Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
  • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2207/00—Modified animals
  • A01K2207/15—Humanized animals
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/0306—Animal model for genetic diseases
  • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• Neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia, affect millions of people worldwide. Neurodegenerative diseases occur when nerve cells in the brain or peripheral nervous system lose function over time and ultimately die. Over the next 40 years, the prevalence of neurodegenerative diseases is estimated to double in the US. Although treatments may help relieve some of the physical or mental symptoms associated with neurodegenerative diseases, there is currently no way to slow disease progression and no known cures. There is a continuing need for non-human animal models that recapitulate predominant disease mechanisms observed in neurodegenerative disease patients in order to develop therapeutic approaches.
• the present disclosure provides, in some aspects, mouse models of Tar DNA-binding protein 43 (TDP-43) proteinopathies, such as amyotrophic lateral sclerosis and frontotemporal dementia. These mouse models are based in part on experimental evidence showing Stathmin-2 (Slmn2) gene disruption in such proteinopathies. This gene undergoes cryptic splicing of its mRNA, resulting in a truncated mRNA.
• TDP-43 proteinopathies such as amyotrophic lateral sclerosis and frontotemporal dementia.
• mice models described herein recapitulate a disease mechanism central to a broad spectrum of neurodegenerative conditions and may be used, for example, to develop therapies in TDP-43 proteinopathies through identification of agents (e.g., antisense oligonucleotides and/or small molecule drugs) that block cryptic (aberrant) splicing of Stmn2 pre-mRNA.
• agents e.g., antisense oligonucleotides and/or small molecule drugs
• TDP-43 proteinopathies comprising a humanized stathmin-2 (Slmn2) gene comprising an exogenous polynucleotide sequence, wherein the exogenous polynucleotide sequence comprises human STMN2 exon 2a sequence.
• the human STMN2 exon 2a sequence has a length of about 200 to about 300 nucleotides, optionally a length of about 220 to about 225, preferably 222 nucleotides.
• the exogenous polynucleotide sequence comprises human genomic DNA flanking the human STMN2 exon 2a sequence.
• the human STMN2 exon 2a sequence comprises a human MS2 stem loop sequence.
• the human MS2 stem loop sequence comprises the sequence of 5’-ACATGAGGATCACCCATGT-3’ (SEQ ID NO: 4).
• the human MS2 stem loop sequence replaces TDP-43 binding sequence.
• the TDP-43 binding sequence comprises the sequence of 5’- TGTGTGAGCATGTGTGCGTGTGTG-3’ (SEQ ID NO: 5).
• the human STMN2 exon 2a sequence is in intron 1 of the mouse Stmn2 gene.
• the mouse model has a C57BL/6 genetic background.
• the genotype of the mouse is C57BL/6J- Stmn2 eni8(STMN2 * )Lutzy /Mmjax.
• the TDP-43 proteinopathies are selected from amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD).
• ALS amyotrophic lateral sclerosis
• FTD frontotemporal degeneration
• aspects provide a method comprising administering a candidate therapeutic agent to the mouse of any one of the preceding claims, and assaying the mouse for a modified phenotype.
• the candidate therapeutic agent blocks aberrant/cryptic splicing of Stmn2 pre-mRNA.
• the candidate therapeutic is an antisense oligonucleotide (ASO) that binds to the human exon2a polynucleotide sequence.
• ASO antisense oligonucleotide
• the candidate therapeutic is a small molecule drug.
• the phenotype is selected from a behavioral phenotype, a pathological phenotype, a cognitive deficit, a motor deficit, motor neuron degeneration, neuromuscular denervation.
• the phenotype is a motor deficit.
• the motor deficit is selected from tremors, paralysis, abnormal gait, or hindlimb clasping.
• the motor deficit is assayed by open field, grip strength, or rotarod analyses.
• the phenotype is a cognitive deficit.
• Yet other aspects provide a method of producing the mouse of any one of claims 1- 11, the method comprising introducing the exogenous polynucleotide sequence into an intron of the endogenous Stmn2 gene of the mouse, optionally using a gene editing technique.
• FIG. 1A shows a schematic of the human STMN2 gene.
• FIG. 1B shows a schematic of the mouse Stmn2 gene.
• FIG. 2A shows a schematic of a mouse Stmn2 allele that includes a 394 base pair segment of intron 1 of the human STMN2. This modified allele is referred to herein as a “humanized mouse Stmn2 allele.”
• FIG. 2B shows a schematic of the humanized mouse Stmn2 allele in which the human TDP-43 binding sites have been replaced by MS2 binding sites, leading to constitutive inclusion of exon 2 (from top to bottom, SEQ ID NOs: 5 and 4).
• FIGs. 3A-3D show a schematic and data relating to the generation and in vitro validation of humanized Stmn2 mice (i.e., mice that include the humanized mouse Stmn2 allele).
• Primary cortical neurons were generated from mouse embryos edited to carry the human exon 2a genomic region within intron 1 of the stathmin-2 mouse gene (Stmn2 Hum/+ mice) and treated during 12 days with 1 or 3 ⁇ M of an RNase-H dependent ASO targeting mouse TDP-43 (FIG. 3A).
