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WO2025026896A1 - Modulation de pathologie présynaptique avec traduction locale de munc13-1 - Google Patents

Modulation de pathologie présynaptique avec traduction locale de munc13-1 Download PDF

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Publication number
WO2025026896A1
WO2025026896A1 PCT/EP2024/071226 EP2024071226W WO2025026896A1 WO 2025026896 A1 WO2025026896 A1 WO 2025026896A1 EP 2024071226 W EP2024071226 W EP 2024071226W WO 2025026896 A1 WO2025026896 A1 WO 2025026896A1
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muncl3
motoneurons
smn
utr
rescue
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Michael Anton Sendtner
Mehri MORADI
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Julius Maximilians Universitaet Wuerzburg
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Julius Maximilians Universitaet Wuerzburg
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4728—Calcium binding proteins, e.g. calmodulin
    • 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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/203—Animal model comprising inducible/conditional expression system, e.g. hormones, tet
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • 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
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/007—Vectors comprising a special translation-regulating system cell or tissue specific
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/80—Vectors comprising a special translation-regulating system from vertebrates
    • C12N2840/85—Vectors comprising a special translation-regulating system from vertebrates mammalian

Definitions

  • Degeneration of neuromuscular synapses is a pathological hallmark of neurodegenerative diseases including spinal muscular atrophy (SMA), Amyotrophic lateral sclerosis (ALS), fragile X syndrome, Charcot-Marie-Tooth disease, multiple sclerosis (MS), parkinson’s disease (PD), muscular dystrophy, Myasthenia Gravis (MG), myopathy, myositis (including polymyositis and dermatomyositis), and peripheral neuropathy.
  • SMA spinal muscular atrophy
  • ALS Amyotrophic lateral sclerosis
  • MS multiple sclerosis
  • PD parkinson’s disease
  • MG Myasthenia Gravis
  • myopathy myositis (including polymyositis and dermatomyositis)
  • peripheral neuropathy including spinal muscular atrophy (SMA), Amyotrophic lateral sclerosis (ALS), fragile X syndrome, Charcot-Marie-Tooth disease, multiple sclerosis (MS), parkinson’s disease (PD), muscular dystrophy
  • AD Alzheimer's disease
  • a neurodegenerative disorder and the most common form of late-onset dementia, affecting a substantial proportion of individuals aged 65 and over. It is characterized by progressive memory loss and is expected to increase dramatically over the coming decades as aging is the main risk factor. AD is caused by the accumulation of insoluble protein aggregates in the brain, including the formation of tau fibrils in neuronal axons, leading to neuron dysfunction and loss, which in turn results in progressive memory loss leading to a reduced ability to execute daily functions.
  • SMA spinal muscular atrophy
  • SMA spinal muscular atrophy
  • SMA is the second most common fatal autosomal recessive genetic disease with an incidence of 1 per 6,000 births.
  • SMA is caused by deletions of the Survival Motor Neuron 1 (SMN1) gene, which lead to degeneration of spinal motoneurons and muscle atrophy.
  • Smn protein is required for the assembly of small nuclear ribonucleoproteins involved in pre-mRNA splicing as well as regulation of the axonal mRNA transport and local translation.
  • Munc 13-1 (mammalian uncoordinated- 13) is an abundant protein isoform in the mammalian brain, and contains a variable N-terminal region with a C2A domain and a calmodulin-binding region (CaMb), as well as a conserved C-terminal region that includes the Ci, C2B, MUN and C2C domains.
  • the C2A domain forms a homodimer and alternatively a heterodimer with the Rab3 effectors called RIMs, thus providing a switch that controls neurotransmitter release and couples exocytosis to diverse forms of Rab3- and RIM-dependent presynaptic plasticity.
  • Muncl3-1 Transcript levels for Muncl3-1 but not Synaptophysin are specifically reduced in axon terminals of Smn-deficient motoneurons. In presynaptic terminals, neurotransmitter release occurs at active zones (AZs). Muncl3-1 mediates the assembly of release sites by docking and priming of synaptic vesicles (SV) onto AZs and activating the SNARE/SM fusion machinery. In addition, Muncl3-1 regulates synaptic plasticity by modulating vesicle release probability and altering the fusion competence of the readily releasable pool.
  • the present disclosure relates to the fields of molecular biology and genetics, as well as to biopharmaceuticals and therapeutics generated from expressible molecules. More particularly, this disclosure relates to methods, structures and compositions for molecules having the ability to be translated into active polypeptides or proteins, for use in vivo and as therapeutics.
  • This disclosure includes structures, compositions and methods for novel molecules having the ability to be translated, which can be used to provide one or more active polypeptides, proteins, or fragments thereof.
  • this disclosure provides methods for ameliorating, preventing, or treating a disease or condition in a subject comprising administering to the subject a composition containing a translatable molecule of this disclosure.
  • the patent or application file contains at least one drawing executed in color, and the same set executed in black and white, with the same numbering.
  • Reference to any figure in the specification should be construed indifferently as a reference to the corresponding drawing in color and as a reference to the corresponding drawing in black and white.
  • FIGs. 1A-1M illustrate that Muncl3-1 local translation is dysregulated in axon terminals of SMA motoneurons.
  • FIG. 1A illustrates cultivation of primary motoneurons in compartmentalized chambers enables axon growth into a separate chamber side.
  • FIG. 1C illustrates that Muncl3-1 transcripts are discernable in distal axons in cultured motoneurons using smFISH.
  • FIG. IF illustrates neuromuscular junctions (NMJs) of TVA muscles from P5 littermates are stained against Muncl3-1. SynPhy antibody labels presynaptic membranes and a -Bungarotoxin labels AChRs at postsynaptic membranes. FIGs.
  • FIG. II is a schematic of puromycin-Muncl3-l PLA assay.
  • FIG. 1J illustrates that locally translated Muncl3-1 molecules are detected in axonal growth cones in cultured motoneurons by Puro-PLA.
  • Mann-Whitney U test one-tailed in FIGs. IB, IL, and IM, and two-tailed in FIGs. ID- IK. Bars represent mean ⁇ SEM.
  • FIGs. 2A-2G illustrate that Muncl3-1 supramolecular release sites undergo rearrangement in response to stimulation.
  • FIG. 2A is a schematic of three viral rescue constructs expressing Muncl3-1.
  • FIG. 2D illustrates that Muncl3-1 becomes locally translated in axonal growth cones in response to Roscovitine stimulation.
  • FIG. 2F illustrates that Lattice-SIM reveals Muncl3-1 supramolecular release sites in axonal growth cones in cultured motoneurons.
  • FIG. 2G illustrates that upon Roscovitine pulse, the number of Muncl3-1 release sites per growth cone increases in control and KO Rescue motoneurons.
  • Mann-Whitney U test one-tailed in FIG. 2B and two-tailed in FIG. 2E. Bars represent mean ⁇ SEM.
  • FIGs. 3A-3F illustrate that ExM demonstrates the nanoscale transition of Muncl3-1 assemblies after induction of neuronal activation.
  • FIG. 3A illustrates that ExM reveals Muncl3-1 nanoassemblies within supramolecular release sites in axonal growth cones of cultured motoneurons before and after stimulation of the neuronal activity.
  • FIG. 3A-3F illustrate that ExM demonstrates the nanoscale transition of Muncl3-1 assemblies after induction of neuronal activation.
  • FIG. 3A illustrates that ExM reveals Muncl3-1 nanoassemblies within supramolecular release sites in axonal growth
  • scale bars are corrected to indicate pre-expansion dimensions. Two-tailed Mann-Whitney U test. Bars represent mean ⁇ SEM.
