Treatment of spinal muscular atrophy (SMA) and other motor neuron diseases by causing presynaptic expression of a hybrid molecule of munc13-1 and synaptophysin3'utr to enhance munc13-1 expression

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

TREATMENT OF SPINAL MUSCULAR ATROPHY (SMA) AND OTHER MOTOR NEURON DISEASES BY CAUSING PRESYNAPTIC EXPRESSION OF A HYBRIDMOLECULE OF MUNC13-1 AND SYNAPTOPHYSIN3’UTR TO ENHANCEMUNC13-1 EXPRESSION

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 516,846, filed July 31, 2023; the disclosure of which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Defective synaptic transmission and synapse degeneration of neuromuscular junctions (NMJs) are associated with diverse neurological diseases such as Amyotrophic lateral sclerosis (ALS) and Spinal muscular atrophy (SMA).

[0003] 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] 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.

[0005] 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. Smn Knockout mice exhibit atrophic and smaller synapses, which associate with impaired neurotransmitter release, disturbed clustering of voltage-gated Ca2+channels (VGCCs), and reduced evoked postsynaptic potential at neuromuscular endplates. In presynaptic terminals, Ca2+entry through VGCCs elicits neurotransmitter release from the active zone (AZ).

[0006] 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.

[0007] Muncl3-1 plays an important role in the synaptic vesicle (SV) docking, priming, and release from the active zones, recruits VGCCs to the presynaptic membrane, and regulates the synaptic plasticity. Loss of Muncl3-1 function arrests both spontaneous and evoked synaptic release events in hippocampal neurons as well as in neuromuscular junctions, and causes severe paralysis that ultimately leads to early postnatal death in knockout mice. In humans, mutations in the Muncl3-1 gene cause microcephaly, cortical hyperexcitability, and fatal myasthenia.

[0008] The synaptic defects in SMA resemble the phenotype of Muncl3-1 knockout mice. Induction of neuronal excitability has been shown to alter cellular transcriptome leading to improved survival in ALS and SMA. Thus, increasing the Muncl3-1 levels in presynaptic nerve terminals might beneficially affect the active zone organization and neurotransmitter release in SMA patients.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] 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.

[0011] Embodiments of the disclosure include mRNA constructs containing one or more 5' UTR sequences along with one or more 3' UTR sequences.

[0012] This disclosure provides a range of mRNA constructs, each of which can produce a protein of interest, or one or more fragments thereof. The protein of interest can be any protein, natural, non-natural, or synthetic. In some embodiments, the protein of interest can be a human protein. In further embodiments, the protein may be a fusion protein, or a chimeric protein. In additional embodiments, the protein may be a globular protein, a fibrous protein, a membrane protein, or a disordered protein.

[0013] In certain embodiments, this disclosure includes a heterologous mRNA construct designed to produce a human protein, or one or more fragments thereof, in mammalian cells.

[0014] 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

[0015] The patent or application file contains a set of drawings 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.

[0016] FIGs. 1A-1C are schematics of different viral constructs expressing Muncl3-1. FIG. 1A is directed to the viral construct harboring the Muncl3-1 coding region fused to the endogenous Muncl3-1 3'UTR, which depends on the Smn protein and is deficient in SMA for the axonal localization. FIG. IB is directed to the viral construct harboring the Muncl3-1 coding region fused to the Synaptophysin 3'UTR, which localizes Muncl3-1 mRNAs in axons independent of the Smn protein. FIG. 1C is directed to the viral construct harboring the Muncl3-1 coding region lacking the axonal localization sequences. RNA was extracted from axon as well as somatodendritic compartments of WT motoneurons transduced with these viral constructs and mRNA levels of Muncl3-1 were analyzed by qRT-PCR. Graphs below each of FIGs. 1A-1C illustrate relative expression of Muncl3-1 mRNAs in axon and somatodendritic compartments.

[0017] FIG. 2A is a schematic of the Cre / loxP cassette expressing the conditional Muncl3- 1 with modified 3'UTR inserted into the mouse ROSA26 locus. FIG. 2B is a schematic of SMA mice expressing NestinCre bred with Muncl3-1 knockin rescue mice (R26Uncl3-ltg / +).

