WO2018008770A1

WO2018008770A1 - Method for controlling peripheral nerve, method for developing function to alter function of nerve cell in organs excluding brain and spinal cord and others, novel method for preventing or treating disease, and medicine for application to peripheral nerves

Info

Publication number: WO2018008770A1
Application number: PCT/JP2017/025468
Authority: WO
Inventors: 厚範神谷
Original assignee: 国立研究開発法人国立循環器病研究センター
Priority date: 2016-07-06
Filing date: 2017-07-05
Publication date: 2018-01-11
Also published as: JPWO2018008770A1

Abstract

Provided is a method for preventing or treating a disease, said method being characterized in that a viral vector having, integrated thereinto, a gene encoding a protein having a function to kill, stimulate or depress peripheral nerves or a function to alter another nerve cell function is injected topically to an organ excluding brain and spinal cord, a tumor or an area that has undergone tumor resection to cause the infection of the nerves with the viral vector and consequently cause the expression of the protein having the above-mentioned function in the infected nerves, whereby the function of the nerves infected with the viral vector is controlled by the protein so that the function of the organ, excluding brain and spinal cord, to which the nerves are connected is altered. The method can be utilized for providing a therapeutic medicine and a treatment means in the medical industry. Particularly in cancer therapies, this method can be utilized widely both domestically and abroad in such a way that the viral vector is injected topically to a remaining cancer tissue postoperatively to suppress the recurrence or metastasis of the cancer.

Description

Methods for manipulating peripheral nerves, methods for generating functions that change neuronal cell functions in organs other than the brain and spinal cord, methods for preventing or treating novel diseases, and pharmaceuticals for peripheral nerve administration

The present invention relates to a method for manipulating peripheral nerves, a method for generating a function for changing nerve cell functions in organs other than the brain and spinal cord, a novel disease prevention or treatment method, and a pharmaceutical for peripheral nerve administration. More specifically, therapeutic agents and methods for preventing and treating diseases such as cancer, hypertension, diabetes, heart failure or obesity using a viral vector incorporating a gene that expresses a protein having biopharmacological activity, etc. About.

The autonomic nervous system (sympathetic and parasympathetic) distributes to all organs of the body and regulates its organ function, but the regulation is not uniform throughout the body, but is performed on an organ-specific or local basis. . The autonomic nervous system plays an important role in maintaining homeostasis such as circulation, respiration, metabolism, and immunity in a healthy state, while in diseases, the neurodynamics of certain organs are irrational, Rather, the disease state may be exacerbated (Non-patent Document 1). In addition, afferent nerves are distributed to all organs of the body to sense local biological information and play an important role in maintaining homeostasis. On the other hand, in diseases, the neurodynamics of specific organs It is irrational and may worsen the disease.

Here, in Non-Patent Document 2, in view of the development of selective introduction of photogenes into specific nerve fibers in brain science, other emerging technologies that selectively manipulate peripheral local nerve inputs for therapeutic purposes. It can be imagined that the technology is described in the hypothetical past (cold evolution). Nevertheless, the fact that it has not been realized to date is a proof that even those skilled in the art could not easily correspond.

That is, there is currently no medical technology that can manipulate the local peripheral nerve function in a form selective to a specific nerve type of a specific organ, and there is no such technical field. First, the drug is a systemic action, and the side effects due to the action on non-target organs cannot be avoided, so the dose and therapeutic effect are limited. For example, when it is desired to administer an appropriate amount of a drug in order to obtain a therapeutic effect on the target organ for treatment, it is necessary to reduce the dose due to side effects in other organs that occur at the same time. The therapeutic effect will decrease.

Also, nerve intervention other than current drugs includes nerve electrical stimulation, cautery cutting and blocking. However, the peripheral nerve is a bundle of nerve fibers, efferent fibers for multiple organs (nerve fibers that transmit electrical information from the central nervous system brain or spinal cord to the organs), and afferents for multiple organs. It contains fibers (conversely, nerve fibers that carry electrical information from the organ to the brain or spinal cord, which is the central nervous system). Therefore, nerve electrical stimulation stimulates multiple nerve fibers (multiple organs, efferent fibers and afferent fibers, and various types of nerves such as sympathetic and parasympathetic nerves) contained in nerve bundles and ganglia. In addition, side effects caused by stimulation of nerve fibers that are not intended cannot be avoided, and medical applications and therapeutic effects are limited. For example, stimulating the cervical vagus nerve to stimulate parasympathetic nerves (efferent fibers) distributed in the heart during heart disease treatment also stimulates afferent nerves distributed in the digestive tract, etc. There is a risk of side effects such as pain and pain. Similarly, nerve cautery cutting also destroys multiple nerve fibers contained in nerve bundles and ganglia, so the side effects of undesired nerve fiber destruction cannot be avoided, limiting the therapeutic effect. obtain. Similarly, nerve blocks (local injection near the nerve, such as anesthetics, nerve destroyers, nerve blockers, and physiological saline, and physical loads such as compression and decompression) are also included in nerve bundles and ganglia. In order to destroy a plurality of nerve fibers, side effects due to destruction of unintended nerve fibers cannot be avoided, so that the therapeutic effect can be limited.

On the other hand, in the central nervous system (brain or spinal cord), gene transfer and gene manipulation are carried out in a form selective to a specific nerve species using a viral vector. At this time, a method of introducing a gene by infecting a neuronal cell body with a viral vector (an antegrade infection, an antegrade gene introduction, and antegrade means the direction from the neuronal cell body to the terminal), In addition, there is a method of infecting a virus vector from a nerve terminal to introduce a gene (retrograde infection, retrograde gene introduction, and retrograde means the direction from the nerve terminal to the cell body). By using a sequence such as a promoter specific to a nerve species (related gene expression regulatory sequences such as a promoter and an enhancer) in a viral vector, gene manipulation specific to the nerve species can be performed. Examples of the gene sequence for neural manipulation include DTA when the nerve is destroyed, photoresponsive channel (optogenetics-related) and bacterial channel NaChBac when stimulating the nerve activity, and Kir2 when suppressing the nerve activity. .1 etc. are used.

Also, in the central nervous system (brain or spinal cord), gene transfer and gene manipulation are performed in a manner selective to a specific nerve pathway using a viral vector. Nerve cells have long axons and wire from one place to another in the central nervous system (brain or spinal cord). In every place, there are many wiring neurons that connect to different places, forming a network of many nerve cells. Among the various wirings in the neural network, two methods on the wiring (an antegrade infection of nerve cell bodies and a retrograde infection of nerve terminals) ) Virus vector technology (double infection method) (Non-patent Document 3) in which two types of viral vectors (driver vector and reporter vector) are locally injected and the gene sequence for manipulation is expressed only in superinfected cells. It has been reported. The driver vector and the reporter vector are set so that both cooperate to regulate gene expression. In Non-Patent Document 3, a tet switch, which is a known gene expression regulation mechanism, is used, tTA is expressed by a driver vector, and a gene sequence for manipulation is expressed in an inducible manner (doxycycline). However, other gene expression regulation mechanisms can be used. For example, Cre recombinase is expressed in a driver vector to change the insertion base sequence of the reporter vector to regulate (induce or stop) the expression of the gene sequence for manipulation. I can do it.

For medical practical use, it is desirable to use a virus vector with excellent safety. What is currently evaluated as having the highest safety and low pathogenicity to the human body (almost no) is an adeno-associated virus vector (Non-Patent Document 4), and in the central nervous system (brain or spinal cord), It is used for clinical treatment of Parkinson's disease (Non-patent Document 5). However, it is known that adeno-associated virus vectors are weak in retrograde infection and retrograde gene transfer into nerves in the central nervous system (brain or spinal cord). However, it has not been known whether retrograde infection or retrograde gene transfer to the autonomic nervous system or afferent nerve of the peripheral nerve is possible. In addition, since adeno-associated virus vectors are known to have a gene size that can be inserted limited to 4.5 kb in length, even if genes can be introduced into peripheral and autonomic nerves, short insertion It has not been known whether the gene can manipulate the nerve function, whether the nerve operation can treat the disease, or the nucleobase sequence of the inserted gene to enable them.

Lentiviral vectors are known to be capable of retrograde infection and retrograde gene transfer into nerves in the central nervous system (brain or spinal cord). However, it has not been known whether retrograde infection or retrograde gene transfer to the autonomic nerve or afferent nerve of the peripheral nerve is possible. Furthermore, it has not been known whether or not these nerve functions can be actually manipulated, whether or not the disease can be treated by the nerve manipulation, and the nucleobase sequence of the inserted gene for enabling them.

That is, no one was aware of the usefulness of local nerve manipulation itself in disease. Up to now, neither a method for realizing a local nerve operation nor an experiment of such a method has been attempted, and in fact, it has not been done to date. The present inventor has noticed the usefulness of local nerve manipulation in a disease, and devised and developed a method for realizing it, and verified it through experiments, and has found a completely new technical field of local nerve manipulation that has never existed before. This local nerve operation selectively manipulates the autonomic and afferent nerves of the peripheral nerve only in specific organs and local tissues, and only specific nerve types (sympathetic nerve, parasympathetic nerve, afferent nerve). That is. The autonomic and afferent nerves of the peripheral nerve work locally, and in the disease, the function of the local nerve is irrational (exacerbation of disease, inhibition of treatment, etc.), but the conventional method Drugs and nerve interventions (electrical stimulation, cautery cutting, blocking) are not only capable of manipulating nerves with such locality, but everyone is aware of the usefulness of such local nerve manipulation itself It wasn't.

More specifically, for example, a renal sympathetic nerve that specifically infects or distributes a kidney by locally injecting a viral vector incorporating a gene for manipulation encoding a protein having a function of stimulating renal sympathetic nerve into a rat kidney. The patient was retrogradely infected and stimulated the renal sympathetic nerve to induce hypertension.
Furthermore, the blood pressure was lowered by removing the renal sympathetic nerve in a drug-induced manner in this hypertensive rat. In addition, a hypertension rat is locally injected with a viral vector incorporating a manipulation gene encoding a protein having a function of stimulating afferent nerves into the baroreceptor region tissue of the cervical artery to stimulate the afferent nerves. Decreased blood pressure.
In addition, a viral vector incorporating a gene for manipulation that encodes a protein having a function of destroying sympathetic nerves in hypertensive rats is locally injected into the kidney, and the renal sympathetic nerve is specifically communicated or distributed to the kidney. By retrogradely infecting and destroying the renal sympathetic nerve, hypertension was improved.

Furthermore, a viral vector incorporating a manipulation gene encoding a protein having a function of stimulating parasympathetic nerves is locally injected into the pancreas of a diabetic model rat, and retrograde to parasympathetic nerves that are specifically communicated or distributed in the pancreas Glucose tolerance in diabetic model rats was improved by infecting (partly antegrade infection) and stimulating the parasympathetic nerve.

Furthermore, it was found that autonomic nerves including sympathetic nerves and parasympathetic nerves are specifically connected to or distributed in breast cancer tissues. The breast cancer tissue is locally injected with a viral vector incorporating a gene for manipulation that encodes a protein having a function of destroying the sympathetic nerve, and retrogradely infects the sympathetic nerve that is specifically connected to or distributed in the breast cancer tissue. By removing sympathetic nerves that are specifically connected to or distributed in the breast cancer tissue, the increase in the breast cancer tissue was suppressed, and the occurrence, metastasis, or recurrence of breast cancer was suppressed. Furthermore, by removing parasympathetic nerves by local injection of a viral vector incorporating a gene for manipulation that encodes a protein having a function of destroying parasympathetic nerves communicating or distributed with breast cancer tissue, breast cancer tissue is increased, And by stimulating the parasympathetic nerves by local injection of a viral vector incorporating a gene for manipulation that encodes a protein having a function of stimulating the parasympathetic nerves that communicates or distributes to the breast cancer tissue, thereby suppressing the increase of the breast cancer tissue. And also suppressed the occurrence, metastasis or recurrence of breast cancer.

Furthermore, it has been found that autonomic nerves are specifically connected to or distributed to skin cancer tissues. The skin cancer tissue is locally injected with a viral vector incorporating a manipulation gene encoding a protein having a function of destroying sympathetic nerves, and is specifically communicated or distributed or distributed to the skin cancer tissue. By retrogradely infecting the sympathetic nerve and removing the sympathetic nerve that specifically communicates or distributes or distributes the skin cancer tissue, the increase in the skin cancer tissue is suppressed, or the recurrence of the skin cancer Was suppressed. Furthermore, by stimulating the parasympathetic nerve by local injection of a viral vector that incorporates a gene for manipulation that encodes a protein having a function of stimulating the parasympathetic nerve that communicates or distributes or distributes to skin cancer tissue, Suppression of cancer tissue growth or skin cancer recurrence was suppressed.

Moreover, for the purpose of selectively stimulating the afferent vagus nerve that communicates or distributes in the stomach, a viral vector incorporating a gene that expresses the Cre recombinant protein is first obtained. Inject locally into the stomach (the body of the stomach) where nerve nerve endings are present to retrogradely infect the afferent vagus nerve, express Cre recombinant protein in the afferent vagus nerve, and stimulate the afferent vagus nerve The gene for the operation that encodes the protein having the function to be expressed is responsive to Cre using the loxP system (more specifically, the gene sequence to be expressed is sandwiched between two pairs of lox sequences (eg, loxP and lox2272)) Inserted double-opened-reading-frame (In a structure of (DIO)), the incorporated viral vector is locally injected into the nodular ganglion with the cell body of the afferent vagus nerve to cause antegrade infection, and the afferent vagus nerve is transmitted by the Cre-loxP system. Stimulation with expressed protein reduced food intake and body weight. Furthermore, by using the Cre-loxP system, an antegrade infection of the afferent vagus nerve with a viral vector incorporating a manipulation gene encoding a protein having a function of suppressing the afferent vagus nerve is performed. Diet and weight were increased.

Furthermore, a viral vector incorporating a manipulation gene encoding a protein having a function of stimulating afferent nerves is locally injected into the baroreceptor region tissue of the cervical artery of a myocardial infarction heart failure model rat. The heart function of heart failure model rats was improved by stimulating baroreflex afferent nerves that specifically communicate or distribute or distribute to the container area tissue.

Furthermore, a viral vector incorporating a gene for manipulating a protein having a function of stimulating afferent nerves is locally injected into the baroreceptor region tissue of the rat cervical artery, and specific to the baroreceptor region tissue of the cervical artery. Blood pressure was reduced by stimulating baroreflex afferent nerves that communicated or distributed.

Furthermore, the femoral bone of the rat is retrogradely infected with a viral vector incorporating a gene for operation that encodes a protein having a function of stimulating parasympathetic nerves, and the parasympathetic nerves that specifically communicate with the femur are stimulated. By doing so, the bone mass was increased, the number of osteoblasts and the bone formation function (bone mineralization rate, bone formation rate) were increased, and at the same time, the bone resorption function of osteoclasts was reduced.

Furthermore, in the rat small intestine, it was discovered that the cholinergic nerves of the enteric nerves take up the antigen and secrete the vesicles containing the antigen to the outside of the cell. Cholinergic activity that exists in the small intestine by retrograde infection (partially antegrade infection) with a viral vector incorporating a nucleotide sequence encoding shRNA that suppresses secretion (eg, shRNA against Neutral sphingomyelinase 2 (smpd3)) By changing the function of the sexual nerve, the secretion of vesicles containing some antigens was suppressed and the immune function was changed.

Furthermore, for all receptors expressed in neurons, a viral vector incorporating a specific receptor gene sequence or a mutant gene sequence thereof, or a shRNA base sequence that suppresses gene expression of the receptor By retrograde infection (partially antegrade infection) of local afferent nerves (or other peripheral nerves) with a viral vector incorporating a miRNA base sequence that changes the gene expression Biological signals such as physical signals in the body (temperature, pressure, mechano stimulation, etc.), chemical signals (pH, etc.), physiologically active substances (ATP, ions, peptides, cytokines, etc.), neurotransmitters (acetylcholine, peptides, etc.) The response characteristics (threshold, sensitivity, etc.) when responding to the response are changed to induce and / or enhance biological responses that contribute to disease treatment Or may be or suppress an undesirable biological response to disease treatment. For example, by changing the response characteristics (threshold, sensitivity, etc.) of airway afferent nerves to airway endocrine fluid, mucus, etc., the activity of the afferent nerves can be suppressed and excessive coughing can be suppressed. In addition, for example, by changing the response characteristics (threshold, sensitivity, etc.) to physiologically active substances related to pain, numbness, etc. of the afferent nerve of the foot, the activity of the afferent nerve is suppressed, and pain, numbness, etc. are suppressed. I can do it.

The object of the present invention is to target peripheral nerves in specific organs and local tissues, and further to specific nerve types (for example, efferent nerves and afferent nerves, sympathetic nerves, parasympathetic nerves, vagus nerves, somatic perceptions). It is intended to provide a novel method for preventing or treating diseases in which genetic manipulation of a nerve, visceral sensory nerve, afferent nerve of special sensory nerve, and enteric nerve) is selectively performed.