  • FIGs. 4A-4H illustrate that Muncl3-1 local translation rescues neuronal excitability in Smn-deficient motoneurons.
  • FIG. 4F illustrates that growth cones were depolarized by a 90 mM KC1 pulse and Ca 2+ transients were measured.
  • FIG. 4H illustrates that % of failure to membrane depolarization is diminished in KO Rescue but not KO RescueA3 ' UTR motoneurons.
  • FIGs. 5A-5O illustrate restoration of Muncl3-1 local translation rescues motor functions in SMA mice.
  • FIG. 5A is a schematic of the Cre/loxP conditional Muncl3-1 rescue cassette inserted into the mouse ROSA26 locus and the breeding schema with SMA mice.
  • FIG. 5B illustrates that R26Uncl3-l tg/+ mice were cross-bred with Nestin-Cre tg/+ .
  • FIG. 5A is a schematic of the Cre/loxP conditional Muncl3-1 rescue cassette inserted into the mouse ROSA26 locus and the breeding schema with SMA mice.
  • FIG. 5B illustrates that R26Uncl3-l tg/+ mice were cross-bred with Nestin-Cre tg/+
  • FIG. 5C illustrates that motoneurons Muriel 3-l +/ ⁇ ,R26Uncl 3-l tg/+ and Munc 13-1 ⁇ ,R26Unc 13- l tg/+ genotypes were transduced with AAV-eFl-Cre virus and cultured for 7 days. Protein product of Muncl3-1 rescue allele is detectable in Muncl3-l ⁇ / ⁇ ,R26Uncl3-l tg/+ motoneurons by Western blot.
  • FIG. 5D illustrates representative images of NMJs of TV A muscles from PIO littermates stained against SynPhy, Neurofilament H (NFH), and AChRs.
  • FIG. 51 and 5J illustrates that grip strength of forelimbs is improved in Smn ⁇ / ⁇ ,Hun ⁇ g/+ ,R26Uncl3-l tg/+
  • FIGs. 6A-6J illustrate that Muncl3-1 expression is not altered in cultured Smn- deficient motoneurons.
  • FIG. 6B are representative images of axonal growth cones of cultured motoneurons stained against Muncl3-1 and Tubulin.
  • FIG. 6D and 6E illustrate immunostaining of Muncl3-1 and Tubulin in somata of cultured motoneurons.
  • FIG. 6G is an illustration of no PLA signal being detectable when only Muncl3-1 or puromycin antibodies are incubated.
  • FIGs. 61 and 6H are representative immunoblots of cortical synaptosome fractions after RNA pulldown reveals comparable amounts of ribosomes in input and IP fractions of control and Smn KO littermates.
  • Mann-Whitney U test one-tailed in FIG. 61- FIG. 6J and two-tailed in FIG. 6A- FIG. 6E. Bars represent mean ⁇ SEM.
  • FIGs. 7A-7F illustrate that rescue construct of Muncl3-l+SynPhy3'UTR drives axonal localization of Muncl3-1 mRNAs in cultured Smn-deficient motoneurons.
  • FIG. 7A is representative images of smFISH in the soma, axon, and axonal growth cone of cultured motoneurons.
  • FIG. 7E and FIG. 7F Following transduction with Rescue and RescueA3'UTR viruses, elevated Muncl3-1 protein levels are detected in the soma (FIG.
  • FIGs. 8A-8F illustrate that formation of Muncl3-1 release sites depends on neuronal activity.
  • FIG. 8E illustrates that motoneurons grown on lamininl l l do not exhibit Muncl3-1 supramolecular release sites.
  • FIG. 8F illustrates that mCLING uptake through SV endocytosis occurs in close vicinity to Muncl3-1 supramolecular release sites. Two-tailed Mann-Whitney U test. Bars represent mean ⁇ SEM.
  • FIGs. 9A-9C illustrate that Muncl3-1 colocalizes with the SNARE complex marker Snap25 within supramolecular release sites.
  • FIG. 9A are representative lattice-SIM merged images of Muncl3-1, RPL8, and Snap25 in supramolecular release sites within axonal growth cones of cultured motoneurons.
  • FIGs. 10A-10E illustrate thatMuncl3-l overexpression restores AZ organization and rescues growth and differentiation defects in Smn-deficient motoneurons.
  • FIG. 10A is representative images of SV recycling assay in CTX and TTX-treated neurons indicate that the uptake of Synaptotagminl antibody depends on neuronal activity.
  • FIG. 10A is representative images of SV recycling assay in CTX and TTX-treated neurons indicate that the uptake of Synaptotagminl antibody depends on neuronal activity.
  • FIG. 10C is representative images of axonal growth cones of cultured motoneurons stained against Act0 and Tau.
  • FIGs. 10D and 10E are illustrating that Smn-deficient motoneurons grown on laminin221/211 display increased growth cone size (FIG.
  • FIGs. 11 A-l IF Validation of Cre/loxP conditional Muncl3-1 knock-in rescue mouse model.
  • FIGs. 11A-11C illustrate that qRT-PCR indicates Cre-dependent expression of R26Uncl3-l rescue allele in cultured motoneurons isolated from R26Uncl3-l tg/+ knock-in mice.
  • FIG. 12 is a table of transcripts of some synaptic proteins are upregulated in soma and axons of Smn knockdown motoneurons compared to control as determined by RNAseq. Transcripts for Snap91, Syt5, and Stx7 are upregulated only in the axonal compartment (table is prepared from previously published RNAseq data.
  • FIGs. 13A-13D are directed to axonal localization of the UNC13A mRNA and protein are impaired in hiPSC-derived motoneurons from SMA patients.
  • FIG. 13 A is an image showing the growing of axons of hiPSC-derived motoneurons cultured in compartmentalized chambers.
  • FIG. 13 A is an image showing the growing of axons of hiPSC-derived motoneurons cultured in compartmentalized chambers.
  • FIG. 13C are representative images of axonal growth cones of cultured DIV25 hiPSC-derived motoneurons from SMA patients and control individuals stained against TuJl, UNCI 3 A, and Ca v 2.2. Representative images of SMA hiPSC-derived motoneurons transduced with UNC13A+SYP3TJTR Rescue virus are included in Fig. 14E.
  • FIG. 14E Representative images of SMA hiPSC-derived motoneurons transduced with UNC13A+SYP3TJTR Rescue virus are included in Fig. 14E.
  • FIGs. 14A-14E are directed to generation of human iPSC-derived motoneurons from SMA patients and control individuals.
  • FIG. 14A is representative images of cultured DIV25 hiPSC-derived motoneurons from three SMA patients and two control individuals.
  • FIG. 14B is an immunoblot of total lysates obtained from hiPSCs reveals reduced SMN levels in SMA patients compared to control.
  • FIG. 14C is representative images of cultured DIV25 hiPSC- derived motoneurons expressing ChAT and TuJl. Nuclei are stained with DAPI.
  • FIG. 14A is representative images of cultured DIV25 hiPSC- derived motoneurons expressing ChAT and TuJl. Nuclei are stained with DAPI.
  • FIG. 14E is representative images of axonal growth cones of cultured DIV25 SMA hiPSC-derived motoneurons from three SMA patients transduced with UNC13A+SYP3'UTR Rescue virus (related to FIGs. 13, C and D).
  • the term “substantially” or “substantial”, is equally applicable when used in a negative connotation to refer to the complete or near complete lack of an action, characteristic, property, state, structure, item, or result.
  • a surface that is “substantially” flat would either be completely at, or so nearly flat that the effect would be the same as if it were completely flat.
  • any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range.
  • reference herein to a range of “at least 50” or “at least about 50” includes whole numbers of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers 50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc.
  • reference herein to a range of “less than 50” or “less than about 50” includes whole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., and fractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1, 49.0, etc.
