression after ICV injection of CO-8178 or aCSF (control) as determined using qPCR Tg hGRN mRNA levels in mice. Fig. 20BhGRNT expression after ICV injection of CO-8178 or aCSF (control) as determined by ELISA Tg hPGRN protein quantification in mice. Fig. 20C Provided are the fold changes (FC) of hPGRN protein in the CSF of mice following ICV injection of CO-8178 or aCSF (control). Fold changes were calculated by normalizing the levels of hPGRN protein CO-8178 treated samples to aCSF treated samples.

[0076] Fig.21A Provided are the expression of hGRNT after ICV injection of the indicated ASOs or aCSF (control), as determined using qPCR. Tg hGRN mRNA levels in mouse brain tissue. Fig.21B Provided are the expression of hGRNT as determined by ELISA following ICV injection of the indicated ASOs or aCSF (control). Tg hGRN protein levels in mouse brain tissue.

[0077] Fig.22A Shown are heterozygous Grn expression after ICV injection of the indicated ASOs or aCSF (control), as determined using qPCR. tm1.1Far mGrn mRNA levels in mouse brain tissue. Fig. 22B Shown are heterozygous Grn after ICV injection of the indicated ASOs or aCSF (control), as determined using ELISA. tm1.1Far Quantification of mPgrn protein in mouse brain tissue.

[0078] Fig.23A , Fig. 23B , Fig.23C and Fig.23D Shown are Grn after ICV injection of the indicated ASOs or aCSF (control), as determined using qPCR. tm1.1Far mGrn mRNA levels in mouse brain tissue: cortex ( Fig.23A )、Seahorse( Fig. 23B ), cerebellum( Fig.23C ) and striatum ( Fig.23D ).

[0079] Fig.24 Shown are Grn after ICV injection of the indicated ASOs or aCSF (control), as determined using ELISA. tm1.1Far mPgrn protein levels in mouse brain tissues (cortex and cerebellum).

[0080] Fig.25A and Fig.25BShown are secreted hPGRN protein in SK-NA-S cells following treatment with the indicated ASOs as determined using ELISA ( Fig.25A ) and intracellular hPGRN protein ( Fig.25B )’s increase.

[0081] Fig.26A and Fig.26B Shown is a dose-dependent upregulation of hGRN mRNA in SK-NA-S cells following treatment with the indicated ASOs at the indicated concentrations, as determined using qPCR.

[0082] Fig. 27 Shown is a dose-dependent upregulation of hGRN mRNA in human iPSC-derived neuronal cells following treatment with the indicated ASOs as determined using qPCR.

[0083] Fig.28A Shown is the upregulation of GRN mRNA expression in iMGL cells treated with PBS or IFNγ and the indicated ASOs as determined using qPCR. Fig.28B and Fig.28C Shown is a reduction in IFNγ-induced chemokine (CCL3 and CCL4) expression in iMGL cells treated with PBS or IFNγ and the indicated ASOs as determined using ELISA. DETAILED DESCRIPTION

[0084] The present disclosure provides antisense oligonucleotides (ASOs) targeting regulatory RNAs (such as promoter-associated RNAs and enhancer RNAs), and methods of using these ASOs to regulate gene expression. These methods can be used to regulate the level of gene products, such as regulating the expression level of progranulin or progranulin (PGRN, encoded by the GRN gene), thereby treating diseases associated with abnormal GRN gene expression in subjects, such as but not limited to frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathic frontotemporal lobar degeneration associated with accumulation of TDP-43 inclusions (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerosis dementia, spinocerebellar ataxia 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 system-dominant age-related TDP-43 encephalopathy (LATE) Gaucher disease, 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.

[0085] In some embodiments, the ASOs of the present disclosure can be used to restore progranulin or granulin expression 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 can be used to restore or increase the level of secretory progranulin or granulin in a subject (e.g., a subject with progranulin haploinsufficiency).

[0086] Various aspects of the multispecific binding proteins described in this application are set forth in the following sections.

[0087] I. Definitions

[0088] To facilitate understanding of this application, a number of terms and phrases are defined below.

[0089] As used herein, the terms "a" or "an" mean "one or more" and include the plural unless the context is inappropriate.

[0090] As used herein, the term "progranulin" or "progranulin" or "PGRN" refers to the protein of UniProt Accession No. P28799 (human) when used in reference to the human form of the protein, and refers to the protein of UniProt Accession No. P28798 (mouse) when used in reference to the mouse form of the protein, as well as related isoforms and orthologs.

[0091] As used herein, the terms "regulatory RNA" and "regRNA" are used interchangeably to refer to non-coding RNA transcribed from a regulatory element of a gene (e.g., a protein-coding gene), wherein the gene itself is not a non-coding RNA. Exemplary regulatory elements include, but are not limited to, promoters, enhancers, and super enhancers. Non-coding RNA transcribed from a promoter in the antisense direction is also referred to as "promoter RNA" or "paRNA". Non-coding RNA transcribed from an enhancer or super enhancer in the sense or antisense direction is also referred to as "enhancer RNA" or "eRNA". It should be understood that a natural antisense transcript (NAT) complementary to at least a portion of a gene transcript is not a regulatory RNA used herein.

[0092] As used herein, the term "nascent RNA" refers to an RNA that is still being transcribed by an RNA polymerase or has just been transcribed by an RNA polymerase and is still tethered to the DNA from which it was transcribed. RNA that is separated from the DNA from which it was transcribed is also referred to as "untethered RNA".

[0093] 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 present disclosure encompasses pharmaceutically acceptable salts of any ASO 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 ASO provided herein is lyophilized and separated into a salt (e.g., a sodium salt).

[0094] As used herein, in some embodiments, the stability of regRNA is inversely correlated with the degradation rate of regRNA. In some embodiments, when ASO increases the stability of regRNA, it reduces the degradation rate of regRNA. In some embodiments, when ASO reduces the stability of regRNA, it increases the degradation rate of regRNA. In some embodiments, the degradation rate of regRNA can be measured by blocking the synthesis of new regRNA and evaluating the half-life of existing regRNA.

[0095] 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, apes, equines, bovines, porcines, canines, felines, etc.), and more preferably include humans.

[0096] 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. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a specific formulation or route of administration. As used herein, the term "treatment" includes any effect, such as mitigation, reduction, regulation, improvement, or elimination, which results in an improvement in an illness, disease, condition, or the like, or improves its symptoms.

[0097] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0098] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (e.g., such as oil / water emulsions or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Martin, Remington's Pharm aceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA (1975).

[0099] Throughout the specification, when compositions are described as having, including, or comprising specific components, or when processes and methods are described as having, including, or comprising specific steps, it is contemplated that, in addition, the compositions described herein consist essentially of or consist of the listed components, and that the processes and methods according to the present application consist essentially of or consist of the listed processing steps.

[0100] Generally, compositions specifying percentages are by weight unless otherwise indicated. In addition, if a variable is not accompanied by a definition, the preceding definition of the variable will prevail.

[0101] II. Antisense Oligonucleotides

[0102] In some embodiments, the antisense oligonucleotides (ASOs) disclosed herein hybridize to regRNA (e.g., regRNA or paRNA) transcribed from a regulatory element of a GRN gene (also referred to herein as "GRN regRNA"). It should be understood that both eRNA and paRNA are regRNAs that regulate (e.g., promote or upregulate) gene expression (FIG. 1). In some embodiments, the GRN regRNA is a murine GRN regRNA. In some embodiments, the GRN regRNA is a human GRN regRNA. In certain embodiments, the target GRN regRNA is an eRNA. In certain embodiments, the target GRN regRNA is a paRNA. eRNA can be identified using methods known in the art, such as assay for transposase accessible chromatin using sequencing (ATAC-seq), global continuous sequencing, precise continuous sequencing, cap analysis gene expression and histone modification analysis (see, for example, Sartorelli & Lauberth, Nat. Struct. Mol. Biol. (2020) 27: 521-28; PCT application publication No. WO2013 / 177248). paRNA is an RNA transcribed from the promoter of the target gene in the antisense direction (the transcript in the sense direction is the mRNA of the target gene). Considering their specific location and orientation, they can be identified by similar methods. The nucleotide sequences of exemplary regRNAs are provided in Table 1 below. Any of these GRN regRNAs is considered as the target GRN regRNA of the ASO disclosed herein.

[0103] Table 1. Exemplary regRNAs

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] The present disclosure describes an ASO that increases the amount or stability of target GRN regRNA, thereby increasing the expression of GRN genes. These ASOs are different from previously described ASOs designed to inhibit eRNA (see, for example, PCT Application Publication No. WO2013 / 177248 and PCT Application Publication No. WO2017 / 075406). Without wishing to be bound by theory, it is assumed that the ability of ASOs to upregulate GRN regRNA is due to the selection of target sequences in regRNA and / or chemical modification of ASOs.

[0113] ASO sequence

[0114] In certain embodiments, the ASOs disclosed herein are complementary to a sequence in a GRN regRNA that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 5' or 3' end of the GRN regRNA. In certain embodiments, the ASOs disclosed herein are complementary to a sequence in a GRN regRNA that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 5' end of the GRN regRNA (i.e., the 5'-most nucleotide of the regRNA sequence that forms a duplex with the ASO is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 5' end of the GRN regRNA). In certain embodiments, an ASO disclosed herein is complementary to a sequence in a GRN regRNA that is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 3' end of the GRN regRNA (i.e., the 3'-most nucleotide of the GRN regRNA sequence that forms a duplex with the ASO is no more than 300, 250, 200, 150, 100, 50, 40, 30, 20, or 10 nucleotides from the 3' end of the GRN regRNA).

[0115] In certain embodiments, the length of the ASO does not exceed 25, 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides. In certain embodiments, the ASO is designed to lack a stable secondary structure formed within itself or between each other, thereby increasing the amount of the ASO in single-stranded form ready to hybridize with the GRN regRNA. Methods for predicting secondary structure are known in the art (see, e.g., Seetin and Mathews, Methods Mol. Biol. (2012) 905: 99-122; Zhao et al., PLoS Comput. Biol. (2021) 17 (8): e1009291) and web-based programs (e.g., RNAfold) are available to public users.

[0116] For example, ASOs have been designed to target human GRN paRNA or GRN eRNA. The nucleotide sequences of some of these hGRN ASOs are provided in Fig.17 Additional ASOs have been designed to target mouse GRN paRNA or eRNA. The nucleotide sequences of some of these mGRNA SOs are provided in Fig.18 In some embodiments, the ASO provided herein comprises a nucleotide sequence of any one of SEQ ID NOs: 1369-4738. In some embodiments, the ASO provided herein comprises a nucleotide sequence as provided in Table 19 below.

[0117] Table 19. Exemplary ASO sequences.

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

[0125]

[0126]

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Hybridization and ΔG

[0158] As used herein, the term "hybridizing" or "hybridizes" is understood to mean the formation of hydrogen bonds between base pairs on opposite strands of two nucleic acid chains (e.g., an oligonucleotide and a target nucleic acid), thereby forming a duplex. The binding affinity between two nucleic acid chains is the strength of the hybridization. It is usually measured in terms of the melting temperature (T m ), the melting temperature is defined as the temperature 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 affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537). The standard state Gibbs free energy ΔG° is a more accurate representation of binding affinity and is related to the dissociation constant (K d ) is related, ΔG°=-RTIn(K d), where R is the gas constant and T is the absolute temperature. Therefore, 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 the reaction in which the water concentration is 1M, the pH is 7 and the temperature is 37°C. The hybridization of the oligonucleotide with the target nucleic acid is a spontaneous reaction, and for a spontaneous reaction, ΔG° is less than zero. ΔG° can be measured experimentally, for example, by using an isothermal titration calorimetry (ITC) method, as described in Hansen et al., 1965, Chem, Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today. Those skilled in the art will know that commercial equipment can be used for ΔG° measurement. ΔG° can also be numerically estimated by using a nearest neighbor model, as described by SantaLucia, 1998, ProcNatlAcedSciUSA.95:1460-1465, using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405. In order to have the possibility of regulating its expected nucleic acid target by hybridization, the oligonucleotides of the present disclosure hybridize with the target nucleic acid, and for oligonucleotides of 10-30 nucleotides in length, the ΔG° value is estimated to be less than -10 kcal / mol. In some embodiments, the degree or intensity of hybridization is measured by the standard state Gibbs free energy ΔG°. The oligonucleotide can hybridize with the target nucleic acid, and for oligonucleotides of 8-30 nucleotides in length, the estimated ΔG° value is below the range of -10 kcal / mol, such as below -15 kcal / mol, such as below -20 kcal / mol and such as below -25 kcal / mol. In some embodiments, the oligonucleotide hybridizes with the target nucleic acid, and the estimated ΔG° value is -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.

[0159] Duplex region

[0160] The phrase "duplex region" refers to a region where two complementary or substantially complementary polynucleotides form base pairs with each other by Watson-Crick base pairing or any other means that allow the formation of a stable duplex between complementary or substantially complementary polynucleotide chains. For example, a polynucleotide chain with 21 nucleotide units can base pair with another polynucleotide chain with 21 nucleotide units, but only 19 bases on each chain are complementary or sufficiently complementary so that the "duplex" has 19 base pairs. For example, the remaining bases can exist as 5' and 3' overhangs. In addition, within the duplex region, 100% complementarity is not required; substantially complementary within the duplex is allowed. Substantially complementary refers to 70% or more complementarity. For example, a mismatch in a duplex region consisting of 19 base pairs results in 94.7% complementarity, making the duplex region substantially complementary. The duplex region can be formed by two separate oligonucleotide chains, as well as by a single oligonucleotide chain that can form a hairpin structure comprising a duplex region.

