EP4638751A2

EP4638751A2 - Compositions and methods for treating hnrnph2-related disorders

Info

Publication number: EP4638751A2
Application number: EP23908389.2A
Authority: EP
Inventors: Rodney BOWLING; Neda GHOUSIFAM
Original assignee: To Cure A Rose Foundation
Current assignee: To Cure A Rose Foundation
Priority date: 2022-12-20
Filing date: 2023-12-20
Publication date: 2025-10-29
Also published as: WO2024137726A3; WO2024137726A2

Abstract

The present disclosure provides compositions and methods for treating conditions associated with pathogenic variants of Heterogeneous Nuclear Ribonucleoprotein HNRNPH2. Such pathogenic variants in various embodiments can be linked to one or more of developmental delay, intellectual disability, language impairment, motor impairment, growth and musculoskeletal disturbances, dysmorphic features, epilepsy, autism spectrum disorder, and cortical visual impairment. According to this disclosure, the composition comprises an effective amount of an antisense oligonucleotide (ASO) that inhibits the expression of heterogeneous nuclear ribonucleoprotein HNRNPH2 Mrna, including a pathogenic variant, and a pharmaceutically acceptable vehicle. In embodiments, HNRNPH1 is not decreased in expression, and in some embodiments may increase in expression.

Description

COMPOSITIONS AND METHODS FOR TREATING HNRNPH2 -RELATED DISORDERS

PRIORITY

This Application claims the benefit of, and priority to, U.S. provisional application no. 63/433,870 filed December 20, 2022, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on December 15, 2023, is named “TCRF-001PC/134401- 5001_SequenceListing” and is 342,000 bytes in size.

BACKGROUND

RNA processing (including RNA splicing, transport, localization, translation, and degradation) is critically important for brain development and function, as neurons are postmitotic cells dependent on RNA expression [2], To successfully regulate RNA processing and protein synthesis, over 500 RNA-binding proteins (RBPs) in humans are abundantly and ubiquitously expressed, and found primarily in the nucleus [3], Although ubiquitous, there are tissue-specific changes, including in alternative splicing, of RBPs resulting in cellspecific phenotypes [4], As RBPs are necessary for many steps of neuronal RNA metabolism, there are multiple opportunities for dysfunction, which is highlighted by the range of neurological phenotypes resulting from variation in RBP-encoding genes, including neurodegenerative diseases, muscular atrophies, and various cancers.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a subfamily (of 33 core and minor members) of RNA binding proteins that form a complex with heterogeneous nuclear RNA (hnRNA) and are implicated in many steps of RNA processing [5], These proteins have distinct nucleic acid binding properties, are associated with pre-mRNAs in the nucleus, and influence pre-mRNA processing and other aspects of mRNA metabolism and transport. While all members of the subfamily are present in the nucleus, some also shuttle between the nucleus and the cytoplasm. Heterogeneous nuclear ribonucleoprotein HNRNPH2 is a member of this subfamily and is encoded on the X-chromosome. It is ubiquitous in expression and is highly colocalized in the nuclear compartments in the brain, gastrointestinal tract, lung, skin, spleen and testes. The protein is also found in the cytoplasm and is suspected to shuttle back and forth from the nucleus. HNRNPH2 harbors three quasi- RNA Recognition Motifs (RRMs) that bind RNA, two glycine-rich domains that aid in homo/heterodimerization, as well as a nuclear localization signal (NLS) that is necessary for its localization to the nuclear compartment.

Variants in the HNRNPH2 gene cause a rare neurodevel opmental syndrome [5], Over 100 individuals (male and female) have been identified with a suspected prevalence of 1:50,000-1.5:100,000 live births. Although there is currently no positively defined phenotype, the main characteristics seem to include: developmental delay/intellectual disability, severe language impairment, motor problems, growth and musculoskeletal disturbances, dysmorphic features, epilepsy, autism spectrum disorder, cortical visual impairment, and rarely, early stroke and premature death [5,6],

The majority of variants (eleven different de novo missense variants) in HNRNPH2 are localized in the NLS but some are located outside the NLS. All of these are either heterozygous (in females) or hemizygous (in males) variants and seem to be gain-of-function with no toxic accumulation. R206N and P209L mutations (in NLS) show dysfunctional nucleocytoplasmic shuttling of proteins. Whereas the R114W mutation (outside NLS, in RRM) exhibits reduced interaction with members of the large assembly of splicing regulators, while shuttling is believed to be intact [7],

Currently, no therapeutics exist for HNRNPH2 -related disorders.

