Treatment of neurological trauma

Abstract

The present invention provides pharmaceutical compositions, combination pharmaceuticals, and vectors such as expression vectors, as well as their use in methods for repairing the central nervous system after neurological injuries such as spinal cord injury (SCI) or stroke, and their use in methods for regenerating spinal cord or brain neurons in subjects suffering from spinal cord injury (SCI), stroke, or other neurotraumatic conditions.

Description

[Technical Field]

[0001] The present invention provides pharmaceutical compositions, pharmaceutical combinations, vectors such as expression vectors, and their use in methods for repairing the central nervous system after neurological injuries such as spinal cord injury (SCI) or stroke, and their use in methods for regenerating spinal cord or brain neurons in subjects suffering from spinal cord injury (SCI), stroke, or other neurotraumatic conditions. [Background technology]

[0002] Spinal cord injury occurs when the axons or nerve fibers of the spinal cord are interrupted, typically by mechanical stress. When the spinal cord is compressed, severed, or contorted, the axons physically or physiologically break down, making it impossible for nerve electrical impulses to be conducted along the length of the affected axon. Ultimately, a large population of axons and their associated cell bodies dies, causing significant loss of transmission between the brain and peripheral nerves, potentially resulting in varying degrees of functional impairment. Currently available treatments have demonstrated only limited success in functional recovery after spinal cord injury.

[0003] Similar neuropathological responses occur after stroke, in which damaged or ruptured blood vessels in the brain break down, often resulting in a large non-neurofibrous lesion core, accompanied by disruption of axonal connections and permanent loss of neurological function.

[0004] The consequences of spinal cord injury are the formation of scar tissue and post-traumatic syringomyelia. The degree of each of these post-injury diseases depends on the severity of the injury. Two types of scar tissue, namely glial and fibrous scar tissue, can be formed. Glial scars consist of a loose network of astrocytic processes connected by tight junctions. Recently, there has been a challenge to the general doctrine that glial scars are considered to be a failure of axonal regrowth in the central nervous system. Instead, astrocytes in spinal cord injury lesions have been found to express a number of axonal growth-promoting molecules that play a role in stimulating axonal regrowth past scar-forming astrocytes. Fibrous scars are composed of extracellular matrix deposition and type IV collagen, forming a strong barrier. In addition to creating a mechanical barrier, both types of scars can potentially impede neuron regeneration. Current tissue engineering research focuses on constructing a permissive environment that supports axonal regrowth at the injury site.

[0005] To date, there is no treatment option that can promote healing, reduce fibrous scarring, alleviate cellular damage after spinal cord injury or stroke, provide neuroprotection, or induce the growth and development of spinal cord cells that can replace injured or dead cells, and any one or all of which can help return an injured patient to normal or near-normal function. Accordingly, an object of the present invention is to provide such treatment options for patients with spinal cord injury or stroke. SUMMARY OF THE INVENTION

[0006] One aspect of the present invention is i) a first agent comprising an exogenous nucleic acid encoding an osteopontin (Spp1) polypeptide as set forth in SEQ ID NO: 1 or SEQ ID NO: 8, an insulin-like growth factor 1 (Igf1) polypeptide as set forth in SEQ ID NO: 2, and a ciliary neurotrophic factor (Cntf) polypeptide as set forth in SEQ ID NO: 3 or SEQ ID NO: 9, and / or ii) A second active substance comprising exogenous nucleic acids encoding fibroblast growth factor 2 (FGF2) polypeptide as shown in SEQ ID NO: 4 or SEQ ID NO: 10, epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and glial-derived neurotrophic factor (GDNF) polypeptide as shown in SEQ ID NO: 6, A combination medicine containing, If both are present, the first active substance and the second active substance described above are administered sequentially or simultaneously, and The first and second active agents described above provide a combination pharmaceutical product that results in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

[0007] Another aspect of the present invention provides a combination pharmaceutical product used in a method for treating incomplete spinal cord injury or stroke in a subject.

[0008] Another aspect of the present invention provides a combination pharmaceutical for use in a method of treating complete spinal cord injury in a subject, comprising a first active substance, a second active substance, and a third active substance.

[0009] Further embodiments of the present invention are defined by the appended claims.

[0010] The above and other purposes, features, and advantages of the subject matter presented herein will become more apparent by considering the following detailed description with reference to the attached figures illustrating several preferred embodiments of the subject matter. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the identification of Vsx2 neurons. It is a clustering dendrogram of neuronal subpopulations in the thoracic spinal cord. VEP Vsx2 neurons undergo the largest transcriptional changes after incomplete SCI. [Figure 2]This figure shows the projection patterns of long-projection Vsx2 neurons. The image shows spinal cord labeled with long-projection Vsx2 neurons traced by the virus and terminating in the lumbar spinal cord. [Figure 3] This figure shows that even when the axon is guided to just before passing the SCI lesion, walking does not recover. (Top) Even when the axon is guided past one segment of the SCI lesion using a Gdnf biomaterial depot, walking does not recover. (Bottom) When the axon is guided past two segments of the SCI lesion using a Gdnf biomaterial depot, it results in longer but lower density growth, and walking still does not recover. [Figure 4] This diagram shows axonal regeneration after complete SCI. Gene therapy-based regenerative intervention allows axons to regrow beyond the SCI lesion and reach the lumbar spinal cord. [Figure 5] This figure shows an experiment involving loss of function of Vsx2 neurons. The virus-induced destruction of regenerated Vsx2 neurons reduces walking ability. [Figure 6] This figure shows the delivery of growth factors by lentivirus after SCI. By delivering the active ingredient via a single lentiviral construct, it is possible to induce axonal regeneration and recovery of walking upon delivery after SCI. [Figure 7] This figure shows axonal growth in moderate SCI. Delivery of AAV-Igf1 / Cntf / Spp1 alone induces spinal cord intrinsic axonal growth after incomplete compression SCI. [Figure 8] This figure shows the acceleration of astrocyte boundaries in aged mice after crush-induced SCI. Lentiviral delivery of Egf / Fgf2 / Vegf limits the size of lesions after SCI and restores walking ability in aged mice. [Figure 9] This figure shows the acceleration of astrocyte boundaries in aged mice after ischemic stroke. Lentiviral delivery of Egf / Fgf2 / Vegf limits the size of lesions after ischemic stroke in aged mice. [Figure 10]This figure shows lentiviral plasmid maps. The plasmid maps are for lentiviral constructs using either the GFAP promoter or the PGK promoter. [Modes for carrying out the invention]

[0012] All documents, patents, patent applications, publications, product descriptions, and protocols referenced throughout this application constitute, by reference, part of this specification for any purpose. Publications and applications discussed herein are provided only with respect to disclosures prior to the filing date of this application. Nothing in this specification should be construed as acknowledging that the present invention does not have prior rights over such publications due to prior inventions. Furthermore, materials, methods, and examples are illustrative and not intended to limit the invention.

[0013] In case of any conflict, this specification, including its definitions, shall prevail. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the subject matter of this specification pertains. The following definitions, used herein, are provided to facilitate the understanding of the invention.

[0014] The term “comprise” is generally used to mean “include,” that is, to allow the presence of one or more features or components. Also, as used herein and in the claims, the word “comprising” may include similar embodiments described with respect to “consisting of” and / or “consisting essentially of.”

[0015] As used herein and in the claims, the singular forms ("a", "an", and "the") refer to multiple subjects unless otherwise explicitly indicated by the context.

[0016] As used herein and in the claims, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B."

[0017] As used herein, “treat,” “treatment,” “treating,” or “amelioration” refers to a therapeutic action aimed at reversing, reducing, relieving, preventing, slowing, or halting the progression or severity of a condition associated with spinal cord injury or stroke. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of spinal cord injury, such as partial or complete paralysis. Generally, a “treatment” is “effective” if one or more symptoms or clinical markers are reduced. Alternatively, a “treatment” is “effective” if the progression of the disease is reduced or halted. That is, a “treatment” includes not only improvement of symptoms or markers compared to what would be expected without treatment, but also interruption, or at least slowing, the progression or worsening of symptoms. Beneficial or desired clinical outcomes include, but are not limited to, the reduction of one or more symptoms, whether detectable or undetectable; a reduction in the severity of the disease; stabilization (i.e., non-exacerbation) of the spinal cord injury or stroke; a delay or slowing of the progression of the spinal cord injury; remission or temporary relief of the injury; remission (either partial or complete); and / or a reduction in mortality. The term “treatment” for spinal cord injury or stroke also includes the relief of symptoms or side effects of the disease (including temporary relief measures).

[0018] As used herein, the term “administering” means placing a therapeutic agent (e.g., an active agent, a viral vector, etc.) or combination drug or pharmaceutical composition disclosed herein into a subject by a method or route that results in at least partial delivery of the active agent to the subject. Combination drugs or pharmaceutical compositions containing the active agents disclosed herein may be administered to a subject by any suitable route that results in an effective treatment.

[0019] As used herein, “subject” means human or animal. Typically, animals are vertebrates such as primates, rodents, domesticated animals, or game animals. Examples of primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques, such as rhesus macaques. Examples of rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Examples of domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canid species such as dogs, foxes, and wolves, avian species such as chickens, emus, and ostriches, and fish such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein. Preferably, the subject is a mammal. Mammals may be, but are not limited to, humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. Non-human mammals may be advantageously used as subjects corresponding to animal models of spinal cord injury. Subjects may be male or female. Subjects may have a spinal cord injury or have been previously diagnosed or identified as having one or more complications associated with such injury, and may optionally have already received treatment for a spinal cord injury or one or more complications associated with such injury. Alternatively, subjects may not have been previously diagnosed with such a spinal cord injury or related complications. For example, subjects may exhibit one or more risk factors for spinal cord injury or stroke, e.g., subjects who participate in activities that may cause spinal cord injury or stroke, e.g., full-contact sports, e.g., American football, or activities that may cause one or more complications associated with spinal cord injury or stroke, or subjects who do not exhibit any risk factors. Subjects with spinal cord injury or stroke, or suspected of having spinal cord injury or stroke, may be identified by a physician using current diagnostic methods for the condition.Symptoms and / or complications of spinal cord injury or stroke that characterize and aid in the diagnosis of such injury are well known in the art and include, but are not limited to, loss or reduction of limb mobility. Tests that aid in the diagnosis of spinal cord injury include, but are not limited to, X-ray, MRI scan, myelography, somatosensory evoked potential (SSEP) testing, or CT scan.

[0020] The methods, combinations of pharmaceuticals, and pharmaceutical compositions described herein are used for the treatment of spinal cord injury or stroke. As used herein, “spinal cord injury,” or “SCI,” refers to any injury to any region of the spinal cord, such as the cervical, thoracic, lumbar, sacral, sacral, or coccyx. “Spinal cord injury” can result in a range of severity levels, from those that do not affect motor function, such as the ability to walk, to paraplegia (e.g., paralysis of the legs and lower body) and quadriplegia (e.g., loss of muscle strength in all four limbs). “Spinal cord injury” can be a complete spinal cord injury, such as an injury resulting in the complete loss of all motor and sensory functions below the site of injury. “Spinal cord injury” can be an incomplete spinal cord injury, such as an injury in which some motor function remains below the site of primary injury. Non-limited examples of incomplete spinal cord injury include, but are not limited to, anterior spinal cord syndrome, central spinal cord syndrome, and Brown-Séquard syndrome. “Spinal cord injury” can be a concussion or contusion, such as an injury that heals spontaneously, for example, in one or two days. Spinal concussion or spinal contusion may be complete or incomplete.

[0021] Complete or incomplete spinal cord injury can be assessed and classified based on the American Spinal Injury Association (ASIA) Disability Scale, or AIS, which describes a person's functional impairment as a result of SCI. This scale indicates the degree of sensation a person feels after light touch and pinprick at multiple points on the body and examines major bilateral movements of the body.

