Modulation of presynaptic pathology with munc13-1 local translation

Abstract

This application includes a mammalian mRNA expression construct for producing a protein or polypeptide, the mammalian mRNA expression construct including a modified Munc13-1 molecule.

Description

MODULATION OF PRESYNAPTIC PATHOLOGY WITH MUNC13-1 LOCAL TRANSLATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 516,842, filed July 31, 2023, the contents of which are incorporated by reference in the present disclosure in their entirety.BACKGROUND

[0002] 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.

[0003] One example of these diseases, Alzheimer's disease (AD), is 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.

[0004] Another example of these diseases, 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.

[0005] 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.

[0006] 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. Loss of Muncl3- 1 arrests spontaneous and evoked synaptic release events in hippocampal neurons and at neuromuscular junctions (NMJs), and causes severe paralysis leading to early postnatal death in knockout (KO) mice. In humans, mutations in UNC13A gene cause microcephaly, cortical hyperexcitability, and fatal myasthenia.

[0007] Local translation participates in synaptogenesis, plasticity, and axon regeneration through rapid modulation of the local proteome in response to extracellular cues. Alterations in local protein synthesis contribute to the pathology of diverse neurodegenerative disorders. A central mechanism of impaired local translation involves perturbed mRNA localization including those encoding synaptic components.

[0008] Nevertheless, the role of locally translated synaptic proteins in presynaptic differentiation and plasticity had remained elusive. In ALS and SMA, defective synaptic transmission contributes to degeneration of NMJs, raising the question of whether perturbed presynaptic synthesis of AZ proteins, in particular Muncl3-1 could underlie the mechanisms of neurotransmission and plasticity defects. The impact of Muncl3-1 is discussed herein.SUMMARY

[0009] 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.

[0010] Exchange of the 3'UTR from Muncl3-1 to Synaptophysin rescues the defective transport of Muncl3-1 transcripts and restores the nanoscale architecture of presynaptic Muncl3-1 release sites, as revealed by super-resolution microscopy. In addition, we observed functional synaptic recovery in cell cultures of Smn-deficient motoneurons after viral delivery of modified Muncl3-1. Similarly, SMA mice bred with a knock-in mouse expressing modified Muncl3-1 from the ROSA26 locus displayed attenuated synapse degeneration and improved motor function, thus representing a new therapeutic approach for SMA.

[0011] Using super-resolution microscopy, the nanoscale supramolecular architecture of Muncl3-1 release sites at axonal presynaptic membranes was resolved, providing evidence that activity-dependent assembly of new release sites depends on Muncl3-1 local translation. This disclosure provides a description for perturbed local synthesis of Muncl3-1 at axonal presynaptic terminals in Smn deficient motoneurons. Viral transduction of cultured motoneurons with a Muncl3-1 construct with modified 3'UTR that could restore the axonal transport and local translation of Muncl3-1 in the absence of Smn leads to improved neurotransmitter release and enhanced excitability in SMA. Moreover, a ROSA26 Cre / loxP conditional Muncl3-1 knock-in rescue mouse model was generated that harbors a Muncl3-1 allele with a 3'UTR from the synaptophysin transcript. SMA mice cross-bred with the knock- in rescue mice displayed diminished synapse degeneration, attenuated muscle weakness, and improved motor function, demonstrating a new therapy approach for counteracting the synaptic dysfunction in SMA.

[0012] 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.

[0013] In additional embodiments, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] 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.

[0015] 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. IB illustrates a qRT-PCR graph reveals significant reduction of Muncl3-1 mRNAs in axons after Smn knockdown (*P = 0.0303, **P = 0.0011; n = 6 experiments). FIG. 1C illustrates that Muncl3-1 transcripts are discernable in distal axons in cultured motoneurons using smFISH. FIGs. ID and IE illustrate graphs showing reduced Muncl3-1 transcripts in axons (ID: **P = 0.0041; n = 89-123 cells) and axonal growth cones (IE: **P = 0.0053; n = 13-21 cells) in Smn- deficient neurons. FIG. IF illustrates neuromuscular junctions (NMJs) of TVA muscles fromP5 littermates are stained against Muncl3-1. SynPhy antibody labels presynaptic membranes and a -Bungarotoxin labels AChRs at postsynaptic membranes. FIGs. 1G and 1H illustrate that quantification of fluorescent signals reveals decreased Muncl3-1, but unaltered SynPhy levels in NMJs of Smn KO littermates< 0.0001; n = 5 experiments). 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. FIG. IK illustrates that Smn-deficient neurons exhibit reduced Puro-PLA signal for Muncl3-1 (**P = 0.0075; n = 49-52 cells). FIG. IL illustrates that RNA-IP shows reduced Muncl3-1 transcripts bound to translating ribosomes in cortical synaptosome fractions from Smn KO mice (*P = 0.0143; n = 4 experiments). FIG. IM illustrates that Muncl3-1 mRNAs is reduced by 25% in the input of Smn KO compared to control mice (*P = 0.0143; n = 4 experiments). Mann-Whitney U test: one-tailed in FIGs. IB, IL, and IM, and two-tailed in FIGs. ID- IK. Bars represent mean ± SEM.

[0016] 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. 2B illustrates that qRT-PCR relevels that 3'UTRs of Muncl3-1 (***P = 0.0003, ***P = 0.0006) and SynPhy (*P = 0.0143, *P = 0.0286) drive Muncl3-1 transcripts into distal axons in control motoneurons, while Muncl3-1 construct lacking 3'UTR does not (**P = 0.004; n = 4-7 experiments). FIG. 2C is an immunoblot showing increased Muncl3-1 protein levels in total lysates from motoneurons transduced with viral constructs shown in FIG. 2 A and FIG. 2B (representative of n = 3 experiments). FIG. 2D illustrates that Muncl3-1 becomes locally translated in axonal growth cones in response to Roscovitine stimulation. FIG. 2E Graph demonstrates increased Munc 13-1 levels in stimulated control and KORescuemotoneurons (***p = 0.0004, ****p < 0.0001; n = 59-81 cells). FIG. 2F illustrates that Lattice-SIM reveals Muncl3-1 supramolecular release sites in axonal growth cones in cultured motoneurons. Insert: Zoom-in shows colocalization of Muncl3-1 with Snap25 in supramolecular release sites. FIG. 2G illustrates that upon Roscovitine pulse, the number of Muncl3-1 release sites per growth cone increases in control and KORescuemotoneurons. Mann-Whitney U test: one-tailed in FIG. 2B and two-tailed in FIG. 2E. Bars represent mean ± SEM.

[0017] 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 ofcultured motoneurons before and after stimulation of the neuronal activity. FIGs. 3B and 3C illustrate that the number of Muncl3-1 nanoassemblies increases upon stimulation< 0.0001; n = 26 cells). FIG. 3D illustrates that the diameter of Muncl3-1 nanoassemblies decreases upon stimulation< 0.0001; n = 115-153 nanoassemblies from 26 cells). FIG. 3E illustrates that upon neuronal stimulation, the center-to-center distance between nearest neighboring Muncl3-1 nanoassemblies decreases (*P = 0.0167; n = 116-154 nanoassemblies from 26 cells). FIG. 3F is representative lattice-SIM images of Muncl3-1 release sites in axonal growth cones. Muncl3-1 colocalizes with ribosomes within supramolecular release sites. In control and KORescuemotoneurons, colocalization between Muncl3-1 and ribosomes increases upon stimulation (**P = 0.0084, ****p < 0.0001; n = 27-42 cells). In FIG. 3A, scale bars are corrected to indicate pre-expansion dimensions. Two-tailed Mann-Whitney U test. Bars represent mean ± SEM.

