JP4494863B2 - VHL peptide - Google Patents

Description

  The present invention relates to an oligopeptide (hereinafter referred to as “VHL peptide”) having a partial amino acid sequence of von Hippel-Lindau (VHL) protein capable of inducing differentiation of stem cells into neurons. Stem cells into which the VHL peptide of the present invention has been introduced induce differentiation into neurons. Differentiated nerve cells are transplanted to the central nervous system or peripheral nerves, and are engrafted and function as nerve cells, or directly administered to the human body to induce differentiation of endogenous stem cells into nerve cells. Thus, it is possible to treat Parkinson's disease, cerebral infarction, Alzheimer's disease, spinal cord injury, cerebral contusion, amyotrophic lateral sclerosis, Huntington's disease or malignant brain tumor.

  Nerve cells (neurons) are the main element that controls the life activity of higher organisms. Traditionally, neurons in the central nervous system do not divide at all after birth, and they never drop and only regenerate. It has been said. However, in the 1990s, neural stem cells before differentiation into neurons were first discovered in the fetal brain, and further, the existence of neural stem cells in the adult cerebrum was proved. As shown, treatments for intractable neurological diseases using tissue stem cells such as neural stem cells and embryonic stem cells (ES cells) are in the spotlight. However, tissue stem cells such as neural stem cells and embryonic stem cells are hardly differentiated into nerve cells (neurons) even if transplanted as they are, and even engraftment is difficult, and even when engrafted, most of them become glial cells (glia) There is a problem of differentiation. In addition, neural stem cells that are most easily induced to differentiate into nerve cells (neurons) are difficult to collect from the brain of the same adult individual, and ethical problems and rejection must be overcome when using fetal brain. In the case of bone marrow stromal cells, skin stem cells, and adipose stem cells that have been reported to differentiate into other nerve cells, it is difficult to induce differentiation into nerve cells, and a technology to induce nerve cells at a high rate in a short time has been established It has not been.

  In view of this problem, the present inventors believe that the VHL gene and VHL protein are specifically expressed in neurons of the central nervous system, and thus have some role from the stage of neuronal development. When the expression of VHL protein in neural stem cells was examined, VHL protein was mainly expressed in the cytoplasm as it differentiated into nerve cells, and when the VHL gene was introduced into neural stem cells using a viral vector, it was introduced into neurons. In contrast, when an antisense oligonucleotide that is the reverse sequence of the messenger ribonucleic acid (RNA) of the VHL gene is used to inhibit the action of the VHL gene, the differentiation into a neural cell is suppressed while remaining as a neural stem cell. From these results, it was revealed that the VHL gene has the ability to induce neural differentiation (Non-patent Document 1). The ability of the VHL gene to induce neuronal differentiation has been confirmed to be a common phenomenon in neuroblastoma (Non-patent Document 2), ES cells, skin stem cells, and bone marrow stromal cells. Nurr 1 and Mash 1 are known as genes showing the ability to induce neuronal differentiation similar to this VHL gene, but until now, only the method of introducing the gene into cells using any vector has been reported. No attempt has been made to induce differentiation into nerve cells by introducing a protein having a strong ability to induce nerve differentiation into stem cells instead of genes. In addition, a method for activating endogenous neural stem cells and promoting nerve regeneration by administering such a protein showing the ability to induce neural differentiation into the body has not been developed. In addition, there has been no report on the preparation of a chemically synthesizable oligopeptide exhibiting the ability to induce neural differentiation, and there has been no report on its use for nerve regeneration.

  In the United States, clinical trials using the aborted fetal brain for the treatment of Parkinson's disease have been conducted and certain effects have been observed. At the level of animal experiments, transplantation of neural stem cells and ES cells into the brain and spinal cord, where they differentiate into neurons, thereby treating not only Parkinson's disease but also intractable neurological diseases such as cerebral infarction and spinal cord injury Is starting to take place. Attempts have been made to regenerate peripheral nerves in the form of nerve fiber bundles in vitro or in vivo. However, since nerve cells do not divide and proliferate in principle, it is difficult to make them into practical nerve bundles. In nerve transplantation for peripheral nerve rupture, treatment is usually performed to remove the autonomic nerve of the lower limb and transplant it, but it succeeded in producing artificial nerves to replace the autonomic nerve for nerve transplantation. Not.