As a result of intensive studies to achieve the above object, the present inventors have found that a part of the viral vector used for nerves in the central nervous system (brain or spinal cord) is an autonomic nerve (sympathetic) of peripheral nerves. Nerve or parasympathetic or vagus nerve), afferent nerves or enteric nerves are retrogradely infected, and local peripheral nerves distributed only in specific organs or local tissues are nerve type specific (sympathetic nerves) It was found that it is possible to selectively operate only the parasympathetic nerve, the vagus nerve, the afferent nerve, or the enteric nerve, and the present invention was completed through further studies.

First, the inventors of the present invention locally inject a virus vector into which a neuron-specific gene expression regulatory mechanism (a combination of a neuron-specific promoter and the like and a therapeutic gene) is inserted into a specific organ or local tissue. By treating the local autonomic nerves distributed there, a therapeutic gene is expressed in a neuron-specific manner (selectively administered to the sympathetic and parasympathetic nerves), and the nerve function is manipulated to treat the disease. New medical technology was developed, and animal experiments demonstrated its therapeutic efficacy (cancer, hypertension, diabetes, increased bone mass).

Next, the present inventors have made retrograde infection by locally injecting a viral vector into a local tissue where only afferent nerves are distributed, or two points of afferent nerves (neuronal cells). Local afferent nerves by locally injecting two types of viral vectors that co-operate in the ganglia and spinal cord where the body exists and in specific organs and local tissues where nerve endings exist) He developed a medical technique to treat a disease by expressing a therapeutic gene in an animal, and demonstrated its therapeutic efficacy in animal experiments (hypertension, heart failure, obesity).
Such a technique capable of infecting the peripheral nerves of autonomic nerves or afferent nerves with a viral vector has been found for the first time in the present invention, and the inventors have further advanced research based on these findings. The present invention has been completed.

Note that the description of the paragraphs [0011] to [0020] described above constitutes not only the explanation of the difference from the known technique but also the [means for solving the problems] of the present invention. [0020] is quoted as it is fully described in paragraph [0026].

That is, the present invention
(1) A viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs, tumors or tumors other than the brain and spinal cord Is injected locally into the excised site, the virus vector is infected into the nerve, the protein having the function is expressed in the infected nerve, the function of the nerve infected with the virus vector is manipulated by the protein, A method for preventing or treating a disease, comprising changing the function of an organ other than the brain and spinal cord to which a nerve communicates.
(2) A viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs, tumors or tumors other than the brain and spinal cord Incorporating a gene that expresses a protein having a function of killing, stimulating, suppressing, or altering other neuronal functions of peripheral nerves, characterized by including a step of locally injecting into the excised region A method for antegrade or retrograde infection of the nerve with a viral vector.
(3) A viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs, tumors or tumors other than the brain and spinal cord A method for locally injecting a tumor to a site where the tumor is excised and generating the function in the organ-specific, the tumor or the site where the tumor is excised.
(4) A viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, and organs, tumors or tumors other than the brain and spinal cord A method for manipulating peripheral nerves, characterized in that local injection is performed at the site of excision.
(5) Any of the above (1) to (4), wherein the peripheral nerve is efferent or afferent sympathetic nerve, efferent or afferent parasympathetic nerve, efferent or afferent vagus nerve The method of crab.
(6) Peripheral nerves are somatosensory nerves (tactile, pressure, cold, warm, pain, itch, numbness), visceral sensory deities or special sensory nerves (olfaction, vision, hearing, balance, taste) The method according to any one of (1) to (4) above, wherein
(7) The method according to any one of (1) to (4), wherein the peripheral nerve is an enteric nerve.
(8) The method according to any one of (1) to (7) above, wherein the viral vector has a neuron-specific promoter sequence.
(9) The method according to (1) above, wherein the disease is a bacterial infection, a viral infection, a mycosis, another infection or a parasitic disease.
(11) Disease is lip, oral cavity, pharynx, esophagus, stomach, small intestine, colon, rectosigmoid junction, rectum, anus, anal canal, liver, intrahepatic bile duct, gallbladder (sac), pancreas, inner ear, larynx , Bronchus, lung, heart, mediastinum, pleura, peripheral nerve, autonomic nerve, retroperitoneum, peritoneum, connective tissue, soft tissue, bone, cartilage, skin, breast, cervix, uterine body, ovary, placenta, penis, Prostate, testis (testicle), kidney excluding renal pelvis, renal pelvis, urinary bladder, thyroid, adrenal gland, endocrine tissue, lymphoid tissue, hematopoietic tissue, eye, ocular appendage, meningeal malignant neoplasm, secondary site of these The method according to (1) above, which is a malignant neoplasm, an intraepithelial neoplasm, a benign neoplasm, a malignant neoplasm of unknown site, or a neoplasm with unknown or unknown properties.
(15) The method according to (1) above, wherein the disease is anemia, coagulation disorder, purpura, hemorrhagic pathology, hyposplenic function, hypersplenism, or sarcoidosis.
(19) The disease is hypothyroidism, thyroid poisoning (hyperthyroidism), thyroiditis, thyroid disorder, insulin-dependent diabetes (IDDM), non-insulin-dependent diabetes (NIDDM), diabetes, hypoglycemia, Parathyroid (hypoparathyroidism) hypofunction, parathyroid (hypoparathyroidism) hyperfunction, parathyroid (parathyroidism) disorder, aldosteronism, hyperadrenocorticism, primary adrenocortical dysfunction (disease), Addison crisis (onset), drug-induced adrenocortical dysfunction (disease), adrenocortical dysfunction (disease), adrenal medulla hyperfunction, adrenal disorder, hyperestrogen (disease), androgen excess (disease), multiple Cystic ovary syndrome, primary ovarian dysfunction (disease), ovarian dysfunction, testicular (testicular) hyperfunction (disease), testicular (testis) hypofunction (disease), testicular (testis) dysfunction, etc. Endocrine disorders, obesity (disease), a hyperalimentation (overfed), amyloidosis (amyloidosis) or metabolic disorders, the method according to (1).
(23) The disease is Alzheimer's disease dementia, vascular dementia, other dementia, personality disorder, mood (emotional) disorder, physical expression disorder, anxiety disorder, obsessive-compulsive disorder (obsessive-compulsive disorder) , Adaptation disorder, schizophrenia, mania, bipolar affective disorder (manic depression), depression, recurrent depressive disorder, eating disorder, sexual dysfunction, organic disorder, panic disorder (insert) Interim paroxysmal anxiety), post-traumatic stress disorder, pervasive developmental disorder, autism, other mental disorder or behavioral disorder.
(27) The disease is Parkinson's disease, secondary Parkinson's syndrome, Alzheimer's disease, sleep disorder, extrapyramidal disorder, neurodegenerative disease, trigeminal nerve disorder, facial nerve disorder, olfactory nerve disorder, Glossopharyngeal nerve disorder, vagus nerve disorder, hypoglossal nerve disorder, other cranial nerve disorders, cerebral palsy, hemiplegia, paraplegia and quadriplegia, other paralytic syndromes, neuropathy, nerve root or plexus disorders The method according to (1), wherein the disorder is an autonomic nervous system disorder, other peripheral nervous system disorder, or other nervous system disorder.
(31) The method according to (1) above, wherein the disease is glaucoma, optic nerve or visual (pathological) tract disorder, visual dysfunction or blindness (blindness), other eye diseases or appendage diseases.
(33) The method according to (1) above, wherein the disease is an outer ear disease, a middle ear disease, a mastoid disease, hearing loss, or an inner ear disease, another ear disease or a mastoid disease.
(36) The disease is essential (primary (primary)) hypertension (disease), hypertensive heart disease, hypertensive kidney disease, hypertensive cardiorenal disease, secondary (secondary) hypertension (disease), narrow Cardiomyopathy, myocardial infarction, other acute ischemic heart disease, chronic ischemic heart disease, cardiomyopathy, arrhythmia, pericarditis, endocarditis, pulmonary embolism, subarachnoid hemorrhage, cerebral hemorrhage, cerebral infarction, stroke, artery The method according to (1) above, which is embolism or thrombosis, arteritis, aortic aneurysm and dissection, venous embolism or thrombosis, phlebitis, varicose veins, and other circulatory diseases.
(40) The disease is tracheitis, pneumonia, influenza, emphysema, chronic obstructive pulmonary disease, asthma, bronchiectasis, pulmonary fibrosis, adult respiratory distress <promotion> constriction syndrome (ARDS), pulmonary edema, allergic rhinitis ( Nasal allergy), pharyngitis, sinusitis, tonsillitis, laryngitis, and other diseases of the respiratory system.
(44) Diseases are esophagitis, esophageal ulcer, gastroesophageal reflux disease, gastric ulcer, duodenal ulcer, Crohn's disease (localized enteritis), ulcerative colitis, paralytic ileus and bowel obstruction, irritable bowel syndrome, constipation , Functional diarrhea, polyp of colon (colon), alcoholic liver disease, toxic liver disease, chronic hepatitis, liver fibrosis or cirrhosis, cholelithiasis, cholecystitis, cholangitis, acute pancreatitis, chronic pancreatitis, etc. The method according to (1) above, which is a digestive system disease.
(48) The disease is skin or subcutaneous tissue infection, blistering, atopic dermatitis, dermatitis, eczema, papule rash (garbage) disorder, urticaria, erythema, rash (1) The method according to (1) above, which is an acne ulcer, other skin disease or subcutaneous tissue disease.
(52) The disease is an infectious joint disorder, inflammatory multiple joint disorder, arthropathy, rheumatoid arthritis, knee injury, soft tissue disorder, bone disorder, cartilage disorder, other musculoskeletal disorder or connective tissue disorder The method according to (1), wherein
(57) The disease is acute nephritic syndrome, rapid progressive nephritic syndrome, chronic nephritic syndrome, nephrotic syndrome, tubulointerstitial nephritis, hydronephrosis, acute renal failure, chronic renal failure, renal stone or ureteral stone, tension Sexual stress (urinary pressure) urinary incontinence, prostatic hypertrophy (symptom), testicular (testicular) torsion, testicular (testicular) inflammation, epididymis (cold testicular) inflammation, male infertility (symptom), fallopianitis, ovitis, intrauterine The method according to (1) above, which is a membranous disorder, amenorrhea, undermenstrual period, excessive menstruation, menopause or other perimenopausal disorders, female infertility, or other diseases of the genitourinary system of the renal urinary tract.
(61) The method according to (1) above, wherein the disease is pregnancy-induced hypertension (symptoms), other maternal disorders related to pregnancy, or a pathological condition that occurred in the perinatal period.
(67) If the disease is cough, nausea, vomiting, heartburn, dizziness (feeling dizzy), or pain, numbness, itching, skin sensory disturbance, olfactory disturbance, taste disorder, sensory disturbance, unknown fever, other symptoms, signs, The method according to (1) above, wherein the method has abnormal clinical findings or abnormal test findings.
(75) The above (1) to (9), (11), (15), (19), (23), wherein the viral vector is an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or an adenoviral vector. , (27), (31), (33), (36), (40), (44), (48), (52), (57), (61) and (67) Method.
(76) A peripheral nerve administration agent comprising a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of a peripheral nerve.
(77) Use of a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or otherwise changing a nerve cell function for the manufacture of a peripheral nerve agent .
(78) A viral vector into which a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve functions is used for peripheral nerve administration. About.

According to the present invention, this function can be changed by manipulating a peripheral nerve of a specific nerve type distributed in a specific organ or local body. According to the present invention, it is possible to perform treatment with fewer side effects (that is, less influence on other organs and other nerve types) than conventional medical techniques (drugs, nerve electrical stimulation, nerve excision cauterization, etc.). I can do it. Furthermore, since this side effect is small, it is possible to strongly intervene in specific organs and local areas of the body, thereby obtaining a greater preventive or therapeutic effect.

