Antisense oligonucleotides for modulating TMEM106B expression

Abstract

The present disclosure relates to antisense oligonucleotides capable of modulating the expression of TMEM106B in target cells. The antisense oligonucleotides hybridize to TMEM106B mRNA. The present disclosure further relates to methods of using the antisense oligonucleotides described herein.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety, said XML copy was created on Jan. 18, 2023, is named 51527-003WO2_SL.xml, and is 450,030 bytes in size.

[0002] Technical Field The present disclosure relates to antisense oligonucleotides that are complementary to transmembrane protein 106B (TMEM106B) messenger RNA (mRNA) and can inhibit expression of TMEM106B protein. Modulation of TMEM106B expression is beneficial for a range of medical disorders, such as neurodegenerative disorders, such as frontotemporal lobar degeneration. [Background technology]

[0003] Transmembrane protein 106B (TMEM106B) is a single-pass type 2 integral membrane glycoprotein that is primarily located within the membranes of endosomes and lysosomes. TMEM106B is expressed in neurons, glia, and endothelial cells. TMEM106B is thought to be involved in dendritic morphogenesis, such as dendritic branching, as well as lysosomal function. Multiple common neurodegenerative disorders, including frontotemporal lobar degeneration (FTLD), have been associated with dysregulation of TMEM106B expression. Neurodegenerative disorders represent a major class of neurological conditions for which curative treatments are lacking. Thus, there is a need for agents that modulate TMEM106B expression for use in therapeutic applications. Summary of the Invention

[0004] The present disclosure provides antisense oligonucleotides (ASOs) that reduce TMEM106B expression in vitro and in vivo. The present disclosure has identified specific ASO compounds that target regions in the pre-mRNA of human TMEM106B that strongly inhibit TMEM106B expression. The present disclosure also provides ASO sequences, modification motifs, compounds, conjugates, and salts that can reduce TMEM106B expression. Further provided by the present disclosure are methods for treating diseases or disorders characterized by neurodegeneration, such as, for example, frontotemporal lobar degeneration (FTLD), Parkinson's disease (or parkinsonism), cerebral leukodystrophy, amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), Alzheimer's disease (AD), motor neuron disease (MND), corticobasal syndrome (CBS), progressive supranuclear palsy (PSP), and neuronal ceroid lipofuscinosis (NCL). Such disorders may be characterized by dysregulation (eg, elevation) of TMEM106B expression or activity.

[0005] In one aspect, the disclosure provides an antisense oligonucleotide, the antisense oligonucleotide comprising: TIFF2025508310000001.tif95170, where non-italicized capital letters are beta-D-oxy LNA nucleosides, italicized capital letters are 2'-O-methyl nucleosides, and lowercase letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages. In some embodiments, the antisense oligonucleotide is compound number 26. In some embodiments, the antisense oligonucleotide is compound number 8_3. In some embodiments, the antisense oligonucleotide is compound number 8_4. In some embodiments, the antisense oligonucleotide is compound number 12. In some embodiments, the antisense oligonucleotide is compound number 24_3. In some embodiments, the antisense oligonucleotide is compound number 24_1. In some embodiments, the antisense oligonucleotide is compound number 29_5. In some embodiments, the antisense oligonucleotide is compound number 8_1. In some embodiments, the antisense oligonucleotide is compound number 10_4. In some embodiments, the antisense oligonucleotide is compound number 6. In some embodiments, the antisense oligonucleotide is compound number 29_3. In some embodiments, the antisense oligonucleotide is compound number 14. In some embodiments, the antisense oligonucleotide is compound number 15.

[0006] In some embodiments, the TMEM106B target nucleic acid is a mammalian TMEM106B target nucleic acid. In some embodiments, the mammalian TMEM106B target nucleic acid is a human TMEM106B target nucleic acid.

[0007] In another aspect, the disclosure provides a conjugate comprising any one of the antisense oligonucleotides described herein and at least one conjugate moiety covalently attached to said antisense oligonucleotide.

[0008] In another aspect, the present disclosure provides a pharma- ceutically acceptable salt of any one of the antisense oligonucleotides described herein.In another aspect, the present disclosure provides a pharma- ceutically acceptable salt of any one of the conjugates described herein.In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the antisense oligonucleotides described herein and a pharma- ceutically acceptable diluent, solvent, carrier, salt or adjuvant.In another aspect, the present disclosure provides a pharmaceutical composition comprising any one of the conjugates described herein and a pharma- ceutically acceptable diluent, solvent, carrier, salt or adjuvant.

[0009] In another aspect, the present disclosure provides an in vivo or in vitro method for modulating TMEM106B expression in a target cell expressing TMEM106B, the method comprising administering to the cell an effective amount of any one of the pharmaceutical compositions described herein.

[0010] In another aspect, the disclosure provides a method for treating or preventing a disease, the method comprising administering a therapeutically or prophylactically effective amount of any one of the pharmaceutical compositions described herein to a subject suffering from or susceptible to the disease.

[0011] In another aspect, the disclosure provides a pharmaceutical composition as described herein for use in the treatment or prevention of a disease.

[0012] definition The terms "antisense oligonucleotide" and "ASO" as used herein are defined as molecules that contain two or more covalently linked nucleosides. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. ASOs are generally produced in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to the sequence of an ASO, it refers to the sequence or order of the nucleobases of the covalently linked nucleotides or nucleosides, or modifications thereof. ASOs of the present disclosure are chemically synthesized and typically purified or isolated. ASOs of the present disclosure may contain one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides or inverted nucleosides, among others. ASOs can hybridize to a target polynucleotide, such as an mRNA molecule (e.g., TMEM106B mRNA), to promote degradation of the polynucleotide through RNA interference. ASOs of the present disclosure can modulate expression of a target gene by hybridizing to a target nucleic acid, particularly a contiguous sequence on the target nucleic acid. ASO is not double-stranded in nature, and therefore is not siRNA or shRNA.ASO of the present disclosure is single-stranded.It is understood that single-stranded ASO of the present disclosure can form hairpin or intermolecular duplex structure (duplex between two molecules of the same ASO) as long as the degree of complementarity over the entire length of the ASO is less than 50%.Advantageously, single-stranded ASO of the present disclosure does not contain RNA nucleosides, because the incorporation of RNA nucleotides reduces nuclease resistance.

[0013] Advantageously, the ASO of the present disclosure comprises one or more modified nucleosides or nucleotides, such as 2' sugar modified nucleosides.Furthermore, it is advantageous for the unmodified nucleoside to be a DNA nucleoside.

[0014] The term "contiguous nucleotide sequence" as used herein refers to a region of an ASO that is complementary to a TMEM106B target nucleic acid or target sequence. This term is used interchangeably herein with "contiguous nucleobase sequence" and "antisense oligonucleotide motif sequence." All of the nucleotides of an ASO may constitute a contiguous nucleotide sequence. An ASO may include a contiguous nucleotide sequence, such as the FG-F' gapmer region described herein, and may optionally include additional nucleotides, such as a nucleotide linker region that may be used to attach a functional group to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to a TMEM106B target nucleic acid. It is understood that the contiguous nucleotide sequence of an ASO cannot be longer than the ASO, and that an ASO cannot be shorter than the contiguous nucleotide sequence.

[0015] Nucleotides are the building blocks of ASOs and polynucleotides, and for purposes of this disclosure, include both naturally occurring and non-naturally occurring nucleotides. Naturally, nucleotides, such as DNA and RNA nucleotides, contain a ribose sugar moiety, a nucleobase moiety, and one or more phosphate groups (not present in nucleosides). Nucleosides and nucleotides may be referred to interchangeably as "units" or "monomers."

[0016] The term "modified nucleoside" or "nucleoside modification" as used herein refers to a nucleoside that is modified relative to an equivalent DNA or RNA nucleoside by the introduction of one or more modifications to the sugar moiety or nucleobase. In some cases, the modified nucleoside includes a modified sugar moiety. The term "modified nucleoside" may also be used interchangeably herein with the term "nucleoside analog" or modified "unit" or modified "monomer."

[0017] The term "modified internucleoside linkage" is defined as a linkage other than a phosphodiester (PO) linkage that covalently links two nucleosides to each other. Thus, the ASOs of the present disclosure include modified internucleoside linkages. Without wishing to be bound by theory, modified internucleoside linkages increase the nuclease resistance of ASOs compared to phosphodiester linkages. In naturally occurring ASOs, the internucleoside linkages include a phosphate group that creates a phosphodiester between adjacent nucleosides. Modified internucleoside linkages are particularly useful in stabilizing ASOs for in vivo use and may function to protect against nuclease cleavage in regions of DNA or RNA nucleosides within the ASOs of the present disclosure, such as within gap region G of a gapmer ASO, and in regions of modified nucleosides, such as regions F and F'.

[0018] The ASOs of the present disclosure may include one or more (e.g., 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, or more) internucleoside linkages modified from natural phosphodiester, such as, for example, one or more modified internucleoside linkages that are more resistant to nuclease attack (e.g., phosphorothioate linkages). Nuclease resistance can be determined by incubating the ASO in serum or by using a nuclease resistance assay (e.g., snake venom phosphodiesterase (SVPD)), both of which are well known in the art. An internucleoside linkage that can increase the nuclease resistance of an ASO is referred to as a nuclease-resistant internucleoside linkage. The ASO agent of the present disclosure may contain at least 50% (e.g., at least 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) modified internucleoside linkages relative to the total number of internucleoside linkages within the ASO. In some cases, all of the internucleoside linkages of the ASO or its contiguous nucleotide sequence are modified. It will be appreciated that the nucleoside that links the ASO of the present disclosure to a non-nucleotidic functional group, such as a conjugate, may be a phosphodiester. In some cases, all of the internucleoside linkages of the ASO or its contiguous nucleotide sequence are nuclease-resistant internucleoside linkages.

[0019] The modified internucleoside linkage may be selected from the group including phosphorothioate, diphosphorothioate, and boranophosphate. In some embodiments, the modified internucleoside linkage is compatible with the recruitment of RNase H to the ASO. The internucleoside linkage may include a sulfur (S) atom, as in the case of phosphorothioate internucleoside linkages. Phosphorothioate internucleoside linkages are particularly useful because they provide nuclease resistance, favorable pharmacokinetics, and ease of manufacture. The ASOs disclosed herein may include at least 50% (e.g., at least 51%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) phosphorothioate linkages. The ASO of the present disclosure may include internucleoside linkages that are all phosphorothioate internucleoside linkages. In some cases, the ASO of the present disclosure includes phosphorothioate internucleoside linkages and at least one phosphodiester linkage, for example, 2, 3, or 4 phosphodiester linkages. The phosphodiester linkage in the gapmer oligonucleotide, if present, is suitably not located between consecutive DNA nucleosides in the gap region G.

[0020] In some embodiments, the ASO comprises one or more natural internucleoside linkages, such as an internucleoside linkage selected from phosphotriester, methylphosphonate, MMI, amide-3, formacetal, or thioformacetal. Additional internucleoside linkages are disclosed in WO 2009 / 124238, which is incorporated herein by reference. In some cases, the internucleoside linkage is selected from the linkers disclosed in WO 2007 / 031091, which is incorporated herein by reference. In particular, the internucleoside bonds are -OP(O)2-O-, -OP(O,S)-O-, -OP(S)2-O-, -SP(O)2-O-, -SP(O,S)-O-, -SP(S)2-O-, -OP(O)2-S-, -OP(O,S)-S-, -SP(O)2-S-, -O-PO(R H)-O-, 0-PO(OCH3)-0-, -O-PO(NR H )-O-, -O-PO(OCH2CH2S-R)-O-, -O-PO(BH3)-O-, -O-PO(NHR H )-O-, -OP(O)2-NR H -, -NR H -P(O)2-O-, -NR H -CO-O-, and -NR H -CO-NR H or the internucleoside linker may be selected from -O-CO-O-, -O-CO-NR H -, -NR H -CO-CH2-, -O-CH2-CO-NR H -, -O-CH2-CH2-NR H -, -CO-NR H -CH2-, -CH2-NR H CO-, -O-CH2-CH2-S-, -S-CH2-CH2-O-, -S-CH2-CH2-S-, -CH2-SO2-CH2-, -CH2-CO-NR H -, -O-CH2-CH2-NR H -CO-, and -CH2-NCH3-O-CH2- (wherein R H may be selected from the group consisting of hydrogen and C1-4-alkyl.

[0021] Nuclease-resistant linkages such as phosphorothioate linkages are particularly useful in regions of the ASO that can recruit nucleases when duplexed with a TMEM106B target nucleic acid, such as region G of a gapmer. Phosphorothioate linkages may also be useful in non-nuclease recruiting or affinity enhancing regions of the ASO, such as regions F and F' within a gapmer. A gapmer ASO can contain one or more phosphodiester linkages in region F or F', or both regions F and F', and all internucleoside linkages in region G can be phosphorothioate.

[0022] In some embodiments, the ASOs of the disclosure contain phosphodiester inverted internucleoside linkages. The terms "inverted nucleoside" or "inverse nucleoside" or "DNA X INV " (X=A, T, C, G) refers to nucleosides containing a dimethoxytrityl (DMT) group and a phosphoramidite group that are inverted from the normal; the DMT group is attached to the 3'-OH of ribose and the phosphoramidite is attached to the 5'-OH of ribose. Synthesis of inverted oligonucleosides is described in the Materials and Methods section herein.

[0023] The term "nucleobase" as used herein refers to the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides, which form hydrogen bonds during nucleic acid hybridization. In the context of the present disclosure, the term nucleobase also encompasses modified nucleobases that may differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, and non-naturally occurring variants. Such variants are described, for example, in Hirao et al., Accounts of Chemical Research 45:2055 (2012) and Bergstrom, Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1 (2009).

[0024] The nucleobase moiety may be a purine or pyrimidine, e.g., a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., 5-methylcytosine, 5-hydroxycytosine, 5-methoxycytosine, N 4 -Methylcytosine, N 3 -Methylcytosine, N 4-ethylcytosine, pseudoisocytosine, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, 5-aminocytosine, 5-ethynylcytosine, 5-propynylcytosine, pyrrolocytosine, 5-aminomethylcytosine, 5-hydroxymethylcytosine, naphthyridine, 5-methoxyuracil, pseudouracil, dihydrouracil, 2-thiouracil, 4-thiouracil, 2-thiothymine, 4-thiothymine, 5,6-dihydrothymine, 5-halouracil, 5-propynyluracil, 5-aminomethyluracil, 5-hydroxymethyluracil, hypoxanthine, 7-deazaguanine, 8-aza-7-deazaguanine, 7-aza-2,6-diaminopurine, thienoguanine, N 1 -Methylguanine, N 2 -Methylguanine, 6-thioguanine, 8-methoxyguanine, 8-allyloxyguanine, 7-aminomethyl-7-deazaguanine, 7-methylguanine, imidazopyridopyrimidine, 7-deazaadenine, 3-deazaadenine, 8-aza-7-deazaadenine, 8-aza-7-deazaadenine, N 1 -Methyladenine, 2-methyladenine, N 6 The nucleic acid may be modified by changing it to a nucleobase selected from -methyladenine, 7-methyladenine, 8-methyladenine, or 8-azidoadenine.

[0025] The nucleobase moieties can be represented by the letter code of each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter can optionally include a modified nucleobase of equivalent function (i.e., complementary). For example, in an exemplary ASO, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

[0026] The term "modified antisense oligonucleotide" describes an ASO that contains one or more sugar-modified nucleosides, modified nucleobases, or modified internucleoside linkages. The term "chimeric ASO" has been used in the literature to describe ASOs with modified nucleosides.

[0027] The term "complementarity" describes the Watson-Crick base pairing ability of nucleosides / nucleotides. Watson-Crick base pairs are guanine (G)-cytosine (C) and adenine (A)-thymine (T) / uracil (U). It will be understood that ASOs may contain nucleosides with modified nucleobases. For example, 5-methylcytosine is often used in place of cytosine, and thus the term "complementarity" encompasses Watson-Crick base pairing between unmodified and modified nucleobases.

[0028] The term "% complementarity" as used herein refers to the percentage of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an ASO of the present disclosure) that are complementary to a reference sequence (e.g., a target sequence or sequence motif). The percentage of complementarity is calculated by counting the number of aligned nucleobases that are complementary between the two sequences (when aligning 5'-3' of the target sequence with 3'-5' of the ASO sequence), dividing that number by the total number of nucleotides in the ASO, and multiplying by 100. In such a comparison, nucleobases / nucleotides that do not align (i.e., do not form base pairs) are referred to as mismatched nucleobases / nucleotides. Insertions and deletions are not taken into account in calculating the percent complementarity of a contiguous nucleotide sequence. In determining complementarity, it will be understood that chemical modifications of nucleobases are disregarded (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity), so long as the functional ability of the nucleobase to form Watson-Crick base pairs is retained (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity). The term "fully complementary" refers to 100% complementarity.

[0029] The term "identity" as used herein refers to the proportion (expressed as a percentage) of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule (e.g., an ASO of the present disclosure) that are identical to a reference sequence (e.g., a sequence motif). The percentage of identity is calculated by counting the number of aligned nucleobases that are identical between the two sequences, dividing that number by the total number of nucleotides in the ASO, and multiplying by 100. Insertions and deletions are not taken into account in calculating the percentage identity of a contiguous nucleotide sequence. It will be understood that in determining identity, chemical modifications of nucleobases are disregarded (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity), so long as the functional ability of the nucleobases to form Watson-Crick base pairs is retained (e.g., 5'-methylcytosine is considered identical to cytosine for purposes of calculating % identity).

