Compositions for inducing direct cross-differentiation of somatic cells into motor neurons and methods of using same - Patents.com

Abstract

The present invention relates to a composition for inducing direct cross-differentiation of somatic cells into motor neurons, motor neurons in which direct cross-differentiation has been induced by introducing the composition into somatic cells, pharmaceutical compositions for preventing or treating nervous system diseases and injuries, compositions for screening cell therapeutic agents and agents for preventing or treating nervous system diseases and injuries, and biomaterial compositions for 3D printer technology for producing artificial tissues. One embodiment of the composition can be used to efficiently induce differentiation of somatic cells into motor neurons without passing through the pluripotency stage of dedifferentiated stem cells through the expression of direct cross-differentiation-inducing factors, enabling effective prevention and treatment of nervous system diseases and injuries. Furthermore, the induced motor neurons of one embodiment can be used as a biomaterial composition for 3D printer technology for producing therapeutic artificial tissues that can treat nervous system diseases and injuries that are previously known to be intractable, and can be widely used in the field of regenerative medicine.

Description

[Technical field]

[0001] The present invention relates to a composition for inducing direct cross-differentiation from somatic cells to motor neurons, motor neurons in which direct cross-differentiation is induced by introducing the composition into somatic cells, a pharmaceutical composition for preventing or treating nervous system diseases and injuries, a cell therapeutic agent, a composition for screening for preventive or therapeutic agents for nervous system diseases and injuries, and a biomaterial composition for 3D printer technology aimed at producing artificial tissues. [Background technology]

[0002] In the existing methods of differentiation of motor neurons using embryonic stem cells and dedifferentiated stem cells, the embryo must be destroyed to establish embryonic stem cells, or somatic cells must be reprogrammed into dedifferentiated stem cells and then differentiated into motor neurons. In addition, the conventional methods may have ethical issues in that they use embryonic stem cells, and in the case of dedifferentiated stem cells, the differentiation step requires time, money, and labor, but the yield is low and it is difficult to artificially control the differentiation ability, making them inefficient. In addition, it is difficult to secure a sufficient number of cells required for drug metabolism and toxicity verification at the in vitro level, and there is a high possibility of teratoma formation derived from undifferentiated cells at the stage of applying cell therapy to regenerate the function of motor neurons, which raises safety issues.

[0003] In order to eliminate the risk of teratoma formation as described above, research is being conducted to directly cross-differentiate somatic cells into somatic cells of other lineages or multipotent stem cells without the intervention of pluripotent stem cells, but the mechanism of such a reprogramming process has only been revealed to a very small extent because the number of genes used in the induction is too large to elucidate the detailed mechanism. In addition, even though reverse differentiation techniques have been established, technically simple and efficient methods must be continuously developed in order to apply them to actual treatments, and any such developed techniques are limited because they must be evaluated in terms of efficiency and safety. Furthermore, a method for producing motor neurons by expressing only a few genes from somatic cells is not yet known.

[0004] While conducting research into a method for direct cross-differentiation of somatic cells into motor neurons, the inventors discovered that by introducing a transcription factor of a specific gene into somatic cells and inducing ectopic expression, somatic cells can be effectively induced to become neuronal cells, thus completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0005] One aspect relates to a composition for inducing direct cross-differentiation of somatic cells into motor neurons, comprising: (1) one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein; (2) a nucleic acid molecule encoding the protein; and (3) one or more vectors into which the nucleic acid molecule has been introduced.

[0006] Another aspect relates to a method for directly cross-differentiating a somatic cell into a motor neuron, comprising the step of contacting or inserting into a somatic cell a composition comprising one or more proteins selected from the group consisting of (1) OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, (2) a nucleic acid molecule encoding the protein, and (3) a vector into which the nucleic acid molecule has been introduced.

[0007] Another aspect relates to a motor neuron in which direct cross-differentiation has been induced by introducing into a somatic cell one or more of the following: (1) a nucleic acid molecule encoding one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box11) protein; and (2) a vector containing the nucleic acid molecule.

[0008] Another aspect relates to a pharmaceutical composition and a cell therapeutic agent for preventing or treating nervous system diseases and injuries, comprising as an active ingredient a motor neuron induced by direct cross-differentiation.

[0009] Another embodiment relates to a composition for screening a preventive or therapeutic drug for nervous system diseases and injuries, comprising as an active ingredient a motor neuron induced by direct cross-differentiation.

[0010] Other aspects relate to biomaterial compositions for 3D printer technology for the creation of artificial tissues for the treatment of nervous system diseases and injuries.

[0011] Another aspect relates to a method for preventing or treating nervous system diseases and injuries, comprising the step of administering the composition for direct cross-differentiation induction or motor neurons induced by direct cross-differentiation to an individual in need thereof. [Means for solving the problem]

[0012] To achieve the above-mentioned object, one embodiment provides a composition for inducing direct cross-differentiation from somatic cells to motor neurons, comprising: (1) one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein; (2) a nucleic acid molecule encoding the protein; and (3) one or more vectors into which the nucleic acid molecule has been introduced.