• stathmin-2 Reduction of stathmin-2 is mediated by abnormal splicing between murine exon 1 and human exon 2a upon TDP-43 loss detected by RT-PCR in neurons from heterozygous Stmn2 Hum/+ but not from wild-type embryos (FIG. 3D).
• FIGs. 4A-4C show data from mouse neuroblastoma cells edited to include human exon 2a with TDP-43 binding sites replaced by MS2 sites, demonstrating constitutional mis- splicing and reduction of Stmn2.
• Quantitative RT-PCR for full length mouse Stmn2 in N2A neuroblastoma clones after CRISPR/Cas9 editing to introduce human exon 2a without TDP- 43 binding sites see FIG. 2B) in one allele (Het) or both alleles (Homo) compared to wild- type (WT) isogenic clones (FIG. 4A) (**p ⁇ 0.001, one way ANOVA).
• FIG. 4B RT-PCR showing constitutive splicing between murine exon 1 and human exon 2a in heterozygous and homozygous clones.
• Constitutive misplicing and reduced levels of stathmin-2 mRNA leads to 50% reduction or complete loss of STMN2 protein, respectively, in heterozygous and homozygous cells (FIG. 4C).
• ALS Amyotrophic lateral sclerosis
• FTD Frontotemporal degeneration
• TDP- 43 disruption represents a common pathological hallmark in both sporadic and familial forms of ALS and FTD including patients with mutations in the genes encoding for TDP-43 (TARDBP) and progranulin (GRN) and patients with G4C2 repeat expansions in the C9ORF72 gene.
• TDP-43 pathology is reported in a large spectrum of neurodegenerative conditions, referred as TDP-43 proteinopathies, that include ALS, FTD, and various forms of parkinsonism and dementia. More than 30% of patients have Alzheimer’s disease or limbic- predominant age-related TDP-43 encephalopathy.
• TDP-43 is an RNA binding protein playing a role in different aspects of RNA metabolism regulation.
• the protein is detectable in the nuclei of unaffected neurons but is cleared from nuclei in neurons containing cytoplasmic aggregations, evidence strongly supporting that TDP-43 loss of function is a key aspect of disease mechanism underlying ALS and FTD pathogenesis.
• Genome-wide approaches have been used to define binding sites of TDP-43 on thousands of RNA targets, with GU-rich sequences as preferred binding sites, and to define the influence of TDP-43 disruption on RNA splicing and expression of mouse and human transcripts.
• TDP-43 is an essential protein, and its ubiquitous deletion in mice is embryonically lethal. Numerous transgenic rodents expressing human TDP-43 have been generated, with or without disease-causing mutations and under various promoters. Overexpression of TDP-43 induces a severe lethal phenotype independent of the presence of mutation. However, mice expressing levels close to endogenous TDP-43 develop mutant and age-dependent neurological phenotypes, including mild motor and cognitive deficits, motor neuron degeneration, and neuromuscular denervation, but without paralysis or reduced lifespan.
• TDP-43 proteinopathies An important caveat for animal modeling of TDP-43 proteinopathies is that the repertoire of RNAs bound by TDP-43 differs between species with RNA processing alterations elicited by TDP-43 dysfunction distinct between mice and humans. Indeed, recent data shows that the human RNA most affected by TDP-43 disruption encodes the neuronal growth-associated factor stathmin-2 (also known as SCG10), but stathmin-2 RNAs are neither bound nor regulated by TDP-43 in rodents.
• stathmin-2 also known as SCG10
• stathmin-2 Abnormal processing of stathmin-2 is not recapitulated in mice expressing TDP-43 transgenes or in TDP-43 deficient mice, as the cryptic poly adenylation signal and three GU-rich TDP-43 binding sites in intron 1 of the human stathmin-2 gene are not found in the corresponding mouse intron (FIGs. 1A and 1B). Consistently, stathmin-2 pre-mRNA is not bound by TDP-43 in mice, and stathmin-2 mRNA level is not altered after siRNA or ASO-mediated reduction of TDP-43 in mouse N2A cells and in the central nervous system of wild-type mice. See Melamed et al. (2019) Nat. Neurosci.. incorporated herein by reference.
• TDP-43 Tar DNA-binding protein 43
• hnRNPs heterogenous nuclear ribonucleoproteins
• TDP-43 pathology is now recognized as a neuropathological feature of a substantial proportion of cases of Alzheimer’s disease as well as Parkinson’s and Huntington’s diseases, and even the degenerative muscle disease inclusion body myopathy (see Glass JD J Clin Invest. 2020 Nov 2; 130(11):5677-5680).
• TDP-43 is a transcription suppressor. In the absence of TDP-43, i.e., when TDP-43 is cleared from the nucleus as it mislocalizes to the cytoplasm, cryptic exons are exposed and transcribed, creating aberrant mRNAs that lead to neurotoxicity in ALS models. Recently, this TDP-43 suppressor function was specifically implicated in suppressing a cryptic early polyadenylation site in the pre-mRNA for the protein stathmin-2 (STMN2). Loss of TDP-43 allows incorporation of a premature poly A tail, resulting in a truncated and nonfunctional form of STMN2 (Klim JR et al., Nat Neurosci.
• STMN2 is a highly conserved cytosolic protein essential for axonal outgrowth and maintenance. STMN2-knockout mice develop late-onset, predominantly motor axonopathy (Liedtke W et al. Am J Pathol. 2002;160(2):469-480) paralleling that seen in human ALS.