[0161] dsRNA includes two RNA chains, which are complementary and hybridized to form a duplex structure under the conditions of using dsRNA. One chain (antisense strand) of dsRNA includes a complementary region that is substantially complementary to the target sequence and is usually completely complementary. The target sequence can be derived from the sequence of GRN regRNA, such as eRNA or paRNA. The other chain (sense strand) includes a region complementary to the antisense strand so that the two chains hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequence of dsRNA can also be included as a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide. Generally, the duplex structure is between 5 and 50 base pairs in length, for example, 5-50, 5-49, 5-48, 5-47, 5-46, 5-45, 5-44, 5-43, 5-42, 5-41, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-28, 5-29, 5-30 -18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-49, 6-48, 6-47, 6-46, 6-45, 6-44, 6-43, 6-42, 6-41, 6-40, 6-39, 6-38, 6-37, 6-36, 6-35, 6-34, 6-33, 6-32, 6-31, 6-30, 6-29, 6-28, 6-27, 6-26, 6-25, 6-24 , 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 8-50, 8-49, 8-48, 8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8 -28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 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、19-32、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, 21-4 9, 21-48, 21-47, 21-46, 21-45, 21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22- 50, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22-35, 22-34, 22-33, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 2 23-29, 23-28, 23-27, 23-26, 23-25 ​​or 23-24 base pairs. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

[0162] Similarly, the region complementary to the target sequence can be between 15 and 50 nucleotides in length, e.g., between 5-50, 5-49, 5-48, 5-47, 5-46, 5-45, 5-44, 5-43, 5-42, 5-41, 5-40, 5-39, 5-38, 5-37, 5-36, 5-35, 5-34, 5-33, 5-32, 5-31, 5-30, 5-29, 5-28, 5-27, 5-26, 5-25, 5-24, 5-23, 5-22, 5-21, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-50, 6-49, 6-48, 6-47, 6-46, 6-45, 6-44, 6-43, 6-42, 6-41, 6-40, 6-39, 6-38, 6-37, 6-36, 6-35, 6-34, 6-33, 6-32, 6-31, 6-30, 6-29, 6-28, 6- 27, 6-26, 6-25, 6-24, 6-23, 6-22, 6-21, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 8-50, 8-49, 8-48, 8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 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、19-32、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、21-41、21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21-33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-24, 21-26, 21-24, 21-26 2, 22-50, 22-49, 22-48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-35, 22-34, 22-34. 32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23-50, 23-49, 23-48, 23-47, 23-46, 23-45, 23-45, 23-45, 23-45, 23-45, 23-45, 23-45 3-41, 23-40, 23-39, 23-38, 23-37, 23-36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23- Between 29, 23-28, 23-27, 23-25, or 23-25 ​​nucleosides. The ranges and lengths intermediate above are also considered to be part of the present disclosure. ,

[0163] Chemical modification of ASO

[0164] In certain embodiments, the ASO consists of more than just DNA. In certain embodiments, the ASO comprises at least one chemical modification relative to a native nucleotide (e.g., ribonucleotide (e.g., 2'-deoxy-2'-ribonucleotide)). Various chemical modifications may be included in the ASO of the present disclosure. Modifications may include one or more modifications in a sugar group (e.g., ribose), one or more modifications in a phosphate group, one or more modifications in a nucleobase, one or more terminal modifications, or combinations thereof. In some embodiments, e.g Fig.17 , Fig.18 or exemplary ASO sequences targeting regRNAs shown in other parts of the present disclosure are chemically modified. Such modifications may be, but are not limited to, 2'-O-(2-methoxyethyl) (2'-MOE), locked nucleic acid (LNA), 5-methyl on cytidine, restriction ethyl (cET) , phosphate (PS) linkage and / or phosphodiester (PO) linkage 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 incorporated herein by reference. In certain embodiments, each cytidine in the ASO provided herein is modified with 5-methyl.

[0165] Various chemical modifications for use with the ASOs of the present disclosure include, but are not limited to, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and nucleotides containing 5'-phosphate mimetics.

[0166] In certain embodiments, ASO comprises RNA polynucleotides chemically modified to resist one or more nucleases (e.g., ribonuclease (e.g., exosome complex or RNase H)). In some embodiments, all nucleotide bases in ASO are modified. In certain embodiments, chemical modification includes β-D-ribonucleotides, 2'-modified nucleotides (e.g., 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3 or 2'-fluoro-arabinose (FANA)), bicyclic sugar-modified nucleotides (e.g., with constrained ethyl or locked nucleic acid (LNA)) and / or one or more modified internucleotide bonds (e.g., phosphorothioate internucleotide linkages). In certain embodiments, chemical modification includes 2'-MOE and phosphorothioate internucleotide bonds. In certain embodiments, at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more continuous nucleotides of ASO are modified by 2'-MOE. In certain embodiments, each nucleotide of the ASO is modified with 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 bonds of the ASO are phosphorothioate internucleotide bonds. In certain embodiments, each internucleotide bond of the ASO is a phosphorothioate internucleotide bond.

[0167] 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) such as dimethylaminophosphoramidate (P(N(CH3)2)), phosphonocarboxylate (P(CH2) n COOR) such as phosphonoacetate "PACE" (P(CH2COO- ))、Thiophosphonocarboxylate ((S)P(CH2) n COOR) such as thiophosphonoacetate "thioPACE" ((S)P(CH2COO - ))、alkylphosphonates (P(C 1-3 alkyl) such as methylphosphonate - P(CH3), borophosphonate (P(BH3)) and dithiophosphate (P(S)2).

[0168] In some embodiments, the ASOs provided herein comprise 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 bonds. In some embodiments, all internucleotide bonds of the ASOs provided herein are PO internucleotide bonds. In some embodiments, the ASOs provided herein do not comprise PO internucleotide bonds. In some embodiments, the ASOs provided herein comprise 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 internucleotide bonds. In some embodiments, all internucleotide bonds of the ASOs provided herein are PS bonds. In some embodiments, the ASOs provided herein do not comprise PS internucleotide bonds.

[0169] In certain embodiments, the ASO comprises one or more chemical modifications at the 5' end, the 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 in 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 in at least 1, 2, 3, 4, or 3 nucleotides at the 5' end of the ASO. In certain embodiments, the ASO comprises one or more chemical modifications in at least 1, 2, 3, 4, or 5 nucleotides at the 5' end of the ASO, and comprises one or more chemical modifications in at least 1, 2, 3, 4, or 5 nucleotides at the 3' end of the ASO.

[0170] Chemical structures may also be described in writing. In such cases, 'M' represents MOE; 'd' represents DNA, 'L' represents LNA, 'm' represents 2'O methyl, '=' represents phosphorothioate (PS) linkage, '-' represents phosphodiester (PO) linkage; '5C' represents 5-methylcytosine, 'ag' represents GalNAc, 'tg' represents Teg-GalNAc, '^' represents FANA, 'BioTeg' represents biotin; 'Palm' represents palmitic acid; and 'C18' represents spacer 18 moiety.

[0171] For the avoidance of doubt, the LNA has the following formula:

[0172]

[0173] Where B is a specific designated base.

[0174] Visual representations of exemplary ASOs with chemical modifications are provided in Fig.17 and Fig.18 In. Additional exemplary ASOs with chemical modifications are provided in Tables 17 and 18. In some embodiments, the ASOs provided herein comprise the nucleotide sequences provided in Table 17 below. In some embodiments, the ASOs provided herein comprise the nucleotide sequences provided in Table 18 below. In some embodiments, the ASOs comprise the nucleotide sequences and / or chemical modifications of any one of the oligonucleotides provided in Tables 17 and 18 below. In some embodiments, the ASOs comprise the nucleotide sequences and / or chemical modifications of any one of SEQ ID NOs: 1-442, 691, 991-1368, or 4743-4915. In some embodiments, the ASOs comprise the nucleotide sequences and / or chemical modifications of any one of SEQ ID NOs: 443-690, 692-990, or 4916.

[0175] Table 17. Exemplary ASOs targeting hGRN regRNA with chemical modifications

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] Table 18. Exemplary ASOs with chemical modifications targeting mGrn regRNA

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] In some embodiments, the ASO comprises a sequence and / or chemical modification selected from the sequence provided in any one of SEQ ID NOs: 10-4916. In some embodiments, the ASO comprises a sequence selected from the sequence provided in any one of SEQ ID NOs: 1369-4738. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the sequence provided in any one of SEQ ID NOs: 10-1368 or 4734-4916. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the sequence provided in any one of SEQ ID NOs: 10-442, 691, 991-1368, or 4743-4915. In some embodiments, the ASO comprises a sequence and a chemical modification selected from the sequence provided in any one of SEQ ID NOs: 443-690, 692-990, or 4916.

[0242] High affinity modified nucleotides

[0243] High affinity modified nucleotides are modified nucleotides that, when incorporated into an oligonucleotide, enhance the affinity of the oligonucleotide for its complementary target, e.g., as determined by the melting temperature (T m ). The high affinity modified nucleotides of the present invention preferably result in an increase in the melting temperature of each modified nucleoside by between +0.5°C and +12°C, such as between +1.5°C and +10°C or between +3°C and +8°C. Many high affinity modified nucleotides are known in the art and include, for example, many 2' substituted nucleosides and locked nucleic acids (LNA) (see, e.g., Freier and Altmann (1997) Nucl. Acid Res. 25:4429-43 and Uhlmann (2000) Curr. Opinion in Drug Development 3(2):203-213, each of which is hereby incorporated by reference).

[0244] Sugar modification

[0245] ASO as described herein may include one or more nucleotides with modified sugar moieties, i.e., the modification of sugar moieties when compared with the ribose sugar moieties found in DNA and RNA. Many nucleotides with modified ribose sugar moieties have been prepared, and the main purpose is to improve certain characteristics of oligonucleotides, such as affinity and / or nuclease resistance. Such modifications include those in which the ribose ring structure is modified, such as by replacing with a hexose ring (HNA) or a dicyclo, which generally has a diradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or generally lacks a non-connected ribose ring (such as UNA) of a key between the C2 and C3 carbons. Other sugar-modified nucleotides include, for example, dicyclohexose nucleic acids (WO2011 / 017521) or tricyclic nucleic acids (WO2013 / 154798), both of which are hereby incorporated by reference. Modified nucleotides also include nucleotides in which the sugar moiety is replaced by a non-sugar moiety, such as in the case of peptide nucleic acids (PNA) or morpholino nucleic acids.

[0246] Sugar modifications also include modifications by changing the substituents on the ribose ring to groups other than hydrogen or changing the 2'-OH group naturally present in RNA nucleosides. The substituents may be introduced, for example, at the 2', 3', 4' or 5' positions.

[0247] 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 carbonyl methylene linkages as repeating units instead of a sugar-phosphate backbone), a locked nucleotide (LNA) (e.g., an oligonucleotide comprising one or more locked ribose sugars, and 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., any of an oligonucleotide comprising a backbone containing one or more phosphorodiamidate morpholino oligomers), a 2'-deoxy-2'-fluoro nucleotide (e.g., an oligonucleotide comprising one or more 2'-fluoro-β-D-arabino nucleotides), a tcDNA (e.g., an oligonucleotide comprising one or more tcDNA modified sugars), a constrained ethyl 2'-4'-bridged nucleic acid (cEt), 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).

[0248] In some embodiments, the oligonucleotide comprises a nucleobase modification selected from the group consisting of 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. uracil, 5,6-dehydrouracil, 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 nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), and isocytosine ("isoC"), glycerol nucleic acid (GNA), thiomorpholino (C4H9NS) or thiophosphoramidite morpholine (TMO). The synthesis of glycerol nucleic acids (GNAs) (also known as diol nucleic acids) is described in Zhang et al., Current Protocols in Nucleic Acid Chemistry 4.40.1-4.40.18, September 2010, hereby incorporated by reference. The synthesis of thiophosphoramidite morpholino oligonucleotides is described in Langer et al. J. Am. Chem. Soc. 2020, 142(38): 16240–253.

[0249] 2' sugar modified nucleotides

[0250] 2' sugar modified nucleotides are nucleotides having a substituent other than H or -OH at the 2' position (2' substituted nucleotides) or nucleotides such as LNA (2'-4' diradical bridged) nucleotides that contain a 2' linked diradical capable of forming a bridge between the 2' carbon and the second carbon in the ribose ring.

[0251] Without wishing to be bound by theory, 2' modified sugars can provide enhanced binding affinity and / or increased nuclease resistance to oligonucleotides. 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, e.g., Freier and Altmann 1997, supra; Uhlmann 2000, supra; and Deleavey and Damha (2012) Chemistry and Biology 19: 937, each of which is hereby incorporated by reference.

[0252] Locked Nucleic Acid Nucleotides (LNA Nucleotides)

[0253] "LNA nucleotides" are 2'-sugar modified nucleotides that contain a C2' and C4' diradical (also referred to as a "2'-4' bridge") connecting the ribose sugar ring of the nucleotide, which restricts or locks the conformation of the ribose ring. In other words, a locked nucleotide is a nucleotide that contains a bicyclic sugar moiety that contains a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose in a 3'-endo conformation. It has been shown that adding locked nucleotides to oligonucleotides can increase 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 sometimes also referred to as bridged nucleic acids or bicyclic nucleic acids (BNA). When LNA is incorporated into an oligonucleotide complementary to an RNA or DNA molecule, the locking of the ribose conformation is associated with enhanced hybridization affinity (duplex stabilization). This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex.Exemplary LNA nucleotides include β-D-oxy-LNA, 6'-methyl-β-D-oxy-LNA such as (S)-6'-methyl-β-D-oxy-LNA (ScET) and ENA.