[0022] LT = Light Contact, PP = Pin Injury, DAP = Deep Anal Pressure, AIS = ASIA Disability Scale, NLI = Neurological Injury Level Grade A = Complete. No sensory or motor function remains in the sacral segments S4-S5. Grade B = Sensory impairment. Sensory function remains below the neurological level, but motor function is absent, including the sacral spinal segments S4-S5 (LT, PP, or DAP in S4-S5), and motor function is absent in any aspect of the body, more than 3 levels below the motor level. Grade C = Motor dysfunction. Motor function remains in the most caudal sacral segment for voluntary contraction of the anus, or the patient meets the criteria for sensory dysfunction (sensory function remains in the most caudal sacral segment (S4-S5) by LT, PP, or DAP), and has some modest motor function on any side of the body that is more than 3 levels below the ipsilateral motor level (this includes major or non-major muscle function to determine motor dysfunction). If AIS is C, less than half of the major muscle functions below a single NLI have a muscle grade of 3 or higher (i.e., they are not strong enough to move against gravity). Grade D = Motor Dysfunction. A state of motor dysfunction as defined above, in which at least half (more than half) of the major muscle functions below a single NLI have a muscle grade of 3 or higher (i.e., the joint can be moved against gravity).

[0023] Therefore, within the scope of the present invention, when "complete spinal cord injury" is referred to herein, it means a Grade A spinal cord injury according to AIS, whereas when "incomplete spinal cord injury" is referred to herein, it means a Grade B to Grade D spinal cord injury according to AIS.

[0024] Spinal cord injury (SCI) severs the axonal connections from the brain to the spinal cord. Because injured axons have a limited ability to regrow beyond the injury, SCI typically leads to permanent neurological loss. In certain types of incomplete SCI, rehabilitation can improve functional recovery by stimulating the reorganization of axons that escaped severance due to the incomplete injury. In contrast, individuals with anatomically complete SCI do not have axons that escaped severance beyond the injury site. Recovery from such complete injury requires stimulating the axons to regrow beyond the complete SCI lesion and to form new connections with neurons below the injury. One object of the present invention is to address this limitation in the art.

[0025] To address the limitations described above, this specification discloses a mechanism-based biological repair strategy that can induce the regeneration of a molecularly defined subpopulation of spinal cord neurons in subjects suffering from complete or incomplete spinal cord injury (SCI) or stroke, guide them to reconnect with their natural topological targets, and restore ground walking ability. The inventors applied projection-specific and relatively mononuclear RNA sequencing to elucidate the transcriptional phenotypes and connectomes of neuronal subpopulations involved in natural spinal cord repair. They identified a molecularly defined subpopulation of excitatory projection neurons in the thoracic spinal cord that extend axons to the lumbar spinal cord where the walking execution center resides. This specification shows that regrowth of axons from these specific neurons beyond anatomically complete SCIs and their reconnection to appropriate target regions in the lumbar spinal cord restores walking ability in mice. These results demonstrate that a mechanism-based repair strategy that replicates the natural topology of a molecularly defined neuronal subpopulation can restore neurological function. The inventors have also demonstrated that malformation of the blood-brain barrier and astrocyte boundary leads to a worsening of the severity of injury. Administration of growth factors that promote the formation of these barriers improves neurological and functional recovery.

[0026] In fact, this specification demonstrates that recovery can be achieved, for example, in mice suffering from anatomically complete spinal cord injury (SCI) through phenotypic mimicry mechanisms underlying spontaneous spinal cord repair. By regrowing axons from specific neuronal subpopulations beyond the anatomically complete SCI and guiding them to reconnect to appropriate target regions in the lumbar spinal cord, walking is restored in mice suffering from complete paralysis. As expected, the treated mice did not walk as well as uninjured mice, but instead exhibited a phenotype comparable to mice that underwent spontaneous spinal cord repair after incomplete SCI. A comprehensive molecular catalog of all neuronal populations, their growth requirements, and subpopulation-specific chemoattractants would facilitate achieving equivalent repair across the entire cellular structure of the spinal cord. Such a catalog could potentially unravel a framework for achieving complete repair of damaged spinal cord, and this principle could potentially facilitate repair after other forms of central nervous system injury and disease.

[0027] Considering the above, one aspect of the present invention is: i) A first active substance comprising an exogenous nucleic acid encoding an osteopontin (Spp1) polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 8, an insulin-like growth factor 1 (Igf1) polypeptide as shown in SEQ ID NO: 2, and a ciliary body-derived neurotrophic factor (Cntf) polypeptide as shown in SEQ ID NO: 3 or SEQ ID NO: 9, and / or ii) A second active substance comprising exogenous nucleic acids encoding fibroblast growth factor 2 (FGF2) polypeptide as shown in SEQ ID NO: 4 or SEQ ID NO: 10, epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and glial-derived neurotrophic factor (GDNF) polypeptide as shown in SEQ ID NO: 6, A combination medicine containing, If both are present, the first active substance and the second active substance are administered sequentially or simultaneously, and The first and second active substances provide a combination pharmaceutical product that results in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

[0028] According to one embodiment of the combination pharmaceutical of the present invention, the second active substance further comprises an exogenous nucleic acid encoding a vascular endothelial growth factor (VEGF) polypeptide represented by SEQ ID NO: 7 or SEQ ID NO: 12.

[0029] According to another embodiment, the combination pharmaceutical of the present invention is iii) A third active substance containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, This further includes, and here, The first active substance, the second active substance, and the third active substance described above are administered sequentially or simultaneously, and The first, second, and third active agents described above result in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

[0030] According to one embodiment of the combination pharmaceutical of the present invention, the first active substance, the second active substance, and the third active substance are a gene editing system, a vector containing one or more nucleic acid sequences encoding one or more polypeptides according to the present invention, or an expression vector.

[0031] According to another embodiment of the combination drug of the present invention, the expression vector is a viral vector selected from the group consisting of retroviruses, lentiviruses, adenoviruses, herpesviruses, poxviruses, alphaviruses, vaccinia viruses, and adeno-associated viruses.

[0032] According to another embodiment of the combination drug of the present invention, the vector is an mRNA vector.

[0033] According to another embodiment of the combination drug of the present invention, the first active agent is a lentivirus expression vector, the second active agent is an adeno-associated virus (AAV) expression vector, and the third active agent is an adeno-associated virus (AAV) expression vector, where the adeno-associated virus (AAV) is selected from the group including AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11.

[0034] According to another embodiment of the combination drug of the present invention, the first active substance and the second active substance are each lentiviral expression vectors. According to yet another embodiment, the first active substance, the second active substance, and the third active substance are each lentiviral expression vectors.

[0035] According to another embodiment of the combination drug of the present invention, the third active ingredient results in the sustained delivery of glial-derived neurotrophic factor (GDNF).

[0036] According to another embodiment, the present invention is i) The first active substance, a. A lentiviral vector containing an exogenous nucleic acid encoding the osteopontin (Spp1) polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 8, b. A lentiviral vector containing an exogenous nucleic acid encoding the insulin-like growth factor 1 (Igf1) polypeptide shown in Sequence ID No. 2, and c. A lentiviral vector containing an exogenous nucleic acid encoding a ciliary body-derived neurotrophic factor (Cntf) polypeptide as shown in SEQ ID NO: 3 or SEQ ID NO: 9, The first active substance consists of, ii) A second active substance, a. A lentiviral vector containing an exogenous nucleic acid encoding the fibroblast growth factor 2 (FGF2) polypeptide shown in SEQ ID NO: 4 or SEQ ID NO: 10, b. A lentiviral vector containing an exogenous nucleic acid encoding an epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and c. A lentiviral vector containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, A second active substance consisting of, iii) A third active ingredient comprising a lentiviral vector containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, The present invention provides a combination pharmaceutical comprising, wherein the first active substance, the second active substance, and the third active substance are administered sequentially or simultaneously, and the first active substance, the second active substance, and the third active substance result in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

[0037] Osteopontin (Spp1) polypeptide (Sequence No. 1) (Sequence of Spp1 (Mus musculus) that is overexpressed): TIFF2026517854000001.tif25170

[0038] Homo sapiens secreted phosphoprotein 1 (SPP1) (SEQ ID NO: 8) MRIAVICFCLLGITCAIPVKQADSGSSEKQLYNKYPDAVATWLNPPDPSQKQNLLAPQNAVSSEETNDFKQETLPSKSNESHDHMDDMDDEDDDDHVDSQDSIDSNDSDDVDDTDDSHQSDESHHSDESDELVTDFPTDLPATEVFTPVVPTVDTYD GRGDSVVYGLRSKSKKFRRPDIQYPDATDEDITSHMESEELNGAYKAIPVAQDLNAPSDWDSRGKDSYETSQLDDQSAETHSHKQSRLYKRKANDESNEHSDVIDSQELSKVSREFHSHEFHSHEDMLVVDPKSKEEDKHLKFRISHELDSASSEVN *

[0039] Insulin-like growth factor 1 (IGF1) polypeptide (SEQ ID NO: 2) (Sequence of overexpressed IGF1 (Homo sapiens)): MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKEVHLKNASRGSAGNKNYRM *

[0040] Ciliary body-derived neurotrophic factor (Cntf) polypeptide (SEQ ID NO: 3) (Sequence of overexpressed Cntf (Mus musculus)): TIFF2026517854000002.tif16170

[0041] Ciliary neurotrophic factor (CNTF) (SEQ ID NO: 9) in Homo sapiens MAFTEHSPLTPHRRDLCSRSIWLARKIRSDLTALTESYVKHQGLNKNINLDSADGMPVASTDQWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHTLLLQVAAFAYQIEELMILLEYKIPRNEADGMPINVGDGGLFEKKLWGLKVLQELSQWTVRSIHDLRFISSHQTGIPARGSHYIANNKKM

[0042] Fibroblast growth factor 2 (FGF2) polypeptide (Sequence code 4) (overexpressed FGF2 (Mus musculus) sequence): MAASGITSLPALPEDGGAAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHVKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTEECFFFERLESNNYNTYRSRKYSSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS *

[0043] Homo sapiens fibroblast growth factor 2 (FGF2) (SEQ ID NO: 10) MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS *

[0044] Epidermal growth factor (EGF) polypeptide (SEQ ID NO: 5) (Sequence of overexpressed Egf (Mus musculus)): *

[0045] Homo sapiens epidermal growth factor (EGF) (SEQ ID NO: 11) *

[0046] Glial cell line-derived neurotrophic factor (GDNF) polypeptide (SEQ ID NO: 6) (sequence of overexpressed GDNF (Homo sapiens)): MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI *

[0047] Vascular endothelial growth factor A (Vegfa) of Mus musculus (SEQ ID NO: 7) MNFLLSWVHWTLALLLYLHHAKWSQAAPTTEGEQKSHEVIKFMDVYQRSYCRPIETLVDIFQEYPDEIEYIFKPSCVPLMRCAGCCNDEALECVPTSESNITMQIMRIKPHQSQHIGEMSFLQHSRCECRPKKDRTKPEKKSVRGKGKGQKRKRKKSRFKSWSVHCEPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR *

[0048] Vascular endothelial growth factor A (VEGFA) of Homo sapiens (SEQ ID NO: 12) MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQLELNERTCRCDKPRR *

[0049] In other embodiments, the polypeptides of the present invention have at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to one or more amino acid sequences of SEQ ID NOs: 1 to 12. In certain embodiments, polypeptides having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to one or more amino acid sequences of SEQ ID NOs: 1 to 12 include substitutions (such as conservative substitutions), insertions, or deletions with respect to the reference sequence, but their biological activity is still maintained. In certain embodiments, one or more sequence numbers 1 to 12 contain substitutions, insertions, and / or deletions of a total of 1 to 10 amino acids, while still maintaining their biological activity. Optionally, the polypeptide sequences of sequence numbers 1 to 12 include post-translational modifications of their sequences.