• stathmin-2 is the most prominently expressed in both mouse and human motor neurons. Indeed, stathmin-2 is among the top 25 most enriched mRNAs in this neuronal population that is specifically affected in ALS. Stathmin-2 has been proposed to play an important role in neurite outgrowth48, most likely by promoting microtubule dynamics in axonal growth cones. It binds ⁇ / ⁇ -tubulin dimers and was shown to be essential for axonal regeneration. Upon axonal injury, stathmin-2 is upregulated and recruited to growth cones of regenerating axons, and it was determined that it accumulates at motor axon termini of neuromuscular junctions (NMJs) in adult mice.
• NMJs neuromuscular junctions
• the mouse (mouse model) provided herein has the genotype C57BL/6J-Stmn2 em8(STMN2*)Lutzy /Mmjax (common name Stmn2 em8(STMN2*) , e.g., MMRRC Strain #069792-JAX).
• Stmn2 em8(STMN2*) is a CRISPR/cas9 generated mutant of the stathmin- like 2 (Slmn2) gene carrying 222 nucleotides of human STMN2 exon 2a.
• the STMN2 sequence is modified to include the human MS2 stem loop sequence and replace the TDP-43 binding element.
• the Stmn2 em8(STMN2*) allele was generated using CRISPR/cas9 endonuclease- mediated genome editing.
• Guide RNAs were selected to target intron 1 of the stathmin-like 2 gene (Stmn2) and insert a modified human SMNT2 exon 2a.
• the humanized/mutant exon 2a in this strain is 222nt in length and is flanked by 1491 bp and 1548 bp of human genomic DNA, located 5’- and 3’- of exon 2a, respectively.
• the 19-nt MS 2 stem loop contains the sequence 5’-ACATGAGGATCACCCATGT-3’ (SEQ ID NO: 4) was used to replace the 24- nt TDP-43 binding sequence 5’-TGTGTGAGCATGTGTGCGTGTGTG-3’ (SEQ ID NO: 5) in the 3’-UTR of the exon2a-containing mouse-human hybrid STMN2 prematurely terminated mRNA transcript.
• the donor plasmid, both guides and cas9 nuclease were introduced into single cell C57BL/6J zygotes and transferred to pseudopregnant females. Note that the flanking mouse intron 1 sequences present in the donor plasmid was derived from N2A cell line.
• the human Exon 2A sequence includes the following sequence:
• the poly A signal sequence is ATT AAA .
• the Mut primer binding sites are and .
• stathmin-2 was the mRNA most affected by reduction in TDP-43, with a corresponding almost complete loss of the 22 kD stathmin-2 protein (Melamed Z, et al. Nat Neurosci. 2019;22(2): 180-190).
• the stathmin-2 gene contains 5 annotated exons.
• GUGUGU Three “GUGUGU” hexamers marking potential binding sites of TDP-43 were identified in a region between the cryptic 3’ splice site and premature polyA site whose use produces exon 2a. Indeed, an analysis of ultraviolet cross-linking and immuno-precipitation (iCLIP) data for TDP-43 in human SH-SY5Y cells confirmed (Fig. le) that TDP-43 does bind to the stathmin-2 pre-mRNA at these three GUGUGU sequences (the only sites of TDP-43 binding to the stathmin-2 pre-mRNA), thus confirming physical interaction of the pre-mRNA with TDP-43.
• iCLIP ultraviolet cross-linking and immuno-precipitation
• mouse and “mouse models” (e.g., surrogates for human conditions). It should be understood that these terms, unless otherwise stated, may be used interchangeably throughout the specification to encompass “rodent” and “rodent models,” including mouse, rat and other rodent species.
• strain symbol conveys basic information about the type of strain or stock used and the genetic content of that strain.
• Rules for symbolizing strains and stocks have been promulgated by the International Committee on Standardized Genetic Nomenclature for Mice. The rules are available on-line from the Mouse Genome Database (MGD; informatics.jax.org) and were published in print copy (Lyon et al. 1996).
• Strain symbols typically include a Laboratory Registration Code (Lab Code). The registry is maintained at the Institute for Laboratory Animal Research (ILAR) at the National Academy of Sciences, Washington, D.C.
• Lab Codes may be obtained electronically at ILAR's web site (nas.edu/cls/ilarhome.nsf). See also Davisson MT, Genetic and Phenotypic Definition of Laboratory Mice and Rats / What Constitutes an Acceptable Genetic- Phenotypic Definition, National Research Council (US) International Committee of the Institute for Laboratory Animal Research. Washington (DC): National Academys Press (US); 1999.
• transgenic mouse models that express an endogenous Stathmin-2 gene that has been genetically modified to include an exogenous human Stamin-2 polynucleotide sequence, referred to herein as a ‘humanized Stathmin-2 gene’.
• a transgenic mouse is a mouse having an exogenous nucleic acid (e.g., transgene or region of a transgene) in (integrated into) its genome.
• transgenic mice Methods of producing transgenic mice are well-known. Three conventional methods used for the production of transgenic mice include DNA microinjection (Gordon and Ruddle, Science 1981: 214: 1244-124, incorporated herein by reference), embryonic stem cell- mediated gene transfer (Gossler et al., Proc. Natl. Acad. Sci. 1986, 83: 9065-9069, incorporated herein by reference) and retrovirus -mediated gene transfer (Jaenisch, Proc. Natl. Acad. Sci. 1976, 73: 1260-1264, incorporated herein by reference), any of which may be used as provided herein.