[0018] FIGs. 4A-4H illustrate that Muncl3-1 local translation rescues neuronal excitability in Smn-deficient motoneurons. FIG. 4A illustrates that SV recycling assay reveals improved neurotransmitter release in KORescuebut not KORescueA3 UTRmotoneurons (*P = 0.0352, *P = 0.0258, ***p = 0.0008; n = 35-66 cells). FIG. 4B illustrates that clustering of Cav2.2 is restored in axonal growth cones of both KORescueand KORescueA3 UTRmotoneurons (*P = 0.0379, **P = 0.0029, ****p < 0.0001; n = 42-91 cells). FIGs. 4C-4E illustrate that Ca2+imaging indicates an increased frequency of spontaneous Ca2+transients in axonal growth cones of KORescueand QRescueA3'UTRmotoneurons(*p = 0.0102, ****p < 0.0001; n = 15-26 cells). The amplitude of spontaneous Ca2+transients is increased only in KORescuebut not KORescueA3 UTRmotoneurons (*P = 0.039, *P = 0.0463, **P = 0.0083; n = 172-489 transients). FIG. 4F illustrates that growth cones were depolarized by a 90 mM KC1 pulse and Ca2+transients were measured. FIG. 4G illustrates that Maximum response to depolarization is lower in Smn KO and KORescueA3 UTRmotoneurons (*P = 0.0111, *P = 0.0122, *P = 0.0464; n = 28-45 cells). FIG. 4H illustrates that % of failure to membrane depolarization is diminished in KORescuebut not KORescueA3'UTRmotoneurons. One-way ANOVA with Dunn’s post-test.

[0019] 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-ltg / +mice were cross-bred with Nestin-Cretg / +. qRT-PCR with cortices indicates that Nestin-Cre drives the expression of Muncl3-1 rescue allele inR26Uncl3-ltg / +mice and its expression increases with animal age (n = 2 experiments). FIG. 5C illustrates that motoneuronsMuriel 3-l+ / ~ ,R26Uncl 3-ltg / +and Munc 13-1 ^ ,R26Unc 13- ltg / +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-ltg / +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. 5E illustrates increased percentage of fully innervated NMJs in TVA muscles of PIO Smn^ ,Huri 'g,R26Uncl 3-ltg / +,Cretg / +mice indicating attenuated degeneration of motor endplates (*P = 0.0121, ****p < 0.0001; n = 3-10 animals). FIGs. 5F and 5G illustrate that Muncl3-1 (**P = 0.0073, ****P < 0.0001) as well as Cav2.1 (***P = 0.0002, ****P < 0.0001; n = 3 experiments) are upregulated in NMJs of TVA muscles from Smir ,Hiingg,1126lhic 13- ltg / +,Cretg / +littermates compared to Smn KO. FIG. 5H illustrates that motoric tests reveal improved time taken to self-right in Smn ^ ,Hun^g / +,R26Unc 13-ltg / +,Cretg / +compared to Smn KO littermates (**P = 0.0018, ****p < 0.0001; n = 9-34 animals). FIG. 51 and 5J illustrates that grip strength of forelimbs is improved in Smn~ / ~,Hun^g / +,R26Uncl3-ltg / +,Cretg / +littermates compared to Smn KO (**P = 0.0018, **P = 0.004, ****p < 0.0001; n = 9-32 animals). FIG. 51 and 5K illustrate that Smn~ / ~,Hun8 / R26Uncl3Mt8 / Cretg / +littermates exhibit improved hind limb clasping posture. FIGs. 5L and 5M illustrate that Kaplan-Meier curve indicates increased survival of Smn ^ ,Hung'g,R26Unc 13-ltg / +,Cretg / +animals (n = 7-32 animals). Two-tailed Mann-Whitney U test in FIGs. 5E, 5H-5M. One-way ANOVA with Dunn’s post-test in FIG. 5F and 5G. Bars represent mean ± SEM. FIG. 5N illustrates ChAT+ motoneurons within L1 / L2 sections of the ventral horn in the spinal cord from P10 littermates. Nuclei are stained with dapi. FIG. 50 illustrates that graph illustrates diminished neurodegeneration of motoneurons within spinal cords of Smn~ / ~,Hung / +,R26Uncl3- ltg / +,Cretg / +mice (*P = 0.020, **P = 0.007; n = 5-7 animals). One-way ANOVA with Dunn’s post-test.

[0020] FIGs. 6A-6J illustrate that Muncl3-1 expression is not altered in cultured Smn- deficient motoneurons. FIG. 6A illustrates that quantification of smFISH indicates similar Muncl3-1 mRNA levels in somata (n = 140-186) of cultured Smn-deficient motoneurons compared to control. FIG. 6B are representative images of axonal growth cones of cultured motoneurons stained against Muncl3-1 and Tubulin. FIG. 6C is a graph of decreased Muncl3- 1 immunoreactivity in axonal growth cones of Smn-deficient motoneurons (***p = 0.0002; n = 62 cells). FIGs. 6D and 6E illustrate immunostaining of Muncl3-1 and Tubulin in somata ofcultured motoneurons. FIG. 6E illustrates that Muncl3-1 protein level is not altered in the soma of cultured Smn-deficient motoneurons (n = 63 cells). FIG. 6F is an immunoblot of total lysates obtained from cultured motoneurons reveals no significant differences in Muncl3-1 protein levels between Smn-deficient and control, calnexin was used as loading control (representative of n = 3 experiments). FIG. 6G is an illustration of no PLA signal being detectable when only Muncl3-1 or puromycin antibodies are incubated. FIG. 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. FIGs. 61 and 6H are quantifications of 18srRNA in input (FIG. 61) and IP (FIG. 6J) fractions of cortical synaptosomes indicates similar ribosomal content in Smn KO and control (n = 4 experiments). Mann-Whitney U test: one-tailed in FIG. 61- FIG. 6J and two-tailed in FIG. 6A- FIG. 6E. Bars represent mean ± SEM.

[0021] 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. 7B illustrates that Muncl 3-1 mRNAs are upregulated in the soma of Rescue and RescueA3'UTR-transduced neurons (**P = 0.0022, ****p = 0.001, ****p < 0.0001; n = 134-186 cells). FIGs. 7C and 7D illustrate that Muncl3-1 mRNA levels are increased in axons (FIG. 7C: *P = 0.0216, ****p < 0.0001; n = 92-124 cells) and axonal growth cones (FIG. 7D: *P = 0.0204, *P = 0.0267, **P = 0.0011; n = 13-27 cells) of Rescue, but not RescueA3'UTR- transduced neurons. (FIG. 7E and FIG. 7F) Following transduction with Rescue and RescueA3'UTR viruses, elevated Muncl3-1 protein levels are detected in the soma (FIG. 7E: *P = 0.0357, **P = 0.0015, **P = 0.007, ***p = 0.0002; n = 52-63 cells) and axonal growth cones (FIG. 7F: **P = 0.003, ****p < 0.0001; n = 62-99 cells) of transduced neurons. Oneway ANOVA with Dunn’s post-test.