[0254] Examples of bicyclic nucleotides for use in the polynucleotides of the present disclosure include, but are not limited to, nucleotides comprising a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the polynucleotide agents of the present disclosure comprise one or more bicyclic nucleotides comprising 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. Pat. No. 7,399,845); 4'-C( 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, e.g., U.S. Pat. Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2', wherein R is H, C1-C 12 alkyl or protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and the like; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the above are hereby incorporated herein by reference.

[0255] Additional representative U.S. patents and U.S. patent publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,3 No. 99,845; No. 7,427,672; No. 7,569,686; No. 7,741,457; No. 8,022,193; No. 8,030,467; No. 8,278,425; No. 8,278,426; No. 8,278,283; US2008 / 0039618; and US2009 / 0012281, the entire contents of each of which are hereby incorporated herein by reference.

[0256] Any of the foregoing bicyclic nucleotides can be prepared with one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see PCT Application Publication No. WO 99 / 14226, the contents of which are incorporated herein by reference).

[0257] Oligonucleotides of the present disclosure can also be modified to comprise one or more constrained ethyl nucleosides.As used herein, "constrained ethyl nucleotides" or "cEt" are locked nucleotides comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-O-2' bridge.In one embodiment, the constrained ethyl nucleotides are in an S conformation, referred to herein as "S-cEt".

[0258] The oligonucleotides of the present disclosure may also contain one or more "conformationally restricted nucleotides" ("CRNs"). CRNs are nucleotide analogs with linkers connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and increase hybridization affinity with RNA (e.g., regRNA or mRNA). The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity, thereby reducing ribose ring puckering.

[0259] Representative publications that teach the preparation of certain of the above-described CRNs include, but are not limited to, US Patent Publication No. 2013 / 0190383; and PCT Application Publication No. WO 2013 / 036868, the entire contents of each of which are hereby incorporated herein by reference.

[0260] In some embodiments, the oligonucleotides of the present disclosure include one or more monomers that are UNA (unlocked nucleotides) nucleotides. UNA is an unlocked acyclic nucleotide in which any bond of a sugar has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the bond between C1'-C4' has been removed (i.e., a covalent carbon-oxygen-carbon bond between C1' and C4' carbons). In another example, the C2'-C3' bond of a sugar (i.e., a covalent carbon-carbon bond between C2' and C3' carbon) has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference).

[0261] Representative U.S. publications that teach UNA preparation include, but are not limited to, U.S. Patent No. 8,314,227; and U.S. Patent Publication Nos. 2013 / 0096289; 2013 / 0011922; and 2011 / 0313020, the entire contents of each of which are hereby incorporated by reference herein.

[0262] Ribose sugar can also be modified with a cyclopropane ring to produce tricyclic deoxynucleic acid (tricyclic DNA). The ribose moiety can be replaced by another sugar, such as 1,5,-anhydrohexitol, threose to produce threose nucleotides (TNA), or arabinose to produce arabinose nucleotides. The ribose molecule can also be replaced by a non-sugar such as cyclohexene to produce cyclohexene nucleotides, or by ethylene glycol to produce ethylene glycol nucleotides.

[0263] Potential stabilizing modifications to the termini of nucleotide molecules may include N-(acetylaminohexanoyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(hexanoyl-4-hydroxyprolinol) (Hyp-C6), N-(acetyl-4-hydroxyprolinol) (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminohexanoyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3"-phosphate, inverted base dT (idT), and others. Disclosure of such modifications may be found in PCT Publication No. WO 2011 / 005861.

[0264] Other alternative chemistries of the oligonucleotides of the present disclosure include a 5' phosphate or a 5' phosphate mimetic, such as a 5'-terminal phosphate or phosphate mimetic of the oligonucleotide. Suitable phosphate mimetics are disclosed, for example, in US Patent Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0265] Additional non-limiting exemplary LNA nucleotides are disclosed in WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071、WO 2009 / 006478、WO 2011 / 156202、WO 2008 / 154401、WO 2009 / 067647、WO 2008 / 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 of which is hereby incorporated by reference.

[0266] In some embodiments, the length of the ASO is 5×n+5 nucleotides (n is an integer of 3 or greater), wherein the nucleotides at positions 5×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

[0267] 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'-arabinose") nucleotides, 2'-F-arabinosyl ("2'-F-arabinose") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acid (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-type sugar ("L-sugar") or 4'-thioribosyl nucleotides.

[0268] Hybrids and spacers

[0269] ASOs can have hybrid and / or spacer structures, e.g. Fig.17 or Fig.18 The mode exposed by ASO in .

[0270] In certain embodiments, ASO is a hybrid. As used herein, the term "hybrid" refers to an oligonucleotide comprising an alternating composition of a DNA monomer and a nucleotide analog monomer on at least a portion of an oligonucleotide sequence. In certain embodiments, ASO is a hybrid based on a spacer structure, which comprises a mixture of DNA nucleotides and 2'-MOE nucleotides in the gap, and an RNA sequence (e.g., 2'-modified RNA sequence) in the flank. Hybrids can be designed to comprise a mixture of affinity-enhancing nucleotide analogs, such as 2'-O-alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, LNA monomers, Arabic nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, HNA monomers, INA monomers, 2'-MOE-RNA (2'-O-methoxyethyl-RNA), 2'-fluoro-DNA and LNA in non-limiting examples. In some embodiments, hybrids can not raise RNase H. In some embodiments, hybrids comprise a type of affinity-enhancing nucleotide analogs and DNA and / or RNA.

[0271] Multiple different modifications can be spaced apart in a mixture. For example, an ASO may comprise LNA modifications in multiple nucleotides and different modifications in some or all of the remaining nucleotides. In some embodiments, any two adjacent LNA-modified nucleotides are spaced apart by at least 1, 2, 3, 4, or 5 nucleotides. The distance between adjacent LNA-modified nucleotides can be constant (e.g., any two adjacent LNA-modified nucleotides are spaced apart by 1, 2, 3, 4, or 5 nucleotides) or variable throughout the ASO. 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), wherein (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) comprising 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) comprising 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) comprising a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions comprise 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), wherein (a) (i) the nucleotide at position 2×m-1 (m is an integer of 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) comprising a first modification (e.g., LNA), or (ii) the nucleotide at position 2×m (m is an integer of 1 to n) is a nucleotide (e.g., ribonucleotide or deoxyribonucleotide) comprising a first modification (e.g., LNA); and (b) the nucleotides at the remaining positions comprise a second different modification (e.g., 2'-O-methoxyethyl). Similar modification patterns are also contemplated, for example, where the first modification is repeated every 4, 5 or more nucleotides.

[0272] 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 consecutive nucleotides of unmodified DNA) flanking an RNA sequence (e.g., a 2'-modified RNA sequence or a 2'-modified ribonucleotide) on both sides. This structure is referred to as a "gap body", in which the DNA region is referred to as a "gap", and the RNA region is referred to as a "wing" (see, e.g., PCT Application Publication No. WO2013 / 177248). It is known that the gap body promotes the degradation of the target RNA by recruiting nucleases (e.g., ribonucleases (e.g., RNase H)). Surprisingly, in some embodiments of the present disclosure, it has been found that the gap body (which has the same sequence as the parent ASO but has different chemical modifications) bound to the regRNA can also increase target gene expression.

[0273] In some embodiments, the ASO spacer 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 nucleotides, while the external RNA 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides. Exemplary spacer 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, wherein the first and third numbers represent the number of outer RNA nucleotides and the second number represents the number of inner DNA nucleotides.

[0274] The ASO can also be a hybrid comprising a DNA region linked to an RNA region. In some embodiments, the hybrid comprises at least 10 DNA nucleotides linked to at least 10 RNA nucleotides, wherein the DNA nucleotides are located at the 5' end of the hybrid or at the 3' end of the hybrid. In some embodiments, the hybrid 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 connected 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 nucleotides, wherein the DNA nucleotides are located at the 5' end of the hybrid or the 3' end of the hybrid. In some embodiments, the RNA region of the spacer or hybrid can include any additional chemical modifications as disclosed herein.

[0275] In certain embodiments, the length of the ASO (e.g., gap body or hybrid) 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 certain embodiments, the length of 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 certain embodiments, the length of one or two wings is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 or more nucleotides. In certain embodiments, one or two RNA regions or wings include RNA modifications, for example, β-D-ribonucleotides, 2'-modified nucleotides (for example, 2'-O-(2-methoxyethyl) (2'-MOE), 2'-O-CH3 or 2'-fluoro-arabinose (FANA)) and bicyclic sugar-modified nucleotides (for example, with constrained ethyl or locked nucleic acid (LNA)). In certain embodiments, each ribonucleotide in a hybrid or spacer is modified by 2'-MOE. In certain embodiments, a hybrid or spacer includes one or more modified internucleotide bonds, such as phosphorothioate (PS) internucleotide linkages. In certain embodiments, every two adjacent nucleotides in a hybrid or spacer are connected by a phosphorothioate internucleotide bond.

[0276] In certain embodiments, the ASO does not comprise 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, or 45 or more consecutive nucleotides of unmodified DNA. In some embodiments, such a DNA sequence is disrupted by modified (e.g., 2'-MOE modified) ribonucleotides every 2, 3, 4, 5 or more nucleotides. In some embodiments, the ASO comprises only ribonucleotides and no deoxyribonucleotides.

[0277] The structural features of hybrids and spacers can be combined. In certain embodiments, the ASO has a structure similar to the hybrid disclosed herein (e.g., a hybrid with a gap modification), except that the second modification in the gap is changed to a third modification (e.g., a deoxyribonucleotide). In certain embodiments, the ASO has a structure similar to the spacer disclosed herein, except that the nucleotides in the gap are modified in a hybrid mode.

[0278] In certain embodiments, the ASO further comprises a ligand moiety, such as a ligand moiety that specifically targets a tissue or organ in a subject. For example, N-acetylgalactosamine (GalNAc) specifically targets the liver. In certain embodiments, the ligand moiety comprises GalNAc. In certain embodiments, the ligand moiety comprises three clusters of GalNAc moieties, generally indicating GalNAc3. Other types of GalNAc moieties are one, two or four clusters of GalNAc, indicated as GalNAc1, GalNAc2 or GalNAc4. In certain embodiments, the ligand moiety comprises GalNAc1, GalNAc2, GalNAc3 or GalNAc4.

[0279] 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).

[0280] III. Pharmaceutical Compositions

[0281] In certain embodiments, the ASO disclosed herein may be present in a pharmaceutical composition. The pharmaceutical composition may be formulated for a variety of drug delivery systems. The composition may also include one or more pharmaceutically acceptable excipients or carriers for appropriate formulation. In some embodiments, pharmaceutically acceptable carriers include sterile saline, sterile water, phosphate buffered saline (PBS) or aCSF. Suitable formulations for the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief review of methods for drug delivery, see, for example, Langer (Science 249: 1527-1533, 1990).

[0282] Exemplary vectors and pharmaceutical formulations suitable for delivery of nucleic acids are described in Durymanov 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 of which is incorporated herein by reference. It should be understood that the presence of a ligand moiety conjugated to an ASO can avoid the need for a vector for delivery to a tissue or organ targeted by the ligand moiety.

[0283] Oligonucleotides of the present invention are delivered to cells, for example, subjects, such as human subjects, for example, subjects in need, such as cells in subjects suffering from GRN related disorders, can be achieved in a variety of different ways. For example, delivery can be carried out by contacting cells with oligonucleotides of the present invention in vitro or in vivo. In vivo delivery can also be carried out directly by administering a composition comprising an oligonucleotide to the subject. These alternatives will be discussed further below.

[0284] In general, any method of delivering nucleic acid molecules (in vitro or in vivo) may be applicable for use with the oligonucleotides of the present disclosure (see, e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2 (5): 139-144 and WO 94 / 02595, the entire contents of which are incorporated herein by reference). For in vivo delivery, factors to be considered for delivery of oligonucleotide molecules include, e.g., the biostability of the delivered molecule, the prevention of nonspecific effects, and the accumulation of the delivered molecule in the target tissue. The nonspecific effects of oligonucleotides can be minimized by topical administration, e.g., by direct injection or implantation in tissues or topical administration of preparations. Topical administration to the treatment site maximizes the local concentration of the agent, limits exposure of the agent to systemic tissues that may be injured by the agent or may degrade the agent, and allows the administration of a lower total dose of oligonucleotide molecules.