[0018] FIGs. 3A-E illustrate spontaneous and evoked Ca2+transients being elevated in growth cones from cultured motoneurons from Smn-deficient mice expressing Muncl3-1 rescue allele which is modified at the 3'UTR. In FIGs. 3A-3C, images of both frequency and amplitude of the spontaneous Ca2+transients are significantly increased in axonal growth cones of the Smn KORescuemotoneurons expressing Muncl3-1 with the modified 3'UTR. However, Smn-deficient motoneurons expressing Muncl3-1 viral construct, which lacks the axonal targeting sequences (FIG. 1C), show reduced spontaneous Ca2+transients, a similar phenotype to Smn KO motoneurons. In FIGs. 3D and 3E, a 90 mM KCI pulse was applied directly to the growth cones during the Ca2+imaging recording to measure the evoked Ca2+transients, maximum response to the depolarization is increased in the growth cones of Smn KORescuecompared to the Smn KO.

[0019] FIGs. 4A-4C are images and illustrations of overexpression of Muncl3-1 harboring Synaptophysin 3'UTR rescues axonal growth and synapse differentiation defects in SMA. Smn-deficient motoneurons grown on laminin221 / 211 display increased growth cone size areshown in FIGs. 4A and 4B, with restored axon length shown in FIG. 4C after transduction with the virus expressing Muncl3-1 with the modified 3'UTR (KORescue).

[0020] FIG. 5A are images and an illustration of the percentage of the fully innervated neuromuscular junctions (NMJs) is increased in the transverse abdominal muscle (TV A) of SMAmice expressing the Muncl 3-1 rescue allele. FIG. 5B is an illustration of Smn' , Hungtg / +, R26Unc 13-ltg / +, Cretg / +mice show improved muscle weakness and motor functions determined by time to right (FIG. 5B), grip test of the forelimbs (FIG. 5C and 5D), and the hindlimbs (FIG. 5C and 5E). Survival of SMA mice expressing the Muncl3-1 rescue allele is increased by 6 days (FIG. 5F and 5G).

[0021] FIG. 6 includes illustrations of a proposed mechanism of the disclosure. Muncl3-1 with the modified 3'UTR (Muncl 3-1 Rescue mRNA) becomes translated locally at the endplate-presynapses in the absence of SMN. Introduction of the Muncl3-1 modified molecule could rescue the synaptic transmission defects and ameliorate the muscle weakness in SMA mouse models and patients.DETAILED DESCRIPTION

[0022] Reduced mRNA levels of the "Muncl3-1" occurs in presynaptic terminals of embryonic mouse motoneurons with Smn deficiency. Deficiency of Muncl3-1 at presynaptic terminals is a main driver of disease pathology in Spinal muscular atrophy (SMA), and restoration of Muncl3-1 could be an effective new treatment of SMA.

[0023] Muncl3-1 is the master regulator of synaptic transmission and plasticity, and also a survival modifier of sporadic ALS patients. Pharmacological induction of neuronal excitability has shown neuroprotective effects on ALS hiPSC-derived motoneurons as well as in SMA mice.

[0024] In the present disclosure, a new viral construct of the Muncl3-1 with a modified 3'UTR was generated that delivers Muncl3-1 mRNAs into neuronal axons in the absence of Smn. Cell culture experiments showed that restoring the mRNA levels of Muncl3-1 in nerve presynaptic terminals of SMA mice leads to an increased intra-axonal synthesis of Muncl3-1, thereby normalizing pathological defects in Smn depleted cultured neurons. SMA mice bred with a knockin rescue mouse model expressing Muncl3-1 with the modified 3'UTR showed improved motor functions, attenuated neuromuscular endplate (neuromuscular junction (NMJ)) degeneration, and increased survival. Thus, this new modified viral construct could offer a new therapeutic approach for SMA patients and / or ALS patients.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] As used herein, terms defined in the singular are intended to include those terms defined in the plural and vice versa.

[0029] 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.

[0030] 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 desired properties 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The terms “individual,” “subject,” “host,” and “patient,” are used interchangeably herein and include any mammalian subject for whom diagnosis, treatment, or therapy is desired.