[0013] Another aspect provides a method for directly cross-differentiating a somatic cell into a motor neuron, comprising the step of contacting or inserting into a somatic cell a composition comprising one or more proteins selected from the group consisting of (1) OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, (2) a nucleic acid molecule encoding the protein, and (3) a vector into which the nucleic acid molecule has been introduced.

[0014] Another aspect provides a motor neuron in which direct cross-differentiation has been induced by introducing into a somatic cell (1) a nucleic acid molecule encoding one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box11) protein, and (2) one or more vectors into which the nucleic acid molecule has been introduced.

[0015] Another aspect is to provide a pharmaceutical composition and a cell therapeutic agent for preventing or treating nervous system diseases and injuries, comprising as an active ingredient a motor neuron induced by direct cross-differentiation.

[0016] Another embodiment provides a composition for screening a preventive or therapeutic agent for nervous system diseases and injuries, comprising as an active ingredient a motor neuron induced by direct cross-differentiation.

[0017] Another embodiment provides biomaterial compositions for 3D printer technology for the creation of artificial tissues for the treatment of nervous system diseases and injuries.

[0018] Another aspect provides a method for preventing or treating nervous system diseases and injuries, comprising administering to an individual in need thereof the composition for inducing direct cross-differentiation or motor neurons induced by direct cross-differentiation.

[0019] The present invention will now be described in further detail.

[0020] The term "Direct Conversion" refers to the process of inducing conversion between mature (completely differentiated) cells with completely different cell types in higher organisms. (Kim, J. et al, Neurobiol. 22, 778-784, 2012) This is different from the conventional technology that requires reprogramming into induced pluripotent stem cells (iPSCs) and then redifferentiating them into the desired cells, in that it directly induces conversion into the desired cells without going through the induced pluripotent stem cell stage. Currently, direct cross-differentiation is recognized as a technology that can be used for disease modeling and new drug development, and can also be applied to gene therapy and regenerative medicine.

[0021] In one embodiment, the composition for direct cross-differentiation induction provided herein comprises one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein; (2) a nucleic acid molecule encoding the protein; and (3) one or more vectors into which the nucleic acid molecule has been introduced.

[0022] In one embodiment, the composition may comprise one or more of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, and / or a nucleic acid molecule encoding one or more of these proteins. For example, the composition can include OCT4, LHX3, ISL1, NKX6.1, NGN2 or SOX11, and can include combinations of OCT4 and LHX3, OCT4 and ISL1, OCT4 and NKX6.1, OCT4 and NGN2, OCT4 and SOX11, OCT4, LHX3 and ISL1, OCT4, LHX3 and NKX6.1, OCT4, LHX3 and NGN2, and OCT4, LHX3 and SOX11.

[0023] In one embodiment, the nucleic acid molecules encoding OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein can be used in a state inserted into a vector expressing OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, which contains the nucleic acid molecule encoding the protein.

[0024] In the present invention, the term "vector" refers to an expression vector capable of expressing a target protein in an appropriate host cell, and may refer to a gene carrier containing the necessary regulatory elements operably linked to express a gene insert.

[0025] The vector of the present invention includes expression regulatory elements such as a promoter, an operator, a start codon, a stop codon, a polyadenylation signal, an enhancer, and a signal sequence or a leader sequence for membrane targeting and secretion, and can be produced in a variety of ways depending on the purpose. The promoter of the vector can be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting host cells containing the vector, and includes a replication origin if it is a replicable expression vector. The vector can be self-replicating or integrated into the host DNA.

[0026] In a specific embodiment, a nucleic acid molecule encoding one or more proteins selected from the group consisting of OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, or a vector containing the nucleic acid molecule, was introduced into somatic cells, and direct cross-differentiation into motor neurons was confirmed.

[0027] Furthermore, the composition of the present invention can introduce a transcription factor capable of directly inducing cross-differentiation of motor neurons via a vector, and the vector that can be used in the present invention may be, for example, one or more vectors selected from the group consisting of a plasmid vector, a cosmid vector, a virus vector, a lentivirus vector, a retrovirus vector, a human immunodeficiency virus (HIV) vector, a murine leukemia virus (MLV) vector, an avian sarcoma / leukosis (ASLV) vector, a spleen necrosis virus (SNV) vector, a rous sarcoma virus (RSV) vector, a mouse mammary tumor virus (MMTV) vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, and an episomal vector.