[0285] For systemic administration of oligonucleotides to treat diseases, oligonucleotides can include alternative nucleobases, alternative sugar moieties and / or alternative internucleotide linkages, or alternatively use a drug delivery system to deliver; both methods prevent oligonucleotides from being rapidly degraded by endonucleases and exonucleases in vivo. The modification of oligonucleotides or drug carriers can also allow oligonucleotide compositions to target target tissues and avoid undesirable off-target effects. Oligonucleotide molecules can be modified by chemical conjugation with lipophilic groups (such as cholesterol) to enhance cellular uptake and prevent degradation. In alternative embodiments, oligonucleotides can be delivered using a drug delivery system, such as nanoparticles, lipid nanoparticles, polymer nanoparticles, liposome complex nanoparticles, dendrimers, polymers, liposomes or cationic delivery systems. Positively charged cationic delivery systems promote the combination of oligonucleotide molecules (negatively charged), and also enhance the interaction at the negatively charged cell membrane to allow cells to effectively take up oligonucleotides. Cationic lipids, dendrimers or polymers can be combined with oligonucleotides, or induce the formation of vesicles or micelles that encapsulate oligonucleotides. When systemically administered, the formation of vesicles or micelles further prevents the degradation of the oligonucleotide. In general, any nucleic acid delivery method known in the art may be applicable to the delivery of the oligonucleotides disclosed herein. Methods for preparing and administering cationic oligonucleotide complexes are well within the capabilities of those skilled in the art (see, e.g., Sorensen, DR, et al. (2003) J.Mol.Biol 327:761-766; Verma, U N. et al., (2003) Clin.Cancer Res.9:1291-1300; Arnold, AS et al., (2007) J.Hypertens.25:197-205, the entire contents of which are incorporated herein by reference).Some non-limiting examples of drug delivery systems that can be used to deliver oligonucleotides systemically include DOTAP (Sor ensen, DR, et al. (2003), supra; Verma, U N. et al. (2003), supra), Oligoectamine, "solid nucleic acid lipid particles" (Zimmermann, TS, 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 ME, et al. (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Ar g-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; Yo o, H. et al., (1999) Pharm. Res. 16: 1799-1804). In some embodiments, the oligonucleotide forms a complex with cyclodextrin for systemic administration. Methods of administration and pharmaceutical compositions of oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, the entire contents of which are incorporated herein by reference. In some embodiments, the oligonucleotides of the present disclosure are delivered via polymer or liposome complex nanoparticles. Methods of administration and pharmaceutical compositions of oligonucleotide and polymer nanoparticles and liposome complex 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 incorporated herein by reference in their entirety.

[0286] In some embodiments, the compounds described herein can be administered in combination with additional therapeutic agents. Examples of additional therapeutic agents include standard of care anti-epileptic drugs, such as quinidine and / or sodium channel blockers. In addition, the compounds described herein can be administered in combination with recommended lifestyle changes such as a ketogenic diet.

[0287] Membrane Molecular Assembly Delivery Methods

[0288] Oligonucleotides disclosed herein can also be delivered using a variety of membrane molecule assembly delivery methods, including polymerization known in the art, biodegradable microparticles or microcapsule delivery devices. For example, colloidal dispersion systems can be used for targeted delivery of oligonucleotide agents as described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. Liposomes are artificial membrane vesicles that can be used as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUVs) with a size range of 0.2-4.0 μm can encapsulate a considerable proportion of aqueous buffers containing macromolecules. Liposomes can be used to transfer and deliver active ingredients to the site of action. Since the liposome membrane is similar to a biological membrane in structure, when liposomes are applied to tissues, the liposome bilayer fuses with the cell membrane bilayer. As the liposomes and cells fuse, the internal aqueous content containing the oligonucleotides is delivered to the cells, and the oligonucleotides can specifically bind to the target RNA in the cells. In some cases, liposomes are also specifically targeted, for example, oligonucleotides are guided to specific cell types. The composition of liposomes is usually a combination of phospholipids, usually in combination with a steroid, especially 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.

[0289] Liposomes containing oligonucleotides can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in a detergent so as to form micelles with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The detergent can have a high critical micelle concentration and can be nonionic. Exemplary detergents include cholate, CHAPS, octyl glucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide preparation is then added to the micelle 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 detergent is removed, for example, by dialysis to produce a liposome preparation of the oligonucleotide.

[0290] If desired, a carrier compound that aids condensation can be added during the condensation reaction, for example by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to facilitate condensation.

[0291] Methods for generating stable polynucleotide delivery vehicles incorporating polynucleotide / cationic liposome complexes as structural components of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of the exemplary methods described in Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. 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. Common techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. When consistently small (50 to 200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are readily adapted for packaging oligonucleotide formulations into liposomes.

[0292] Liposomes are divided into two major categories. Cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complex binds to the negatively charged cell surface and is internalized into endosomes. Due to the acidic pH in the endosome, the liposomes rupture and release their contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147: 980-985).

[0293] pH-sensitive or negatively charged liposomes encapsulate nucleic acids rather than complex with them. Since nucleic acids and lipids are similarly charged, repulsion rather than complex formation occurs. However, some nucleic acids are encapsulated in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding thymidine kinase genes to monolayer cells in culture. The expression of exogenous genes was detected in target cells (Zhou et al. (1992) Journal of Controlled Release, 19: 269-274).

[0294] The liposome composition of a kind of main type comprises the phospholipids except the phosphatidylcholine of natural origin.Neutral liposome composition can be formed by dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) for example.Anionic liposome composition is usually formed by dimyristoylphosphatidylglycerol, and anionic fusion liposome is mainly formed by dioleoylphosphatidylethanolamine (DOPE).Another type of liposome composition is formed by phosphatidylcholine (PC), such as, for example soybean PC and egg PC.Another type is formed by the mixture of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0295] Examples of other methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 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.

[0296] Nonionic liposomal systems have also been examined to determine their utility in delivering drugs to the skin, particularly systems containing nonionic surfactants and cholesterol. TM I (Dilaurin / Cholesterol / Polyoxyethylene-10-Stearyl Ether) and NOVASOME TM A nonionic liposome formulation of II (distearin / cholesterol / polyoxyethylene-10-stearyl ether) was used to deliver cyclosporin-A into the dermis of mouse skin. The results showed that this nonionic liposome system can effectively promote the deposition of cyclosporin A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4 (6): 466).

[0297] The liposomes may also be sterically stabilized liposomes comprising one or more specialized lipids which result in an increased circulation life relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which a portion (A) of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids, such as monosialoganglioside G M1 , or (B) those derivatized with one or more hydrophilic polymers such as polyethylene glycol (PEG) moieties. While not wishing to be bound by any particular theory, it is believed in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelins, or PEG-derivatized lipids, the extended circulation half-life of these sterically stabilized liposomes results from reduced uptake by cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0298] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., (1987), 507:64) reported monosialoganglioside G M1 , galactocerebroside sulfate and phosphatidylinositol improve the blood half-life of liposomes. Gabizon et al. (Proc. Natl. Acad. Sci. USA, (1988), 85:6949) described these findings. U.S. Patent No. 4,837,028 and WO 88 / 04924 to Allen et al. disclose liposomes containing (1) sphingomyelin and (2) ganglioside G M1 Liposomes containing sphingomyelin or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0299] In one embodiment, cationic liposomes are used. Cationic liposomes have the advantage of being able to fuse with cell membranes. Although non-cationic liposomes cannot effectively fuse with plasma membranes, they are absorbed by macrophages in vivo and can be used to deliver oligonucleotides to macrophages.

[0300] Other advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a variety of water-soluble and lipid-soluble drugs; liposomes can protect oligonucleotides encapsulated in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," ​​Lieberman, Rieger and Banker (eds.), 1988, Vol. 1, p. 245). Important considerations in preparing liposome formulations are lipid surface charge, vesicle size, and the water volume of the liposomes.

[0301] 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 to form lipid-nucleic acid complexes that are able to fuse with negatively charged lipids of the cell membrane of tissue culture cells, resulting in delivery of oligonucleotides (see, e.g., Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417 and U.S. Pat. No. 4,897,355 describing DOTMA and its use with DNA).

[0302] The DOTMA analog 1,2-bis(oleoyloxy)-3-(trimethylamino)propane (DOTAP) can be used in combination with phospholipids to form DNA complexed vesicles. TM Bethesda Research Laboratories (Gaithersburg, Maryland) is an effective agent for delivering highly anionic nucleic acids to living tissue culture cells, wherein the cells contain positively charged DOTMA liposomes, which spontaneously interact with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complex prepared in this way spontaneously attaches to the negatively charged cell surface, fuses with the plasma membrane, and effectively delivers functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylamino)propane ("DOTAP") (BoehringerMannheim, Indianapolis, Ind.) is different from DOTMA in that the oleoyl moiety is connected by an ester rather than an ether linkage.

[0303] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties, including, for example, carboxyspermine, which has been conjugated to one of two types of lipids, and including such compounds as 5-carboxyarginylglycine dioctanoylamide ("DOGS") (TRANSFECTA M TM, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyarginylamide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0304] Another cationic lipid conjugate includes lipids derivatized with cholesterol ("DC-Chol"), which have been formulated into liposomes in combination with DOPE (see, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, prepared by conjugating polylysine to DOPE, is reported to be effective in transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more effective transfection than compositions containing DOTMA. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSP A) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0305] Liposome formulations are particularly suitable for topical administration, and liposomes have multiple advantages compared to other formulations. These advantages include reducing the side effects associated with the high systemic absorption of the administered drug, increasing the accumulation of the administered drug at the desired target, and the ability to administer the oligonucleotide to the skin. In some implementations, liposomes are used to deliver oligonucleotides to epidermal cells and are also used to enhance the penetration of oligonucleotides into dermal tissues, such as into the skin. For example, liposomes can be applied topically. It has been documented that drugs formulated as liposomes are delivered topically to the skin (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, Vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18: 259-265; Mannino, RJ 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, RM and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, CY and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0306] Nonionic liposome systems were also examined to determine their effectiveness in delivering drugs to the skin, particularly systems containing nonionic surfactants and cholesterol. 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 drugs to the dermis of mouse skin. Such formulations containing oligonucleotides can be used to treat skin diseases.

[0307] The targeting of liposomes may also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art. In the case of a liposome targeted delivery system, a lipid group can be incorporated into the lipid bilayer of the liposome to maintain the stable binding of the targeting ligand to the liposome bilayer. Various linking groups can be used to connect the lipid chain to the targeting ligand. Other methods are known in the art and are described, for example, in U.S. Patent Application Publication No. 20060058255, whose linking groups are incorporated herein by reference.

[0308] Liposomes containing oligonucleotides can be made highly deformable. This deformability can make liposomes penetrate holes less than the average radius of liposomes. For example, transfer bodies are another type of liposomes, and are highly deformable lipid aggregates, as attractive candidates for drug delivery carriers. Transfer bodies can be described as lipid droplets, which are highly deformable, so that they can easily penetrate holes smaller than lipid droplets. Transfer bodies can be prepared by adding surface edge activators (usually surfactants) to standard liposome compositions. Transfer bodies containing oligonucleotides can be delivered, for example, by infecting subcutaneous delivery to deliver oligonucleotides to keratinocytes in the skin. In order to pass through intact mammalian skin, lipid vesicles must pass through a series of pores less than 50nm in diameter under the influence of a suitable transdermal gradient. In addition, due to lipid properties, these transfer bodies can be self-optimized (adapting to the shape of the hole, such as the shape of the hole in the skin), self-repaired, and can often reach their targets without fragmentation, and often self-loaded. Transfer bodies have been used to deliver serum albumin to the skin. Transfer body-mediated serum albumin delivery has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0309] Other formulations suitable for use in the present disclosure are described in PCT Publication Nos. WO 2009 / 088891, WO 2009 / 132131, and WO 2008 / 042973, which are hereby incorporated by reference in their entireties.

[0310] 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 natural and synthetic surfactants is to use the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also referred to as the "head") provides the most useful method for classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, page 285).

[0311] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants have a wide range of applications in pharmaceuticals and cosmetics and can be used over a wide pH range. Generally speaking, 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, glycerol esters, polyglycerol esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers also belong to this category. Polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0312] If the surfactant molecule carries a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, sulfates such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are alkyl sulfates and soaps.

[0313] If the surfactant molecule carries 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 commonly used members of this class.

[0314] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkyl amides, N-alkyl betaines, and phospholipids.

[0315] The use of surfactants in pharmaceutical products, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0316] Oligonucleotides used in the methods of the present disclosure can also be provided as micellar formulations. Micelles are a special type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure so that all hydrophobic portions of the molecules face inwards, while the hydrophilic portions are in contact with the surrounding water. If the environment is hydrophobic, the opposite arrangement exists.

[0317] Lipid nanoparticle-based delivery methods

[0318] Oligonucleotides of the present disclosure can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNP) or other nucleic acid-lipid particles. LNP can be used for systemic applications because they show prolonged circulation life after intravenous (iv) injection and accumulate in distal positions (such as positions physically separated from the application site). LNP includes "pSPLP", which includes encapsulated condensing agent-nucleic acid complexes, as described in PCT Publication No. WO 00 / 03683. Particles of the present disclosure generally have an average diameter of about 50nm to about 150nm, more generally about 60nm to about 130nm, more generally about 70nm to about 110nm, most generally about 70nm to about 90nm, and substantially nontoxic. In addition, when present in nucleic acid-lipid particles of the present disclosure, nucleic acid resists the degradation of nuclease in aqueous solution. Nucleic acid-lipid particles and methods for making them are disclosed in, for example, 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. WO 96 / 40964.

[0319] Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N--(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N--(1-(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-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), and 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA). MA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-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 (DLin-TMA.Cl ), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), or 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane Cyclic (DLin-K-DMA) or its analogs, (3aR, 5s, 6aS) -N, N-dimethyl-2,2-di ((9Z, 12Z) -octadecane-9,12-diene tetrahydro-3aH-cyclopenta [d] [1,3] dioxol-5-amine (ALN100), (6Z, 9Z, 28Z, 31Z) -triacontria-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (MC3), 1,1'- (2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) ethyl) piperazine-1-yl ethyl azabifolia docosane-2-ol (Tech G1) or mixtures thereof. The cationic lipid may account for, for example, about 20 mol % to about 50 mol % or about 40 mol % of the total lipids present in the particle.