[0035] 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 ofthe present disclosure can be administered through any modality of administration. 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 intravenous, subcutaneous, pulmonary, intramuscular, intraperitoneal, dermal, oral, inhalation or nasal administration.

[0036] As used herein, the term “subj ect’ ’ refers to human and non-human animals. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be a mammal. A subject may be a primate, including non-human primates and humans.

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] This disclosure provides methods and compositions for novel molecules having the ability to be translated, which can be used to provide one or more active polypeptides and proteins, or fragments thereof. Embodiments of the disclosure can be directed to mRNA constructs comprising 5'UTR sequences in combination with 3 'UTR sequences, not previously used in the context of heterologous mRNA constructs, to efficiently produce human proteins, or fragments thereof, in mammalian cells or animals.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] This disclosure further encompasses DNA templates for making an mRNA construct above by in vitro transcription.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] As seen in FIGs. 1A-1C, this mechanism has been generated by cell culture models with new lentivirus constructs that allow the transport of Muncl3-1 mRNAs into axonal nerve terminals of motoneurons in an SMN-independent manner. These viral constructs were generated based on the observation that while mRNA levels of the Muncl3-1 weredramatically reduced in presynapses of Smn-depleted motoneurons, the mRNA levels of the synaptophysin were elevated. Hence, fusion of the Muncl3-1 coding region to the 3'UTR of Synaptophysin will target Muncl3-1 mRNAs into axons in the absence of SMN protein.

[0062] To validate the cell culture data with Smn-deficient motoneurons, a new Cre / loxP conditional Muncl3-1 knockin rescue mouse model (R26Uncl3-l 19'+) was generated, wherein a cassette harboring the modified Muncl3-1 with Synaptophysin3'UTR was inserted into the ROSA26 locus on the mouse chromosome 6 through the CRISPR-Cas9 technology. This insertion is illustrated in FIG. 2A.

[0063] Due to the presence of loxP-flanked stop sequences, the SMA mouse model expressing the conditional Muncl3-1 rescue allele was crossed with a NestinCre-driver line, as illustrated in FIG. 2B.

[0064] Expression of the modified Muncl3-1 molecules improves the assembly of Active Zones (AZs) in Smn-depleted motoneurons, and ameliorates the clustering of voltage-gated Ca2+channels (VGCCs) in vitro, leading to increased Ca2+transients in growth cones of Smn- deficient motoneurons, as shown in FIGs. 3A-3E.

[0065] Overexpression of Muncl3-1 with the modified 3'UTR rescued the axonal growth and synapse differentiation defects that were prominent in primary cultured motoneurons with Smn deficiency, as shown in FIGs. 4A-4C. SMA mice expressing the modified Muncl3-1 rescue allele showed diminished synapse degeneration, attenuated muscle weakness and improved motor functions, which beneficially affected the survival of the animals, as shown in FIGs. 5 A- 5G.

[0066] Introduction of the Muncl3-1 rescue allele through gene therapy approaches could prevent the synapse degeneration in SMA patients, thereby representing a treatment for SMA and other neurological disorders with synapse dysfunction such as ALS and PD in the future. A proposed mechanism for such introduction is shown in FIG. 6, in the right-most figure. In FIG. 6 the left-most figure represents a healthy control synapse, with the center figure representing an untreated, diseased, SMA mouse model synapse.