[0050] In further embodiments, the present invention also provides variants of the polypeptides of the present invention as described herein. As used herein, the term "variant" refers to naturally occurring genetic variants and recombinant variants that include one or more changes in their amino acid sequence compared to the reference polypeptide Spp1, Igf1, Cntf, FGF2, EGF, GDNF, or VEGF, such as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12, respectively. Such changes include modifications of one or more amino acid residues by substitution, addition, or deletion of amino acids. The term “mutant” includes orthologs of human Spp1, Igf1, Cntf, FGF2, EGF, VEGF, and / or GDNF, including, but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cattle, pigs, birds, and, but not limited to, mammalian Spp1, Igf1, Cntf, FGF2, EGF, VEGF, and / or GDNF orthologs from rodents such as mice and rats. In non-limiting examples, the mouse Spp1, Igf1, Cntf, FGF2, and / or EGF exemplified herein as the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 7 are orthologs of human Spp1, Igf1, Cntf, FGF2, and / or EGF. In some cases, preferred variants have at least 60%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0051] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (for example, a gap can be introduced in the first amino acid sequence or nucleic acid sequence to achieve optimal alignment with the second amino acid sequence or nucleic acid sequence). Then, the amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical overlapping positions / total number of positions × 100%). In one embodiment, the two sequences are the same length. Determining the percentage identity between two sequences can also be achieved using a mathematical algorithm. A preferred non-restrictive example of a mathematical algorithm used to compare two sequences is the algorithm in Karlin and Altschul, PNAS, 87:2264-2268 (1990), which has been modified as in Karlin and Altschul, PNAS, 90:5873-5877 (1993). Such algorithms are incorporated into the NBLAST and XBLAST programs in Altschul et al., J. Mol. Biol., 215:403 (1990). A BLAST nucleotide search is performed using NBLAST nucleotide program parameters set, for example, score=100 and word length=12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. A BLAST protein search is performed using XBLAST program parameters set, for example, score=50 and word length=3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparative purposes, use Gapped BLAST as described by Altschul et al. (Nucleic Acids Res., 25:3389-3402 (1997)). Alternatively, use PSI BLAST to perform repeated searches to detect distant relationships between molecules.When using the BLAST, Gapped BLAST, and PSI Blast programs, use the default parameters for each program (e.g., XBLAST and NBLAST) (see, for example, the NCBI website). Another preferred non-restrictive example of a mathematical algorithm used for sequence comparison is the algorithm by Myers and Miller (CABIOS, 4:11-17 (1988)). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, use the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties. Percent identicality between two sequences is determined with or without gaps using the same techniques as above. When calculating percentage identicality, typically only perfect matches are counted.

[0052] In certain embodiments, the nucleic acid encodes a biologically active fragment of the polypeptide of the present invention as described herein.

[0053] The term "polypeptide" includes, but is not limited to, osteopontin (Spp1) polypeptide, insulin-like growth factor 1 (Igf1) polypeptide, ciliary body-derived neurotrophic factor (Cntf) polypeptide, fibroblast growth factor 2 (FGF2) polypeptide, epidermal growth factor (EGF) polypeptide, glial-derived neurotrophic factor (GDNF) polypeptide, vascular endothelial growth factor (VEGF), and other biologically active fragments thereof, such as the fragments of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which are operable in the combination pharmaceuticals and methods described herein, as well as their variants.

[0054] The polypeptides and nucleic acids of the present invention can be isolated from natural sources such as cells of the brain or cell lineage of an organism expressing the polypeptide. Alternatively, polypeptides or nucleic acids can be recombinantly produced in vitro or in vivo by expression using an expression construct. Polypeptides and nucleic acids can also be synthesized by well-known methods. The polypeptides included in the combination pharmaceuticals and methods described herein can be produced using recombinant nucleic acid technology. Recombinant production involves introducing a recombinant expression vector containing a DNA sequence encoding the polypeptide into a host cell. Thus, in some embodiments, the nucleic acid encoding the polypeptide of the present invention, introduced into a host cell to produce the polypeptide of the present invention, may encode SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, SEQ ID NOs: 10, SEQ ID NOs: 11, and SEQ ID NOs: 12, their biologically active fragments, or their variants.

[0055] It will be understood that the first, second, and third active agents of the combination drug of the present invention may be administered simultaneously in the same formulation, or separately or sequentially in different pharmaceutical formulations. Accordingly, in one embodiment, the first, second, and third active agents are administered simultaneously. In such an embodiment, administration in combination is achieved by combining the first, second, and third active agents in a single dosage form. In another embodiment, the first, second, and third active agents are administered sequentially or separately. In such an embodiment, the first active agent should be administered first, the second active agent second, and the third active agent last. In yet another embodiment, the first, second, and third active agents are administered via the same or different routes. In some embodiments, the period between the administration of the first active substance and the administration of the second active substance may be about 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, or 30 days. In some embodiments, the second and third active substances are administered simultaneously. In further embodiments, the period between the administration of the second and third active substances may be about 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 28 days, or 30 days. The same applies, with necessary modifications, to combination drugs used in the treatment of incomplete SCI or stroke in which only the first and second active agents are present.

[0056] In some embodiments, the combination pharmaceuticals of the present invention further include a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” means a compound, material, composition, and / or dosage form that is suitable for use in contact with human and animal tissues in proportion to a reasonable benefit-to-risk ratio, without excessive toxicity, irritation, allergic response, or other problems or complications, within the bounds of sound medical judgment. As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition, or vehicle involved in transporting or carrying the compound of interest from one organ or part of body to another organ or part of body, such as a liquid or solid filler, diluent, additive, manufacturing aid (e.g., lubricant, talc, magnesium stearate, calcium stearate, or zinc stearate, or stearic acid), or solvent encapsulation material. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation and is not harmful to the patient.Some examples of materials that can function as pharmaceutically acceptable carriers include, but are not limited to, (1) sugars such as lactose, glucose, and sucrose, (2) starches such as corn starch and potato starch, (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate, (4) tragacanth powder, (5) malt, (6) gelatin, (7) lubricants such as magnesium stearate, sodium lauryl sulfate, and talc, (8) additives such as cocoa butter and suppository wax, (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil, (10) glycols such as propylene glycol, ( 11) Polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) Esters such as ethyl oleate and ethyl laurate; (13) Agar; (14) Buffers such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethyl alcohol; (20) pH buffer solution; (21) Polyesters, polycarbonates, and / or polyanhydrides; (22) Expanders such as polypeptides and amino acids; (23) Serum components such as serum albumin, HDL, and LDL; (22) C2-C12 alcohols such as ethanol; and (24) Other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, binders, fillers, lubricants, colorants, disintegrants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, water, salt solutions, alcohol, antioxidants, polyethylene glycol, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone, etc. may also be present in the formulation. Terms such as "additives," "carriers," and "pharmaceutically acceptable carriers" are used interchangeably in this specification.

[0057] In other embodiments, the combination pharmaceutical of the present invention further comprises an active substance that facilitates passage across the blood-brain barrier. In one embodiment, the pharmaceutically acceptable active substance facilitates passage across the blood-brain barrier or has the ability to pass across the blood-brain barrier.

[0058] As used herein, the term “vector” refers to a nucleic acid construct designed for delivery to or transfer between different host cells. In embodiments of the present invention, the vector is a viral vector. The term “vector” includes any genetic element that is capable of replicating when associated with appropriate regulatory elements and can transfer a gene sequence into a cell. Examples of vectors include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, mRNA, cosmids, artificial chromosomes, viruses, and virions.

[0059] As used herein, the term “expression vector” refers to a vector that ligates a transcriptional regulatory sequence on the vector to induce the expression of RNA or polypeptides from nucleic acid sequences contained within the vector (e.g., osteopontin (Spp1) polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 8, insulin-like growth factor 1 (Igf1) polypeptide shown in SEQ ID NO: 2, ciliary body-derived neurotrophic factor (Cntf) polypeptide shown in SEQ ID NO: 3 or SEQ ID NO: 9, fibroblast growth factor 2 (FGF2) polypeptide shown in SEQ ID NO: 4 or SEQ ID NO: 10, epidermal growth factor (EGF) polypeptide shown in SEQ ID NO: 5 or SEQ ID NO: 11, glial-derived neurotrophic factor (GDNF) polypeptide shown in SEQ ID NO: 6, or vascular endothelial growth factor (VEGF) polypeptide shown in SEQ ID NO: 7 or SEQ ID NO: 12). The expressed sequences are often, but not always, heterogeneous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thus enabling maintenance in two organisms, for example, human cells for expression and a prokaryotic host for cloning and amplification. The term “expression” refers to cellular processes, where applicable, that are involved in the production of RNA and proteins, and, if necessary, the secretion of proteins, including, but not limited to, transcription, transcription product processing, translation, and protein folding, modification, and processing. “Expression products” include RNA transcribed from genes and polypeptides obtained by the translation of mRNA transcribed from genes. The term “gene” means a nucleic acid sequence (DNA) that, when operably ligated to appropriate regulatory sequences, is transcribed into RNA in vitro or in vivo. A gene may or may not include the regions before and after the coding region, such as the 5' untranslated (5'UTR) sequence or “leader” sequence, and the 3'UTR sequence or “trailer” sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0060] Embedding vectors have the RNA / DNA they deliver that is permanently integrated into the chromosomes of the host cell. Non-embedding vectors remain in the episome, meaning that the nucleic acids contained in the vector are never integrated into the chromosomes of the host cell. Examples of embedding vectors include retroviral vectors, lentiviral vectors, hybrid adenovirus vectors, and herpes simplex virus vectors.

[0061] One example of a non-integrated vector is a non-integrated viral vector. Non-integrated viral vectors do not integrate the genome of integrated retroviruses into the host DNA, thus eliminating the risks associated with integrated retroviruses. One example is the Epstein-Barr oriP / nuclear antigen-1 ("EBNA1") vector, which is capable of limited self-replication and is known to function in mammalian cells. Because it contains two elements from the Epstein-Barr virus, namely oriP and EBNA1, when the EBNA1 protein binds to the viral replicon region oriP, the plasmid remains as an episome for a relatively long period in mammalian cells. This special characteristic of the oriP / EBNA1 vector makes it ideal for generating non-integrated iPSCs. Other non-integrated viral vectors include adenovirus vectors and adeno-associated virus (AAV) vectors. Yet another non-integrated viral vector is the RNA Sendai virus vector, which can produce proteins without entering the nucleus of infected cells. F-deficient Sendai virus vectors persist in the cytoplasm of infected cells for a few passages, but are rapidly diluted and completely lost after several passages (e.g., 10 passages). A further example of a non-integrated vector is the minicircle vector. A minicircle vector is a circularized vector from which the plasmid backbone has been removed, leaving only the eukaryotic promoter and cDNA (or more) to be expressed.

[0062] In various embodiments, the vector crosses the blood-brain barrier. In other embodiments, any active ingredient described herein is formulated to cross the blood-brain barrier. The blood-brain barrier is a highly selective semipermeable barrier separating the circulating blood from the extracellular fluid of the brain within the central nervous system (CNS). For therapeutic agents that need to be delivered to the CNS, a skilled clinician can deliver the therapeutic agent, such as the active ingredient or combination of pharmaceuticals of the present invention, directly into the spinal canal. In the case of direct administration into the spinal canal, the active ingredients and combination of pharmaceuticals described herein would be administered by a skilled clinician via intrathecal administration. Intrathecal administration is a route of drug administration in which a drug is injected directly into the spinal canal or subarachnoid space, allowing the drug to reach the cerebrospinal fluid (CSF) directly. The vector may be packaged with at least a second active ingredient that allows it to cross the blood-brain barrier. Those skilled in the art can determine, for example, whether the vector has crossed the blood-brain barrier by determining, for example, whether the vector is detected in the cerebrospinal fluid after administration.

[0063] In some embodiments, the vector is messenger RNA (mRNA). In recent years, messenger RNA therapy has become an increasingly important option for treating a variety of diseases and conditions, particularly those associated with the deficiency of one or more proteins. The term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. As used herein, mRNA includes both modified and unmodified RNA. mRNA may contain one or more coding and uncoding regions. mRNA may be purified from natural sources, produced using recombinant expression systems and optionally purified, or chemically synthesized. Where appropriate, for example, in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs such as chemically modified bases or sugars, or analogs with skeletal modifications. Unless otherwise indicated, mRNA is represented in the 5' to 3' direction. In some embodiments, mRNA is a natural nucleoside (e.g., adenosine, guanosine, cytidine, uridine), a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine) , C5-iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), modified phosphate groups (e.g., phosphorothioates, and 5'-N-phosphoamidite linkages), and any combination of the foregoing, or comprising them. The mRNA according to the present invention may contain one or more modified nucleotides, for example, to enhance its stability or to avoid or reduce its degradation upon delivery to a subject.