DETAILED DESCRIPTION

The present disclosure provides compositions (e.g., pharmaceutical compositions) and methods for treating conditions associated with pathogenic variants of Heterogeneous Nuclear Ribonucleoprotein HNRNPH2. Such pathogenic variants in various embodiments can be linked to one or more of developmental delay, intellectual disability, language impairment, motor impairment, growth and musculoskeletal disturbances, dysmorphic features, epilepsy, autism spectrum disorder, and cortical visual impairment. According to this disclosure, the composition comprises an effective amount of an antisense oligonucleotide (ASO) that inhibits the expression of heterogeneous nuclear ribonucleoprotein HNRNPH2 mRNA (e.g., including a pathogenic variant) and a pharmaceutically acceptable vehicle.

In various embodiments, the antisense oligonucleotide comprises 10 to 40 linked nucleotides, or from 10 to 35 linked nucleotides, or from 10 to 30 linked nucleotides, and has a sequence that is complementary to HNRNPH2 mRNA. In some embodiments, the oligonucleotide is at least 12 nucleotides in length, or is at least 14 nucleotides in length, or is at least 16 nucleotides in length, or at least 18 nucleotides in length. In various embodiments, the oligonucleotide is from 10 to 24 nucleotides in length, or is from 16 to 24 nucleotides in length. In various embodiments, the oligonucleotide is 16, 17, 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, the oligonucleotide is 20 nucleotides in length.

In various embodiments, the oligonucleotide can hybridize specifically (and hence specifically inhibit) a pathogenic variant of HNRNPH2, thereby not significantly or substantially inhibiting expression of a non-pathogenic allele (e.g., in females). In these or other embodiments, the ASO does not significantly or substantially inhibit expression of the related HNRNPH1 mRNA and/or protein. HNRNPH1 is a highly conserved paralog of HNRNPH2 with over 95% sequence homology at the DNA level. Individuals with missense mutations in HNRNPH1 display overlapping phenotypic characteristics to those carrying mutations in HNRNPH2. Assuming that HNRNPH1 is functional in individuals with the HNRNPH2-related disorder, functional HNRNPH1 may be able to partially compensate for disrupted HNRNPH2. In some embodiments, the oligonucleotide reducing expression of HNRNPH2 surprisingly results in increased expression of HNRNPH1.

In various embodiments, the oligonucleotide hybridizes specifically to a pathogenic variant of HNRNPH2 mRNA, and wherein the pathogenic variant encodes a mutant of the RRM, such as R114W (see SEQ ID NO: 57), or encodes a mutant of the NLS (such as R206N or P209L). In still other embodiments, the pathogenic variant encodes a mutant of a glycine-rich domain. In some embodiments, the oligonucleotide does not hybridize to a specific pathogenic variant of HNRNPH2 mRNA, and is thus agnostic as to the pathogenic variant. In these embodiments, HNRNPH1 (which is increased in expression in some embodiments) is believed to compensate for the loss of HNRNPH2 expression.

In various embodiments, the oligonucleotide comprises at least 8 contiguous nucleobases of any nucleotide sequence or oligonucleotide described herein (in Tables 1-4; SEQ ID NOS: 1-56 and 60-390), and the oligonucleotide is substantially (e.g., at least 90% or at least 95%) or entirely complementary to an equal length segment of HNRNPH2 mRNA (SEQ ID NO: 58). In some embodiments, the oligonucleotide comprises at least 10 contiguous nucleobases of any one of SEQ ID NOs: 1-56 or 60-390, and the oligonucleotide is substantially (e.g., at least 90% or at least 95%) or entirely complementary to an equal length segment of HNRNPH2 mRNA (SEQ ID NO: 58). In various embodiments, the oligonucleotide comprises at least 12, or at least 14, or at least 16, or at least 18 contiguous nucleobases of any nucleotide sequence selected from SEQ ID NOS: 1-56 or 60-390. In some embodiments, the oligonucleotide comprises or consists of a nucleobase sequence of any one of SEQ ID NOS: 1-56 and 60-390. For simplicity nucleotide sequences may be shown herein using DNA nucleotide sequences (i.e., including T nucleobases) or as RNA nucleotide sequences (i.e., including U nucleobases). It is understood from the context that when a nucleotide or sequence is intended to be RNA, T nucleotides are substituted with U (or modified U such as pseudouridine or 1 -methylpseudouridine); and when the nucleotide or sequence is intended to be DNA, T nucleotides are employed.