[0067] The present disclosure is also directed to mouse models and methods of screening with those mouse models, including screening with the Cre / loxP conditional Muncl3-1 knockin rescue mouse model (R26Uncl3-l 19'+) by contacting them with a candidate agent of interest and the effect of the candidate agent is assessed by monitoring one or more output parameters. These output parameters may be reflective of the viability of the cells, e.g. the total number of neuronal cells, and / or the total number of hematopoietic cells or the number of cells of aparticular hematopoietic cell type, or of the apoptotic state of the cells, e.g. the amount of DNA fragmentation, the amount of cell blebbing, the amount of phosphatidyl serine on the cell surface, and / or the length of axons and the composition of presynaptic active zones for neurotransmitter release, and the like by methods that are known in the art. Alternatively or additionally, the output parameters may be reflective of the differentiation capacity of the cells, e.g. the proportions of differentiated cells and differentiated cell types, e.g., functionally active neurons such as motoneurons, T cells and / or NK cells. Alternatively, or additionally, the output parameters may be reflective of the function of the cells, e.g., the cytokines and chemokines produced by the cells, the ability of the cells to home to and extravasate to a site of challenge, the ability of the cells to modulate, i.e., promote or suppress, the activity of other cells in vitro or in vivo, etc. Other output parameters may be reflective of the extent of pathogen infection in the animal, e.g., muscle innervation and the extent of neurotransmission at specific synapses such as neuromuscular junctions, the titer of pathogen in the non-human animal, e.g., mouse, etc.

[0068] Parameters are quantifiable components of cells, particularly components that can be accurately measured, desirably in a high throughput system. A parameter can be any cell component or cell product including synaptic vesicle, cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, neurotransmitter, carbohydrate, organic or inorganic molecule, nucleic acid, e.g., mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi -quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

[0069] Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, vaccines, antibiotics or other agents suspected of having antibiotic properties, peptides, polypeptides, antibodies, antigen-binding proteins, agents that have been approved pharmaceutical for use in a human, etc. An important aspect of the invention is to evaluate candidate drugs, including toxicity testing; and the like.

[0070] Candidate agents include organic molecules including functional groups necessary for structural interactions, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, frequently at least two of the functional chemical groups. The candidate agents often include cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules, including peptides, polynucleotides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Included are pharmacologically active drugs, genetically active molecules, etc. Compounds of interest include chemotherapeutic agents, hormones or hormone antagonists, etc.

[0071] Candidate agents of interest for screening also include nucleic acids, for example, nucleic acids that encode siRNA, shRNA, antisense molecules, or miRNA, nucleic acids that interact with mRNAs and the 3 UTR of mRNAs, and / or nucleic acids that encode polypeptides. Many vectors useful for transferring nucleic acids into target cells are available. 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.

[0072] 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 ecotropicenvelope protein, e.g., MMLV, are capable of infecting most murine and rat cell types, and are generated by using ecotropic packaging cell. Retroviruses bearing amphotropic envelope protein are capable of infecting most mammalian cell types, including human, dog and mouse, and are generated by using amphotropic packaging cell lines such as PA12; PA317; GRIP. Retroviruses packaged with xenotropic envelope protein, e.g., AKR env, are capable of infecting most mammalian cell types, except murine cells. The appropriate packaging cell line may be used to ensure that the cells of interest — in some instance, the engrafted cells, in some instance, the cells of the host are targeted by the packaged viral particles.

[0073] 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.

[0074] Candidate agents of interest for screening also include polypeptides. Such polypeptides may optionally be fused to a polypeptide domain that increases solubility of the product. The domain may be linked to the polypeptide through a defined protease cleavage site, e.g., a TEV sequence, which is cleaved by TEV protease. The linker may also include one or more flexible sequences, e.g., from 1 to 10 glycine residues. In some embodiments, the cleavage of the fusion protein is performed in a buffer that maintains solubility of the product, e.g., in the presence of from 0.5 to 2 M urea, in the presence of polypeptides and / or polynucleotides that increase solubility, and the like. Domains of interest include endosomolytic domains, e.g., influenza HA domain; and other polypeptides that aid in production, e.g., IF2 domain, GST domain, GRPE domain, and the like. Additionally, or alternatively, such polypeptides may be formulated for improved stability. For example, the peptides may be PEGylated, where the polyethyleneoxy group provides for enhanced lifetime in the blood stream. The polypeptide may be fused to another polypeptide to provide for added functionality, e.g., to increase the in vivo stability. Generally, such fusion partners are a stable plasma protein, which may, for example, extend the in vivo plasma half-life of the polypeptide when present as a fusion, inparticular wherein such a stable plasma protein is an immunoglobulin constant domain. In most cases where the stable plasma protein is normally found in a multimeric form, e.g., immunoglobulins or lipoproteins, in which the same or different polypeptide chains are normally disulfide and / or noncovalently bound to form an assembled multichain polypeptide, the fusions herein containing the polypeptide also will be produced and employed as a multimer having substantially the same structure as the stable plasma protein precursor. These multimers will be homogeneous with respect to the polypeptide agent they include, or they may contain more than one polypeptide agent.