• DNA microinjection Gibdon and Ruddle, Science 1981: 214: 1244-124, incorporated herein by reference
• embryonic stem cell- mediated gene transfer Gossler et al., Proc. Natl. Acad. Sci. 1986, 83: 9065-9069, incorporated herein by reference
• retrovirus -mediated gene transfer Jaenisch,
• Genomic editing methods using, for example, clustered regularly interspace palindromic repeats (CRISPR/Cas) nucleases, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) are described elsewhere herein.
• CRISPR/Cas clustered regularly interspace palindromic repeats
• TALENs transcription activator-like effector nucleases
• ZFNs zinc finger nucleases
• nucleic acids Following delivery of nucleic acids to a fertilized embryo (e.g., a single-cell embryo (e.g., a zygote) or a multi-cell embryo (e.g., a developmental stage following a zygote, such as a blastocyst), the fertilized embryo is transferred to a pseudopregnant female, which subsequently gives birth to offspring.
• a pseudopregnant female e.g., a developmental stage following a zygote, such as a blastocyst
• the presence or absence of a nucleic acid encoding humanized Stathmin-2 gene may be confirmed, for example, using any number of genotyping methods (e.g., sequencing and/or genomic PCR).
• the nucleic acids provided herein, in some embodiments, are engineered.
• An engineered nucleic acid is a nucleic acid (e.g., at least two nucleotides covalently linked together, and in some instances, containing phosphodiester bonds, referred to as a phosphodiester backbone) that does not occur in nature.
• Engineered nucleic acids include recombinant nucleic acids and synthetic nucleic acids.
• a recombinant nucleic acid is a molecule that is constructed by joining nucleic acids (e.g., isolated nucleic acids, synthetic nucleic acids or a combination thereof) from two different organisms (e.g., human and mouse).
• a synthetic nucleic acid is a molecule that is amplified or chemically, or by other means, synthesized.
• a synthetic nucleic acid includes those that are chemically modified, or otherwise modified, but can base pair with (bind to) naturally occurring nucleic acid molecules.
• Recombinant and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing.
• An engineered nucleic acid may comprise DNA (e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA), RNA or a hybrid molecule, for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.
• DNA e.g., genomic DNA, cDNA or a combination of genomic DNA and cDNA
• RNA or a hybrid molecule for example, where the nucleic acid contains any combination of deoxyribonucleotides and ribonucleotides (e.g., artificial or natural), and any combination of two or more bases, including uracil, adenine, thymine, cytosine
• a nucleic acid is a complementary DNA (cDNA).
• cDNA is synthesized from a single- stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by reverse transcriptase.
• mRNA messenger RNA
• miRNA microRNA
• Engineered nucleic acids of the present disclosure may be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press).
• nucleic acids are produced using GIBSON ASSEMBLY® Cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated by reference herein).
• GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: 5' exonuclease, the 3' extension activity of a DNA polymerase and DNA ligase activity.
• the 5' exonuclease activity chews back the 5' end sequences and exposes the complementary sequence for annealing.
• the polymerase activity then fills in the gaps on the annealed domains.
• a DNA ligase then seals the nick and covalently links the DNA fragments together.
• the overlapping sequence of adjoining fragments is much longer than those used in Golden Gate Assembly, and therefore results in a higher percentage of correct assemblies.
• Other methods of producing engineered nucleic acids may be used in accordance with the present disclosure.
• a gene is a distinct sequence of nucleotides, the order of which determines the order of monomers in a polynucleotide or polypeptide.
• a gene typically encodes a protein.
• a gene may be endogenous (occurring naturally in a host organism) or exogenous (transferred, naturally or through genetic engineering, to a host organism).
• An allele is one of two or more alternative forms of a gene that arise by mutation and are found at the same locus on a chromosome.
• a gene in some embodiments, includes a promoter sequence, coding regions (e.g., exons), non-coding regions (e.g., introns), and regulatory regions (also referred to as regulatory sequences).
• a mouse comprising a human gene is considered to comprise a human transgene.
• a transgene is a gene exogenous to a host organism. That is, a transgene is a gene that has been transferred, naturally or through genetic engineering, to a host organism. A transgene does not occur naturally in the host organism (the organism, e.g., mouse, comprising the transgene).
• a promoter is a nucleotide sequence to which RNA polymerase binds to initial transcription (e.g., ATG). Promoters are typically located directly upstream from (at the 5' end of) a transcription initiation site. In some embodiments, a promoter is an endogenous promoter. An endogenous promoter is a promoter that naturally occurs in that host animal.