[0028] The OCT4 (Octamer-binding transcription factor 4) protein, the LHX3 (LIM homeobox 3) protein, the ISL1 (ISL LIM homeobox 1) protein, the NKX6.1 (NK6 homeobox 1) protein, the NGN2 (neurogenin 2) protein, and the SOX11 (SRY-box 11) protein may be provided in the form of a nucleic acid molecule encoding the protein, or a vector into which the nucleic acid molecule has been introduced. OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein may include all OCT4 (Octamer-binding transcription factor 4) proteins, LHX3 (LIM homeobox 3) proteins, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) proteins from mammals such as humans, horses, sheep, pigs, goats, camels, antelopes, and dogs.Furthermore, the nucleic acid molecules encoding the OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein that can be used in the present invention include not only proteins having wild-type amino acid sequences, but also OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, which contain nucleic acid molecules encoding the above proteins. The nucleic acid molecules encoding the ISL1 (ISL LIM homeobox 1) protein, the NKX6.1 (NK6 homeobox 1) protein, the NGN2 (neurogenin 2) protein, and the SOX11 (SRY-box 11) protein may include one or more protein variants (e.g., subtypes of each protein) selected from the group consisting of the OCT4 (Octamer-binding transcription factor 4) protein, the LHX3 (LIM homeobox 3) protein, the ISL1 (ISL LIM homeobox 1) protein, the NKX6.1 (NK6 homeobox 1) protein, the NGN2 (neurogenin 2) protein, and the SOX11 (SRY-box 11) protein, which comprise the nucleic acid molecules encoding the proteins.

[0029] The variants of OCT4 (Octamer-binding transcription factor 4), LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2), and SOX11 (SRY-box 11) proteins refer to proteins that have a sequence that differs from the native amino acid sequence of OCT4 (Octamer-binding transcription factor 4), LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2), and SOX11 (SRY-box 11) proteins by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, and that maintain the inherent biological function of the wild-type protein. The variants may be functional equivalents that exhibit the same biological activity as the native protein, or may be variants in which the physicochemical properties of the protein have been altered as necessary, such as variants with increased structural stability against physical and chemical environments or increased physiological activity.

[0030] The OCT4 (Octamer-binding transcription factor 4) protein, the LHX3 (LIM homeobox 3) protein, the ISL1 (ISL LIM homeobox 1) protein, the NKX6.1 (NK6 homeobox 1) protein, the NGN2 (neurogenin 2) protein, and the SOX11 (SRY-box 11) protein or variants thereof may be isolated from nature, recombinantly or synthetically produced (non-naturally occurring).

[0031] Furthermore, nucleic acids encoding OCT4 (Octamer-binding transcription factor 4), LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2), and SOX11 (SRY-box 11) proteins are base sequences encoding said proteins in wild-type or mutant form as described above, in which one or more bases may be mutated by substitution, deletion, insertion, or a combination thereof, and may be isolated from nature or produced using chemical synthesis methods. Nucleic acids having base sequences encoding OCT4 (Octamer-binding transcription factor 4), LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2), and SOX11 (SRY-box 11) proteins may be single-stranded or double-stranded and may be DNA molecules (genomic, cDNA) or RNA (mRNA) molecules.

[0032] In a specific embodiment, the direct cross-differentiation factors OCT4 or OCT4 and LHX3 were introduced into fibroblasts selected as representative examples of somatic cells via lentivirus, and the factors were ectopically expressed to directly cross-differentiate the fibroblasts into motor neurons. In addition, reverse transcription polymerase chain reaction (RT-PCR) was performed on the induced motor neurons, and it was confirmed that the induced motor neurons showed motor neuron markers different from those of the fibroblasts before the direct cross-differentiation factors were introduced.

[0033] The term "somatic cells" may refer to any cell except germ cells, and may be derived from or isolated from mammals, such as humans, horses, sheep, pigs, goats, camels, antelopes, and dogs. The composition of the present invention may also induce direct cross-differentiation of somatic cells into motor neurons, and the somatic cells may be one or more selected from the group consisting of fibroblasts, epithelial cells, muscle cells, nerve cells, hair cells, hair root cells, hair follicle cells, oral epithelial cells, somatic cells extracted from urine, gastric mucosa cells, goblet cells, G cells, B cells, pericytes, astrocytes, blood cells, neural stem cells, hematopoietic stem cells, umbilical cord blood stem cells, and mesenchymal stem cells, but are not limited thereto. However, in a specific embodiment of the present invention, the somatic cells were used as fibroblasts.

[0034] The term "motor neuron" refers to a type of neuron that is part of the nervous system and transmits signals from the central nervous system, such as the spinal cord, to effectors, such as muscles and glands, to perform functions. Additionally, "induced motor neuron" can refer to a motor neuron that is induced from a somatic cell, for example, by direct cross-differentiation according to one embodiment.

[0035] In one embodiment, the direct cross-differentiation method can include the steps of culturing somatic cells in a medium, transfecting the cultured somatic cells with vectors containing OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein genes, and culturing the infected somatic cells under culture conditions capable of inducing direct cross-differentiation. In addition, the direct cross-differentiation method may further include a step of transfecting somatic cells with OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein genes.

[0036] The medium used for culturing the somatic cells includes all media commonly used in the art for culturing somatic cells. The medium used for culturing generally contains a carbon source, a nitrogen source and trace element components. In a specific embodiment of the present invention, a medium containing protamine sulphate is used, but is not limited thereto.

[0037] Furthermore, the culture conditions capable of inducing direct cross-differentiation of the somatic cells may include media and / or conventional culture conditions that are commonly used in the art for inducing direct cross-differentiation of somatic cells.