In some embodiments, the binding (e.g., hybridization) of the antisense oligonucleotide to the target mRNA leads to degradation of the target mRNA or blocks translation of the target mRNA. In some embodiments, the binding of the antisense oligonucleotide to the target mRNA creates a duplex nucleic acid molecule, which then recruits an endogenous nuclease for degradation of the mRNA. In some embodiments, the antisense oligonucleotide has a stretch of DNA nucleotides sufficient to recruit RNaseH, and thereby trigger degradation of the target mRNA. For example, the antisense oligonucleotide may have a stretch (e.g., a central stretch) of at least 6 or at least 8 DNA nucleotides, and which is optionally a stretch of 9 or 10 DNA nucleotides. In some embodiments, the antisense oligonucleotide has a stretch of 10 DNA nucleotides, 11 DNA nucleotides, or 12 DNA nucleotides. In some embodiments, the antisense oligonucleotide has a stretch of 10 DNA nucleotides. In some embodiments, one or more DNA nucleotides comprise a 2' chemical modification independently selected from 2'-Fluoro, 2'-Methyl, and 2'-Ethyl. In some embodiments, the DNA nucleotides do not contain a 2' modification. For example, the antisense oligonucleotide may be a gapmer having a 5' and a 3' segment, each of the 5' and 3' segments being (independently) from 2 to 6 nucleotides or from 2 to 5 nucleotides (e.g., selected from 3, 4, or 5 nucleotides), and where the 5' and 3' segments do not contain DNA nucleotides. In some embodiments, the 5' and 3' wing segments each have 5 nucleotides. In some embodiments, the gapmer is a 5-10-5 gapmer, having a central bock of 10 DNA nucleotides and 5' and 3' segments of 5 RNA nucleotides each. In other embodiments, the gapmer is a 5-10-4, 4-10-5, or 4-10-4 gapmer, having a central block of 10 DNA nucleotides and a 5' and 3' segments of 4 or 5 RNA nucleotides each. In some embodiments, one or more nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents, optionally where all of the nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents. Exemplary 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxy ethyl (MOE), and a bridged nucleotide (e.g., a locked or bi-cyclic nucleotide) having a 2' to 4' bridge. In some embodiments, the bridged nucleotide has a methylene bridge (locked nucleic acid or LNA) or a constrained ethyl bridge (cEt). In embodiments, the antisense oligonucleotides are 5-10-5 gapmers having MOE, LNA, or a mix of MOE and LNA modifications in 5' and 3' wing segments (e.g., in an alternating pattern). Exemplary chemical modification patterns are shown in Tables 1, 2, 3, 4A, and 4B.

The term “gapmer” refers to an oligonucleotide having a central block of deoxynucleotides (also referred to herein as “DNA nucleotides”) with 5' and 3' segments (of at least 2 nucleotides) of RNA nucleotides. As used herein, the term “DNA nucleotide” refers to a nucleotide that is not an RNA nucleotide. DNA nucleotides typically have a 2' H, but may alternatively have various 2' chemical modifications, including 2'-halo and 2'-lower alkyl (e.g., Cl-4). In some embodiments, the 2' chemical modifications of DNA nucleotides are independently selected from 2'-Fluoro, 2'-Methyl, and 2'-Ethyl.

Locked nucleic acid (LNA) or “locked nucleotides” are described, for example, in U.S. Patent Nos. 6,268,490; 6,316,198; 6,403,566; 6,770,748; 6,998,484; 6,670,461; and 7,034,133, all of which are hereby incorporated by reference in their entireties. LNAs are modified nucleotides that contain a bridge between the 2' and 4' carbons of the sugar moiety resulting in a “locked” conformation, and/or bicyclic structure. Other suitable locked nucleotides that can be incorporated in the oligonucleotides of this disclosure include those described in U.S. Pat. Nos. 6,403,566 and 6,833,361, both of which are hereby incorporated by reference in their entireties. In exemplary embodiments, the locked nucleotides are independently selected from a 2' to 4' methylene bridge and a constrained ethyl (cEt) bridge (see, US Patent Nos. 7,399,845 and 7,569,686, which are hereby incorporated by reference in their entireties).