Kanno H et al .: Cancer Res 60: 2820-4, 2000 Murata H, et al.: Cancer Res 62: 7004-7011, 2002

  The problem to be solved is that the introduction of the VHL gene, which shows the ability to induce neural differentiation to promote the differentiation of neural stem cells into neural cells, involves many dangers.

  The present invention relates to endogenous stem cells by introducing a VHL peptide capable of inducing differentiation of neural stem cells into neural cells into neural stem cells and inducing differentiation of neural stem cells into neural cells, or by directly administering VHL peptide into the human body. Regenerative medicine treats intractable neurological diseases such as Parkinson's disease, cerebral infarction, Alzheimer's disease, spinal cord injury, brain contusion, amyotrophic lateral sclerosis, Huntington's disease, malignant brain tumor It is characterized by doing.

  By introducing the VHL peptide of the present invention without gene transfer, neural differentiation of tissue stem cells becomes possible. Further, when the VHL peptide is fused with TAT (amino acid sequence of SEQ ID NO: 2: YGRKKRRQRRRD) or ANT (amino acid sequence of SEQ ID NO: 3: KKWKMRRNQFWVKVQRGK) that facilitates cell membrane penetration, almost no cytotoxicity is observed. Furthermore, there is no risk associated with infection as seen in viral vectors with peptide introduction. Therefore, the synthesized peptide has an advantage that it can be used in the same manner as a normal drug.

The invention disclosed herein encompasses the following inventions.
(1) A VHL peptide containing a partial amino acid sequence of von Hippel-Lindau protein that induces differentiation of stem cells into neurons.
(2) The VHL peptide according to (1), comprising 15 amino acids from the 157th to the 171st amino acid sequence of the von Hippel-Lindau protein.

(3) The VHL peptide according to (2) in the amino acid sequence of SEQ ID NO: 1 (TLKERCLQVVRSLVK).
(4) The VHL peptide according to (3), wherein TAT of the amino acid sequence of SEQ ID NO: 2 or ANT of the amino acid sequence of SEQ ID NO: 3 is fused to the N-terminus or C-terminus thereof.
(5) The VHL peptide according to any one of (1) to (4), which is introduced into stem cells.
(6) The VHL peptide according to (1), wherein the stem cells are selected from the group consisting of neural stem cells, skin stem cells, bone marrow stromal cells, adipose stem cells and embryonic stem cells.

(7) A nerve cell induced to differentiate from a stem cell into which the VHL peptide according to any one of (1) to (4) has the following properties.
(A) As a nerve cell-specific protein, a neurofilament and a minute related protein 2 are expressed.
(B) An electrical signal can be transmitted as a nerve to form a neural circuit.
(C) After being cultured and proliferated in a test tube, it is engrafted when transplanted into the central nervous system or peripheral nerve, functions as a nerve cell, and can be used for the treatment of a disease associated with impaired nerve function.

(8) The nerve cell according to (7), wherein the stem cell is selected from the group consisting of a neural stem cell, a skin stem cell, a bone marrow stromal cell, an adipose stem cell, and an embryonic stem cell.
(9) The nerve cell according to (7), wherein the disease is selected from the group consisting of Parkinson's disease, cerebral infarction, Alzheimer's disease, spinal cord injury, brain contusion, amyotrophic lateral sclerosis, Huntington's disease, and malignant brain tumor.