  • reference herein to a range of from “5 to 10” includes whole numbers of 5, 6, 7, 8, 9, and 10, and fractional numbers 5.1, 5.2, 5.3, 5,4, 5,5, 5.6, 5.7, 5.8, 5.9, etc.
  • the tern “about” indicates that the value listed may be somewhat altered, as long as the alteration does not result in nonconformance of the process or structure to the illustrated embodiment.
  • the term “about” can refer to a variation of ⁇ 0.1%, for other elements, the term “about” can refer to a variation of ⁇ 1% or ⁇ 10%, or any point therein.
  • the terms “treat”, “treatment”, “treating” and the like are used herein to generally include obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment as used herein include any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and include any mammalian subject for whom diagnosis, treatment, or therapy is desired.
  • a translatable molecule of this disclosure may be used for ameliorating, preventing or treating a disease.
  • a composition comprising a translatable molecule of this disclosure can be administered to regulate, modulate, or increase the concentration or effectiveness of the natural enzyme in a subject.
  • the enzyme can be an unmodified, natural enzyme for which the patient has an abnormal quantity.
  • the molecules of the present disclosure can be administered through any modality of administration that can result in transduction of the molecules into structures of interest.
  • the molecules of the present disclosure can be administered systemically to the patients, for example, by oral administration, injection or infusion, intravenous injection or infusion, and/or intramuscular injection.
  • the molecules of the present disclosure can be administered through any appropriate route.
  • administration may be bronchial (including by bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, pulmonary and vitreal.
  • administration may be intratumoral or peritumoral.
  • administration may involve intermittent dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • the expressed protein or polypeptide may be natural or non-natural, or can be an antibody or antibody fragment, or an immunogen or toxoid for use in a vaccine, or a fusion protein, or a globular protein, a fibrous protein, a membrane protein, or a disordered protein.
  • the protein may be a human protein, or a fragment thereof, or be deficient in a rare human disease, such as Amyotrophic lateral sclerosis (ALS) and/or Spinal muscular atrophy (SMA), among others noted herein, including fragile X syndrome, Charcot-Marie-Tooth disease, multiple sclerosis (MS), parkinson’s disease (PD), muscular dystrophy, Myasthenia Gravis (MG), myopathy, myositis (including polymyositis and dermatomyositis), and peripheral neuropathy.
  • ALS Amyotrophic lateral sclerosis
  • SMA Spinal muscular atrophy
  • fragile X syndrome including fragile X syndrome, Charcot-Marie-Tooth disease, multiple sclerosis (MS), parkinson’s disease (PD), muscular dystrophy, Myasthenia Gravis (MG), myopathy, myositis (including polymyositis and dermatomyositis), and peripheral neuropathy.
  • a synthetic mRNA construct may have a coding sequence for encoding the protein or polypeptide having alternative codons as compared to a native human protein or polypeptide.
  • the coding sequence for encoding the protein or polypeptide may have a high codon adaptation index.
  • Embodiments of this disclosure contemplate synthetic mRNA constructs having from 50 to 15,000 nucleotides.
  • a synthetic mRNA construct may comprise one or more chemically- modified nucleotides.
  • a synthetic mRNA construct may have at least 50% increased translation efficiency in vivo as compared to a native mRNA.
  • a translatable molecule or transgene of this disclosure can be a modified mRNA.
  • a modified mRNA can encode one or more biologically active peptides, polypeptides, or proteins.
  • a modified mRNA can comprise one or more modifications as compared to wild type mRNA. Modifications of an mRNA may be located in any region of the molecule, including a coding region, an untranslated region, or a cap or tail region.
  • translatable may be used interchangeably with the term “expressible.” These terms can refer to the ability of polynucleotide, or a portion thereof, to provide a polypeptide, by transcription and/or translation events in a process using biological molecules, or in a cell, or in a natural biological setting. In some settings, translation is a process that can occur when a ribosome creates a polypeptide in a cell. In translation, a messenger RNA (mRNA) can be decoded by a ribosome to produce a specific amino acid chain, or polypeptide.
  • mRNA messenger RNA
  • a translatable polynucleotide can provide a coding sequence region (usually, CDS), or portion thereof, that can be processed to provide a polypeptide, protein, or fragment thereof.
  • Translatable molecules are also referred to herein as transgenes.
  • a translatable oligomer or polynucleotide of this disclosure can provide a coding sequence region, and can comprise various untranslated sequences, such as a 5' cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and a tail region.
  • various untranslated sequences such as a 5' cap, a 5' untranslated region (5' UTR), a 3' untranslated region (3' UTR), and a tail region.
  • a translatable molecule of this disclosure may comprise a coding sequence that is at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identical to a portion of a reference mRNA sequence, such as a human wild type mRNA sequence.
  • a reference mRNA sequence can be a rare disease mRNA.
  • a translatable molecule of this disclosure may comprise a coding sequence that has one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty or more synonymous or non-synonymous codon replacements as compared to a reference mRNA sequence, such as a human wild type mRNA sequence.
  • a non-coding polynucleotide template sequence that is transcribable to provide a translatable molecule of this disclosure when transcribed may provide a translatable molecule that is at least 40%, or 50%, or 60%, or 70%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% identical to a portion of a reference mRNA sequence, such as a human wild type mRNA sequence.
  • a non-coding polynucleotide template sequence that is transcribable to provide a translatable molecule of this disclosure when transcribed may provide a translatable molecule that has one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty or more synonymous or non-synonymous codon replacements as compared to a reference mRNA sequence, such as a human wild type mRNA sequence.
  • a translatable molecule of this disclosure may be used to express a polypeptide that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to a portion of a reference polypeptide or protein sequence, such as a human wild type protein sequence.
  • a reference polypeptide or protein sequence can be a rare disease protein sequence.
  • a translatable molecule of this disclosure may be used to express a polypeptide that has one, or two, or three, or four, or five, or six, or seven, or eight, or nine, or ten, or fifteen, or twenty or more variant amino acid residues as compared to a reference polypeptide or protein sequence, such as a human wild type protein sequence.
  • a translatable molecule of the disclosure may encode a fusion protein comprising a full length, or fragment or portion of a native human protein fused to another sequence, for example by N or C terminal fusion.
  • the N or C terminal sequence can be a signal sequence or a cellular targeting sequence.
  • an mRNA construct of this disclosure can be homologous or heterologous.
  • the term “homologous mRNA construct” is a class of expressible polynucleotides, where the sequences of the polynucleotides are derived from a human gene.
  • heterologous mRNA construct is a class of expressible polynucleotides wherein at least one of the untranslated region sequences of the polynucleotide is derived from a non-human gene, and the coding region of such construct is derived from a human gene.
  • This disclosure provides methods and compositions for novel molecules having the ability to be translated, which can be used to provide one or more active polypeptides and proteins, or fragments thereof.
  • Embodiments of the disclosure can be directed to mRNA constructs comprising 5'UTR sequences in combination with 3'UTR sequences, not previously used in the context of heterologous mRNA constructs, to efficiently produce human proteins, or fragments thereof, in mammalian cells or animals.
  • This disclosure further contemplates methods for delivering one or more vectors comprising one or more translatable molecules to a cell. In further embodiments, the disclosure also contemplates delivering or one or more translatable molecules to a cell.
  • one or more translatable molecules can be delivered to a cell, in vitro, ex vivo, or in vivo. Viral and non-viral transfer methods as are known in the art can be used to introduce translatable molecules in mammalian cells.
  • Translatable molecules can be delivered with a pharmaceutically acceptable vehicle, or for example, with nanoparticles or liposomes.