In some embodiments, the antisense oligonucleotide has a modified backbone or modified internucleotide linkages. The term “internucleotide linkage” refers to the linkage between two adjacent nucleosides in a polynucleotide molecule. Naturally, the intemucleotide linkage is a phosphodiester bond that forms between two oxygen atoms of the phosphate group and an oxygen atom of the sugar (either at 3' or 5' position) to form two ester bonds bridging between the two adjacent nucleosides. Modification of the intemucleotide linkage may provide different characteristics, including but not limited to enhanced stability. For example, phosphorothioate or phosphorodithioate linkages increase the resistance of the intemucleotide linkage to nucleases. Another example is phosphoacetate linkage (PACE), which improves transfection characteristics and enhances nuclease resistance. Intemucleotide linkages and oligonucleotide backbone modifications which may be employed in the oligonucleotides of the present description include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, phosphoramidite, phosphorodiamidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, peptide nucleic acid, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone intemucleoside linkages. In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides.

In some embodiments, the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate intemucleotide linkages. In some embodiments, phosphorothioate or phosphorodithioate bonds can be introduced between the last three to five nucleotides at the 5'- and/or 3'-end of the oligonucleotide to reduce exonuclease degradation. In some embodiments, the antisense oligonucleotide has a combination of phosphodiester and phosphorothioate/phosphorodithioate linkages. In some embodiments, the antisense oligonucleotide contains at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten phosphorothioate or phosphorodithioate intemucleotide linkages. In some embodiments, the antisense oligonucleotide comprises substantially alternating phosphodiester and phosphorothioate intemucleotide linkages. In some embodiments, the antisense oligonucleotide is fully phosphorothioate/phosphorodithioate linked (i.e., all bonds are either phosphorothioate or phosphorodithi oate) .

In some embodiments, DNA intemucleotide linkages, in addition to intemucleotide linkages at 5' and 3' ends, are phosphorothioate linkages. In such embodiments, other intemucleotide linkages in the wing segments between RNA nucleotides can be phosphodiester (e.g., unmodified backbone linkage).

In some embodiments, particularly where RNaseH recruitment is not desired, the antisense oligonucleotide has a morpholino backbone. Morpholino oligonucleotides do not generally trigger the degradation of their target RNA molecules, and can be effective for steric blocking of a target RNA sequence. Morpholino oligonucleotides and their synthesis are disclosed generally in US Patent No. 11,028,386, US Patent No. 10,947,533, and US Patent No. 10,927,378, each of which is hereby incorporated by reference in its entirety. In some embodiments, the antisense oligonucleotide comprises thiomorpholino nucleotides and/or other substituted or modified nucleotides such as those described, for example, in International Patent Application Publication No. WO/2019/060522 and International Patent Application Publication No. WO/2018/057430, each of which is hereby incorporated by reference in its entirety. For example, Langner et al. describe methods for synthesizing oligonucleotide analogs dubbed thiophosphoramidate morpholino oligonucleotides (TMOs) which incorporate morpholino nucleosides and phosphorothioate linkages (“Synthesis and characterization of thiophosphoramidate morpholino oligonucleotides and chimeras.” JACS 142.38 (2020): 16240-16253; see also Dumbovic, Gabrijela, et al. “Nuclear compartmentalization of TERT mRNA and TUG1 IncRNA is driven by intron retention.” Nature Communications 12.1 (2021): 1-19; both of which are hereby incorporated by reference in their entireties). Thus, the antisense oligonucleotides described herein may comprise full or partial TMO-modified nucleotides, or may comprise chimeras of TMO- modified nucleotides and unmodified nucleotides and/or other nucleotides comprising different modifications (e.g., LNAs).

In some embodiments, the antisense oligonucleotide may contain one or more modified bases. In some embodiments, cytosine is replaced with 5-methylcytosine, which may enhance base pairing. Other modified bases (particularly of cytosine or guanine) can be employed to reduce immunogenicity, where needed. Other modified bases are described in US Patent No. 10,064,959, which is hereby incorporated by reference. In various embodiments, cytidine nucleobases in the antisense oligonucleotide are 5-methyl cytidine. Thus, it will be understood by the skilled person that where a sequence includes a cytidine nucleobase (“C”), the term includes 5-methyl C. Further, it is understood that where uracil bases are included in a sequence, the term “U” includes pseudouridine and Nl- methylpseudouridine.