• An open reading frame is a continuous stretch of codons that begins with a start codon (e.g., ATG), ends with a stop codon (e.g., TAA, TAG, or TGA), and encodes a polypeptide, for example, a protein.
• An open reading frame is operably linked to a promoter if that promoter regulates transcription of the open reading frame.
• An exon is a region of a gene that codes for amino acids.
• An intron (and other non- coding DNA) is a region of a gene that does not code for amino acids.
• a nucleotide sequence encoding a product in some embodiments, has a length of 200 base pairs (bp) to 100 kilobases (kb).
• the nucleotide sequence in some embodiments, has a length of at least 10 kb.
• the nucleotide sequence may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, or at least 35 kb.
• the nucleotide sequence has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb.
• Any one of the nucleic acids provided herein may have a length of 200 bp to 500 kb, 200 bp to 250 kb, or 200 bp to 100 kb.
• a nucleic acid in some embodiments, has a length of at least 10 kb.
• a nucleic acid may have a length of at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 50 kb, at least 100 kb, at least 200 kb, at least 300 kb, at least 400 kb, or at least 500 kb.
• a nucleic acid has a length of 10 to 500 kb, 20 to 400 kb, 10 to 300 kb, 10 to 200 kb, or 10 to 100 kb.
• a nucleic acid has a length of 10 to 100 kb, 10 to 75 kb, 10 to 50 kb, 10 to 30 kb, 20 to 100 kb, 20 to 75 kb, 20 to 50 kb, 20 to 30 kb, 30 to 100 kb, 30 to 75 kb, or 30 to 50 kb.
• a nucleic acid may be circular or linear.
• the nucleic acids described herein, in some embodiments, include a modification.
• a modification with respect to a nucleic acid, is any manipulation of the nucleic acid, relative to the corresponding wild-type nucleic acid (e.g., the naturally-occurring nucleic acid).
• a genomic modification is thus any manipulation of a nucleic acid in a genome (e.g., in a coding region, non-coding region, and/or regulatory region), relative to the corresponding wild-type nucleic acid (e.g., the naturally-occurring (unmodified) nucleic acid) in the genome.
• Non-limiting examples of nucleic acid (e.g., genomic) modifications include deletions, insertions, “indels” (deletion and insertion), and substitutions (e.g., point mutations).
• a deletion, insertion, indel, or other modification in a gene results in a frameshift mutation such that the gene no longer encodes a functional product (e.g., protein).
• Modifications also include chemical modifications, for example, chemical modifications of at least one nucleobase.
• nucleic acid modification for example, those that result in gene inactivation, are known and include, without limitation, RNA interference, chemical modification, and gene editing (e.g., using recombinases or other programmable nuclease systems, e.g., CRISPR/Cas, TALENs, and/or ZFNs).
• a nucleic acid such as an allele or alleles of a gene, may be modified such that it does not produce a detectable level of a functional gene product (e.g., a functional protein).
• an inactivated allele is an allele that does not produce a detectable level of a functional gene product (e.g., a functional protein).
• a detectable level of a protein is any level of protein detected using a standard protein detection assay, such as flow cytometry and/or an ELISA.
• an inactivated allele is not transcribed.
• an inactivated allele does not encode a functional protein.
• Vectors used for delivery of a nucleic acid include minicircles, plasmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes. It should be understood, however, that a vector may not be needed.
• a circularized or linearized nucleic acid may be delivered to an embryo without its vector backbone.
• Vector backbones are small ( ⁇ 4 kb), while donor DNA to be circularized can range from > 100 bp to 50 kb, for example.
• Methods for delivering nucleic acids to mouse embryos for the production of transgenic mice include, but are not limited to, electroporation (see, e.g., Wang W et al.
• Engineered nucleic acids such as guide RNAs, donor polynucleotides, and other nucleic acid coding sequences, for example, may be introduced to a genome of an embryo using any suitable method.
• the present application contemplates the use of a variety of gene editing technologies, for example, to introduce nucleic acids into the genome of an embryo to produce a transgenic rodent.
• Non-limiting examples include programmable nuclease-based systems, such as clustered regularly interspaced short palindromic repeat (CRISPR) systems, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., Carroll D Genetics.
• CRISPR clustered regularly interspaced short palindromic repeat
• ZFNs zinc-finger nucleases
• TALENs transcription activator-like effector nucleases
• a CRISPR system is used to edit the genome of mouse embryos provided herein. See, e.g., Harms DW et al., Curr Protoc Hum Genet. 2014; 83: 15.7.1-15.7.27; and Inui M et al., Sci Rep. 2014; 4: 5396, each of which are incorporated by reference herein).
• Cas9 mRNA or protein, one or multiple guide RNAs (gRNAs), and/or a donor nucleic acid can be delivered, e.g., injected or electroporated, directly into mouse embryos at the one-cell (zygote) stage or a later stage to facilitate homology directed repair (HDR), for example, to introduce an engineered nucleic acid (e.g., donor nucleic acid) into the genome.