[0022] FIGs. 8A-8F illustrate that formation of Muncl3-1 release sites depends on neuronal activity. FIG. 8A illustrates that treatments with nocodazole do not block Muncl3-1 local translation in response to Roscovitine pulse (**P = 0.0025; n = 38-42 cells). Treatments with anisomycin block Muncl3-1 local translation (n = 39-41 cells). FIG. 8B illustrates that the number of Muncl 3-1 releases sites increases significantly in axonal growth cones of stimulated control and KORescue(**P = 0.0068, ***p = 0.0002; n = 21-31 cells), but not in stimulated Smn-deficient and KORescueA3 UTRmotoneurons. FIG. 8C and 8D illustrate that CTX, andanisomycin treatments inhibit the dynamic formation of Muncl3-1 supramolecular release sites (n = 21-24 cells). 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.

[0023] 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. FIG. 9B illustrates that following Roscovitine stimulation, colocalization between Muncl3-1 and Snap25 increases significantly in control and KORescuemotoneurons (*P = 0.0354, **P = 0.009; n = 27-40 cells). FIG. 9C illustrates that anisomycin- treated neurons do not exhibit stimuli-dependent increment in Muncl3-1 and RPL8 colocalization in axonal growth cones (n = 23-33 cells). Two-tailed Mann-Whitney U test. Bars represent mean ± SEM.

[0024] 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. 10B illustrates that RIM1 / 2 (***P = 0.0006, ****P < 0.0001; n = 79-85 cells), Piccolo (**P = 0.0022, ***P = 0.0001; n = 80-85 cells), and Bassoon (*P = 0.0103, ****p < 0.0001; n = 69-92 cells) are restored in axonal growth ones of Smn-deficient motoneurons following expression of Muncl3-1 Rescue construct. 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. 10D: **P = 0.0025, ***p = 0.0003; n = 112-134 cells) and diminished axon length (FIG. 10E: ****p < 0.0001; n = 214-324 cells) following transduction with Rescue virus. One-way ANOVA with Dunn’s post-test.

[0025] 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-ltg / +knock-in mice. For Cre expression, motoneurons were transduced with eFl-Cre virus (FIG. 11 A: **P = 0.004; n = 5 experiments), and cross-bred with (FIG. 11B) ChAT-IRES-Creigl+, or (FIG. 11C) Nestin-Cretg / +mice (n = 2 experiments). FIGs. 11D-11F illustrate that the expression ofR26Uncl3-l rescue allele was assessed by qRT-PCR following LMD in motoneurons within the lumbar spinal cords in R26Uncl3-lt^ Nestin-Crets / +and R26Uncl3-lts / +,ChAT-IRES- Crev° mice (n = 2 experiments in FIG. 1 ID and FIG. 1 IE, and n = 1 experiment in FIG. 1 IF). One-tailed Mann-Whitney U test. Bars represent mean ± SEM.

[0026] 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.

[0027] 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. 13B are graphical representations of results of qRT-PCR indicating reduced mRNA levels of UNC13A in distal axons (left panel) of hiPSC-derived motoneurons from two type I and one type II SMA patients compared to two healthy individuals (*P = 0.0286 for C88iCTR versus C13iSMA and C77iSMA, *P = 0.0143 for C88iCTR versus C84iSMA from n = 3-4 independent experiments). UNC13A mRNA levels are increased in both somatodendritic and axonal compartments in SMA hiPSC-derived motoneurons transduced with Rescue lentivirus expressing UNC13 A+SYP3'UTR (n = 2 replicates / SMA line). 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 Cav2.2. Representative images of SMA hiPSC-derived motoneurons transduced with UNC13A+SYP3TJTR Rescue virus are included in Fig. 14E. FIG. 13D are graphical representations of UNC13A and Cav2.2 protein levels reduced in axonal growth cones of cultured SMA hiPSC-derived motoneurons compared to the control (****p < 0.0001; from n = 4 independent experiments). In SMA hiPSC-derived motoneurons, UNC13A and Cav2.2 levels are restored in axonal growth cones upon expression of UNC13A+SYP3TJTR Rescue construct (from n = 4 independent experiments). One-tailed Mann-Whitney U test in FIG. 13B and One-way ANOVA with Dunn’s post-test in FIG. 13D. Bars represent mean ± SEM.

[0028] 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. 14D isimmunoblots showing increased UNC13A protein levels in total lysates from human control motoneurons transduced with Rescue lentivirus expressing UNC13A+SYP3'UTR (representative of n = 2 experiments). 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).DETAILED DESCRIPTION

[0029] To facilitate the understanding of this disclosure a number of terms of in quotation marks are defined below. It is noted that the drawings of the present application are provided for illustrative purposes only and, as such, the drawings are not drawn to scale.

[0030] In the following description, numerous specific details are set forth, such as particular structures, components, materials, dimensions, processing steps and techniques, in order to provide an understanding of the various embodiments of the present application. However, it will be appreciated by one of ordinary skill in the art that various embodiments of the present application may be practiced without these specific details. In other instances, well-known structures or processing steps have not been described in detail in order to avoid obscuring the present application.

[0031] As used herein, 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. For example, 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.

[0032] As used herein, terms defined in the singular are intended to include those terms defined in the plural and vice versa.

[0033] As used in this specification and its appended claims, terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration, unless the context dictates otherwise. The terminology herein is used to describe specific embodiments of the disclosure, but their usage does not delimit the disclosure, except as outlined in the claims.

[0034] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weights, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present disclosure. At the very least, and without limiting the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters describing the broad scope of the disclosure are approximations, the numerical values in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains standard deviations that necessarily result from the errors found in the numerical value's testing measurements.

[0035] Thus, reference herein to any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range. To illustrate, 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. In a further illustration, 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. In yet another illustration, 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.

[0036] In the discussion and claims herein, 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. For example, for some elements 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.

[0037] 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.

[0038] The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and include any mammalian subject for whom diagnosis, treatment, or therapy is desired.

[0039] A translatable molecule of this disclosure may be used for ameliorating, preventing or treating a disease. In these embodiments, 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. In some aspects, 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. In some embodiments, 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. In some embodiments, the molecules of the present disclosure can be administered through any appropriate route. For example, in some embodiments, 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. In some embodiments, administration may be intratumoral or peritumoral. In some embodiments, administration may involve intermittent dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0040] In a synthetic mRNA construct of this disclosure, 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. In certain embodiments, 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.

[0041] 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.In certain embodiments, the coding sequence for encoding the protein or polypeptide may have a high codon adaptation index.

[0042] 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.

[0043] In some embodiments, 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.

[0044] As used herein, the term “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. 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.

[0045] 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.

[0046] In some embodiments, 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. In some embodiments, a reference mRNA sequence can be a rare disease mRNA.

[0047] In some embodiments, 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.

[0048] In some embodiments, a non-coding polynucleotide template sequence that is transcribable to provide a translatable molecule of this disclosure, when transcribed mayprovide 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.

[0049] In some embodiments, 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.

[0050] In some embodiments, 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. In some embodiments, a reference polypeptide or protein sequence can be a rare disease protein sequence.

[0051] In some embodiments, 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.

[0052] In some embodiments, 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. In some embodiments, the N or C terminal sequence can be a signal sequence or a cellular targeting sequence.

[0053] In some aspects, an mRNA construct of this disclosure can be homologous or heterologous. As used herein, the term “homologous mRNA construct” is a class of expressible polynucleotides, where the sequences of the polynucleotides are derived from a human gene.

[0054] As used herein, the term “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.

[0055] 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 previouslyused in the context of heterologous mRNA constructs, to efficiently produce human proteins, or fragments thereof, in mammalian cells or animals.

[0056] 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.

[0057] In some embodiments, 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.

[0058] 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. In preferred embodiments, AAV based vectors provided herein comprise capsids from one or more of AAV8, AAV9, AAV10, AAV11, AAVrhlO or AAVrh20 serotypes.