[0064] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminus (5') and a "tail" to the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" protects mRNA from exonuclease degradation.

[0065] In some embodiments, mRNA is delivered to neurons located in the brain. In some embodiments, mRNA is delivered to neurons located in the spinal cord. In some embodiments, mRNA is delivered to motor neurons. In some embodiments, mRNA is delivered to upper motor neurons and / or lower motor neurons.

[0066] In some embodiments, mRNA is administered intrathecally to a subject so that administration of the composition results in intracellular delivery of mRNA to neurons in the brain and / or spinal cord. Intracellular delivery of mRNA results in the expression of the protein encoded by the mRNA. In some embodiments, the encoded protein is expressed in the cytoplasm of the neuron. In some embodiments, the encoded protein is expressed and then secreted extracellularly from the neuron.

[0067] mRNA may be delivered to the CNS as naked (unpackaged) RNA or via a delivery carrier. In this specification, “delivery carrier,” “import carrier,” “nanoparticle,” or grammatically equivalent terms are used without distinction.

[0068] In some embodiments, mRNA may be delivered via a single delivery carrier. In some embodiments, mRNA may be delivered via one or more delivery carriers, each having a different composition. Suitable delivery carriers, according to various embodiments, include, but are not limited to, polymer-based carriers such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, naturally derived and synthetic exosomes, naturally derived, synthetic and semi-synthetic lamellar bodies, nanogranules, calcium phosphate nanogranules, silicon dioxide nanogranules, nanocrystalline granules, semiconductor nanogranules, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides, and other vector tags.

[0069] In some embodiments, the therapeutically effective dose of mRNA is, for example, about 0.001 mg / kg(body weight) to 10 mg / kg(body weight), about 0.005 mg / kg(body weight) to 10 mg / kg(body weight), about 0.01 mg / kg(body weight) to 10 mg / kg(body weight), about 0.01 mg / kg(body weight) to 9 mg / kg(body weight), about 0.01 mg / kg(body weight) to 8 mg / kg(body weight), about 0.01 mg / kg(body weight) to 7 mg / kg(body weight), about 0.01 mg / kg(body weight) to 6 mg / kg(body weight), about 0.01 mg / kg(body weight) to 5 mg / kg(body weight), The range may be approximately 0.01 mg / kg (body weight) to 4 mg / kg (body weight), approximately 0.01 mg / kg (body weight) to 3 mg / kg (body weight), approximately 0.01 mg / kg (body weight) to 2 mg / kg (body weight), approximately 0.01 mg / kg (body weight) to 1 mg / kg (body weight), approximately 0.01 mg / kg (body weight) to 0.5 mg / kg (body weight), approximately 0.1 mg / kg (body weight) to 10 mg / kg (body weight), approximately 0.1 mg / kg (body weight) to 5 mg / kg (body weight), approximately 0.5 mg / kg (body weight) to 10 mg / kg (body weight), or approximately 0.5 mg / kg (body weight) to 5 mg / kg (body weight).

[0070] In fact, surprisingly, the inventors have demonstrated that the combination of the first, second, and third active substances according to the present invention is not only advantageous for the functional and anatomical recovery of complete SCI, but also that administration of the first active substance alone, the second active substance alone, or a combination thereof to subjects has a remarkable effect and functional benefit on less severe injuries such as neuronal damage after stroke, and enables improved recovery of walking after incomplete SCI. The results also showed that the sustained expression of exogenous nucleic acids of various active substances, either alone or in combination, is important for achieving spinal cord repair, axonal regeneration, and improved limb movement function.

[0071] Accordingly, another aspect of the present invention provides a combination pharmaceutical of the present invention used in a method for treating incomplete spinal cord injury or stroke in a subject. According to this aspect of the present invention, the combination pharmaceutical comprises a combination of a first active substance and a second active substance, or the first active substance alone, or the second active substance alone.

[0072] Another aspect of the present invention provides a method for treating incomplete spinal cord injury or stroke in a subject, comprising administering a combination pharmaceutical of the present invention, comprising a first active substance, a second active substance, or a combination thereof, to the subject.

[0073] In a preferred embodiment of a method for treating incomplete spinal cord injury or stroke, the combination drug of the present invention or the first active agent and / or the second active agent is administered after the incomplete spinal cord injury or stroke.

[0074] Another aspect of the present invention provides a combination pharmaceutical for use in a method of treating complete spinal cord injury in a subject, comprising a first active substance, a second active substance, and a third active substance.

[0075] Another aspect of the present invention provides a method for treating a complete spinal cord injury in a subject, comprising administering a combination pharmaceutical of the present invention, comprising a first active substance, a second active substance, or a combination thereof, to the subject.

[0076] In a preferred embodiment of a method for treating complete spinal cord injury, the combination drug of the present invention or the first, second, and third active agents of the present invention are administered after the complete spinal cord injury.

[0077] According to some embodiments of the combination pharmaceuticals used in the present invention and the treatment methods of the present invention, the treatment method further includes an initial assessment step to determine whether the subject has incomplete spinal cord injury, complete spinal cord injury, or stroke. In one embodiment, the initial assessment step includes an analysis of spinal cord injury symptoms or stroke symptoms selected from the group including one or more of the following: loss or impairment of motor function(s), loss or impairment of sensory function(s), and loss or impairment of autonomic nervous system function(s).

[0078] Another aspect of the present invention is a method for treating neurological trauma in a subject, If the neurological injury is an incomplete spinal cord injury or stroke, the subject requiring treatment should The first active substance, or, A second active substance, or A pharmaceutical product comprising the first active substance and the second active substance described above, Administering one of these, or In cases of complete spinal cord injury, administer a combination drug containing a first active agent, a second active agent, and a third active agent to the subject requiring treatment. Including, here, The first active substance described above includes an exogenous nucleic acid encoding an osteopontin (Spp1) polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 8, an insulin-like growth factor 1 (Igf1) polypeptide shown in SEQ ID NO: 2, and a ciliary body-derived neurotrophic factor (Cntf) polypeptide shown in SEQ ID NO: 3 or SEQ ID NO: 9. The second active substance described above includes exogenous nucleic acids encoding fibroblast growth factor 2 (FGF2) polypeptide as shown in SEQ ID NO: 4 or SEQ ID NO: 10, epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and glial-derived neurotrophic factor (GDNF) polypeptide as shown in SEQ ID NO: 6. The third active substance described above includes an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide shown in Sequence ID No. 6, a) The first active substance, the second active substance, and the third active substance induce the sustained expression and secretion of an effective amount of polypeptide for at least three days, b) In the case of combinations, the present invention provides a method in which the active substances are administered sequentially or simultaneously.

[0079] In a preferred embodiment of the method for treating neurological trauma, the combination drug of the present invention, or the first active agent, the second active agent, and the third active agent (depending on the injury), is administered after incomplete spinal cord injury, stroke, or complete spinal cord injury.

[0080] Advantageously, according to some embodiments, the second active ingredient further comprises an exogenous nucleic acid encoding a vascular endothelial growth factor (VEGF) polypeptide as shown in SEQ ID NO: 7 or SEQ ID NO: 12.

[0081] In some embodiments, the treatment method of the present invention further includes an initial assessment step to determine whether the neurological injury is an incomplete spinal cord injury, a complete spinal cord injury, or a stroke. In one embodiment, the initial assessment step includes an analysis of spinal cord injury symptoms or stroke symptoms selected from the group including one or more of the following: loss or impairment of motor function(s), loss or impairment of sensory function(s), and loss or impairment of autonomic nervous system function(s).

[0082] According to another embodiment of the present invention, a complete or incomplete spinal cord injury is a spinal cord injury of the cervical, thoracic, and / or lumbar spinal cord.

[0083] According to another embodiment of the present invention, the treatment of stroke involves neuronal repair, functional repair, and anatomical repair of the brain after stroke.

[0084] In one embodiment, the present invention provides the ability of severed axons to form functional connections with their natural target regions within the spinal cord to improve gait after anatomically complete spinal cord injury (SCI). According to this embodiment, a method for treating complete spinal cord injury comprises administering lentiviral vectors (LV) to three different sites: (1) Administering a cocktail of three lentiviral vectors to the rostral side of the spinal cord injury, wherein each lentiviral vector expresses a different major growth factor that upregulates the dormant developmental growth program, selected from Igf1, Cntf, and Spp1 (disclosed herein); (2) Administering a cocktail of three lentiviral vectors at the spinal cord injury, wherein two lentiviral vectors express different growth factors that manipulate the injury microenvironment, selected from FGF2 and EGF (disclosed herein), and the third lentiviral vector expresses a chemoattractant-activating factor, namely GDNF (disclosed herein). (3) A lentiviral vector expressing GDNF (as disclosed herein) is administered caudally to the spinal cord injury. According to this embodiment, better treatment of complete spinal cord injury is possible by replacing the adenovirus vector (AAV) with a combination of lentiviral vectors (LV) expressing individual growth factors. This individual delivery of Igf1, Cntf, Spp1, FGF2, EGF, and GDNF at three different locations of the spinal cord injury resulted in vigorous axonal regeneration through anatomically complete spinal cord injury (SCI). The consequence of this regeneration was a gradual recovery of walking.

[0085] Therefore, according to another embodiment, the present invention is a method for treating complete spinal cord injury in a subject, i. On the rostral side of the spinal cord injury, the first active substance is, a. A lentiviral vector containing an exogenous nucleic acid encoding the osteopontin (Spp1) polypeptide shown in SEQ ID NO: 1 or SEQ ID NO: 8, b. A lentiviral vector containing an exogenous nucleic acid encoding the insulin-like growth factor 1 (Igf1) polypeptide shown in Sequence ID No. 2, and c. A lentiviral vector containing an exogenous nucleic acid encoding a ciliary body-derived neurotrophic factor (Cntf) polypeptide as shown in SEQ ID NO: 3 or SEQ ID NO: 9, The process consists of administering a first active substance, ii. In spinal cord injury, the second active substance is: a. A lentiviral vector containing an exogenous nucleic acid encoding the fibroblast growth factor 2 (FGF2) polypeptide shown in SEQ ID NO: 4 or SEQ ID NO: 10, b. A lentiviral vector containing an exogenous nucleic acid encoding an epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and c. A lentiviral vector containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, The process consists of administering a second active substance, iii. A step of administering a third active substance, consisting of a lentiviral vector containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, to the caudal side of the spinal cord injury, This provides a method that includes [something].

[0086] In a preferred embodiment of the method for treating complete spinal cord injury, the first active agent, the second active agent, and the third active agent are administered after the complete spinal cord injury.

[0087] According to one embodiment of the present invention, the treatment method includes sequentially or simultaneously administering a first active substance, a second active substance, and a third active substance according to the present invention to target neurons expressing Vsx2 and / or Zfhx3 (projection V2a neurons) to perform spinal cord repair in the cervical, thoracic, and / or lumbar spinal cords. To perform spinal cord repair in the cervical, thoracic, and / or lumbar spinal cords after complete SCI, sequential or simultaneous administration of the first active substance, the second active substance, and the third active substance according to the present invention is envisioned.

[0088] According to one embodiment of the method of the present invention, the method includes sequentially or simultaneously administering a first active substance and / or a second active substance according to the present invention to target V1 neurons, V0 neurons, V2b neurons, CSF contact neurons, Vglut2 neurons, Vgat neurons, Chat neurons, Vsx2 and / or Zfhx3 neurons, thereby performing spinal cord repair in the cervical, thoracic, and / or lumbar spinal cord, and / or regenerating brain neuronal function. To perform spinal cord repair in the cervical, thoracic, and / or lumbar spinal cord after incomplete SCI, and / or regenerating brain neuronal function after stroke, sequential or simultaneous administration of the first active substance and / or the second active substance according to the present invention is anticipated.