[0038] Through the step of introducing the composition for direct cross-differentiation induction into somatic cells, ectopic expression of direct cross-differentiation factors such as OCT4 (Octamer-binding transcription factor 4), LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2), and SOX11 (SRY-box 11) can be induced. Ectopic expression means that a gene is expressed outside the tissue or cell in which it is originally expressed, or is expressed at a time different from the time when it is originally expressed. According to the method of the present invention, motor neurons can be effectively produced from somatic cells.

[0039] In one embodiment, the motor neurons induced by the disclosed method and composition are characterized by not expressing markers derived from original cells, but simply expressing markers expressed in motor neurons.In one embodiment, to obtain motor neurons induced to directly cross-differentiate, a composition comprising OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein can be utilized to induce direct cross-differentiation. Direct cross-differentiation into motor neurons was confirmed with a composition containing a nucleic acid molecule encoding OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, or a vector into which the nucleic acid molecule was introduced. In a specific embodiment, a composition containing a nucleic acid molecule encoding OCT4 (Octamer-binding transcription factor 4) protein or a vector into which the nucleic acid molecule was introduced further contains a nucleic acid molecule encoding LHX3 (LIM homeobox protein 3) or a vector into which the nucleic acid molecule was introduced, and direct cross-differentiation from somatic cells to motor neurons by the composition was confirmed.In addition, motor neurons in which direct cross-differentiation has been induced can be provided by one or more of the following proteins contained in the composition: OCT4 (Octamer-binding transcription factor 4) protein, LHX3 (LIM homeobox 3) protein, ISL1 (ISL LIM homeobox 1) protein, NKX6.1 (NK6 homeobox 1) protein, NGN2 (neurogenin 2) protein, and SOX11 (SRY-box 11) protein, and / or a nucleic acid molecule encoding one or more of these proteins.

[0040] The term "prophylaxis" refers to any action that prevents nervous system diseases and injuries by eliminating their etiology or detecting their early stages.

[0041] The term "treatment" refers to any action in which the symptoms of nervous system diseases and injuries are ameliorated or beneficially altered by the compositions of the present invention.

[0042] The nervous system disease and injury that can be prevented or treated may be, for example, one or more selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic-ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Alzheimer's disease, congenital metabolic nervous system disease, and traumatic brain injury, but is not limited thereto, and may include all diseases and / or pathological symptoms caused by decreased, lost, and / or abnormal function of the nervous system.

[0043] The composition may further comprise one or more known active ingredients that have preventive or therapeutic effects on nervous system diseases and injuries in addition to induced motor neurons.

[0044] The composition may further include a pharma- ceutically acceptable additive, and in this case, examples of the pharma- ceutically acceptable additive that can be used include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, and persaccharose. The pharma- ceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight relative to the composition, but is not limited thereto.

[0045] The composition can be administered in various oral or parenteral formulations in actual clinical administration, and can be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used when formulating, and it is preferable to use suitable formulations known in the art that are disclosed in the literature (Remington's Pharmaceutical Science, recently published by Mack Publishing Company, Easton PA). Carriers, excipients, and diluents that can be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0046] The preferred dosage of the composition varies depending on the individual's condition and body weight, the degree of the disease, the drug form, the administration route and period, but can be appropriately selected by those skilled in the art. For a preferred effect, the motor neurons in which direct cross-differentiation is induced according to one embodiment can be administered once a day to several times a day at regular intervals, for example, about 1,000 to 10,000 cells / time, 1,000 to 100,000 cells / time, 1,000 to 1,000,000 cells / time, 1,000 to 10,000,000 cells / time, 1,000 to 100,000,000 cells / time, 1,000 to 1,000,000,000 cells / time, or 1,000 to 10,000,000,000 cells / time, based on an adult patient weighing 70 kg.

[0047] The composition can be administered to an individual by various routes, all manner of administration being contemplated, for example it can be administered orally, rectally or by intravenous, intramuscular or subcutaneous injection, it can be administered by application to the skin or any other route.

[0048] The term "individual" refers to a subject in need of treatment for nervous system diseases and injuries, and more specifically refers to mammals, such as human or non-human primates, mice, rats, dogs, cats, horses and cows.

[0049] In one embodiment, the composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to prevent or treat nervous system diseases and injuries. The pharmaceutical composition comprising the directly cross-differentiated motor neurons can further include HB9 protein.

[0050] The term "cell therapy" refers to a therapy using autologous cells, allogenic cells, or xenogenic cells to restore tissue function and to be used in the suppression of cancer. When the motor neurons in which direct cross-differentiation has been induced are included as an active ingredient, they can be used as a cell therapy for the treatment or prevention of nervous system diseases and injuries.

[0051] The cell therapy agent may further include a pharma- ceutically acceptable carrier, which may be, for example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, HSA (human serum albumin), or a mixture of one or more of these components, and may contain other conventional additives such as antioxidants, buffers, and bacteriostatic agents, if necessary.

[0052] The cell therapeutic agent may be further supplemented with, for example, a diluent, a dispersant, a surfactant, a binder, and a lubricant, and may be formulated into an injectable preparation such as an aqueous solution, suspension, or emulsion.