In embodiments, the melting temperature of the antisense oligonucleotide hybridized to its target sequence is at least about 35 °C. The T_m of an oligonucleotide is the temperature at which 50% of the oligonucleotide is duplexed with its perfect complement and 50% is free in solution. The Tm can be determined experimentally by measuring the absorbance change of the oligonucleotide with its complement as a function of temperature. The T_m can also be estimated using known publicly available T_m calculators. In some embodiments, the Tm of the oligonucleotide hybridized to its target sequence is at least about 40°C, or at least about 45°C, or at least about 50°C. In some embodiments, the T_m of the oligonucleotide hybridized to its target sequence is from about 35°C to about 60°C. In some embodiments, the T_m of the oligonucleotide hybridized to its target sequence is from about 40°C to about 60°C, or from about 50°C to about 60°C.

In various embodiments, the antisense oligonucleotide is selected from an oligonucleotide shown in Tables 1, 2, 3, 4A, or 4B. In some embodiments, the composition or oligonucleotide further comprises a targeting or cell penetrating moiety that increases distribution or accumulation of the oligonucleotides in certain cells or tissues (e.g., brain, neurons). For example, such a targeting or cell penetrating moiety may be conjugated directly or indirectly to the 3' end of the oligonucleotides, optionally though a linker which may be biologically cleavable. In some embodiments, the cell penetrating moiety is conjugated to a transfection component or delivery vehicle. In some embodiments, the composition comprises a sterol conjugate (e.g., cholesterol conjugate) or fatty acid conjugate such as a palmitoyl or stearyl lipid conjugate, which is optionally conjugated to the 3' end of the antisense oligonucleotide. These moi eties can enhance cell penetration. See US 9,012,225, which is hereby incorporated by reference in its entirety.

In various embodiments, the targeting or cell penetrating moiety comprises an antibody or antigen-binding fragment thereof, a peptide, a biological ligand (e.g., including a glycoconjugate), aptamer, lipid, sterol, cholesterol or derivative thereof, integrin, RGD peptide, or cell-penetrating peptide (CPP). More specifically, the targeting moiety may be selected from a single-domain antibody, a single chain antibody, a bi-specific antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin, a Tetranectin, an Affibody; a Transbody, an Anticalin, an AdNectin, an Affilin, a Microbody, a phylomer, a stradobody, a maxibody, an evibody, a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody, a pepbody, a vaccibody, a UniBody, a DuoBody, a Fv, a Fab, a Fab', a F(ab')2, and a peptide mimetic molecule. Various ligand-binding platforms are described in US Patent Nos. or Patent Publication Nos. US 7,417,130, US 2004/132094, US 5,831 ,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446, and/or US 7,166,697, the contents of which are hereby incorporated by reference in their entireties.

In some embodiments, the composition comprises an encapsulation or transfection reagent or vehicle. In some embodiments, the antisense oligonucleotide is encapsulated in a particle. In various embodiments, the particle is a liposome, polymeric nanoparticle, or lipid nanoparticle. Exemplary polymeric nanoparticles can be formed of PLA, PLGA, or PEG copolymers thereof. In some embodiments, the particle comprises poly(β amino ester) polymers. In various embodiments, the LNPs comprise a cationic or ionizable lipid, a neutral lipid, a cholesterol or cholesterol moiety, and aPEGylated lipid. In exemplary embodiments, a targeting moiety is conjugated to the termini of a portion of PEG groups that form a hydrophilic outer sheath.

In some embodiments, the lipid nanoparticle (or LNP) comprises a structural lipid. Exemplary structural lipids can be selected from one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, and tocopherols (e.g., alpha tocopherol). In some embodiments, the structural lipid is cholesterol.

In some embodiments, the LNP comprises one or more phospholipids. Exemplary phospholipids are selected from cardiolipins, sterol modified lipids (modified with a cholesterol moiety attached at the sn-2 carbon of the glycerol backbone), mixed-acyl glycerophospholipids, and symmetrical acyl glycerophospholipids. Head groups for acyl glycerophospholipids include, for example, phosphatidic acid, lysophosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphoinositides, and phosphatidyl serine. Exemplary phospholipids are selected from 1,2-dilinoleoyl-sn- glycero-3 -phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3- phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3 -phosphocholine (18:0 Diether PC), 1-oleoyl — 2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1- hexadecyl-sn-glycero-3 -phosphocholine (Cl 6 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3- phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl- sn-glycero-3 -phosphocholine, 1,2-dioleoyl-sn-glycero-3 -phosphoethanol amine (DOPE),

1.2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3- phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

In some embodiments, the lipid nanoparticle composition further comprises one or more PEG lipids. A PEG lipid is a lipid modified with polyethylene glycol. Exemplary PEG lipids are selected from one or more of a PEG-modified phosphatidylethanolamine, a PEG- modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG- modified diacylglycerol, and a PEG-modified dialkylglycerol. A PEG lipid may be selected from PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG- Cholesterol, PEG tocopherol, or a PEG- DSPE lipid.