• HDR homology directed repair
• the CRISPR/Cas system is a naturally occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided-DNA-targeting platform for gene editing.
• Engineered CRISPR systems contain two main components: a guide RNA (gRNA) and a CRISPR-associated endonuclease (e.g., Cas protein).
• the gRNA is a short synthetic RNA composed of a scaffold sequence for nuclease-binding and a user-defined nucleotide spacer (e.g., ⁇ 15-25 nucleotides, or -20 nucleotides) that defines the genomic target (e.g., gene) to be modified.
• a user-defined nucleotide spacer e.g., ⁇ 15-25 nucleotides, or -20 nucleotides
• the genomic target e.g., gene
• the Cas9 endonuclease is from Streptococcus pyogenes (NGG PAM) or Staphylococcus aureus (NNGRRT or NNGRR(N) PAM), although other Cas9 homologs, orthologs, and/or variants (e.g., evolved versions of Cas9) may be used, as provided herein.
• RNA-guided nucleases that may be used as provided herein include Cpfl (TTN PAM); SpCas9 D1135E variant (NGG (reduced NAG binding) PAM); SpCas9 VRER variant (NGCG PAM); SpCas9 EQR variant (NGAG PAM); SpCas9 VQR variant (NGAN or NGNG PAM); Neisseria meningitidis (NM) Cas9 (NNNNGATT PAM); Streptococcus thermophilus (ST) Cas9 (NNAGAAW PAM); and Treponema denticola (TD) Cas9 (NAAAAC).
• the CRISPR-associated endonuclease is selected from Cas9, Cpf1, C2c1, and C2c3.
• the Cas nuclease is Cas9.
• a guide RNA comprises at least a spacer sequence that hybridizes to (binds to) a target nucleic acid sequence and a CRISPR repeat sequence that binds the endonuclease and guides the endonuclease to the target nucleic acid sequence.
• each gRNA is designed to include a spacer sequence complementary to its genomic target sequence. See, e.g., Jinek et al., Science, 2012; 337: 816-821 and Deltcheva et al., Nature, 2100; 471: 602-607, each of which is incorporated by reference herein.
• RNA-guided nuclease and the gRNA are complexed to form a ribonucleoprotein (RNP), prior to delivery to an embryo.
• RNP ribonucleoprotein
• the concentration of RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease may vary. In some embodiments, the concentration is 100 ng/ ⁇ l to 1000 ng/ ⁇ l. For example, the concentration may be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng/ ⁇ l. In some embodiments, the concentration is 100 ng/ ⁇ l to 500 ng/ ⁇ l, or 200 ng/ ⁇ l to 500 ng/ ⁇ l. The concentration of gRNA may also vary. In some embodiments, the concentration is 200 ng/ ⁇ l to 2000 ng/ ⁇ l.
• the concentration may be 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1700, 1900, or 2000 ng/ ⁇ l.
• the concentration is 500 ng/ ⁇ l to 1000 ng/ ⁇ l.
• the concentration is 100 ng/ ⁇ l to 1000 ng/ ⁇ l.
• the concentration may be 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 ng/ ⁇ l.
• the ratio of concentration of RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease to the concentration of gRNA is 2:1. In other embodiments, the ratio of concentration of RNA-guided nuclease or nucleic acid encoding the RNA-guided nuclease to the concentration of gRNA is 1:1.
• a donor nucleic acid typically includes a sequence of interest flanked by homology arms.
• Homology arms are regions of the ssDNA that are homologous to regions of genomic DNA located in a genomic locus.
• One homology arm is located to the left (5') of a genomic region of interest (into which a sequence of interest is introduced) (the left homology arm) and another homology arm is located to the right (3') of the genomic region of interest (the right homology arm).
• These homology arms enable homologous recombination between the ssDNA donor and the genomic locus, resulting in insertion of the sequence of interest into the genomic locus of interest (e.g., via CRISPR/Cas9-mediated homology directed repair (HDR)).
• HDR homology directed repair
• each homology arm may have a length of 20 nucleotide bases to 1000 nucleotide bases.
• each homology arm has a length of 20 to 200, 20 to 300, 20 to 400, 20 to 500, 20 to 600, 20 to 700, 20 to 800, or 20 to 900 nucleotide bases.
• each homology arm has a length of 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 nucleotide bases.
• the length of one homology arm differs from the length of the other homology arm.
• one homology arm may have a length of 20 nucleotide bases, and the other homology arm may have a length of 50 nucleotide bases.
• the donor DNA is single stranded.
• the donor DNA is double stranded.
• the donor DNA is modified, e.g., via phosphorothioation. Other modifications may be made.
• the mouse models provided herein may be used, in some embodiments, to determine how stathmin-2 contributes to neuronal function.
• the mouse models may be used to develop therapeutic approaches for treating neurodegenerative diseases, such as TDP-43 proteinopathies that include, for example, ALS, FTD, and various forms of parkinsonism.
• methods comprising administering to a mouse model a candidate therapeutic molecule, and assaying for a modified phenotype in the mouse.
• ASOs antisense oligonucleotides
• ASOs have since been utilized for targeting genetic causes of several neurological diseases including Huntington’s disease, C9ORF72-ALS/FTD67-70, Taupathies, sporadic ALS and spinocerebellar ataxia, Batten’s disease, and Pelizaeus-Merzbacher disease.