[0089] According to one embodiment of the present invention, spinal cord repair of the cervical, thoracic, and / or lumbar spinal cord, and / or functional and anatomical repair of brain neurons, is achieved by targeting V1 neurons, V0 neurons, V2b neurons, CSF contact neurons, Vglut2 neurons, Vgat neurons, Chat neurons, Vsx2 and / or Zfhx3 (projection V2a neurons).

[0090] According to one embodiment of the method of the present invention, spinal cord repair consists of axonal regeneration and restoration of neurological function.

[0091] According to one embodiment of the method of the present invention, spinal cord repair consists of the regeneration of the blood-brain barrier and the astrocyte barrier, and the restoration of neurological function.

[0092] According to one embodiment of the method of the present invention, the symptoms of spinal cord injury or stroke are selected from the group including one or more of the following: loss or impairment of motor function(s), loss or impairment of sensory function(s), and loss or impairment of autonomic function(s), in the cervical, thoracic, lumbar, or sacral segments, and / or in general the central nervous system, particularly the brain.

[0093] Another aspect of the present invention provides a method for regenerating spinal nerve neurons in a subject suffering from complete or incomplete spinal cord injury (SCI), comprising administering the combination drug of the present invention to the spinal cord, wherein the spinal nerve neurons are a group of one or more neurons present in the spinal cord.

[0094] Another aspect of the present invention provides a method for regenerating brain neurons in a subject who has survived a stroke, comprising administering the combination drug of the present invention into the brain, wherein the brain neurons are a group of one or more brain neurons.

[0095] According to one embodiment of the method of the present invention, the spinal cord neurons are dorsal, medial, and / or ventral.

[0096] According to one embodiment of the method of the present invention, the spinal cord neurons and brain neurons are V1 neurons, V0 neurons, V2b neurons, CSF contact neurons, Vglut2 neurons, Vgat neurons, Chat neurons, Vsx2, and Zfhx3 neurons.

[0097] According to one embodiment of the spinal cord repair method and the spinal cord neuron or brain neuron regeneration method of the present invention, the combination drug of the present invention is administered by injection into the cervical, thoracic, lumbar, or sacral spinal cord of a subject via methods such as stereotactic injection, or by injection into the brain of a subject, or by intravenous infusion or intravenous injection.

[0098] According to one embodiment of the spinal cord repair method and spinal cord neuron regeneration method of the present invention, stereotactic injection is guided by identifying the location of the spinal cord injury core by a method selected from ultrasound guidance, electrophysiology, or molecular guidance.

[0099] According to one embodiment of the spinal cord repair method and the spinal cord neuron or brain neuron regeneration method of the present invention, the subject is a mammal, preferably a human.

[0100] In some embodiments, an effective amount of the first active agent, the second active agent, and the third active agent described herein, and / or a combination of pharmaceuticals described herein, is administered to a subject if applicable and appropriate in accordance with this disclosure.

[0101] As used herein, the term “effective dose” means the amount of a pharmaceutical composition or combination of the first, second, and third active agents that can be administered to a subject suffering from or diagnosed with a spinal cord injury or stroke, and is necessary to alleviate at least one symptom of the spinal cord injury or stroke, as applicable and appropriate in accordance with this disclosure. Accordingly, the term “therapeutic effective dose” means the amount of a pharmaceutical composition or combination of the first, second, and third active agents that, when administered to a typical subject, is sufficient to produce a specific anti-spinal cord injury effect or anti-stroke effect, as applicable and appropriate in accordance with this disclosure. As used in various contexts herein, effective amounts also include amounts of a pharmaceutical composition or combination of a first, second, and third active ingredient sufficient to delay the onset of symptoms of spinal cord injury or stroke, alter the course of symptoms of spinal cord injury or stroke (e.g., slow the progression of loss of sensation or motor function in the limbs), or reverse the symptoms of spinal cord injury or stroke (e.g., restore previously reduced or lost sensation or motor function in the limbs), where applicable and appropriate in accordance with this disclosure. Therefore, specifying an exact “effective amount” is generally not feasible. However, in any case, those skilled in the art can determine an appropriate “effective amount” using only routine experiments.

[0102] In one embodiment, a combination pharmaceutical of the first active ingredient, the second active ingredient, and the third active ingredient described herein, if appropriate and suitable in accordance with this disclosure, may be used at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, and at least 12 hours after the onset of spinal cord injury or stroke. It is administered at least 18 hours later, at least 24 hours later, at least 36 hours later, at least 48 hours later, at least 60 hours later, at least 72 hours later, at least 96 hours later, at least 5 days later, at least 6 days later, at least 1 week later, at least 2 weeks later, at least 3 weeks later, at least 1 month later, at least 2 months later, at least 3 months later, at least 4 months later, at least 5 months later, at least 6 months later, at least 7 months later, at least 8 months later, at least 9 months later, at least 10 months later, at least 11 months later, at least 12 months later, at least 2 years later, at least 3 years later, at least 4 years later, or at least 5 years later, or later.

[0103] The effective dose, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. Dosage may vary depending on the dosage form used and the route of administration utilized. The dose-to-toxicity ratio is the therapeutic index and can be expressed as the LD50 / ED50 ratio. Compositions and methods exhibiting a large therapeutic index are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Alternatively, doses can be formulated in animal models to achieve a circulating plasma concentration range including the IC50 (i.e., the concentration of the active ingredient that achieves 50% inhibition of symptoms) determined in cell cultures or appropriate animal models. Plasma levels can be measured, for example, by high-performance liquid chromatography. The effects of any specific dosage can be monitored by appropriate bioassays, such as measurements.

[0104] The results of the spinal cord repair methods described herein, as well as the spinal cord neuron or brain neuron regeneration methods described herein, are the recovery of walking or other neurological functions. Furthermore, the advantage of the combination medicines described herein is that they guide neurons to their proper locations, deliver all growth factors instead of using biomaterial depots, improve growth factor expression, and activate the intrinsic neuronal growth capacity.

[0105] Those skilled in the art will understand that the inventions described herein are subject to modifications and alterations other than those specifically described. It should be understood that the present invention includes all such modifications and alterations without departing from the spirit or essential characteristics of the invention. The present invention also includes all the steps, features, compositions, and compounds mentioned or indicated herein, individually or collectively, and all combinations or any two or more of the above steps or features. Therefore, this disclosure is illustrative in all aspects and should not be considered restrictive, and the scope of the invention is indicated by the appended claims, and all variations that fall within the meaning and scope of the doctrine of equivalents are intended to be included therein.

[0106] The detailed explanation above will be better understood by referring to the following examples. However, these examples are illustrative of ways in which the present invention can be carried out and are not intended to limit the application and scope of the present invention. [Examples]

[0107] Neurons involved in natural repair The initial objective was to determine the transcriptional characteristics of neuronal subgroups involved in the spontaneous repair mechanism for gait recovery after severe but incomplete spinal cord injury (SCI). In human and animal models (Brown-Séquard syndrome) where essential superior spinal cord input from the gait execution center on one side of the spinal cord is deprived after unilateral hemisection, spontaneous gait recovery occurs. Some previous studies have shown that neurons located in the mid-thoracic spinal segment can relay commands from the superior spinal cord across the lateral hemisection, thereby restoring gait. Even after temporally and spatially separated hemisection lesions that completely interrupt direct projections from the brain to the gait execution center, these neurons can still relay sufficient superior spinal cord input, restoring voluntary, unassisted walking. While destruction of these neurons did not alter gait in undamaged cases, the spontaneous gait recovery observed after lateral hemisection completely disappeared. Therefore, the objective was to clarify the molecular and anatomical characteristics of the neuronal subgroups underlying this recovery.

[0108] To identify neuronal subpopulations with projections to the locomotion center, rAAV2 encoding eGFP fused to the nuclear membrane protein KASH was injected into the lumbar spinal cord of undamaged mice (data not shown). This strategy labeled the nuclei of neurons with direct projections to the locomotion center throughout the central nervous system, including relay neurons in the mid-thoracic spinal cord (data not shown). This strategy enabled fluorescence-activated nuclear sorting coupled with single-nuclear RNA sequencing (snRNA-seq) of projection-specific neuronal subpopulations (data not shown).

[0109] 122 eGFPON The nucleus and 2823 eGFP OFF High-quality single-nucleus transcription profiles were obtained from the nuclei (data not shown). Unsupervised clustering identified all major cell types in the mouse spinal cord (data not shown). A second clustering of neurons identified 28 neuronal subpopulations expressing classical marker genes (Figure 1). Taxonomy parsed the major spinal cord neuron classes into subpopulations of motor-sensory neurons, local-long-range neurons, and excitatory-inhibitory neurons (data not shown). Surprisingly, eGFP was found. ON Neurons are primarily a single ventral neuronal subgroup (spinal cord (SC)) that express the marker Vsx2 and Zfhx3, the major marker for long-range projection neurons. Vsx2::Zfhx3→腰髄 It was found within neurons (data not shown).

[0110] Thoracic neurons are essential for walking after severe incomplete spinal cord injury (SCI), but not in the absence of damage, and therefore, SCI occurs after spontaneous spinal cord repair. Vsx2::Zfhx3→腰髄 We investigated whether neurons are also perturbed transcriptionally. Neuronal nuclei from undamaged mice and mice with restored walking ability (data not shown) were compared after temporally and spatially separated lateral hemisectomy (SCI) (data not shown). High-quality transcriptional profiles were obtained from 9264 nuclei representing all major cell types in the mouse spinal cord (data not shown) (data not shown). These were then subjected to a second clustering of neurons. This data was integrated with projection-specific snRNA-seq experiments, where the same 28 neuronal subpopulations were identified and evaluated (data not shown). Cell type prioritization was used to identify SC across all neuronal subpopulations embedded in the thoracic spinal segments of mice with restored walking ability. Vsx2::Zfhx3→腰髄 Neurons were found to exhibit the largest transcriptional perturbations, which is consistent with their involvement in natural repair (data not shown).

[0111] SC Vsx2::Zfhx3→腰髄 Anatomical characteristics of neurons Based on the results obtained so far, SC Vsx2::Zfhx3→腰髄 The neurons were suggested to be putative neurons that restore walking after natural spinal cord repair. Therefore, it was hypothesized that these neurons must possess anatomical features that are suited to the requirements for walking after paralysis.

[0112] Visualizing the projections from neurons embedded in the mid-thoracic spinal cord revealed dense projections across the entire lumbar spinal cord where the walking execution center resides (data not shown). Cross-genetics and Vsx2 were used to identify neuronal subpopulations that possessed a combination of this projection and transcriptional phenotypes consistent with prioritized neuronal subpopulations (data not shown). Cre Using virus tracking in mice, Vsx2 ON The distribution and connectome of neurons were compared. Vsx2 located in the mid-thoracic spinal cord. ON Neurons were found to account for 5.9% of the neurons in this region (data not shown), which was consistent with the neuronal distribution identified in our snRNA-seq data (data not shown). Mid-thoracic spinal cord Vsx2 ON By tracking neurons, it was revealed that dense projections exist throughout the entire walking execution center, as expected (data not shown).

[0113] Next, mid-thoracic spinal cord Vsx2 ON We analyzed whether neuronal populations can be stratified into subpopulations that project locally and subpopulations that project over long distances. In the spinal cord, neurons with local projections and neurons with long-range projections can be distinguished by the expression of Zfhx3 (data not shown). Long-range projection Vsx2 ON To label neurons, use rAAV2-Ef1α-DIO-Flpo in Vsx2. Cre The lumbar spinal cord was injected into the mice, followed by an injection of AAV5-Con / Fon-eYFP into the thoracic spinal cord (Figure 2). This cross-following strategy projected Vsx2 to the walking execution center. ONIt became possible to label only neurons (Figure 2). Zfhx3 and Vsx2 are neurons that project to this region (SC Vsx2::Zfhx3→腰髄 Since it was found to co-localize only to (data not shown), Zfhx3 is confirmed to be an accurate marker targeting a subpopulation of spinal cord neurons with long-range projections. Local projection (Vsx2 ON Zfhx3 OFF )Vsx2 ON Long-range projection of neurons (Vsx2 ON Zfhx3 ON )Vsx2 ON Quantification of neurons revealed that these two subgroups are distributed almost evenly throughout the mid-thoracic spinal cord (data not shown). In summary, these findings suggest that Vsx2 implanted in the mid-thoracic spinal cord ON A subset of neurons was found to co-express Zfhx3 and to extend dense projections to the lumbar spinal cord, where the walking execution center is located.