[0059] However, other viral vectors may be used, 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.

[0060] This disclosure further encompasses DNA templates for making an mRNA construct above by in vitro transcription.

[0061] This disclosure includes compositions containing an mRNA construct above and a pharmaceutically acceptable carrier. The carrier may comprise a transfection reagent, a nanoparticle, or a liposome. A nanoparticle may include a lipid nanoparticle. In some embodiments, a composition of this disclosure may include lipid nanoparticles comprising a thiocarbamate or carbamate-containing lipid molecule.

[0062] 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.

[0063] 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. 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. As disclosed, the target Muncl3-1 mRNA can be any RNA, whether native or unknown, synthetic or derived from a natural source. In other embodiments, the target RNA can include UNA molecules composed of nucleotides and UNA monomers, and optionally chemically modified nucleotides.

[0064] Muncl3-1 local translation is impaired in SMA

[0065] 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. 12, whereas transcripts for Muncl3-1 are decreased by about 75%, as seen in FIGs. 1A, and IB. Muncl3-lmRNA levels in the somatodendritic compartment appear normal, as seen in FIG. IB, indicating that axonal translocation of this transcript is specifically defective in SMA.

[0066] Thus, Muncl3-1 loss of functions could contribute to the disease pathology by affecting active zone organization and neurotransmitter release in SMA. smFISH with cultured motoneurons was performed, which also revealed diminished localization of Muncl3-1 transcripts in axons as well as axonal growth cones of Smn-deficient motoneurons, as seen in FIGs. 1C-1E, despite normal mRNA levels in the soma, as seen in FIG. 6A. This alteration is associated with reduced protein levels of Muncl3-1 in axonal growth cones in cultured motoneurons, as seen in FIGs. 6B and 6C, as well as in NMJs of TVA muscles from P5 control and SMA mice of the methods and materials section of the present disclosure, as revealed by immunofluorescence analysis, as seen in FIGs.lF-H.

[0067] Muncl3-1 protein levels were not altered in somata, as seen in FIGs. 6D and 6E. Since somata represent the main part of these neurons in culture, total Muncl3-1 protein content also appears unaltered in lysates from Smn-deficient cultured motoneurons as seen in FIG. 6F, indicating that the presynaptic protein and mRNA localization of Muncl3-1 but not the nuclear transcription and processing of the Muncl3-1 transcript are altered in SMA. Next, puromycin proximity ligation analysis (Puro-PLA) was performed to detect Muncl3-1 translation in axonal growth cones of cultured motoneurons in situ, as seen in FIG. II. A decreased Puro-PLA signal in growth cones of Smn-deficient motoneurons was found, as seen in FIGs. 1 J and IK.

[0068] The specificity of the PLA signal was verified in control experiments, where only Muncl3-1 or puromycin antibodies were incubated, as seen in FIG. 6C. 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. Similarly, RNA-IP revealed reduced levels of Muncl3-1 mRNA bound to ribosomes in immunoprecipitated fractions of Smn KO, as shown in FIG. IL. This reduction correlated with the reduced Muncl3-1 mRNA levels in synapses in relation to total Muncl3-1 transcript levels, as shown by the assessment of input fractions, as seen in FIG. IM. This phenotype was not caused by altered levels of ribosomes in synaptic fractions from Smn KO animals, as shown in FIGs. 6H-6J. These data indicate that axonal translocation of Muncl3-1 mRNAs as well as its presynaptic synthesis depend on normal Smn protein levels, indicating that dysregulated local translation of Muncl3-1 could be responsible for the synaptic defects in SMA.

[0069] Axonal localization of Muncl3-1 mRNA depends on its 3'UTR

[0070] 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. In contrast, 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 axonaltargeting 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.

[0071] To validate these constructs, 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.

[0072] To examine whether the Rescue construct with the modified 3'UTR could translocate Muncl3-1 mRNAs into distal axons in the absence of Smn, smFISH was performed with Smn- deficient motoneurons. Muncl3-1 mRNAs were significantly increased in the soma of Smn- deficient motoneurons after transduction with both Rescue and RescueA3'UTR viruses, as seen in FIG. 7A. Nevertheless, the axonal localization of Muncl3-1 mRNAs was restored only in Smn-deficient motoneurons following the expression of Rescue, but not RescueA3'UTR construct, as seen in FIGs. 7B and 7C. 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.

[0073] However, 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.

[0074] Muncl3-1 local translation is crucial for the formation of release sites upon induction of neuronal activity.

[0075] To elucidate the specific role of Muncl3-1 local translation in activity-dependent synaptic modulation, a pulse stimulation was applied with Roscovitine, which is a drug that activates the opening of Cav2.1 / Cav2.2 channels and thus improves neurotransmitter release in Smn deficient motoneurons. Remarkably, following Roscovitine stimulation, a significant increase in Muncl3-1 immunoreactivity was detectable in stimulated control motoneurons, as seen in FIG. 2D, which was impeded upon pretreatment of neurons with anisomycin that blocks local protein synthesis, and not influenced by nocodazole treatment which disrupts microtubule and thus axonal transport of proteins that are synthesized in the somatic compartment, as seen in FIG. 8A.

[0076] Similar to control, stimulation elicited the local axonal translation of Muncl3-1 in KORescuemotoneurons, as seen in FIG. 2D. In contrast, in stimulated KORescueA3'UTRand in Smn- deficient motoneurons, elevated Muncl3-1 levels were not observed, as seen in FIG. 2D. Thus, replacing the 3'UTR domain of Muncl3-1 with the SynPhy3'UTR restores the axonal translocation of the Muncl3-1 mRNA and the local presynaptic synthesis of the corresponding protein in response to stimulation in Smn-depleted motoneurons.

[0077] Next, super-resolution microscopy to assess the dynamic changes in the nanoscale architecture of Muncl3-1 release sites at axonal presynaptic membranes using lattice-SIM was performed. A ring-like Muncl3-1 supram olecular architecture was identified that tightly overlapped with the active zone marker Snap25, as seen in FIG. 2F. In cultured control motoneurons, one or two of such supramolecular assemblies were identified in each presynaptic membrane, which displayed a diameter range between about 170 to about 1250 nm. Strikingly within 5 min stimulation, these supramolecule assemblies underwent a complete rearrangement to form new structures, as seen in FIG. 2G.

[0078] These highly organized supramolecular assemblies are Muncl3-1 containing release sites for synaptic vesicles, since treatment with co-conotoxin (CTX), an inhibitor of Cav2.1 / Cav2.2 channels completely blocked the formation of such supramolecule architectures, as seen in FIG. 8C, with FIG. 8B showing that the that the number of Muncl3-1 releases sites increases significantly in axonal growth cones of stimulated control and KORescue(**P = 0.0068, ***p = 0.0002; n = 21-31 cells), but not in stimulated Smn-deficient and KORescueA3 UTRmotoneurons. Notably, Muncl3-1 release sites were preserved after inhibition of the translation by anisomycin treatment. However, the formation of new release sites in response to stimulation was impeded, as seen in FIG. 8D. These data indicate that Muncl3-1 local translation is required for the formation of such release sites for synaptic vesicles. Moreover,motoneurons cultured on a non-muscle-specific laminin isoform (lamininl l l) did not form such organized release sites indicating that these structures are specific to presynaptic membranes, as seen in FIG. 8E.