[0030] The terms "hybridizing" or "hybridize" as used herein refer to two nucleic acid strands (e.g., an ASO and a target nucleic acid) that form hydrogen bonds between base pairs on opposite strands, thereby forming a duplex between the two strands. The affinity of binding between two nucleic acid strands is the strength of hybridization. This is measured by the melting temperature (T), which is defined as the temperature at which half of the ASO becomes duplexed with the TMEM106B target nucleic acid. m ) is often described. Under physiological conditions, T m is not strictly proportional to affinity (Mergny and Lacroix, Antisense oligonucleotides 13:515-537 (2003)). The Gibbs free energy ΔG° at standard conditions more accurately represents the binding affinity, ΔG° = -RTln(K d ) to calculate the dissociation constant (K d), where R is the gas constant and T is the absolute temperature. Thus, the extremely low ΔG° of the reaction between the ASO and the TMEM106B target nucleic acid reflects the strong hybridization between the ASO and the target nucleic acid. ΔG° is the energy associated with a reaction in which the solute is at 1M in water, pH is 7, and temperature is 37°C. The hybridization of the ASO to the target nucleic acid is a spontaneous reaction with a negative ΔG° value, and is therefore thermodynamically favorable. ΔG° can be measured experimentally, for example, by using the isothermal titration calorimetry (ITC) method described in Hansen et al., Chem. Comm. 36-38 (1965) and Holdgate et al., Drug Discov Today (2005). Those skilled in the art will appreciate that commercially available instruments are available for measuring ΔG°. ΔG° can also be estimated numerically by using the nearest neighbor model described in Santa Lucia, PNAS 95:1460-1465 (1998) using appropriately derived thermodynamic parameters described in Sugimoto et al., Biochemistry 34:11211-11216 (1995) and McTigue et al., Biochemistry 43:5388-5405 (2004). To bind to an intended nucleic acid target by hybridization, the ASOs of the present disclosure hybridize to the target nucleic acid with an estimated ΔG° value of less than -10 kcal for ASOs of 10-30 nucleotides in length. The degree or strength of hybridization can be measured by ΔG°. The AOS may hybridize to a target nucleic acid with an estimated ΔG° value in the range of less than -10 kcal, e.g., less than -15 kcal, e.g., less than -20 kcal, e.g., less than -25 kcal, for an AOS of 8 to 30 nucleotides in length. In some cases, the ASO hybridizes to a target nucleic acid with an estimated ΔG° value of -10 to -60 kcal, e.g., -12 to -40, e.g., -15 to -30 kcal, or -16 to -27 kcal, e.g., -18 to -25 kcal.

[0031] The term "target nucleic acid" as used herein refers to a nucleic acid encoding mammalian (e.g., human) TMEM106B, which may be, for example, a gene, an RNA, a mature mRNA, a pre-mRNA, or a cDNA sequence. Thus, a target may be referred to as a "TMEM106B target nucleic acid," or simply "TMEM106B." Oligonucleotides of the present disclosure may target, for example, an exon of a mammalian TMEM106B RNA, or may target an intron in a TMEM106B pre-mRNA (see Table 1). Oligonucleotides may target an exon-exon boundary of an mRNA, or may target a pre-mRNA exon-intron boundary.

[0032] TIFF2025508310000002.tif101170

[0033] Suitably, the TMEM106B target nucleic acid encodes a TMEM106B protein, in particular a mammalian TMEM106B protein, such as a human TMEM106B protein. Tables 2 and 3 show the mRNA and pre-mRNA sequences of human and monkey TMEM106B.

[0034] In some embodiments, the TMEM106B target nucleic acid is selected from the group consisting of SEQ ID NO: 1, 2, 3, and 4, or naturally occurring variants thereof, including SNP variants. Known single nucleotide polymorphisms (SNPs) of the TMEM106B sequence of SEQ ID NO: 1 are shown in Table 4. When the oligonucleotides of the present disclosure are used for research or diagnostic purposes, the TMEM106B target nucleic acid may be a cDNA or a synthetic nucleic acid derived from DNA or RNA.

[0035] The ASOs of the present disclosure are typically capable of inhibiting expression of a TMEM106B target nucleic acid in a cell expressing the TMEM106B target nucleic acid. The contiguous sequence of nucleobases of the oligonucleotides of the present disclosure is typically complementary (fully or partially) to a TMEM106B target nucleic acid when measured over the entire length of the ASO, optionally excluding one or two mismatches, and optionally excluding a nucleotide-based linker region that may link the ASO to an optional functional group, such as a conjugate or other non-complementary terminal nucleotide (e.g., region D' or D''). In some embodiments, the TMEM106B target nucleic acid may be a mature mRNA or a pre-mRNA.

[0036] In some embodiments, the TMEM106B target nucleic acid is an RNA encoding a mammalian TMEM106B protein, such as a human TMEM106B pre-mRNA sequence, such as that disclosed as SEQ ID NO: 1, or a human mature mRNA, such as that disclosed in SEQ ID NO: 2. Exemplary target nucleic acids are shown in Tables 1-3.

[0037] TIFF2025508310000003.tif62170Fwd = forward strand. Rv = reverse strand. Genomic coordinates provide the pre-mRNA sequence (genomic sequence). NCBI reference sequence provides the mRNA sequence (cDNA sequence).

[0038] TIFF2025508310000004.tif54170

[0039] The term "target sequence" as used herein refers to a sequence of nucleotides present in a TMEM106B target nucleic acid, including a nucleobase sequence that is complementary to an oligonucleotide of the present disclosure. In some embodiments, the target sequence consists of a region on a TMEM106B target nucleic acid having a nucleobase sequence that is complementary to a contiguous nucleotide sequence of an oligonucleotide of the present disclosure. This region of the TMEM106B target nucleic acid may be referred to interchangeably as a target nucleotide sequence, a target sequence, or a target region. In some embodiments, the target sequence is longer than the complementary sequence of a single oligonucleotide, and can represent a preferred region of a TMEM106B target nucleic acid that may be targeted by, for example, multiple oligonucleotides of the present disclosure.

[0040] The target sequence to which the ASO is complementary or typically hybridizes comprises a contiguous nucleobase sequence of at least 10 nucleotides (e.g., at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or more nucleotides). In some embodiments, the contiguous nucleotide sequence is between 10 and 50 nucleotides, e.g., 10 to 30 nucleotides, e.g., 14 to 20, e.g., 15 to 18 contiguous nucleotides.

[0041] The term "target cell" as used herein refers to a cell expressing a TMEM106B target nucleic acid. In some embodiments, the target cell may be in vivo or in vitro. In some embodiments, the target cell is a mammalian cell, such as a rodent cell, e.g., a mouse cell or a rat cell, or a primate cell, e.g., a monkey cell or a human cell. Exemplary target cells may include neuronal cells, e.g., neuronal cells expressing TMEM106B, e.g., astrocytes, oligodendrocytes, microglia, and ependymal cells. The target cell may express TMEM106B mRNA, e.g., TMEM106B pre-mRNA or TMEM106B mature mRNA. The polyA tail of TMEM106B mRNA is typically ignored for ASO targeting. Where tests are being conducted to assess the knockdown efficacy of the ASOs of the present disclosure, the term "target cells" may include human SK-N-BE(2) neuroblastoma cells, neurons or glial cells derived from induced pluripotent stem cells (iPSCs), or retinal pigment epithelial cells.

[0042] The term "naturally occurring variant" as used herein refers to a variant of the TMEM106B gene or transcript thereof, which may differ, for example, due to alternative splicing of pre-mRNA, or the degeneracy of the genetic code resulting in a multiplicity of codons encoding the same amino acid due to the presence of polymorphisms, such as SNPs and allelic variants. Naturally occurring variants may have at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more) homology to a mammalian TMEM106B target nucleic acid, such as a target nucleic acid selected from the group consisting of SEQ ID NOs: 1-4. Naturally occurring variants of TMEM106B may also include the SNPs set forth in Table 4.

[0043] TIFF2025508310000005.tif255170TIFF2025508310000006.tif255170TIFF2025508310 000007.tif255170TIFF2025508310000008.tif255170TIFF2025508310000009.tif25517 0TIFF2025508310000010.tif255170TIFF2025508310000011.tif255170TIFF2025508310 000012.tif255170TIFF2025508310000013.tif255170TIFF2025508310000014.tif48170

[0044] The term "modulation of expression" as used herein should be understood as a general term for the ability of an ASO to change the amount of TMEM106B when compared to the amount of TMEM106B before administration of the ASO. Alternatively, modulation of expression can be determined by reference to a control experiment. The control is generally understood to be an individual or target cell treated with a saline composition, or an individual or target cell treated with a non-targeting ASO (scrambled or mock control). One type of modulation is the ability of an ASO to inhibit, downregulate, reduce, suppress, eliminate, stop, block, prevent, reduce, decrease, or terminate the expression of TMEM106B, for example, by degradation of mRNA or suppression of transcription. Another type of modulation is the ability of an ASO to restore, increase, or enhance the expression of TMEM106B, for example, by repairing a splice site or preventing splicing, or by removing or blocking an inhibitory mechanism, for example, suppressing microRNA. In the context of the present disclosure, the ASOs described herein act to reduce or suppress TMEM106B expression.

[0045] As used herein, a "high affinity modified nucleoside" when incorporated into an ASO, e.g., m A high affinity modified nucleoside of the present disclosure is a modified nucleotide that enhances the affinity of the ASO for its complementary target as measured by T mmay be increased by about +0.5 to +12° C., +1.5 to +10° C., or +3 to +8° C. per modified nucleoside. A number of high affinity modified nucleosides are known in the art, including, for example, 2′-substituted nucleosides, as well as locked nucleic acids (LNAs) (see Freier et al., Nucl. Acid Res. 25:4429-43 (1997) and Uhlmann, Curr. Opinion in Drug Development 3:293-213 (2000)).

[0046] The oligomers of the present disclosure may include one or more nucleosides with modified sugar moieties. A number of nucleosides containing modifications of the ribose sugar moiety have been created with the primary goal of improving certain properties of ASOs, such as affinity, nuclease resistance, or pharmacokinetics. Such modifications include those in which the ribose ring structure is modified, for example, by replacement with a bicyclic ring (LNA) or a hexose ring (HNA), which typically have a biradical bridge between the C2 and C4 carbons on the ribose ring, or an unlinked ribose ring (e.g., UNA), which typically lacks a bond between the C2 and C3 carbons. Other sugar-modified nucleosides include, for example, bicyclohexose nucleic acids (WO 2011 / 017521) or tricyclonucleic acids (WO 2013 / 154798). Modified nucleosides also include nucleosides in which a sugar moiety is replaced with a non-sugar moiety, for example, in the case of peptide nucleic acids (PNAs) or morpholino nucleic acids.

[0047] Sugar modifications also include modifications made by changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group that occurs naturally in DNA and RNA nucleosides. Substituents can be introduced at the 2', 3', 4', or 5' positions of the sugar moiety.

[0048] As used herein, a "2' sugar modified nucleoside" is a nucleoside that has a substituent other than H or -OH at the 2' position (e.g., a 2'-substituted nucleoside) or contains a 2'-linked biradical that can form a bridge between the 2' carbon of the ribose ring and a second carbon, such as an LNA (2'-4' biradical bridge) nucleoside. A number of 2'-substituted nucleosides have been found to have beneficial properties when incorporated into ASOs. For example, a 2'-modified sugar may enhance binding affinity or increase the nuclease resistance of the ASO. Examples of 2'-substituted modified nucleosides include 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA,

[0049] 2'-fluoro-RNA, and 2'-F-ANA nucleosides. For further examples, see, for example, Freier et al., Nucl.Acid Res.25:4429-4443(1997) and Uhlmann Curr.Opinion in Drug Development 3:293-313(2000), and Deleavey et al., Chemistry and Biology 19:937(2012). Below is a description of some 2'-modified nucleosides. TIFF2025508310000015.tif75170

[0050] "LNA nucleosides" are 2'-modified nucleosides that contain a biradical (also called a "2'-4' bridge") linking C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or locks the conformation of the ribose ring. These nucleosides are also called bridged nucleic acids or bicyclic nucleic acids (BNAs) in the literature. Ribose locking is associated with increased affinity of hybridization (duplex stabilization) when LNAs are incorporated into ASOs. This can be routinely determined by measuring the melting temperature of the antisense oligonucleotide / target duplex.

[0051] Non-limiting examples of LNA nucleosides include those described 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. al., Bioorg.Med.Chem.Lett.12:73-6(2002), Seth et al., J.Org.Chem.75:1569-81(2010), Mitsuoka et al., Nucleic Acid Res.37:1225-38(2009), and Wan et al., J.Med.Chem.59:9645-67(2016). Other exemplary LNA nucleosides are disclosed in Scheme 1 below.

[0052] Scheme 1 TIFF2025508310000016.tif138170

[0053] The LNA nucleoside may be beta-D-oxy-LNA, 6'-methyl-beta-D-oxy-LNA, such as (S)-6'-methyl-beta-D-oxy-LNA (ScET), and ENA. A particularly advantageous LNA is beta-D-oxy-LNA.

[0054] Example nucleosides using HELM annotation notation: DNA nucleosides TIFF2025508310000017.tif42170

[0055] Beta-D-oxy-LNA nucleosides TIFF2025508310000018.tif44170

[0056] 2'-O-Methyl Nucleosides TIFF2025508310000019.tif44170

[0057] Inverted nucleosides (when incorporated into oligonucleotides containing phosphodiester bonds) TIFF2025508310000020.tif42170

[0058] DNA 7-deaza-8-aza-guanine nucleoside JPEG2025508310000021.jpg39170

[0059] Representation of exemplary phosphorothioate internucleoside linkages according to HELM annotation [sP] TIFF2025508310000022.tif17170

[0060] Representative phosphodiester internucleoside linkages as per HELM annotation [P] TIFF2025508310000023.tif16170

[0061] The dashed lines represent the covalent bond between each nucleoside and the 5' or 3' phosphorothioate internucleoside linkage. In the 5' terminal nucleoside, the 5' dotted line represents the bond to a hydrogen atom (forming the 5' terminal -OH group). In the 3' terminal nucleoside, the 3' dotted line represents the bond to a hydrogen atom (forming the 3' terminal -OH group).

[0062] Nuclease-mediated degradation refers to ASOs that can mediate the degradation of complementary nucleotide sequences when they form a duplex with the complementary nucleotide sequence. In some embodiments, ASOs can function through nuclease-mediated degradation of TMEM106B target nucleic acid when the ASO of the present disclosure can recruit nucleases, particularly endonucleases, preferably endoribonucleases (RNases), such as RNase H. An example of an ASO design that acts through a nuclease-mediated mechanism is an ASO that typically comprises a region of at least 5 or 6 consecutive DNA nucleosides, flanked on one or both sides by affinity-enhancing nucleosides, such as gapmers, headmers and tailmers.

[0063] RNase H activity of an ASO refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine RNase H recruitment ability. Typically, an ASO is considered to be capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate, measured in pmol / l / min, of at least 5%, such as at least 10%, or more than 20%, of the initial rate determined when using an ASO that has the same base sequence as the modified ASO being tested, but contains only DNA monomers with phosphorothioate linkages between all monomers in the ASO, and using the methodology provided by Examples 91-95 of WO 01 / 23613 (incorporated herein by reference). Recombinant human RNase H1 is available from Lubio Science GmbH, Lucerne, Switzerland for use in determining RNase H activity.

[0064] The ASO of the present disclosure, or its contiguous nucleotide sequence, may be a gapmer, also called a gapmer ASO or gapmer design. Antisense gapmers are generally used to inhibit target nucleic acids via RNase H-mediated degradation. Gapmer ASOs contain at least three distinct structural regions in the '5->3' direction: a 5' flanking region, a gap, and a 3' flanking region (FGF). The "gap" region (G) contains a stretch of contiguous DNA nucleotides that allows the ASO to recruit RNase H. The gap region is flanked by a 5' flanking region (F) that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides, and a 3' flanking region (F') that contains one or more sugar-modified nucleosides, advantageously high affinity sugar-modified nucleosides. One or more sugar-modified nucleosides in regions F and F' enhance the affinity of the ASO for a TMEM106B target nucleic acid (i.e., are affinity-enhancing sugar-modified nucleosides). In some embodiments, one or more sugar-modified nucleosides in regions F and F' are high affinity 2' sugar modifications independently selected from 2' sugar-modified nucleosides, e.g., LNA and 2'-MOE.

[0065] In a gapmer design, the nucleosides at the 5' and 3' ends of the gap region are DNA nucleosides, positioned adjacent to sugar-modified nucleosides in the 5' (F) or 3' (F') regions, respectively. The flanking portions may be further defined by having at least one sugar-modified nucleoside at the end furthest from the gap region, i.e., the 5' end of a 5' flanking portion and the 3' end of a 3' flanking portion.

[0066] The regions FG-F' form a contiguous nucleotide sequence. An ASO of the disclosure, or a contiguous nucleotide sequence thereof, may comprise a gapmer region of the formula FG-F'.

[0067] The overall length of the gapmer design FG-F' may be, for example, from 12 to 32 nucleosides, such as from 13 to 24, such as from 14 to 22 nucleosides, such as from 14 to 17, such as from 16 to 18 nucleosides, such as from 16 to 20 nucleotides.

[0068] By way of example, a gapmer ASO of the disclosure can be represented by the formula: 1-8 -G 6-16 -F' 2-8 , e.g., F 2-8 -G 6-14 -F' 2-8 , e.g., F 3-8 -G 6-14 -F' 2-8 with the proviso that the overall length of the gapmer region FG-F' is at least 10, such as at least 12, such as at least 14 nucleotides in length.

[0069] In one aspect of the disclosure, the ASO or contiguous nucleotide sequence thereof consists of or comprises a gapmer of the formula 5'-FG-F'-3', where regions F and F' independently comprise or consist of 1-8 nucleosides, 2-4 of which are 2' sugar modified, and define the 5' and 3' ends of the F and F' regions, and G is a region between 6-16 nucleosides capable of recruiting RNase H. Regions F, G, and F' are further defined below and can be incorporated into the formula FG-F'.

[0070] The region G (gap region) of a gapmer is a region of nucleosides that allows the ASO to recruit RNase H, e.g. human RNase H1, typically a DNA nucleoside. RNase H is a cellular enzyme that recognises a duplex between DNA and RNA and enzymatically cleaves the RNA molecule. Suitably, a gapmer may have a gap region (G) of at least 5 or 6 consecutive DNA nucleosides, such as 5-16 consecutive DNA nucleosides, such as 6-15 consecutive DNA nucleosides, such as 7-14 consecutive DNA nucleosides, such as 8-12 consecutive DNA nucleotides, such as 8-12 consecutive DNA nucleotides in length. The gap region G in some embodiments consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 consecutive DNA nucleosides. Cytosine (C) DNA in the gap region may be methylated, and such residues are designated as 5-methyl-cytosine ( me Cytosine DNA methylation within the gap is advantageous when a CG dinucleotide is present within the gap to improve therapeutic safety, and the modification does not significantly affect the efficacy of the ASO. 5' substituted DNA nucleosides, such as 5' methyl DNA nucleosides, have been reported for use in DNA gap regions (EP2742136).