[0320] The ionizable / non-cationic lipid can be an anionic lipid or a neutral lipid, including but not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid may be, for example, from about 5 mol % to about 90 mol %, about 10 mol %, or about 60 mol % (if cholesterol is included) of the total lipid present in the particle.

[0321] The conjugated lipid that inhibits particle aggregation can be, for example, polyethylene glycol (PEG)-lipids, including but not limited to PEG-diacylglycerol (DAG), PEG-dialkoxypropyl (DAA), PEG-phospholipids, PEG-ceramide (Cer), or mixtures thereof. The PEG-DAA conjugate can be, for example, PEG-dilauroyloxypropyl (Cer). 12 ), PEG-dimyristoyloxypropyl (C 14 ), PEG-dipalmitoyloxypropyl (C 16 ) or PEG-distearoyloxypropyl (C 18 ). The conjugated lipid that prevents aggregation of the particles may be, for example, 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particles.

[0322] In some embodiments, the nucleic acid-lipid particle further comprises cholesterol, eg, from about 10 mol % to about 60 mol % or about 50 mol % of the total lipid present in the particle.

[0323] ASOs can also be delivered in lipidoid form. The synthesis of lipidoids has been widely described, and formulations containing these compounds are particularly suitable for delivering 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 US A. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011 108: 12996-3001; all of which are incorporated herein in their entirety).

[0324] Lipid compositions for RNA delivery are disclosed in WO2012170930A1, WO2013149141A1 and WO2014152211A1, each of which is incorporated herein by reference.

[0325] IV. Therapeutic Applications

[0326] The disclosure provides a method for treating diseases and disorders related to reduced GRN gene expression and other diseases and disorders. In some embodiments, the method adopts an ASO hybridized with the GRN regRNA transcribed from the regulatory element of the GRN gene or a pharmaceutical composition comprising the ASO. Oligonucleotide compositions as described herein can be used in the method of the present disclosure, and although not bound by theory, it is believed that the level of PGRN protein (and its GRN peptide protein hydrolysate) and / or GRN mRNA and / or the state or activity of GRN can be regulated by it (for example, by increasing the level of PGRN protein in the subject (for example, mammal, mouse, hamster, non-human primate (for example, monkey) or mankind) cell) to play its desired effect.

[0327] One aspect of the present disclosure relates to a method for treating a GRN-related disorder (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) to increase the expression of GRN in cells of the subject. In some embodiments, the GRN-related disorder is frontotemporal dementia (FTD) (e.g., GRN-FTD, also referred to as FTD-GRN) or frontotemporal lobar degeneration (e.g., GRN-related frontotemporal lobar degeneration). In some embodiments, the subject comprises progranulin haploinsufficiency.

[0328] Another aspect of the disclosure relates to methods of treating a disease or condition (e.g., a disease or condition provided herein) in a subject in need thereof, the method comprising administering an ASO of the disclosure (or a pharmaceutical composition comprising the ASO), thereby treating the disease or condition in the subject. In some embodiments, the disease or disorder is selected from frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathic frontotemporal lobar degeneration associated with accumulation of TDP-43 inclusions (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerosis dementia, spinocerebellar ataxia 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 (CLN 11), limbic system-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 selected from a sphingolipid disorder (e.g., GM2 gangliosidosis, type A (also known as Tay-Sachs disease); GM2 gangliosidosis, type O (also known as Sandhoff disease); 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., classic Fabry disease and late-onset Fabry disease); metachromatic leukodystrophy; globoid cell leukodystrophy (also known as Krabbe disease) disease); LSDs of GM1 gangliosidosis (e.g., GM1 gangliosidosis type 1, GM1 gangliosidosis type 2, and GM1 gangliosidosis type 3); and multiple sulfatase deficiency); oligosaccharidosis disorder (e.g., alpha mannosidosis, Schindler disease, aspartylglucosaminuria, and fucosidosis);mucopolysaccharidosis (MPS) (e.g., Hurler syndrome, Scheie syndrome, Hurler-Schwein 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 (Battendisease) (e.g., CLN1, CLN2, CLN 3, CLN4, CLN5, CLN6, CLN7, CLN8, CLN9, CLN10, CLN11, CLN12, CLN13, and CLN 14); sialic acid disorders (e.g., galactosialidosis, infantile sialic acid storage, Salladisease, and sialiduria); 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. Lysosomal storage diseases are generally described in Rajkumar and Dumpa, "Lysosomal Storage Disease," In: Stat Pearls [International]. Treasure Island (FL): Stat Pearls. Publishing; January-March 2023, available at: ncbi.nlm.nih.gov / books / NBK563270 / , which is hereby incorporated by reference in its entirety. ;

[0329] Another aspect of the present disclosure includes a method of increasing the level of PGRN protein or GRN mRNA in 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.

[0330] Another aspect of the present disclosure includes a method of increasing expression of a GRN gene in cells of a subject (e.g., a subject suffering from a disease or condition described herein), the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby increasing expression of the GRN gene in cells of the subject.

[0331] Another aspect of the disclosure relates to a method of treating a disease or condition in a subject in need thereof, the method comprising administering an ASO of the disclosure (or a pharmaceutical composition comprising the ASO), thereby treating the disease or condition in the subject.

[0332] Yet another aspect of the present disclosure includes methods of increasing expression of GRN in cells of a subject, the method comprising administering an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO), thereby treating a disease or disorder in the subject.

[0333] In yet another aspect, the disclosure provides an ASO of the disclosure (or a pharmaceutical composition comprising the ASO) for use as a medicament. In addition, the disclosure provides an ASO of the disclosure (or a pharmaceutical composition comprising the ASO) for use in treatment.

[0334] Another aspect of the present disclosure includes a method for regulating (e.g., increasing or decreasing) the expression of a GRN gene in a cell (e.g., in vivo, ex vivo, or in vitro), the method comprising contacting the cell with an 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. The method may include contacting the cell with an ASO of the present disclosure (or a pharmaceutical composition comprising the ASO) that effectively regulates (e.g., increases) the expression of GRN in the cell, thereby increasing the expression of PGRN protein or GRN mRNA in the cell. In some embodiments, the cell is contacted with an ASO (or a pharmaceutical composition comprising the ASO) to regulate (e.g., increase) the amount of GRN mRNA in the cell. In some embodiments, the cell is contacted with an ASO (or a pharmaceutical composition comprising the ASO) to regulate (e.g., increase) the amount of PGRN protein in the cell.

[0335] Based on the above methods, further aspects of the present disclosure include oligonucleotides of the present disclosure or compositions comprising such oligonucleotides, which are used for treatment, or as medicines, or for treating a disease or disorder (e.g., a GRN-related disorder or FTD) of a subject in need, or for increasing the level of PGRN in a subject's (e.g., a subject identified as suffering from a GRN-related disorder) cell, or for increasing the expression of GRN in a subject's cell. The use includes contacting a cell with an oligonucleotide that is effective in increasing 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 may also be applied to these further aspects.

[0336] Contacting cells with ASOs can be performed in vitro, in vitro or in vivo. Contacting cells with ASOs in vivo includes contacting cells or cell groups in a subject (e.g., a human subject) with oligonucleotides. Combinations of in vitro, in vitro, and in vivo methods of contacting cells are also possible. As described above, contacting cells can be direct or indirect. In addition, contacting cells can be accomplished by targeting ligands, including any ligands described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand that directs the oligonucleotide to a site of interest. The cell can be a CNS cell, such as a neuron or brain cell, a microglia.

[0337] ASO or pharmaceutical composition disclosed herein can be administered intravenously, intraarterially, intraperitoneally, intramuscularly, subcutaneously, intrapleurally, intrathecally, intracavitaryly, by catheter perfusion or by direct intralesional injection to a subject. In certain embodiments, ASO or pharmaceutical composition are administered systemically. In certain embodiments, ASO or pharmaceutical composition are administered by parenteral route. For example, in certain embodiments, ASO or pharmaceutical composition are administered intravenously (for example, by intravenous infusion), for example, using a pre-filled bag, a pre-filled pen or a pre-filled syringe. In other embodiments, ASO or pharmaceutical composition are administered locally to an organ or tissue (for example, liver or brain tissue (for example, cortex, hypothalamus, hippocampus, cerebellum and coronary brain tissue)) where target gene expression is required to increase.

[0338] In some embodiments, the oligonucleotide is applied to the subject so that the oligonucleotide is delivered to a specific site in the subject. This targeted delivery can be achieved by systemic administration or topical administration. The increase in GRN expression can be evaluated using the measured value of the level of GRN mRNA or PGRN protein or the change of the level in a sample (e.g., blood, tissue or CNS sample) derived from a specific site in the subject. In certain embodiments, the method includes a clinically relevant increase in GRN expression, for example, as demonstrated by the clinically relevant results after treating a subject with an agent that reduces GRN expression.

[0339] In other embodiments, the oligonucleotide is administered in an amount and for a duration effective to reduce (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) one or more symptoms of a disease or disorder (e.g., a GRN-related disorder), such as deterioration in behavior or personality, language disorders, disturbances or changes in muscle or motor function, memory loss, cognitive dysfunction, tremors, seizures, and dizziness.

[0340] Increased GRN expression levels

[0341] In some embodiments, the therapeutic methods disclosed herein using ASOs targeting GRN regRNA are designed to increase GRN expression in a subject. Increasing the expression of a GRN gene includes an increase in any level of a GRN gene, such as at least a partial increase in GRN gene expression. Increased expression can be assessed by an increase in the absolute or relative level of one or more of these variables compared to a control level. The control level can be any type of control level used in the art, such as a baseline level before administration, or a level determined from a similar subject, cell or sample that has never been treated or treated with a control (such as, for example, only a buffer (vehicle) control or an inactive agent control). In certain embodiments, the method causes a clinically relevant increase in GRN expression, for example, as demonstrated by clinically relevant results after treating a subject with an agent that increases GRN expression.

[0342] 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 pre-dose, pre-administration, or pre-exposure baseline levels. 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 pre-dose, pre-application, or pre-exposure baseline levels. In certain embodiments, the subject has a GRN expression defect, and the methods disclosed herein restore GRN expression levels 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 a species of similar age and sex.

[0343] In some embodiments, the ASOs of the present disclosure may enhance production of GRN mRNA (e.g., in a cell or in a cell, tissue, or sample of a subject) 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%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, In some embodiments, the ASOs of the present disclosure can enhance the production of GRN mRNA (e.g., in a cell or in a cell, tissue, or sample of a subject) 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 pre-dose, pre-administration, or pre-exposure baseline levels.

[0344] In some embodiments, the ASOs of the present disclosure can enhance the production of PGRN protein (e.g., in a cell or in a cell, tissue, or sample of a subject) 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%, relative to pre-dose, pre-administration, or pre-exposure baseline levels. %, 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%, 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. In some embodiments, the ASO of the present disclosure can enhance the production of PGRN protein (e.g., in cells or in cells, tissues or samples of subjects) 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 the pre-dose, pre-application or pre-exposure baseline level.

[0345] The expression of the GRN gene can be assessed based on the level of any variable associated with the expression of the GRN gene (e.g., GRN mRNA level or PGRN protein level). In certain embodiments, the expression level or activity of GRN herein refers to the average expression level or activity in neuronal cells or brain (e.g., brain cells in the brain regions described herein).

[0346] In certain embodiments, surrogate markers can be used to detect an increase in the level of GRN expression. For example, effective treatment of a GRN-related disorder with an agent that increases GRN expression can be understood as demonstrating a clinically relevant increase in GRN, as demonstrated by accepted diagnostic and monitoring criteria.

[0347] An increase in GRN gene expression can be manifested by an increase in the amount of GRN mRNA expressed by a first cell or group of cells (such cells can be present, for example, in a sample derived from a subject) in which the GRN gene is transcribed and has been treated (e.g., by contacting one or more cells with an oligonucleotide of the disclosure, or by administering an oligonucleotide of the disclosure to a subject in which the cell is or was present) such that expression of the GRN gene is increased compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but has not been so treated (one or more control cells that have not been treated with an oligonucleotide or have not been treated with an oligonucleotide targeting a gene of interest).

[0348] In other embodiments, the increase in GRN gene expression can be assessed based on an increase in a parameter functionally related to GRN gene expression (e.g., PGRN protein expression, granulin peptide levels, or PGRN activity). The increase in GRN expression can be determined in any cell expressing the GRN (whether endogenous or heterologous to the expression construct) and by any assay known in the art.

[0349] The increase in GRN expression can be manifested as an increase in the level of PGRN protein (or its proteolytic granulin peptide product) expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject) relative to a control cell or cell control group. The increase in GRN expression can also be manifested as an increase in the level of GRN mRNA in the treated cell or cell group relative to a control cell or cell control group.

[0350] Control cells or cell groups that can be used to assess the increase in GRN gene expression include cells or cell groups that have not been contacted with the oligonucleotides of the present disclosure. For example, control cells or cell groups can be derived from individual subjects (e.g., human or animal subjects) before treating the subject with the oligonucleotides.