FIG. 1 is a graph showing potential changes in HEK293T cells into which a neural stimulation gene NaChBac and its mutant NaChBac G229A have been introduced. This result showed that the neural activity of cells can be increased by gene transfer of the neural stimulation gene NaChBac and its mutant NaChBac G229A. FIGS. 2a to 2c show the electrical properties of renal sympathetic nerves of kidneys locally injected with TH-NaChBac-2A-turboRFP AAV vector or TH-NaChBac G229A-2A-turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml or more). It is a figure which shows the measurement result of a signal and blood pressure. As a result, in the pseudonerve stimulation vector group, the renal sympathetic nerve activity on the vector side is the same as the renal sympathetic nerve activity on the contralateral side (FIG. 2a), whereas in the NaChBac nerve stimulation vector group, the kidney on the vector side Sympathetic nerve activity increases compared to the contralateral side (FIG. 2b, 182% on the contralateral side), and in the NaChBac G229A nerve stimulation vector group, the renal sympathetic nerve activity on the vector side is even greater compared to the contralateral side. An increase was shown (FIG. 2c, 251% contralateral). FIGS. 3a to 3c show the electrical properties of renal sympathetic nerves of kidneys locally injected with TH-NaChBac-2A-turboRFP AAV vector or TH-NaChBac G229A-2A-turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml or more). It is a figure which shows the quantitative result of a signal and the norepinephrine content of this kidney. FIG. 3 d shows systolic and diastolic blood pressure of rats locally injected with TH-NaChBac-2A-turboRFP AAV vector or TH-NaChBac G229A-2A-turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml or more). It is a figure which shows the value which quantified the measurement result. As a result, in the NaChBac nerve stimulation vector group (N = 6), the amount of renal sympathetic nerve activity on the vector side (FIG. 3a) and the firing frequency per pulse (FIG. 3b) increased compared to the contralateral side (contralateral side). In the NaChBac G229A neural stimulation vector group (N = 6), these were shown to increase even more (251% contralateral). Moreover, it was shown that such an increase in neural activity does not occur in the pseudo-nerve stimulation vector group (N = 6). Renal norepinephrine levels agreed well with the results of neural activity. That is, it was shown that the NaChBac nerve stimulation vector group (N = 6) was higher in the vector-side kidney than the contralateral side, and the NaChBac G229A nerve stimulation vector group (N = 6) was higher (FIG. 3c). Furthermore, it was shown that the systolic blood pressure and the diastolic blood pressure increase in the NaChBac nerve stimulation vector group (N = 6) to become hypertension, and the NaChBac G229A nerve stimulation vector group (N = 6) becomes more severe hypertension. (FIG. 3d, #P <0.05 vs. contralateral sympathetic nerve activity, * P <0.05 NaChBac nerve stimulation vector group vs. NaChBac G229A nerve stimulation vector group). 4a to 4d are sections of kidney and celiac ganglion locally injected with TH-NaChBac-2A-turboRFP AAV vector or TH-NaChBac G229A-2A-turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml or more). It is a figure which shows the immuno-staining image. FIG. 4e is a diagram showing an immunostained image of a section of the kidney not locally injected with the AAV vector. The arrows in FIG. 4 indicate fluorescent protein positive signals or each marker positive signal. However, the arrow in the RFP diagram of FIG. 4e indicates that there is no fluorescent protein positive signal. From this result, it was shown that by locally injecting the prepared AAV vector into the kidney, nerves distributed retrogradely in the kidney can be infected and the inserted gene can be expressed specifically in the sympathetic nerve. Figure 5 shows TH-NaChBac G229A-2A-turboRFP AAV vector, TH-CreER AAV vector, and Flex-DTA AAV vector simultaneously injected locally, and drug-induced renal sympathetic nerves are removed 9 weeks after the local injection. It is a figure which shows the time-dependent change of the systolic blood pressure and diastolic blood pressure of the rat which was made. According to this result, in the control group (N = 8, the control AAV vector solution was locally injected into the kidney), the blood pressure did not change until 24 weeks (FIG. 5, ▽), whereas the nerve stimulation vector group (N = 8 ) Showed that the blood pressure increased until 9 weeks after the local injection of the vector and became hypertension, and the hypertension persisted after 9 weeks (FIG. 5, Δ). Further, in the nerve removal vector group after nerve stimulation (N = 8), blood pressure increased to hypertension as in the nerve stimulation vector group (N = 8) until 9 weeks after the local injection of the vector. After tamoxifen administration, blood pressure gradually decreased and returned to the level before vector local injection at 24 weeks (Fig. 5, ●). Therefore, local injection of viral vector to remove kidney sympathetic nerves improves hypertension It was shown that a therapeutic effect can be obtained. (#P <0.05 vs. control group, * P <0.05 nerve stimulation vector group vs. nerve removal vector group after nerve stimulation) FIG. 6 shows TH-NaChBac G229A-2A-turboRFP AAV vector, TH-CreER AAV vector and Flex-DTA AAV vector simultaneously stained with immunostained images (right figure), and the three AAV mixed vectors. Is a diagram showing an immunostained image (left figure) of a section of a kidney from which renal sympathetic nerve is removed in a drug-induced manner after local injection. The arrow in FIG. 6 indicates a TH positive signal. According to this result, TH-positive renal sympathetic nerves were observed in the kidney in the nerve stimulation vector group, but the kidney sympathetic nerves disappeared in the nerve removal vector group after nerve stimulation. It has been shown that local injection of viral vectors can actually remove sympathetic nerves. FIG. 7 is a graph showing changes over time in blood glucose levels of rats locally injected with ChAT-NaChBac T220A-2A-turboRFP AAV vector, ChAT-turboRFP AAV vector, or PBS into the pancreas. As a result, in the parasympathetic nerve stimulation vector group (N = 8), compared with the control group (N = 8) and the parasympathetic pseudostimulation vector group (N = 8), the fasting blood glucose level is low, and the glucose tolerance test The blood glucose level (0,15,30,60,120 minutes value) was low, so that a local injection of a viral vector that stimulates the parasympathetic nerve of the pancreas provides a therapeutic effect that improves glucose tolerance in diabetes It was shown that. (#P <0.05 vs. before glucose load, * P <0.05 nerve stimulation vector group vs. nerve pseudostimulation vector group and control group) FIG. 8 is a diagram showing immunostained images in the vagus nerve dorsal motor nucleus and suspected nucleus of rat medulla obtained by locally injecting ChAT-NaChBac T220A-2A-turboRFP AAV vector or ChAT-turboRFP AAV vector into the pancreas. The arrows in FIG. 8 indicate RFP positive signals. According to this result, in the parasympathetic nerve stimulation vector group, RFP positive neurons were present in the vagus nerve dorsal motor nucleus and suspicion nucleus of the medulla and not observed in the control group. When injected locally into the pancreas, it was shown that the parasympathetic nerve distributed retrogradely in the pancreas was infected, and the insertion gene (turboRFP) was expressed specifically in the parasympathetic nerve. FIG. 9 shows the time course measurement of changes in the size of a rat breast cancer tissue in which a breast cancer sympathetic nerve was removed by drug induction after locally injecting the TH-CreER AAV vector and Flex-DTA AAV vector into the rat breast cancer tissue. It is a figure which shows a result. As a result, in the control group (N = 8) and the sympathetic false removal vector group (N = 8), the cancer increased markedly, but in the sympathetic removal vector group (N = 8), the cancer Since the increase was strongly suppressed, it was shown that a therapeutic effect that suppresses the increase in breast cancer can be obtained by viral vector local injection that removes sympathetic nerves distributed in breast cancer. (#P <0.05 vs. control group) FIG. 10 is a diagram showing the measurement results of norepinephrine content in a rat breast cancer tissue in which the breast cancer sympathetic nerve was removed after the TH-CreER AAV vector and Flex-DTA AAV vector were locally injected into the rat breast cancer tissue. is there. This result showed that the amount of breast cancer tissue norepinephrine was significantly reduced in the sympathetic denervation vector group compared to the control group and the sympathetic pseudoremoval vector group. FIG. 11 is a view showing an immunostained image of a breast cancer tissue section of a rat from which breast cancer sympathetic nerves were removed after drug injection after the TH-CreER AAV vector and Flex-DTA AAV vector were locally injected into the rat breast cancer tissue. . An arrow in FIG. 11 indicates a TH positive signal, and an arrow head in FIG. 11 indicates a Lectin positive signal. This result shows that TH-positive sympathetic nerve fibers are observed in the breast cancer tissue in the control group and sympathetic false removal vector group, but the sympathetic nerve in the breast cancer tissue disappears in the sympathetic nerve removal vector group. It was done. FIG. 12 is a diagram showing the results of measuring the change in the size of breast cancer tissue of rats locally injected with ChAT-NaChBac T220A-2A-GCaMP6f AAV vector or ChAT-GCaMP3 AAV vector. As a result, cancer increased to the same extent in the control group (N = 8) and the parasympathetic pseudostimulation vector group (N = 8), but the increase in cancer was suppressed in the parasympathetic nerve stimulation vector group (N = 8). From these results, it was shown that a therapeutic effect that suppresses breast cancer growth can be obtained by local injection of a viral vector that stimulates parasympathetic nerves distributed in breast cancer. (#P <0.05 vs. control group) FIG. 13 is a view showing an immunostained image of a breast cancer tissue section of a rat locally injected with ChAT-NaChBac T220A-2A-GCaMP6f AAV vector or ChAT-GCaMP3 AAV vector. As a result, it was shown that in the parasympathetic nerve stimulation vector group and the parasympathetic nerve pseudostimulation vector group, the VAChT-positive parasympathetic nerve was GFP-positive, and gene expression to the parasympathetic nerve by the retrograde virus vector was possible. FIG. 14 is a diagram showing the results of measuring the change in the size of skin cancer tissue of a mouse locally injected with TH-DTA AAV vector or TH-turboRFP AAV vector over time. As a result, cancer was markedly increased in the control group (N = 8) and the sympathetic false removal vector group (N = 8), but in the sympathetic removal virus vector group (N = 8), the cancer Since the increase in the amount of the virus was strongly suppressed, it was shown that a therapeutic effect that suppresses the increase in skin cancer can be obtained by viral vector local injection that removes sympathetic nerves distributed in skin cancer. (#P <0.05 vs. control group) FIG. 15 is a diagram showing the results of measuring the change in the size of skin cancer tissue of a mouse locally injected with ChAT-NaChBac T220A-2A-GCaMP6f AAV vector or ChAT-GCaMP3 AAV vector over time. As a result, cancer increased to the same extent in the control group (N = 8) and the parasympathetic pseudostimulation vector group (N = 8), but the increase in cancer was suppressed in the parasympathetic nerve stimulation vector group (N = 8). Therefore, it was shown that a therapeutic effect that suppresses the increase in skin cancer can be obtained by local injection of a viral vector that stimulates parasympathetic nerves distributed in skin cancer. (#P <0.05 vs. control group) FIGS. 16a and 16b respectively show the amount of food intake of rats in which HiRet-Cre lentiviral vector was locally injected into the stomach and FLEX-NaChBac G229A-2A-turboRFP AAV vector was locally injected into the right ganglion (FIG. 16a). It is a figure which shows a time-dependent change of body weight (FIG. 16b). From this result, the nerve stimulation vector group (N = 8) decreased food intake compared to the control group (N = 8) (FIG. 16a), and the weight gain was suppressed (FIG. 16). 16b), it was shown that the effect of suppressing feeding and obesity can be obtained by local injection of a viral vector that stimulates afferent nerves distributed in the stomach. (#P <0.05 vs. control group) 17a and 17b respectively show the nodular ganglia of rats in which HiRet-Cre lentiviral vector was locally injected into the stomach and FLEX-NaChBac G229A-2A-turboRFP AAV vector was locally injected into the right ganglion (Fig. 17). It is a figure which shows the immuno-staining image of 17a) and a medulla section (FIG. 17b). As a result, in the nerve stimulation vector group, there is a red fluorescent nerve cell body due to turboRFP expression in the nodal ganglion (FIG. 17a), and in the solitary nucleus of the medulla oblongata (FIG. 17b), red color due to turboRFP expression. Due to the presence of fluorescent nerve endings (the neuronal cell bodies did not express turboRFP), it was shown that gene expression can be selectively induced only in the afferent nerves of the stomach by the double vector method.

The present invention relates to a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs other than brain and spinal cord, tumors or Injecting the tumor locally into the excised site, infecting the nerve with the viral vector, expressing the protein having the function in the infected nerve, manipulating the function of the nerve infected with the viral vector by the protein, Provided is a method for preventing or treating a disease, which comprises changing the function of an organ other than the brain and spinal cord to which the nerve communicates.

The present invention also provides a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other functions of peripheral nerves, organs other than brain and spinal cord, tumors or tumors Including a step of locally injecting into the excised region, and urging the nerve with a viral vector incorporating a gene expressing a protein having a function of killing, stimulating, or suppressing peripheral nerves Provide a method of sexual or retrograde infection.

Furthermore, the present invention relates to a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs other than brain and spinal cord, tumors Alternatively, a method of locally injecting a tumor at a site where the tumor is excised and generating the function at the organ-specific site where the tumor or tumor is excised is provided.

The present invention relates to a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of peripheral nerves, organs other than brain and spinal cord, tumors or Provided is a method for genetic manipulation of a peripheral nerve, which is characterized by locally injecting into a site where a tumor is excised.

In the present invention, the peripheral nerve is not particularly limited, but in particular, autonomic nerves such as sympathetic nerves and parasympathetic nerves (or vagus nerves), somatosensory nerves (tactile sensation, pressure sensation, cold sensation, warm sensation, pain sensation, numbness, numbness, etc. ), Visceral sensory nerves, special sensory nerves (olfactory, visual, auditory, balanced sensations, sensations involving the cranial nerves such as taste), afferent nerves (nerves that transmit biological information in peripheral tissues to the center), enteric nerves, etc. It is preferable to be a system (cholinergic nerve such as Dogiel type I, Dogiel type II, Dogiel type III, serotonin nerve, etc.).

In the present invention, the function of killing peripheral nerves is not particularly limited, but refers to, for example, the function of killing peripheral nerve cells by apoptosis or necrosis. Further, it refers to a function of killing peripheral nerve cells by apoptosis or necrosis by administering a drug solution that kills cells in cooperation with the gene after gene expression.

In the present invention, the function of stimulating peripheral nerves is not particularly limited. For example, peripheral nerve cells (single cells) can be produced by, for example, expressing an ion channel or an ion pump in the peripheral nerve to easily cause a depolarization response. A function of increasing the occurrence frequency of action potentials at the level of nerve fibers) or increasing the amount, magnitude, and frequency of electrical nerve activity at the level of peripheral nerve cells (nerve fiber groups). In addition, the function to increase the release of neurotransmitters from nerve endings caused by nerve electrical activity, the function to increase neurotransmitter concentration by suppressing the reuptake of released neurotransmitters, etc. Say etc.

In the present invention, the function of suppressing peripheral nerves is not particularly limited. For example, the peripheral nerve can be expressed by changing the repolarization response, the hyperpolarization response, and the rectification by expressing an ion channel or an ion pump in the peripheral nerve. Functions that reduce the frequency of action potentials at the level of nerve cells (single nerve fibers), or reduce the amount, size, and frequency of electrical nerve activity at the level of peripheral nerve cells (nerve fibers) Say. It also reduces the neurotransmitter concentration by, for example, promoting the function of suppressing the release of neurotransmitters from nerve endings and the reuptake of released neurotransmitters, which occurs in response to the electrical activity of nerves. Say functions.

In the present invention, the function of changing the nerve cell function of the peripheral nerve is not particularly limited. For example, specific gene expression using RNA interference by miRNA, PIWI interacting RNA (piRNA), short hairpin RNA (shRNA) or the like. The function etc. which suppresses or changes. In addition, it refers to a function of expressing a specific gene or a specific gene variant, or regulating the amount and timing of the expression. Furthermore, it refers to a function of changing cell functions such as nerve cell metabolism, information transmission and secretion by suppressing or regulating the expression of these genes. Examples of shRNA include shRNA against Neutral sphingomyelinase 2 (smpd3).

In the present invention, the protein only needs to have a function of killing, stimulating, suppressing, or changing other functions of the peripheral nerve. Examples of the protein having a function of killing peripheral nerves include diphtheria toxin (DTA), diphtheria toxin receptor (Diphtheria Toxin Receptor; 1 DTR), and interleukin 2 receptor (IL2Rα). Proteins having a function of stimulating peripheral nerves include, for example, photoresponsive channels (optogenetics), bacterial-derived channels NaChBac (NaChBac T220A, NaChBac T229A, NaChBac G219A, channel rhodopsin 2 (ChR2), mutant channel rhodopsin 2 ( ChR2 / H134R, ChR2 / C128X (X is T, A or S) ChR2 / D156A, ChR2 / E123T (ChETA), chimeric ChR, etc.), Volbox channel rhodopsin 1, etc. It has a function of suppressing peripheral nerves. Examples of the protein include Kir2.1, EGFP-eTeNT-PEST, halorhodopsin, arkyodopsin 3, and arkyodopsin T. Other nerve cells of peripheral nerves Examples of the protein having a function of changing the ability include gp130 that changes a neurophenotype and a neurotransmitter, a JAK-STAT pathway and a MAP kinase pathway signaling protein, and variants thereof. All the receptors expressed are mentioned, for example, Protease-Activated receptors (PAR, in particular PAR1, PAR2, PAR3, PAR4), bradykinin receptors (in particular B1, B2), prostanoid receptors, prostaglandins D receptor, prostaglandin E receptor (especially EP1, EP2, EP3, EP4), prostaglandin F receptor, prostaglandin I receptor, prostaglandin T receptor, transient receptor potential proto in (TRP) receptors (especially TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, TRPA1), adenosine receptors (especially A1, A2A, A2B, A3), ATP receptors (especially P2X1 , P2X2, P2X3, P2X4, P2X5, P2X6, P2X7, P2X8, P2Y1, P2Y2, P2Y3, P2Y4, P2Y5, P2Y6, P2Y7, P2Y8, P2Y9, P2Y10, P2Y11, P2Y12, P2Y13 (especially serotonin receptor, HT1A, 5HT1B, 5HT1D, 5HT1E, 5HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT4, 5-HT5A, 5-HT5B, 5-HT6, 5-HT7), histamine receptor (In particular, H1 , H2, H3, H4), adrenergic receptors (especially α1A, α1B, α1D, α2A, α2B, α2C, α2D, β1, β2, β3), neurotrophic factor receptors (especially TrkA, TrkB, GFRα1, AMPK) Type glutamate receptor (particularly GluR1-4 or GluRα1-α4)), kainate type glutamate receptor (particularly GluR5-7 or GluRβ1-β3)), NMDA type glutamate receptor (particularly NA2A-D) , Or GluRε1-ε4), NR1 (GluRζ), NR3A, 3B (GluRκ1)), metabotropic glutamate receptors (especially mGluR1, mGluR2, mGluR3, mGluR4, mGluR5, mGluR6, mGluR7, BAm receptor, particularly BABA) , GABAA, GABAB, G BAC), glycine receptor, opioid receptor, sigma receptor, cannabinoid receptor (especially CB1 receptor, CB2 receptor), tachykinin receptor (especially NK1, NK2, NK3 receptor), CGRP receptor, VIP Receptor, PAC receptor, somatostatin receptor (especially sst1, sst2A, sst2B, sst3, sst4, sst5), CCK receptor (especially CCK1, CCK2), galanin receptor (GalR1, GalR2), NPY receptor ( In particular, Y1, Y2, Y3, Y4, Y5, Y6), nicotinic acetylcholine receptor, muscarinic acetylcholine receptor, dopamine receptor, endothelin receptor, orexin receptor, cytokine receptor (particularly IL1 receptor, IL2 Receptor, IL3 receptor, IL4 receptor, IL5 receptor Body, IL6 receptor, IL7 receptor, IL8 receptor, IL9 receptor, IL10 receptor, IL11 receptor, IL12 receptor, IL13 receptor, IL14 receptor, IL15 receptor, IL16 receptor, IL17 receptor, IL18 receptor, IL19 receptor, IL20 receptor, LIF receptor, OSM receptor, CNTF receptor, CT-1 receptor, PRL receptor, GH receptor, EPO receptor, TPO receptor, GCSF receptor, IFN-α, IFN-β, IFN-γ, TNF receptor, Fas receptor, CD28 receptor, CD30 receptor, CD40 receptor, SCF receptor, EDF receptor, FGF receptor, PDGF receptor, M- CSF receptor, insulin IGF receptor, TGFβ receptor, activin receptor, chemokine receptor, MP-1 receptor, RANTES receptor Angiotensin receptor (especially AT1, AT2), angiotensin (1-7) receptor, platelet membrane glycoprotein receptor, CRF receptor, glucocorticoid receptor, Toll-like receptor (especially TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), retinoic acid receptor, IP3 receptor, ryanodine receptor, RIG-I-like receptor, NOD-like receptor, Acid-Sensing Ion Channels (ASIC, In particular, ASIC1, ASIC1a, ASIC1b, ASIC2, ASIC2a, ASIC2b, ASIC3, ASIC4, ENaC / Deg superfamily (ENaC is an episodic sodium channel) (in particular, α NaC, βENaC, γENaC), or the like, or variants thereof.
Further, when the protein used in the present invention is used for human therapy, it may be, for example, a protein that functions in a human living body (such as a protein derived from a mammal such as a human, mouse, rat, etc.). It is preferably a protein derived from.