[0071] In some embodiments, the gap region G can consist of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive phosphorothioate linked DNA nucleosides. In some embodiments, all internucleoside linkages within the gap are phosphorothioate linkages.

[0072] Although traditional gapmers have a DNA gap region, there are numerous examples of modified nucleosides that allow for the recruitment of RNase H when used within the gap region. Modified nucleosides reported to be capable of recruiting RNase H when included within the gap region include, for example, alpha-L-LNA, C4' alkylated DNA (International Application EP 2009 / 050349 and Vester et al., Bioorg. Med. Chem. Lett. 18 (2008) 2296-2300, both of which are incorporated herein by reference), nucleosides derived from arabinose such as ANA and 2'F-ANA (Mangos et al. 2003 J. AM. CHEM. SOC. 125, 654-661), UNA (unlocked nucleic acid) (Fluiter et al., Mol. Biosyst., 2009, 10, 1039, incorporated herein by reference). UNAs are typically unlocked nucleic acids in which the bond between C2 and C3 of the ribose has been removed to form an unlocked "sugar" residue. The modified nucleosides used in such gapmers can be nucleosides that adopt a 2'-endo (DNA-like) structure when introduced into the gap region, i.e., a modification that allows for RNase H recruitment. In some embodiments, the DNA gap region (G) described herein can optionally contain one to three sugar-modified nucleosides that adopt a 2'-endo (DNA-like) structure when introduced into the gap region.

[0073] Alternatively, there have been numerous reports of inserting modified nucleosides that confer a 3'-end conformation in the gap region of a gapmer while retaining some RNase H activity. Such gapmers with a gap region containing one or more 3'-end modified nucleosides are called "gap breaker" or "gap-disrupting" gapmers. See, for example, WO 2013 / 022984. Gap breaker ASOs retain a sufficient region of DNA nucleosides within the gap region to allow RNase H recruitment. The RNase H recruitment ability of gap breaker ASO designs is sequence-specific or, in some cases, compound-specific (see Rukov et al. 2015 Nucl.Acids Res.Vol.43 pp.8476-8487, which discloses "gap breaker" ASOs that recruit RNase H, which in some cases provides more specific cleavage of target RNA). Modified nucleosides used within the gap region of a gap breaker ASO can be, for example, a modified nucleoside that confers a 3' endo conformation, such as a 2'-O-methyl (OMe) or 2'-O-MOE (MOE) nucleoside, or a beta-D LNA nucleoside (in which the bridge between the C2' and C4' of the ribose sugar ring of the nucleoside is in a beta conformation), such as beta-D-oxy LNA or ScET nucleoside.

[0074] Similar to the gapmers containing region G above, the gap region of a gap breaker or gap disrupting gapmer has a DNA nucleoside at the 5' end of the gap (adjacent to the 3' nucleoside of region F) and a DNA nucleoside at the 3' end of the gap (adjacent to the 5' nucleoside of region F'). Gapmers containing disrupted gaps typically retain a region of at least three or four contiguous DNA nucleosides at either the 5' or 3' end of the gap region.

[0075] Exemplary designs for Gap Breaker ASOs include: 1-8 -[D 3-4 -E1-D 3-4 ] - F' 1-8 ;F1-8 -[D 1-4 -E1-D 3-4 ]-F' 1-8 ; and F 1-8 -[D 3-4 -E1-D 1-4 ]-F' 1-8 where the region G is the bracket [D n -E r -D m ] within the gapmer region FG-F', D is a contiguous sequence of DNA nucleosides, E is a modified nucleoside (gap breaker or gap disrupting nucleoside), and F and F' are flanking regions as defined herein, with the proviso that the overall length of the gapmer region FG-F' is at least 12, e.g., at least 14, nucleotides in length. In some embodiments, region G of the gap disrupting gapmer comprises at least 6 DNA nucleosides, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 DNA nucleosides. As above, the DNA nucleosides may be contiguous or, optionally, intermixed with one or more modified nucleosides, with the proviso that the gap region G is capable of mediating RNase H recruitment.

[0076] Region F is located immediately adjacent to the 5' DNA nucleoside of region G. The 3'-most nucleoside of region F is a sugar-modified nucleoside, such as a high affinity sugar-modified nucleoside, such as a 2'-substituted nucleoside, such as an MOE nucleoside or an LNA nucleoside.

[0077] Region F' is located immediately adjacent to the 3' DNA nucleoside of region G. The 5'-most nucleoside of region F' is a sugar-modified nucleoside, such as a high affinity sugar-modified nucleoside, such as a 2'-substituted nucleoside, such as an MOE nucleoside or an LNA nucleoside.

[0078] Region F is 1 to 8 contiguous nucleotides in length, such as 2 to 6, such as 3 to 4 contiguous nucleotides in length. Advantageously, the 5'-most nucleoside of region F is a sugar-modified nucleoside. In some embodiments, the two 5'-most nucleosides of region F are sugar-modified nucleosides. In some embodiments, the 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are LNA nucleosides. In some embodiments, the two 5'-most nucleosides of region F are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 5'-most nucleosides of region F are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 5'-most nucleosides of region F are 2'-substituted nucleosides, such as MOE nucleosides.

[0079] Region F' is 2 to 8 contiguous nucleotides in length, such as 3 to 6, such as 4 to 5 contiguous nucleotides in length. Advantageously, in embodiments, the 3'-most nucleoside of region F' is a sugar-modified nucleoside. In some embodiments, the two 3'-most nucleosides of region F' are sugar-modified nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the 3'-most nucleosides of region F' are LNA nucleosides. In some embodiments, the two 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as two 3'MOE nucleosides. In some embodiments, the 3'-most nucleosides of region F' are 2'-substituted nucleosides, such as MOE nucleosides. It should be noted that when the length of region F or F' is 1, it is advantageously an LNA nucleoside.

[0080] In some embodiments, regions F and F' independently consist of or comprise a contiguous sequence of sugar-modified nucleosides. In some embodiments, the sugar-modified nucleosides of region F can be independently selected from 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, LNA units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments, regions F and F' independently comprise both LNA and 2'-substituted modified nucleosides (mixed wing designs).

[0081] In some embodiments, regions F and F' are either MOE only or beta - D - It consists of only one type of sugar-modified nucleoside, such as only oxyLNA or only ScET. Such designs are also called uniform flanking or uniform gapmer designs.

[0082] In some embodiments, all nucleosides of regions F or F' are LNA nucleosides, e.g., independently selected from beta-D-oxyLNA, ENA, or ScET nucleosides. In some embodiments, region F consists of 1 to 5, e.g., 2 to 4, e.g., 3 to 4, e.g., 1, 2, 3, 4, or 5 contiguous LNA nucleosides. In some embodiments, all nucleosides of regions F and F' are beta-D-oxyLNA, ENA, or ScET nucleosides. - It is a D-oxy LNA nucleoside.

[0083] In some embodiments, all of the nucleosides of region F or F' are 2'-substituted nucleosides, such as OMe or MOE nucleosides. In some embodiments, region F consists of 1, 2, 3, 4, 5, 6, 7 or 8 contiguous OMe or MOE nucleosides. In some embodiments, only one of the flanking regions can consist of a 2'-substituted nucleoside, such as an OMe or MOE nucleoside. In some embodiments, it is the 5' (F) flanking region that consists of a 2'-substituted nucleoside, such as an OMe or MOE nucleoside, and the 3' (F') flanking region comprises at least one LNA nucleoside, such as a beta-D-oxy LNA nucleoside or a cET nucleoside. In some embodiments, it is the 3' (F') flanking region that consists of a 2' substituted nucleoside, such as an OMe or MOE nucleoside, and the 5' (F) flanking region comprises at least one LNA nucleoside, such as a beta-D-oxy LNA nucleoside or a cET nucleoside.

[0084] In some embodiments, all of the modified nucleosides in regions F and F' are independently selected from LNA nucleosides, e.g., beta-D-oxy LNA, ENA, or ScET nucleosides, and regions F or F' may optionally include DNA nucleosides (alternating flanks; see these definitions for details). In some embodiments, all of the modified nucleosides in regions F and F' are beta-D-oxy LNA nucleosides, and regions F or F' may optionally include DNA nucleosides (alternating flanks; see these definitions for details). In some embodiments, the 5'-most and 3'-most nucleosides in regions F and F' are LNA nucleosides, e.g., beta-D-oxy LNA nucleosides or ScET nucleosides.

[0085] In some embodiments, the internucleoside linkage between region F and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between region F' and region G is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage between the nucleosides of regions F or F', F and F' are phosphorothioate internucleoside linkages.

[0086] An "LNA gapmer" is a gapmer in which one or both of regions F and F' comprise or consist of LNA nucleosides. A beta-D-oxy gapmer is a gapmer in which one or both of regions F and F' comprise or consist of beta-D-oxy LNA nucleosides. In some embodiments, an LNA gapmer has the formula: [LNA] 1-5 -[Area G]-[LNA] 1-5 and region G is as defined in the definition of gapmer region G. In one embodiment, the LNA gapmer has the formula [LNA]4-[region G] 10-12 -[LNA]4.

[0087] An MOE gapmer is a gapmer in which regions F and F' consist of MOE nucleosides. In some embodiments, an MOE gapmer has the design [MOE] 1-8 -[Area G] 5-16 -[MOE] 1-8 , e.g. [MOE] 2-7 -[Area G] 6-14 -[MOE] 2-7 , e.g. [MOE] 3-6 -[Area G] 8-12 -[MOE] 3-6 and region G is as defined in the gapmer definition. MOE gapmers with the 5-10-5 design (MOE-DNA-MOE) are widely used in the art.

[0088] A "mixed wing gapmer" is an LNA gapmer in which one or both of regions F and F' comprise a 2'-substituted nucleoside, e.g., an MOE nucleoside, independently selected from the group consisting of 2'-O-alkyl-RNA units, 2'-O-methyl-RNA, 2'-amino-DNA units, 2'-fluoro-DNA units, 2'-alkoxy-RNA, MOE units, arabinonucleic acid (ANA) units, and 2'-fluoro-ANA units. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least one LNA nucleoside, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some embodiments in which at least one of regions F and F', or both of regions F and F' comprise at least two LNA nucleosides, the remaining nucleosides in regions F and F' are independently selected from the group consisting of MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides. Mixed wing gapmer designs are disclosed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated herein by reference.

[0089] The flanking regions may contain both LNA and DNA nucleosides and are referred to as "alternating flanking regions" because they contain alternating motifs of LNA-DNA-LNA nucleosides. Gapmers containing such alternating flanking regions are referred to as "alternating flanking region gapmers." An "alternating flanking region gapmer" is an LNA gapmer ASO in which at least one of the flanking regions (F or F') contains DNA in addition to one or more LNA nucleosides. In some embodiments, at least one of the regions F or F', or both regions F and F', contain both LNA and DNA nucleosides. In such embodiments, the flanking regions F or F', or both F and F', contain at least three nucleosides, and the 5'-most and 3'-most nucleosides of the F or F' region are LNA nucleosides. Alternating flanking region LNA gapmers are disclosed in WO 2016 / 127002. The alternating flanking region may contain up to 3 consecutive DNA nucleosides, e.g., 1 to 2, or 1 or 2 or 3 consecutive DNA nucleosides. The alternating flanking region may be annotated as a series of integers representing the number of LNA nucleosides (L) followed by the number of DNA nucleosides (D). For example: [L] 1-3 -[D] 1-4 -[L] 1-3 Or [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2 .

[0090] In ASO design, these are usually represented numerically, such that 2-2-1 represents 5'[L]2-[D]2-[L]3' and 1-1-1-1-1 represents 5'[L]-[D]-[L]-[D]-[L]3'. The length of the flanking parts (regions F and F') in ASOs with alternating flanking parts can be independently 3 to 10 nucleosides, such as 4 to 8, such as 5 to 6 nucleosides, such as 4, 5, 6 or 7 modified nucleosides. In some embodiments, only one of the flanking parts in a gapmer ASO is alternating, the other being composed of LNA nucleotides. It can be advantageous to have at least two LNA nucleosides at the 3' end of the 3' flanking part (F') to confer additional exonuclease resistance. In one embodiment, the flanking parts in an alternating flanking gapmer have a total length of 5 to 8 nucleosides, of which 3 to 5 are LNA nucleosides. Some examples of ASOs with alternating flanking parts are: [L] 1-5 -[D] 1-4 -[L] 1-3 -[G] 5-16 -[L] 2-6 [L] 1-2 -[D] 2-3 -[L] 3-4 -[G] 5-7 -[L] 1-2 -[D] 2-3 -[L] 2-3 [L] 1-2 -[D] 1-2 -[L] 1-2 -[D] 1-2 -[L] 1-2 -[G] 5-16 -[L] 1-2 -[D] 1-3 -[L] 2-4 [L] 1-5 -[G] 5-16 -[L]-[D]-[L]-[D]-[L]2; and [L]4-[G] 6-10 -[L]-[D]3-[L]2; wherein the overall length of the gapmer is at least 12 nucleotides in length, such as at least 14 nucleotides in length.

[0091] The ASOs of the present disclosure, in some embodiments, comprise or consist of a contiguous nucleotide sequence of the ASO that is complementary to a TMEM106B target nucleic acid, such as a gapmer FG-F', and may further comprise 5' or 3' nucleosides. The 5' or 3' nucleosides may or may not be fully complementary to the TMEM106B target nucleic acid. Such 5' or 3' nucleosides are sometimes referred to herein as regions D' and D''.

[0092] The addition of region D' or D" can be used to attach a contiguous nucleotide sequence, such as a gapmer, to a conjugate moiety or another functional group. When used to attach a contiguous nucleotide sequence to a conjugate moiety, region D' or D" can function as a biodegradable linker. Alternatively, region D' or D" can be used to provide exonuclease protection or to facilitate synthesis or manufacturing. Regions D' and D" can be attached to the 5' end of region F or the 3' end of region F', respectively, to generate designs of the following formula D'-FG-F', FG-F'-D" or D'-FG-F'-D" In this example, FG-F' is the gapmer portion of the ASO and region D' or D" constitutes another portion of the ASO.

[0093] Region D' or D'' may independently comprise or consist of 1, 2, 3, 4 or 5 additional nucleotides, which may be complementary or non-complementary to the TMEM106B target nucleic acid. The nucleotides adjacent to the F or F' region are not sugar-modified nucleotides. The D' or D' region may function as a nuclease-sensitive biodegradable linker. In some embodiments, the additional 5' or 3' terminal nucleotide is linked with a phosphodiester bond and is DNA or RNA. Nucleotide-based biodegradable linkers suitable for use as region D' or D'' are disclosed in WO 2014 / 076195, which includes, by way of example, phosphodiester-linked DNA dinucleotides. The use of biodegradable linkers in poly-ASO constructs for use in linking multiple antisense constructs (e.g., gapmer regions) within a single ASO is described in WO 2015 / 113922. In some embodiments, the ASO of the disclosure comprises a region D' or D'' in addition to the contiguous nucleotide sequence that makes up the gapmer.

[0094] In some embodiments, the ASO of the disclosure can be represented by the formula: FG-F'; in particular F 2-8 -G 6-16 -F' 2-8 ;D'-FG-F', especially D' 2-3 -F 1-8 -G 6-16 -F' 2-8 ;FG-F'-D'', especially F 2-8 -G 6-16 -F' 2-8 -D'' 1-3 or D'-FG-F'-D'', especially D' 1-3 -F 2-8 -G 6-16 -F' 2-8 -D'' 1-3 In some embodiments, the internucleoside linkage located between region D' and region F is a phosphodiester linkage. In some embodiments, the internucleoside linkage located between region F' and region D'' is a phosphodiester linkage.

[0095] The term "conjugate" as used herein refers to an ASO covalently linked to a non-nucleotide moiety (conjugate moiety, region C, or third region). Conjugation of the ASO of the present disclosure to one or more non-nucleotide moieties may improve the pharmacology of the ASO, for example, by affecting the activity, cellular distribution, cellular uptake, or stability of the ASO. The conjugate moiety may modify or enhance the pharmacokinetic properties of the ASO by improving cellular distribution, bioavailability, metabolism, excretion, permeability, or cellular uptake. In particular, the conjugate may target the ASO to a particular organ, tissue, or cell type, thereby enhancing the efficacy of the ASO in that organ, tissue, or cell type. At the same time, the conjugate may function to reduce the activity of the ASO in non-target cell types, tissues, or organs, such as off-target activity or activity in non-target cell types, tissues, or organs. ASO conjugates and their synthesis are reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, STCrooke, ed., Ch. 16, Marcel Dekker, Inc., 2001, and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103, each of which is incorporated herein by reference in its entirety.

[0096] The non-nucleotide conjugate moiety can be selected from the group consisting of carbohydrates (e.g., GalNAc), cell surface receptor ligands, drug substances, hormones, lipophiles, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof. For example, the conjugate can be an antibody or antibody fragment having specific affinity for the transferrin receptor, e.g., as disclosed in WO 2012 / 143379, which is incorporated herein by reference. In other examples, the non-nucleotide moiety can be an antibody or antibody fragment, e.g., an antibody or antibody fragment that facilitates delivery across the blood-brain barrier, e.g., an antibody or antibody fragment that targets the transferrin receptor.