[0351] The level of GRN mRNA expressed by a cell or group of cells 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 a transcribed polynucleotide or portion thereof (e.g., mRNA of a GRN gene). RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNA extraction). TM B; Biogenesis), RNeasy TM RNA preparation kit (Qiagen) or (PreAnalytix, Switzerland). Typical assay formats utilizing RNA hybridization include nuclear run assays, RT-PCR, RNAse protection assays, Northern blots, in situ hybridization, and microarray analysis. Circulating GRN mRNA can be detected using the methods described in PCT Publication WO 2012 / 177906, the entire contents of which are hereby incorporated herein by reference. In some embodiments, nucleic acid probes are used to determine the expression level of GRN. As used herein, the term "probe" refers to any molecule that can selectively bind to a specific GRN or PGRN sequence (e.g., mRNA or polypeptide). The probe can be synthesized by a person skilled in the art, or derived from an appropriate biological preparation. The probe may be specially designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0352] In some embodiments, the therapeutic methods disclosed herein using ASOs targeting GRN regRNA are designed to reduce the expression level of immune response genes in subjects. 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, GRN ASOs reduce the expression of IL-8, IL-6, CCL4, and CCL2 in cells or subjects.

[0353] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern blot analysis, polymerase chain reaction (PCR) analysis, and probe arrays. A method for measuring mRNA levels includes contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with GRN mRNA. In one embodiment, the mRNA is fixed on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane (such as nitrocellulose). In an alternative embodiment, one or more probes are fixed on a solid surface and the mRNA is contacted with one or more probes, for example in an AFFYMETRIX gene chip array. Those skilled in the art can easily adopt known mRNA detection methods for measuring the level of GRN mRNA.

[0354] Alternative methods for determining the expression level of GRNs in a sample involve amplification of nucleic acids such as mRNA in a sample and / or reverse transcriptase (to prepare cDNA) by, for example, RT-PCR (experimental embodiments described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustaining 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-β replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for detecting nucleic acid molecules if the number of such molecules is very small. In a specific aspect of the present disclosure, the expression level of GRN is measured by quantitative fluorescent RT-PCR (i.e., TAQMAN TM system) or DUAL- Luciferase assay was used to determine the

[0355] The expression level of GRN mRNA can be monitored using membrane blots (such as for hybridization analysis, such as Northern blots, Southern blots, spots, etc.) or microwells, sample tubes, gels, beads or fibers (or any solid support containing bound nucleic acids). See U.S. Patents 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 the use of nucleic acid probes in solution.

[0356] In some embodiments, branched DNA (bDNA) assays or real-time PCR (qPCR) are used to assess GRN mRNA expression levels. Such methods can also be used to detect GRN nucleic acids.

[0357] The level of PGRN protein expression and the level of granulin peptide can be measured using any method known in the art for measuring protein level. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), super diffusion chromatography, fluid or gel precipitation reaction, absorption spectrum, colorimetric determination, spectrophotometry, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blot, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc. Such assays can also be used to detect proteins indicating the presence of PGRN protein and granulin peptide.

[0358] Example

[0359] Example 1: Synthesis and in vitro characterization of ASOs targeting human GRN regRNA

[0360] Six GRN regRNAs, one paRNA, and five eRNAs were identified in the human genome. 177 spatial ASOs targeting GRN regRNAs were designed and synthesized. 29 ASOs were selected for tiling after the first screening. 52 ASOs were designed and synthesized for fine-tuning. Among them, 5 ASOs are spacers, 27 have PO / PS bonds, 5 are hybrids, and 15 contain LNA.

[0361] To evaluate the expression of hGRN paRNA and mRNA in iPSC-derived neurons and iMGL cells, the following experiments were performed. After the cells were exposed to 1uM or 3uM of the histone deacetylase (HDAC) inhibitor vorinostat (VOR) (which is known to increase the expression of GRN mRNA) for 24 hours, real-time quantitative PCR (qPCR) was used to evaluate the expression of hGRN paRNA and mRNA in these cells. As a control, the cells were exposed to the DMSO vehicle control. For this analysis, the qPCR reference gene was the geometric mean of GAPDH and PPIA normalized for iPSC-derived neurons treated with DMSO samples. n = 3.

[0362] hGRN mRNA was detected in iPSC-derived neurons and iMGL cells ( Figure 2A ) and paRNA( Figure 2B ). hGRN mRNA and paRNA were 15- to 20-fold more abundant in microglia (iMGL cells) compared to iPSC-derived neurons. Vorinostat treatment induced both hGRN mRNA and paRNA levels.

[0363] To test whether hGRN paRNA is also expressed in human cortical tissue, similar qPCR analysis was performed using human cortical tissue. Figure 3 As shown, hGRN paRNA was detected in human cortical tissue.

[0364] To evaluate the ability of ASOs targeting hGRN paRNA to regulate human GRN mRNA expression, 110 ASOs targeting hGRN paRNA (-strand) were tested in the hepatocellular carcinoma cell line HepG2. Briefly, HepG2 cells were transfected with 100 nM ASOs as indicated in Table 2. 48 hours after transfection, cells were collected for mRNA analysis by qPCR. The expression level of hGRN mRNA in cells treated with each ASO is provided in Table 2.

[0365]

[0366]

[0367] The best ASO based on the increased GRN mRNA fold change was selected for further characterization in a dose titration study. Briefly, human neuroblastoma SK-N-AS cells were transfected with ASOs CO-3423, CO-3431, CO-3463, and CO-3503 at 20 to 160 nM. A spatial non-targeting control ASO (sNTC) was used as a control. Cells were collected 48 hours later for mRNA analysis, and GRN mRNA was quantified by qPCR. The housekeeping genes used for normalization were GAPDH and PPIA, and the mRNA fold change (FC) was normalized for sNTC. Compared with the same dose of sNTC, CO-3423 upregulated GRN mRNA by approximately 2.5-fold ( Figure 4 ). CO-3431 down-regulated GRN levels by approximately 50%.

[0368] Thirty-six additional ASOs with different modifications were designed and base walking was performed around the selected ASOs. The effects of increasing doses of these ASOs were evaluated in the human neuroblastoma cell line SK-N-AS after transfection. Relative GRN mRNA levels in treated SK-N-AS cells were assessed 48 hours after transfection and data from exemplary ASOs are shown in Figure 5A and Figure 5B middle.

[0369] CO-3423 was subjected to additional chemical modifications, including LNA modification. SK-N-AS cells were transfected with increasing concentrations of LNA / PS modified ASOs, and GRN mRNA was quantified by qPCR. Different LNA modified ASOs including CO-3423 had similar efficacy (i.e., similar increase in GRN mRNA expression). The GRN mRNA fold changes induced by each ASO are shown in Table 3. GRN mRNA was normalized to the average fold change of cells treated with two spatial non-targeting control ASOs (CO-3772 and CO-1589 (sNTC1)).

[0370]

[0371] The effect of ASO length on the regulation of GRN gene expression was also investigated. SK-N-AS cells were transfected with increasing concentrations of longer ASOs and GRN mRNA was quantified by qPCR. As shown in Table 4, increasing the length of CO-3423 increased the potency of the ASO. The mRNA was targeted to the average fold change of cells treated with two spatial non-targeting control ASOs (CO-3772 and CO-1589 (sNTC1)).

[0372]

[0373]

[0374] To determine whether changes in PO / PS internucleotide linkages 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 mixed PO / PS linkage ASOs as described in Tables 5 and 6, and GRN mRNA was quantified by qPCR. ASOs containing mixed PO / PS internucleotide linkages did not affect the efficacy of CO-3423 or CO-3431.

[0375] The GRN mRNA fold changes following treatment with the mixed PO / PS bond form of CO-3423 are shown in Table 5. mRNA was normalized to the average fold change of cells treated with spatial non-targeting controls (CO-3772, CO-1589 (sNTC1), and CO-1929 (sNTC3)).

[0376]

[0377] As shown in Table 5, the PO / PS mixed version of CO-3423 had similar efficacy as the parent ASOCO-3423.

[0378] The fold changes in GRN mRNA following treatment with mixed PO / PS bond forms of CO-3431 are shown in Table 6. mRNA was normalized as described above.

[0379]

[0380] Similarly, the PO / PS blended version of CO-3431 has similar efficacy to CO-3431.

[0381] To further evaluate the effect of additional modifications on the ability of the CO-3423 parent ASO to regulate GRN gene expression, additional LNA tiling and PO bond modifications were incorporated into CO-3423. SK-N-AS cells were transfected with increasing concentrations of modified ASOs and GRN mRNA was quantified by qPCR. CO-3423 was used for comparison. GRN mRNA was normalized to the average fold change of cells treated with the spatial non-targeting control ASO CO-1589 (sNTC1). The GRN mRNA fold changes induced by each ASO are shown in Table 7.

[0382]

[0383] ASOs containing additional modifications had similar efficacy to ASOs containing LNA residues. CO-4452, CO-5268, and CO-5269 had similar or higher efficacy than CO-4113.

[0384] To further evaluate the effect of the modification on the ability of the CO-3463 ASO to regulate GRN gene expression, an ASO based on this ASO incorporating additional LNA modifications was prepared and compared with CO-3462. SK-N-AS cells were transfected with increasing concentrations of ASOs and GRN mRNA was quantified by qPCR. GRN mRNA was normalized to the average fold change of cells treated with the spatial non-targeting control ASO CO-1589 (sNTC1). The fold changes in GRN mRNA induced by each ASO are shown in Table 8.

[0385]

[0386]

[0387] ASOCO-3462, CO-5288, and CO-5289 upregulated GRN mRNA 2-fold compared to the control ASOCO-1589.

[0388] PGRN protein levels after ASO treatment were also assessed. To assess the effect of ASO treatment on PGRN protein expression, the following experiment was performed. 120 nM of various ASOs (CO-3423, CO-3431, CO-4113) were transfected into SK-N-AS cells. As a control, cells were treated with a spatial non-targeting control ASO CO-1589 (sNTC1). 48 h after transfection, proteins were extracted using RIPA lysis and extraction buffer (Thermo Fisher Scientific) and assayed using GRN ELISA (PGRN ELISA kit, PGRN protein levels were determined using the lysate from cells treated with sNTC1. Figure 6 As shown, CO-3423 and CO-4113 increased PGRN protein by 1.5- to 3-fold.

[0389] ASOs CO-3462, CO-3463, CO-41113, CO-4359, and CO-5269 also upregulated GRN mRNA in iMGL cells. To test the ability of ASOs targeting GRN paRNA to regulate GRN gene expression in iMGL cells, the following experiments were performed. In brief, iMGL cells were nuclear transfected with ASOs CO-3462, CO-3463, CO-4113, CO-4359, CO-5269, two spatial non-targeting control ASOs (CO-3772 and CO-1589 (sNTC1)) and negative control ASOs CO-5075 (described in Laudisi et al. (2019) Mol. Oncol. 13 (10): 2142-59). iMGL cells were collected 72 hours after nuclear transfection for mRNA quantification 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. Fig. 7A As shown, all tested ASOs (i.e., CO-3462, CO-3463, CO-4113, CO-4359, and CO-5269) upregulated GRN mRNA in iMGL cells compared to the control CO-1589. ASOs CO-4113, CO-4359, and CO-5269 also increased secreted PGRN protein in iMGL cells ( Figure 7B ).

[0390] ASOCO-3431, CO-3463 and CO-4113, CO-4359 and CO-5269 also up-regulated iPSC wild-type or GRN M1LTo test the regulation of GRN mRNA by ASO targeting GRN paRNA in wild-type iPSC-derived neurons and iPSC-derived neurons including those with GRN M1L missense mutation (GRN M1L To investigate the ability of iPSC-derived neurons to express GRN genes in sNTC1 (sNTC1; CO-1589) neurons, the following experiments were performed. Briefly, iPSC-derived neurons and GRN genes were expressed in sNTC1 (sNTC1; CO-1589) neurons using the indicated ASOs or a spatial non-targeting control as a negative control. M1L Neurons were nucleofected. Cells were collected 120 hours after nucleofection for mRNA quantification using qPCR and normalized to cells treated with sNTC1. Fig. 8A and 8B As shown, the tested ASOs upregulated wild-type iPSC neurons and GRNs M1L GRN mRNA in neurons.

[0391] CO-4113 rescues staurosporine-induced toxicity in GRN-FTD patient-derived neurons. GRN-FTD patient-derived neurons were treated with 10uM CO-4113 or sNTC (CO-1589) ASO. As positive controls, cells were treated with recombinant human PGRN protein (2.5nM) or brain-derived neurotrophic factor (BDNF) protein (1nM). After one week of treatment, neurons were treated with DMSO, 10nM or 100nM staurosporine. The culture medium was collected 24 hours later and LDH-Glo was used according to the manufacturer's instructions. TM Cytotoxicity was measured using a cytotoxicity assay (Promega Corp.). TM X-100 treated cells served as positive control. All values ​​were normalized to the positive control cells to calculate the percentage of cytotoxicity. The assay timeline is provided in Fig. 9A In. Fig. 9B As shown, treatment with ASOCO-4113 reduced the cytotoxicity of GRN-FTD patient-derived neuronal cells induced by 10 nm or 100 nM staurosporine.

[0392] 68 additional ASOs were designed to target intergenic enhancers (eRNAs). SK-N-AS cells were transfected with 120 nM ASOs as indicated in Table 9. 48 hours after transfection, cells were collected for GRN mRNA analysis using qPCR. CO-1589 (spatial non-targeting control ASO) was used as a control. GRN mRNA was normalized to cells treated with CO-1589. The GRN mRNA fold changes induced by each ASO are shown in Table 9 below.