In the present invention, the gene may encode a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of the peripheral nerve, or killing the peripheral nerve. It may have a function of stimulating, suppressing, or changing other neuronal functions and does not encode a protein. For example, a gene encoding diphtheria toxin (represented by the sequence shown in SEQ ID NO: 1), a gene encoding a diphtheria toxin receptor, a gene encoding an interleukin 2 receptor, a gene encoding a light-responsive channel A gene encoding NaChBac (represented by a sequence represented by SEQ ID NO: 2), a gene encoding NaChBac T220A (represented by a sequence represented by SEQ ID NO: 3), a gene encoding NaChBac T229A (sequence) A gene encoding NaChBac G219A, a gene encoding channel rhodopsin 2, a mutant channel rhodopsin 2 (ChR2 / H134R, ChR2 / C128X (X is T, A or S)) ChR2 / D156A, ChR2 / E123T ( hETA) a gene encoding a chimeric ChR, etc.), a gene encoding Volbox channel rhodopsin 1, a gene encoding Kir2.1 (represented by sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, etc.), EGFP-eTeNT -A gene encoding PEST (represented by the sequence shown in SEQ ID NO: 7, etc.), a gene encoding halorhodopsin, a gene encoding arkyhodopsin 3, a gene encoding a gene encoding arkyodopsin T, a neuronal gene Examples include gp130 that changes phenotypes and neurotransmitters, JAK-STAT pathway and MAP kinase pathway signaling proteins, and genes encoding mutant proteins thereof.
The nucleotide sequence having a function of changing other nerve cell functions of peripheral nerves is, for example, the nucleotide sequence of shRNA that suppresses vesicular secretion of cells (for example, shRNA against Neutral sphingomyelinase 2 (smpd3)), Examples include shRNA base sequences that individually suppress all expressed receptors, and miRNA base sequences that change them, such as Protease-Activated receptors (PAR, in particular PAR1, PAR2 , PAR3, PAR4), bradykinin receptor (especially B1, B2) prostanoid receptor, prostaglandin D receptor, prostaglandin E receptor (especially EP1, EP2, EP3, EP4), prostaglandin F Receptor, prostagland Receptor, prostaglandin T receptor, transient receptor potential protein (TRP) receptor (especially TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, TRPA1), adenosine receptor (especially A1) , A2A, A2B, A3), ATP receptors (especially P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, P2X7, P2X8, P2Y1, P2Y2, P2Y3, P2Y4, P2Y5, P2Y6, P2Y7, P2Y8, P2Y9, P2Y10 P2Y11, P2Y12, P2Y13), serotonin receptors (especially 5-HT1A, 5HT1B, 5HT1D, 5HT1E, 5HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5 -HT3,5-HT4,5-HT5A, 5-HT5B, 5-HT6,5-HT7), histamine receptor (especially H1, H2, H3, H4), adrenergic receptor (especially α1A, α1B, α1D) , Α2A, α2B, α2C, α2D, β1, β2, β3), neurotrophic factor receptor (particularly TrkA, TrkB, GFRα1, AMPK type glutamate receptor (particularly GluR1-4 or GluRα1-α4)), kainin Acid-type glutamate receptor (especially GluR5-7 or GluRβ1-β3)), NMDA-type glutamate receptor (particularly NA2A-D or GluRε1-ε4), NR1 (GluRζ), NR3A, 3B (GluRκ1)) , Metabotropic glutamate receptors (particularly mGluR1, mGluR2, mGluR3, mGluR4, m luR5, mGluR6, mGluR7, mGluR8), GABA receptors (especially GABAA, GABAB, GABAC), glycine receptors, opioid receptors, sigma receptors, cannabinoid receptors (especially CB1 receptors, CB2 receptors) tachykinin receptors Body (particularly NK1, NK2, NK3 receptor), CGRP receptor, VIP receptor, PAC receptor, somatostatin receptor (particularly sst1, sst2A, sst2B, sst3, sst4, sst5), CCK receptor (particularly, CCK1, CCK2), galanin receptor (GalR1, GalR2), NPY receptor (especially Y1, Y2, Y3, Y4, Y5, Y6), nicotinic acetylcholine receptor, muscarinic acetylcholine receptor, dopamine receptor, endothelin Receptor, orexi Receptor, cytokine receptor (in particular, IL1 receptor, IL2 receptor, IL3 receptor, IL4 receptor, IL5 receptor, IL6 receptor, IL7 receptor, IL8 receptor, IL9 receptor, IL10 receptor, IL11 receptor, IL12 receptor, IL13 receptor, IL14 receptor, IL15 receptor, IL16 receptor, IL17 receptor, IL18 receptor, IL19 receptor, IL20 receptor, LIF receptor, OSM receptor, CNTF receptor Body, CT-1 receptor, PRL receptor, GH receptor, EPO receptor, TPO receptor, GCSF receptor, IFN-α, IFN-β, IFN-γ, TNF receptor, Fas receptor, CD28 receptor Body, CD30 receptor, CD40 receptor, SCF receptor, EDF receptor, FGF receptor, PDGF receptor, M-CSF receptor, insulin I F receptor, TGFβ receptor, Activin receptor, chemokine receptor, MP-1 receptor, RANTES receptor), angiotensin receptor (especially AT1, AT2), angiotensin (1-7) receptor, platelet membrane sugar Protein receptor, CRF receptor, glucocorticoid receptor, Toll-like receptor (especially TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), retinoic acid receptor, IP3 receptor, Ryanodine receptor, RIG-I-like receptor, NOD-like receptor, Acid-Sensing Ion Channels (ASIC, ASIC1, ASIC1a, ASIC1b, ASIC2, ASIC2a, ASIC2b, ASIC3, ASIC4, ASIC4, ENaC / eg superfamily (ENaC is, epithelial sodium channel) (in particular, αENaC, βENaC, γENaC), etc. - the nucleotide sequence and the inhibit shRNA and also like nucleotide sequence of the miRNA varying these.
As the gene for function manipulation, a commercially available gene can be used, and a commercially available gene mutated by a known method or a method known per se may be used.

In the present invention, the method for expressing a gene is not particularly limited, but includes a method for expressing a gene under a promoter, a Cre-loxP system (for example, Cre added with a nuclear translocation signal represented by SEQ ID NO: 8, SEQ ID NO: 9 and A method of expressing a gene using a loxP sequence represented by SEQ ID NO: 10 or a method of manipulating gene expression in a drug-induced manner (for example, a tet-on system (TRE promoter (SEQ ID NO: 11) and rtTA ( SEQ ID NO: 12)), tet-off system (TRE promoter (SEQ ID NO: 11) and tTA (SEQ ID NO: 13))) and the like.

In the present invention, the viral vector is not particularly limited, but adeno-associated virus (AAV) vector, lentivirus (HIV) vector, retrovirus vector, adenovirus vector, box virus vector, herpes virus vector, herpes simplex virus vector, Sendai Examples thereof include viral vectors, Epstein-Barr virus (EBV) vectors, vaccinia virus vectors, poliovirus vectors, simpis virus vectors, and SV40 vectors.

More preferably, adeno-associated virus (AAV) vector, lentivirus (HIV) vector, retrovirus vector, adenovirus vector and the like can be mentioned. There are various serotypes in adeno-associated virus (AAV) vectors. In the present invention, it is preferable to use type 2 or type 5 adeno-associated virus, but other serotypes may be used. Adeno-associated virus vectors are known to be (almost) not integrated into the host cell genome and have no pathogenicity, and safety is ensured. From this viewpoint, adeno-associated virus (AAV) vectors It is further preferable to use Lentiviral vectors and retroviral vectors have integration of the transgene into the host genome, but can be used, for example, when targeting a lethal disease such as cancer. Although various serotypes exist in adenovirus, the serotype is not particularly limited in the present invention.

The viral vector is preferably a replication-deficient viral vector in which the viral gene is completely or almost completely deleted. The adenoviral vector is preferably non-functional at least in the E1 region. Other regions may also be modified, in particular the E3 region (WO95 / 0267), E2 region (WO94 / 28938), E4 region (WO94 / 28152, WO94 / 12649, WO95 / 02697) or the late genes L1 to L5. Any can be modified. A modified viral vector such as a replication-deficient virus can be prepared by a method known per se. The modified virus vector can be recovered (and further purified) by a method known per se.

The animal infected with the viral vector is not particularly limited, and examples thereof include mammals, birds, reptiles, amphibians and the like. Among them, mammals such as humans, monkeys, horses, dogs, cats, pigs, sheep, goats, rats, mice, rabbits and cows are preferable, rats, mice or humans are more preferable, and humans or mice are more preferable. Such animal cells are preferred as the cells to be infected with the viral vector.

In the present invention, the organ is not particularly limited as long as it is an organ other than the brain and spinal cord, for example, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, cecum, appendix, large intestine, rectum, anus, liver, gallbladder, Pancreas, spleen, lymph node, lymphatic vessel, tonsils, larynx, glottis, trachea, bronchi, lungs, diaphragm, heart, blood vessels (including arteries and veins), kidney, adrenal gland, ureter, urethra, bladder, prostate, testis, Epididymis, penis, ovary, fallopian tube, uterus, vagina, bone, bone marrow, cartilage, muscle, tendon, ligament, skin, adipose tissue, thyroid, parathyroid, nose, eye, ear (outer ear, middle ear, and inner ear) It may be the tongue, face, and the like, and may be the inside, membrane, or surrounding tissue of these organs, and particularly the baroreceptor region tissue of the renal cortex, pancreas, stomach, and cervical artery.

In the present invention, the tumor may be a benign tumor or a malignant tumor, for example. The tumor may be, for example, a primary tumor that has developed in the organ described in [0046], or a metastatic tumor that has metastasized from another organ to the organ described in [0046]. Tumors include oral cancer, pharyngeal cancer, esophageal cancer, stomach cancer, small intestine tumor, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, lymphoma, laryngeal cancer, lung cancer, kidney Cancer, adrenal cancer, bladder cancer, prostate cancer, testicular cancer, esophageal cancer, stomach cancer, liver cancer, pancreatic cystic tumor, biliary tract cancer, pancreatic cancer, colon cancer, rectal cancer Breast cancer, lung cancer, bone soft tissue tumor, prostate cancer, bladder cancer, testicular cancer, kidney cancer, renal pelvic and ureteral cancer, penile cancer, retroperitoneal tumor, adrenal cancer, head and neck cancer, thyroid gland Cancer, uterine cancer, ovarian cancer, skin cancer, sarcoma (occurring in muscles, nerves, bones, etc.), leukemia, mediastinal tumor, metastatic lung cancer, metastatic lung tumor, metastatic liver cancer, metastatic It may be a malignant tumor of a liver tumor or a metastatic bone tumor, or may be a benign tumor of these.

In the present invention, the site where the tumor is excised can also be referred to as the tissue after excision of part or all of the cancer.

In the present invention, examples of a method for excising a tumor include surgery.

In the present invention, the term “incorporating a gene” means that the gene is incorporated into a host cell chromosome with a promoter, a regulator, etc., if necessary, or the gene is incorporated into an expression vector with an appropriate promoter, etc. It is meant to be contained in a host cell in a state of being prepared.

In the present invention, the method of locally injecting a viral vector is performed by directly injecting or spraying an injection or a spray into an organ other than the brain and spinal cord, a tumor, or a site where the tumor is excised, or a sustained-release preparation (depot). The agent is preferably implanted in an organ other than the brain and spinal cord, a tumor, or a site where the tumor is excised. The local injection amount is appropriately selected according to the local injection target, the local injection organ, the dosage form, the degree of disease, the local injection method, and the like. Usually, the local injection amount of the virus vector solution is per 100 mm ^{3 of} tissue. , 0.5 μl to 5 μl at a time. The genome copy concentration of the virus vector contained in the virus vector solution to be injected locally may be 1-5 × 10 ¹¹ GC / ml or more. In addition, the number of local injections is appropriately selected according to the dosage form, the degree of disease, age, or the like, and can be a local injection once or a continuous administration at a certain interval. In the case of continuous local infusion, the local infusion interval may be once a day to once every several months.

Specific local injection methods include, for example, genes encoding proteins that kill neurons such as DTA, diphtheria toxin receptors (Diphtheria Toxin Receptor; DTR), interleukin 2 receptors (IL2Rα, etc.), etc. , NaChBac and its mutants (NaChBac T220A, NaChBac G229A, NaChBac G219A, etc.), a gene encoding a protein having a neurostimulatory effect, channel rhodopsin 2 (ChR2), its mutant channel rhodopsin 2 (ChR2 / H134R, ChR2 / C128X (X is T, A or S) ChR2 / D156A, ChR2 / E123T (ChETA), chimeric ChR, etc.), activated by light stimulation such as Volbox channel rhodopsin 1 Genes that encode proteins with neurostimulatory activity, genes that encode proteins with neurosuppressive activity such as Kir2.1, eTeNT, and neurostimulatory activity that are activated by light stimulation such as halorhodopsin, arkyodopsin 3 and arkyodopsin T A specific tissue, tumor or tumor was excised from a gene of a certain protein, shRNA (for example, shRNA to Neutral sphingomyelinase 2 (smpd3)), a viral vector in which a nucleotide sequence having a gene expression regulating action such as miRNA was incorporated. Diphtheria toxin receptor (Diphtheria Toxin Receptor), a gene that encodes a protein that kills nerve cells such as DTA and removes tissue from the lesion site of the patient. DTR), genes encoding interleukin 2 receptors (such as IL2Rα), NaChBac and its mutants (NaChBac T220A, NaChBac G229A, NaChBac G219A, etc.), and the like. (ChR2), its mutant channel rhodopsin 2 (ChR2 / H134R, ChR2 / C128X (X is T, A or S) ChR2 / D156A, ChR2 / E123T (ChETA), chimeric ChR, etc.), Volbox channel rhodopsin 1, etc. Genes encoding proteins with neurostimulatory activity activated by light stimulation, genes encoding proteins with neurosuppressive activity such as Kir2.1, eTeNT, halorhodopsin, arkyrodpsin 3 and arkylo Viruses in which a base sequence having a gene expression regulating action such as a gene of a protein that is activated by light stimulation such as pussin T and has a neurosuppressive action, shRNA (for example, shRNA for Neutral sphingomycinase 2 (smpd3)), miRNA, etc. Examples thereof include a method of transplanting the transformed cells after introduction of a vector and transformation into a specific tissue, tumor or tumor excision site of a patient.
In the present invention, nerve cells may be killed by administration of a drug solution after gene expression. For example, a diphtheria toxin receptor gene may be expressed and then a diphtheria toxin-containing drug solution may be administered to kill neurons, or an interleukin 2 receptor gene may be expressed and then an immunotoxin-containing drug solution may be administered to You may kill it.

In the present invention, the local injection amount of the viral vector solution may be 0.5 μl to 5 μl of the viral vector solution at a time per 100 mm ^{3 of} tissue. The volume of the tissue can be measured using a known method.

In the present invention, the genome copy concentration of the virus vector contained in the virus vector solution to be locally injected may be 1-5 × 10 ¹¹ GC or more per 1 mL of the cell culture solution. The genome copy concentration of the viral vector can be measured using a known method.

In the present invention, infecting peripheral nerves with a viral vector means introducing a foreign gene incorporated into a viral vector into a cell or a living body. Infections include antegrade or retrograde infections. An antegrade infection refers to, for example, infecting a peripheral nerve in the direction from the cell body to the terminal. Retrograde infection refers to, for example, infection in the direction from the peripheral nerve terminal to the cell body. A method for introducing an exogenous gene including a step of infecting an in vitro cell or animal with such a viral vector is also included in the present invention.

In the present invention, it is preferable to retrogradely infect peripheral nerves with a viral vector from within a peripheral organ, it is preferable to infect peripheral nerves antegradely with a viral vector in a peripheral organ, and in combination with these, It is preferable to infect peripheral nerves antegradely with a viral vector outside the peripheral organ.

In the present invention, by infecting a peripheral nerve with a viral vector, a protein having a function of killing, stimulating, suppressing, or changing other functions of the peripheral nerve in the infected peripheral nerve can be expressed. it can.

In the present invention, operating the peripheral nerve means, for example, promoting or suppressing the function of the peripheral nerve, killing the peripheral nerve or the like, or changing other nerve cell functions. Depending on the purpose of this operation, a gene to be incorporated into the viral vector can be appropriately selected.

In the present invention, the disease is not particularly limited as long as it requires prevention or treatment. For example, an international statistical classification of diseases and related health problems (WHO) prepared by the World Health Organization (WHO) based on the World Health Organization Charter ( It may be a disease described in the basic classification table based on ICD-10 (2003 version) which is International Statistical Classification of Diseases and Related Health Problems (hereinafter abbreviated as “ICD”) (http: // www. .Go.jp / toukei / sippei /). In this case, the diseases described in Chapters 1-19 include brain and spinal cord diseases. However, in the case where peripheral organs are involved in this disease state, etiology or symptom, it is an object of the present invention.

A. ICD-10 (2003 edition) compliant Basic classification table

Bacteria, viruses and other pathogens (B95-B97) usually cause diseases. Since diseases caused by these bacteria, viruses and other pathogens (B95-B97) have been well studied and well known to those skilled in the art, they may be followed in the present invention.