[0097] As used herein, a "linkage" or "linker" is a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. The conjugate moiety can be attached to the ASO directly or through a linking moiety (e.g., a linker or tether). The linker functions to covalently connect a third region, e.g., the conjugate moiety (region C), to a first region, e.g., an ASO complementary to a TMEM106B target nucleic acid (region A) or a contiguous nucleotide sequence complementarity. The conjugates of the present disclosure may optionally include a linker region (second region or region B or region Y) located between the ASO or its contiguous nucleotide sequence (region A or first region) and the conjugate moiety (region C or third region). Region B refers to a biodegradable linker that includes or consists of a physiologically labile bond that is cleavable under physiological conditions. Conditions under which a physiologically labile linker undergoes chemical cleavage include chemical conditions, such as pH, temperature, oxidative or reductive conditions or agents, and physiological salt concentrations. Such physiological conditions may include mammalian intracellular conditions, such as enzymatic activities normally present in mammalian cells, such as proteolytic or hydrolytic enzymes or nucleases. In some embodiments, the biodegradable linker is susceptible to S1 nuclease cleavage. The nuclease-sensitive linker may comprise between 1 and 10 nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, such as between 2 and 6 nucleosides, such as between 2 and 4 linked nucleosides, and may further comprise at least two consecutive phosphodiester bonds, such as at least 3, 4, or 5 consecutive phosphodiester bonds. Preferably, the nucleosides are naturally occurring DNA or RNA, or modified DNA or RNA. Phosphodiester-containing biodegradable linkers are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0098] Region Y refers to a linker that is not necessarily biodegradable, but functions primarily to covalently connect the conjugate moiety to the ASO. Region Y linkers can include chain structures or oligomers of repeating units such as ethylene glycol, amino acid units, or aminoalkyl groups. The ASO conjugates of the present disclosure can be constructed from the following region elements AC, ABC, ABYC, AYBC, or AYC. The linker can be an aminoalkyl, such as a C2-C36 aminoalkyl group, including, for example, a C6 to C12 aminoalkyl group. In some embodiments, the linker is a C6 aminoalkyl group.

[0099] The term "treatment" as used herein refers to both treatment of an existing disease (e.g., a disease or disorder described herein) or prevention of disease, i.e., prophylaxis. One of ordinary skill in the art will recognize that "treatment" can be prophylactic. Treatment can be performed on a patient diagnosed with a neurological disorder, for example, a neurological disorder selected from the group consisting of dysregulation of TMEM106B expression or activity, Alzheimer's disease (AD), limbic-predominant age-related TDP-43 encephalopathy (LATE), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), FTD with parkinsonism linked to chromosome 17 (FTDP-17), and Pick's disease (PiD). As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness and properties of the free base or free acid, and is not biologically or otherwise undesirable. Salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, or N-acetylcysteine. In addition, these salts can be prepared from the addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, or magnesium salts. Salts obtained from organic bases include, but are not limited to, primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, and polyamine resin salts.The ASO agents disclosed herein may also exist in zwitterionic form.Some pharma- ceutically acceptable salts of the ASO compounds disclosed herein may be salts of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or methanesulfonic acid.

[0100] The term "protecting group" as used herein, alone or in combination, refers to groups that selectively block reactive sites of a multifunctional compound so that chemical reactions can be selectively carried out at otherwise unprotected reactive sites. Protecting groups can be removed. Exemplary protecting groups are amino-protecting groups, carboxy-protecting groups, or hydroxy-protecting groups. [Brief description of the drawings]

[0101] [Figure 1] The structural formula of compound number 29_3 is shown. Its pharmaceutical salts include monovalent cations such as Na+, K+, and Ca2+, or mixtures thereof. The figure discloses SEQ ID NO: 29. The illustrated compound is shown in protonated form (the S atom on the phosphorothioate bond is protonated), and it will be understood that the presence of a proton will depend on the acidity of the molecule's environment and the presence of another cation (e.g., when the antisense oligonucleotide (ASO) is in salt form). Protonated phosphorothioates exist in tautomeric forms. [Diagram 2] The structural formula of compound number 29_5 is shown. Its pharmaceutical salts include monovalent cations such as Na+, K+, and Ca2+, or mixtures thereof. The figure discloses SEQ ID NO: 25. The illustrated compound is shown in protonated form (the S atom on the phosphorothioate bond is protonated), and it will be understood that the presence of a proton will depend on the acidity of the molecule's environment and the presence of another cation (e.g., when the antisense oligonucleotide (ASO) is in salt form). Protonated phosphorothioates exist in tautomeric forms. [Diagram 3]FIG. 1 shows the structural formula of compound No. 26. Its pharmaceutical salts include monovalent cations, such as Na+, K+, and Ca2+, or mixtures thereof. SEQ ID NO: 19 is disclosed in the figure. The illustrated compound is shown in protonated form (the S atom on the phosphorothioate bond is protonated), and it will be understood that the presence of a proton will depend on the acidity of the molecule's environment and the presence of another cation (e.g., when the antisense oligonucleotide (ASO) is in salt form). Protonated phosphorothioates exist in tautomeric forms. [Figure 4] Scatter plot showing the degree of human TMEM106B messenger RNA (mRNA) knockdown induced by 613 novel ASO compounds of the present disclosure administered in vitro to SK-N-BE(2) glioblastoma cells at a concentration of 2.5 μM for 3 days. 22 published TMEM106B-targeting ASOs, including compound numbers 25_1, 20, and 31, were also tested and used as benchmarks for the potency (i.e., knockdown) of the newly developed compounds of the present disclosure. The horizontal dashed line shows the knockdown of human TMEM106B mRNA induced by compound number 31, a previously published anti-TMEM106B ASO. Novel compounds compound numbers 15, 24_3, 12, 29_3, and 26 exhibited greater knockdown compared to compound number 31. The x-axis shows the nucleotide position within human TMEM106B mRNA of SEQ ID NO: 1. The y-axis shows the percent knockdown relative to cells treated with a PBS control solution. [Diagram 5]Scatter plot showing the degree of human TMEM106B mRNA knockdown induced by 908 novel ASO compounds of the present disclosure administered in vitro to SK-N-BE(2) glioblastoma cells at a concentration of 0.5 μM for 3 days. 22 published TMEM106B-targeting ASOs, including compound numbers 25_1, 20, and 31, were also tested and used as a benchmark for the potency (i.e., knockdown) of the newly developed compounds of the present disclosure. The horizontal dashed line shows the knockdown of human TMEM106B mRNA induced by compound number 31, a previously published anti-TMEM106B ASO. Novel compounds compound numbers 8_1, 8_3, 24_1, 6, 10_4, 29_5, 15, 24_3, 12, 29_3, 26, and 8_4, along with others, exhibited greater knockdown compared to compound number 31. The x-axis shows the nucleotide position within human TMEM106B mRNA of SEQ ID NO:1. The y-axis indicates the percent knockdown relative to cells treated with a PBS control solution. [Figure 6] Scatter plot showing the degree of human TMEM106B mRNA knockdown induced by 312 novel ASO compounds of the present disclosure administered to SK-N-BE(2) glioblastoma cells in vitro at a concentration of 0.25 μM for 3 days. 22 published TMEM106B-targeting ASOs, including compound number 25_1 and compound number 20, were also tested and used as a benchmark for the potency (i.e., knockdown) of the newly developed compounds of the present disclosure. Novel compounds compound numbers 8_1, 8_3, 24_1, 6, 8_4, and 29_5, along with others, exhibited greater knockdown compared to compound number 25_1. The x-axis shows the nucleotide position within human TMEM106B mRNA of SEQ ID NO:1. The y-axis shows the percent knockdown relative to cells treated with a PBS control solution. [Figure 7]Shown is the IC50 curve for compound numbers 29_5, 10_13, and 14. Human iPSC-derived neurons were treated with ASOs of compound numbers 29_5, 10_13, and 14 in a dose response curve from 10 to 0.001 μM in two replicate wells for 7 days. Taqman assay was used to determine KD (%) of TMEM106B mRNA levels relative to PBS control wells. IC50 curves were determined for each ASO shown in the table (μM). [Figure 8] Shown are IC50 curves for compound numbers 12, 29_3, and 24_3. Human iPSC-derived neurons were treated with compound numbers 12, 29_3, and 24_3 ASOs in a dose response curve from 10 to 0.001 μM in two replicate wells for 7 days. Taqman assay was used to determine KD (%) of TMEM106B mRNA levels relative to PBS control wells. IC50 curves were determined for each ASO shown in the table (μM). [Figure 9] IC50 curves for compound numbers 8_2, 26, and 10_6 are shown. Human iPSC-derived neurons were treated with compound numbers 8_2, 26, and 0_6 ASOs in a dose response curve from 10 to 0.001 μM in two replicate wells for 7 days. Taqman assay was used to determine KD (%) of TMEM106B mRNA levels relative to PBS control wells. IC50 curves were determined for each ASO shown in the table (μM). [Figure 10A]Figure 10 shows a functional assay of human retinal pigment epithelium (RPE) cells with ASO compounds Compound Nos. 29_3, 15, 26, 14, and control ASO 10_13. Human RPE cells were treated with ASOs Compound Nos. 29_3, 15, 26, 14, and control ASO 10_13 in triplicate wells at concentrations ranging from 10 μM to 0.00001 μM for 5 days. IC50 curves for the indicated ASOs are shown in Figure 10A. At 10 μM, ASO Compound Nos. 29_3, 15, and 26 reduced TMEM106B mRNA by approximately 80%. Human RPE cells were treated with ASOs, Compounds Nos. 29_3, 15, 26, 14, and control ASO 10_13, over a 5-day period in four replicate wells at concentrations ranging from 10 μM, 5 μM, and 1 μM, and assayed for their effects on lysosomal function using a self-quenching form of bovine serum albumin (BSA)-BODIPY, which fluoresces upon proteolytic dissolution inside lysosomes. The average BSA degradation activity was normalized to the number of cells per well and then to a well without ASO treatment as a control. Compounds Nos. 29_3, 15, 26, and 14 reduced lysosomal activity by approximately 50%. [Figure 10B] Figure 1 shows a functional assay of human retinal pigment epithelium (RPE) cells for ASO compounds Compound Nos. 29_3, 15, 26, 14, and control ASO 10_13. Human RPE cells were treated with ASOs Compound Nos. 29_3, 15, 26, 14, and control ASO 10_13 in triplicate wells at concentrations ranging from 10 μM to 0.00001 μM for 5 days. Negative control Compound No. 10_13 did not reduce BSA degradation (n=4, + / - standard error of the mean (SEM)=+ / -3). Four asterisks denote statistical significance at the p<0.0001 level, two asterisks denote statistical significance at the p<0.01 level, and a single asterisk denote statistical significance at the p<0.05 level (ns=not significant) as assessed by Dunnett's post-hoc test. [Figure 10C]A shows a functional assay of human retinal pigment epithelium (RPE) cells for SO compounds compound nos. 29_3, 15, 26, 14, and control ASO 10_13. Human RPE cells were treated with ASO compounds nos. 29_3, 15, 26, 14, and control ASO 10_13 in triplicate wells at concentrations ranging from 10 μM to 0.00001 μM for 5 days. Fluorescence images of human RPE cells. White dots indicate proteolytically cleaved BSA-BODIPY fragments. Cells treated with compound no. 29_3 show reduced lysosomal activity. [Figure 11] 1 shows a bar graph of human TMEM106B (hTMEM106B) mRNA measured by qPCR from brain tissue of transgenic mice expressing hTMEM106B treated in vivo with ASOs of the present disclosure (compounds no. 14, 15, 29_3, 26) or the negative control (NC) ASO of compound no. 10_13. Also shown is hTMEM106B expression from untreated non-transgenic (NTG) mice as a control. The newly developed ASO agents of compound nos. 29_3 and 26 show superior inhibition of hTMEM106B expression in vivo compared to the published prior art ASO of compound no. 31. Asterisks denote statistical significance at the p<0.0001 level (ns=not significant) as assessed by Dunnett's post-hoc test. [Figure 12] 1 shows a bar graph of hTMEM106B mRNA measured by qPCR from brain tissue of transgenic mice expressing hTMEM106B treated with ASOs of the present disclosure (compound numbers 8_1, 24_1, 6, 10_4) or the published prior art anti-TMEM106B ASO, compound number 25_1. Also shown is hTMEM106B expression from saline-treated NTG mice as a control. Asterisks denote statistical significance at the p<0.0001 level as assessed by Dunnett's post-hoc test (ns=not significant). [Figure 13]1 shows a bar graph of hTMEM106B mRNA measured by qPCR from brain tissue of transgenic mice expressing hTMEM106B treated in vivo with an ASO of the present disclosure (compound no. 29_3 (25 μg and 100 μg) and compound no. 26 (25 μg and 100 μg)), a previously published anti-TMEM106B ASO of compound no. 31, or a negative control ASO of compound no. 10_13 (100 μg). Also shown is hTMEM106B expression from a non-transgenic mouse treated with 100 μg of the negative control ASO of compound no. 10_13 as a control. The newly developed ASO agents of compound nos. 29_3 and 26 show superior inhibition of hTMEM106B expression in vivo compared to the published prior art ASO of compound no. 31. Asterisks denote statistical significance at the p<0.0001 level as assessed by Dunnett's post-hoc test. [Figure 14] 1 shows a bar graph of hTMEM106B mRNA measured by qPCR from transgenic mice expressing hTMEM106B treated in vivo with an ASO of the disclosure (compound number 29_5 (25 μg and 100 μg)) or compound number 13 negative control ASO (100 μg). Also shown is hTMEM106B expression from non-transgenic mice treated with 100 μg of compound number 13 negative control ASO as a control. Asterisks denote statistical significance at the level of ***p<0.001 or ****p<0.0001 as assessed by Dunnett's post-hoc test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0102] Described herein are antisense oligonucleotide (ASO) agents that target, bind to, and promote the degradation of transmembrane protein 106B (TMEM106B) mRNA, and pharmacologic acceptable compositions containing the same. Such agents and compositions may be particularly useful for treating neurodegenerative disorders (e.g., frontotemporal lobar degeneration (FTD), Parkinson's disease (PD), Alzheimer's disease (AD), hippocampal sclerosis, hypomyelination (HML), amyotrophic lateral sclerosis (ALS), corticobasal syndrome (CBS), limbic-predominant age-related TDP-43 encephalopathy (LATE), progressive supranuclear palsy (PSP), and neuronal ceroid lipofuscinosis (NCL)). For example, a subject (e.g., a human) having or at risk of developing a neurodegenerative disorder can be administered one or more ASO agents or compositions containing the same by any route to treat the disorder of the subject. The present disclosure encompasses the treatment of any disease or condition that is associated with elevated levels of TMEM106B and that may benefit from the reduction of the protein in a subject.

[0103] Transmembrane protein 106B Transmembrane protein 106B (TMEM106B) is a single-pass type 2 integral membrane glycoprotein that is primarily located within the membranes of endosomes and lysosomes. TMEM106B is expressed in neurons as well as glial and endothelial cells. TMEM106B is thought to be primarily involved in lysosomal function. Overexpression and knockdown of TMEM106B in cell lines can cause an increase in lysosomal size, number, and acidification. TMEM106B has been associated with multiple common neurodegenerative disorders, including frontotemporal lobar degeneration (FTLD) (Nicholson and Rademakers, Acta Neuropathol. 132:639-651 (2016)).

[0104] For example, neurodegenerative disorders including frontotemporal lobar degeneration (FTLD), Parkinson's disease (or parkinsonism), cerebral leukodystrophy, amyotrophic lateral sclerosis (ALS), Alzheimer's disease, hippocampal sclerosis, aging, Alzheimer's disease (AD), corticobasal syndrome, limbic-predominant age-related TDP-43 encephalopathy (LATE), progressive supranuclear palsy (PSP), and neuronal ceroid lipofuscinosis (NCL) represent a large unmet medical need, and there is clear genetic and experimental evidence indicating specific TMEM106B allelic variants and altered TMEM106B expression in neurodegenerative disorders. Thus, there is a need for agents that inhibit the expression of TMEM106B for use in research and therapeutic applications.

[0105] Neurodegenerative disorders Neurodegenerative disorders are associated with dysregulated TMEM106B expression or activity. Exemplary disorders associated with dysregulated expression of TMEM106B can include neurodegenerative disorders such as, for example, frontotemporal lobar degeneration (FTLD), Parkinson's disease (or parkinsonism; PD), FTD with parkinsonism linked to chromosome 17 (FTDP-17), hypomyelination (HML), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), hippocampal sclerosis, corticobasal degeneration (CBD), corticobasal syndrome (CBS), chronic traumatic encephalopathy (CTE), Pick's disease (PiD), limbic-predominant age-related TDP-43 encephalopathy (LATE), progressive supranuclear palsy (PSP), and neuronal ceroid lipofuscinosis (NCL). Given that several of these disorders are associated with dysfunction of the frontal cortex, compounds of the present disclosure may be used for the treatment of age-related or disease-related changes in the frontal cortex.

[0106] Therefore, the ASO described herein may be suitable for use in the treatment of FTLD.In particular, the ASO or pharmaceutical composition of the present disclosure may be advantageous in the treatment of FTLD (FTLD-U) characterized by nuclear or cytoplasmic accumulation of ubiquitinated proteins, such as FTLD-TDP (TDP-43) characterized by the presence of ubiquitinated TAR DNA-binding protein 43 deposits in frontal and temporal brain regions, as well as other TDP-43 proteinopathies (e.g., ALS, Alzheimer's disease, cerebral age-related TDP-43 with sclerosis (CARTS), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), limbic-predominant age-related TDP-43 encephalopathy (LATE), and hippocampal sclerosis).

[0107] Frontotemporal Lobar Degeneration FTLD is a clinical syndrome characterized by progressive neurodegeneration in the frontal and temporal lobes of the cerebral cortex. The manifestation of FTLD is complex and heterogeneous and can manifest as one of three clinically distinct variants: 1) behavioral frontotemporal dementia (BVFTD), characterized by behavioral and personality changes, apathy, social withdrawal, perseveration, attention deficits, disinhibition, and prominent degeneration of the frontal lobe; 2) semantic dementia (SD), characterized by verbal amnesic aphasia, progressive loss of semantic knowledge of words, objects, and concepts, and prominent degeneration of the anterior temporal lobe; or 3) progressive non-verbal aphasia (PNA), characterized by motor impairments in speech, reduced language expression, and prominent degeneration of the parasylvian cortex. Neuronal loss in the brains of FTLD patients is associated with one of three distinct neuropathologies: 1) the presence of tau-positive neuronal and glial inclusions; 2) ubiquitin (ub)-positive and TAR DNA-binding protein 43 (TDP43)-positive but tau-negative inclusions; or 3) ub and fusion in sarcoma (FUS)-positive but tau- and TDP-43-negative inclusions. These neuropathologies are believed to be important in the pathogenesis of FTLD.

[0108] Nearly half of FTLD patients have a first-degree family member with dementia, ALS, or Parkinson's disease, suggesting a strong genetic link in the etiology of the disease. Multiple mutations on chromosome 17q21 have been associated with the development of FTLD symptoms.