[0114] Therefore, in order to function as a relay for commands from above the spinal cord, SC Vsx2::Zfhx3→腰髄 It was hypothesized that neurons must also receive direct projections from major neurons involved in the recovery of walking after paralysis. To elucidate this connectome, AAV5-CAG-gComet was used with Vsx2 Cre::tdTomato It was injected into the ventral giant cell nucleus (vGi) of mice, because vGi neurons are essential for this recovery (data not shown). Vsx2 located in the mid-thoracic spinal cord. ON Zfhx3 ON Neurons were found to receive high-density projections from vGi (data not shown).

[0115] In summary, these results suggest that, within the diverse population of cells in the middle thoracic spinal cord, SC Vsx2::Zfhx3→腰髄 It was pointed out that these neurons represent the most perturbed neuronal subgroup during spontaneous repair and exhibit relevant projectome and receptor profiles that relay higher spinal cord commands mediating gait recovery after incomplete SCI.

[0116] SC after complete SCI Vsx2::Zfhx3→腰髄 Regeneration of neurons to their natural targets Previous studies have shown that factors essential for developing axonal growth can support spinal cord axonal regeneration to viable neuronal tissue located in nodes below the injury beyond anatomically complete SCI lesions, but this regrowth does not restore walking. Based on the above findings, the restoration of walking after anatomically complete SCI requires the restoration of walking after anatomically incomplete SCI, and therefore, SC Vsx2::Zfhx3→腰髄 It was thought necessary to replicate the projections that cause neurons to regrow into their natural targets within the walking execution center.

[0117] To test this concept, firstly, we reactivated the intrinsic growth capacity of neurons located above the SCI using viral overexpression of osteopontin (Spp1), insulin-like growth factor 1 (Igf1), and ciliary body-derived neurotrophic factor (Cntf) (AAV-OIC). Secondly, we induced the formation of axon growth support substrates by transient delivery of fibroblast growth factor 2 (FGF2) and epidermal growth factor (EGF). Thirdly, we delivered a biomaterial depot of glial-derived neurotrophic factor (GDNF) as a chemoattractant to sites within and below the injury at regular intervals. In fact, snRNA-seq data showed that SC Vsx2::Zfhx3→腰髄 The expression of Gfra1 and Ret, both Gdnf receptors in neurons necessary for proper Gdnf signaling, was revealed, and Vsx2 was tracked using AAV5-Con / Fon-eYFP. ON Immunohistochemical analysis of axons further verified Gdnf receptor expression within cells and along the entire length of the axon, tracked using AAV5-Con / Fon-eYFP (data not shown).

[0118] Consistent with previous observations, stimulated, supported, and chemoattracted axons were again found to vigorously regrow through astrocyte boundaries, across fibrous scars, and into viable neuronal tissue beneath anatomically complete SCIs (Figure 3). Nevertheless, when regenerating axons terminated just one segment below the injury where the most distal GDNF-containing biomaterial depot was injected, no recovery could be detected even by high-precision behavioral assessments performed four weeks after the injury (data not shown). This observation suggests that axons terminating within the walking execution center, located several segments distal to the SCI, may not be viable. Vsx2::Zfhx3→腰髄 This was in contrast to the remarkable recovery of walking observed up to four weeks after spontaneous spinal cord repair, including the inclusion of neurons (data not shown).

[0119] Therefore, it was inferred that for walking to be restored after anatomically complete SCI, the regenerated axons must replicate the topology of natural spinal cord repair and reach the lumbar spinal cord. To achieve such long-distance regeneration, an additional depot of chemoattractive GDNF was placed in the lumbar spinal cord (data not shown). However, this additional depot attracted a relatively small number of axons to the targeted lumbar spinal cord region (data not shown), and high-precision behavioral assessments again failed to detect any recovery of walking (data not shown). Thus, it was inferred that the relatively slow time course of long-distance axon growth, maturation, and synapse formation may require more sustained and higher concentrations of chemoattractive growth factor delivery than that provided by biomaterial depots. To achieve this, a lentivirus was designed to provide sustained GDNF delivery. Replacing biomaterial depots with lentivirus-mediated Gdnf expression enabled significant axonal regrowth to their natural topological targets (Figure 4), further demonstrating that appropriate chemoattraction gradients can guide long-distance axonal regeneration as well as development.

[0120] The regenerated axon is SC Vsx2::Zfhx3→腰髄To determine if the eGFP originated from neurons, rAAV2-hSyn-KASH-eGFP was injected into the lumbar spinal cord, and eGFP was retrogradely expressed only in neurons with axons that had regrown sufficiently to reach the walking execution center (data not shown). eGFP located above the injury site. ON Neuronal nuclei were sorted for snRNAseq, and the resulting single-neuron transcriptional profiles were integrated into an atlas of overall cell types and neuronal subpopulations in the thoracic spinal cord (data not shown). eGFP ON By comparing the distribution of neurons with the distribution of neuronal subpopulations in the undamaged spinal cord, SC Vsx2::Zfhx3→腰髄 Neurons were identified as the primary virus-labeled subpopulation, thus confirming that this particular neuronal subpopulation successfully regenerated to their natural topological targets (data not shown). Retrograde follow-up coupled with immunohistochemical analysis of Vsx2 confirmed these results (data not shown).

[0121] Finally, we investigated whether it was possible to detect commands from the upper spinal cord below the anatomically complete SCI. We found that micro-stimulation of vGi induced large motor evoked potentials in the lower limb muscles, indicating that the upper spinal cord centers regained access to the walking execution center (data not shown).

[0122] These results are SC Vsx2::Zfhx3→腰髄 We have shown that by manipulating neurons, electrophysiologically active axons can be regrown in their natural topological target regions in the lumbar spinal cord where the walking execution center resides.

[0123] Walking restored by repair based on the anatomically complete SCI mechanism. Longitudinal quantification of whole-body movement during walking is combined with anatomically complete SCI (Scalar Combination). Vsx2::Zfhx3→腰髄The study was conducted in three separate cohorts of mice that underwent targeted regrowth of neurons in the lumbar spinal cord. These evaluations revealed that complete crushing SCI rendered all mice unable to walk, with no signs of recovery even four weeks after SCI (data not shown). In mice that received complete treatment, gradual recovery of walking was observed approximately three to four weeks after SCI (data not shown). The final evaluation was performed at eight weeks. In Cohort 1, five out of six mice with anatomically complete SCI and complete treatment exhibited a gait pattern similar to that quantified in mice with spontaneous spinal cord repair after incomplete SCI (data not shown). When these experiments were repeated in two subsequent cohorts, 13 out of 15 mice (or a total of 18 out of 21) showed similar results (data not shown).

[0124] Thoracic spinal cord Vsx2 in the recovery of walking ON To evaluate the causal relationships of neurons, Vsx2 was used in Cohort 3. Cre These neurons were destroyed by injecting AAV5-hSyn-flex-DTR into the thoracic spinal cord of mice to induce expression of the diphtheria toxin receptor (DTR) (Figure 5). Eight weeks after the repair strategy in addition to SCI, all four mice in Cohort 3 regained the ability to walk with a gait pattern similar to the gait pattern quantified in mice that underwent spontaneous repair (Figure 5). Administration of diphtheria toxin caused paralysis again in all mice tested (Figure 5). Postmortem anatomical analysis revealed Vsx2 in the thoracic spinal cord. ON It was confirmed that the neurons were almost completely destroyed (data not shown).

[0125] In summary, these results indicate that the SC has regenerated axons that have reached their lumbar spinal cord targets beyond the anatomically complete SCI. Vsx2::Zfhx3→腰髄 It has become clear that neurons are necessary and sufficient for the recovery of voluntary walking after complete paralysis.

[0126] In an attempt to demonstrate the usefulness of the single active ingredient according to the present invention in the functional recovery of impaired SCI, the inventors conducted two additional exemplary experiments described below.

[0127] Use of the first active agent of the present invention alone: ​​The data shown in Figure 7 demonstrates that reactivation of the growth program alone (first active agent (Igf1 / Spp1 / Cntf)) can improve neurological recovery and impaired SCI. The inventors observed that when the first active agent was used alone, a significantly larger number of axons sprouted beyond incomplete injury, resulting in a doubling of functional recovery (BMS score, observational scoring system).

[0128] Use of the second active agent alone: ​​In the context of incomplete SCI, the inventors have also demonstrated that acute intervention with viruses expressing Egf, Fgf2, Gdnf, and Vegf (the second active agent) has the ability to promote blood-brain barrier reformation, astrocyte barrier formation, prevent immune infiltration, and improve neurological recovery (Figures 8 and 9).

[0129] Restoration of walking using a single LV construct: In a situation of anatomically complete SCI, the inventors also demonstrated that functional restoration of walking in tested mice could be achieved by injection of lentiviral vectors (LVs) at three different locations. The inventors injected a cocktail of three LVs (first active agent) rostrally to the lesion, each expressing one major growth factor (Igf1, Cntf, and Spp1, respectively) that upregulates the developmental growth program during dormancy. At the lesion, the inventors injected a second active agent containing three LVs, two of which express different growth factors (Fgf2 and Egf, respectively) that manipulate the lesion's microenvironment, and a third LV that expresses a chemoattraction-activating factor (Gdnf). At the caudal side of the lesion, the inventors injected a third active agent containing only an LV expressing Gdnf. These results indicate that this treatment can be delivered after SCI by replacing AAV with a combination of LV expressing individual growth factors. When this manipulation was delivered in a single surgery, it was found to lead to robust axonal regeneration through anatomically complete SCI (see Figure 6). The consequence of this regeneration was a gradual recovery of walking in all tested mice.

[0130] method Mouse model. All experiments used adult male or female C57BL / 6 mice (15g-25g body weight, 8-15 weeks old) or transgenic mice. Vsx2 Cre (MMMRRC 36672, Chx10 Cre Transgenic mouse strains (also known as transgenic mice) were crossbred and maintained on a mixed genetic background (C57BL / 6). All rearing, surgery, behavioral experiments, and euthanasia were carried out in accordance with Swiss veterinary law guidelines. Throughout the experiment, manual urination and all other animal care were performed twice daily. All procedures and surgeries were approved by the Geneva Cantonal Veterinary Office (Switzerland, approval numbers GE / 25 / 17 and GE / 109 / 20).

[0131] Viral vectors and vector production. The viruses used in this study were either commercially obtained or produced at the EPFL core facility. The following AAV plasmids were used: AAV5-CMV-TurboRFP (Addgene number 105548), AAV9-CAG-IGF1, AAV9-CAG-Spp1, AAV9-CAG-CNTF (Igf1, Spp1, and Cntf), AAV5-CAG-COMET-GFP, AAV-CAG-flex-tdTomato, AAV-CAG-flex-Human Diphtheria Toxin Receptor (DTR, rAAV2-hSyn-KASH-GFP (Addgene number 60231)), AAV5-hSyn-Con / Fon-eYFP (Addgene number 55650), rAAV2-EF1a-DIO-Flpo (Addgene number 87306), and SIN-cPPT-GFAP-GDNF-WPRE. See also Figure 10. The injection volume, coordinates, and experimental objectives are explained below.

[0132] SCI model. Spinal cord compression has been described previously. Alternating half-section SCI was performed as previously described. In the case of alternating half-section SCI, laminectomy was performed at the mid-thoracic level (T12), and the lateral half of the spinal cord was severed using a microscalpel. Eight weeks after the first half-section, a second mid-thoracic half-section (T7) was performed on the opposite side of the first half-section.