In the present invention, diseases include, for example, cardiovascular diseases and related diseases (heart failure, hypertension, arrhythmia, ischemic heart disease (myocardial infarction, angina pectoris), valvular heart disease, peripartum cardiomyopathy, stroke, chronic Kidney disease, renal failure, thoracic aortic aneurysm, abdominal aortic aneurysm, aortic dissection, obstructive arteriosclerosis, obstructive thrombotic vasculitis, primary pulmonary hypertension, arteriosclerosis, diabetes), obesity, hyperlipidemia, high Uricemia, gout, infection, sepsis, infectious respiratory disease (cold syndrome, influenza, acute bronchitis, bacterial pneumonia, lung abscess, pulmonary tuberculosis, nontuberculous pulmonary mycobacterial disease, pulmonary mycosis, pulmonary parasitism Insect disease, opportunistic infections (pneumocystis pneumonia, cytomegalovirus pneumonia, etc.), aspiration pneumonia, airway obstructive disease (chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis), allergic lung disease ( Bronchial asthma, hypersensitivity pneumonia Eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, drug-induced pneumonia, eosinophilic polyangiitis granulomatosis), interstitial lung disease (idiopathic interstitial pneumonia, radiation pneumonia, sarcoidosis, idiopathic Organized pneumonia, collagen disease lung), pulmonary vascular lesion (pulmonary thromboembolism, pulmonary arterial hypertension, pulmonary edema), pleural disease (pleuritis, empyema, pneumothorax), respiratory failure (acute respiratory failure, ARDS, chronic respiratory disease) Failure), bronchiectasis, pneumoconiosis, primary alveolar hypoventilation syndrome, hyperventilation syndrome, sleep apnea syndrome, lymphangioleiomyomatosis, lung transplantation, allergic disease (hay fever, allergic rhinitis, bronchitis, Bronchial asthma, reflex syndrome, stomatitis, dietary allergic gastritis, enteritis (allergic diarrhea), ulcerative colitis, endocarditis, nodular periarteritis, obstructive arteritis, urticaria, quinke edema, Nodular red , Eczema, contact dermatitis, allergic keratitis, conjunctivitis, exchangeable ophthalmitis, etc.), autoimmune diseases (Guillain-Barre syndrome, myasthenia gravis, chronic gastritis, chronic atrophic gastritis, autoimmune hepatitis, Primary biliary cirrhosis, ulcerative colitis, Crohn's disease, primary sclerosing cholangitis, autoimmune pancreatitis, Takayasu arteritis, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia , Autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, chronic discoid lupus erythematosus, localized scleroderma , Pemphigus, pustular psoriasis, psoriasis vulgaris, pemphigoid, gestational herpes zoster, linear IgA bullous dermatosis, acquired epidermolysis bullosa, alopecia areata, vulgaris vulgaris, Sutton acquired efferent vitiligo Sutton's nevus, Harada disease, autoimmune optic neuropathy , Autoimmune inner ear disorder, idiopathic azoospermia, habitual abortion, rheumatoid arthritis, systemic lupus erythematosus, antiphospholipid antibody syndrome, polymyositis, dermatomyositis, scleroderma, Sjogren's syndrome, IgG4-related disease, vasculitis Syndrome, mixed connective tissue disease), gastrointestinal disease (gastroesophageal reflux disease, Barrett esophagus, esophageal achalasia, esophageal / gastric varices, Mallory-Weiss syndrome, acute gastritis, acute gastric mucosal lesions, chronic gastritis, functional dyspepsia, Gastric / duodenal ulcer, gastric MALT lymphoma, gastric malignant lymphoma, gastric adenoma, gastric polyp, infectious enteritis, drug-induced enteritis, appendicitis, intestinal obstruction (ileus), ischemic enteritis, Crohn's disease, ulcerative colitis, irritable bowel Syndrome, sigmoid colon torsion, colonic diverticulosis, colon polyp, epilepsy, etc.), liver disease (acute hepatitis, chronic hepatitis B, chronic hepatitis C, Cirrhosis, drug-induced liver disorder, alcoholic liver disorder, autoimmune hepatitis, primary biliary cirrhosis, metabolic liver disorder (Wilson's disease, hemochromatosis), liver abscess), gallbladder stone, cholecystitis, common bile duct stone , Intrahepatic stone, cholangitis, gallbladder polyp, gallbladder adenomatosis, acute pancreatitis, chronic pancreatitis, pancreatic cyst, pancreatic cystic tumor, peritonitis, gastrointestinal hernia, bone loss, osteoporosis, fracture, dislocation, deformability Examples include knee arthropathy, endocrine disease, numbness, pain, mental illness (bipolar disorder, depression), epilepsy, and cancer.

Malignant neoplasms (also referred to as cancer in the present invention) include oral cancer, pharyngeal cancer, esophageal cancer, stomach cancer, small intestine tumor, colon cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreas. , Pancreatic cystic tumor, lymphoma, laryngeal cancer, lung cancer, kidney cancer, adrenal cancer, bladder cancer, prostate cancer, testicular cancer, esophageal cancer, stomach cancer, liver cancer, biliary tract cancer, pancreas Cancer, colorectal cancer, rectal cancer, breast cancer, lung cancer, bone and soft tissue tumors, prostate cancer, bladder cancer, testicular cancer, kidney cancer, renal pelvic and ureteral cancer, penile cancer, retroperitoneal tumor, Adrenal cancer, head and neck cancer, thyroid cancer, uterine cancer, ovarian cancer, skin cancer, sarcoma (occurring in muscles, nerves and bones), leukemia, mediastinal tumor, metastatic lung cancer, metastatic lung Examples include malignant neoplasms such as tumors, metastatic liver cancer, metastatic liver tumors, and metastatic bone tumors.

In the present invention, “prevention” of a disease refers to, for example, preventing the complete development of the disease, and “treatment” of the disease refers to, for example, amelioration of symptoms caused by the disease or complete cure.

In the present invention, a promoter is an untranslated sequence located upstream of a translation initiation codon and controls transcription of specific DNA.
The promoter used in the present invention is preferably a nerve-specific promoter, and is further a peripheral autonomic nerve, afferent nerve, vagus nerve, afferent nerve, or enteric nervous system (eg, serotonin nerve) -specific promoter. Preferably, for example, the sequence shown in SEQ ID NO: 14, SEQ ID NO: 15, etc., Molecular Brain Research 2003 Volume 112 (1-2): 8-23. A TH promoter represented by a sequence described in The human thyrosine hydroxylene gene promoter, etc., a sequence represented by SEQ ID NO: 16, etc., J Biological Chem. 1992 vol. 267 (28): 20392-20399. Gene expression of Mouse Chloline Acetyltransferase Alternative splicing and identification of a high active promoter region, which is described in the array as represented by C, etc. Promoter, TpH-2 promoter represented by the sequence shown in SEQ ID NO: 18, etc., Gene Ther. 2009 16: 681-688. TpH-3 promoter represented by the sequence described in the sequence described in Targeting central serotoneuronics with lentiral vectors based on a transcriptional strategy etc. 20, an NSE promoter represented by SEQ ID NO: 21 and the like, a DBH promoter represented by a sequence represented by SEQ ID NO: 22 and the like, a sequence represented by SEQ ID NO: 23 and the like, Human Gene Therapy 2004.12 (14): 1731-1740 . A High-Efficiency Synthetic Promoter That Drives Transgene Expression Selectively in Noradrenergic Neurons, in Journal of Molecular and Cellular Cardiology 41 (2006) 364-370 Noradrenergic neuron-specific overexpression of nNOS in cardiac sympathetic nerves decreases neurotransmission sequences are described in such like PRS × 8 promoter represented, first half sequence (sequence shown by SEQ ID NO: 24, SEQ ID NO: 25, etc.) + Functional manipulation gene sequence + second half sequence SEQ ID NO: 26, the sequence shown in the sequence) of SEQ ID NO: 27, etc., Cell 1995.83 (2): 269-278. Overexpression of the neurological growth-associated protein GAP-43, induces next spreading in the absolute system of transgenemics, EMBO. 1990.9 (3): 833-840. Thy-1.2 cassette promoter represented by sequences described in Tissue-specific control elements of the The-1 gene, etc., Thy1S promoter represented by sequences represented by SEQ ID NO: 28, SEQ ID NO: 29, etc. , CR-I (sequence represented by SEQ ID NO: 30) + promoter represented by the sequence represented by transcription initiation sequence (SEQ ID NO: 31), CR-II (sequence represented by SEQ ID NO: 32) + transcription initiation sequence ( A promoter represented by a sequence represented by SEQ ID NO: 31), a promoter represented by a sequence represented by CR-III (sequence represented by SEQ ID NO: 33) + a transcription initiation sequence (SEQ ID NO: 31), CR-IV (Sequence represented by SEQ ID NO: 34) + represented by the transcription sequence (SEQ ID NO: 31) A promoter represented by a promoter, CR-V (sequence represented by SEQ ID NO: 35) + sequence represented by a transcription initiation sequence (SEQ ID NO: 31), or the like, preferably a SERT promoter, particularly SEQ ID NO: 14, SEQ ID NO: 15 Et al., Molecular Brain Research 2003 Volume 112 (1-2): 8-23. A TH promoter represented by a sequence described in The human thyrosine hydroxylene gene promoter, etc., a sequence represented by SEQ ID NO: 16, etc., J Biological Chem. 1992 vol. 267 (28): 20392-20399. Gene expression of Mouse Choleline Acetyltransferase Alternate splicing and identification of a high active promoter region, which is described in the array as represented by C, etc. A promoter is preferred.
In addition, when the promoter used in the present invention is used for human therapy, for example, a promoter that functions nerve-specifically in the human body (such as a nerve-specific promoter derived from a mammalian genomic sequence such as human, mouse, rat, etc.) Among them, a nerve-specific promoter derived from human genome sequence information is preferable.
In addition, as a promoter used in the present invention, when a viral vector is ingested into an organ other than the brain and spinal cord, a tumor or a nerve specifically connected to a site from which the tumor has been removed, Any one capable of self-replication can be used, more preferably a CAG promoter or a CMV (cytomegalovirus) promoter, and particularly preferably a CAG promoter.
A commercially available product can be used as the promoter, and a commercially available promoter mutated by a known method or a method known per se may be used.

The fluorescent protein that is added to or incorporated in the gene for functional manipulation, the recombinant expression vector, or the viral vector is not particularly limited. For example, turboRFP represented by the sequence represented by SEQ ID NO: 36, SEQ ID NO: Examples include GCaMP3 represented by a sequence represented by 37 and the like, GCaMP6f represented by a sequence represented by SEQ ID NO: 38, GFP, EGFP, mCherry, RFP, YFP, and the like.
A commercially available fluorescent protein can be used as the fluorescent protein, and a gene encoding a commercially available fluorescent protein mutated by a known method or a method known per se may be used.

Examples of plasmids for preparing virus vectors generally include plasmids for preparing DNA or RNA virus vectors that can express proteins in bacteria, yeast, filamentous fungi, plant bacteria, mammalian cells, and the like. In the present invention, a vector can be prepared by incorporating a promoter and a gene for function manipulation into a specific plasmid by a known method or a method known per se. When confirming the level of gene expression, gene expression site, and the like, a fluorescent protein-encoding gene may be appropriately incorporated into the plasmid. In the present invention, combinations of plasmids, promoters, and functional manipulation genes (and optionally fluorescent protein expression genes) include all combinations of the aforementioned plasmids, promoters, and functional manipulation genes (and fluorescent protein expression genes). To do. As the plasmid, a commercially available product can be used, and a commercially available plasmid obtained by mutating it by a known method or a method known per se may be used.

The present invention also provides a peripheral nerve administration agent comprising a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other functions of peripheral nerves. The peripheral nerve agent of the present invention is very useful as a drug for local nerve manipulation. In the present invention, the local region can also be referred to as an organ other than the brain and spinal cord, a tumor, or a site where the tumor is excised.

In the present invention, the peripheral nerve agent can be administered by directly injecting or spraying an injection or spray directly into an organ other than the brain and spinal cord, a tumor, or a site where the tumor is excised, or a sustained-release preparation ( And a method of implanting a depot agent) in an organ other than the brain and spinal cord, a tumor, or a site where the tumor is excised. The dose is appropriately selected according to the administration subject, administration organ, dosage form, degree of disease, administration method, and the like. Usually, the dosage of the peripheral nerve administration agent is 0 at a time per 100 mm ^{3 of} tissue. .5 μl to 5 μl. The genome copy concentration of the virus vector contained in the virus vector solution may be 1-5 × 10 ¹¹ GC / ml or more. In addition, the number of administrations is appropriately selected according to the dosage form, the degree of disease, age, or the like, and can be administered once or continuously at a certain interval. In the case of continuous administration, the administration interval may be once a day to once every several months.

Specific administration methods include, for example, genes encoding proteins that kill neurons such as DTA, diphtheria toxin receptor (DTR), interleukin 2 receptors (IL2Rα and the like), and the like, Genes encoding proteins with neurostimulatory effects such as NaChBac and its variants (NaChBac T220A, NaChBac G229A, NaChBac G219A, etc.), channel rhodopsin 2 (ChR2), its mutant channel rhodopsin 2 (ChR2 / H134R, ChR2 / C128X) (X is T, A or S) ChR2 / D156A, ChR2 / E123T (ChETA), chimeric ChR, etc.), activated by light stimulation such as Volbox channel rhodopsin 1 A gene encoding a protein having a stimulating action, a gene encoding a protein having a neurosuppressive action such as Kir2.1 or eTeNT, and a neurosuppressive action by being activated by light stimulation such as halorhodopsin, arkyodopsin 3 and arkyodopsin T To a specific tissue, tumor or tumor excision site of a gene of a certain protein, shRNA (for example, shRNA for Neutral sphingomyelinase 2 (smpd3)), a viral vector incorporating a nucleotide sequence having a gene expression regulating action such as miRNA A method of local injection, or a gene encoding a protein that causes a nerve cell such as DTA to be killed by removing tissue from a lesion site of a patient, and diphtheria toxin receptor (DTH) R), genes encoding interleukin 2 receptors (such as IL2Rα), NaChBac and mutants thereof (NaChBac T220A, NaChBac G229A, NaChBac G219A, etc.), genes encoding proteins with neurostimulatory effects, channel rhodopsin 2 (ChR2), its mutant channel rhodopsin 2 (ChR2 / H134R, ChR2 / C128X (X is T, A or S) ChR2 / D156A, ChR2 / E123T (ChETA), chimeric ChR, etc.), Volbox channel rhodopsin 1, etc. Genes encoding proteins with neurostimulatory activity by light stimulation, genes encoding proteins with neurosuppressive activity such as Kir2.1, eTeNT, halorhodopsin, arkyrodpsin 3 and arkydop A combination of a gene sequence that regulates gene expression, such as a gene of a protein that is activated by light stimulation such as T, and has a neurosuppressive effect, shRNA (for example, shRNA for Neutral sphingomyelinase 2 (smpd3)), miRNA, etc. Examples thereof include a method in which a transformed expression vector is introduced and transformed, and then the transformed cells are transplanted into a specific tissue, tumor or tumor excision site of a patient.
In the present invention, nerve cells may be killed by administration of a drug solution after gene expression. For example, a diphtheria toxin receptor gene may be expressed and then a diphtheria toxin-containing drug solution may be administered to kill neurons, or an interleukin 2 receptor gene may be expressed and then an immunotoxin-containing drug solution may be administered to You may kill it.
The present invention also provides a virus incorporating a gene encoding a protein having a function of killing, stimulating, or suppressing peripheral nerves or changing other nerve cell functions for the manufacture of a peripheral nerve administration agent. Provide the use of vectors.

In the present invention, the dosage of the peripheral nerve agent may be 0.5 μl to 5 μl of a viral vector solution at a time per 100 mm ^{3 of} tissue.

In the present invention, the genome copy concentration of the virus vector contained in the virus vector solution may be 1-5 × 10 ¹¹ GC / ml or more.

The present invention is characterized in that (B) a viral vector incorporating a gene is administered to (A) an organ, a tumor, or a tumor excision site of a mammal including a human (or to a peripheral nerve located there). (C) The disease prevention or treatment method is included.
The present invention includes (B) a medicine containing (B) above, wherein (A) a viral vector incorporating the gene (B) is injected into an organ, tumor or tumor excision site (or into a peripheral nerve there) To do.
(B) Containing disease (C) characterized by injecting (B) a viral vector incorporating the gene (B) into an organ, tumor or tumor excision site (or into a peripheral nerve there) For the prevention or treatment of
The present invention includes (A) a viral vector incorporating a gene (B) used for injection into an organ, tumor or tumor excision site (or into peripheral nerves there).
(C) a gene used for the prevention or treatment of a disease (A) an organ, a tumor, or a tumor excised site (or into a peripheral nerve located therein) (B) a gene Including a virus vector-containing pharmaceutical into which is incorporated.
The present invention includes the use of (A) an organ, a tumor or a viral vector incorporating the gene (B) for producing an injection for the site of excision of the tumor (or the peripheral nerve there).
The present invention includes (A) an organ, a tumor, or a site where the tumor has been excised (or into the peripheral nerves there) (C) a virus incorporating a gene (B) for producing a preventive or therapeutic agent for disease. Includes vector-containing drugs.
The gene (B) is selected from the viewpoint of how to change the function of which peripheral nerve from any mechanism of the target disease, and the present invention incorporates the gene (B) thus selected. Embodiments include injecting a viral vector into a (A) organ, tumor or tumor excision site of a mammal including humans (or into peripheral nerves there) for the treatment or prevention of the disease.
The present inventor has confirmed the (C) disease prevention or treatment effect in the combination of (A), (B) and (C) shown in Table 20 above.
For example, the second row from the top of Table 20 shows that hypertension was treated by injecting a viral vector incorporating a gene encoding a protein that kills sympathetic nerve function into the kidney. The same applies to the other stages.