[0109] Alzheimer's Disease Alzheimer's disease is a neurodegenerative disorder characterized by progressive neuronal loss in the frontal, temporal, and parietal lobes of the cerebral cortex, as well as in subcutaneous tissues such as the basal forebrain cholinergic system and the locus coeruleus within the brainstem. Clinical symptoms of Alzheimer's disease are progressive decline in multiple cognitive functions, including short-term and long-term memory, spatial navigation, verbal fluency, and impulse control, as well as anhedonia and social withdrawal. Neuronal atrophy in the brains of Alzheimer's disease patients is associated with the accumulation of extracellular and intracellular protein inclusions. Aggregates of insoluble amyloid-β (Aβ) protein are often found in the extracellular space, while neurofibrillary tangles (NFTs) of hyperphosphorylated tau protein are usually found in the intracellular compartment of diseased neurons. Such neuropathology is thought to be important in the etiology of Alzheimer's disease.

[0110] The clinical management of Alzheimer's disease employs pharmacological and behavioral interventions to alleviate symptoms of the disorder. For example, acetylcholine has been found to be depleted in Alzheimer's disease patients, and acetylcholinesterase inhibitors have been used to increase the levels of this neurotransmitter in the brain as a means of improving the cognitive impairment of Alzheimer's disease. In addition, negative-type antipsychotics are commonly prescribed to Alzheimer's disease patients for behavioral management. However, such strategies target improving symptoms of the disease without addressing its onset and progression. Unlike such treatments, the compositions and methods described herein offer the advantage of treating different biochemical phenomena that may underlie the onset of Alzheimer's disease. Thus, the compositions and methods described herein target the physiological causes of the disease, offering the potential for a cure.

[0111] Cerebral leukodysplasia Cerebral hypomyelination refers to a set of primarily genetic disorders characterized by degeneration of white matter in the brain. Cerebral hypomyelination may be characterized by the presence of white matter degeneration, loss of axons and myelin, the presence of axonal spherules, and in some cases, cystic bone lesions in the distal limbs (e.g., Nasu-Kohara disease). Leukodystrophies usually present around infancy and early childhood and may be characterized by hyperexcitability, hypersensitivity to the environment, muscular rigidity, head-back behavior, reduced or lost hearing and vision, and epilepsy. Non-limiting examples of leukodystrophies include Nasu-Kohara disease (PLOSL), metachromatic leukodystrophy, Krabbe disease, X-linked adrenoleukodystrophy, Canavan disease, and Alexander disease.

[0112] Amyotrophic lateral sclerosis ALS, also known as Lou Gehrig's disease, is the most fatal neurodegenerative disorder characterized by the loss of motor neurons in the primary motor cortex, brainstem, and spinal cord. The loss of motor neurons leads to severe impairment of basic motor control, including breathing, and causes death within 2-5 years after diagnosis. ALS patients typically require respiratory assistance during advanced stages of the disease. These patients also present with weakness in striated muscles, as well as smooth muscles, such as those involved in swallowing. Some patients may also develop frontotemporal dementia at the same time.

[0113] Two common forms of ALS have been described: sporadic ALS (sALS), which is the most common form of ALS in the United States and accounts for 90-95% of all diagnosed cases, and familial ALS (fALS), which exhibits dominant inheritance and accounts for approximately 5-10% of all cases in the U.S. These two forms of ALS are clinically indistinguishable.

[0114] At the molecular level, ALS is associated with disruption of cellular processes subsequent to disease initiation, including, for example, endoplasmic reticulum stress, reactive oxygen species generation, mitochondrial dysfunction, protein aggregation, apoptosis, inflammation, and glutamate excitotoxicity, particularly in motor neurons. Despite these findings, the causes of ALS are complex and diverse. In general, ALS is considered to be a complex genetic disorder in which multiple genes interact with environmental factors to increase susceptibility to disease. More than a dozen genes have been linked to the pathology of ALS, including Cu 2+ / Zn 2+ These include superoxide dismutase (SOD-1), TARDBP, TAR DNA-binding protein-43 (TDP-43), sarcoma fusions / translocations (FUS), angiogenin (ANG), ataxin-2 (ATXN2), valosin-containing protein (VCP), optineurin (OPTN), and a noncoding GGGGCC hexanucleotide repeat expansion in chromosome 9, open reading frame 72 (C9ORF72). However, the exact mechanism of motor neuron degeneration remains unclear.

[0115] There is currently no curative treatment for ALS available. The only FDA-approved drug is riluzole, which is an antagonist of TTX-sensitive sodium channels, kainate, and NMDA receptors, and a potentiator of GABAa receptors. However, only an extension of life span of about 3 months has been reported for early-stage ALS patients, and no therapeutic effect has been observed in late-stage ALS patients.

[0116] multiple system atrophy MSA is a neurodegenerative disorder that typically presents with dysregulation of autonomic function, tremor, hypokinesia, muscle rigidity, parkinsonism, and ataxia. Patients with MSA usually survive 6-10 years after diagnosis. At the cellular level, MSA corresponds to an insidious loss of neurons within the basal ganglia, inferior olivary nucleus, and cerebellum. The presence of modified α-synuclein is thought to be a possible cause of MSA. Lewy bodies have also been found in brain tissue from MSA patients. Another common feature of MSA is gliosis, astrocytic proliferation, glial scarring, Papp-Lantos bodies composed of α-synuclein, and tau protein.

[0117] Onset of MSA is typically accompanied by a hypokinetic syndrome (62% of cases), as well as balance disorders, genitourinary symptoms, and falls. Disease progression is characterized by progressive parkinsonism, urinary incontinence or retention, impotence, constipation, vocal cord paralysis, dry mouth and skin, impaired thermoregulation, respiratory distress, visual impairment, and cognitive impairment.

[0118] MSA is usually divided into two subtypes: parkinsonism-predominant MSA (MSA-P) and MSA with cerebellar manifestations (MSA-C). MSA-P is characterized by extrapyramidal movement disorders and may involve degeneration in the striatonigral system. MSA-C is characterized by cerebellar ataxia and may be accompanied by sporadic olivopontocerebellar atrophy.

[0119] Treatment of MSA is largely symptomatic, and no curative therapy has yet been developed to treat this devastating disorder.

[0120] Motor neuron disease MND belongs to a group of neurological disorders caused by destruction of motor neurons in the central nervous system and degenerative changes within the motor neuron circuitry. MND is a progressive degenerative disorder that typically affects upper and lower motor neurons, leading to successive generalized muscle denervation. MND is often identified in middle age, but a wide age range has been observed for the onset of symptomatic disease, from 18 to 85 years. MND symptoms often include difficulty swallowing, limb weakness, slurred speech, gait disturbance, facial muscle weakness, and muscle spasms. In advanced stages of the disease, the respiratory musculature loses its motor innervation, which is the ultimate cause of death in ALS patients. The etiology of most MNDs is unknown, but environmental, toxic, viral, and genetic factors are thought to be involved in the pathology of the disease.

[0121] Motor neurons, including upper and lower motor neurons, affect voluntary muscles, stimulating them to contract. Upper motor neurons originate from the cerebral cortex and send axonal fibers through the brainstem and spinal cord to control lower motor neurons. Lower motor neurons are located in the brainstem and spinal cord and innervate the musculoskeletal system. Lower motor neuron diseases are diseases involving the degeneration of lower motor neurons. Degeneration of lower motor neurons is associated with disconnection of neurons and muscles, causing muscle weakness and reduced reflexes. Loss of upper or lower motor neurons results in weakness, muscle atrophy (wasting), and painless wasting is the clinical hallmark of MND.

[0122] Corticobasal syndrome Corticobasal syndrome is a progressive atypical parkinsonian syndrome associated with tauopathy and FTLD. Corticobasal syndrome can present in one of three forms: frontal-behavioral dysexecutive-spatial syndrome (FBS), incompetent / agrammatic variant of primary progressive aphasia, and progressive supranuclear palsy syndrome (PSPS). Common symptoms usually include apraxia, alien limb phenomenon, frontal deficits, and extrapyramidal motor symptoms, including myoclonus or rigidity. Corticobasal syndrome usually leads to death within 6.5 years after diagnosis. As of now, there is no curative treatment available for the condition.

[0123] progressive supranuclear palsy PSP is a progressive neurodegenerative disease characterized by falls, vertical ophthalmoplegia, bradykinesia, prominent ocular dysfunction, and frontal subcortical dementia. Loss of independent ambulation (inability to stand unassisted) occurs within 5 years of disease onset. PSP is associated with severe neuronal cell loss in the substantia nigra, globus pallidus, subthalamic nucleus, midbrain, and pontine reticular formation, as well as the presence of neurofibrillary tangles composed of tau fibers. In addition to the presence of widespread multifocal neuropathological changes, there are also abnormalities of multiple neurotransmitters, including dysfunction of the dopamine, acetylcholine, GABA, and noradrenergic systems.

[0124] The name "progressive supranuclear palsy" describes the main feature of the disease, which is the progressive failure of voluntary eye movements. The onset of the disease is usually between the ages of 50 and 70. Many patients initially report persistent dizziness and loss of balance or persistent falls, typically backwards. The reduction in voluntary eye movements impairs the ability to read, climb stairs, and drive a car. Other early symptoms may include personality changes, for example irritability or loss of impulse control. Some patients lose interest in daily activities and hobbies. Mood changes and depression are quite common, even early in the disease.

[0125] The areas of the brain that control eye movement are located near the areas that control the tongue and muscles for swallowing. Patients usually speak slowly and in a low tone. Swallowing of liquids and food also becomes impaired as the disease progresses, leading to life-threatening pneumonia, which is the leading cause of death in progressive PSP. To date, there is no cure for the disease.

[0126] Neuronal ceroid lipofuscinosis NCL is an umbrella term that refers to a collection of at least eight clinically recognized lysosomal storage diseases caused by the accumulation of lipofuscin inside the cells of the body, such as neuronal cells, liver cells, spleen cells, cardiac muscle cells, and kidney cells. NCL presents clinically with severe neurodegeneration and progressive and irreversible loss of motor and cognitive abilities, although the severity and clinical manifestations of the disease may depend on the particular NCL variant.

[0127] Known variants of NCL include infantile variant, also known as Santovuori-Haltia disease (SHD), late infantile variant, also known as Jansky-Bielschowsky disease (JBD), Finnish late infantile variant (FLI), variant late infantile (VLI), CLN7 variant (CLN7), CLN8 variant (CLN8), Turkish late infantile variant (TLI), type 9 variant (T9), CLN10 variant (CLN10), juvenile variant, also known as Batten disease (BD), and adult variant, also known as Kufs disease (KD). SHD is an early visual disorder that progresses to complete retinal blindness by age 2 years, a vegetative state by age 3 years, and brain death by age 4 years. This variant is also associated with spontaneous occurrence of epileptic seizures. The JBD variant appears between 2 and 4 years of age and is associated with ataxia, epileptic seizures, progressive cognitive decline, and abnormalities in language development, typically leading to death by age 8. BD typically appears between 4 and 10 years of age and includes symptoms of vision loss, epileptic seizures, cognitive impairment, and premature death. NCL patients with the KD variant usually have milder symptoms than the SHD and BD variants and have a life expectancy of approximately 40 years.

[0128] Antisense oligonucleotides The present disclosure relates to ASO compounds capable of modulating the expression of TMEM106B, e.g., inhibiting TMEM106B. Modulation is achieved by contacting a target nucleic acid (e.g., pre-mRNA or mRNA) encoding TMEM106B with an ASO capable of hybridizing to the TMEM106B target nucleic acid and promoting its degradation by promoting recruitment of RNase H and by inhibiting translation (e.g., through steric hindrance). The TMEM106B target nucleic acid can be a mammalian TMEM106B mRNA sequence, e.g., a sequence selected from the group consisting of SEQ ID NOs: 1-4.

[0129] The ASO compounds of the present disclosure comprise a nucleoside sequence that is at least substantially complementary or fully complementary to a region of a sequence of TMEM106B mRNA (e.g., any one of SEQ ID NOs: 1-4) or a variant thereof, and the complementarity is sufficient to cause specific binding under intracellular conditions. For example, the present disclosure contemplates that the ASO has an antisense sequence that is complementary to at least 7 (e.g., at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or more) consecutive nucleotides of one or more regions of TMEM106B mRNA. In certain embodiments, the ASO has an antisense sequence that is complementary to 7 consecutive nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 8 consecutive nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 9 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 10 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 11 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 12 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 13 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 14 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 15 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another example, the ASO has an antisense sequence complementary to 16 contiguous nucleotides of one or more regions of TMEM106B mRNA.In another embodiment, the ASO has an antisense sequence that is complementary to 17 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 18 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 19 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 20 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 21 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 22 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 23 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 24 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 25 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 26 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 27 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 28 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 29 contiguous nucleotides of one or more regions of TMEM106B mRNA. In another embodiment, the ASO has an antisense sequence that is complementary to 30 contiguous nucleotides of one or more regions of TMEM106B mRNA.In yet another embodiment, the ASO has an antisense sequence that is 100% complementary to the nucleotides of one or more regions of TMEM106B mRNA.

[0130] The present disclosure contemplates ASO compounds that, upon binding to one or more regions of TMEM106B mRNA (e.g., any one of the regions of TMEM106B mRNA set forth in SEQ ID NOs: 1-4), form a duplex with TMEM106B mRNA that is between 7-22 (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) nucleotides in length. For example, the duplex between the ASO compound and TMEM106B mRNA can be 7 nucleotides in length. In another example, the duplex between the ASO compound and TMEM106B mRNA can be 8 nucleotides in length. In another example, the duplex between the ASO compound and TMEM106B mRNA can be 9 nucleotides in length. In another example, the duplex between the ASO compound and TMEM106B mRNA can be 10 nucleotides in length. In another example, the duplex between the ASO compound and TMEM106B mRNA can be 11 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 12 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 13 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 14 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 15 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 16 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 17 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 18 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 19 nucleotides in length. In another embodiment, the duplex between the ASO compound and TMEM106B mRNA may be 20 nucleotides in length. In another example, the duplex between the ASO compound and TMEM106B mRNA can be 21 nucleotides in length.In yet another embodiment, the duplex between the ASO compound and TMEM106B mRNA can be 10 nucleotides in length.

[0131] According to the disclosed methods and compositions, the duplex structure formed by an ASO (e.g., an agent having at least 90% (at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to a nucleic acid sequence of any one of SEQ ID NOs: 5-50) and one or more regions of TMEM107B mRNA may contain at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) mismatch, which may be an insertion of a nucleobase not present in TMEM107B mRNA, a deletion of a nucleobase present in TMEM106B mRNA, or a nucleobase that is not complementary to TMEM106B mRNA. For example, the duplex structure may contain one mismatch. In another embodiment, the duplex structure contains two mismatches. In another embodiment, the duplex structure contains three mismatches. In another embodiment, the duplex structure comprises 4 mismatches. In another embodiment, the duplex structure comprises 5 mismatches. In another embodiment, the duplex structure comprises 6 mismatches. In another embodiment, the duplex structure comprises 7 mismatches. In another embodiment, the duplex structure comprises 8 mismatches. In another embodiment, the duplex structure comprises 9 mismatches. In another embodiment, the duplex structure comprises 10 mismatches. In another embodiment, the duplex structure comprises 11 mismatches. In another embodiment, the duplex structure comprises 12 mismatches. In another embodiment, the duplex structure comprises 13 mismatches. In another embodiment, the duplex structure comprises 14 mismatches. In yet another embodiment, the duplex structure comprises 15 mismatches.

[0132] In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 95% (e.g., at least 96%, 97%, 98%, 99% or more) sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having the nucleic acid sequence of SEQ ID NO:1.

[0133] In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having the nucleic acid sequence of SEQ ID NO:2.

[0134] In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:3. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having the nucleic acid sequence of SEQ ID NO:3.

[0135] In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 85% (e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having at least 95% (e.g., at least 96%, 97%, 98%, 99%, or more) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the TMEM106B target nucleic acid is a polynucleotide having the nucleic acid sequence of SEQ ID NO:4.

[0136] In some embodiments, the ASOs of the disclosure can modulate expression of a TMEM106B target nucleic acid by inhibiting or downregulating expression of the TMEM106B target nucleic acid (TMEM106B pre-mRNA or mRNA) in a cell. Such modulation inhibits expression of the TMEM106B target nucleic acid by at least 10% (e.g., at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) compared to the expression level of the target in a cell not treated with the ASO. Modulation of TMEM106B expression can occur in vitro (e.g., in primary cell cultures, cell lines, or tissue organoids) or in vivo (e.g., upon administration to an animal, e.g., a mammal, e.g., a human). In some embodiments, the ASOs of the present disclosure can inhibit expression levels of TMEM106B mRNA in primary neuronal cells by at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) in vitro following application of 5 μM ASO to the primary neuronal cells. In some embodiments, the ASOs of the disclosure can inhibit expression levels of TMEM106B protein in primary neuronal cells by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) in vitro following application of 0.5 μM of the ASO to the primary neuronal cells. Suitably, the Examples provide assays that can be used to measure inhibition of TMEM106B RNA or protein (e.g., Examples 1, 2, and 5).

[0137] Modulation of the TMEM106B target nucleic acid may be triggered by hybridization between the contiguous nucleotide sequence of the ASO and the TMEM106B target nucleic acid. In some embodiments, the ASO of the present disclosure includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, or more) mismatches between the ASO and the TMEM106B target nucleic acid. Hybridization to the TMEM106B target nucleic acid may still be sufficient to indicate the desired modulation of TMEM106B expression even in the absence of one or more mismatches. The reduced binding affinity caused by the mismatches may be advantageously compensated for by increasing the number of nucleotides in the ASO or by increasing the number of modified nucleosides present within the ASO sequence that can increase binding affinity to the target, such as 2' sugar modified nucleosides (e.g., LNAs).

[0138] In some embodiments, the ASO comprises a contiguous sequence of 10 to 30 nucleotides in length that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) complementary to a region of a TMEM106B target nucleic acid. An ASO of the present disclosure, or a contiguous nucleotide sequence thereof, can be fully complementary (i.e., 100% complementary) to a region of a TMEM106B target nucleic acid.