[0079] Further, experiments were conducted with mCLING-ATTO647N uptake, a dye specially developed for super-resolution microscopy. Notably, depolarization of presynaptic membranes concomitants with the endocytosis of mCLING at distinct membrane domains that clearly overlapped with Muncl3-1 release sites, as seen in FIG. 8F. Strikingly, similar to control, Roscovitine stimulation triggered a complete, or substantially complete, rearrangement of Muncl3-1 release sites in KORescueSMA motoneurons, leading to increased number of release sites, as seen in FIG. 8F. On the contrary, these dynamic changes were not evident in stimulated-Smn-deficient and KORescueA3 UTRmotoneurons, as seen in FIG. 2G, suggesting that activity-dependent formation of new release sites requires Muncl3-1 local production.

[0080] To gain insights into individual Muncl3-1 nanoassemblies within these supramol ecul ar release sites, structures of cultured motoneurons were expanded by 7.5-fold and axon terminals were imaged with lattice-SIM, as seen in FIG. 3A. Such Muncl3-1 nanoassemblies have been described in dendritic synapses of cultured hippocampal neurons. In unstimulated growth cones, on average, 6 individual Muncl3-1 nanoassemblies were identified within each release site, which exhibited a diameter range between 18 and 92 nm, as seen in FIGs. 3A-3D. The center-to-center distance between nearest neighboring assemblies was 118.8 ± 27.8 nm, as seen in FIG. 3E.

[0081] Significantly, the average number of individual Muncl3-1 nanoassemblies within release sites increased from 6 to 8 upon stimulation, as seen in FIGs. 3A-3C. As shown in Fig. 3D, this coincided with a decrease in the average diameter of Muncl3-1 nanoassemblies from 49.6 nm in unstimulated to 41.9 nm in stimulated neurons. Further, a significant reduction in the center-to-center distance between nearest neighboring assemblies in stimulated neurons, 107 ± 31.7 nm, as seen in FIG. 3E, was noted. Collectively, the super-resolution microscopy data indicates that Muncl3-1 release sites undergo a dynamic rearrangement upon induction of neuronal activity, thus providing evidence of a role of Muncl3-1 in modulation of presynaptic plasticity in motoneurons at the nanoscale.

[0082] Overexpression of Muncl3-1 lacking the 3'UTR domain does not rescue the stimuli- dependent formation of new release sites in Smn-deficient neurons, as seen in FIG. 2G. Likewise, the stimuli-dependent formation of new release sites depends on the proteinsynthesis as shown in FIG. 8C. It appeared that Muncl3-1 becomes translated at ribosomes directly at release sites and becomes immediately recruited into such new presynaptic release sites.

[0083] Interestingly, upon stimulation, in control motoneurons, the colocalization between Muncl3-1 and ribosomal marker RPL8 increased within the release sites, as seen in FIG. 3F, which was accompanied with enhanced colocalization with Snap25, as seen in FIGS. 9A and 9B. Motoneurons pretreated with anisomycin did not exhibit increased colocalization between Muncl3-1 and ribosomes, as seen in FIG. 9C. Remarkably, the colocalization between Muncl3-1 and ribosomes as well as Snap25 increased significantly also in stimulated-KORescueneurons. Smn-deficient and KORescueA3 UTRmotoneurons failed to respond to the stimulation, as revealed by unaltered Muncl3-l / ribosomes and Muncl3-1 / Snap25 colocalization, as seen in FIG. 3F, FIG. 9 A and FIG. 9B.

[0084] These data indicate a novel function for the locally translated Muncl3-1 in the modulation of presynaptic release sites and plasticity and the relevance of this mechanism for the disease pathology in SMA.Muncl3-1 local translation rescues neuronal excitability and recovers motor function in SMA.

[0085] 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 KORescue, but not in KORescueA3 UTRwas measured, indicating that locally translated Muncl3-1 is needed for neurotransmitter release at presynaptic membranes, as seen in FIG. 4A.

[0086] Treatment of neurons with CTX and TTX completely, or substantially completely, blocked the Synaptotagminl antibody uptake in KORescuemotoneurons, as seen in FIG. 10 A. Consistent with this, restoring the Muncl3-1 local translation improved the presynaptic assembly of AZ markers including RIM1 / 2, Piccolo, and Bassoon, as seen in FIG. 10B, and rescued the axon growth and synapse differentiation defects in cultured Smn-deficient motoneurons, as seen in FIGs. 10C-10E.

[0087] Interestingly, expression of both Rescue and RescueA3'UTR ameliorated clustering of Cav2.2 channels, leading to increased spontaneous Ca2+transients, in growth cones of Smn-deficient motoneurons, as shown by Ca2+imaging, as seen in FIGs. 4B-4D. Nevertheless, the lower amplitude of spontaneous Ca2+spikes in KORescueA3 UTRneurons suggests that only local Ca2+transients, but not action potential-dependent transients are restored, as seen in FIG. 4E. This was further confirmed by measuring the evoked response after depolarization with 90 mM KC1, which revealed a prevalent failure response to depolarization in Smn-deficient and QRescueA3'UTRneurons,as seenjnFIGS4F-4H. Based on these data, impaired excitability and neurotransmitter release in SMA are caused by impaired local translation of Muncl3-1.

[0088] To establish whether restoring Muncl3-1 local translation could rescue motor defects in SMA mice in vivo, a Cre / loxP conditional Muncl3-1 knock-in rescue mouse model was generated. The rescue cassette harboring a conditional Muncl3-l+SynPhy3'UTR cassette, as seen in FIG. 5 A, was inserted into the ROSA26 locus. Due to the presence of loxP-flanked stop sequences, SMA mice were cross-bred with conditional Muncl3-1 knock-in as well as a Nestin-Cre driver line. The selection of Nestin-Cre was based on the observation that Smn loss of function in all neurons and glia affects neuromuscular transmission, thereby leading to animal death.

[0089] As shown in FIG. 5B, the expression of R26Uncl3-l rescue allele increases with age in cortical tissues from Muncl3-1 knock-in mice cross-bred with Nestin-Cre. Muncl3-1 knock-in mice were also cross-bred with MundS-M mice and prepared cultured motoneurons from E13.5 embryos. The R26Uncl 3-1 -derived transgenic protein was detected by Western blot in lysates of Mund 3- 1 ^ motoneurons transduced with a Cre-expressing AAV, as seen in FIG. 5C. Smn ^ ,Hung" ,R26Und 3-ltg / +,Cretg / +animals displayed diminished synapse degeneration of NMJs in vulnerable TVA muscles, as seen in FIGs. 5D and 5E, which correlated with an upregulation of Muncl3-1 and Cav2.1 in NMJs, as seen in FIGs. 5F and 5G.

[0090] In addition, improved motor function and attenuated muscle weakness, as determined by time to right, as seen in FIG. 5H, and muscle strength measurements of forelimbs and hind limbs, as seen in FIGs. 5I-5K, was determined. Also, the survival of these animals was increased, as seen in FIGs. 5L-5M. Additionally, the Smn^ ,Hung" ,R26Und 3- ltg / +,Cretg / +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. Thus, 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

[0091] The importance of UNC13A synaptic function on SMA pathophysiology was investigated, including the role of SMN in axonal translocation and translation of UNC13A in human induced pluripotent stem cell-derived (hiPSC) motoneurons from two type I and one type II SMA patients (FIGs. 14, A-C). To this end, hiPSC-derived motoneurons were cultured in compartmentalized chambers and the mRNA levels of UNC13A in axonal compartments were analyzed by qRT-PCR (FIG. 13A). Importantly, marked reduction in UNC13A mRNA levels in axons of cultured hiPSC-derived motoneurons was present in all three SMA patient lines, while UNC13A mRNA levels were not significantly altered in the somata (FIG. 13B). Then, a rescue lentivirus construct expressing the human UNC13A coding region fused to the 3'UTR of human Synaptophysinl mRNA (UNC13A+SYP3'UTR) was generated. Western blot analysis revealed increased UNC13A expression in total lysates of transduced cultured hiPSC- derived motoneurons (FIG. 14D). In agreement with data with SMA mice, transduction of SMA hiPSC-derived motoneurons with the UNCI 3 A Rescue lentivirus restored the attenuated translocation of UNC13A mRNA in axons (FIG. 13B).