[0053] The composition for screening a preventive or therapeutic agent for nervous system diseases and injuries can be usefully used in screening a therapeutic agent for nervous system diseases and injuries by the method of confirming the reactivity of the motor neurons whose differentiation has been induced in the present invention in the presence and absence of a candidate substance for treating nervous system diseases and injuries. For example, the motor neurons whose differentiation has been induced in one embodiment can be used to evaluate the toxicity or efficacy of a candidate substance as an important cell for the recovery or treatment of nervous system diseases and injuries.

[0054] The toxicity is evaluated in the presence and absence of a therapeutic candidate substance of one embodiment, or by IC 50The efficacy can be evaluated according to a method generally used in the art for determining toxicity, etc. Furthermore, the efficacy can be evaluated according to a method that can be used in the art to confirm the efficacy of the treatment of nervous system diseases and injury diseases, such as promoting the regeneration of injured nerves by motor neurons induced to differentiate in the present invention in the presence and absence of one embodiment of a therapeutic candidate substance.

[0055] The 3D printing technology is a technology for printing three-dimensional (3D) solid materials, and the biomaterial composition of the 3D printer technology refers to biocompatible polymers, natural polymers, biomolecules, bioactive substances, and cells that have characteristics such as biomimetic, small tissues, and autonomous self-organization.

[0056] According to one aspect, motor neurons induced by direct cross-differentiation can be additively manufactured into desired shapes or patterns, enabling the fabrication of artificial tissues for the treatment of nervous system diseases and injuries, which can be widely used in the field of regenerative therapy.

[0057] Duplicate content will be omitted in consideration of the complexity of this specification, and terms not specifically defined in this specification have the meanings commonly used in the technical field to which this invention belongs. Effect of the Invention

[0058] By using the composition of one embodiment, it is possible to efficiently induce differentiation from somatic cells into motor neurons without going through the pluripotency stage of reverse differentiation stem cells through the direct expression of cross-differentiation inducers, and by using this, it is possible to effectively prevent and treat nervous system diseases and injuries. In addition, the induced motor neurons of one embodiment can be used as a therapeutic pharmaceutical composition that can treat nervous system diseases and injuries that are known to be intractable diseases, and as a biomaterial composition for 3D printer technology for producing artificial tissues, and can be widely used in the field of regenerative medicine. [Brief description of the drawings]

[0059] [Figure 1A]FIG. 1A is a schematic diagram of the process of direct cross-differentiation of fibroblasts into motor neurons. [Figure 1B] FIG. 1B relates to the results of identifying the histological morphology of induced motor neurons prepared by 3D culture. [Diagram 2] FIG. 2 shows the results of confirming the expression of the HB9 gene in motor neurons induced by single or combined direct cross-differentiation factor genes (FIG. 2A), and the results of confirming the efficiency of direct cross-differentiation of motor neurons induced by the factors (FIG. 2B). [Diagram 3] FIG. 3 shows the results of immunofluorescence staining of induced motor neurons. [Figure 4] FIG. 4 shows the results of confirming the motor neuron markers expressed in induced motor neurons (induced motor neuron intermediate cells, induced motor neuron cells produced by OCT4 infection, and induced motor neuron cells infected with OCT4 and then treated with LHX3). [Diagram 5] FIG. 5 shows the results of confirming the relative difference in gene expression levels observed in induced motor neurons compared with fibroblasts, the cells from which the neurons were derived. [Figure 6] Figure 6 shows the results of comparing the therapeutic effect on nerve damage in spinal cord injury model mice with that in the control group of mice, based on the BBB score, an index that can be used to confirm the accuracy of motor function recovery (Figure 6A), and the results of H&E staining of the spinal cord sections (Figure 6B). MODE FOR CARRYING OUT THEINVENTION

[0060] The present invention will be described in detail below with reference to examples and experimental examples to aid in understanding the present invention. However, the following examples and experimental examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples and experimental examples. The examples and experimental examples of the present invention are provided to more completely explain the present invention to those having average knowledge in the art.

[0061] (Experimental Example 1) <Cloning and packaging of lentiviral plasmids>

[0062] Transcription factors (OCT4 and LHX3) were amplified by polymerase chain reaction (PCR) using HepG2 cDNA. The transcription factors were then introduced into lentiviral vectors and confirmed by sequence analysis. Lentiviral packaging was performed using XtremeGENE 9 DNA transfection reagent (Roche, 06365787001) by adding lentiviral transfer plasmid, packaging plasmid (psPAX2), and envelope plasmid (VSV-G) in a ratio of 3:2:1, respectively. The mixture was then added to DMEM to a total volume of 200 μl and transfected into 50% confluent 293T cells for 48 h. The cell culture medium was centrifuged at 80,000 g for 1 hour and 30 minutes in an ultracentrifuge, after which the lentiviral pellet obtained in the centrifugation supernatant was diluted in 1 ml of DMEM (Gibco, 10313-021) and used for transformation.

