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WO2024137726A2 - Compositions et méthodes de traitement de troubles liés à la hnrnph2 - Google Patents

Compositions et méthodes de traitement de troubles liés à la hnrnph2 Download PDF

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Publication number
WO2024137726A2
WO2024137726A2 PCT/US2023/084987 US2023084987W WO2024137726A2 WO 2024137726 A2 WO2024137726 A2 WO 2024137726A2 US 2023084987 W US2023084987 W US 2023084987W WO 2024137726 A2 WO2024137726 A2 WO 2024137726A2
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composition
nucleotides
oligonucleotide
hnrnph2
antisense oligonucleotide
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WO2024137726A3 (fr
Inventor
Rodney BOWLING
Neda GHOUSIFAM
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To Cure A Rose Foundation
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To Cure A Rose Foundation
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Priority to EP23908389.2A priority Critical patent/EP4638751A2/fr
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Publication of WO2024137726A3 publication Critical patent/WO2024137726A3/fr
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/33—Chemical structure of the base
    • C12N2310/334—Modified C
    • C12N2310/3341—5-Methylcytosine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/341—Gapmers, i.e. of the type ===---===

Definitions

  • RNA processing is critically important for brain development and function, as neurons are postmitotic cells dependent on RNA expression [2].
  • RBPs RNA-binding proteins
  • Heterogeneous nuclear ribonucleoproteins 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.
  • 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.
  • RRMs quasi- RNA Recognition Motifs
  • NLS nuclear localization signal
  • HNRNPH2 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],
  • compositions e.g., pharmaceutical compositions
  • 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.
  • 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.
  • ASO antisense oligonucleotide
  • 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.
  • 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.
  • the oligonucleotide is from 10 to 24 nucleotides in length, or is from 16 to 24 nucleotides in length.
  • the oligonucleotide is 16, 17, 18, 19, 20, 21, or 22 nucleotides in length.
  • the oligonucleotide is 20 nucleotides in length.
  • 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).
  • 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.
  • HNRNPH1 is functional in individuals with the HNRNPH2-related disorder
  • functional HNRNPH1 may be able to partially compensate for disrupted HNRNPH2.
  • the oligonucleotide reducing expression of HNRNPH2 surprisingly results in increased expression of HNRNPH1.
  • 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).
  • the pathogenic variant encodes a mutant of a glycine-rich domain.
  • the oligonucleotide does not hybridize to a specific pathogenic variant of HNRNPH2 mRNA, and is thus agnostic as to the pathogenic variant.
  • HNRNPH1 (which is increased in expression in some embodiments) is believed to compensate for the loss of HNRNPH2 expression.
  • 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).
  • 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.
  • the oligonucleotide comprises or consists of a nucleobase sequence of any one of SEQ ID NOS: 1-56 and 60-390.
  • 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.
  • 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.
  • 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.
  • the antisense oligonucleotide has a stretch of DNA nucleotides sufficient to recruit RNaseH, and thereby trigger degradation of the target mRNA.
  • 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.
  • the antisense oligonucleotide has a stretch of 10 DNA nucleotides, 11 DNA nucleotides, or 12 DNA nucleotides.
  • the antisense oligonucleotide has a stretch of 10 DNA nucleotides.
  • one or more DNA nucleotides comprise a 2' chemical modification independently selected from 2'-Fluoro, 2'-Methyl, and 2'-Ethyl.
  • the DNA nucleotides do not contain a 2' modification.
  • 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.
  • the 5' and 3' wing segments each have 5 nucleotides.
  • 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.
  • 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.
  • 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.
  • the bridged nucleotide has a methylene bridge (locked nucleic acid or LNA) or a constrained ethyl bridge (cEt).
  • 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.
  • 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.
  • 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 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.
  • 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).
  • the antisense oligonucleotide has a modified backbone or modified internucleotide linkages.
  • internucleotide linkage refers to the linkage between two adjacent nucleosides in a polynucleotide molecule.
  • 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.
  • phosphorothioate or phosphorodithioate linkages increase the resistance of the intemucleotide linkage to nucleases.
  • PACE phosphoacetate linkage
  • 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.
  • the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate nucleotides.
  • the antisense oligonucleotide comprises one or more phosphorothioate or phosphorodithioate intemucleotide linkages.
  • 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.
  • the antisense oligonucleotide has a combination of phosphodiester and phosphorothioate/phosphorodithioate linkages.
  • 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) .
  • DNA intemucleotide linkages in addition to intemucleotide linkages at 5' and 3' ends, are phosphorothioate linkages.