[0092] Next, the protein levels of UNC13A as well as Cav2.2 in axonal growth cones of cultured SMA hiPSC-derived motoneurons was assessed (FIG. 13C). As illustrated in FIG. 13D, SMA hiPSC-derived motoneurons showed reduced levels of UNC13A and Cav2.2, which is consistent with SMA mice. Similarly, expression of the UNC13A Rescue construct resulted in increased levels of both UNC13A and Cav2.2 in the axonal growth cones in cultured SMA hiPSC-derived motoneurons (FIG. 13D and FIG. 14E).

[0093] Collectively, these data indicate that similar to the mouse model, in human motoneurons, axonal mRNA transport and local translation of UNC13A depend on SMN and are diminished in SMA.

[0094] The role of locally translated proteins in activity-dependent modification of synaptic plasticity as well as their contribution to synapse degeneration is being studied. Loss of dendritic synthesis of calcium / calmodulin-dependent protein kinase II a (CaMIIKa) abolishes the induction of long-term plasticity (LTP), thereby affecting learning and memory. Previous studies have focused on the role of de novo synthesized proteins in dendritic presynapticfunctions and postsynaptic plasticity, elucidating the specific function of locally translated proteins, in particular, AZ components, at axonal presynapses remains challenging.

[0095] 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. In most synapses, 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.

[0096] In cultured hippocampal neurons, 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. Here, using 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.

[0097] Pharmacological induction of neuronal excitability shows neuroprotective effects on ALS hiPSC-derived motoneurons. In SMA mice, treatment of animals with a potassium channel blocker that increases neuronal activity improves motor function, and treatment with Roscovitine that enhances Ca2+influx and transmitter release beneficially affects survival of Smn deficient mice. Polymorphisms in the UNC13A gene have been identified as survival modifiers in sporadic ALS and FTD patients. Cellular mechanisms by which single nucleotide polymorphisms (SNPs) in UNC13A increase the risk for ALS and FTD involve inclusion of a cryptic exon in UNC13A that leads to mis-splicing of corresponding transcripts and subsequent degradation as a result of Tdp-43 loss of function.

[0098] In the present disclosure it is shown that Muncl3-1 axonal mRNA localization and its presynaptic translation are dysregulated in SMA, 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. SMA mice crossbred with a conditional Muncl3-1 knock-in construct expressing the Smn-independent locally translatable Muncl3-1 display improved motor functions, ameliorated NMJ pathology, and increasedsurvival. 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. 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 local translation and treatment of Alzheimer’s Disease

[0099] 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.

[0100] 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. Also, 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.

[0101] 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.

[0102] Additionally, 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.

[0103] 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 newpromising 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.

[0104] Vector Systems

[0105] Many 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.

[0106] Methods for contacting cells, e.g., cells in culture or cells in a human animal or nonhuman animal, e.g., mouse, with nucleic acid vectors, such as electroporation, calcium chloride transfection, and lipofection, are well known in the art. Alternatively, 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. In other words, 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. Commonly used 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. To generate viral particles including nucleic acids of interest, 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, e.g., MMLV, are capable of infecting most murine and rat cell types, and are generated by using ecotropic packaging cell. Retroviruses bearing amphotropicenvelope 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.

[0107] 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. 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. By 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. In addition, 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.Materials and Methods

[0108] To generate these results herein, the following materials and methods were used.Animals

[0109] Laboratory mice were housed in the animal facility of the Institute of Clinical Neurobiology at the University Hospital Wuerzburg following the regulations on animal protection of the German federal law and of the Association for Assessment and Accreditation of Laboratory Animal care. SMA litters, Smtr ,Hiing':’ . (referred to as Smn-deficient or Smn KO in the text), and control litters, Smn ,Hung:’ . (referred to as control in the text) were offspring of two mouse strains (I) Smn+Athat is hemizygote for the SmntmIHungtargeted mutation, and (II) Smn ^ ,Hurig" '" that is homozygote for the SmntmIHungtargeted 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-ltg / +knock-in mouse was designed and cloned by M. M. and generated at Czech Centre for Phenogenomicsin 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.Primary mouse motoneuron culture and viral transduction

[0110] Primary mouse motoneuron culture was applied as previously described. Motoneurons were isolated from E13.5 mouse embryos, enriched via p75NTRantibody 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. For immunofluorescence, SmFISH, and ExM, motoneurons were plated onto glass coverslips, for Western blot and qRT-PCR onto 24-well plates, for Ca2+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.R-Roscovitine stimulation and immunocytochemistry

[0111] For stimulation experiments, 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. Following stimulation, 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. After three times washing with TBST, secondary antibodies diluted 1 :500 in PBS were added and incubated for 1 hour at RT. Coverslips were embedded in Aqua Poly / Mount (Polysciences, 18606-20). For P-actin (ActP) immunostaining, motoneurons were first exposed to ice-cold methanol for 5 min at -20 °C and then permeabilized with 0.1% Triton X-100 for 5 min at RT. In control experiments, prior to as well as during Roscovitine stimulation, cells were treated with 10 pM nocodazole for 2 hours, or 100 ng / mL anisomycin for 1 hour in order to inhibit axonal transport and local translation, respectively. For the mCLING labeling assay, 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 wasquenched 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. Following primary antibodies were used: 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-Ca2+channel N-type alpha-lB (Cav2.2) (Synaptic System, 152305, 1 :250), mouse monoclonal anti-P-actin (GeneTex, GTX26276, 1 : 1000), goat polyclonal anti-ribosomal protein L8 (RPL8) (Sigma- Aldrich, SAB2500882, 1 :500), guinea pig monoclonal recombinant IgG anti-Snap25 (Synaptic systems, 111308, 1 :250), and guinea pig polyclonal antiserum anti -Synapsinl / 2 (Synaptic systems, 111308, 1 :500). Secondary antibodies are as followed: donkey anti-mouse IgG (H+L) (Alexa Fluor 488, Jackson ImmunoResearch, 715-545-150), donkey anti-rabbit IgG (H+L) AffiniPure (Alexa Fluor 488, Jackson ImmunoResearch, 711-545-152), donkey anti -rabbit IgG (H+L) AffiniPure (Cy3, Jackson ImmunoResearch, 711-165-152), and donkey anti-guinea pig IgG (H+L) AffiniPure (Cy5, Jackson ImmunoResearch, 706-175-148).Single molecule fluorescence in situ hybridization (smFISH)

[0112] 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. For the amplification of FISH signal, preamplifier, amplifier, and label probe oligonucleotides (diluted 1 :25 in respective amplification buffers) were incubated each for 1 hour at 40 °C. After the washing steps, cells were immunostained against Tau for visualization of the neurite boundaries.Immunohistochemistry