[0393]

[0394]

[0395] The best hits of these ASOs were tested in a dose escalation study. Briefly, SK-N-AS cells were transfected with increasing concentrations of ASOs (3.75 nM to 120 nM) as indicated in Table 10. The cells were incubated for 48 hours. Subsequently, cells were collected for mRNA quantification by RT-qPCR. ASOs CO-3431, CO-4113, and CO-4124 targeting GRN paRNA were used as positive controls. GRN mRNA was normalized for cells treated with CO-1589. The GRN mRNA fold changes induced by each ASO are shown in Table 10 below.

[0396]

[0397] ASOs CO-4611, CO-4613, CO-4619, CO-4622 and CO-4631, CO-4637, CO-4649 were identified as initial hits. ASOs CO-4606 and CO-4619 showed greater than 1.3-fold upregulation of GRN mRNA.

[0398] 26 additional ASOs were designed to target intragenic enhancer RNAs (eRNAs). SK-N-AS cells were treated with 120 nM ASOs as shown in Table 11 for 48 hours. Cells were collected 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 changes in GRN mRNA induced by each ASO are shown in Table 11 below.

[0399]

[0400]

[0401] ASOCO-6411, CO-6445, and CO-6452 showed greater than 1.3-fold upregulation of GRN mRNA.

[0402] Additional ASOs based on the parent ASO CO-3462 were designed. SK-N-AS cells were transfected with increasing concentrations of ASOs based on CO-3462 designed to contain 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 GRN mRNA fold changes induced by each ASO are shown in Table 12.

[0403]

[0404] Additional ASOs based on the parental ASOs CO-4363 and CO-4364 were designed. SK-N-AS cells were transfected with increasing concentrations of ASOs based on CO-4363 and CO-4364 designed to contain additional chemical modifications, and GRN mRNA was quantified using qPCR. GRN mRNA was normalized to cells treated with CO-1589. The GRN mRNA fold changes (FC) induced by each ASO are shown in Table 13.

[0405]

[0406] ASOs CO-6416, CO-6417, CO-6420, CO-6423, and CO-6224 showed greater than 2-fold upregulation of GRN mRNA.

[0407] The IFNγ-induced immunosuppressive activity of the selected ASO was evaluated. In order to evaluate the ability of ASO targeting GRN regRNA to alleviate the immunosuppression induced by IFNγ, the following experiment was performed. In brief, iMGL cells were nuclear transfected with CO-4113, CO-4359 or CO-5269. Spatial non-targeted control ASO was used as a control. After ASO nuclear transfection, IFNγ was added to the culture medium of iMGL cells treated with ASO to induce an immune response. As an additional control, cells treated with spatial non-targeted control ASO were treated with phosphate-buffered saline (PBS) only. Cells and supernatants were collected for qPCR and ELISA quantification of secreted proteins (PGRN, IL-8 and CCL4) and gene expression (IL-6mRNA, CCL4 mRNA and CCL2 mRNA).

[0408] like Fig.19 As shown in Figure 2, treatment of iMGL cells with ASO targeting GRN regRNA upregulated secreted PGRN protein levels and significantly reduced the expression of IFNγ-mediated immune response genes IL-8 and CCL4, as shown by the reduced secretion of IL-8 and CCL4 proteins. Treatment of iMGL cells with ASO targeting GRN regRNA also significantly reduced the expression of IFNγ-mediated immune response genes IL-6, CCL4, and CCL2, as shown by their respective reduced mRNA levels ( Fig.19 ).

[0409] Example 2: Synthesis and in vitro characterization of ASO targeting mouse GRN regRNA

[0410] To determine whether GRN paRNA and mRNA are expressed in mouse CNS tissues, CNS tissues from C57 / BL6 mice were lysed and stained with TRIzol TM RNA was extracted using RT-PCR reagent (Thermo Fisher Scientific) and analyzed using qPCR. cDNA was synthesized with and without reverse transcriptase (RT) to ensure that the amplified products were derived from RNA in the samples. Fig.10 As shown in , GRN mRNA and paRNA were detected and expressed in the mouse cortex, hippocampus, striatum, cerebellum, and spinal cord.

[0411] To assess the levels of Pgrn protein in mouse samples, the following experiment was performed. Briefly, mouse plasma, CSF, and brain tissue from C57 / BL6 mice were obtained. Brain tissue (cortical or mixed brain regions) was homogenized in RIPA buffer containing protease inhibitors. Pgrn was expressed by ELISA (Mouse Pgrn ELISA Kit, Mouse Pgrn protein levels were quantified using the Biotinylation System (Biotinylation® Life Sciences; catalog number AG-45A-0019). Serum and brain samples were evaluated at different dilutions, and all sample results were normalized to total protein. Fig.11A , Fig. 11B and Fig. 11C As shown in , this assay detected mouse Pgrn in serum, CSF, cortex, and mixed brain region lysates.

[0412] 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, cells were exposed to 0.3uM, 1uM or 3uM vorinostat (VOR) or DMSO control for 24 hours, and total RNA was extracted from Neuro2a cells using the Qiagen RNeasy kit and cDNA was synthesized using random hexamers. Mouse Grn paRNA was detected by real-time quantitative PCR (qPCR) using two different primer sets (1F / 1R or 3F / 3R). Fig.12 As shown, mouse Grn paRNA levels increased with vorinostat treatment.

[0413] 91 spatial ASOs targeting mGrn regRNA were designed and synthesized. 33 ASOs were selected for tiling after the first screening. 88 ASOs were designed and synthesized for fine-tuning. Among them, 5 ASOs are spacers, 11 are spacers, 28 have PO / PS bonds, and 44 are hybrids.

[0414] 57 ASOs targeting the Grn promoter (-strand) were screened in Neuro2a cells. Neuro2a cells were transfected with 100 nM of each ASO listed in Table 14 and cells were harvested 48 hours later for Grn mRNA quantification using qPCR. The housekeeping genes used for normalization were Gapdh and Ppia. Relative Grn mRNA levels were normalized to mRNA extracted from cells treated with sNTC3 (spatial non-targeting control). Fig.13 As shown, ASOCO-3544 and CO-3595 targeting Grn regRNA upregulated mGrn mRNA in a dose-dependent manner and up to 1.5-fold and 1.8-fold, respectively.

[0415] The mGrn mRNA fold changes (FC) induced by 100 nM of each ASO are shown in Table 14.

[0416]

[0417]

[0418] Next, 26 ASOs based on the parent ASOs CO-3544 and CO-3595 with different modifications and base walking were designed. Neuro2a cells were transfected with 80, 120 or 160 nM of each ASO listed in Table 15, and cells were harvested 48 hours later for mGrn mRNA quantification using qPCR. As a control, cells were treated with sNTC3 or no ASO. The housekeeping genes used for normalization were Gapdh and Ppia. Relative Grn mRNA levels were normalized to mRNA extracted from cells treated with sNTC3. Four new ASOs (CO-4082, CO-4083, CO-4084, CO-4085) upregulated mGRN mRNA in Neuro2a cells by 1.5 to 2.5 times ( Fig.14 Similar upregulation was observed with these ASOs when used to treat primary mouse neuronal cells (1.5-6 μM by free uptake, Fig.15 ).

[0419] CO-3544 was modified to contain PO / PS bonds and the efficacy in upregulating mGrn mRNA was determined. As shown in Table 15 below, CO-3544 with up to 9 PO to PS internucleotide bond substitutions effectively upregulated mGrn mRNA in Neuro 2a cells at 40, 80 and 160 nM after transfection.

[0420]

[0421]

[0422] Similarly, CO-3595 was modified to contain different PO / PS internucleotide bonds, and the efficacy of these ASOs in upregulating mGrn mRNA was determined in Neuro2A cells. As shown in Table 16 below, CO-3595-based ASOs containing up to 13 PO / PS internucleotide bond substitutions effectively upregulated mGrn mRNA at 40, 80, and 160 nM after transfection.

[0423]

[0424] Example 3: In vivo regulation of mouse GRN expression using ASO targeting regRNA

[0425] Determine the upregulation of progranulin in different brain regions mediated by ASO in vivo.ASOCO-3544 (300 μg) in PBS was injected into the right lateral ventricle of 8-week-old C57 / BL6 mice.Use spatial non-targeted control ASO (CO-1929) and vehicle (PBS) control as control.Mice were killed on the 28th day after injection.Collect tissue samples (cortex, hippocampus and striatum) and process for total RNA and protein extraction as described above.Mouse Grn mRNA quantification was performed using real-time qPCR assay.

[0426] like Fig.16 As shown, treatment with CO-3544 increased Grn mRNA levels in different brain regions by more than 1.5-fold. Thus, this ASO exhibits in vivo therapeutic efficacy as shown by its upregulation of Grn mRNA levels in mouse CNS tissues.

[0427] Example 4: hGRNT Tg In vivo regulation of PGRN in mice

[0428] To evaluate the ability of ASOs targeting hGRN paRNA to upregulate hGRN in vivo, experiments using a human GRN transgenic mouse model were performed as follows.

[0429] Material

[0430] CO-4359 was modified to remove the 3' and 5' terminal nucleotides to generate CO-8178. CO-8178 was characterized in SK-N-AS, HEK293T, and Vero-76 cell lines as well as NGN2 neurons and iMGL cells as described previously (data not shown).

[0431] B6.Cg-Grn tm1.2Blrl Hprt1tm1(GRN)Blrl / J mice (hGRN Tg ; The Jackson Laboratory, strain number 036240) was used to evaluate the efficacy of ASOCO-8178 in upregulating human GRN in vivo. These mice express an X-linked transgenic human GRN gene (Hprttm1(GRN)Blrl) and have a knockout of exons 3 and 4 in the mouse Grn gene (Grntm1.2Blrl).

[0432] 5 μL of aCSF (vehicle control) or 100 μg CO-8178 were injected into the mouse intracerebroventricular (ICV) at a rate of 1 μl / min. Animals were sacrificed 3 weeks after administration for analysis. Mouse brain sections were processed to analyze PGRN protein and mRNA levels. GRN mRNA and intracellular and secreted PGRN were quantified using the above method.

[0433] hGRN was repeated as described above with ASOs CO-4452, CO-8883, CO-8903, CO-8879, CO-8871, CO-8873, CO-8873, CO-3462, and CO-6424 Tg In vivo assay in mice. aCSF and spatial non-targeted (NTC) were used as controls. Mouse brain slices were processed to measure PGRN protein and mRNA. GRN mRNA and intracellular and secreted PGRN protein were quantified using the above method.

[0434] result

[0435] CO-8178 upregulated GRN mRNA in SK-N-AS, HEK293T and Vero-76 cell lines as well as NGN2 neurons and iMGL cells (data not shown). CO-8178 also inhibited IFNγ-induced immune responses in iMGL cells (data not shown). Without wishing to be bound by theory, CO-8178 is an 18-mer, so it can be better distributed in tissues and is more likely to escape from endosomes.

[0436] like Fig. 20A and Fig. 20B As shown, in vivo treatment with CO-8178 upregulated GRN mRNA and protein in all CNS tissues evaluated. Fig. 20A Provides hGRN mRNA quantification, and Fig. 20B hGRNT TghPGRN protein quantification in mice. The bar graphs on the left of each tissue show samples from aCSF-treated control mice, and the bar graphs on the right of each tissue show samples from CO-8178-treated mice. GRN mRNA and protein expression levels correlated with treatment with CO-8178. r = 0.8158, 95% CI 0.7079-0.8899, r 2 =0.6699. P value <0.0001. In addition, after CO-8178 treatment, secreted PGRN protein in CSF was also upregulated ( Fig. 20C ). Thus, CO-8178 upregulated GRN mRNA and PGRN protein in human GRN mice in all CNS tissues measured. CO-8178 upregulated secreted PGRN protein in CSF. Furthermore, the upregulation of GRN mRNA and PGRN protein was significantly correlated.

[0437] like Fig.21A and Fig.21B As shown, 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 Provides hGRN mRNA quantification, and Fig.21B hGRNT Tg Quantification of human PGRN protein in mouse brain tissue.

[0438] Example 5: Grn + / - In vivo regulation of Grn mRNA in haploinsufficient mice

[0439] To evaluate mouse model analogs of human GRN haploinsufficiency, the following experiments were performed using Grn heterozygous knockout mice.

[0440] Materials and methods

[0441] Grn tm1.1Far (B6.129S4(FVB)-Grn tm1.1Far / Mmjax; The Jackson Laboratory, catalog number MMRRC strain #036771-JAX) heterozygous mice were used to evaluate the efficacy of CO-3544 and CO-10691 in upregulating mGrn in vivo. These mice have a deletion of exons 2-13 of the mouse Grn gene. CO-3544 and CO-10691 target the mouse Grn paRNA.

[0442] Mice were injected ICV with 5 μL artificial CSF (aCSF; vehicle control), 300 μg of ASOCO-3544 or 200 ug of ASOCO-10691 (each in aCSF) at a rate of 1 μL / min. For ASOCO-3544, mice were sacrificed 4 weeks after dosing for analysis. For CO-10691, mice were sacrificed at weeks 2 and 4 after dosing for analysis. Mouse brain sections were processed as described above, and Pgrn protein and Grn mRNA were quantified.