The present invention enables prevention and treatment by local nerve manipulation in a disease in which the action of a local nerve in the disease is known or can be predicted. In addition, the present invention is useful as an experimental technique for investigating the action of a local nerve in a disease in which the action of the local nerve is unknown or unclear at the present time. After understanding the function of, the prevention and treatment by local nerve operation becomes possible. Therefore, prevention and treatment that stimulate local nerves of specific organs in each disease, prevention and treatment that suppresses, prevention and treatment that removes, and prevention and treatment that changes nerve function according to the disease. Depending on the degree of understanding of the nerve function in the disease, any method can be selected.

Hereinafter, the present invention will be described in detail by way of examples. However, these examples are examples of the present invention, and the technical scope of the present invention is not limited thereto.

<Example 1> Construction of a recombinant adeno-associated virus (AAV) vector RNA was extracted from a cultured cell derived from human tissue, a cultured cell derived from mouse or rat tissue or tissue by a known method, and then subjected to a reverse transcription reaction. A cDNA library derived from each organism was prepared. Using these cDNA libraries as templates, the functional manipulation genes listed in paragraph [0040] were amplified by PCR. Cre recombinase and loxP sequences, fluorescent protein genes listed in paragraph [0085] were obtained by PCR amplification. Promoter sequences were obtained by PCR using genomic DNA from cultured cells derived from human tissue, cultured cells or tissue derived from mouse or rat tissue. A promoter sequence, a functional manipulation gene, and a fluorescent protein gene obtained by the above method are selected, and an AAV vector plasmid (pAAV-MCS Expression Vector (SEQ ID NO: 41)): CELL BIOLABS, INC., Catalog No. VPK-410-SER2 PAAV-TH-NaChBac-2A-turboRFP, pAAV-TH-NaChBac G229A-2A-turboRFP, pAAV-TH-CreER, pAAV-TH-turboRFP, pAAV-TH-DTA, pAAV-ChAT-NaChBac 2A-turboRFP, pAAV-ChAT-NaChBac T220A-2A-GCaMP6f, pAAV-ChAT-turboRFP, pA AV-ChAT-GCaMP3, pAAV-Flex-NaChBac G229A-2A-turboRFP, and pAAV-Flex-DTA were prepared. In this example, the promoter represented by the sequence represented by SEQ ID NO: 15 was used as the TH promoter, and the promoter represented by the sequence represented by SEQ ID NO: 16 was used as the ChAT promoter. Any one or more of these were mixed with pAAV-RC and pHelper in a 50 mL falcon tube (see Example 2 and later for specific combinations). The day before transfection, 293AAV cells seeded at 3 × 10 ⁶ cells / 10 cm dish were transfected by the calcium phosphate method. The amount of each plasmid used for transfection is shown below.
pAAV-RC2 (SEQ ID NO: 39) 6 μg / 10 cm dish
pHelper (SEQ ID NO: 40) 6 μg / 10 cm dish
pAAV-MCS Expression Vector (SEQ ID NO: 41) -specific promoter-functional manipulation gene 6 μg / 10 cm dish

After transfection, 293AAV cells were cultured in Dulbecco's modified Eagle's medium for 48 hours at 37 ° C., and the supernatant was collected. AAV was purified and concentrated using AAV Purification Mega Kit (Cell biolabs) (1-5 × 10 ¹¹ GC / Ml).

<Example 2> Confirmation of effect of expression of gene for nerve stimulation Experiments for confirming the effect of retrograde AAV virus vector infection and gene expression, and the actual nerve activity increasing effect of nerve stimulation gene NaChBac and its mutants I did it. First, Irie K1, Shimomura T, Fujiyoshi Y. et al. The C-terminal helical bundle of the tetrameric prokaryotic sodium channel accelerates the inactivation rate. Nat Commun. 2012 Apr 24; 3: 793. doi: 10.1038 / ncomms 1797. And Irie K, Kitagawa K, Nagara H, Imai T, Shimomura T, Fujiyoshi Y. et al. Comparative study of the gating motif and C-type inactivation in pro- valento-voltage-gated sodium channels. In accordance with the method described in J Biol Chem 285, 3585-3694 (2009), NaChBac and its mutant NaChBac G229A were introduced into the human fetus 12 hours to 36 hours after gene introduction by the calcium phosphate method (calcium phosphate transfection kit, Invitrogen). Potential of whole kidney patch (clamped by the whole cell patch method) (Dulbecco's modified Eagle's medium medium, Sigma-Aldrich, St. Louis, MO, 10% fetal bovine serum, BioWhittaker, Switzerland) (Amplifier device, EPC-10, manufactured by HEKA Corporation is used). FIG. 1 shows the measurement results of neural activity. The composition of the solution in the electrode was 105 mM CsF, 35 mM NaCl, 10 mM EGTA, and 10 mM HEPES (pH 7.4). The bath solution contained 150 mM NaCl, 1.5 mM CaCl ₂ , 1 mM MgCl ₂ , 2 mM KCl, 10 mM HEPES (pH 7.4), and 10 mM glucose. All experiments were performed at 25 ± 2 ° C.

From the results shown in FIG. 1, changes in cell potential were observed by transfecting cells with the neural stimulation gene NaChBac and its mutant NaChBac G229A. From this result, it was confirmed that the neural stimulation gene NaChBac and its mutant NaChBac G229A have an action of increasing neural activity.

Further, for the purpose of sympathetic nerve stimulation, TH-NaChBac-2A-turboRFP AAV vector and TH-NaChBac G229A-2A-turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml or more) were used (TH is a Tyrosin Hydroxylas promoter). means). As a control experiment vector, TH-turboRFP (1-5 × 10 ¹¹ GC / ml or more) having no nerve stimulating action was used. This expresses only the red fluorescent protein RFP for gene expression confirmation under the control of a sympathetic nerve-specific TH promoter. In cells transfected with TH-NaChBac G229A-2A-turboRFP AAV vector, NaChBac or NaChBac G229A (NaChBac mutant) is expressed as a nerve stimulation gene under the control of a sympathetic nerve-specific TH promoter. At the same time, both of the red fluorescent protein RFP (turboRFP) for gene expression confirmation are expressed.
Sprague Dawley rats (Crl: CD (SD) rats) (body weight 300-400 g, sputum) under anesthesia (inhalation anesthesia Frocene (Halothane), Takeda Pharmaceutical, 0.5-1%) The left kidney was dissected from the peritoneal incision. In the left kidney (mainly cortex), the above-mentioned TH-NaChBac-2A-turboRFP vector solution is used in the NaChBac nerve stimulation vector group (N = 6), and the above TH-NaChBac-2A is used in the NaChBac G229A nerve stimulation vector group (N = 6). -TurboRFP vector solution was injected 10ul locally using syringe (7105, Hamilton) in the pseudo-nerve stimulation vector group (N = 6) using syringe (7105, Hamilton). Thereafter, the incised portion was sutured to awaken from anesthesia, and was bred.

Eighteen weeks after topical injection of vector, Kamiya A, Kawada T, Shimizu S, Sugimachi M. et al. Closed-loop Spontaneous Baroflex Transfer Function is Inappropriate for System Identification of the Natural Arc Butly Peripherate for Peripheral Peripherate. J Physiol 2011 Apr 1; 589 (Pt 7): 1769-90. The left and right renal sympathetic nerve activity and body blood pressure were measured according to the method described in PMID: 21486939. Under anesthesia (inhalation anesthesia Frocene (Halotan), Takeda Pharmaceutical Co., Ltd., 0.5-1%), the area of the left renal artery (including the renal nerve) was dissected from the retroperitoneal incision on the right supine position. Two stainless steel wire electrodes (Bioflex wire AS633, Cooner Wire, Chatsworth, Calif.) Are attached to the left renal nerve, and silicon gel (Silicon Low Viscosity, KWIK-SIL, World Precision Instrument, Inc., Saras., Saras., Saras.). ), The electrode was fixed, and a surgical thread was tied to the distal side of the electrode mounting portion. Next, a stainless steel wire electrode (same as above) was also attached to the right renal nerve from the retroperitoneal incision on the left lateral position. The electrical signals of the left and right kidney sympathetic nerves (multiunit, postganglionicity) thus obtained are passed through a band-pass filter (150-1000 Hz) (biological amplifiers MEG-6108 and AB-610J, Nihon Kohden Co., Ltd.). Rectified and integrated, A / D converted, recorded at 200 Hz, and stored. 2 and 3a, 3b, 3c show the results of neural activity.
On the other hand, the right common carotid artery is dissected by incising the right cervical skin, and a high-precision pressure transducer (SPR-320, Millar Instruments; Houston, TX) is inserted downward (in the direction toward the heart) from the right common carotid artery. Body blood pressure was measured using a blood pressure bio-amplifier (AP641G, Nihon Kohden). FIG. 3d shows the results of measurement of body blood pressure. Thereafter, half of the right and left kidneys were removed, homogenized (BRANSON, SONIFER 250), and the amount of norepinephrine in the kidney was measured (Labor Diagnostics Nova, Germany). Reflux fixation and post-fixation with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, left and right remaining kidneys and celiac ganglia removed, frozen sections prepared, and immunostaining analysis Went. The antibodies and dyes used were DAPI (4 ', 6-diamidino-2-phenylindole, staining of cell nuclei, 102236276001 ROCHE, Roche Life Sciences), RFP antibody (ab286664, Abcam), TH antibody (Tyrosin Hydroxylase, sc- 7847, Santa cruz biotechnology), Tuj-1 antibody (beta tubulin III, sc-58888, Santa cruz biotechnology). The tissue morphology was observed with a confocal microscope (FV1000, Olympus). FIG. 4 shows the results of immunostaining analysis.

2. From the results of FIG. 2, in the pseudo-nerve stimulation vector group, the renal sympathetic nerve activity on the vector side was the same as the contralateral renal sympathetic nerve activity (FIG. 2a). In the NaChBac nerve stimulation vector group, renal sympathetic nerve activity on the vector side increased compared to the contralateral side (FIG. 2b, 182% on the contralateral side). In the NaChBac G229A nerve stimulation vector group, renal sympathetic nerve activity on the vector side was further increased compared to the contralateral side (FIG. 2c, 251% on the contralateral side).

From the results of FIG. 3, in the NaChBac nerve stimulation vector group (N = 6), the amount of renal sympathetic nerve activity on the vector side (FIG. 3a) and the firing frequency per pulse (FIG. 3b) increased compared to the contralateral side ( In the NaChBac G229A nerve stimulation vector group (N = 6), these increased further (251% on the contralateral side). In the group of pseudo nerve stimulation vectors (N = 6), such increase in neural activity did not occur. Renal norepinephrine levels agreed well with the results of neural activity. That is, in the NaChBac nerve stimulation vector group (N = 6), it was higher in the vector side kidney than in the contralateral side, and in the NaChBac G229A nerve stimulation vector group (N = 6), it was higher (FIG. 3c). Further, in the NaChBac nerve stimulation vector group (N = 6), systolic blood pressure and diastolic blood pressure increased to become hypertension, and in the NaChBac G229A nerve stimulation vector group (N = 6), more severe hypertension (#P <0.05 vs. contralateral sympathetic nerve activity, * P <0.05 NaChBac nerve stimulation vector group vs. NaChBac G229A nerve stimulation vector group).

From the results of FIG. 4, in the NaChBac G229A nerve stimulation vector group, TH-NaChBac G229A-2A-turboRFP AAV vector had TH positive RFP positive (FIGS. 4b, 4c, 4d) in the left kidney on the side injected locally, This RFP positive nerve was certainly a nerve because it was Tuj1 positive. On the other hand, there was no RFP-positive sympathetic nerve in the contralateral right kidney where the vector was not locally injected (FIG. 2e). In the celiac ganglion, there was also a TH-positive RFP-positive sympathetic cell body (FIG. 4a), which appeared to be a left kidney sympathetic cell body. From the above, it was confirmed that when the prepared AAV vector was locally injected into the kidney, it retrogradely infects the nerve distributed in the kidney and expresses the insertion gene (turboRFP) specifically in the sympathetic nerve.
From the above results, it was confirmed that NaChBac and NaChBac G229A actually have an action of increasing neural activity.

<Example 3> Confirmation of therapeutic effect on hypertension AAV vector (TH-NaChBac G229A-2A-turboRFP AAV vector) prepared in Example 1 (1-5 × 10 ¹¹ GC / ml or more) for the purpose of sympathetic nerve stimulation, and The AAV vector (TH-CreER AAV vector, Flex-DTA AAV vector) prepared in Example 1 (1-5 × 10 ¹¹ GC / ml or more) was used for the purpose of drug-induced sympathetic nerve removal. In cells transfected with the TH-CreER AAV vector, CreER (a fusion protein of Crerecombinase and human estrogen receptor ligand-binding domain) is expressed under the control of the sympathetic nerve-specific promoter TH. When the Flex-DTA AAV vector is duplicated and introduced into the gene, CreER is transferred to the nucleus by tamoxifen administration, and Cre recombinase is activated by the flex switch (Cre-loxp system) to express DTA (diphtheria toxin). The cell dies. Equivalent amounts of these three AAV vectors: TH-NaChBac G229A-2A-turboRFP for nerve stimulation or TH-turboRFP without nerve stimulation and TH-CreER and Flex-DTA for drug-induced denervation ( 10 ul / animal) were mixed to prepare three AAV mixed vector solutions for animal administration (10 ul for each vector for a total of 30 ul / animal).
Sprague Dawley rats (Crl: CD (SD) rats) (body weight 300-400 g, sputum) under anesthesia (inhalation anesthesia Frocene (Halothane), Takeda Pharmaceutical, 0.5-1%) The left kidney was dissected from the peritoneal incision. In the nerve stimulation vector group (N = 8) and the post-neural stimulation nerve removal vector group (N = 8), three types of AAV mixed vector solutions (TH-NaChBac G229A-2A-turboRFP, TH- CreER and Flex-DTA) were injected locally at 30 ul each using syringe (7105, Hamilton) (3 ul in 10 locations). In the control group (N = 8), the same amount of the control AAV vector solution (TH-turboRFP, TH-CreER and Flex-DTA) was locally injected in the same manner. Next, the right kidney was dissected from a retroperitoneal incision on the left lateral position, and the nerve stimulation vector group (N = 8) and the nerve removal vector group after nerve stimulation (N = 8) were placed in the right kidney (its cortex). Three AAV mixed vector solutions were locally injected in the same amount as in the case of the left kidney in the control group (N = 8). Thereafter, the incised portion was sutured to awaken from anesthesia, and was kept up to 24 weeks after local injection of the vector (or PBS).
After blood pressure measurement 9 weeks after local injection of the vector, in the post-neural stimulation nerve removal vector group (N = 8), tamoxifen (Tamoxifen T5648, Wako) was used as dimethylsulfoxide (DMSO), etc. (4% DMSO (046-21981, Wako), 6% Ethanol (057-00456, Wako), 90% Sesame oil (SesameOil, 25620-65, nacalai tesque)) was prepared and administered intraperitoneally (tamoxifen 130 mg / kg body weight). On the other hand, in the nerve stimulation vector group (N = 8), the same amount of PBS was intraperitoneally administered as a control. Blood pressure (systolic blood pressure and diastolic blood pressure) was measured from the tail artery every 3 weeks from immediately after the local injection of the vector to 24 weeks (non-heated non-invasive blood pressure monitor for mice and rats MK-2000ST, Muromachi Machinery Corporation). FIG. 5 shows the blood pressure measurement results. In addition, 24 weeks after the local injection of the vector, the animals were reflux-fixed with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, and the left and right kidneys and celiac ganglion were removed. After fixing, frozen sections were prepared and immunostaining analysis was performed. The antibodies and dyes used were DAPI (4 ', 6-diamidino-2-phenylindole, staining of cell nuclei, 102236276001 ROCHE, Roche Life Science), TH antibody (Tyrosin Hydroylase, sc-7847, Santa Cruz biotechnology). It was. The tissue morphology was observed with a confocal microscope (FV1000, Olympus). FIG. 6 shows the results of immunostaining.