  • the recombinant vector used for delivering the translatable molecule or transgene includes non-replicating recombinant adeno-associated virus vectors (“rAAV”).
  • rAAVs are particularly attractive vectors for a number of reasons — they can transduce non-replicating cells, and therefore, can be used to deliver the transgene to tissues where cell division occurs at low levels, such as the CNS; they can be modified to preferentially target a specific organ of choice; and there are hundreds of capsid serotypes to choose from to obtain the desired tissue specificity, and/or to avoid neutralization by pre-existing patient antibodies to some AAVs.
  • Such rAAVs include but are not limited to AAV based vectors comprising capsid components from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrhlO or AAVrh20.
  • AAV based vectors provided herein comprise capsids from one or more of AAV8, AAV9, AAV10, AAV11, AAVrhlO or AAVrh20 serotypes.
  • viral vectors including but not limited to lentiviral vectors; vaccinia viral vectors, or non-viral expression vectors referred to as “naked DNA” constructs. Expression of the transgene can be controlled by constitutive or tissue-specific expression control elements.
  • This disclosure further encompasses DNA templates for making an mRNA construct above by in vitro transcription.
  • compositions containing an mRNA construct above and a pharmaceutically acceptable carrier may comprise a transfection reagent, a nanoparticle, or a liposome.
  • a nanoparticle may include a lipid nanoparticle.
  • a composition of this disclosure may include lipid nanoparticles comprising a thiocarbamate or carbamate-containing lipid molecule.
  • This disclosure further contemplates methods for ameliorating, preventing or treating a disease or condition in a subject in need thereof, by administering to the subject a composition containing an mRNA construct.
  • a composition may be for use in medical therapy, or for use in preparing or manufacturing a medicament for preventing, ameliorating, delaying onset or treating a disease or condition in a subject in need.
  • the present disclosure includes a new therapeutic mechanism in that restoration of Muncl3-1 neosynthesis in axon terminals can slow down disease progression and normalize synaptic transmission at neuromuscular junctions (NMJs) that are defective in SMA.
  • NMJs neuromuscular junctions
  • This can be achieved by viral transduction of a modified Muncl3-1 mRNA, and by other means such as oligonucleotides or substances that lead to normalized Muncl3-1 expression in presynaptic terminals in patients with spinal muscular atrophy.
  • the target Muncl3-1 mRNA can be any RNA, whether native or unknown, synthetic or derived from a natural source.
  • the target RNA can include UNA molecules composed of nucleotides and UNA monomers, and optionally chemically modified nucleotides.
  • Smn deficient mice that represent a model for SMA exhibit atrophic and smaller neuromuscular synapses, which associate with impaired synaptic transmission and degeneration. Since the Smn protein plays an important role in axonal mRNA transport and local translation, it was hypothesized that Smn is required for axonal mRNA translocation and intra-axonal synthesis of transcripts encoding components of the active zone. mRNAs encoding SV proteins including Synaptophysin, Synaptotagmin, Synapsin, and SV2 are not reduced in the axonal compartment of cultured Smn-deficient motoneurons, as shown in the Table of FIG.
  • RNA immunoprecipitation (RNA-IP) assays were conducted, using ribosome pulldown with cortical synaptosome fractions isolated from P5 Smn KO and control littermates to verify the local translation of Muncl3-1 in synapses in vivo.
  • RNA-IP revealed reduced levels of Muncl3-1 mRNA bound to ribosomes in immunoprecipitated fractions of Smn KO, as shown in FIG. IL.
  • Muncl3-1 binds to hnRNP R via its 3'UTR binding domain. Impaired axonal localization of Muncl3-1 could be a consequence of reduced hnRNP R in axons of Smn- deficient motoneurons.
  • transcripts such as Synaptophysinl (SynPhy) are not reduced in axon terminals, as seen in the table of FIG. 12, indicating that their translocation depends on other transport proteins, which are not dysfunctional in SMA. Therefore, it was investigated whether the exchange of the 3'UTR of Muncl3-1 transcripts with the 3'UTR from SynPhy mRNA could rescue defective Muncl3-1 axonal localization in SMA.
  • Three lentivirus rescue constructs were generated: (i) a construct that harbors the coding and the 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors the coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and (iii) a construct that lacks the axonal targeting domains in the 3'UTR (RescueA3'UTR), as shown in FIG. 2A.
  • These three lentiviral rescue constructs which include the modified Muncl3-1 molecules as noted above, can be included as a component of a mammalian mRNA expression construct. Additionally, these three rescue constructs are described as being used in conjunction with a lentiviral vector, in other embodiments, any suitable vector capable of transduction of the modified Muncl3-1 molecules into any target cell can be used.
  • qRT-PCR was used for motoneurons grown in compartmentalized chambers in which axons are separated from somata, using primers that detect the endogenous Muncl3-1 as well as all rescue variants. Elevated Muncl3-1 transcript levels in axons of wild type (wt) motoneurons were found following viral transduction of Smn- dependent and Rescue, but not the construct lacking the 3'UTR domain, as seen in FIG. 2B. Moreover, upregulated Muncl3-1 protein levels were detected in total lysates of cultured motoneurons after transduction with these constructs, as seen in FIG. 2C.
  • This modification of a defective Muncl3-1 axonal localization is a method that includes administering a vector system that includes one or more of the modified Muncl3-1 molecules noted herein, including the rescue constructs noted herein. Additionally, the vector system of this method can be a lentiviral vector system, and/or in other embodiments, any suitable vector capable of transduction of the modified Muncl3-1 molecules into any target cell can be used.
  • Muncl3-1 protein levels were increased in the soma and axonal growth cones of control and Smn-deficient motoneurons transduced with Rescue or RescueA3'UTR constructs, as shown by immunostaining in FIGs. 7D and 7E. These data demonstrate that axonal localization of Muncl3-1 mRNA requires Cis-elements within its 3'UTR, which are recognized by Smn-dependent RNA binding proteins.
  • Muncl3-1 local translation is crucial for the formation of release sites upon induction of neuronal activity.
  • Roscovitine is a drug that activates the opening of Ca v 2.1/Ca v 2.2 channels and thus improves neurotransmitter release in Smn deficient motoneurons.
  • Roscovitine stimulation a significant increase in Muncl3-1 immunoreactivity was detectable in stimulated control motoneurons, as seen in FIG.
  • Deficiency of Muncl3-1 at presynaptic terminals could contribute to disease pathology in SMA, and that restoration of Muncl3-1 could be an effective new treatment for this disease.
  • An SV recycling assay was performed using an antibody against the Synaptotagminl luminal domain to assess neurotransmitter release in Smn-deficient motoneurons following transduction with the Rescue construct. Increased SV release only in KO Rescue , but not in KO RescueA3 UTR was measured, indicating that locally translated Muncl3-1 is needed for neurotransmitter release at presynaptic membranes, as seen in FIG. 4A.
  • mice demonstrated a diminished neurodegeneration of motoneurons within the spinal cord, including ChAT+ motoneurons within L1/L2 sections of the ventral horn in the spinal cord of the mice, as seen in FIGs. 5N-5O.
  • restoring the Muncl3-1 presynaptic translation through 3'UTR modifications beneficially affect synapse, motor functions, and neurodegenerati on .
  • Axonal localization of UNC13A mRNA and protein are perturbed in hiPSC-derived motoneurons from SMA patients
  • the present disclosure describes a novel function for the local synthesis of Muncl3- 1 in the assembly of presynaptic supramolecular release sites that modulate the synaptic plasticity upon induction of neuronal activity. This occurs directly at ribosomes within these release sites, leading to the recruitment of neosynthesized Muncl3-1 molecules into newly assembled active zone structures.