[0063] (Experimental Example 2) <Cell culture medium and preparation of induced motor neurons (iMN)>

[0064] Dermal fibroblasts were cultured at 3 × 10 on gelatin-coated 6-well plates in 10% FBS culture medium. 4They were dispensed with cells. One day later, fibroblasts were infected with pMX retroviral vectors expressing OCT4, LHX3, ISL1, NKX6.1, NGN2, and SOX11 respectively, OCT4 and LHX3, OCT4 and ISL1, OCT4 and NKX6.1, OCT4 and NGN2, OCT4 and SOX11, OCT4, LHX3 and ISL1, OCT4, LHX3 and NKX6.1, OCT4, LHX3 and NGN2, OCT4, LHX3 and SOX11 in a medium containing 6 μg / ml protamine sulfate to induce motor neurons. Thereafter, the virus supernatant was removed and replaced with fresh medium. After infecting fibroblasts with the vector, the prepared iMNs were plated on a PDL / laminin-coated 4-well plate which is a motor neuron differentiation medium. After plating, the cells were cultured, the neuron cell groups were mechanically separated, and the mature iMNs were picked from the gelatin-coated plate or trypsinized for subculture.

[0065] (Experimental Example 3) <RNA Extraction and cDNA Synthesis>

[0066] Total RNA of cell lysates was extracted with RiboEX (Geneall, 301 - 001). RNA extraction was performed according to the manufacturer's protocol. 500 ng of RNA and oligo dT primer were added to a total of 20 μl of reaction mixture, and cDNA was synthesized with M-MuLV reverse transcriptase (NEB, M0253L).

[0067] (Experimental Example 4) <RT-PCR Analysis>

[0068] Reverse transcription polymerase chain reaction (RT-PCR) was performed at 58°C for 35 cycles using Taq polymerase (Invitrogen, 10342-020) and primer pairs. PCR products were subjected to gel electrophoresis at 100V for 25 min on 1% agarose gels, which were performed in triplicate for motor neuron marker genes and motor neuron precursor and neuroectoderm markers, and normalized with the housekeeping gene Gapdh. Gene expression was measured and analyzed by the Ct value calculation method, and all experiments were performed according to the manufacturer's protocol.

[0069] (Experimental Example 5) <Immunofluorescence staining>

[0070] Cells were fixed with 4% paraformaldehyde (Tech&Innovation, BPP-9004) for 10 min at room temperature. Fixed cells were washed three times with Dulbecco′s Phosphate-Buffered Saline (DPBS, Corning, 21-031-CV). Then, cells were permeabilized with DBPS containing 0.1% Triton X-100 (Sigma, T9284) for 10 min at room temperature. After washing three times with DPBS, nonspecific binding was blocked by reacting with DPBS containing 4% FBS for 1 h at room temperature. Cells were then incubated with primary antibodies for 1 h at room temperature, followed by washing three times with DPBS containing 0.05% Tween 20 (Sigma, P7949). Secondary fluorescent antibodies were then treated with samples and incubated for 1 h in the dark. If double staining was required, an additional blocking step was performed for 30 min before treating with primary antibodies following the procedure described above.

[0071] (Experimental Example 6) <Microarray Analysis>

[0072] Microarray analysis was used to profile and compare the total gene expression of fibroblasts, fibroblast-induced motor neuron intermediate cells (iMN intermediate cells) after OCT4 infection, induced motor neuron cells produced by OCT4 infection alone (Mock_iMN), and induced motor neuron cells produced by OCT4 infection followed by LHX3 treatment (LHX3_iMN). Total RNA was isolated using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Samples were hybridized to Affymetrix arrays. Normalization was performed using the Robust Multi-array Analysis (RMA) algorithm. Data processing and graphics were performed in Matlab, which was developed in-house. Hierarchical clustering of genes and samples was performed by the one minus correlation metric and the unweighted average distance (UPGMA) linkage method.

[0073] (Experimental Example 7) <Production of a spinal cord injury model and transplantation of induced motor neurons>

[0074] The spinal cord injury model was created in 6-week-old male rats by using an air delivery device to injure the spinal cord at thoracic level 9 (T9). One week after injury, the harvested iMNs were labeled with DiI and injected with 1 × 10 5 iMNs / rat were stereotaxically injected into the spinal cord at thoracic level 8 (T8) and thoracic level 10 (T10), respectively. Rats were given water and food regularly, and their bladders were massaged for voiding. The animal experiment process was approved by the animal facility and the IBR committee of Ulsan University of Science and Technology. The animal experiment process was in accordance with the National Institutes of Health guidelines for animal research.

[0075] Example 1. Direct cross-differentiation of induced motor neurons (iMNs)

[0076] To induce motor neurons, skin fibroblasts were planted in culture medium and transformed with OCT4, a transcription factor that induces motor neuron induction, via a lentivirus expression system for 24 hours to produce induced motor neuron intermediate cells (iMN intermediate cells). LHX3 was then further introduced into the cells via a lentivirus expression system to produce induced motor neurons, or induced motor neurons without LHX3 were produced. The skin tissue was then attached to a gelatin-coated culture dish and cultured for use. The experimental process is shown diagrammatically in Figure 1A, and the histological morphology of the induced motor neurons in 3D culture is shown in Figure 1B.