From the results of FIG. 5, in the control group (N = 8, the control AAV vector solution was locally injected into the kidney), the blood pressure did not change until 24 weeks (FIG. 5, ▽). In the nerve stimulation vector group (N = 8), the blood pressure increased until 9 weeks after the local injection of the vector and became hypertension (hypertensive model animals were created) after 9 weeks (preparation of hypertensive model animals) (FIG. 5, Δ). In the nerve removal vector group after nerve stimulation (N = 8), blood pressure increased to hypertension as in the nerve stimulation vector group (N = 8) until 9 weeks after the local injection of the vector, but tamoxifen at 9 weeks. After administration, blood pressure gradually decreased and returned to the level before local vector injection at 24 weeks (FIG. 5, ●) (#P <0.05 vs. control group, * P <0.05 nerve stimulation vector group vs. nerve removal vector group after nerve stimulation). Moreover, it was confirmed from the result of FIG. 5 that the hypertension model animal was able to be produced.

From the results of FIG. 6, TH-positive renal sympathetic nerves were observed in the kidney in the nerve stimulation vector group, but the kidney sympathetic nerves disappeared in the nerve removal vector group after nerve stimulation. From the above, the sympathetic nerve was actually removed by local injection of a viral vector that removes the sympathetic nerve of the kidney.
From the above results, a therapeutic effect that improves hypertension was confirmed by local injection of a viral vector that removes the sympathetic nerves of the kidney.

<Example 4> Confirmation of therapeutic effect on diabetes The AAV vector (ChAT-NaChBac T220A-2A-turboRFP AAV vector) (1-5 × 10 ¹¹ GC / ml) prepared in Example 1 was used for the purpose of parasympathetic nerve stimulation. In cells transfected with this ChAT-NaChBac T220A-2A-turboRFP AAV vector, under the control of the parasympathetic nerve-specific ChAT promoter, NaChBac T220A (a variant of NaChBac) as a gene for neural stimulation, Both of the red fluorescent proteins turboRFP for expression confirmation are expressed. For the purpose of control experiments, the AAV vector (ChAT-turboRFP AAV vector) prepared in Example 1 was used. In cells transfected with this ChAT-turboRFP AAV vector, a red fluorescent protein turboRFP for gene expression confirmation is expressed. Each of these two AAV vectors was prepared as an AAV vector solution for animal administration (5 ul / animal).
Sprague Dawley rats (Crl: CD (SD) rats) (body weight 300-450 g, sputum) were subjected to streptozocin (streptozocin, 0.5-1% under inhalation anesthesia, halothane, Takeda Pharmaceutical Co., Ltd.). S0130, Sigma) was administered intraperitoneally (twice at 2-day intervals, 30 mg / kg body weight each time). From the day of the first intraperitoneal administration, the diet for diabetes obesity research (High Fat Diet32, the ratio of fat-derived calories in the total calories (Fat kcal%) is about 60%, CLEA Japan) for 8 weeks. Feeded (food intake was free of animals).

Eight weeks after administration of the streptozocin high-fat diet, under anesthesia (inhalation anesthesia Frocene (Hallown), Takeda Pharmaceutical Co., Ltd., 0.5-1%), the pancreas was dissected through a midline abdominal incision, and parasympathetic nerve stimulation vector group (N = 8), the ChAT-NaChBac T220A-2A-turboRFP AAV vector solution, the parasympathetic pseudostimulation vector group (N = 8), the ChAT-turboRFP AAV vector solution (10 ul / mouse), and the control group (N = 8) PBS (10 ul / animal) was locally injected 10 ul at a time using syringe (7105, Hamilton) (2 ul in 5 locations). Thereafter, the incised portion was sutured to awaken from anesthesia. The animals were kept up to 4 weeks after the local injection of the vector (or PBS). In the meantime, the diet for the study of diabetes obesity (High Fat Diet 32, Claire Japan) was fed (the amount of food consumed was free of animals).
At 4 weeks after local injection of AAV vector or PBS, a fasting glucose tolerance test was performed. First, it was fasted for 13 hours from 21:00 on the day before measurement. On the morning of the measurement day, under anesthesia (inhalation anesthesia Frocene (Halothane), Takeda Pharmaceutical Co., Ltd., 0.5-1%), the neck skin at the top of the clavicle is dissected and the jugular vein is dissected, and the inguinal skin is further dissected The femoral vein was then dissected. At 10:00 in the morning, a 26-gauge needle (TERUMO) was punctured shallowly into the jugular vein and femoral vein to cause a small amount of bleeding, and a glucose kit for examination (GLUCOCARD G BLACK, GT-1830, manufactured by arkray, and Gsensor, arkray) ) To measure fasting blood glucose, and calculate the average of the two values. Next, a glucose tolerance test was performed and blood glucose level was measured. 2 g / kg body weight of glucose (50% glucose injection solution, manufactured by Otsuka Pharmaceutical Co., Ltd.) was intraperitoneally administered, and blood glucose levels were measured from the jugular vein and femoral vein in the

same manner

0, 15, 30, 60, and 120 minutes later. And the average value of two values was calculated. FIG. 7 shows the blood glucose level measurement results.
Thereafter, the animals were fixed by refluxing with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, and the medulla was extracted and fixed, frozen sections were prepared, and immunostaining analysis was performed. The antibodies and dyes used were RFP antibody (ab28664, Abcam) and DAPI (4 ′, 6-diamidino-2-phenylindole, staining of cell nuclei, 10236276001 ROCHE, Roche Life Science). The tissue morphology was observed with a confocal microscope (FV1000, Olympus). FIG. 8 shows the results of immunostaining.

From the results shown in FIG. 7, the parasympathetic nerve stimulation vector group (N = 8) has a lower fasting blood glucose than the control group (N = 8) and the parasympathetic pseudostimulation vector group (N = 8). The blood glucose level (0, 15, 30, 60, 120 minute value) in the performance test was low (#P <0.05 vs. before glucose load, * P <0.05 nerve stimulation vector group vs. nerve pseudostimulation vector group) And control group).

From the results of FIG. 8, in the parasympathetic nerve stimulation vector group, RFP-positive neurons were present in the vagus dorsal motor nucleus and suspicion nucleus of the medulla. This was not observed in the control group. From the above, it was confirmed that when the prepared AAV vector was locally injected into the pancreas, the parasympathetic nerve distributed retrogradely in the pancreas was infected and the inserted gene (turboRFP) was expressed specifically in the paranerve.
Based on the above, a therapeutic effect that improves glucose tolerance in diabetes was observed by local injection of a viral vector that stimulates parasympathetic nerves in the pancreas.

<Example 5> Confirmation of therapeutic effect against cancer (1) Confirmation of therapeutic effect against rat breast cancer (when using a sympathetic nerve manipulation virus vector)
The AAV vector (TH-CreER AAV vector, Flex-DTA AAV vector) (1-5 × 10 ¹¹ GC / ml) prepared in Example 1 was used for the purpose of drug-induced sympathetic nerve removal. In cells transfected with the TH-CreER AAV vector, CreER (Crerecombinase and human estrogen receptor ligand-binding domain) is expressed under the control of a sympathetic nerve-specific TH promoter. When this cell is infected with the Flex-DTA AAV vector in duplicate and transduced, CreER is transferred to the nucleus by tamoxifen administration, and Cre recombinase is activated by the flex switch (Cre-loxp system) to produce DTA (diphtheria toxin). Expressed and the cell dies. These two types of AAV vectors were mixed in equal amounts (5 ul / mouse) to prepare an AAV vector solution for animal administration (both AAV vectors in total 10 ul / mouse).
Carcinogenic N-methyl-N-nitrosourea (MNU) 50 mg / kg body weight was intraperitoneally administered to Hras128 rats (Japan Claire, body weight 250-400 g, rabbit) to induce breast cancer. Produced. The presence or absence of breast cancer was examined from the body surface by visual inspection and palpation. When breast cancer was observed, the size was measured with calipers (Mitsutoyo Corporation). When the size of the breast cancer reaches 800 mm ³ or more and 1000 mm ³ or less, the above AAV vector solution (sympathetic nerve removal vector group) under anesthesia (inhalation anesthesia flow sen (Halotan), Takeda Pharmaceutical, 0.5-1%) N = 8, sympathetic mock removal vector group N = 8) or PBS (control group, N = 8) was locally injected into the breast cancer tissue using syringe (7105, Hamilton). Furthermore, in the sympathetic denervation vector group (N = 8), tamoxifen (Tamoxifen T5648, Wako) was converted to Dimethylsulfoxide (DMSO) and the like (4% DMSO (046-21981, Wako), 6% Ethanol (Ethanol 99.5, 057). -00456, Wako), 90% sesame oil (Sesame Oil, 25620-65, nacalai tesque)) was prepared and administered intraperitoneally (tamoxifen 130 mg / kg body weight), while sympathetic sham removal group (N In = 8), the same amount of PBS was intraperitoneally administered as a control. Thereafter, they were awakened from anesthesia, and were fed up to 14 weeks after the local injection of vector (or PBS). After vector local injection, the size of breast cancer was measured with calipers every 2 weeks up to 14 weeks. FIG. 9 shows the measurement results of changes in the size of breast cancer. Thereafter, half of the breast cancer tissue was excised and homogenized (BRANSON, SONIFIER 250), and the amount of tissue norepinephrine was measured (Labor Diagnostics Noka, Germany). FIG. 10 shows the measurement results of the amount of breast cancer tissue norepinephrine. Thereafter, the animals were reflux fixed with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, the remaining breast cancer tissues were removed and fixed, frozen sections were prepared, and immunostaining analysis was performed. went. The antibodies and dyes used were DAPI (4 ', 6-diamidino-2-phenylindole, staining of cell nuclei, 102236276001 ROCHE, Roche Life Sciences), Lycopersicon esculentum Lectin (DL1174, DyLight 488 Conjugate, VECTORLA, BECTORLA, INVORTH) The antibody (Tyrosin Hydroylase, sc-7847, Santa Cruz biotechnology). The tissue morphology was observed with a confocal microscope (FV1000, Olympus). FIG. 11 shows the results of immunostaining.

From the results of FIG. 9, cancer was markedly increased in the control group (N = 8) and the sympathetic false removal vector group (N = 8), whereas in the sympathetic removal vector group (N = 8) The increase in cancer was strongly suppressed (#P <0.05 vs. control group).

From the results of FIG. 10, the amount of breast cancer tissue norepinephrine was significantly reduced in the sympathetic nerve removal vector group compared to the control group and the sympathetic false removal vector group.

From the results of FIG. 11, in the immunostaining analysis (20-um thick frozen section), TH-positive sympathetic nerve fibers were observed in the breast cancer tissue in the control group and the sympathetic false removal vector group, whereas the sympathetic nerve removal vector group Then, it was observed that the sympathetic nerve in the breast cancer tissue disappeared.
From the above, a therapeutic effect that suppresses breast cancer growth was confirmed by local injection of a viral vector that removes sympathetic nerves distributed in breast cancer.

<Example 6> Confirmation of therapeutic effect on cancer (2) Confirmation of therapeutic effect on rat breast cancer (when using parasympathetic nerve operation virus vector)
The AAV vector (ChAT-NaChBac T220A-2A-GCaMP6f AAV vector) (1-5 × 10 ¹¹ GC / ml) prepared in Example 1 was used for the purpose of parasympathetic nerve stimulation. In cells transfected with this ChAT-NaChBac T220A-2A-GCaMP6f AAV vector, NaChBac T220A (NaChBac mutant) and a gene as a neurostimulatory gene under the control of a parasympathetic nerve-specific ChAT promoter, Both the green fluorescent protein GCaMP6f for expression confirmation and nerve activity measurement are expressed. For the purpose of control experiments, ChAT-GCaMP3 AAV vector was used (1-5 × 10 ¹¹ GC / ml). In cells transfected with this ChAT-GCaMP3 AAV vector and transfected, a green fluorescent protein GCaMP3 for gene expression confirmation and nerve activity measurement is expressed. Each of these two AAV vectors was prepared as an AAV vector solution for animal administration (5 ul / animal).
The breast cancer model animal produced in Example 5 was used. The presence or absence of breast cancer was examined from the body surface by visual inspection and palpation. When breast cancer was observed, the size was measured with calipers (Mitsutoyo Corporation). When the size of the breast cancer reaches 800 mm ³ or more and 1000 mm ³ or less, the parasympathetic nerve stimulation vector group (N = 8) under anesthesia (inhalation anesthesia Frocene (Halotan), Takeda Pharmaceutical, 0.5-1%) Then, the ChAT-NaChBac T220A-2A-GCaMP6f AAV vector solution (5 ul / animal), the parasympathetic pseudostimulation vector group (N = 8), the ChAT-GCaMP3 AAV vector solution (5 ul / animal), and the control group ( In N = 8), PBS (5 ul / animal) was locally injected into the breast cancer tissue using syringe (7105, Hamilton). Thereafter, they were awakened from anesthesia, and were fed up to 14 weeks after the local injection of vector (or PBS). After vector local injection, the size of breast cancer was measured with calipers every 2 weeks up to 14 weeks. FIG. 12 shows the measurement results of changes in the size of breast cancer. Thereafter, the animals were reflux fixed with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, the remaining breast cancer tissues were removed and fixed, frozen sections were prepared, and immunostaining analysis was performed. went. The antibodies and dyes used were DAPI (4 ', 6-diamidino-2-phenylindole, staining of cell nuclei, 102236276001 ROCHE, Roche Life Sciences), GFP antibody (Green fluorescence protein, ab 13970, Abcam, Vachycinel antibody) The tissue morphology was observed with a confocal microscope (FV1000, Olympus), and the results of immunostaining are shown in FIG.

From the results of FIG. 12, cancer increased to the same extent in the control group (N = 8) and the parasympathetic pseudostimulation vector group (N = 8), but increased in the parasympathetic stimulation vector group (N = 8). Was suppressed (#P <0.05 vs. control group).

From the results of FIG. 13, in the parasympathetic nerve stimulation vector group and the parasympathetic pseudostimulation vector group, the VAChT-positive parasympathetic nerve was GFP-positive, and gene expression to the parasympathetic nerve by the retrograde virus vector was shown.
From the above, a therapeutic effect that suppresses breast cancer growth by local injection of a viral vector that stimulates parasympathetic nerves distributed in breast cancer was observed.

<Example 7> Confirmation of therapeutic effect on cancer (3) Confirmation of therapeutic effect on mouse skin cancer (when using sympathetic nerve manipulation virus vector)
The AAV vector (TH-DTA AAV vector) (1-5 × 10 ¹¹ GC / ml) prepared in Example 1 was used for the purpose of removing sympathetic nerves. In cells transfected with a TH-DTA AAV vector, DTA (diphtheria toxin) is expressed under the control of a sympathetic nerve-specific TH promoter, and the cells die. For the purpose of control experiments, TH-turboRFP AAV vector was used (1-5 × 10 ¹¹ GC / ml). For each AAV vector, an AAV vector solution (5 ul / animal) for animal administration was prepared.
rasH2 mice (Jic: CB6F1-TgrasH2 @ Jcl mice, CLEA Japan, body weight 27-33 g, rabbit and rabbit) were administered intraperitoneally with carcinogenic N-methyl-N-nitrosourea (MNU) 50 mg / kg body weight. Then, skin cancer was induced to produce a skin cancer model animal. The presence or absence of cancer was examined by palpation from the body surface, and when skin cancer was observed, the size was measured with calipers (Mitsutoyo Corporation). When the size of the skin cancer reaches 40 mm ³ or more and 60 mm ³ or less, the skin cancer tissue is sympathized under anesthesia (inhalation anesthesia flow sen (halothane), Takeda Pharmaceutical, 0.5-1%). In the nerve removal vector group (N = 8), the TH-DTA AAV vector solution, in the sympathetic false removal vector group (N = 8), the TH-turboRFP AAV vector solution, in the control group (N = 8), PBS, and syringe 5ul local injection using (7105, Hamilton). Thereafter, they were awakened from anesthesia, and were fed up to 16 weeks after the local injection of vectors. After the local injection of the vector, the size of the skin cancer was measured with a caliper every 2 weeks up to 16 weeks. FIG. 14 shows the measurement results of changes in the size of skin cancer.

From the results of FIG. 14, cancer was markedly increased in the control group (N = 8) and the sympathetic false removal vector group (N = 8), whereas the sympathetic removal virus vector group (N = 8). Then, the increase in cancer was strongly suppressed (#P <0.05 vs. control group).
From the above, a therapeutic effect that suppresses the increase in skin cancer was confirmed by local injection of a viral vector that removes sympathetic nerves distributed in skin cancer.