  • the AZs contain one or more release sites, with discrete domains believed to mediate the fusion of a single SV. Within these ⁇ 29 nm nanodomains six molecules of Muncl3-1 assemble under a single SV.
  • Muncl3-1 molecules form multiple and discrete supramolecular self-assemblies that serve as independent vesicular release sites by recruiting syntaxin- 1. Within these supramolecular self-assemblies, the number of Muncl3-1 molecules directly determines the quantal release, enabling a stable synaptic weight on neuronal circuits.
  • ExM the molecular Muncl3-1 assemblies within presynaptic release sites were resolved at the nanoscale and provide support that stimulation of the neuronal activity increases the Muncl3-1 nanoassemblies from 6 in unstimulated neurons to 8, and decreases the distance between the neighboring nanoassemblies.
  • SNPs single nucleotide polymorphisms
  • This modified Muncl3-1 construct could thus represent a new promising therapeutic target for SMA patients and possibly other neurodegenerative diseases with synapse degeneration.
  • These methods of improving synaptic function and/or treating patients diagnosed with SMA include administering a vector system that includes one or more of the modified Muncl3-1 molecules noted herein, including the rescue constructs noted herein.
  • the vector system of this method can be a lentiviral vector system, and/or in other embodiments, any suitable vector capable of transduction of the modified Muncl3-1 molecules into any target cell can be used.
  • Muncl3-1 Deficiency of Muncl3-1 could contribute to disease pathology in Alzheimer’s Disease such that restoration of Muncl3-1 could be an effective new treatment for this disease.
  • Muncl3-1 is involved in the maintenance of synaptic plasticity through its modulation of long-term potentiation and serves an important role in hippocampal glutamatergic neurotransmission.
  • Muncl3-1 plays a role in regulating amyloid precursor protein (APP) processing and synaptic function, with Muncl3-1 contributing to the regulation of secretory APP metabolism and affecting the production of P-amyloid peptides, which are a constituent of senile plaques in the brains of patients diagnosed with Alzheimer’s disease.
  • shedding of the Alzheimer APP ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Muncl3-1.
  • PPC protein kinase C
  • Phorbol esters which mimic the effects of diacylglycerol (DAG), can stimulate non- amyloidogenic processing of APP.
  • Muncl3-1 is a receptor for phorbol esters and DAG, indicating that Muncl3-1 plays a role in APP processing and therefore administration of Muncl3-1 can be a useful treatment for patients diagnosed with Alzheimer’s disease.
  • Muncl3-1 plays a role in synaptic vesicle priming, which is important for proper neurotransmitter release.
  • Muncl3 proteins regulates the priming step in the transport of synaptic vesicles, thus, enhancing synaptic vesicle priming could improve synaptic transmission in the brains of patients diagnosed with Alzheimer’s disease.
  • Muncl3-1 axonal mRNA localization and its presynaptic translation can be dysregulated in Alzheimer’s disease, revealing an Smn-dependent transport mechanism. This is reinforced by experiments wherein viral transduction of a Muncl3-1 construct with modified 3'UTR restores Muncl3-1 mRNA levels and local translation in the absence of Smn, leading to improved synaptic functions. This modified Muncl3-1 construct could thus represent a new promising therapeutic target for Alzheimer’s disease patients and possibly other neurodegenerative diseases with synapse degeneration.
  • These methods of improving synaptic function and/or treating patients diagnosed with Alzheimer’s disease include administering a vector system that includes one or more of the modified Muncl3-1 molecules noted herein, including the rescue constructs noted herein. Additionally, the vector system of this method can be a lentiviral vector system, and/or in other embodiments, any suitable vector capable of transduction of the modified Muncl3-1 molecules into any target cell can be used.
  • vector systems useful for transferring nucleic acids into target cells are available and can be used in conjunction with transferring any nucleic acids of the present disclosure to any target cells, and for any treatment method disclosed herein.
  • the vectors may be maintained episomally, e.g., as plasmids, minicircle DNAs, virus-derived vectors such cytomegalovirus, adenovirus, etc., or they may be integrated into the target cell genome, through homologous recombination or random integration, e.g., retrovirus derived vectors such as MMLV, HIV-1, ALV, etc.
  • Vectors may be provided directly to the subject cells. In other words, the pluripotent cells are contacted with vectors including the nucleic acid of interest such that the vectors are taken up by the cells.
  • nucleic acid vectors such as electroporation, calcium chloride transfection, and lipofection
  • the nucleic acid of interest may be provided to the cells via a virus and/or a peptide that is capable of translocation through the cell membrane.
  • the cells are contacted with viral particles and/or peptides including the nucleic acid of interest.
  • Retroviruses for example, lentiviruses, are particularly suitable to the method of the invention.
  • retroviral vectors are “defective”, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line.
  • the retroviral nucleic acids including the nucleic acid are packaged into viral capsids by a packaging cell line.
  • Different packaging cell lines provide a different envelope protein to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells.
  • Envelope proteins are of at least three types, ecotropic, amphotropic and xenotropic.
  • Retroviruses packaged with ecotropic envelope protein are capable of infecting most murine and rat cell types, and are generated by using ecotropic packaging cell.
  • Retroviruses bearing amphotropic envelope protein are capable of infecting most mammalian cell types, including human, dog and mouse, and are generated by using amphotropic packaging cell lines such as PA12; PA317; GRIP.
  • Retroviruses packaged with xenotropic envelope protein, e.g., AKR env are capable of infecting most mammalian cell types, except murine cells.
  • the appropriate packaging cell line may be used to ensure that the cells of interest — in some instance, the engrafted cells, in some instance, the cells of the host are targeted by the packaged viral particles.
  • Vectors used for providing nucleic acid of interest to the subject cells will typically include suitable promoters for driving the expression, that is, transcriptional activation, of the nucleic acid of interest.
  • suitable promoters for driving the expression that is, transcriptional activation, of the nucleic acid of interest.
  • This may include ubiquitously acting promoters, for example, the CMV-b-actin promoter, or inducible promoters, such as promoters that are active in particular cell populations or that respond to the presence of drugs such as tetracycline.
  • transcriptional activation it is intended that transcription will be increased above basal levels in the target cell by at least about 10-fold, by at least about 100-fold, more usually by at least about 1000 fold.
  • vectors used for providing reprogramming factors to the subject cells may include genes that must later be removed, e.g., using a recombinase system such as Cre/Lox, or the cells that express them destroyed, e.g., by including genes that allow selective toxicity such as herpesvirus TK, bcl-xs, etc.
  • mice (referred to as control in the text) were offspring of two mouse strains (I) Smn +A that is hemizygote for the Smn tmIHung targeted mutation, and (II) Smn ⁇ ,Hurig" '" that is homozygote for the Smn tmIHung targeted mutation as well as for the transgenic Hung allele, Tg(SMN2)2Hung.
  • Muncl3-1 KO mice (Mund 3-M) were originally obtained from Goettingen, Germany and cross-bred in-house.
  • Nestin-Cre transgenic mice (B6.Cg(Nes-cre)lKln/J) were cross-bred in-house.
  • R26Uncl3-l tg/+ knock-in mouse was designed and cloned by M. M. and generated at Czech Centre for Phenogenomics in Prague, Czech Republic (https://www.phenogenomics.cz). Smn +/ ⁇ ,R26Uncl 3-1'" and Smrr ,Hung” ! ",Neslin-(3 re'" mice were cross-bred from parents and generated in-house.
  • Motoneurons were isolated from E13.5 mouse embryos, enriched via p75 NTR antibody panning, transduced with lentiviral particles for 10 min at RT, and plated onto precoated poly ornithine and laminin211/221 (Biolamina, LN211-0501, and LN221-0501) cell culture dishes. This muscle-specific laminin isoform induces the differentiation of axonal growth cones into presynaptic structures in cultured motoneurons. Cells were grown in presence of 3 ng/ml BDNF for 6 days.