[0139] ASOs of the disclosure can comprise or consist of 10 to 35 nucleotides in length (e.g., 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, or 35 nucleotides in length). In some embodiments, the ASO comprises or consists of 16 to 22 (e.g., 16, 17, 18, 19, 20, 21, or 22) nucleotides in length. In some embodiments, the ASO comprises or consists of 16 to 20 (e.g., 16, 17, 18, 19, 20) nucleotides in length. In some embodiments, the ASO or a contiguous nucleotide sequence thereof comprises or consists of 22 nucleotides or less (e.g., 22, 21, 20, 19, 18, 17, 16 nucleotides or less in length). Any range provided herein should be understood to include the end points of the range. For example, when an ASO is said to comprise 10 to 30 nucleotides, both 10 and 30 nucleotides are included. In some embodiments, the contiguous nucleotide sequence comprises or consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides. In some embodiments, the ASO comprises or consists of 16, 17, 18, 19, or 20 nucleotides.

[0140] In some embodiments, the ASO or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to a sequence selected from the group consisting of SEQ ID NOs: 5-50 (see motif sequences listed in Table 5 in the Materials and Methods section). In some embodiments, the ASO or contiguous nucleotide sequence comprises or consists of 10 to 30 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length having at least 90% identity, preferably 100% identity, to a sequence selected from the group consisting of SEQ ID NOs: 5-50 (motif sequences listed in Table 5). In some embodiments, the ASO or contiguous nucleotide sequence comprises or consists of 10 to 30 nucleotides in length that have at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) complementarity to a sequence of SEQ ID NOs: 5-50 (see target sequences listed in Table 5).

[0141] Contiguous nucleobase sequences can be modified, for example, to increase nuclease resistance or binding affinity to TMEM106B target nucleic acid. The pattern in which modified nucleosides (such as high affinity modified nucleosides) are incorporated into ASO sequences is generally referred to as ASO design. The ASO of the present disclosure is designed using modified nucleosides and DNA nucleosides. Advantageously, high affinity modified nucleosides are used.

[0142] In some embodiments, the ASO contains at least one modified nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 modified nucleosides. In some embodiments, the ASO contains from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modified nucleosides. Suitable modifications are described, among others, in the "Definitions" section under "Modified Nucleosides," "High Affinity Modified Nucleosides," "Sugar Modifications," "2' Sugar Modifications," "Locked Nucleic Acids (LNAs)," and "Inverted Nucleotides."

[0143] In some embodiments, the ASO comprises one or more sugar-modified nucleosides, such as 2' sugar-modified nucleosides. Preferably, the ASO of the present disclosure comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) 2' sugar-modified nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA (e.g., 2'-O-methyl-RNA), 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, albino nucleic acid (ANA), 2'-fluoro-ANA, and LNA nucleosides. It is advantageous if one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) of the modified nucleosides are locked nucleic acids (LNA).

[0144] In some embodiments, the ASO of the disclosure comprises at least one LNA nucleoside, e.g., at least 1, 2, 3, 4, 5, 6, 7, or 8 LNA nucleosides, e.g., 2 to 6 (e.g., 2, 3, 4, 5, or 6) LNA nucleosides, e.g., 3 to 7 (e.g., 3, 4, 5, 6, or 7) LNA nucleosides, 4 to 8 (e.g., 4, 5, 6, 7, or 8) LNA nucleosides, or 3, 4, 5, 6, 7, or 8 LNA nucleosides. In some embodiments, at least 75% (e.g., at least 76%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the modified nucleosides in the ASO are LNA nucleosides, e.g., beta-D-oxy LNA or ScET. In some embodiments, all modified nucleosides in the ASO are LNA nucleosides. In some embodiments, the ASO may comprise beta-D-oxy-LNA and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) of the following LNA nucleosides: thio-LNA, amino-LNA, oxy-LNA, ScET, or ENA, in either beta-D or alpha-L configuration or a combination thereof. In some embodiments, all of the LNA cytosine nucleosides are 5-methylcytosine nucleosides. The ASO or contiguous nucleotide sequence may have at least one LNA nucleoside at the 5' end and at least two LNA nucleosides at the 3' end of the nucleotide sequence to confer nuclease resistance to the ASO. In some embodiments, the ASO of the present disclosure may recruit RNase H.

[0145] In some embodiments, ASOs can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) inverted nucleosides (see Materials and Methods). In some embodiments, ASOs can contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) DNA 7-deaza-8-aza-guanine pyrazolo[3,4-d]pyrimidine (PPG) nucleosides.

[0146] In the context of the present disclosure, it may be advantageous to use ASO structural designs that are gapmer designs described in the "Definitions" section, e.g., "Gapmers", "LNA Gapmers", "MOE Gapmers", "Mixed Wing Gapmers", and "Alternating Flanking Gapmers". Gapmer designs include gapmers with uniform flanking parts, mixed wing flanking parts, alternating flanking parts, and gap breaker designs. It can be advantageous if the ASO of the disclosure is a gapmer of the FG-F' design, a particular gapmer of the formula 5'-FG-F'-3', where regions F and F' independently comprise 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) nucleosides, of which 2 to 5 (e.g., 2, 3, 4, or 5) are 2' sugar modifications and define the 5' and 3' ends of the F and F' regions, and G is a region of between 6 to 16 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) nucleosides capable of recruiting RNase H, such as a region comprising 6 to 16 (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) DNA nucleosides. In some embodiments, the gapmer is an LNA gapmer. In some embodiments, the LNA gapmers are selected from the following uniform flanking designs: 4-10-4, 3-11-4, 4-11-4, 4-12-4, or 4-14-2, with the general formula 5' flanking-central block-3' flanking. In some embodiments, the LNA gapmers are selected from the following alternating flanking designs: 3-1-3-10-2, 1-3-4-6-1-3-2, 1-2-1-2-2-8-4, or 3-3-1-8-2-1-2. Table 5 lists exemplary designs for each motif sequence.

[0147] In all examples, the FG-F' design may further include region D' or D" as described in "Region D' or D'' of ASOs" in the "Definitions" section. In some embodiments, the ASOs of the disclosure have one, two, or three phosphodiester linked nucleoside monomers, e.g., DNA monomers, at the 5' or 3' end of the gapmer region.

[0148] The ASO may also include at least one (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) modified internucleoside linkage. Suitable internucleoside modifications are described in the "Definitions" section under "Modified Internucleoside Linkages." In some embodiments, at least 75% (e.g., at least 76%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) of the internucleoside linkages within a contiguous nucleotide sequence are phosphorothioate or boranophosphate internucleoside linkages. In some embodiments, all of the internucleoside linkages in the ASO or in a contiguous sequence thereof are phosphorothioate linkages.

[0149] In some embodiments of the present disclosure, the ASO is selected from the group of ASO compounds having compound numbers 6; 8_1; 8_3; 8_4; 10_4; 12; 14; 15; 24_1; 24_3; 26; 29_3; and 29_5.

[0150] Particularly advantageous ASOs in the context of the present disclosure are ASO compounds selected from the group consisting of: TIFF2025508310000024.tif95170 where non-italicized capital letters are beta-D-oxy LNA nucleosides, italicized capital letters are 2'-O-methyl nucleosides, lowercase letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.

[0151] Method of production The present disclosure further provides a method for producing the ASO described herein, comprising reacting nucleotide units under suitable conditions and for a time to form a covalently linked contiguous nucleotide polymer contained in the ASO. The production method can use phosphoramidite chemistry (see, for example, Caruthers et al., Methods in Enzymology 154:287-313 (1987)). The method can further comprise reacting the contiguous nucleotide sequence with a conjugate moiety under suitable conditions and for a time to covalently link the conjugate moiety to the ASO. Thus, the present disclosure provides a method for producing the ASO compound of the present disclosure or a composition comprising the same, comprising mixing the ASO or conjugated ASO of the present disclosure with a pharma- ceutically acceptable diluent, solvent, carrier, salt or adjuvant.

[0152] Pharmaceutically acceptable salts The compounds according to the present disclosure may be in the form of pharma- ceutically acceptable salts. The term "pharma- ceutically acceptable salts" refers to conventional acid or base addition salts that retain the biological effectiveness and properties of the compounds of the present disclosure and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Acid addition salts include those derived from inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides, such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a technique well known to medicinal chemists to obtain improved physical and chemical stability, hygroscopicity, flowability, and solubility of the compounds. Such methods are described, for example, in Bastin, Organic Process Research & Development 4:427-35(2000) or in Ansel, In:Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed.(1995), pp.196 and 1456-7. For example, a pharma- ceutically acceptable salt of a compound provided herein can be a sodium salt.

[0153] Thus, the present disclosure provides a pharma- ceutically acceptable salt of the ASO or a conjugate thereof. In some embodiments, the pharma- ceutically acceptable salt is a sodium or potassium salt. The present disclosure further provides a pharmaceutical composition comprising the ASO or a salt or conjugate thereof.

[0154] Pharmaceutical Compositions The present disclosure further provides pharmaceutical compositions comprising any of the aforementioned ASOs or salts or conjugates thereof and a pharma- ceutically acceptable diluent, carrier, salt, or adjuvant. The pharma- ceutically acceptable diluent may include, for example, phosphate buffered saline (PBS), such as sterile PBS. The pharma- ceutically acceptable salts may include, but are not limited to, sodium and potassium salts. In some embodiments, the ASO is used in a solution of a pharma- ceutically acceptable diluent at a concentration of 1 μM to 10 mM (e.g., 1 μM to 8 mM, 1 μM to 6 mM, 1 μM to 4 mM, 1 μM to 2 mM, 1 μM to 500 μM, 1 μM to 300 μM, 300 μM to 10 mM, 500 μM to 10 mM, 2 mM to 10 mM, 4 mM to 10 mM, 6 mM to 10 mM, and 8 mM to 10 mM).

[0155] Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, for example, Langer, Science 249:1527-1533, (1990). WO 2007 / 031091 provides further suitable examples of pharma- ceutically acceptable diluents, carriers and adjuvants (herein incorporated by reference). Suitable doses, formulations, routes of administration, compositions, dosage forms, combinations with other therapeutic agents, prodrug formulations are also provided in WO 2007 / 031091, herein incorporated by reference.

[0156] The ASO of the present disclosure may be mixed with pharma- ceutically acceptable active or inactive substances for the preparation of pharmaceutical compositions or formulations. The composition and method for the preparation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, the route of administration, the extent of the disease, or the dose to be administered.

[0157] These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged as is, or lyophilized. The lyophilized preparations may be combined with sterile aqueous carriers prior to administration. The pH of the preparation will typically be between 3 and 11 (e.g. 3, 4, 5, 6, 7, 8, 9, 10 or 11), for example between 5 and 9 (e.g. 5.5, 6, 6.5, 7, 7.5, 8, 8.5 or 9), for example between 6 and 8 (e.g. 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8), for example between 7 and 8 (e.g. 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8), for example from 7 to 7.5 (e.g. 7.1, 7.2, 7.3, 7.4 or 7.5). The resulting composition in solid form may be packaged into a number of single-dose units, each containing a fixed amount of one or more of the aforementioned agents, for example in a sealed package of tablets or capsules.

[0158] In some embodiments, the ASO or conjugate thereof of the present disclosure is a prodrug.

[0159] Pharmaceutical Use The ASO of the present disclosure can be used as a research reagent, for example, for diagnosis, treatment and prevention.In the context of research reagent, such ASO can be used to specifically regulate the synthesis of TMEM106B protein in cells (e.g., in vitro cell cultures or tissue organoids) and experimental animals, thereby facilitating the functional analysis of target genes or facilitating the preclinical evaluation of their usefulness as targets for therapeutic intervention.When using the ASO of the present disclosure or its conjugate in research or diagnosis, the TMEM106B target nucleic acid can be cDNA or synthetic nucleic acid obtained from DNA or RNA.

[0160] The present disclosure provides an in vivo or in vitro method for modulating TMEM106B expression in a target cell expressing TMEM106B, the method comprising administering to the cell an effective amount of an oligonucleotide of the present disclosure. In some embodiments, the target cell is a neuronal cell. In some embodiments, the target cell is a glial cell (e.g., a microglia, an astrocyte, an oligodendrocyte, or a Schwann cell). In some embodiments, the target cell is a mammalian cell, e.g., a human cell. The target cell can be an in vivo cell forming part of an in vitro cell culture or a tissue organoid or a mammalian tissue. The target cell can be in the central nervous system, e.g., the brain or spinal cord. In some embodiments, the cell is a brain cell. In some embodiments, the cell is a frontal cortex cell or a frontotemporal lobe cell. In some embodiments, the target cell is a cell of the thalamus, hippocampus, striatum, retina, or spinal cord.

[0161] It will be appreciated that for in vitro uses, such as, for example, assessing TMEM106B expression or inhibition thereof in target cells, cells can be isolated or derived from tissues (e.g., established or immortalized cell lines) such as central nervous system tissue, brain tissue, frontal cortex, frontotemporal lobe tissue, thalamic tissue, hippocampal tissue, striatal tissue, retinal tissue, or spinal cord tissue. Cells isolated from target tissues are referred to as primary cells.

[0162] In the context of their use for diagnostic purposes, the ASOs of the present disclosure can be used to detect and quantitate TMEM106B expression in cells and tissues by Northern blotting, in-situ hybridization, real-time quantitative polymerase chain reaction (qRT-PCR), RNA sequencing, or other techniques suitable for quantifying mRNA expression.

[0163] In the context of therapeutic applications, the ASOs of the present disclosure can be administered to animals, such as humans, suspected of having a disease or disorder that can be treated by modulating the expression of TMEM106B. Exemplary conditions that can benefit from treatment with the TMEM106B targeting compounds of the present disclosure are described herein. Thus, the present disclosure provides a method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of the ASOs of the present disclosure or pharmaceutical compositions comprising the same to a subject suffering from or suspected of a disease referred to herein. In addition, the present disclosure relates to the ASOs of the present disclosure or pharmaceutical compositions comprising the same for use as a medicament. The ASOs or pharmaceutical compositions of the present disclosure can be administered in an effective amount. Still further, the present disclosure provides the use of the ASOs of the present disclosure for the manufacture of a medicament for the treatment of a disorder referred to herein or for a method of treating a disorder referred to herein.

[0164] The disease or disorder may be associated with dysregulation of TMEM106B expression or activity. In some embodiments, the disease or disorder is associated with a mutation in the TMEM106B gene or a gene whose protein product is associated with or interacts with (e.g., is a binding partner of) TMEM106B. Thus, the TMEM106B target nucleic acid may be a mutated form of the TMEM106B sequence. In some embodiments, the TMEM106B target nucleic acid is a regulator of TMEM106B activity or expression. Thus, the disclosure further provides the use of an ASO or a pharmaceutical composition thereof for the manufacture of a medicament for the treatment of abnormal (e.g., elevated) levels or activity of TMEM106B. Exemplary disorders associated with dysregulation of TMEM106B expression or activity may include, for example, neurodegenerative disorders such as frontotemporal lobar degeneration (FTLD), Parkinson's disease (or parkinsonism; PD), hypomyelination (HML), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), hippocampal sclerosis, corticobasal syndrome (CBS), limbic-predominant age-related TDP-43 encephalopathy (LATE), progressive supranuclear palsy (PSP), and neuronal ceroid lipofuscinosis (NCL). Given that more than one of these disorders is associated with dysfunction of the frontal cortex, the compounds of the present disclosure may be used for the treatment of age-related or disease-related changes in the frontal cortex.

[0165] Therefore, the ASO or pharmaceutical composition containing the ASO described herein may be suitable for use in the treatment of FTLD. In particular, the ASO or pharmaceutical composition of the present disclosure may be advantageous in the treatment of FTLD characterized by nuclear or cytoplasmic accumulation of ubiquitinated proteins (FTLD-U), such as FTLD-TDP characterized by the presence of ubiquitinated TAR DNA-binding protein 43 (TDP-43) accumulation in the frontal and temporal brain regions, as well as other TDP-43 proteinopathies (e.g., ALS, Alzheimer's disease, cerebral age-related TDP-43 with sclerosis (CARTS), limbic-predominant age-related TDP-43 encephalopathy (LATE), chronic traumatic encephalopathy (CTE), and hippocampal sclerosis). In some embodiments, the pharmaceutical composition described herein may be useful in the treatment or prevention of coronavirus (e.g., COVID-19) infection.

[0166] Antisense Oligonucleotides for In Vivo Administration The compounds and compositions disclosed herein can be administered to a subject (e.g., a subject identified as having a disease or disorder described herein) using standard methods. For example, the compounds and compositions disclosed herein can be administered by any of a number of different routes, including, for example, systemic administration. Non-limiting examples of systemic administration include enteral (e.g., oral) or parenteral (e.g., intravenous, intraarterial, transmucosal, intraperitoneal, epicutaneous, intramucosal (e.g., intranasal or sublingual), intramuscular, or transdermal) administration. Further routes of administration can include intradermal, subcutaneous, and transdermal injections.

[0167] The compositions disclosed herein can also be administered using a method suitable for local delivery of ASO or compositions comprising ASO. Non-limiting examples of local administration include epicutaneous (e.g., topical), intra-articular, and inhalation routes. In particular, the disclosed compositions can be administered locally to the subject's brain tissue (e.g., nervous system cells, such as, for example, neurons or glia).

[0168] In particular, the compounds and compositions of the present disclosure may be administered locally to a subject's brain tissue, for example, a brain tissue determined to be responsible for the subject's underlying pathology. Local administration to the brain typically includes any method suitable for delivery of an ASO or a composition comprising an ASO to brain cells (e.g., nervous system cells) such that at least a portion of the cells of a selected, synaptically connected cell population are contacted with the composition. The ASO may be delivered to any cell of the central nervous system, including neurons, glia, or both. Typically, the ASO is delivered to cells of the central nervous system, including, for example, cells of the spinal cord, brainstem (medulla, pons, and midbrain), cerebellum, diencephalon (e.g., thalamus and hypothalamus), telencephalon (striatum, cerebral cortex (e.g., occipital, temporal, parietal, or frontal cortex), or combinations thereof, or any suitable subpopulation of cells therein. Further sites for delivery include neurons of the red nucleus, amygdala, entorhinal cortex, and ventral lateral or anterior nucleus of the thalamus.