Hereinafter, the present invention will be described in detail.
The VHL gene, which is a causative gene of von Hippel-Lindau disease, which is a genetic disease that causes brain tumor (angioblastoma) and renal cancer, is a kind of tumor suppressor gene. This gene was isolated from human chromosome 3 in 1993 by a group of Dr. Zbar et al. It has been reported that this gene and protein are expressed in nerve cells (neurons), but the function of this gene with respect to the nervous system is unknown. Therefore, the present inventors thought that this gene might be involved when the nervous system is formed at the developmental stage of the fetus, and examined the differentiation of neural stem cells isolated from the rat fetal brain over time, It has been found that as neural stem cells differentiate into neurons, VHL protein is expressed in neurons. This suggests that the VHL protein is involved in differentiation into nerve cells, and the present inventors further introduced the VHL gene into neural stem cells using a herpes simplex vector to express the VHL protein. Whether or not to differentiate into nerve cells was examined. As a result, when the VHL gene was introduced and the VHL protein was expressed, neural stem cells differentiated into neurons (neurons). That is, it became clear that the VHL protein has a function of inducing neural differentiation. On the contrary, when the VHL gene function of the neural stem cell is suppressed by the reverse sequence (antisense) of the messenger RNA of the VHL gene, the neural stem cell is not differentiated into a neural cell, and differentiation is suppressed as it is. (Non-Patent Document 1). The function of this VHL protein is shown not only for neural stem cells but also for neuroblastoma cells, which are a type of cancer cell. By introducing the VHL gene into neuroblastoma cells, the function of VHL protein is increased. It has been found that blastoma cells differentiate into functional neurons (Non-Patent Document 2). Furthermore, it was found that skin stem cells are also induced to differentiate into neurons (neurons) (FIG. 1).

In the present invention, rather than the expression of VHL protein in cells transfected with VHL gene, it induces neuronal differentiation by introducing a VHL protein itself into tissue stem cells such as neural stem cells. The VHL protein is a comparatively small protein consisting of 213 amino acids in its full length, but a smaller one is more advantageous for introduction into cells, and synthesis is also easier. Of the full length of the VHL protein, as a part having a function of inducing neuronal differentiation, one of the binding proteins, elongin, from the three-dimensional structure of the VHL protein, which binds to the ligand (binding protein) of the VHL protein (Elongin) The 15 amino acid sequence (VHL157-171) of the site | part couple | bonded with B and C was identified as a site | part which shows the most important function in connection with the function of neural differentiation.

  VHL oligopeptides at amino acids 157 to 171 which are binding sites for elongin, which is an important binding protein of VHL protein, were prepared by a solid phase synthesis method based on the Fmoc method. Oligopeptides were subjected to solid-phase synthesis based on the Fmoc method. As the solid support, Fmoc-SAL-PEG resin (Watanabe Chemical) was used. By using this resin, an oligopeptide in which the carboxyl (C) terminal is aminated can be obtained. The resin was washed with DMF (N, N-dimethylformamide), and then the Fmoc group, which is an amino-protecting group, was removed with 20% piperidine / DMF. After washing with DMF, 3 equivalents Fmoc-AA (amino acid) -OH / 3 equivalents HATU (O- (7-azabenzotriazol-1-yl) -1,1,3 with respect to the amino groups on the resin , 3-tetramethyluronium-hexafluorophosphate) / 6 equivalent DIEA (diisopropylethylamine) was added to conduct an amino acid condensation reaction, and the progress of the condensation reaction was confirmed by a ninhydrin reaction. After coupling of all amino acids, the Fmoc group was removed with 20% piperidine / DMF, and amino (N) terminal acetylation was performed with acetic anhydride. The synthesized resin is dried in vacuum, m-cresol / ethanedithiol / thioanisole / TFA (trifluoroacetic acid) is added, and the mixture is stirred at room temperature for 1 hour to cleave the oligopeptide from the resin and amino acid side chain ( p) was deprotected (FIG. 2). The filtrate was ether precipitated to obtain a crude peptide. The crude peptide was purified by reverse phase HPLC to obtain the desired oligopeptide. The molecular weight of the synthesized peptide was confirmed by MADLI-TOF mass spectrometry, and the peptide concentration was calculated from the extinction coefficient in the UV region of aromatic amino acid residues.