<Example 8> Confirmation of therapeutic effect on cancer (4) Confirmation of therapeutic effect on mouse skin cancer (when using parasympathetic nerve operation virus vector)
The AAV vector (ChAT-NaChBac T220A-2A-GCaMP6f AAV vector) (1-5 × 10 ¹¹ GC / ml) prepared in Example 1 was used for the purpose of parasympathetic nerve stimulation. In cells transfected with this ChAT-NaChBac T220A-2A-GCaMP6f AAV vector, NaChBac T220A (NaChBac mutant) and a gene as a neurostimulatory gene under the control of a parasympathetic nerve-specific ChAT promoter, Both the green fluorescent protein GCaMP6f for expression confirmation and nerve activity measurement are expressed. For the purpose of control experiments, ChAT-GCaMP3 AAV vector was used (1-5 × 10 ¹¹ GC / ml). In cells transfected with this ChAT-GCaMP3 AAV vector and transfected, a green fluorescent protein GCaMP3 for gene expression confirmation and nerve activity measurement is expressed. Each of these two AAV vectors was prepared as an AAV vector solution for animal administration (5 ul / animal).
The skin cancer model animal produced in Example 7 was used. The presence or absence of cancer was examined by palpation from the body surface, and when skin cancer was observed, the size was measured with calipers (Mitsutoyo Corporation). When the size of the skin cancer reaches 40 mm ³ or more and 60 mm ³ , the anesthesia (inhalation anesthesia Frocene (halothane), Takeda Pharmaceutical, 0.5-1%), the skin cancer tissue, parasympathetic nerve In the stimulation vector group (N = 8), the ChAT-NaChBac T220A-2A-GCaMP6f AAV vector solution, in the parasympathetic pseudostimulation vector group (N = 8), the ChAT-GCaMP3 AAV vector solution, and the control group (N = In 8), PBS was locally injected into skin cancer tissue using syringe (7105, Hamilton). Thereafter, they were awakened from anesthesia, and were fed up to 16 weeks after the local injection of vectors. After the local injection of the vector, the size of the skin cancer was measured with a caliper every 2 weeks up to 16 weeks. FIG. 15 shows the measurement results of changes in the size of skin cancer.

From the results of FIG. 15, the control group (N = 8) and the parasympathetic pseudo-stimulation vector group (N = 8) increased cancer to the same extent, but the parasympathetic nerve stimulation vector group (N = 8) increased cancer. Was suppressed. (#P <0.05 vs. control group)
From the above, a therapeutic effect that suppresses the increase in skin cancer was confirmed by local injection of a viral vector that stimulates parasympathetic nerves distributed in skin cancer.

<Example 9> Construction of a lentiviral vector Using a known phage library as a template, the full length of Cre recombinase was amplified by PCR, and a nuclear localization signal (NlS: nuclear localization signal) was added to the N-terminal side of Cre recombinase Into the HiRet vector, a HiRet-Cre plasmid was prepared.
The produced HiReT-Cre vector, envelope, and packaging plasmid were transfected into human fetal kidney 293 cells (HEK293T) by the calcium phosphate method. Lentiviral particles were pelleted by ultracentrifugation (50,000 × g, 90 minutes) and then suspended in PBS. Further, for injection, the vector particles were centrifuged at 10,000 g for 16-18 hours and suspended in PBS. The vector particles were loaded onto a Sepharose Q FF ion exchange column (GE Healthcare, Little Charlotte, UK) and eluted with a linear gradient of 0.0-1.5 M NaCl while monitoring at an absorbance of 260/280 nm. Thereafter, HEK293T, Neuro2A, and N1E-115 seeded in a 10 cm culture dish were transduced with appropriate amounts, and flow cytometry (FACSCalibur, Nippon, Becton Dickinson Co., Tokyo, Japan), and the vector stock solution of viral RNA pinc. RNA virus kit (Clontech, Mountain View, CA) was used for isolation, and RNA genome copy number was determined using a lenti-X qRT-PCR titration kit (Clontech).

<Example 10> Confirmation of therapeutic effect on obesity and / or overeating The lentiviral vector (HiRet-Cre lentiviral vector) prepared in Example 9 and the AAV vector (FLEX-NaChBac G229A-2A-) prepared in Example 1 The turboRFP AAV vector (1-5 × 10 ¹¹ GC / ml) was used for the purpose of stimulation of afferent nerves by the double infection method. In cells infected with the HiRet-Cre lentiviral vector, Cre recombinase is expressed under the control of CAG promoter composed of cytomegalovirus enhancer and chicken β-actin. In cells that are double-infected with the HiRet-Cre lentiviral vector and the FLEX-NaChBac G229A-2A-turboRFP AAV vector, Cre recombinase is activated by the flex switch (Cre-loxp system) to express NaChBac G229A with neurostimulatory action. . In this example, the HiRet-Cre lentiviral vector was locally injected into the stomach, and the FLEX-NaChBac G229A-2A-turboRFP AAV vector was locally injected into the nodose ganglion. It is only the afferent nerve of the stomach which exists and has a cell body in the nodular ganglion. Thus, neural manipulation was designed so that NaChBac G229A was expressed only in the afferent nerve of the stomach and selectively stimulated the activity of the afferent nerve of the stomach.
Sprague Dawley rats (Crl: CD (SD) rats, Charles River, Japan) (body weight 400-460 g, rabbit) were used. First, in the nerve stimulation vector group (N = 8), under anesthesia (inhalation anesthesia flow sen (halothane), Takeda Pharmaceutical Co., Ltd., 0.5-1%), the stomach was dissected through a midline abdominal incision, and the above-mentioned HiRet was applied to the stomach body. -Cre lentiviral vector was injected locally 20ul using syringe (7105, Hamilton) (2ul in 10 places). Further, the right nodular ganglion was dissected by midline cervical incision, and 2 ul of the FLEX-NaChBac G229A-2A-turboRFP AAV vector was locally injected using syringe (7015, Hamilton), and then the left nodose ganglion was autopsied. The same vector was similarly injected locally in the same amount. In the control group (N = 8), the same amount of PBS was locally injected into the stomach and bilateral nodular ganglia instead of the viral vector. Following local injection of the vector, the incision was sutured to wake up from anesthesia. The animals were kept up to 12 weeks after the local injection of the vector (or PBS). Each week, food intake (daily food intake, using individual cages, measured with EK-200i, manufactured by A & D) and body weight (SK-1000, manufactured by A & D) were measured. FIG. 16 shows the result of eating behavior. Thereafter, the animals were reflux fixed with 4% paraformaldehyde / phosphate buffer (163-20145, Wako) for tissue fixation, the ganglion and medulla were removed and fixed, and frozen sections were prepared. Were observed with a confocal microscope (FV1000, Olympus). FIG. 17 shows the results of tissue morphology observation.

From the results of FIG. 16, the nerve stimulation vector group (N = 8) had a lower food intake (FIG. 16a) and a reduced body weight gain compared to the control group (N = 8) (FIG. 16). 16b) (#P <0.05 vs. control group).

From the results of FIG. 17, in the nerve stimulation vector group, a red fluorescent nerve cell body was present in the nodal ganglion (FIG. 17a) due to turboRFP expression. Further, in the solitary nucleus of the medulla oblongata (FIG. 17b), there was a red fluorescent nerve ending due to the expression of turboRFP (the nerve cell body did not express turboRFP). Therefore, gene expression could be selectively induced only in the afferent nerve of the stomach by the double vector method.
As mentioned above, the effect which suppresses feeding and obesity by the viral vector local injection which stimulates the afferent nerve distributed in the stomach was recognized.

It can be used as a therapeutic agent and therapeutic means in the medical industry. In particular, in cancer medical care, it can be widely used at home and abroad in such a manner that a viral vector is locally injected into a residual tissue of cancer after surgery to suppress recurrence and metastasis.

Claims

Organs, tumors, or tumors other than the brain and spinal cord were excised with viral vectors that incorporated a gene encoding a protein that has the function of killing, stimulating, suppressing, or altering other nerve cell functions. Local injection into the site, infecting the virus vector with the virus vector early, expressing the protein having the function in the infected nerve, manipulating the function of the nerve infected with the virus vector by the protein, the nerve contact A method for preventing or treating a disease, comprising changing the function of an organ other than the brain and spinal cord.
Organs, tumors, or tumors other than the brain and spinal cord were excised with viral vectors that incorporated a gene encoding a protein that has the function of killing, stimulating, suppressing, or altering other nerve cell functions. By a viral vector incorporating a gene expressing a protein having a function of killing, stimulating, suppressing, or changing other neuronal functions of peripheral nerves, characterized by including a step of locally injecting into a site A method of antegrade or retrograde infection of the nerve.
Organs, tumors, or tumors other than the brain and spinal cord were excised with viral vectors that incorporated a gene encoding a protein that has the function of killing, stimulating, suppressing, or altering other nerve cell functions. A method of locally injecting a site and generating the function in the organ-specific, the tumor or the site where the tumor has been excised.
Organs, tumors, or tumors other than the brain and spinal cord were excised with viral vectors that incorporated a gene encoding a protein that has the function of killing, stimulating, suppressing, or altering other nerve cell functions. A method for manipulating a peripheral nerve, characterized by local injection into a site.
5. The method according to claim 1, wherein the peripheral nerve is efferent or afferent sympathetic nerve, efferent or afferent parasympathetic nerve, efferent or afferent vagus nerve.
Peripheral nerves are somatosensory nerves (tactile, pressure, cold, warm, painful, itching, numbness), visceral sensory nerves, or special sensory nerves (olfaction, vision, hearing, balance, taste) The method according to any one of claims 1 to 4, characterized in that:
5. The method according to claim 1, wherein the peripheral nerve is an enteric nerve.
The method according to any one of claims 1 to 7, wherein the viral vector has a neuron-specific promoter sequence.
The method according to claim 1, wherein the disease is a bacterial infection, a viral infection, a mycosis, another infection or a parasitic disease.
Disease is lip, oral cavity, pharynx, esophagus, stomach, small intestine, colon, rectosigmoid junction, rectum, anus, anal canal, liver, intrahepatic bile duct, gallbladder (sac), pancreas, inner ear, larynx, bronchus, Lung, heart, mediastinum, pleura, peripheral nerve, autonomic nerve, retroperitoneum, peritoneum, connective tissue, soft tissue, bone, cartilage, skin, breast, cervix, uterine body, ovary, placenta, penis, prostate, testis (Testis), kidney excluding renal pelvis, renal pelvis, urinary tract, bladder, thyroid, adrenal gland, endocrine tissue, lymphoid tissue, hematopoietic tissue, eye, ocular appendage, meningeal malignant neoplasm, malignant neoplasm of these secondary sites The method according to claim 1, wherein these are intraepithelial neoplasms, benign neoplasms, malignant neoplasms with unknown sites, or neoplasms with unknown or unknown properties.
The method according to claim 1, wherein the disease is anemia, coagulation disorder, purpura, hemorrhagic condition, hyposplenic function, hypersplenism, or sarcoidosis.
Diseases include hypothyroidism, thyroid poisoning (hyperthyroidism), thyroiditis, thyroid disorders, insulin-dependent diabetes mellitus (IDDM), non-insulin-dependent diabetes mellitus (NIDDM), diabetes, hypoglycemia, parathyroid gland ( Parathyroidism), hypoparathyroidism (parathyroidism) hyperfunction, parathyroid (parathyroidism) disorder, aldosteronism, adrenocortical hyperfunction, primary adrenocortical dysfunction (disease), Addison (Addison) ) Crisis (onset), drug-induced adrenocortical dysfunction (disease), adrenocortical dysfunction (disease), adrenal medullary hyperfunction, adrenal disorder, hyperestrogenism (disease), androgen excess (disease), polycystic ovary Syndrome, primary ovarian dysfunction (symptom), ovarian dysfunction, testicular (testis) hyperfunction (symptom), testicular (testis) hypofunction (symptom), testicular (testis) dysfunction Disorders, obesity (disease), a hyperalimentation (overfed), amyloidosis (amyloidosis) or metabolic disorders, the method according to claim 1.
The disease is dementia of Alzheimer's disease, vascular dementia, other dementia, personality disorder, mood (emotional) disorder, physical expression disorder, anxiety disorder, obsessive-compulsive disorder (obsessive-compulsive disorder), adjustment disorder , Schizophrenia, mania, bipolar affective disorder (manic depression), depression, recurrent depressive disorder, eating disorder, sexual dysfunction, organic disorder, panic disorder (interstitial seizure) The method according to claim 1, which is sexual anxiety), post-traumatic stress disorder, pervasive developmental disorder, autism, other mental disorders or behavioral disorders.
Diseases are Parkinson's disease, secondary Parkinson's syndrome, Alzheimer's disease, sleep disorder, extrapyramidal disorder, neurodegenerative disease, trigeminal nerve disorder, facial nerve disorder, olfactory nerve disorder, glossopharyngeal nerve Disorders, vagus nerve disorders, hypoglossal nerve disorders, other cranial nerve disorders, cerebral palsy, hemiplegia, paraplegia and quadriplegia, other paralytic syndromes, neuropathy, nerve root or plexus disorders, autonomic nerves The method according to claim 1, wherein the disorder is a system disorder, other peripheral nervous system disorder, or other nervous system disorder.
The method according to claim 1, wherein the disease is glaucoma, optic nerve or visual (pathological) tract disorder, visual dysfunction or blindness (blindness), other eye diseases or appendage diseases.
2. The method according to claim 1, wherein the disease is an outer ear disease, a middle ear disease, a mastoid disease, a hearing loss, or an inner ear disease, another ear disease or a mastoid disease.
The disease is essential (primary (primary)) hypertension (disease), hypertensive heart disease, hypertensive kidney disease, hypertensive cardiorenal disease, secondary (secondary) hypertension (disease), angina, Myocardial infarction, other acute ischemic heart disease, chronic ischemic heart disease, cardiomyopathy, arrhythmia, pericarditis, endocarditis, pulmonary embolism, subarachnoid hemorrhage, cerebral hemorrhage, cerebral infarction, stroke, arterial embolism Or thrombosis, arteritis, aortic aneurysm and dissection, venous embolism or thrombosis, phlebitis, varicose veins, or other cardiovascular diseases.
Diseases include tracheitis, pneumonia, influenza, emphysema, chronic obstructive pulmonary disease, asthma, bronchiectasis, pulmonary fibrosis, adult respiratory distress <promotion> constriction syndrome (ARDS), pulmonary edema, allergic rhinitis (nasal allergy) The method according to claim 1, which is pharyngitis, sinusitis, tonsillitis, laryngitis, and other respiratory diseases.
Diseases are esophagitis, esophageal ulcer, gastroesophageal reflux disease, gastric ulcer, duodenal ulcer, Crohn's disease (localized enteritis), ulcerative colitis, paralytic ileus and bowel obstruction, irritable bowel syndrome, constipation, functional Diarrhea, polyp of colon (colon), alcoholic liver disease, toxic liver disease, chronic hepatitis, liver fibrosis or cirrhosis, cholelithiasis, cholecystitis, cholangitis, acute pancreatitis, chronic pancreatitis, other digestive organs The method of claim 1, wherein the method is a systemic disease.
Diseases include skin or subcutaneous tissue infections, blistering, atopic dermatitis, dermatitis, eczema, papule (debris) (rinse (scale)) disorder, hives, erythema, decubitus 2. The method of claim 1, which is an acne ulcer, other skin disease or subcutaneous tissue disease.
The disease is an infectious joint disorder, inflammatory multiple joint disorder, arthropathy, rheumatoid arthritis, knee injury, soft tissue disorder, bone disorder, cartilage disorder, other musculoskeletal disorder or connective tissue disorder, The method of claim 1.
The disease is acute nephritis syndrome, rapid progressive nephritis syndrome, chronic nephritis syndrome, nephrotic syndrome, tubulointerstitial nephritis, hydronephrosis, acute renal failure, chronic renal failure, kidney stone or ureteral stone, tension (abdominal) Pressure) urinary incontinence, prostate enlargement (disease), testicular (testicular) torsion, testicular (testicular) inflammation, epididymal (cold testicular) inflammation, male infertility (disease), fallopianitis, ovitis, endometriosis, The method according to claim 1, which is amenorrhea, undermenstrual period, excessive menstruation, menopause or other perimenopausal disorders, female infertility, or other diseases of the genitourinary system.
The method according to claim 1, wherein the disease is pregnancy-induced hypertension (symptoms), other maternal disorders related to pregnancy, or a pathological condition that occurred in the perinatal period.
The disease is cough, nausea, vomiting, heartburn, dizziness (feeling dizzy), or pain, numbness, itching, skin sensory disturbance, olfactory disturbance, taste disorder, sensory disturbance, unknown fever, other symptoms, signs, abnormal clinical findings Or the method according to claim 1, wherein there is an abnormality test finding.
The method according to any one of claims 1 to 24, wherein the virus vector is an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, or an adenovirus vector.
A peripheral nerve administration agent comprising a viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions of a peripheral nerve.
Use of a viral vector in which a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve cell functions is used for the manufacture of a peripheral nerve administration agent.
A viral vector incorporating a gene encoding a protein having a function of killing, stimulating, suppressing, or changing other nerve functions, used for peripheral nerve administration.