[0113] 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 mMCaCh, 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. 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. 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). For immunofluorescence staining of motoneurons within the spinal cord, 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. The following primary and secondary antibodies were used: guinea pig polyclonal anti-Synaptophysinl (Synaptic Systems, 101004, 1 : 1000), rabbit polyclonal anti-Ca2+channel P / Q-type alpha-1 A (Cav2.1) (Synaptic Systems, 152203, 1 :500), rabbit polyclonal anti-Muncl3-l (Synaptic System, 126103, 1 :500), chicken polyclonal anti-Neurofilament H (Merck, AB5539, 1 : 1000), goat anti-CholineAcetyltransferase (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).Cloning and generation of Muncl3-1 rescue constructs and virus production

[0114] For cloning of mouse Muncl3-1 and human UNC13A lentivirus rescue constructs, 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 ofMuncl3-1 using NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs, E5520S) and inserted into a lentivirus backbone vector with the Ubiquitin promotor. For RescueA3'UTR, only the coding region of Muncl3-1 was inserted into the lentivirus backbone vector. 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 HEK293Tcells using TransIT-293 (Minis, MIR2706) for transfection. For lentivirus packaging, pCMV-VSVG and pCMVAR8.91 helper plasmids, 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 NSC34cells using standard methods with serial dilutions.Generation of Cre / loxP conditional Muncl3-1 rescue mouse model

[0115] The coding sequence of 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 HEK293cells, which were transfected with the vector expressing the targeting cassette and an eFl-Cre expressing vector. For the generation of R26Uncl 3 -ltg / +knock-in mice, 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‘-CTCTGCTGTGTCTGTGACGT-3‘, and for ROSA26 wt allele: ROSA-reverse: 5’- GGCTC AGTTGGGCTGTTTTG-3 ’ .Ca2+imaging and data quantification

[0116] Ca2+imaging was performed using the calcium indicator Oregon Green™ 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 Ca2+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 Ca2+indicator diluted in the Ca2+imaging buffer for 15 min at 37 °C in a CO2 incubator. Cells were washed again twice with Ca2+imaging buffer and imaged in 2 ml of Ca2+imaging buffer in the presence of 3 ng / ml BDNF. For time-lapse imaging, 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 Ca2+spikes and over 2 min for pulse experiments. 16-bit images of 1.024 x 1.024-pixel resolution were acquired with a 2 x 2 binning. For pulse experiments, 10 pl of 90 mM KC1 was applied to the imaging cell at 1 min post-imaging. For the quantification of Ca2+spikes, first, a region of interest (ROI) was defined within growth cones using Fiji. Next, intensity values were generated from all time-lapse frames using dynamic Z-axis profile. For spontaneous Ca2+spikes, the average of the first 20 frames was considered F0, and in pulse experiments, the average of the first 10 frames immediately before KC1 application was considered F0. For data normalization, all intensity values were divided to F0 and plotted (F / F0). BAR Plugin was used for counting the Ca2+spikes.Synaptic vesicle recycling assay

[0117] To investigate the synaptic vesicle recycling in motoneurons, a Cy 3 -conjugated monoclonal antibody directed against the intravesicular domain of Synaptotagmin 1 (Synaptic Systems, 105103C3) was used. DIV5 cultured motoneurons were incubated with the antibody overnight (diluted 1 :400 in the cell culture medium) in a cell culture incubator. On the next day, cells were washed twice with pre-warmed PBS and fixed with 4% PFA for 5 min at RT. Neurons were imaged using a standard confocal microscope. In control experiments, cells were cultured for 6 days in presence of 30 nM CTX (a selective blocker of Cav2.1 / 2.2), as well as60 nM TTX (a selective blocker of voltage-gated Na+channels) and fed with Synaptotagminl antibody afterward.Puromycin proximity ligation assay (Puro-PLA)

[0118] 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. Cells were then permeabilized with 0.2% Triton X-100 for 10 min at RT and incubated with Duolink blocking solution for 1 hour at 37 °C. PLA assay was conducted using Duolink® In Situ Detection Reagents Orange (Sigma-Aldrich, DU092007). Primary antibodies, rabbit polyclonal anti-Muncl3-l (Synaptic System, 126103, 1 :500) and mouse monoclonal anti-puromycin (Merck Millipore, MABE343, 1 : 1000) were diluted in the Duolink antibody diluent and incubated with cells for 1 hour at RT. Cells were washed first several times and then 2 x 5 min with 1 x Wash Buffer A (Sigma-Aldrich, DUO82049) at RT. PLA probes; anti -mouse MINUS (DU092004) and anti-rabbit PLUS (DU092002); were diluted 1 :50 in Duolink antibody diluent and incubated with cells for 1 hour at 37 °C. After multiple washing steps with 1 x Wash Buffer A, ligase (diluted 1 :40 in ligation buffer) was added to the cells and incubated for 30 min at 37 °C. Cells were washed again several times with 1 x Wash Buffer A and incubated with polymerase (diluted 1 :80 in amplification buffer) for 100 min at 37 °C. The amplification step was stopped by 2 x 10 min wash at RT with 1 x Wash Buffer B (Sigma-Aldrich, DUO82049). Finally, cells were washed for 1 min with 0.01 x Wash Buffer B and mounted using Duolink mounting medium (Sigma-Aldrich, DU082040) and sealed using nail polish. All the incubation steps at 37 °C were carried out in a dark / humid chamber using a dry incubator (Binder BD 23).Cortical synaptosome fractionation and RNA immunoprecipitation (RNA-IP)

[0119] Crude synaptosome fractions were prepared from cortices of P5 Sniri ,Hurig" and Smn ^ ,Hiin '" mice by sequential centrifugation steps in a sucrose buffer (0.32 M sucrose, 5 mM HEPES, 1 x protease inhibitor cocktail (Roche)) as described herein. In brief, cortices were isolated and mechanically homogenized in 500 pl cold lysis buffer and centrifuged at 1000 x g for 10 min at 4 °C. Pellets (Pl) containing the nuclei were discarded and supernatants (SI) were centrifuged at 12000 x g for 20 min at 4 °C. Resulting supernatants (S2) containingthe 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. To prevent the disassembly of ribosomal 80S complexes with their bound transcripts, 100 pg / ml cycloheximide was added into the fractionation buffer as well as into the IP buffer for all the sequential IP steps. For the pulldown of 80S ribosome complexes, a mouse monoclonal rRNA (Y10B) antibody (ThermoFisher Scientific, MAI-16628) and normal mouse IgG control (Santa Cruz Biotechnology, sc-2025) were used. First, 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. For qRT-PCR, 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. To determine ribosome levels in the input and IP fractions, proteins were eluted with 1 x Laemmli buffer (125 mM Tris, pH 6.8, 10% SDS, 50% glycerol, 25% P-mercaptoethanol, and 0.2% bromophenol blue) and subsequently analyzed by Western blot.Differentiation of human induced pluripotent stem cells into motoneurons

[0120] To generate the results shown in FIGs. 13A-13D and FIGs. 14A-14E, 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). For the induction of Embryoid Bodies (EBs), 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, 3pM), and Purmorphamine (PMA) (Cayman Chemical Company, 10009634, 0.5 pM). On day 2, 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. On day 4, 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)). For induction of Neuronal Progenitor Cells (NPCs), EBs were collected from the suspension on the day 6 and plated on Matrigel-coated dishes. In order to achieve pure NPC cultures, cells were splitted for at least 20 passages once a week using Accutase (Thermo Fisher, 07920). For 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). For motoneuron differentiation, on day 9, NPCs were harvested and plated on polyornithine / laminin211 / 221 -coated coverslips or microfluidic chambers. 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.Quantitative RT-PCR (qRT-PCR)