  • other intemucleotide linkages in the wing segments between RNA nucleotides can be phosphodiester (e.g., unmodified backbone linkage).
  • 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.
  • 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.
  • 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.
  • 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).
  • the antisense oligonucleotide may contain one or more modified bases.
  • cytosine is replaced with 5-methylcytosine, which may enhance base pairing.
  • Other modified bases 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.
  • cytidine nucleobases in the antisense oligonucleotide are 5-methyl cytidine.
  • a sequence includes a cytidine nucleobase (“C”)
  • the term includes 5-methyl C.
  • U includes pseudouridine and Nl- methylpseudouridine.
  • 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.
  • 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.
  • 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.
  • the antisense oligonucleotide is selected from an oligonucleotide shown in Tables 1, 2, 3, 4A, or 4B.
  • 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).
  • 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.
  • the cell penetrating moiety is conjugated to a transfection component or delivery vehicle.
  • 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.
  • a sterol conjugate e.g., cholesterol conjugate
  • fatty acid conjugate such as a palmitoyl or stearyl lipid conjugate
  • 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).
  • a biological ligand e.g., including a glycoconjugate
  • aptamer e.g., including a glycoconjugate
  • 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 VHH
  • the composition comprises an encapsulation or transfection reagent or vehicle.
  • the antisense oligonucleotide is encapsulated in a particle.
  • the particle is a liposome, polymeric nanoparticle, or lipid nanoparticle.
  • Exemplary polymeric nanoparticles can be formed of PLA, PLGA, or PEG copolymers thereof.
  • the particle comprises poly( ⁇ amino ester) polymers.
  • the LNPs comprise a cationic or ionizable lipid, a neutral lipid, a cholesterol or cholesterol moiety, and aPEGylated lipid.
  • a targeting moiety is conjugated to the termini of a portion of PEG groups that form a hydrophilic outer sheath.
  • the lipid nanoparticle 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).
  • the structural lipid is cholesterol.
  • 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
  • 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.
  • DOPG 1,2- dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt
  • DOPG 1,2- dioleoyl-s
  • 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.
  • the liposomes or nanoparticles further comprise a targeting moiety as described.
  • the composition is formulated for parenteral administration.
  • 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.
  • administration is intranasal.
  • 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.
  • 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.
  • an effective amount of a composition of the present disclosure is administered to the subject.
  • the subject has a pathogenic variant located in the RRM of HNRNPH2, such as the R114W variant.
  • the subject has a pathogenic variant located in the NLS of HNRNPH2, such as R206N or P209L.
  • the subject has a pathogenic variant located in a glycine-rich domain of HNRNPH2.
  • the subject is female, and the subject is heterozygous for the pathogenic variant.
  • the subject is male.
  • the therapeutic methods described herein are initiated when the subject is a neonate (4 weeks of age or less) or pediatric age.
  • 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.
  • the compounds or compositions described herein are administered parenterally, such as by intravenous or intrathecal infusion.
  • the compounds or compositions are administered by direct infusion to target tissue (eg., brain, central nervous system).
  • target tissue eg., brain, central nervous system.
  • 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.
  • the compositions are administered at least once daily to at least once quarterly.
  • 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.
  • the composition may be administered at least once per month or at least once per week.
  • 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.
  • HNRNPH1 is a paralog of HNRNPH2 with only 15 amino acids separating them.
  • 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* 10 4 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.
  • Nomenclature is the same as Tables 1, 2, and 3.
  • d refers to DNA nucleotide
  • L refers to locked nucleic acid (LNA) (but may optionally be cEt);
  • M refers to 2' -MOE.
  • C nucleotides are 5 methyl C.

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Abstract

La présente divulgation propose des compositions et des méthodes de traitement d'affections associées à des variants pathogènes de la ribonucléoprotéine nucléaire hétérogène HNRNPH2. De tels variants pathogènes dans divers modes de réalisation peuvent être liés à un ou à plusieurs éléments parmi un retard de développement, une déficience intellectuelle, un trouble du langage, une déficience motrice, des troubles musculosquelettiques et de croissance, des caractéristiques dysmorphiques, l'épilepsie, le trouble du spectre autistique et une déficience visuelle corticale. Selon cette divulgation, la composition comprend une quantité efficace d'un oligonucléotide antisens (ASO) qui inhibe l'expression de l'ARNm de la ribonucléoprotéine nucléaire hétérogène HNRNPH2, comprenant un variant pathogène, et un véhicule pharmaceutiquement acceptable. Dans des modes de réalisation, l'expression de la HNRNPH1 n'est pas réduite, et, dans certains modes de réalisation, peut augmenter.
PCT/US2023/084987 2022-12-20 2023-12-20 Compositions et méthodes de traitement de troubles liés à la hnrnph2 Ceased WO2024137726A2 (fr)

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