  • motoneurons were plated onto glass coverslips, for Western blot and qRT-PCR onto 24-well plates, for Ca 2+ imaging on p- dishes (Ibidi, 81156), and for lattice-SIM onto 8-well chambers with 1.5 high-performance cover glasses (Cellvis, C8-1.5H-N). Culturing of motoneurons in compartmentalized microfluidic chambers was performed as previously described.
  • DIV6 motoneurons received a 5 pM R-Roscovitine (referred to as Roscovitine in the text) (Merck, R7772) pulse for 5 min at 37 °C using a hot plate.
  • Roscovitine referred to as Roscovitine in the text
  • cells were fixed with 4% Paraformaldehyde (PFA) (ThermoFisher Scientific, 28908) for 10 min at RT and permeabilized with 0.1% Triton X-100 for 5-10 min.
  • Cells were incubated with block solution (2% BSA, 100 pg/mL saponin, and 0.25% sucrose in PBS) for 1 hour at RT.
  • Primary antibodies were diluted in block solution and incubated at 4 °C overnight.
  • motoneurons were first incubated with 0.2 nmol mCLING-ATTO 647N (Synaptic Systems, 710 006AT1) for 1 min followed by a depolarization step with 90 mM KC1 for 7 min. Neurons were then fixed with 4% PFA, 0.2% glutaraldehyde for 20 min on ice followed by 10 min incubation at RT. The fixation buffer was quenched in 100 mM glycine solution for 20 min at RT and cells were subsequently immunostained against Muncl3-1 and Synapsinl/2. In no-pulse control group, cells were immediately fixed after 1 min incubation with mCLING and treated as described above.
  • rabbit polyclonal anti-Tau (Sigma-Aldrich, T6402, 1 : 1000), mouse monoclonal anti-a-Tubulin (Sigma- Aldrich, T5168, 1 : 1000), mouse monoclonal purified IgG anti-Basoon (Synaptic Systems, 141011, 1 :500), guinea pig polyclonal antiserum anti-Piccolo (Synaptic systems, 142104, 1 :500), rabbit polyclonal purified anti-RIMl/2 (Synaptic Systems, 140213, 1 :500), rabbit polyclonal anti-Muncl3-l (Synaptic System, 126103, 1 :500), guinea pig polyclonal purified anti-Ca 2+ channel N-type alpha-lB (Ca v 2.2) (Synaptic System, 152305, 1 :250), mouse monoclonal anti-P-actin (Gene
  • smFISH was conducted as previously described and following the manufacturer’s instructions (ThermoFisher Scientific). Motoneurons were fixed with paraformaldehyde lysine phosphate (PLP) buffer (4% PF A, 5.4% glucose, and 10 mM sodium metaperiodate, pH 7.4) for 10 min at RT and permeabilized with a supplied detergent solution for 4 min at RT. mRNAs were unmasked by proteinase K digestion, which was applied for 4 min at 1 :8000 dilution. Hybridization probes specific to the Muncl3-1 mRNA coding region were diluted 1 : 100 in the hybridization buffer and incubated at 40 °C overnight.
  • PBP paraformaldehyde lysine phosphate
  • preamplifier, amplifier, and label probe oligonucleotides were incubated each for 1 hour at 40 °C. After the washing steps, cells were immunostained against Tau for visualization of the neurite boundaries.
  • mice were sacrificed at P5 or P 10 and T VA muscles were collected in an extracellular physiological solution (135 mM NaCl, 12 mM NaHCO3, 5 mM KC1, 1 mM MgCh, 2 mM CaCh, 20 mM glucose). Muscles were fixed with 4% PFA at 4 °C for 90 min, washed with 0.1 M glycine on a shaker for 30 min, and permeabilized with PBS-T (1% Triton X-100) twice for 5 min, twice for 10 min, and twice for 30 min.
  • PBS-T 1% Triton X-100
  • Muscles were then blocked with 5% BSA in PBS-T (0.1% Triton X-100) at RT for 3 hours, and then with primary antibodies diluted in block solution for two nights at 4 °C on a shaker. Then, preparations were washed with PBS- T (0.1% Triton X-100) at RT 3 x 15 min on a shaker, and secondary antibodies along with a- Bungarotoxin (ThermoFisher Scientific, B 13422, 1 : 1000) were incubated at RT for 1 hour. After 3 x wash with PBS-T, preparations were rinsed in water and embedded using Aqua- Poly/Mount.
  • PBS-T 0.1% Triton X-100
  • Postsynaptic membranes in the NMJs were labeled with Alexa Fluor 488- conjugated a-Bungarotoxin, which binds to the a-subunits of nicotinic acetylcholine receptors (AChRs).
  • AChRs nicotinic acetylcholine receptors
  • naive spinal cords were isolated from P10 mice and fixed in 4% PFA overnight. L1-L2 regions of the spinal cord were embedded in warm 5% Agar and serial sections of 45 pm were cut on a Vibratome. Sections were first incubated in 0.1 M glycine for 15 min and blocked in 5% Donkey serum, 0.3% Triton X-100 at RT for 2 hours.
  • guinea pig polyclonal anti-Synaptophysinl (Synaptic Systems, 101004, 1 : 1000)
  • rabbit polyclonal anti-Ca 2+ channel P/Q-type alpha-1 A (Ca v 2.1)
  • rabbit polyclonal anti-Muncl3-l (Synaptic System, 126103, 1 :500)
  • chicken polyclonal anti-Neurofilament H Merck, AB5539, 1 : 1000
  • goat anti-Choline (Synaptic Systems, 101004, 1 : 1000)
  • rabbit polyclonal anti-Ca 2+ channel P/Q-type alpha-1 A (Ca v 2.1)
  • rabbit polyclonal anti-Muncl3-l (Synaptic System, 126103, 1 :500)
  • chicken polyclonal anti-Neurofilament H (Merck, AB5539, 1 : 1000)
  • Acetyltransferase (Millipore, AB 144P, 1 :250), donkey anti-rabbit IgG (H+L) AffmiPure (Cy3, Jackson ImmunoResearch, 711-165-152, 1 :500), donkey anti-guinea pig IgG (H+L) AffmiPure (Cy5, Jackson ImmunoResearch, 706-175-148, 1 :500), donkey anti-chicken IgY (H+L) AffmiPure (Cy5, Jackson ImmunoResearch, 703-175-155, 1 :500), and donkey anti-goat IgG (H+L) AffmiPure (Cy3, Jackson ImmunoResearch, 705-165-147, 1 :500).
  • plasmids harboring the coding region (cDNA) of mouse endogenous Muncl3-1 and human UNC13A waere purchased from GenScript.
  • the 3'UTR of mouse endogenous Muncl3-1 was synthesized and purchased from GenScript.
  • the 3'UTR of mouse Synaptophysinl (SynPhy)and human Synaptophysinl (SYP) were amplified by PCR using cDNA from motoneurons as template and PfuUltra II Fusion HotStart DNA Polymerase (Agilent, 600670).
  • the coding region of Muncl3-1 was fused to the 3'UTR of Synaptophysinl or 3'UTR of Muncl3-1 using NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs, E5520S) and inserted into a lentivirus backbone vector with the Ubiquitin promotor.
  • NEBuilder® HiFi DNA Assembly Cloning Kit New England Biolabs, E5520S
  • eF 1 -Cre expressing vector was provided from a third party. The expression of all rescue constructs was validated in cultured motoneurons by Western blot and qRT-PCR.