[0077] As shown in Figure 1B, aggregates were formed about 20 days after transfection with lentivirus, and after 5 days of 3D culture, a typical motor neuron morphology was observed. From the above results, it was confirmed that motor neurons can be effectively generated by treating somatic fibroblasts with OCT4 and LHX3 to induce ectopic expression of the factors. In particular, it was confirmed that the motor neurons induced by the above process exhibited the characteristic morphology seen in actual motor neurons.

[0078] Example 2: Direct cross-differentiation of induced motor neurons (iMNs)

[0079] An experiment was carried out to confirm whether crossover efficiency is effectively achieved when direct crossover differentiation is induced into motor neurons by introducing a single gene of a direct crossover differentiation factor and a combination of two or more genes into somatic cells, and the direct crossover efficiency between the combination of the direct crossover differentiation factors. Motor neurons were produced using a single gene of a direct crossover differentiation factor and its combination under the same conditions as in Experimental Example 2, and the expression of HB9, a motor neuron marker for direct crossover differentiation, was confirmed in the produced motor neurons. The results are shown in Figure 2A, and the results of measuring the efficiency of direct crossover differentiation between a single gene and its combination are shown in Figure 2B.

[0080] As shown in Figure 2A, the expression of the motor neuron marker HB9 was confirmed to be lower in the following order for single direct cross differentiation factors: OCT4, LHX3, ISL1, NKX6.1, NGN2, and SOX11. For combination factors, the expression level of the motor neuron marker HB9 was confirmed to be more than 500 times higher than that of fibroblasts in the combinations of OCT4 and LHX3, OCT4, LHX3 and ISL1, OCT4, LHX3 and NKX6.1, OCT4, LHX3 and NGN2, and OCT4, LHX3 and SOX11. In addition, as shown in Figure 2B, it was confirmed that, for the genes of single direct cross differentiation factors, cells expressing HB9 protein, a motor neuron marker, had the highest direct cross differentiation efficiency. In addition, it was confirmed that the efficiency of all combinations of combined direct cross differentiation factors was 60% or higher, confirming that the efficiency of direct cross differentiation was higher than that of single factors.

[0081] Example 3: Identification of the characteristics of motor neurons induced by direct cross-differentiation

[0082] In order to confirm whether the induced motor neurons (iMNs) in Examples 1 and 2 exhibit characteristics of actual motor neurons, not just morphology, we performed Western blotting and RT-PCR analysis, and simultaneously performed immunofluorescence staining. Experiments were performed according to the methods disclosed in Experimental Examples 4 to 6. The results of immunofluorescence staining are shown in Figure 3, the results of confirming gene markers expressed through Western blotting are shown in Figure 4, and the results of confirming the difference in relative gene expression levels appearing in the induced motor neurons compared to the fibroblasts from which they were derived are shown in Figure 5.

[0083] As shown in Figure 3, the iMNs produced strongly expressed the motor neuron markers HB9, ISLT1, NKX6.1, MAP2, and TUJ1, which were not expressed in fibroblasts. As shown in Figure 4, the intermediate cells of the induced motor neurons (iMN intermediate cells) expressed the ISLT1 and MAP2 markers, and the group in which motor neurons were induced with OCT4 alone (+Mock_iMN) expressed ISLT1 and MAP2 as well as LIM1. The group of motor neurons induced by further introducing LHX3 (+LHX3_iMN) expressed all of LIM1, HB9, ISLT1, NKX6.1, and MAP2.

[0084] As shown in Figure 5, the expression of ISLT1 was significantly increased in the intermediate cells of the induced motor neurons, and the expression of ISLT1, MAP2, and LIM1 was confirmed to increase by more than 10-fold in Mock_iMN, a group in which motor neurons were induced with OCT4 alone. The expression of LIM1, HB9, ISLT1, NKX6.1, SCSL1, and MAP2 was confirmed to increase by more than 10-fold in LHX3_iMN, a group of motor neurons induced by further introducing LHX3.

[0085] From the above results, it was confirmed that the iMNs we created were identical to actual motor neurons not only in morphology but also in the clear expression of motor neuron markers. However, as shown in Figures 3 and 4, it was confirmed that the expression patterns of motor neuron markers that can be confirmed depending on the combination of directly cross-reactive transcription factors used may be slightly different.

[0086] Therefore, taking the above results together, it was confirmed that motor neurons induced from fibroblasts are identical to actual motor neurons, and that motor neurons can be efficiently induced according to the method disclosed in the above examples.

[0087] Example 4 Identification of the therapeutic effect of iMN administration to spinal cord injury animal models

[0088] An experiment was conducted to confirm whether the motor neurons prepared according to the above embodiment have a therapeutic effect when administered to a spinal cord injury animal model. The experimental method was the same as in Experimental Example 7, and the therapeutic effect of nerve injury in a mouse spinal cord injury model administered with iMN and additionally induced iMN and HB9 was compared with that in a control group of mice. The results were confirmed by BBB score, which is an index that can confirm the degree of recovery of motor recovery function, and the results of H&E staining of the spinal cord are shown in Figure 6A and Figure 6B, respectively.