[0169] The ASO or composition of the present disclosure may be delivered directly into the parenchyma or ventricles of the central nervous system by means of stereotactic injection or microinjection. In certain examples, the ASO of the present disclosure may be delivered directly to one or more epileptogenic foci in the brain of a subject. For example, a subject may be administered the ASO of the present disclosure or a pharmaceutical composition comprising the same by direct stereotactic injection into one or both hemispheres of the anisocortex (e.g., hippocampus) or neocortex (e.g., frontal lobe). In certain examples, a subject may be administered the ASO of the present disclosure by direct stereotactic injection into one or both hemispheres of the brain (e.g., frontal cortex). Alternatively, the ASO of the present disclosure may be administered by intravenous injection. In another example, a subject may be administered the ASO of the present disclosure intrathecally, and the ASO may be administered directly into the cisterna magna.

[0170] To specifically deliver the ASO of the present disclosure or pharmaceutical composition comprising the same to a specific region and a specific population of cells in the central nervous system, the ASO can be administered by stereotaxic microinjection. For example, a subject may have a stereotaxic frame base fixed (screwed to the skull) in the appropriate position by surgery. The brain with the stereotaxic frame base (e.g., an MRI-compatible stereotaxic frame base with fiducial markings) is imaged using high-resolution MRI. The MRI images are then transferred to a computer running stereotaxic software. A series of circular, sagittal, and axial images are used to determine the target injection site and trajectory of the cannula or injection needle used to inject the composition of the present disclosure into the brain. The software transfers the trajectory directly into the appropriate three-dimensional coordinates in the stereotaxic frame. A hole is drilled above the entry site, and the stereotaxic device is aligned with the injection needle implanted at a given depth. The composition (e.g., the composition disclosed herein) is injected into the target site.

[0171] Additional routes of administration may include local application of the ASO under direct visualization, e.g., to the superficial cortex, or other non-stereotactic application. The ASO may be delivered intrathecally (e.g., directly into the cisterna magna), intraventricularly (e.g., using intracerebroventricular (ICV) injection), or by intravenous injection.

[0172] In one embodiment, the method of the present disclosure includes intracerebral or intraventricular administration through stereotactic injection. However, other known delivery methods may also be adapted according to the present disclosure. For example, to distribute the composition broadly throughout the central nervous system, the composition may be injected into the cerebrospinal fluid, for example, by lumbar puncture. To direct the composition to the peripheral nervous system (PNS), the composition may be injected into the spinal cord, one or more peripheral ganglia, or under the skin (subcutaneously or intramuscularly) of the body part of interest. In some circumstances, the composition may be administered via an intravascular approach. For example, the composition may be administered into an artery (carotid artery) with or without disruption of the blood-brain barrier. Additionally, for more global delivery, the composition may be administered during the "opening" of the blood-brain barrier, which is achieved by injection of a hypertonic solution containing mannitol.

[0173] The optimal route of administration in a given case will depend on the particular composition being administered, the subject, the particular epilepsy being treated, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the subject's age, weight, sex, the severity of the disease being treated, the subject's diet, and the subject's excretion rate. EXAMPLES

[0174] The following examples are provided to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the invention and are not intended to limit the scope of what the inventors perceive as their invention.

[0175] Materials and Methods ASO motif sequences and ASO compounds Table 5 shows a list of ASO nucleotide sequences (represented by SEQ ID NO), specific ASO compounds (represented by compound number), and HELM annotations designed by the inventors for targeting and degradation of TMEM106B mRNA. The chemical structures of these ASOs are shown in Figures 1-3.

[0176] TIFF2025508310000025.tif251170TIFF2025508310000026.tif252170TIFF2025508310000027.tif252170TIFF2025508310000028.tif40170

[0177] Helm annotation keys: [LR](G) is a beta-D-oxy-LNA guanine nucleoside; [LR](T) is beta-D-oxy-LNA thymine nucleoside; [LR](A) is a beta-D-oxy-LNA adenine nucleoside; [LR] ([5meC]) is beta-D-oxy-LNA 5-methylcytosine nucleoside; [dR](G) is a DNA guanine nucleoside, [dR](T) is a DNA thymine nucleoside, [dR](A) is a DNA adenine nucleoside, [dR] ([C] is a DNA cytosine nucleoside, [dR] ([PPG]) is a DNA 7-deaza-8-aza-guanine nucleoside; [idR](G) is an inverted DNA guanine nucleoside; [idR](T) is an inverted DNA thymine nucleoside; [idR](A) is an inverted DNA adenine nucleoside; [idR](C) is an inverted DNA cytosine nucleoside, [mR](G) is 2'-O-methyl RNA guanine nucleoside; [mR](U) is 2'-O-methyl RNA uracil nucleoside; [mR](A) is 2'-O-methyl RNA adenine nucleoside; [mR](C) is a 2'-O-methyl RNA DNA cytosine nucleoside; [sP] is a phosphorothioate internucleoside linkage, P is a phosphodiester internucleoside linkage.

[0178] The antisense oligonucleotide sequence represents the contiguous sequence of nucleobases present in the ASO. The design refers to a gapmer design, e.g., FG-F'. In a classical gapmer design, e.g., 3-10-3 (5' flanking nucleotides-center block nucleotides-3' flanking nucleotides), all of the flanking nucleotides (F and F') are composed of the same 2'-sugar modified nucleoside (e.g., LNA, cET, or MOE), and the stretch of DNA in the center block forms a gap (G). In a gapmer with alternating flanking design, the flanking parts of the ASO are annotated as a series of integers, representing multiple 2' sugar modified nucleosides (M) followed by multiple DNA nucleosides (D). For example, the flanking parts of the 2-2-1 motif represent 5'[M]2-[D]2-[M]3', and the 1-1-1-1-1 motif represents 5'[M]-[D]-[M]-[D]-[M]3'. Both flanking parts have 2' sugar modified nucleosides at the 5' and 3' ends. A gap region (G) containing DNA nucleosides (typically between 6 and 16) is located between the flanking parts.

[0179] Synthesis of antisense oligonucleotides ASO synthesis is generally known in the art. Described below are exemplary protocols that can be used to generate ASOs with desired sequences and modifications. ASOs are synthesized on a uridine universal support at a 1 μmol scale using a phosphoramidite approach on an Oligomaker 48. At the end of the synthesis, the ASOs are cleaved from the solid support by treating the ASOs with aqueous ammonia at 60° C. for 5-16 hours. The ASOs are purified by reversed-phase HPLC (RP-HPLC) or by solid-phase extraction, characterized by ultra-performance liquid chromatography (UPLC), and the molecular weight is further confirmed by electrospray ionization mass spectrometry (ESI-MS).

[0180] Extension of antisense oligonucleotides Coupling of β-cyanoethyl-phosphoramidites (e.g. DNA-A(Bz), DNA-G(ibu), DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), or LNA-T) was carried out by using a 0.1 M solution of the 5'-O-DMT-protected amidite in acetonitrile and DCI (4,5-dicyanoimidazole) in acetonitrile (0.25 M) as an activator. For the final cycle, the phosphoramidite with the desired adjustment was used (e.g. C6 linker for attaching the conjugate group or the conjugate group itself). Thiolation to introduce phosphorothioate bonds was carried out using hydrogenated xanthan (0.01 M in acetonitrile / pyridine at a ratio of 9:1). Phosphodiester bonds were introduced using 0.02 M iodine in THF / pyridine / water at a molar ratio of 7:2:1.

[0181] For conjugation after solid-phase synthesis, a commercially available C6 amino linker phosphoramidite was used in the final cycle of solid-phase synthesis. After deprotection and cleavage from the solid support, the amino-linked and deprotected ASO was isolated. The conjugation moiety was introduced via activation of functional groups using standard synthetic methods.

[0182] Purification by reversed-phase high-performance liquid chromatography The crude compound was purified by preparative RP-HPLC on a Phenomenex Jupiter 10 μm C18 150×10 mm column. 0.1 M ammonium acetate (pH 8) and acetonitrile were used as buffers at a flow rate of 5 mL / min. Collected fractions were lyophilized to obtain the purified compound, which typically appears as a white solid.

[0183] Abbreviation: DCI: 4,5-dicyanoimidazole DCM: dichloromethane DMF: Dimethylformamide DMT: 4,4'-dimethoxytrityl THF: Tetrahydrofuran Bz: Benzoyl Ibu: Isobutyryl

[0184] Synthesis of inverted oligonucleotides The term "inverted nucleoside" or "reverse nucleoside" (DNA X INV ) refers to a nucleoside that contains a dimethoxytrityl (DMT) group and a phosphoramidite group that are inverted from the typical orientation; the DMT group is attached to the 3'-OH of the ribose moiety, and the phosphoramidite is attached to the 5'-OH of the ribose moiety.

[0185] DNA X INV (X=A, T, C, or G) were introduced into oligonucleotides as phosphoramidites during phosphoramidite oligonucleotide synthesis (see Scheme 1 below). This inverted configuration allows oligonucleotide synthesis in the 5' to 3' direction (rather than the standard 3' to 5' direction) and allows for 5'-5' or 3'-3' nuclease-resistant linkages instead of the natural 3'-5' linkage (see Scheme 2 below). See also Ortigao et al., Antisense Res. Dev., 2:129-146 (1992). Exemplary inverted nucleotides are shown in Scheme 2.

[0186] To improve the safety profile of the designed ASO compounds, the phosphorothioate backbone was modified with inverted phosphodiester bonds to reduce the phosphorothioate content and consequently reduce the chiral complexity of the ASO while retaining resistance to exonucleases.

[0187] As an example, for the synthesis of ASO No. 10_4 containing a 3'-3' phosphodiester bond at the 3' terminus, the appropriate Bz-dA-5'-CE phosphoramidite was first attached to the universal support during the first cycle of phosphoramidite oligonucleotide synthesis, followed by the addition of a second monomer, i.e., Bz-LNA A-3'-CE phosphoramidite, in the standard 3'-5' orientation during the second cycle of phosphoramidite oligonucleotide synthesis to create the corresponding terminal 3'-3' bond. Having a single inverted base at the 3' terminus with a 3'-3' bond confers oligonucleotide exonuclease resistance and prevents extension by polymerase, since there is no free 3' hydroxyl group to initiate synthesis.

[0188] Scheme 1: DNA 5' phosphoramidites for the synthesis of inverted 3'-3' or 5'-5' internucleoside linkages TIFF2025508310000029.tif90170

[0189] Scheme 2: Design of phosphorothioate internucleoside linkages and inverted phosphodiester internucleoside linkages TIFF2025508310000030.tif74170

[0190] Example 1: Antisense oligonucleotides reduce TMEM106B mRNA expression in human SK-N-BE(2) neuroblastoma cells As an initial screen, over 1000 designed TMEM106B-targeting ASOs were tested for their ability to reduce TMEM106B mRNA expression in human SK-N-BE(2) neuroblastoma cells (ECACC; Catalog No. 95011815). SK-N-BE(2) cells were grown in cell culture medium of Minimum Essential Medium (MEM) (Sigma, Cat. No. M2279) mixed with Nutrient Mixture F-12 Ham (Sigma, Cat. No. N4888) at a 1:1 (v / v) ratio and supplemented with 1 mM glutamine (Sigma, Cat. No. G7513), 0.025 mg / mL gentamicin (Sigma, Cat. No. G1397), 15% fetal bovine serum (FBS) (Sigma, Cat. No. F7524), and 10% FBS for all subsequent weeks. Cells were trypsinized every 5 days by washing with PBS (Sigma, Cat. No. 14190-094), followed by addition of 0.25% trypsin-EDTA solution (Sigma, Cat. No. T3924) and incubation for 2-3 min at 37°C. Cells were then subjected to trituration before cell seeding. Cells were maintained in culture for up to 15 passages.

[0191] As a next step, 20,000 cells per well were seeded in 96-well plates (Nunc, Cat. No. 167008) in 190 μL of growth medium. ASOs dissolved in PBS were added approximately 24 hours after the cells were seeded to reach the final concentration. Cells were incubated for 3 days without medium change. After incubation, cells were harvested by removal of the medium, followed by addition of 125 μL of RLT lysis buffer (Qiagen, Cat. No. 79216) and 125 μL of 70% ethanol. RNA was purified according to the manufacturer's instructions (Qiagen RNease 96 kit), eluted in a final volume of 50 μL of water, and then further diluted 10-fold in water prior to qPCR.

[0192] The RNA was heat shocked for 40 seconds at 90°C to denature the RNA:LNA duplex, transferred directly to ice, and spun down before use. For one-step qPCR reactions, the qPCR mixture, qScript TM XLE 1-step RT-qPCR TOUGHMIX® Low ROX (QauntaBio, Cat. No. 95134-500) was mixed with two IDT probes with a concentration of 1X to generate a Mastermix. The Taqman probes were obtained from IDT:TMEM:Hs.PT.58.3604573 (primer-probe ratio 2, FAM); POLR2A:Hs.PT.39.a19639531 (primer-probe ratio 2, HEX / VIC). Then, 4 μL of RNA with a concentration of 1-2 ng / μL was mixed with 6 μL of Mastermix in a qPCR plate (MICROAMP® Optical 384-well, Cat. No. 4309849). After sealing, the plate was given a spin at 1000 g for 1 minute at room temperature and transferred to a ViiaTM 7 system (Applied Biosystems, Thermo). The following PCR conditions were used: 50° C. for 15 min, 95° C. for 3 min, 40 cycles of 95° C. for 5 s followed by a temperature decrease of 1.6° C. / s, followed by 60° C. for 45 s. Data were analyzed using QuantStudio™ real-time PCR software.

[0193] qPCR data was captured and quality control of the raw data was performed using Quantstudio 7 software. The data was then imported into E-Workbook, where the BioBook template was used to capture and analyze the data. The following steps were used to analyze the data: 1. RNA quantity was calculated by the delta Ct method (quantity = 2^(-Ct) * 1,000,000,000). 2. The amount of RNA was normalized to the amount calculated for running a housekeeping gene assay in the same well: Relative target amount = amount_target / amount_housekeeping. 3. The degree of RNA knockdown was calculated for each well by dividing the average of all PBS-treated wells on the same plate. Normalized target amount = (relative target amount / [average] relative target amount]_PBS_well) * 100 4. Final data was expressed as a percentage of RNA relative to untreated (PBS) wells. 5. For concentration-response experiments, curves were fitted from the RNA knockdown values ​​for each compound using 8 or 10 concentrations depending on the dilution model (steps 3-4). Curves are fitted using a 4-parameter sigmoidal dose-response model in Biobook.

[0194] In one set of experiments, 613 novel human TMEM106B target targeting ASO compounds were tested at a concentration of 2.5 μM for 3 days. The experiments also included 22 published human TMEM106B targeting compounds, including compound number 25_1 and compound number 20. These compounds were previously described in U.S. Patent Application Publication No. 2021 / 0054383, which is incorporated herein by reference in its entirety. Knockdown of human TMEM106B induced by the designed compounds was measured and quantified, and specific ASO sequences were mapped onto their regions of hybridization within the human TMEM106B transcript of SEQ ID NO:1 (FIG. 4).

[0195] In another set of experiments, 908 novel human TMEM106B ASO compounds were tested at a concentration of 0.5 μM for 3 days. The experiments also included 22 published human TMEM106B targeting compounds, including compound number 25_1 and compound number 20. These compounds were previously described in U.S. Patent Application Publication No. 2021 / 0054383, which is incorporated herein by reference in its entirety. The knockdown of human TMEM106B induced by the novel compounds was quantified and the specific ASO sequences were mapped to their regions of hybridization within the human TMEM106B transcript of SEQ ID NO:1 (FIG. 5). Compounds Nos. 8_1, 8_3, 24_1, 6, 10_4, 29_5, 15, 24_3, 12, 29_3, 26, and 8_4, along with others, exhibited greater knockdown compared to compound No. 31, and along with other ASOs, showed better inhibition of TMEM106B expression compared to compound No. 31.

[0196] In another experiment, 311 novel human TMEM106B targeting compounds were tested at a concentration of 0.25 μM. The experiment also included two published human TMEM106B targeting compounds, including compound number 25_1 and compound number 46. These compounds were previously described in U.S. Patent Application Publication No. 2021 / 0054383, which is incorporated by reference in its entirety. The knockdown of human TMEM106B induced by the novel compounds was quantified and the specific ASO sequences were mapped onto their regions of hybridization within the human TMEM106B transcript of SEQ ID NO:1 (FIG. 6). Compounds No. 8_1, No. 8_3, No. 6, No. 8_4, and No. 29_5, along with other ASOs, showed superior inhibition of TMEM106B expression compared to compound number 25_1. The results of the above three experiments are detailed in Table 6 below.

[0197] TIFF2025508310000031.tif231170

[0198] Example 2: In vitro efficacy of antisense oligonucleotides targeting human TMEM106B Determination of IC50 values ​​of antisense oligonucleotides targeting TMEM106B Human TMEM106B-targeted ASO compounds were first screened in the human neuroblastoma cell line, SK-N-BE(2), as described in Example 1. To have better translation to human patients, the efficacy of candidate ASO compounds against human TMEM106B was tested in human neurons derived from induced pluripotent stem cells (iPSCs) and co-cultured with human primary astrocytes.

[0199] Neurons derived from human iPSCs were harvested in RLT buffer. RNA extraction was completed using Qiagen RNeasy 96 kit. RNA sample quality was measured and samples were subsequently subjected to RNAseq. DNA libraries were prepared from samples and sequenced in batches of up to 48 samples at a time. ASOs were diluted from stocks in intermediate 96-well plates in serial dilutions in culture-based media buffer. The serial dilutions were designed to yield concentrations of 10X of the final designated concentrations to reduce media dilution after addition to neurons. On day 7, cells were harvested for Taqman assay using Cells to Ct reagent (ThermoFisher) according to the manufacturer's instructions. Relative expression levels obtained using the Taqman assay were normalized to control wells and data was imported into Graph Pad Prism software for IC50 analysis, where analysis was completed using the software's curve fit function.

[0200] After 7 days of treatment, 27 ASOs from all the tested compounds were selected for characterization with IC50 curves. Two prior art published ASOs, namely compound numbers 31 and 25_1, were included as benchmarks for the performance of the newly designed ASO compounds of the present disclosure. The newly designed ASOs were found to have IC50 concentrations ranging from 4.6 nM to 175 nM (Table 7 and Figures 7-9), indicating high potency of the tested compounds. Furthermore, near complete suppression of human neuronal TMEM106B mRNA was observed, indicating promising therapeutic potential. From this screen, ASOs were identified with improved potency compared to compound number 25_1, including compound numbers 8_3, 8_4, 8_1, 29_3, and 12, along with other ASOs. All 27 ASO compounds had improved IC50 compared to compound number 31.