  • Lentiviruses and AVVs were packaged in HEK 293T cells using TransIT-293 (Minis, MIR2706) for transfection.
  • TransIT-293 Minis, MIR2706
  • pCMV-VSVG and pCMVAR8.91 helper plasmids were used for lentivirus packaging, and for AAV packaging, Rep/Capin, pAAV-mGly, and pHGTI-adenol AVV helper plasmids were used.
  • Viral supernatants were harvested by ultracentrifugation 60-72 hours post-transfection.
  • Virus titer was determined in NSC 34 cells using standard methods with serial dilutions.
  • mouse endogenous Muncl3-1, 3'UTR of mouse endogenous Synaptophysinl, and the SV40pA sequence were first amplified by PCR using available plasmids as templates (see the cloning section). PCR products were then assembled into one fragment and inserted into an expression vector using NEBuilder® HiFi DNA Assembly Cloning Kit. Next, the assembled fragments were excised from the expression vector by Xhol restriction enzymes and inserted into a backbone vector harboring the CAG-loxP-Stop-loxP cassette.
  • the resulting cassette including CAG-loxP-Stop-loxP-Muncl3-l+SynPhy3'UTR- CV40pA was excised from this vector by Sall and ligated into a Sall linearized ROSA26 donor vector.
  • the Cre-dependent expression of Muncl3-1 was validated by qRT-PCR and Western blot with HEK 293 cells, which were transfected with the vector expressing the targeting cassette and an eFl-Cre expressing vector.
  • the targeting cassette was inserted into the ROSA26 locus through CRISPR-Cas9 technology at Czech Centre for Phenogenomics in Prague, Czech Republic (https://www.phenogenomics.cz).
  • Three founders were obtained and the transgenic cassette was validated by sequencing as well as genotyping PCR.
  • the Cre-dependent expression of the transgenic Muncl3-1 allele was verified by qRT-PCR using Nestin-Cre and ChAT-Cre driver lines (FIG. 5B and FIGs. 11A-11F), as well as by Western blot in Muncl3-1 KO mice (FIG. 5C). Following primers were used for genotyping of R26Uncl3-l knock-in allele: ROSAext- forward: 5‘-TGCCATGAGTCAAGCCAGTC-3’, SynPhy3'UTR -reverse 5‘-
  • Ca 2+ imaging was performed using the calcium indicator Oregon GreenTM 488 BAPTA-1, AM, cell-permeant (ThermoFisher Scientific, 06807).
  • Calcium indicator was dissolved in Pluronic F-127/DMSO in an ultrasonic bath for 2 min to prepare a 5 mM stock solution.
  • Motoneurons were first washed twice with prewarmed Ca 2+ imaging buffer (135 mM NaCl, 6 mM KC1, 1 mM MgCh, 1 mM CaCh, 10 mM HEPES, and 5.5 mM glucose) and incubated with 5 pM Ca 2+ indicator diluted in the Ca 2+ imaging buffer for 15 min at 37 °C in a CO2 incubator.
  • Cells were washed again twice with Ca 2+ imaging buffer and imaged in 2 ml of Ca 2+ imaging buffer in the presence of 3 ng/ml BDNF.
  • a TE2000 Nikon inverted epifluorescence microscope was used that was equipped with a 60* 1.4-NA objective, a perfect focus system, Orca Flash 4.0 V2 camera (Hamamatsu Photonics), an LED fluorescence light for excitation at 470 nm, and Nikon Element image software.
  • Cells were imaged at 37 °C in the presence of 5% CO2 using a TOKAI HIT CO, LTD heated stage chamber. Cells were imaged at 500 ms intervals over a total period of 7 min for spontaneous Ca 2+ spikes and over 2 min for pulse experiments.
  • Proximity ligation assay was conducted as previously described with minor modifications. Shortly, 10 pg/ml puromycin (Merck, 540222-25MG) and 100 pg/ml cycloheximide (Merck, 01810-1G) were added to the cells and incubated for 5 min at 37 °C. The puromycylation reaction was stopped through washing with PBS-MC (1 x PBS pH 7.4, 1 mM MgCh, 0.1 mM CaCh) and cells were fixed with 4% PF A, 4% sucrose in PBS-MC buffer for 10 min at RT.
  • PBS-MC 1 x PBS pH 7.4, 1 mM MgCh, 0.1 mM CaCh
  • RNA-IP Cortical synaptosome fractionation and RNA immunoprecipitation
  • Resulting supernatants (S2) containing the light membrane fraction and soluble enzymes were discarded and pellets (P2) containing crude synaptosomes were resuspended in 700 pl IP buffer (20 mM Tris pH 7.5, 2 mM MgCh, 150 mM KC1, 0.1% Nonidet P-40, 1 x protease inhibitor cocktail) and incubated on ice for 15 min.
  • 100 pg/ml cycloheximide was added into the fractionation buffer as well as into the IP buffer for all the sequential IP steps.
  • a mouse monoclonal rRNA (Y10B) antibody (ThermoFisher Scientific, MAI-16628) and normal mouse IgG control (Santa Cruz Biotechnology, sc-2025) were used.
  • 1 pg Y10B or IgG control antibodies and 10 pl protein G magnetic dynabeads (ThermoFisher Scientific, 10003D) were added to 100 pl IP buffer and incubated with rotation for 1 hour at RT.
  • Synaptosome fractions were then added into pre-washed magnetic protein G/antibody beads and incubated with rotation for 2 hours at 4 °C. Resulting immunocomplexes were washed twice for 5 min with rotation at 4 °C.
  • RNAs were eluted from magnetic beads with ethanol precipitation and purified using NucleoSpin RNA Clean-up kits (Machery-Nagel, 740948.50) following the manufacturer’s instructions. Purified RNA was resuspended in 20 pl RNase-free water and 10 pl was reverse transcribed with random primers using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, K1621). Relative binding of Muncl3-1 transcripts to ribosomes as well as 18srRNA levels were determined by qRT-PCR.
  • the hiPSC lines used were purchased from Cedars-Sinai Biomanufacturing Center and are as followed: CS84iSMA (type I), CS77iSMA (type I), CS13iSMA (type II), CS83iCTR-33n, and CS88iCTR.
  • Motoneurons were differentiated according to Reinhardt et al,, with some modifications as described herein.
  • iPSCs were grown on Matrigel -coated (Corning, 356234, 1 : 100) dishes and expanded in mTeSR Plus medium (Stemcell Technologies, 05825).
  • iPSCs were seeded into non-coated low adherent 12-well plates (Greiner, M9187) and grown in mTeSR Plus medium supplemented with small molecules; SB431542 (AdooQ BioScience, A10826-50, 10 pM), dorsomorphin homolog 1 (DMH1) (R&D Systems, 4126, 1 pM), CHIR99021 (Cayman Chemical Company, 13122, 3 pM), and Purmorphamine (PMA) (Cayman Chemical Company, 10009634, 0.5 pM).
  • SB431542 AdooQ BioScience, A10826-50, 10 pM
  • DMH1 dorsomorphin homolog 1
  • CHIR99021 Cayman Chemical Company, 13122, 3 pM
  • PMA Purmorphamine
  • mTeSR Plus medium was replaced with neuronal medium (Neurobasal medium (Gibco, 21103049), Dulbecco’s modified Eagle’s medium F-12 (DMEM/F-12) (Gibco, 21331046), N- 2 Supplement (Gibco, 17502048), Penicillin/Streptomycin/Glutamax (Gibco, #10378016, 100 pg/mL)) supplemented with the same small molecules as above.
  • medium was replaced with expansion medium (neuronal medium supplemented with 3 pM CHIR99021, 0.5 pM PMA, and 150 pM Ascorbic acid (AA) (Sigma, A92902)).