[0089] As shown in Figure 6A, the group injected with iMN showed a significant increase in the degree of motor function recovery over time, starting from the 5th week after injection, compared to the control group. In addition, the group administered with HB9 showed a steady increase in motor function recovery from the 1st week after injection, and especially from the 3rd week onwards, the motor function recovery was significantly improved. In addition, as shown in Figure 6B, when HB9 was administered in combination with the induced iMN, the amount of damaged tissue in the damaged area of ​​the spinal cord was significantly reduced, compared to the control group.

[0090] Based on the above results, it was confirmed that administration of induced motor neurons can effectively treat nervous system diseases and injuries, and that they can be used as a therapeutic composition to prevent or treat the above diseases.

Claims

1. A method for producing a cell-free nucleic acid comprising the steps of: (a) detecting a cell-free nucleic acid molecule encoding an OCT4 (Octamer-binding transcription factor 4) protein; and (b) detecting a cell-free nucleic acid molecule encoding the OCT4 protein. one or more proteins selected from the group consisting of LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2) and SOX11 (SRY-box 11), a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule has been introduced; 23. A composition for inducing direct cross-differentiation of somatic cells into motor neurons, comprising:

2. The composition comprises: one or more selected from the group consisting of an OCT4 protein, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule has been introduced; one or more selected from the group consisting of an LHX3 protein, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule has been introduced; one or more proteins selected from the group consisting of ISL1, NKX6.1, NGN2, and SOX11, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule has been introduced; 2. A composition for inducing direct cross-differentiation of somatic cells into motor neurons according to claim 1, comprising:

3. The composition for inducing direct cross-differentiation of somatic cells into motor neurons according to claim 1, characterized in that the somatic cells are at least one selected from the group consisting of fibroblasts, epithelial cells, muscle cells, nerve cells, hair cells, hair root cells, hair follicle cells, oral epithelial cells, somatic cells extracted from urine, gastric mucosal cells, goblet cells, G cells, B cells, pericytes, astrocytes, blood cells, neural stem cells, hematopoietic stem cells, umbilical cord blood stem cells, and mesenchymal stem cells.

4. The vector may be a plasmid vector, a cosmid vector, a virus vector, a lentivirus vector, a retrovirus vector, a human immunodeficiency virus (HIV) vector, a murineleukemia virus (MLV) vector, an avian sarcoma / leukosis (ASLV) vector, a spleen necrosis virus (SNV) vector, a Rous sarcoma virus (RSV) vector, a mouse mammary tumor virus (MMTV) vector, an adenovirus vector, an adeno-associated virus (AAVV) vector, or a human immunodeficiency virus (HIV) vector. The composition for inducing direct cross-differentiation from somatic cells to motor neurons according to claim 1, characterized in that the vector is one or more selected from the group consisting of a herpes simplex virus vector, a herpes simplex virus vector, and an episomal vector.

5. A method for producing a cell-free nucleic acid comprising the steps of: (a) detecting a cell-free nucleic acid molecule encoding an OCT4 (Octamer-binding transcription factor 4) protein; and (b) detecting a cell-free nucleic acid molecule encoding the OCT4 protein. one or more proteins selected from the group consisting of LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2) and SOX11 (SRY-box 11), a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule has been introduced; A method for directly cross-differentiating somatic cells into motor neurons, comprising the step of contacting or inserting a composition comprising the compound into somatic cells in vitro.

6. One or more selected from the group consisting of an OCT4 protein, a nucleic acid molecule encoding the protein, and a vector into which the nucleic acid molecule has been introduced; one or more proteins selected from the group consisting of LHX3 (LIM homeobox 3), ISL1 (ISL LIM homeobox 1), NKX6.1 (NK6 homeobox 1), NGN2 (neurogenin 2) and SOX11 (SRY-box 11), a nucleic acid molecule encoding said protein, and a vector into which said nucleic acid molecule has been introduced; Motor neurons in which direct cross-differentiation was induced by introducing the into somatic cells.

7. A pharmaceutical composition for preventing or treating nervous system diseases and injuries, comprising the composition of claim 1 or the motor neuron of claim 6 in which direct cross-differentiation has been induced as an active ingredient.

8. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 7, wherein the nervous system diseases and injuries are selected from the group consisting of spinal cord injury, Parkinson's disease, stroke, amyotrophic lateral sclerosis, motor nerve injury, peripheral nerve injury due to trauma, ischemic brain injury, neonatal hypoxic-ischemic brain injury, cerebral palsy, epilepsy, refractory epilepsy, Alzheimer's disease, congenital metabolic nervous system diseases and traumatic brain injury.

9. A cell therapeutic agent for preventing or treating nervous system diseases and injuries, comprising the motor neuron according to claim 6 as an active ingredient.

10. The pharmaceutical composition for preventing or treating nervous system diseases and injuries according to claim 7, further comprising HB9 protein.

11. A composition for screening a preventive or therapeutic agent for nervous system diseases and injuries, comprising the motor neuron induced by direct cross-differentiation according to claim 6 as an active ingredient.

12. A biomaterial composition using 3D printer technology for producing artificial tissue for treating nervous system diseases and injuries, comprising the motor neurons induced by direct cross-differentiation according to claim 6 as an active ingredient.

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