• RNase H a superoxide dismutase 1
• the SMA drug Spinraza (also known as Nusinersen) is a chemically modified ASO-based drug that binds its targeted mRNA without recruiting the RNase H enzyme, leading to splicing modulation but not mRNA degradation.
• mice models provided herein may be used to develop an antisense ASO- based therapy targeted to restore stathmin-2 levels by blocking the aberrant splicing of stathmin-2 pre-mRNA. Utilizing an approach reminiscent of the ASO-mediated alteration in splicing of the SMN2 pre-mRNA in SMA, for example, these mouse models may be used to test the therapeutic potential of stathmin-2 ASOs in sporadic and familial ALS/FTD.
• Candidate therapeutic molecules of the present disclosure also include putative or known small-molecule modulators (e.g., inhibitors, drugs) of stathmin-2 pre-mRNA or mRNA splicing.
• the phenotype may be, for example, a behavioral phenotype or a pathological (e.g., neuropathological) phenotype (see, e.g., Kang J. et al. STAR Protoc. 2021 Jul 7;2(3):100654). See also Janus C et al. Methods Mol Biol. 2010;602:323-45; Choi J. et al. NMR Biomed. 2007;20:26-237; van der Staay F. et al. Behav Brain Funct. 2009 Feb 25 ;5: 11.
• the phenotype is a motor deficit.
• motor deficits include tremors, paralysis, abnormal gait, and hindlimb clasping.
• the motor deficit is assayed by open field, grip strength, or rotarod analyses.
• the phenotype is a cognitive deficit (see, e.g., Holter S. et al. Curr Protoc Mouse Biol. 2015 Dec 2;5(4):331-358).
• phenotypes that may be assessed include motor neuron degeneration and/or neuromuscular denervation.
• a mouse comprising a nucleic acid encoding a truncated Stathmin-2 messenger ribonucleic acid (mRNA).
• mRNA messenger ribonucleic acid
• a mouse comprising a nucleic acid comprising a region of a human Stathmin-2 gene.
• a mouse comprising an endogenous Stathmin-2 gene locus, wherein the endogenous Stathmin-2 gene locus comprises a nucleic acid comprising a human Stathmin-2 exon 2a polynucleotide sequence and optionally a polyadenylation sequence.
• the human Stathmin-2 exon 2a polynucleotide sequence has a length of 350-450 nucleotide base pairs.
• a method comprising: administering to the mouse of any one of embodiments 1-13 a candidate therapeutic molecule; and assaying for a modified phenotype in the mouse.
• phenotype is a behavioral phenotype or a pathological phenotype.
• a method of producing the mouse of any one of embodiments 1-13 comprising introducing the nucleic acid into the genome of the mouse.
• stathmin-2 In order to determine how stathmin-2 contributes to neuronal function and to develop therapeutic approaches based on restoration of stathmin-2 levels in ALS and FTD, the field needs more faithful animal models where processing of the stathmin-2 transcript is dependent on TDP-43 function, as has demonstrated to be true in human.
• ALS/FTD mouse models were generated in which a portion of intron 1 of the stathmin-2 gene has been humanized. This approach can be used to evaluate the degree of TDP-43 dysfunction in different ALS/FTD models and establish whether abnormal stathmin-2 mRNA processing contributes to neurodegeneration.
• the mouse Stmn2 gene does not contain an exact homolog to human exon 2a. It does, however, contain a pseudo exon 2a region within intron 1 of the Stmn2 gene that contains a putative (but unused) splice sequence, but it lacks both TDP-43 binding sequences and a polyadenylation signal.
• CRISPR/Cas9 genome-editing was used to replace a 479 nucleotide (nt) region of mouse intron 1 containing the pseudo exon 2a with a 394 nt human sequence that contains the 227 bases of human exon 2a with 75 and 92 flanking bases (FIG. 2A). Correct genome editing was verified by short and long-range PCR followed by sequencing. It was then determined that both heterozygously and homozygously humanized Stmn2 mice (herein referred as Stmn2 Hum/+ and Stmn2 Hum/Hum ) are viable and fertile.
• FIG. 3A Using cultured primary cortical neurons from these Stmn2 Hum/+ mice (FIG. 3A), the processing of the humanized Stmn2 pre-mRNA was validated as dependent upon TDP-43. Indeed, ASO-mediated reduction of TDP- 43 in these humanized primary neurons (FIG. 3B) elicited reduction in full length stathmin-2 mRNA (FIG. 3C), with both cryptic splice and poly adenylation sites used to produce a truncated Stmn2 mRNA (in which mouse exon 1 was spliced into human exon 2a - FIG. 3D).
• testing was performed to determine whether mutating the TDP-43 binding sites led to stathmin-2 misprocessing in mouse N2a neuronal cells engineered to carry a humanized exon 2a.
• a CRISPR/Cas9 engineering approach was used to replace the 24 nucleotides containing the three GU-motifs bound by TDP-43 with a 19 nucleotide MS2 aptamer sequence known to adopt an RNA stem-loop structure which is bound with high affinity by the MS2 coat protein (MCP) (FIG. 2B).
• MCP MS2 coat protein
• stathmin-2 misprocessing led to chronic production of truncated stathmin-2 transcripts (FIG. 4B) and reduced levels of full length stathmin-2 mRNA (FIG. 4A) and protein (FIG. 4C).
• FIG. 4B truncated stathmin-2 transcripts
• FIG. 4A full length stathmin-2 mRNA
• FIG. 4C protein