[0075] The candidate polypeptide agent may be produced from eukaryotic cells, or may be produced by prokaryotic cells. It may be further processed by unfolding, e.g., heat denaturation, DTT reduction, etc., and may be further refolded, using methods known in the art. Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acylation, acetylation, carboxylation, amidation, etc. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine. The polypeptides may have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g., D-amino acids or non- naturally occurring synthetic amino acids. D-amino acids may be substituted for some or all of the amino acid residues.

[0076] The candidate polypeptide agent may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. Alternatively, the candidate polypeptide agent may be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will include at least 20% byweight of the desired product, at least about 75% by weight, at least about 95% by weight, and for therapeutic purposes, at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.

[0077] In some cases, the candidate polypeptide agents to be screened are antibodies or antigen-binding proteins. The term “antibody” or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure and its specific epitope is sometimes referred to as a “lock and key” fit. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g., human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be “antibodies.” Antibodies utilized in the present invention may be either polyclonal antibodies or monoclonal antibodies. Antibodies are typically provided in the media in which the cells are cultured. Besides antibodies, antigen-binding proteins encompass polypeptides that are also designed to bind an antigen of interest and elicit a response, e.g., an immunological reaction. Antigen-binding fragments known in the art (including, e.g., Fab, Fab' F(ab')2, Fabc, and scFv) are also encompassed by the term “antigen-binding protein”. The terms “antibody” and “antigenbinding protein” also include one or more immunoglobulin chains or fragments that may be chemically conjugated to, or expressed as, fusion proteins with other proteins, single chain antibodies, and bispecific antibodies.

[0078] Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0079] Candidate agents are screened for biological activity by administering the agent to at least one and usually a plurality of samples, sometimes in conjunction with samples lackingthe agent. The change in parameters in response to the agent is measured, and the result evaluated by comparison to reference cultures, e.g., in the presence and absence of the agent, obtained with other agents, etc. In instances in which a screen is being performed to identify candidate agents that will prevent, mitigate or reverse the effects of a toxic agent, the screen is typically performed in the presence of the toxic agent, where the toxic agent is added at the time most appropriate to the results to be determined. For example, in cases in which the protective / preventative ability of the candidate agent is tested, the candidate agent may be added before the toxic agent, simultaneously with the candidate agent, or subsequent to treatment with the candidate agent. As another example, in cases in which the ability of the candidate agent to reverse the effects of a toxic agent is tested, the candidate agent may be added subsequent to treatment with the candidate agent. As mentioned above, in some instances, the sample is the Cre / loxP conditional Muncl3-1 knockin rescue mouse model (R26Uncl3-l 19'+) that has been engrafted with cells, i.e., a candidate agent is provided to that mouse that has been engrafted with cells. In some instances, the sample is the cells to be engrafted, i.e., the candidate agent is provided to cells prior to transplantation.

[0080] If the candidate agent is to be administered directly to the non-human animal, e.g., mouse, the agent may be administered by any of a number of well-known methods in the art for the administration of peptides, small molecules and nucleic acids. For example, the agent may be administered orally, mucosally, topically, intradermally, or by injection, e.g., intraperitoneal, subcutaneous, intramuscular, intravenous, or intracranial injection, and the like. The agent may be administered in a buffer, or it may be incorporated into any of a variety of formulations, e.g., by combination with appropriate pharmaceutically acceptable vehicle. “Pharmaceutically acceptable vehicles” may be vehicles approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, such as humans. The term “vehicle” refers to a diluent, adjuvant, excipient, or carrier with which a compound of the invention is formulated for administration to a mammal. Such pharmaceutical vehicles can be lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, saline; gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents may be used. Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The agent may besystemic after administration or may be localized by the use of regional administration, intramural administration, or use of an implant that acts to retain the active dose at the site of implantation. The active agent may be formulated for immediate activity or it may be formulated for sustained release. For some conditions, particularly central nervous system conditions, it may be necessary to formulate agents to cross the blood-brain barrier (BBB). One strategy for drug delivery through the blood-brain barrier (BBB) entails disruption of the BBB, either by osmotic means such as mannitol or leukotrienes, or biochemically by the use of vasoactive substances such as bradykinin. A BBB disrupting agent can be co-administered with the agent when the compositions are administered by intravascular injection. Other strategies to go through the BBB may entail the use of endogenous transport systems, including Caveolin- 1 mediated transcytosis, carrier-mediated transporters such as glucose and amino acid carriers, receptor-mediated transcytosis for insulin or transferrin, and active efflux transporters such as p-glycoprotein. Active transport moi eties may also be conjugated to the therapeutic compounds for use in the invention to facilitate transport across the endothelial wall of the blood vessel.