[0443] result

[0444] like Fig.22A and Fig. 22B As shown, ASOCO-3544 significantly reduced the expression of heterozygous Grn compared with aCSF vehicle control. tm1.1Far In vivo treatment of mice resulted in a 1.5- to 2.0-fold increase in Pgrn protein expression in the hippocampus, striatum, and cerebellum brain regions ( Fig. 22B Treatment with ASOCO-3544 increased Grn mRNA expression by up to 1.5-fold compared to aCSF vehicle control ( Fig.22A ).

[0445] ASOCO-10691 also upregulated mGrn mRNA in the cortex, hippocampus, striatum, and cerebellum brain regions compared to aCSF vehicle control ( Fig.23A , Fig. 23B , Fig.23C and Fig.23D ) and mouse Pgrn protein ( Fig.24 )Express.

[0446] Example 6: Synthesis and in vitro characterization of additional ASOs targeting human GRN regRNA

[0447] Other hGRN regRNA-targeting ASOs with additional chemical modifications were designed, synthesized, and characterized in vitro.

[0448] Materials and methods

[0449] Secreted and intracellular GRN levels

[0450] SK-N-AS cells were transfected with 90 nM ASOs CO-4359, CO-4452, CO-5268, CO-5269, CO-6424, and CO-8178. Spatially non-targeted control ASOs (sNTCs) were used as controls. Cells were collected 48 hours after transfection and analyzed by ELISA as described in Example 1 above. Protein levels were normalized to total protein and cells treated with sNTCs.

[0451] mRNA expression assay

[0452] SK-N-AS cells were transfected with selected ASOs at 3.75 to 90 nM. Scrambled ASOs (sNTCs) were used as controls. mRNA of cells was collected 48 hours after transfection. Housekeeping genes were GAPDH and PPIA, and mRNA fold changes were normalized to cells treated with sNTCs.

[0453] iPSC-derived neurons were nuclear transfected with 20 μM of ASO 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-targeted control ASOs (NTC-ASO-1 and NTC-ASO-2) were used as controls. Cells were harvested 5 days after nuclear transfection for GRN mRNA quantification. The qPCR reference genes were GAPDH and PPIA. Relative GRN mRNA levels were normalized for cells treated with one of the two non-targeted control ASOs.

[0454] Chemokine assay

[0455] iMGL cells were nuclear transfected with 5uM of CO-4452, CO-8865, CO-8866, and CO-8883. Non-targeting control ASO (NTC) was used as a control. After treatment, IFNγ was added to ASO-treated iMGL cells to induce an immune response. Cells and supernatants were collected for qPCR and ELISA quantification of GRN, CCL3, and CCL4 gene expression.

[0456] result

[0457] like Fig.25A and Fig.25B As shown, ASOs CO-4359, CO-4452, CO-5268, CO-5269, CO-6424, and CO-8178 upregulated secretion in SK-N-AS cells ( Fig.25A ) and intracellular ( Fig.25B 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 ( Fig.26A and Fig.26B ).

[0458] Upregulation of GRN mRNA in iPSC-derived neuronal 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 ( Fig. 27 ).

[0459] like Fig.28A , Fig.28B and Fig.28C As shown, ASOCO-4452, CO-8865, CO-8866, CO-8873, and CO-8883 also upregulated GRN mRNA expression ( Fig.28A ) and decreased the expression of chemokines (CCL3 and CCL4) induced by IFNγ in iMGL cells (respectively Fig.28B and 28C ).

[0460] Incorporated by Reference

[0461] Unless otherwise indicated to the contrary, the entire disclosure of each patent document and scientific article cited herein is incorporated by reference for all purposes.

[0462] Equivalent solutions

[0463] Without departing from the spirit or essential features of the present invention, the present invention may be implemented in other specific forms. Therefore, the foregoing embodiments should be considered illustrative in all respects, rather than limiting the present invention described herein. Therefore, the scope of the present invention is indicated by the appended claims rather than by the above description, and all changes within the meaning and scope of the equivalence of the claims are intended to be included herein.

Claims

1. An antisense oligonucleotide (ASO) complementary to at least 5 consecutive nucleotides of a GRN regulatory RNA (regRNA), wherein the regRNA has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-9.

2. The ASO of claim 1, wherein the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides away from the 3' end of the regRNA.

3. The ASO of claim 1, wherein the ASO is complementary to a sequence in the regRNA that is no more than 200 nucleotides away from the 5' end of the regRNA.

4. The ASO of claims 1-3, wherein the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 1369-4738.

5. The ASO of claims 1-3, wherein the regRNA has a nucleotide sequence of SEQ ID NO: 1, and the ASO comprises a nucleotide sequence of any one of SEQ ID NO: 10-268, 691, 991-1368, or 4743-4915.

6. The ASO of claims 1-3, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 2, and the ASO comprises the nucleotide sequence of any one of SEQ ID NO: 269-279.

7. The ASO of claims 1-3, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 3, and the ASO comprises the nucleotide sequence of any one of SEQ ID NO: 280-291 or 336-359.

8. The ASO of claims 1-3, wherein the regRNA has a nucleotide sequence of SEQ ID NO: 4, and the ASO comprises a nucleotide sequence of any one of SEQ ID NO: 292-313 or 360-380.

9. The ASO of claims 1-3, wherein the regRNA has a nucleotide sequence of SEQ ID NO: 5, and the ASO comprises a nucleotide sequence of any one of SEQ ID NO: 314-335 or 381-416.

10. The ASO of claims 1-3, wherein the regRNA has the nucleotide sequence of SEQ ID NO: 6, and the ASO comprises the nucleotide sequence of any one of SEQ ID NOs: 417-442.

11. The ASO of any one of claims 1-4, wherein the ASO comprises an RNA polynucleotide comprising one or more chemical modifications.

12. The ASO of claim 11, wherein each nucleotide in the ASO comprises a ribonucleotide having one or more chemical modifications.

13. The ASO of claim 11, wherein at least 3, 4 or 5 nucleotides at the 5' end and at least 3, 4 or 5 nucleotides at the 3' end of the ASO comprise ribonucleotides having one or more chemical modifications.

14. The ASO of claims 11-13, wherein the one or more chemical modifications include nucleotide sugar modifications including 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" or "NH3"), ), 2'-arabinosyl ("2'-arabinose") nucleotides, 2'-F-arabinosyl ("2'-F-arabinose") nucleotides, 2'-locked nucleic acid ("LNA") nucleotides, 2'-amide bridged nucleic acids (AmNA), 2'-unlocked nucleic acid ("ULNA") nucleotides, L-type sugar ("L-sugar"), 4'-thioribosyl nucleotides, constrained ethyl (cET), 2'-fluoro-arabinosyl (FANA) or thiomorpholino.

15. The ASO of any one of claims 11-14, wherein the one or more chemical modifications comprise an internucleotide linkage modification comprising a phosphorothioate ("PS" or (P(S))), a phosphoramidite (P(NR1R2) such as dimethylaminophosphoramidite (P(N(CH3)2)), a phosphonocarboxylate (P(CH2) n COOR) such as phosphonoacetate "PACE" (P(CH2COO - ))、Thiophosphonocarboxylate ((S)P(CH2) n COOR) such as thiophosphonoacetate "thioPACE" ((S)P(CH2COO - ))、alkylphosphonates (P(C 1-3 alkyl) such as one or more of methylphosphonate - P(CH3), borophosphonate (P(BH3)) and dithiophosphate (P(S)2).

16. The ASO of any one of claims 11-15, wherein the one or more chemical modifications comprise a nucleobase modification comprising 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-hydroxymethyl One or more of cytosine, 5-hydroxymethyluracil, 5,6-dehydrouracil, 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 nucleotides, Z bases, P bases, unstructured nucleic acids ("UNA"), isoguanine ("isoG"), isocytosine ("isoC"), glycerol nucleic acid (GNA), glycerol nucleic acid (GNA) or thiophosphoramide morpholine (TMO).

17. The ASO of any one of claims 11-16, wherein the one or more chemical modifications comprise biotin, palmitic acid, or a C18 moiety attached to the 5' end or the 3' end of the ASO.

18. The ASO of any one of claims 11-17, wherein the one or more chemical modifications comprise a 2'-O-methoxyethyl group, a 5-methyl group on cytidine, a locked nucleic acid (LNA), a phosphodiester (PO) internucleotide bond, or a phosphorothioate (PS) internucleotide bond.

19. The ASO of any one of claims 11-14, wherein the ASO does not comprise 10 or more consecutive nucleotides of unmodified DNA.

20. The ASO of claim 19, wherein the ASO does not comprise deoxyribonucleotides.

21. The ASO of any one of claims 11-20, wherein the ASO does not comprise unmodified ribonucleotides.

22. The ASO of any one of claims 11-21, wherein each ribonucleotide of the ASO is modified with a 2'-O-methoxyethyl group.

23. The ASO of any one of claims 11-21, wherein the length of the ASO is 3×n+10 nucleotides (n is an integer of 4 or greater), wherein the nucleotides at positions 3×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

24. The ASO of any one of claims 11-21, wherein the length of the ASO is 2×n+4 nucleotides (n is an integer of 8 or greater), wherein the nucleotides at positions 2×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

25. The ASO of any one of claims 11-21, wherein the length of the ASO is 3×n+2 nucleotides (n is an integer of 6 or greater), wherein the nucleotides at positions 3×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

26. The ASO of any one of claims 11-21, wherein the length of the ASO is 4×n+4 nucleotides (n is an integer of 4 or greater), wherein the nucleotides at positions 4×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

27. The ASO of any one of claims 11-21, wherein the length of the ASO is 5×n+5 nucleotides (n is an integer of 3 or greater), wherein the nucleotides at positions 5×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

28. The ASO of any one of claims 11-21, wherein the length of the ASO is 5×n+3 nucleotides (n is an integer of 3 or greater), wherein the nucleotides at positions 5×m are LNA-modified ribonucleotides (m is an integer of 1 to n), and the nucleotides at the remaining positions are 2'-O-methoxyethyl-modified ribonucleotides.

29. The ASO of any one of claims 11-21, wherein the length of the ASO is 2×n+8 nucleotides (n is an integer of 8 or greater), wherein the nucleotide at position 2×m is a LNA-modified ribonucleotide (m is an integer of 1 to n), and the nucleotide at position 2×m+1 is a 2'-O-methoxyethyl-modified ribonucleotide.

30. The ASO of any one of claims 11-21, wherein the length of the ASO is 2×n+8 nucleotides (n is an integer of 8 or greater), wherein the nucleotide at position 2×m+1 is a LNA-modified ribonucleotide (m is an integer of 1 to n), and the nucleotide at position 2×m is a 2'-O-methoxyethyl-modified ribonucleotide.

31. The ASO of any one of claims 11-30, wherein the ASO comprises at least one phosphodiester bond.

32. The ASO of any one of claims 11-14, wherein the ASO comprises 10 or more consecutive nucleotides of unmodified DNA flanked by at least 3 nucleotides of modified ribonucleotides at each of the 5' end and the 3' end.

33. The ASO of any one of claims 11-32, wherein each cytidine in the ASO is modified with a 5-methyl group.

34. The ASO of any one of claims 11-14, wherein the ASO comprises 2 or more consecutive nucleotides of unmodified DNA flanked by at least 3 nucleotides of modified ribonucleotides at each of the 5' end and the 3' end.

35. The ASO of any one of claims 1-34, wherein the regRNA is an eRNA.

36. The ASO of any one of claims 1-34, wherein the regRNA is paRNA.

37. A pharmaceutical composition comprising the ASO of any one of claims 1-36 and a pharmaceutically acceptable carrier.

38. A method of increasing transcription of a GRN in a human cell, the method comprising contacting the cell with the ASO of any one of claims 1-36 or the pharmaceutical composition of claim 37.

39. The method of claim 38, wherein the cell is a neuron.

40. The method of claim 38 or 39, wherein the ASO increases the amount of regulatory RNA in the cell.

41. The method of any one of claims 38-40, wherein the ASO increases the stability of a regulatory RNA in the cell.

42. The method of any one of claims 38-41, wherein the ASO increases the stability of PGRN mRNA in the cell.

43. The method of any one of claims 38-42, wherein the ASO increases the amount of PGRN protein in the cell.

44. A method of treating a disease or condition in a subject, the method comprising administering to a subject in need thereof an effective amount of the ASO of any one of claims 1-35 or the pharmaceutical composition of claim 37.

45. The method of claim 44, wherein the disease or condition is selected from the group consisting of frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), neuroinflammation, myopathy, familial frontotemporal dementia with neuropathic frontotemporal lobar degeneration associated with accumulation of TDP-43 inclusions (FTLD-TDP), Down syndrome, Huntington's disease, hippocampal sclerosis dementia, spinocerebellar ataxia 3, chronic traumatic encephalopathy, Alzheimer's disease (AD), myasthenia gravis, ALS, Gaucher disease (GD) and Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL) type 11 (CLN11), limbic system-dominant age-related TDP-43 encephalopathy (LATE), 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.

46. ​​The method of claim 45, wherein the disease or disorder is frontotemporal dementia (FTD).

47. The method of claims 44-46, wherein the ASO increases the amount of GRN regRNA in cells of the subject.

48. The method of any one of claims 44-47, wherein the ASO increases the stability of GRN regRNA in cells of the subject.

49. The method of any one of claims 44-48, wherein the ASO increases the amount of GRN mRNA in the cells of the subject.

50. The method of any one of claims 44-49, wherein the ASO increases the amount of PGRN protein in the cells of the subject.

51. The method of any one of claims 47-50, wherein the cell is a neuron.

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