[0133] Biological repair intervention. The general surgical procedure has been previously described in detail. The surgery was performed under sterile conditions and general anesthesia with 1%-2% isoflurane in an oxygen stream of 0.5 L / min-1 L / min. The surgery was performed in EPFL under general anesthesia with isoflurane in oxygen-concentrated air, using a surgical microscope (Zeiss) and a rodent stereotactic device (David Kopf) as previously described. AAV injection was administered two weeks prior to SCI, anticipating the time of molecular expression, and after laminectomy of a single vertebra, spinal cord intrinsic neurons located rostral to segments 1 and 2 relative to the planned location of the SCI lesion were targeted. AAV was administered using a glass micropipette connected to a 10 μl syringe via a high-pressure tube (Kopf) under the control of a microinfusion pump, at two sites 0.6 mm below the surface (one on each side of the spinal cord), with 0.25 μl [AAV2 / 9 Spp1:1×10 13 pieces, Igf1:5×10 12 pieces, Cntf:5×10 12One genome copy (per 1 ml of sterile saline) was injected at a rate of 0.1 μl per minute. Severe crushing SCIs were created at the T12 / T13 level after laminectomy of a single vertebra by completely compressing the entire spinal cord from both sides for 5 seconds using a No. 5 Dumont forceps (Fine Science Tools) with a tip width of 0.5 mm without a spacer. Two days after SCI, hydrogel depots were injected at a rate of 0.15 μl per minute using a glass micropipette connected to a 10 μl syringe via a high-pressure tube (Kopf) under the control of a microinjection pump, in a stereotactic manner into the center of the SCI lesion 0.6 mm below the surface. In animals that received two hydrogel depots, the second depot was placed 1.5 mm caudal to the SCI 9 days after SCI. In animals that received three hydrogel depots, the third depot was placed 2.5 mm below the SCI 16 days after SCI. Animals injected with a lentiviral vector encoding human GDNF (LV-GDNF: 600 μg of P24 per 1 mL) received vector injections in the L2 and L4 spinal segments two days after spinal cord infiltration (SCI). Conduction pathway tracing was performed by injecting 4 × 0.25 μl of AAV2 / 5 RFP red fluorescent protein (RFP, University of Pennsylvania Vector Core, 2.612 × 10¹⁶ per 1 mL) into the rostral segment of the SCI nine days after SCI. 13 The procedure was performed by injection of (1 genome copy). After surgery, the mice were awakened in an incubator. The analgesic buprenorphine (Essex Chemie AG, Switzerland, 0.01 mg / kg~0.05 mg / kg(sc)) or rimadyl (5 mg / kg(sc)) was administered twice daily for 2-3 days post-surgery. After randomly assigning numbers to the animals, they were evaluated without knowing the experimental conditions. Seven days after SCI, all mice were evaluated in open field, and all animals that showed any hindlimb movement were not studied further.

[0134] Hydrogel depot containing growth factors. Biomaterial depots were prepared using well-characterized diblock copolypeptide hydrogels as previously described and loaded with growth factors. Human recombinant FGF2, human recombinant EGF, and human recombinant GDNF were purchased from Peprotech: (i) Human FGF2 (FGF-basic) (154 amino acids) catalog number 100-18B-100UG, lot number 091608 C0617, (ii) Human EGF catalog number AF-100-15-100UG, lot number 0816AFC05 B2317, (iii) Human GDNF catalog number 405-10-100UG, lot number 0606B64 A2517. Lyophilized K 180 L 20 The powder was reconstituted to 3.0% (weight / volume) in sterile PBS containing a combination of FGF2 (1.0 μg / μl), EGF (1.0 μg / μl), and GDNF (1.0 μg / μl).

[0135] Retrograde labeling by spinal injection. To retrogradely label neurons for fluorescence-activated nuclear sorting and subsequent snRNA-seq, partial laminectomy was performed at the L2 spinal cord level, and two sets of rAAV2-hSyn-KASH-GFP were injected bilaterally (0.15 μl per injection) at two depths (0.8 mm and 0.4 mm below the posterior side) with a 1 mm gap. To label regenerating neurons, animals were injected with the virus 4 weeks after SCI and after repair intervention. Animals were perfused 3 weeks after virus injection. To retrogradely label regenerating neurons for histological evaluation, one set of CTB 647 (Thermofisher C34778) was injected bilaterally (0.15 μl per injection) at two depths (0.8 mm and 0.4 mm below the posterior side). Animals were perfused 3 days after CTB injection.

[0136] Long-distance Vsx2 via spinal injection ON Labeling of neurons only. Vsx2 in the mid-thoracic spinal cord with long-range projections to the lumbar executive center. ON A Boolean virus strategy was used to label only neurons. (Vsx2) CrePartial laminectomy was performed on the T10 and L2 spinal segments of mice. Two sets of AAV5-hSyn-Con / Fon-eYFP (Addgene number 55650) were injected bilaterally (0.25 μl per injection) across the T10 spinal segment at a depth of 0.6 mm below the dorsal side and 1 mm apart. Two sets of rAAV2-EF1a-DIO-Flpo (Addgene number 87306) were injected bilaterally (0.15 μl per injection) at two depths (0.8 mm below the dorsal side and 0.4 mm below the dorsal side) and 1 mm apart. After 3 weeks, the animals were perfused.

[0137] Neuron subpopulation-specific destruction. Vsx2 is used to perform destruction experiments using diphtheria toxin. Cre Mice were subjected to biological repair as described above. Two sets of bilateral injections of AAV5-CAG-FLEX-DTR (0.25 μl per injection) were administered across the T10 spinal segment at a depth of 0.6 mm below the posterior side and a distance of 1 mm apart. Two weeks after spinal injection, mice were intraperitoneally injected with diphtheria toxin (Sigma, D0564) diluted in physiological saline (100 μg / kg) to destroy Vsx2 neurons. Mice were examined immediately before and one week after destruction.

[0138] Brainstem injection. An incision was made across the skull. Bregma was identified to target descending neurons in the ventral giant cell nucleus (vGi), and craniotomy was performed 5-6 mm dorsally and 0-2 mm lateral to the bregma. 100 nL of AAV5-CAG-greenComet was injected at a rate of 0.15 μl per minute, 0.3 mm bilaterally in the medial-lateral direction, -5.8 mm and -6.2 mm rostral-caudal, and 5.6 mm from the brain surface in the dorsal-ventral direction. After 3 weeks, the animals were perfused.

[0139] Behavioral evaluation. All behavioral procedures have been previously described in detail. During ground walking, the kinematics of both legs were recorded using 12 infrared cameras from Vicon Motion Systems (Oxford, UK), tracking reflective markers attached to the neck, hip, knee, ankle joints, and distal toes. The limbs were modeled as a chain of interconnected segments, and a total of 80 gait parameters were calculated from the recordings. Multistage multifactor analysis was performed based on principal component analysis, as previously described in detail, not only to evaluate the differences between experimental conditions but also to identify the most relevant parameters explaining these differences. Linear discriminant analysis classification was performed to evaluate the similarity between mice that regained walking after treatment and other experimental groups, including mice that underwent spontaneous repair. Using holdout regenerative treatment mice, a classifier was trained for the motor parameters of each step for each mouse in each experimental group. This process was completed 50 times with repeated subsampling of kinematic features. The trained classifier was then used to predict experimental labels using the holdout motor parameters from regenerative treatment mice. This process was performed using Vsx2 Cre We also repeated the loss-of-function experiments in mice.

[0140] Perfusion. Mice were perfused at the end of the experiment. Deep anesthesia was induced in the mice by intraperitoneal injection of 0.2 mL of pentobarbital sodium (50 mg / mL). The mice were perfused transcardiacly with PBS, followed by PBS with 4% paraformaldehyde. Tissue was removed, fixed overnight in 4% paraformaldehyde, and then transferred to PBS or cryoprotected in PBS with 30% sucrose.

[0141] Immunohistochemical testing. Immunohistochemical testing was performed as previously described. Perfused postmortem tissue was cryopreserved for 48 hours in 30% sucrose in PBS, then embedded in cryomatrix (Tissue Tek OCT, Sakura Finetek Europe BV) and frozen. 30 μm thick transverse or horizontal sections of spinal cord were cut in a cryostat (Leica), immediately mounted on glass slides and dried, or placed in free-floating wells containing PBS + 0.03% sodium azide. The primary antibodies were rabbit anti-GFAP (1:1000, Dako), guinea pig anti-NeuN (1:300, Millipore), rabbit anti-GDNF-α (GDNF receptor alpha) (1:1000 Abcam), guinea pig anti-homer1 (1:600, Synaptic Systems GmbH), rabbit anti-synaptophysin (1:600, Dako), chicken anti-RFP (1:500, Novus Biologicals), goat anti-GFP (1:1000, Novus Biologicals), chicken anti-GFP (1:500 Life Technologies), rabbit anti-Chx10 (also known as Vsx2) (1:500, Novus Biologicals), and sheep anti-Zfhx3 (1:500, Novus Biologicals). Fluorescent secondary antibodies were conjugated to Alexa 488 (green), Alexa 405 (blue), Alexa 555 (red), or Alexa 647 (far-red) (Thermo Fisher Scientific, USA). Nuclear staining: 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI, 2 ng / ml, Molecular Probes). Sections were digitally imaged using a slide scanner (Olympus VS-120 slide scanner) or a confocal microscope (Zeiss LSM880 + Airy high-speed module with ZEN 2 Black software (Zeiss, Germany, Oberkochen)). Images were digitally processed using ImageJ (ImageJ NIH) software or Imaris (Bitplane, v.9.0.0).

[0142] Tissue clearing (CLARITY). The sample was incubated in X-CLARITY hydrogel solution (Logos Biosystems Inc., South Korea) at 4°C for 24 hours with gentle shaking. The sample was degassed and polymerized using the X-CLARITY polymerization system (Logos Biosystems Inc., South Korea), followed by washing in 0.001 M PBS at room temperature for 5 minutes. Next, the sample was placed in the X-CLARITY Tissue Clearing System (Logos Biosystems Inc., South Korea) set to 1.5 A, 100 RPM, and 37°C for 29 hours. The clearing solution was prepared in-house by adding dH2O to 4% sodium dodecyl sulfate (SDS) and 200 mM boric acid, and adjusting the pH to 8.5. Subsequently, the sample was washed in 0.1 M PBS solution containing 0.1% Triton X-100 at room temperature for at least 24 hours with gentle shaking to remove excess SDS. Finally, the samples were incubated in 40 g of Histodenz dissolved in 30 mL of 0.02 M PB (pH 7.5) and 0.01% sodium azide (refractive index 1.465) at room temperature for at least 24 hours with gentle shaking before imaging.

[0143] 3D imaging. Imagery of cleared tissue was performed using either a customized mesoSPIM or a light-sheet microscope (COLM) optimized for clarity. The central nervous system was fixed in a chamber filled with RIMS using a custom-made sample holder. Samples were imaged using one or two light sheets illuminating the sample from both sides, with 1.25x or 2.5x objective lenses for mesoSPIM and 4x or 10x objective lenses for COLM. The voxel resolution in the x, y, and z directions was 5.3 μm × 5.3 μm × 5 μm at 1.25x acquisition and 2.6 μm × 2.6 μm × 3 μm at 2.5x acquisition. The voxel resolution for COLM was 1.4 μm × 1.4 μm × 5 μm at 4x acquisition and 0.59 μm × 0.59 μm × 3 μm at 10x acquisition. The images were generated as 16-bit TIFF files, which were then stitched together using Arivis Vision4D (Arivis AG, Munich, Germany). 3D reconstructions of the raw images and optical sections were generated using Imaris (bitplane, V.9.8) software.

[0144] Axonal quantification. A custom image analysis pipeline was executed, including preprocessing, registration, and combination of histological images from different sections, to align all sections in a common coordinate space. In short, all preprocessing was performed in Fiji, and all registration procedures were performed in R using the image analysis package "imageR" and the medical image registration package "RNiftyReg". Images were aligned to template spinal cord sections. Axonal density was calculated for every 200 μm bin, starting from the center of the lesion.

[0145] Cell counting. To quantify the proportion of neurons expressing Vsx2 and / or Zfhx3, the number of NeuN-positive neurons expressing Vsx2 and / or Zfhx3 was counted. Cell counting was performed using the image analysis software Imaris (bitplane, V.9.8).