Lipid particle formulations that find use with embodiments of the present disclosure include those described in US 9,738,593; US 10,221,127; US 10,166,298, which are hereby incorporated by reference in their entirety. In some embodiments, the liposomes or nanoparticles further comprise a targeting moiety as described.

In other embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition for parenteral administration comprises encapsulation in a particle (e.g., lipid nanoparticle) as described. The composition in various embodiments is formulated for parenteral administration, such as intravenous, subcutaneous, intramuscular, intrathecal, or intraventricular administration. In some embodiments, administration is intranasal. In various embodiments, administration is directly to the central nervous system (e.g., by parenteral route), which in some embodiments involves administration of naked (e.g., unencapsulated) antisense oligonucleotides.

In another aspect, the present disclosure provides a method for treating a subject having a pathogenic variant of HNRNPH2. The method comprises administering an antisense oligonucleotide targeting HNRNPH2 to said subject, wherein said antisense oligonucleotide does not substantially target and reduce expression of HNRNPH1, and in some embodiments expression of HNRNPH1 is increased. In some embodiments, an effective amount of a composition of the present disclosure is administered to the subject. In some embodiments, the subject has a pathogenic variant located in the RRM of HNRNPH2, such as the R114W variant. In some embodiments, the subject has a pathogenic variant located in the NLS of HNRNPH2, such as R206N or P209L. In some embodiments, the subject has a pathogenic variant located in a glycine-rich domain of HNRNPH2. In some embodiments, the subject is female, and the subject is heterozygous for the pathogenic variant. In some embodiments, the subject is male. In some embodiments, the therapeutic methods described herein are initiated when the subject is a neonate (4 weeks of age or less) or pediatric age. In some embodiments, the subject is 18 years of age or less, 16 years of age or less, 12 years of age or less, 10 years of age or less, 8 years of age or less, or 6 years of age or less, or 4 years of age or less.

In various embodiments, the compounds or compositions described herein are administered parenterally, such as by intravenous or intrathecal infusion. In some embodiments, the compounds or compositions are administered by direct infusion to target tissue (eg., brain, central nervous system). In some embodiments, the compound or compositions are administered directly to the central nervous system, for example, including but not limited to intrathecal injection.

Dosing and administration schedules can vary, e.g., depending on the weight and age of the patient, a particular targeted variant, and the sequence and chemistry of the compound. In various embodiments, the compositions are administered at least once daily to at least once quarterly. In embodiments, the compositions are administered about weekly, about bimonthly (i.e., about every other week), about monthly, or about quarterly. Dosing and administration schedules can further include varying dosing and administration frequency based on the patient’s response. According to embodiments of this disclosure, the composition may be administered at least once per month or at least once per week.

As used herein, the term “about”, unless the context requires otherwise, means ±10% of an associated value.

Other aspects and embodiments of the present disclosure will be apparent from the following Examples. EXAMPLES

This disclosure proposes to target and knockdown the HNRNPH2 mRNA, either in an allele-specific manner (e.g., the R114W allele) or non-allele-specific manner to slow down or reverse pathogenic phenotypes relating to HNRNPH2 mutation. Anitsense oligonucleotides (ASOs) according to this disclosure can be tested in cell lines harboring mutations of interest (such as but not limited to iPSCs and cells or organoids derived therefrom, such as neural precursor cells and neurons), as well as in animal models.

ASOs disclosed below recruit and initiate the RNAse Hl machinery for HNRNPH2 mRNA cleavage and degradation. It is hypothesized that substantial degradation of the pathogenic HNRNPH2 protein will rescue the pathogenic phenotype. For example, HNRNPH1 is a paralog of HNRNPH2 with only 15 amino acids separating them. However, their mRNA sequences are sufficiently divergent to allow for ASO design specific for HNRNPH2 while avoiding HNRNPH1 off-target inhibition.