[0081] If the agent(s) are provided to cells prior to transplantation, the agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0082] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0083] An analysis of the response of cells in a Cre / loxP conditional Muncl3-1 knockin rescue mouse model (R26Uncl3-l 19'+) to the candidate agent may be performed at any time following treatment with the agent. For example, the cells may be analyzed 1, 2, or 3 days,sometimes 4, 5, or 6 days, sometimes 8, 9, or 10 days, sometimes 14 days, sometimes 21 days, sometimes 28 days, sometimes 1 month or more after contact with the candidate agent, e.g., 2 months, 4 months, 6 months or more. In some embodiments, the analysis includes analysis at multiple time points. The selection of the time point(s) for analysis will be based upon the type of analysis to be performed, as will be readily understood by the ordinarily skilled artisan.

[0084] The analysis may include measuring any of the parameters described herein or known in the art for measuring cell viability, cell proliferation, cell identity, cell morphology, and cell function, particularly as they may pertain to axonal cells. For example, flow cytometry may be used to determine the total number of hematopoietic cells or the number of cells of a particular hematopoietic cell type Ca2+transients in or near axonal growth cones. Histochemistry or immunohistochemistry may be performed to determine the apoptotic state of the cells, e.g., terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) to measure DNA fragmentation, or immunohistochemistry to detect Annexin V binding to phosphatidyl serine on the cell surface. ELISAs, Westerns, and Northern blots may be performed to determine the levels of Localized Muncl3-1 mRNAs in axons, expressed in the Cre / loxP conditional Muncl3-1 knockin rescue mouse model (R26Uncl3-l 19'+).

[0085] 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 for producing a protein or polypeptide, the mammalian mRNA expression construct comprising: a modified Muncl3-1 molecule.

2. A method for the expression of a mammalian mRNA expression construct, the method comprising: administering the mammalian mRNA expression construct to a patient, wherein the mammalian mRNA expression construct comprises a modified Muncl3-1 molecule, wherein the modified Muncl3-1 molecule is configured for local translation at axons of the patient independent of a Survival Motor Neuron 1 (SMN1) protein.

3. The method of claim 2, wherein the modified Muncl3-1 molecule is configured to increase the Muncl3-1 mRNA levels in a presynaptic terminal of motoneurons in the patient.

4. The method of claim 2, wherein the patient is diagnosed with spinal muscular atrophy(SMA) or Amyotrophic Lateral Sclerosis (ALS).

5. 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 modified Muncl3- 1 mRNA molecule.

6. A method for screening a candidate agent for modulating MUNC13-1 expression in pre-synaptic terminals of human motoneurons, the method comprising: administering the agent to motoneurons from control or a genetically modified mouse, wherein the genetically modified mouse is either aodel of SMA or ALS or comprises in its genome a Synaptophysin 3'UTR inserted into the ROSA26 locus on chromosome 6; and assessing effect of the agent on MUNC13-1 expression in axons in the genetically modified mouse.

7. The method of claim 6, wherein assessing the effect of the agent comprises assessing the restoration of MUNC13-1 in axon terminals of the genetically modified mouse models.

8. A genetically modified mouse comprising: a Synaptophysin 3'UTR inserted into the ROSA26 locus on chromosome 6, wherein the genetically modified mouse comprises Muncl3-1 knockin.

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