[0121] 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:RatioFollowing primers were used for qRT-PCR: mouse Gapdh (forward) 5’-AACTCCCACTCTTCCACCTTC-3’ and (reverse) 5’-GGTCCAGGGTTTCTTACTCCTT- 3’, mouse Muncl3-1 coding region (forward) 5’-CACCACGCCCACCTACTGCTA-3’ and (reverse) 5’-TTGCGCTCGCGGATCT-3', mouse 18srRNA (forward) 5’-CGCGGTTCTATTTTGTTGGT-3’ and (reverse) 5’-AGTCGGCATCGTTTATGGTC-3’, mouse Muncl 3- l+SynPhy3'UTR (forward) 5’-GGTGCTGCAACTGCGAGA-3’ and (reverse) 5’-TCACTGACCAGACTAGGGCG-3’, human GAPDH (forward) 5’-GCAAATTCCATGGC ACC-3’ and (reverse) 5’-CGCCAGTGGACTCCACGAC-3’, humanUNC13A (forward) 5’- GGACGTGTGGT AC AACCTGG-3 ’ and (reverse) 5’-GTGTACTGGACATGGTACGGG-3Western blotting

[0122] For Western blotting with motoneurons, 200,000 cells were plated and grown for 7 days. After a short wash with prewarmed PBS, cells were lysed in 1 * Laemmli buffer, lysates were boiled at 99°C for 5 min and centrifuged briefly. Protein extracts were loaded onto 4-12% gradient SDS-PAGE gels and blotted onto PVDF membranes. For Western blot with the IP obtained from synaptosome fractions, equal volumes of protein extracts from input, IP, and IgG fractions were loaded. Calnexin was used as loading control. Primary antibodies were incubated overnight at 4 °C, washed the next day and secondary antibodies were incubated for 1 hour at RT. 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).Motor assessments

[0123] All motor tests were carried out with P10 mice. For the righting reflex, 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 ascore 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.Lattice-SIM

[0124] For lattice-SIM, 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. Afterwards, 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. Thereafter the following secondary antibodies were incubated for 1 hour at RT in blocking solution: Alexa Fluor 647 donkey F(ab’)2 anti-rabbit IgG (H+L) (1 :300, abl81347, Abeam), Alexa Fluor 488 donkey anti guinea pig (1 :300, 140967, Dianova) and CF568 donkey anti-goat IgG (H+L) (1 :500, 20106, Biotium). The samples were post-fixed for 15 min at RT with 4% PF A and washed 3 times with PBS.Expansion microscopy (ExM)

[0125] 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. Afterwards, 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). On the next day, after 3 x 15 min washing steps with 0.1% PBST, gels were incubated for 3 hours at 37 °C with the following secondary antibodies in 5% BSA in PBS: Alexa Fluor 647 donkey F(ab’)2 anti-rabbit IgG (H+L) (1 : 100, abl81347, Abeam), CF568 donkey anti-goat IgG (H+L) (1 : 100, 20106, Biotium), CF568 goat anti-mouse IgG(H+L) (1 : 100, SAB4600312, Sigma Aldrich) and FluoTag-X4 anti-rabbit IgG ATTO643 (1 : 100, N2404, Nanotag). Gels were washed again 3 * for 15 min with 0.1% PBST and treated with Alexa Fluor 488 NHS (20 pg / ml, Ih / RT, A20000, ThermoFisher) in 100 mM NaHCO3. The nuclei were stained by adding Hoechst 34580 (1 :500, H21486, ThermoFisher) to the pan staining for 25 min at RT. For expansion, gels were placed in petri dishes. Water was exchanged every 20 min until the gel reached its final expansion factor. For imaging, gels were transferred to Lab-TecTM chambers coated with PDL.Image acquisition and processing

[0126] For 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. For lattice-SIM, five z-stack images of 110 nm were taken, and maximum projection images were shown as representative images. For Lattice-SIM and ExM, 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. For proper overlay of the different channels, a channel alignment was performed on the reconstructed images using fiducial markers (TetraSpeck™ 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.Data analysis

[0127] For the quantification of immunofluorescence signals in growth cones and somata, mean gray values of unprocessed raw images were measured using Fiji following background subtraction. For the quantification of immunofluorescence signals in neuromuscular junctions (NMJs), first average projections of multiple z-stacks images were created, and then mean gray values were measured within SynPhy positive area. Signal intensities of SynPhy were comparable between Smn KO and control and were therefore used for normalization of Muncl3-1 intensities. For the quantification of smFISH and PLA signal, the number of dots was calculated within somata or axonal growth cones using Analyze Particle Plugin of Fiji. 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.Statistical analysis

[0128] GraphPad Prism 9 was used for graph illustration and statistical analyses. Data are depicted as bar graphs, with error bars representing mean ± SEM, or as violin plots, with median indicated as dashed lines. To determine the statistical significance between two groups Mann Whitney t-test, and between multiple groups one-way analysis of variance (ANOVA) Kruskal -Wallis test with Dunn’s Multiple Comparison post-hoc test were used. Data were obtained from at least three independent experiments except otherwise stated.

[0129] This detailed description is to be understood as being in every respect illustrative and exemplary, but not restrictive, and the scope of the inventive concept disclosed herein should be interpreted according to the full breadth permitted by the patent laws. It is to be understood that the embodiments shown and described herein are only illustrative of the principles of the inventive concept and that various modifications may be implemented by those skilled in the art without departing from the scope and spirit of the inventive concept. Those skilled in the art could implement various other feature combinations without departing from the scope and spirit of the inventive concept.

Claims

CLAIMS1. A mammalian mRNA expression construct, the mammalian mRNA expression construct comprising: a vector system comprising a modified Muncl3-1 molecule.

2. The method of claim 1, wherein the modified Muncl3-1 molecule is selected from the group consisting of (i) a construct that harbors a coding and a 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors a coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and combinations thereof.

3. The method of claim 1, wherein the vector system is a lentiviral vector system.

4. A method of modifying a defective Munl3-1 axonal localization, the method comprising administering a vector system comprising a modified Muncl3-1 molecule.

5. The method of claim 4, wherein the modified Muncl3-1 molecule is selected from the group consisting of (i) a construct that harbors a coding and a 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors a coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and combinations thereof.

6. The method of claim 4, wherein the vector system is a lentiviral vector system.

7. A method of increasing synaptic function in a subject, the method comprising administering a vector system comprising a modified Muncl3-1 molecule.

8. The method of claim 7, wherein the modified Muncl3-1 molecule is selected from the group consisting of (i) a construct that harbors a coding and a 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors a coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and combinations thereof.

9. The method of claim 7, wherein the vector system is a lentiviral vector system.

10. A method of treating a patient diagnosed with spinal muscular atrophy (SMA), the method comprising:administering to a subject in need thereof an effective amount of a vector system comprising a modified Muncl3-1 molecule.

11. The method of claim 10, wherein the modified Muncl3-1 molecule is selected from the group consisting of (i) a construct that harbors a coding and a 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors a coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and combinations thereof.

12. The method of claim 10, wherein the vector system is a lentiviral vector system.

13. A method of treating a patient diagnosed with Alzheimer’s disease, the method comprising: administering to a subject in need thereof an effective amount of a vector system comprising a modified Muncl3-1 molecule.

14. The method of claim 13, wherein the modified Muncl3-1 molecule is selected from the group consisting of (i) a construct that harbors a coding and a 3'UTR sequences of Muncl3-1 (Smn-dependent), (ii) a construct that harbors a coding region of Muncl3-1 fused to the 3'UTR of SynPhy (Rescue), and combinations thereof.

15. The method of claim 13, wherein the vector system is a lentiviral vector system.

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