Table 1: Antisense Oligonucleotides Targeting HNRNPH2 * “d” refers to DNA nucleotide; “L” refers to locked nucleic acid; “M” refers to 2'- OMe. Intemucleotide linkages in Table 1 are phosphodiester, but one or more intemucleotide linkages may be optionally modified (e.g., phosphorothioate). One or more (or all) 2’-0Me modifications may be optionally 2 -MOE. LNA may optionally be cEt. C may optionally be 5-methyl C.

IPS-derived-Gaba-Neurons were used to shortlist 56 ASOs for their ability to knockdown expression of HNRNPH2 from both alleles agnostically. Gaba-Neurons were plated in 96 well plates (4* 10⁴ cells per well) and allowed to grow and connect for 2 weeks. Cells were fed via 50% media replacement every 2 days. ASOs were diluted to 20uM concentration. ASOs were added to wells containing Gaba-Neurons via an every 2 day 50% media exchange feeding cycle (final concentration lOuM). Ten wells of cells were left untreated by ASO to establish expression baselines for HNRNPH1, HNRNPH2 and house- keeping genes. Cells were harvested following Qiagen RNEasy 96 well plate protocol. The Invitrogen Superscript 4 VILO kit was used to conduct reverse transcription on all samples.

A Taqman assay using specific primer and probes to HNRNPH1, HNRNPH2, and a housekeeping gene were run to quantify degree of knockdown mediated by ASO exposure. The A ACT method was used to quantify expression relative to untreated negative controls. Results are presented in Table 2. The results show reduced expression of HNRNPH2, with Day 6 post-exposure showing the most dramatic change. Certain oligonucleotides further show no knockdown of HNRNPH1 expression, or even increased expression.

Table 2: Results showing knockdown of HNRNPH2 Expression

Additional antisense oligonucleotides targeting HNRNPH2 mRNA are shown in Tables 3 and 4A,B.

Table 3: Antisense Oligonucleotides Targeting HNRNPH2

Z1

Nomenclature is same as for Tables 1 and 2.

Table 4A: Antisense Oligonucleotides targeting HNRNPH2 mRNA

Nomenclature is the same as Tables 1, 2, and 3.

Table 4B: Antisense Oligonucleotides targeting HNRNPH2 mRNA

“d” refers to DNA nucleotide; “L” refers to locked nucleic acid (LNA) (but may optionally be cEt);

“M” refers to 2' -MOE.

“s” refers to phosphorothioate linkage

“o” refers to phosphodiester linkage

“C” nucleotides are 5 methyl C.

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12. Crooke ST (2021) A call to arms against ultra-rare diseases. Nat Biotechnol 39:671-677. 13. Goyal N, Narayanaswami P (2018) Making sense of antisense oligonucleotides: A narrative review. Muscle Nerve 57:356-370.

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HNRNPH2 SEQUENCES d)

Claims

CLAIMS:

1. A composition comprising: an effective amount of an antisense oligonucleotide that inhibits the expression of heterogeneous nuclear ribonucleoprotein HNRNPH2 mRNA and a pharmaceutically acceptable vehicle.

2. The composition of claim 1, wherein the antisense oligonucleotide comprises 10 to 30 linked nucleotides and has a sequence that is complementary to HNRNPH2 mRNA.

3. The composition of claim 2, wherein the oligonucleotide is at least 12 nucleotides in length.

4. The composition of claim 3, wherein the oligonucleotide is at least 14 nucleotides in length.

5. The composition of claim 2, wherein the oligonucleotide is from 10 to 24 nucleotides in length, or is from 16 to 24 nucleotides in length.

6. The composition of claim 5, wherein the oligonucleotide is 16, 17, 18, 19, 20, 21, or 22 nucleotides in length.

7. The composition of claim 5, wherein the oligonucleotide is 20 nucleotides in length.

8. The composition of any one of claims 1 to 7, wherein the oligonucleotide hybridizes specifically to a pathogenic variant of HNRNPH2 mRNA, and wherein the pathogenic variant encodes a mutant selected from R114W, R206N, and P209L.

9. The composition of any one of claims 1 to 7, wherein the oligonucleotide does not hybridize specifically to a pathogenic variant of HNRNPH2 mRNA, optionally wherein the expression of HNRNPH1 mRNA is increased.

10. The composition of claim 9, wherein the oligonucleotide comprises at least 8 contiguous nucleobases of any one of SEQ ID NOs: 1 to 56 or 60 to 390.

11. The composition of claim 10, wherein the oligonucleotide comprises or consists of a nucleobase sequence of any one of SEQ ID NOs: 1 to 56.