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Health & Medical Sciences (AREA)
• Organic Chemistry (AREA)
• Zoology (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Wood Science & Technology (AREA)
• Biophysics (AREA)
• Biochemistry (AREA)
• Molecular Biology (AREA)
• General Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Environmental Sciences (AREA)
• Analytical Chemistry (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Biomedical Technology (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Gastroenterology & Hepatology (AREA)
• Biodiversity & Conservation Biology (AREA)
• Toxicology (AREA)
• Animal Husbandry (AREA)
• Animal Behavior & Ethology (AREA)
• Immunology (AREA)
• Plant Pathology (AREA)
• Pathology (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Peptides Or Proteins (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Priority Applications (6)

Applications Claiming Priority (2)

Publications (1)

Family

ID=86397888

Family Applications (1)

Country Status (7)

Families Citing this family (1)

Citations (1)

Family Cites Families (1)

• 2022
  • 2022-11-21 IL IL312770A patent/IL312770A/en unknown
  • 2022-11-21 WO PCT/US2022/080224 patent/WO2023092118A1/en not_active Ceased
  • 2022-11-21 CN CN202280077093.0A patent/CN118284707A/zh active Pending
  • 2022-11-21 US US18/711,686 patent/US20250040523A1/en active Pending
  • 2022-11-21 EP EP22896792.3A patent/EP4436586A4/en active Pending
  • 2022-11-21 JP JP2024529784A patent/JP2024539458A/ja active Pending
  • 2022-11-21 KR KR1020247020449A patent/KR20240116486A/ko active Pending

Patent Citations (1)

Non-Patent Citations (4)

Also Published As

Similar Documents

Legal Events