[0146] Synapse detection. To detect the number of synapses in contact with a neuron, the cell surface was first reconstructed with a resolution of 10 μm using the surface reconstruction module of Imaris (bitplane, V9.8). Then, synapses were identified using spot detection in Imaris. To identify synapses adjacent to the target neuron, a MATLAB algorithm was used to isolate synapses located between 0 μm and 1 μm from the reconstructed surface of the neuron.

[0147] Electrophysiology. Animals were anesthetized with ketamine / xylazine, small trepanation holes were drilled in the skull to reach vGi, and needle electrodes were inserted into the tibialis anterior muscles of both hind limbs. A platinum / iridium concentric bipolar electrode (PI-SNE-100, Microprobes USA) was inserted into vGi (coordinates: -6.0 AP, 0.3 ML, -5.7 DV relative to bregma). Electrical stimulation (STG4000, Multi Channel Systems) was transmitted to vGi as a 5 × 200 μs square wave pulse train at 500 Hz, repeated once every 5 seconds. Stimulation was transmitted at 20 μA, 40 μA, 60 μA, 80 μA, and 100 μA, with each intensity repeated 10 times. Evoked EMG recordings were amplified (1000x) and filtered (300Hz high-pass, 5kHz low-pass) using a differential amplifier (Model 1700, AM Systems), then digitized and recorded (PowerLab 8 / 35, AD Instruments). The average peak-to-peak amplitude of the evoked response was calculated offline (LabChart Pro, AD Instruments) and compared between groups.

[0148] Statistics, power calculation, group size, and reproducibility. Statistical evaluation of repeated measures was performed by post-hoc independent pairwise analysis along with one-way ANOVA according to TukeyHSD. Power calculation was performed using G*Power software v.3.1.9.245. A group size was used that was calculated to obtain at least 80% power when quantifying histologically obtained neuroanatomical outcomes such as axonal density, using the following parameters: probability of type I error (α) = 0.05, a conservative effect size of 0.25, and 3 to 10 treatment groups from which numerous measurements were obtained per repeat. All graphs show individual values ​​as dot plots, as well as the mean ± standard error. All bar graphs are overlaid with dot plots, where each dot represents a value for one animal. Experiments testing axonal regrowth beyond SCI lesions in animals were independently repeated at least twice in different groups of mice, yielding similar results. Similar results were obtained when staining experiments were independently repeated using tissues from at least four, and in most cases six, different animals for all microscopic images of histological tissues.

[0149] Mononuclear RNA sequencing. Mononuclear dissociation of mouse lumbar spinal cord was performed according to the inventors' established procedure. After euthanasia by isoflurane inhalation and cervical dislocation, the lumbar spinal cord region was immediately dissected and frozen on dry ice. The spinal cord was immersed in 500 μl of sucrose buffer (0.32 M sucrose, 10 mM HEPES [pH 8.0], 5 mM CaCl2, 3 mM Mg acetate, 0.1 mM EDTA, 1 mM DTT) and 0.1% Triton X-100 using a Kontes Dounce Tissue Grinder. Then, 2 mL of sucrose buffer was added and the mixture was filtered through a 40 μm cell strainer. The lysate was centrifuged at 3200 g for 10 minutes at 4°C. Then, the supernatant was decanted and 3 mL of sucrose buffer was added to the pellet for 1 minute. The pellet was homogenized using Ultra-Turrax, and 12.5 mL of density buffer (1 M sucrose, 10 mM HEPES [pH 8.0], 3 mM Mg acetate, 1 mM DTT) was added below the nucleus layer. The tube was centrifuged at 3200 g at 4°C, and the supernatant was discarded. The nuclei in the lower half of the tube wall were collected using 100 μl of PBS containing 0.04% BSA and 0.2 U / μl of RNase inhibitor. Finally, the nuclei were resuspended through a 30 μm strainer to adjust the concentration to 1000 nuclei per μl.

[0150] Projection-specific snRNA-seq. To perform the projection-specific snRNA-seq experiment, the nuclei were first resuspended in 500 μl of Pre-FACS buffer (1 × PBS containing 1% BSA and 0.2 U / μl of SUPERaseIn RNase inhibitor) and filtered through a 35 μm cell strainer. The samples were processed in a Sony Corporation SH800 cell sorter equipped with a 100 mm sorting tip. The nuclei were analyzed for forward scattering, side scattering, and DRAQ5. + Measurements were used for gating to ensure the doublets were gated out. After this initial gating, a 2D scatter plot was used to analyze the GFP. + The nucleus was identified. GFP + / DRAQ5 +The nuclei were collected into a 1.5 ml centrifuge tube containing 10 μl of Pre-FACS buffer. Approximately 500 GFP cells were collected from the undamaged spinal cord. + We collected nuclei (pooling these nuclei from n=5 mice) and extracted approximately 800 GFP from regenerating axons. + The nuclei were collected (these nuclei were pooled from n=13 mice). Then, GFP was applied. + The nucleus was directly loaded into a Chromium Single Cell Processor (10x Genomics) and RNA from a single nucleus was barcoded.

[0151] Library preparation. snRNA-seq libraries were prepared using the 10x Genomics Chromium Single Cell Kit (version 3). Nuclear suspensions were added to the Chromium RT mix to achieve loading numbers of 2000–5000. Downstream cDNA synthesis (13 PCR cycles), library preparation, and sequencing were performed according to the manufacturer's instructions.

[0152] Read alignment. Reads were aligned to the latest Ensembl release (GRCm38.93) using Cell Ranger, and a matrix of unique molecular identifiers (UMIs) was obtained. Quality control metrics were calculated for each cell barcode using Seurat, including the number of genes detected, the number of UMIs, and the percentage of reads aligned to mitochondrial genes. Low-quality cells were filtered out by removing cells expressing fewer than 200 genes or cells with fewer than 5% mitochondrial reads. Genes expressed in fewer than 3 cells were similarly removed.

[0153] Clustering and Integration. Prior to clustering analysis, batch effect correction and data integration across two different experimental conditions were performed as previously described. Gene expression data were normalized using a regularized negative binomial model and then integrated across batches using the data integration workflow within Seurat. The normalized and integrated gene expression matrices were then clustered to identify cell types in the integrated dataset, again using the default Seurat workflow. Cell types were manually annotated based on marker gene expression, guided by previous studies of mouse spinal cord. Local and projecting neuronal subpopulations were annotated based on Nfib expression and Zfhx3 expression, respectively. Subpopulation annotation was performed after reintegrating each subsequent experiment with this dataset following our projection-specific snRNA-seq experiments in undamaged mice. This allowed for the identification of the same 28 neuronal subpopulations across three different experiments.

[0154] Cell type prioritization using Augur. Our machine learning method, Augur, was used to identify neuronal subpopulations perturbed during natural repair. Augur was run with default parameters for all comparisons. Augur was applied at various clustering resolutions to evaluate the robustness of cell type prioritization against the resolution at which neuronal subtypes are defined in snRNA-seq data. The resulting cell type prioritization was visualized both as a hierarchical clustering tree of neuronal subtypes and as a UMAP progression. The primary assumption underlying Augur is that, within the highly multidimensional space of gene expression, cell types that respond more strongly to perturbations should be separable than less affected cell types. In short, Augur trains a random forest classifier to predict the conditions under which each cell was acquired after retaining some sample labels. The accuracy of this prediction from single-cell gene expression measurements is then evaluated in cross-validation and quantified using the area under the receiver operating characteristic curve (AUC).

[0155] Cell type proportions. Normalized proportions of each neuron subpopulation were calculated to compare proportions within and across datasets. These distributions were compared to the proportions expected in undamaged datasets using a chi-square test.

Claims

1. i) A first active substance comprising an exogenous nucleic acid encoding osteopontin (Spp1) polypeptide as shown in SEQ ID NO: 1 or SEQ ID NO: 8, insulin-like growth factor 1 (Igf1) polypeptide as shown in SEQ ID NO: 2, and ciliary body-derived neurotrophic factor (Cntf) polypeptide as shown in SEQ ID NO: 3 or SEQ ID NO: 9, and / or ii) A second active substance comprising exogenous nucleic acids encoding fibroblast growth factor 2 (FGF2) polypeptide as shown in SEQ ID NO: 4 or SEQ ID NO: 10, epidermal growth factor (EGF) polypeptide as shown in SEQ ID NO: 5 or SEQ ID NO: 11, and glial-derived neurotrophic factor (GDNF) polypeptide as shown in SEQ ID NO: 6, A combination medicine containing, If both are present, the first active substance and the second active substance are administered sequentially or simultaneously, and The first and second active substances are a combination pharmaceutical product that results in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

2. The combination pharmaceutical according to claim 1, wherein the second active substance further comprises an exogenous nucleic acid encoding a vascular endothelial growth factor (VEGF) polypeptide as shown in SEQ ID NO: 7 or SEQ ID NO:

12.

3. iii) A third active substance containing an exogenous nucleic acid encoding a glial-derived neurotrophic factor (GDNF) polypeptide as shown in Sequence ID No. 6, This further includes, and here, The first active substance, the second active substance, and the third active substance are administered sequentially or simultaneously, The combination pharmaceutical according to claim 1 or 2, wherein the first active substance, the second active substance, and the third active substance result in the sustained expression and secretion of an effective amount of the polypeptide for at least three days.

4. The combination pharmaceutical according to any one of claims 1 to 3, wherein the first active substance, the second active substance, and the third active substance are a gene editing system, a vector or expression vector comprising one or more nucleic acid sequences encoding one or more polypeptides according to any one of claims 1 to 3.

5. The combination pharmaceutical according to claim 4, wherein the expression vector is a viral vector selected from the group consisting of retroviruses, lentiviruses, adenoviruses, herpesviruses, poxviruses, alphaviruses, vaccinia viruses, and adeno-associated viruses.

6. A combination pharmaceutical product according to claim 1 or 2, used in a method for treating incomplete spinal cord injury or stroke in a subject.

7. A combination pharmaceutical according to claim 3, used in a method for treating complete spinal cord injury in a subject, comprising the first active substance, the second active substance, and the third active substance.

8. The combination pharmaceutical used according to claim 6 or 7, wherein the aforementioned complete or incomplete spinal cord injury is a spinal cord injury of the cervical, thoracic, and / or lumbar spinal cord.

9. The combination pharmaceutical used according to claim 6, wherein the treatment for stroke is neuronal repair, functional repair, and anatomical repair of the brain after stroke.

10. The combination pharmaceutical used according to claim 8 or 9, wherein the spinal cord repair of the cervical, thoracic, and / or lumbar spinal cord, and / or functional and anatomical repair of brain neurons are obtained by targeting V1 neurons, V0 neurons, V2b neurons, CSF contact neurons, Vglut2 neurons, Vgat neurons, Chat neurons, Vsx2 and / or Zfhx3 (projection V2a neurons).

11. The spinal cord repair comprises axonal regeneration and restoration of neurological function, as described in claim 8 or 10.

12. The combination pharmaceutical used according to any one of claims 6 to 11, wherein the treatment method further includes an initial evaluation step of determining whether the subject has incomplete spinal cord injury, complete spinal cord injury, or stroke.

13. The combination pharmaceutical used according to claim 12, wherein the initial evaluation step includes an analysis of spinal cord injury symptoms or stroke symptoms selected from the group including one or more of the following: loss or impairment of motor function(s) in the cervical, thoracic, lumbar, or sacral spinal cord segments and / or the brain; loss or impairment of sensory function(s); and loss or impairment of autonomic nervous system function(s).

14. The combination pharmaceutical used according to any one of claims 6 to 13, wherein the first active substance, the second active substance, and the third active substance of the combination pharmaceutical, or the combination pharmaceutical, is administered by injection into the cervical, thoracic, lumbar, or sacral spinal cord of a subject via stereotactic injection, or by injection into the brain, or by intravenous infusion or intravenous injection.

15. The stereotactic injection is guided by the location of the core of the spinal cord injury by a method selected from ultrasound guidance, electrophysiology, or molecular guidance, as described in claim 14.

16. The subject is a mammal, preferably a human, in the combination pharmaceutical used according to any one of claims 6 to 15.

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