12. The composition of claim 1, wherein the oligonucleotide comprises a nucleobase sequence of any one of SEQ ID NOs: 14, 16, 33, 39, 51, 52, and 55.

13. The composition of claim 1, wherein the oligonucleotide consists of a nucleobase sequence of any one of SEQ ID NOs: 14, 16, 33, 39, 51, 52, and 55.

14. The composition of any one of claims 1 to 13, wherein the antisense oligonucleotide has a stretch of at least 6 DNA nucleotides sufficient to recruit RNaseH.

15. The composition of claim 14, wherein one or more DNA nucleotides comprise a 2' chemical modification independently selected from 2'-Fluoro, 2'-Methyl, and 2'-Ethyl.

16. The composition of claim 14, wherein DNA nucleotides do not comprise a 2' chemical modification.

17. The composition of any one of claims 1 to 16, wherein the antisense oligonucleotide is a gapmer having a 5' and a 3' segment, each of the 5' and 3' segments being from 2 to 6 nucleotides or from 3 to 5 nucleotides, and where the 5' and 3' segments do not contain DNA nucleotides.

18. The composition of claim 17, wherein the 5' and 3' segments are each 3, 4, or 5 nucleotides in length, and the 5' and 3' segments flank an internal sequence of 10 DNA nucleotides.

19. The composition of claim 18, wherein the gapmer is a 5-10-5 gapmer.

20. The composition of claim 18 or 19, wherein one or more nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents, optionally where all of the nucleotides of the 5' segment and the 3' segment comprise 2'-O substituents.

21. The composition of claim 20, wherein the 2'-O substituents are independently selected from 2'-O methyl, 2'-O ethyl, 2'-O methoxyethyl (MOE), and a bridged nucleotide having a 2' to 4' bridge.

22. The composition of claim 21, wherein the bridged nucleotide has a methylene bridge (LNA) or a constrained ethyl bridge (cEt).

23. The composition of any one of claims 1 to 22, wherein the antisense oligonucleotide has a modified backbone.

24. The composition of claim 23, wherein the antisense oligonucleotide a comprises one or more phosphorothioate or phosphorodi thioate nucleotides.

25. The composition of claim 24, wherein the oligonucleotide is fully phosphorothioate or phosphorodithioate linked.

26. The composition of claim 23, wherein DNA nucleotides and terminal nucleotides are phosphorothioate linked.

27. The composition of any one of claims 1 to 26, wherein cytidine nucleobases in the antisense oligonucleotide are 5-methyl cytidine.

28. The composition of any one of claims 1 to 27, wherein the antisense oligonucleotide hybridizes to its target sequence with a T_m of at least about 35 °C, or at least about 40°C, or at least about 45°C, or at least about 50°C.

29. The composition of claim 28, wherein the antisense oligonucleotide hybridizes to its target sequence with a T_m is from about 35°C to about 60°C, or from about 40°C to about 60°C, or from about 50°C to about 60°C.

30. The composition of any one of claims 1 to 29, wherein the antisense oligonucleotide has a sequence and structure as shown in one of Tables 1, 2, 3, 4A, and 4B.

31. The composition of claim 1, wherein the antisense oligonucleotide has a sequence and structure as shown in Table 4A or 4B.

32. The composition of any one of claims 1 to 31, further comprising a cell penetrating moiety.

33. The composition of any one of claims 1 to 31, wherein the antisense oligonucleotide is encapsulated in a particle.

34. The composition of claim 33, wherein the particle is a liposome, polymeric nanoparticle, or lipid nanoparticle.

35. The composition of any one of claims 1 to 34, wherein the composition is formulated for parenteral administration.

36. A method for treating a subject having a pathogenic variant of heterogeneous nuclear ribonucleoprotein HNRNPH2, comprising administering an antisense oligonucleotide targeting HNRNPH2 to said subject, wherein said antisense oligonucleotide does not substantially target and reduce expression of HNRNPH1, and optionally wherein HNRNPH1 expression is increased.

37. The method of claim 36, wherein a composition of any one of claims 1 to 35 is administered to the subject.

38. The method of claim 36, wherein the subject has a pathogenic variant is selected from R114W, R206N, and P209L.

39. The method of claim 37 or 38, wherein the subject is female.

40. The method of any one of claims 36 to 39, wherein the composition is administered at least once per month or at least once per week.

41. The method of claim 40, wherein the composition is administered parenterally.