  • NPCs Neuronal Progenitor Cells
  • EBs were collected from the suspension on the day 6 and plated on Matrigel-coated dishes.
  • cells were splitted for at least 20 passages once a week using Accutase (Thermo Fisher, 07920).
  • NPC differentiation into motoneurons cells were seeded on Matrigel-coated dishes and expanded for two days in neuronal medium supplemented with 1 pM PMA. On day 2, medium was exchanged with neuronal medium supplemented with 1 pM PMA and 1 pM Retinoic acid (Stemcell Technologies, 72264).
  • Motoneurons were differentiated for 25 days in neuronal medium supplemented with 5 ng/mL glia-derived neurotrophic factor (GDNF) (Alomone Labs, G-240), 5 ng/mL brain-derived neurotrophic factor (BDNF) (PeproTech, 450-02), and 500 pM dibutyryl -c AMP (dbcAMP) (Stemcell Technologies, 73886). For all steps, medium was exchanged every other day.
  • GDNF glia-derived neurotrophic factor
  • BDNF brain-derived neurotrophic factor
  • dbcAMP dibutyryl -c AMP
  • qRT-PCR was performed on a LightCycler 1.5 thermal cycler (Roche) using Luminaris HiGreen qPCR Master Mix (ThermoFisher Scientific, K0992).
  • the relative expression of target genes was measured according to the AACt method. Gapdh was used as internal control and for data normalization. The following equation was used to determine the relative number of Muncl3-1 transcripts bound to ribosomes in RNA-IP experiments using 18srRNA as reference:
  • Membranes were developed using ECL systems (GE Healthcare). The following antibodies were used: rabbit polyclonal anti-Calnexin (Enzo Life Sciences, ADI-SPA-860-F, 1 :6000), goat polyclonal anti-ribosomal protein L8 (SAB2500882, Sigma-Aldrich; 1 :5000), rabbit polyclonal anti-Muncl3-l (Synaptic Systems, 126103, 1 :4000), peroxidase AffiniPure donkey anti-goat IgG (H + L) (Biozol, 705-035-003, 1 : 10000), and peroxidase AffiniPure goat anti-rabbit IgG (H + L) (Biozol, 111-035-144, 1 : 10000).
  • rabbit polyclonal anti-Calnexin Enzo Life Sciences, ADI-SPA-860-F, 1 :6000
  • goat polyclonal anti-ribosomal protein L8 SAB2500882, Sigma-Aldrich
  • mice were placed on their back and the time that they took to turn to the prone position was recorded. An average of three attempts with a maximum of 30 s designated the “time delay to right” score.
  • For the grip strength test of forelimbs mice were placed on a thin glass tube with their forelimbs and the time it took for them to fall was measured.
  • For the hind limb clasping test mice were put on a glass cup and the splay was recorded when the animals climbed into the cup. The observed splay was scored between 0 and 4. A score of 4 was assigned to a healthy splay of both hind limbs. A score of 3 was given to a weak splay of both hind limbs.
  • a score of 2 was assigned to a clasping. A score of 1 was assigned when no splay was observed, and a score of 0 was received for mice that crossed both hind limbs over each other. Animal waiters who carried out the motor tests were blinded to the animal genotypes.
  • DIV6 motoneurons were first stimulated with 5 pM Roscovitine for 5 min and then fixed for 10 min at RT with 4% PF A. Following three washing steps with PBS, cells were permeabilized for 10 min at RT with 0.1% Triton X-100 in PBS.
  • cells were blocked for 1 hour at RT with 5% BSA in PBS and incubated overnight at 4 °C with following primary antibodies: polyclonal rabbit anti-Muncl3-l (126 103, Synaptic Systems), monoclonal guinea pig anti-Snap25 (111 308, Synaptic Systems), and polyclonal goat anti- Ribosomal Protein L8 (RPL8) (SAB2500882, Sigma Aldrich) diluted 1 :250 in the blocking solution. On the next day, cells were washed again trice with PBS.
  • Motoneurons cultured on glass coverslips were first treated with 5 pM Roscovitine for 5 min. Immediately after that treated and untreated control motoneurons were incubated in a humidified chamber for 5 hours at 37 °C in 0.7% formaldehyde and 1% acrylamide diluted in PBS. Thereafter, coverslips with cultured motoneurons were transferred upside down to a 60 pl droplet of the TREx monomer solution on parafilm in a humidified chamber on ice. For the polymerization, the monomer solution was incubated for at least 2 hours at RT.
  • gels were incubated for 1 hour at 95 °C in denaturation buffer (200 mM SDS, 200 mM NaCl and 50 mM Tris, pH 6.8) and washed 2 x with PBS. Gels were then incubated overnight at RT in 5% BSA in PBS with a 1 :200 dilution of the following primary antibodies: polyclonal rabbit anti-Muncl3-l (126 103, Synaptic Systems), polyclonal goat anti-Ribosomal Protein L8 (RPL8) (SAB2500882, Sigma Aldrich) and polyclonal anti-mouse Synaptobrevin 2 (104 202, Synaptic Systems).
  • denaturation buffer 200 mM SDS, 200 mM NaCl and 50 mM Tris, pH 6.8
  • axon length measurements single stack images were acquired with a Keyence BZ-8000K fluorescence microscope with a 20* 0.7-NA objective, and axon length measurements were conducted using Fiji. Motoneurons with an axon length of ⁇ 100 pm were excluded. Confocal images were acquired as 16-bit images with 800 x 800-pixel resolution with an Olympus Fluoview 1000 microscope equipped with a 60 1.35-NA oil objective. For cultured motoneurons, confocal images of a single z-stack were taken. For NMJs, confocal images of multiple z-stacks of 0.5 pm were taken and maximum projection images were shown for representative images.
  • lattice-SIM For lattice-SIM, five z-stack images of 110 nm were taken, and maximum projection images were shown as representative images.
  • images were acquired using an ELYRA 7 SIM Zeiss equipped with either a Plan-Apochromat 63 x NA-1.4 oil objective (for unexpanded samples) or a C-Apochromat 63 x 1.2-NA water immersion objective (used for expanded samples) and 405 nm diode (50mW), 488 nm OPSL (500 mW), 561 nm OPSL (500 mW) and 642 nm diode (500 mW) excitation lasers.
  • the laser power was adjusted between 2 and 5% with an integration time of 200 ms.
  • the acquired 16- bit raw images were processed with a commercial software package from Zeiss (ZEN 3.0 SR FP2 black) to reconstruct super-resolution images.
  • a channel alignment was performed on the reconstructed images using fiducial markers (TetraSpeckTM Microspheres, 0.2 pm, fluorescent blue/green/orange/dark red, T7280, ThermoFisher Scientific). Images were processed and analyzed in Fiji. For better visibility, linear contrast enhancement was implemented to all representative images using Adobe Photoshop 24.2.0.
  • the dots were manually counted in the axon and the number of dots was normalized to pm axon length. Signal intensities of Smn-deficient and control cells were normalized to the mean intensities of the control group within the same experiment. For pulse experiments, all signal intensities were normalized to the mean intensities of the non-stimulated corresponding genotype. Colocalization analysis of lattice-SIM data was performed with single optical sections of raw 16-bit images using Fiji. All immunostaining experiments including smFISH and PLA were carried out and analyzed blindly.

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Abstract

La demande concerne une construction d'expression d'ARNm de mammifère permettant de produire une protéine ou un polypeptide, la construction d'expression d'ARNm de mammifère contenant une molécule Munc13-1 modifiée.
PCT/EP2024/071226 2023-07-31 2024-07-26 Modulation de pathologie présynaptique avec traduction locale de munc13-1 Pending WO2025026896A1 (fr)

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