[0201] Off-target analysis Potential off-target activity of candidate ASO compounds was evaluated in a set of 18 ASOs selected from IC50 experiments. The results of these studies are summarized in Table 7 below. Cells were treated with 5 μM, 0.5 μM, and 0.05 μM ASOs and bulk RNAseq was performed. ASO concentrations were selected at 1-5X, 10X, and approximately 100-1000X IC50 to stress test ASO compounds for off-target effects. This had only 1-2 mismatched, inserted, or deleted base pairs between the ASO and the off-target gene, and therefore activity may have been weak and large amounts of ASO concentration may have been required for observation. At 5 μM, potential ASO off-target effects were detected. This concentration was 100X-1000X IC50 of the ASO tested; such concentrations would not normally occur in vivo. ASO-treated samples were compared to a non-targeting control ASO (compound no. 13) to identify changes in expression of transcripts other than the intended TMEM106B target. Volcano plots were generated and analyzed to determine potential off-target effects based on bioinformatics predictions of 1 or 2 base pair mismatches. This group of ASOs was predicted to have between 56 and 1069 potential protein-coding off-target genes identified through bioinformatics studies. However, several of the ASOs tested (compound nos. 10_4, 29_3, 24_3, and 29_5, along with other ASOs) were found to have less than 5 off-target genes that showed greater than 50% mRNA reduction at this non-physiological concentration. No off-target genes were identified at ASO concentrations of 0.5 μM or 0.05 μM, which are closer to the physiological concentration of ASO administered to subjects. These findings indicate that some of these ASOs may have therapeutically favorable safety profiles. Several of the ASOs tested showed off-target binding to between 6 and 15 observed genes at 5 μM. These off-target effects were significantly reduced when the ASO concentration was reduced to and including 0.5 μM.Significantly, compound No. 24_3 had no detectable off-target effects on human neurons even at a non-physiological ASO concentration of 5 μM. In contrast, other ASOs, including compound No. 8_1, were found to bind to more than 27 off-target genes. The number of off-target genes targeted by these ASOs was reduced as the ASO concentration was decreased. In summary, when the ASO was at a concentration of 0.05 μM, which is close to the IC50 concentration, almost all of the identified off-target genes were not detected or were reduced. The above findings are summarized in Table 7 for different ASO compounds. These findings indicate that carefully selected ASO concentrations can confer selective inhibition of TMEM106B expression and reduced off-target effects.

[0202] TIFF2025508310000032.tif239170

[0203] Description of Table 7: Table 7 summarizes the ASOs selected from the screen for further characterization. Previously identified ASOs of compound numbers 25_1 and 31 were included for comparison. IC50 values ​​are listed. Mm1 gene: 1 base pair mismatch gene. Mm2 gene: 2 base pair mismatch gene. Mm1 protein coding: 1 base pair mismatch gene encoding a protein. Mm2 protein coding: 2 base pair mismatch gene encoding a protein. Off-targets observed at 5 μM: off-target genes with >50% mRNA reduction in human iPSC neurons at 5 μM ASO concentration. Off-target genes at 5 μM: name of first 10 off-target genes. Off-targets observed at 0.5 μM: off-target genes with >50% mRNA reduction observed in human iPSC neurons at 0.5 μM ASO concentration. Off-targets observed at 0.05 μM: Off-target genes with greater than 50% mRNA reduction observed in human iPSC neurons at an ASO concentration of 0.05 μM.

[0204] Example 3: Functional assay of lysosomal protein degradation in human retinal pigment epithelial cells To determine whether the TMEM106B-targeting ASO of the present disclosure affects lysosomal function, a functional assay was performed in human retinal pigment epithelial (RPE) cells in vitro. Human (RPE) cells were selected as a model for this experiment due to their flat cell morphology, which facilitates visualization of lysosomes using known imaging techniques. Human RPE cells were purchased from ATCC and cultured according to the manufacturer's instructions.

[0205] Cells were treated with self-quenching BSA, which can be easily internalized and transported to lysosomes. This was performed by plating human RPE cells at 1000 cells / well in 1% Geltrex-coated 384-well plates. Cells were treated with ASO for 5 days in 0.5% serum medium to prevent overgrowth. 200 nM bafilomycin was used as a positive control. BSA self-quenching BODIPY-FL conjugate was purchased from BioVision (7932-10) and the experiment was performed according to the manufacturer's suggested protocol. SA was added at a final concentration of 20 μg / mL and incubated overnight. Cells were fixed with 4% PFA and 4% sucrose for 20 min at room temperature using Bravo automation. The fixed cells were then washed twice with PBS using a Biotek 406 microplate washer (Beckman Coulter), followed by permeabilization and blocking by incubation with a solution containing 1× PBS, 0.1% Triton X-100, 2% donkey serum, and 1% BSA at room temperature for 30 min. The blocking solution was removed and the cells were incubated with primary antibodies in blocking solution overnight at 4° C. The cells were blocked and then stained for Hoechst (Sigma 1:10,000) and LAMP1 (BD 1:500). After washing six times with PBS on a Biotek 406 cell plate washer (Beckman Coulter), the cells were incubated with fluorophore-conjugated secondary antibodies (Jackson Immuno Research Laboratories, Inc) in the dark for 1 h at room temperature to avoid photobleaching. The cells were then washed another six times with PBS before imaging. Fluorescence images were captured using an InCell6000 confocal microscope (GE Healthcare Life Sciences). Specifically, 16 fields per well were imaged at a magnification of 40x. Each treatment condition was represented by 4 wells. BSA signals were segmented and total intensity was summed from all 16 fields as a total calculation per well.Image analysis of the BSA signal was performed using InCell 6000 analysis software. Quadruplicates were averaged and plotted with error bars representing the standard error of the mean (SEM).

[0206] Upon degradation, the degraded and separated BSA fragments emitted fluorescence, allowing visualization and quantification of lysosomal proteolytic activity (FIG. 10C, white dots). Using the assay, it was observed that ASOs of compounds no. 29_3, 15, 26, and 14 effectively reduced the degradation activity of BSA by about 50%. These findings indicate that lysosomal function was reduced by ASO-mediated degradation of TMEM106B (FIGS. 10B and 10C).

[0207] A select panel of TMEM106B-targeting ASOs was confirmed to have robust TMEM106B knockdown activity in RPE cells. Multiple ASOs, namely compound numbers 29_3, 15, and 26, reduced TMEM106B mRNA by approximately 80% at a concentration of 10 μM (FIG. 10A). ASO compound number 14 reduced TMEM06b mRNA by approximately 50% at a concentration of 10 μM. ASO compound number 10_13 showed almost no reduction of TMEM106B and was therefore used as a negative control for ASOs (FIG. 10A). An increase in the ASO concentration required to cause the same level of knockdown was observed in human RPE cells compared to human iPSC-derived neurons. This is likely due to continued proliferation of RPE cells during the 5-day ASO treatment, and therefore higher ASO concentrations are required to cause TMEM106B knockdown.

[0208] Example 4: Caspase assay to determine the safety of TMEM106B-targeted antisense oligonucleotides To measure the in vitro toxicity profile of the ASOs described herein, a caspase assay was performed to measure apoptosis in treated cells. NIH 3T3-L1 cells (ATCC cat. CL-173) were transfected with 100 nM ASOs using Lipofectamine2000 (Thermo Fisher 11668-019). Activation of caspase 3 and caspase 7 was measured after 24 hours as a surrogate for apoptosis according to the protocol described below, adapted from Dieckmann et al., Mol. Ther. Nucl. Acids 10:45-54 (2018). Experiments were performed in triplicate, with each run performed in a 96-well plate format. 24 hours prior to transfection, cells were trypsinized and seeded at 4000 cells / well in 96-well plates (ViewPlate-96, PerkinElmer, cat 6005181) in 100 μL of Dulbecco's Modified Eagle Medium (DMEM) growth medium (Sigma, Cat. No. D0819), 10% fetal bovine serum (Sigma, Cat. No. F7524), 1 mM sodium pyruvate (Sigma, Cat. No. S8636), and 0.025 mg / mL gentamicin (Sigma, Cat. No. G1397) at 37°C and 5% CO2.

[0209] ASO was diluted in Dulbecco's Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, Cat. No. 14190250) to a final concentration of 5 μM. A negative control was also prepared with oligonucleotide at a concentration of 0 μM. 9.6 μL of diluted ASO was gently mixed with 110.4 μL of Opti-MEM (Thermo Fisher Scientific, Cat. No. 31985047). 30 μL of LOM solution containing Lipofectamine™ 2000 (Thermo Fisher Scientific, Cat. No. 11668019) mixed at a ratio of 1:99 (v / v) with Opti-MEM (Thermo Fisher Scientific, Cat. No. 31985047) was used immediately after preparation and added to each well of an empty 96-well plate. Oligonucleotide diluted in 30 μL of Opti-MEM was added to the LOM mix, followed by a 20-minute incubation. Media was removed from NIH 3T3-L1 cell cultures and 50% media containing DMEM, 20% fetal bovine serum, and 1 mM sodium pyruvate and 50 μL of LNA oligonucleotide in LOM solution were added.

[0210] Then, 24 hours after adding ASO to the cells, 100 μL of Caspase-Glo® 3 / 7 Reagent (Promega, Cat. No. G8093), prepared according to the manufacturer's protocol, was added to the cells. The plate was shaken at 500 rpm for 30 seconds and incubated at room temperature for 1 hour, followed by blocking the underside of the plate with BackSeal (PerkinElmer, Cat. No. 6005199). Luminescence was then measured using an EnSight Multimode Plate Reader (PerkinElmer, Cat. No. HH34000000).

[0211] The median of the raw measurements for wells flagged as "Native" (no oligonucleotide) was calculated. Each measurement within that plate was then normalized by the median. The median of all "Native" and "Positive" wells (Positive = known cytotoxic oligonucleotide) was calculated separately within the same plate. Each well was scaled as ((Value-Median(Native)) / (Median(Positive)-Median(Native))*100. Using the values ​​calculated in the previous step, the median of the triplicates (3 plates) was calculated and reported for each well. The results of the caspase assay are summarized in Table 8.

[0212] TIFF2025508310000033.tif177170

[0213] Example 5: In vivo efficacy of antisense oligonucleotides targeting human TMEM106B mRNA A select panel of ASO agents described in Example 1 was tested for their ability to reduce TMEM106B expression in mouse brain in vivo. Transgenic mice expressing the human TMEM106B gene were generated via bacterial artificial chromosome (BAC) technology. Male and female mice (23-32 g) received 5 μL of ASO compounds formulated in 0.9% saline and delivered by freehand injection into the right lateral ventricle at stereotaxic coordinates of -0.5 AP, 1.0 ML, 2.5 DV. Under isoflurane anesthesia, an incision was made in the skin just above the skull. Mice received an injection directly into the ventricle through the bone and the incision was closed using surgical glue. Animals were allowed to recover on a heating pad and monitored until fully awake.

[0214] Four in vivo studies were performed. Study 1 tested a single dose of 100 μg of four ASOs, compound numbers 14, 15, 29_3, and 26, a negative control ASO of compound number 10_13, or saline (Figure 11). Study 2 tested a single dose of 100 μg of five ASOs, compound numbers 25_1, 8_1, 24_1, 6, and 10_4, and saline (Figure 12). Study 3 tested two dose levels for each of compound numbers 31, 29_3, and 26, a negative control ASO of compound number 10_13, or saline (Figure 13). Study 4 tested two dose levels for compound number 29_5. The same methodology was applied to all four studies. 14 days after injection, mice were sacrificed with an overdose of isoflurane followed by decapitation, and the brains were placed on an ice-cold mouse brain slicer matrix (Stoelting) and sliced ​​(2 mm) (starting at 0.26 mm from bregma) into two circular sections weighing approximately 30-40 mg. Tissues were immersed in RNALater solution (ThermoFisher Scientific, cat.AM7021) and stored at 4°C. RNA was isolated using the RNeasy Mini Kit (Qiagen, cat.74116) using a QIAcube robotic workstation (Qiagen). Briefly, brain fragments were transferred from RNALater solution to 1 mL of RLT buffer in a 1.5 mL Eppendorf tube containing 3 mm tungsten carbide beads (Qiagen, cat 69997). Tissues were lysed using a TissueLyser II (Qiagen; 3 min at 30 Hz) and centrifuged at 20,000 g for 3 min. 550 μL of lysate (no foaming, no sediment) was transferred to a 2 mL Eppendorf tube and placed in a QIAcube pre-filled with all required reagents. RNA was purified according to the program "RNeasy Mini - Animal tissues and cells - Large volume samples (version 2)" with elution in 50 μL H2O. RNA concentration was determined using a NanoDrop The concentration was measured using a ThermoFisher Scientific 8000 spectrophotometer and adjusted to 2 ng / μL with H2O.

[0215] Expression levels of TMEM106B mRNA on sections from the right brain were measured as described in Example 1 using TMEM106B Hs00998849_ml as a probe (Thermo Fisher Scientific, cat.4351370) and a GAPDH probe (cat.4351309, Applied Biosystems) for normalization. Data were analyzed using QuantStudioTM real-time PCR software and duplicate readings were averaged. Statistical analysis of TMEM106B mRNA reduction was analyzed using one-way ANOVA in Graphpad Prism 5.0.

[0216] All TMEM106B-targeted oligonucleotides tested reduced TMEM106B mRNA levels two weeks after a single injection (Figures 11-14 and Table 9 below). Data are presented as a percentage of the average control sample (saline-treated mice; lower values ​​indicate greater inhibition). In Study 1, all four ASOs tested, namely compound numbers 14 (60% remaining), 15 (44%), 29_3 (25%), and 26 (27%), significantly reduced TMEM106B compared to saline-treated mice (F(6,32)=104.0, p<0.00001, Dunnett's post-hoc test). The negative control ASO (compound number 10_13) had no effect on TMEM106B levels. In study 2, all five ASOs significantly reduced TMEM106B transcripts compared to saline-treated mice (Figure 12; (F(6,33) = 30.19, p < 0.0001, Dunnett post-hoc test). Compounds 25_1 remained at 29%, 8_1 at 34%, 24_1 at 33%, 6 at 56%, and 10_4 at 47%. In study 3, all ASOs significantly reduced TMEM106B mRNA (F(8,45) = 64.57, p < 0.0001, Dunnett post-hoc test). Compounds #29_3 and #26 ASOs produced greater knockdown at doses of 25 and 100 μg compared to compound #31 at the same dose level (Figure 13), with a 4-fold greater in In vivo potency was shown. (%KD for Study 3: Negative control ASO (100ug) was not significantly different from saline treated mice (93% remaining), Compound #31 (25ug) 94%, Compound #31 (100ug) 50%, Compound #29_3 (25ug) 53%, Compound #29_3 (100ug) 22%, Compound #26 (25ug) 65%, and Compound #26 (100ug) 28% remaining). Minimal side effects were observed in mice treated with the ASOs described herein (Figure 14).In study 4, the negative control ASO, CMPD #13, had no effect on TMEM106B mRNA, but CMPD #29_5 significantly reduced mRNA with 68% remaining at 25ug (p<.001) and 32% remaining at 100ug (p<.0001) (F(3,20)=41.4, p<.0001, Dunnett's post-hoc test). As expected, the non-transgenic controls did not express human TMEM106b mRNA.

[0217] TIFF2025508310000034.tif151170

[0218] Other embodiments Various modifications and variations of the invention described will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the description for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

[0219] Other embodiments are within the scope of the claims.

Claims

1. 1. An antisense oligonucleotide comprising: is a compound selected from the group consisting of An antisense oligonucleotide where non-italicized capital letters are beta-D-oxy LNA nucleosides, italicized capital letters are 2'-O-methyl nucleosides, lower case letters are DNA nucleosides, all LNA C's are 5-methylcytosines, and all internucleoside linkages are phosphorothioate internucleoside linkages.

2. The antisense oligonucleotide of claim 1, which is compound number 26.

3. The antisense oligonucleotide of claim 1, which is compound number 8_3.

4. The antisense oligonucleotide of claim 1, which is compound number 8_4.

5. The antisense oligonucleotide of claim 1, which is compound number 12.

6. The antisense oligonucleotide of claim 1, which is compound number 24_3.

7. The antisense oligonucleotide of claim 1, which is compound number 24_1.

8. The antisense oligonucleotide of claim 1, which is compound number 29_5.

9. The antisense oligonucleotide of claim 1, which is compound number 8_1.

10. The antisense oligonucleotide of claim 1, which is compound number 10_4.

11. The antisense oligonucleotide of claim 1, which is compound number 6.

12. The antisense oligonucleotide of claim 1, which is compound number 29_3.

13. The antisense oligonucleotide of claim 1, which is compound number 14.

14. The antisense oligonucleotide of claim 1, which is compound number 15.

15. 15. The antisense oligonucleotide of claim 1, wherein the TMEM106B target nucleic acid is a mammalian TMEM106B target nucleic acid.

16. The antisense oligonucleotide of claim 15, wherein the mammalian TMEM106B target nucleic acid is a human TMEM106B target nucleic acid.

17. 17. A conjugate comprising an antisense oligonucleotide according to any one of claims 1 to 16 and at least one conjugate moiety covalently attached to the antisense oligonucleotide.

18. 17. A pharma- ceutically acceptable salt of the antisense oligonucleotide of any one of claims 1 to 16.

19. 20. A pharma- ceutically acceptable salt of the conjugate of claim 18.

20. 17. A pharmaceutical composition comprising an antisense oligonucleotide according to any one of claims 1 to 16 and a pharma- ceutically acceptable diluent, solvent, carrier, salt or adjuvant.

21. 20. A pharmaceutical composition comprising the conjugate of claim 17 and a pharma- ceutically acceptable diluent, solvent, carrier, salt or adjuvant.

22. An in vivo or in vitro method for regulating expression of TMEM106B in a target cell expressing TMEM106B, comprising administering to the cell an effective amount of a pharmaceutical composition described in claim 20 or 21.

23. 22. A method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of a pharmaceutical composition according to claim 20 or 21 to a subject suffering from or susceptible to the disease.

24. 22. A pharmaceutical composition according to claim 20 or 21 for use in the treatment or prevention of a disease.

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