  The oligopeptide prepared by the above method is applied to a neural stem cell derived from an adult rat hippocampus at a concentration of 10 to 1000 ng / mL, and BioPORTER Reagent (BioPORTER protein delivery reagent: Gene Therapy Systems, Inc., San Diego, CA, USA) is used. And introduced into a test tube. As a result, the neural stem cells tended to differentiate into neurons (neurons) after 4 hours at a concentration of 10 ng / mL or more, and neurite outgrowth was observed. At 8 hours after introduction, the morphological change became more prominent than in the control group (FIG. 3). When fluorescent immunostaining was performed 24 hours after introduction at a concentration of 100 ng / mL, it was confirmed that 72% of them were differentiated into neurons (neurons) and 12% were differentiated into glial cells (glia) (Fig. 4). As a result, it became clear that VHL can induce neuronal differentiation not only by gene transfer but also by peptide transfer.

Next, it was examined whether or not the symptoms improved by transplanting neural stem cells into which VHL (157-171) oligopeptide was introduced into the central nervous system of animals with neurological diseases. A rat model of Parkinson's disease as a neurological animal was prepared by microinjecting 6-hydroxydopamine into the brain of the rat to deplete dopaminergic cells on one side. After 2 weeks, it was examined by the number of rotations induced by apomorphine. Rats rotating at least 7 times per minute were used as Parkinson's disease models. Neural stem cells into which 10 ⁵ VHL (157-171) oligopeptides were introduced were transplanted into the rat cerebral linear body. Apomorphine-induced rotational speed was measured every week after transplantation. As a result, about 30% of the rats had an apomorphine-induced rotational speed of 0 after 2 months (FIG. 5). For behavioral analysis, in addition to this apomorphine-induced rotation, a paw reaching test (a test to examine the ability to stretch out the leg on one side of the obstacle and take out the pellet placed on the stairs and eat it) was conducted. -171) An oligopeptide was introduced (FIG. 6). In addition, Parkinson's disease model rats transplanted with neural stem neural stem cells into which VHL (157-171) oligopeptide was introduced were compared with the control group in Parkinson's disease model rats transplanted with cells that showed significant improvement in these behavioral analyses. When the brain was examined histopathologically, about 70% of the transplanted cells were engrafted in the brain, and most of them were neurofilament positive neurons (FIG. 7).

  Furthermore, a fusion protein TAT or ANT having an effect of easily penetrating the cell membrane is synthesized by binding to a VHL oligopeptide to form TAT-VHL (157-171) or VHL (157-171) -ANT, and BioPORTER The peptide synthesized without using Reagent was introduced at a concentration of 10 ng / mL into neural stem cells and differentiated into neural cells. Compared to the case of using BioPORTER Reagent, this method of binding a transmembrane fusion protein did not show the cytotoxicity of BioPORTER Reagent and was considered safe to administer in vivo. Actually, when a therapeutic experiment was conducted by administering an oligopeptide conjugated with a TAT fusion protein into a living body, TAT-VHL (157-171) or VHL (157-171) -ANT can be used as a drug as it is. The possibility was suggested. When this TAT-VHL (157-171) or VHL (157-171) -ANT is actually added to the culture medium of neural stem cells in a test tube, it exhibits physiological activity of the ability to induce neuronal differentiation at a concentration of 10 ng / mL or more, It was proved to be a functional peptide. As a control group, TAT-VHL (104-121) showing tumor suppressive activity and TAT alone were compared, but TAT-VHL (157-171) showed the highest neuronal differentiation inducing activity. From this, it became clear that VHL (157-171) has nerve differentiation-inducing activity, and TAT-VHL (157-171) was found to be a functional peptide having a function of inducing nerve differentiation.

  Next, by examining the types of cells, it was examined whether tissue stem cells exhibit similar physiological activity against skin stem cells and bone marrow stromal cells. Skin stem cells were cultured after chopping the dermal layer of rat newborn and human skin, passing through a filter. Details of this culture method are described in Nature Cell Biology 2001 Sep; 3 (9): 778-84. The medium was DMEM / F12 (Gibco) supplemented with N2 supplement (Gibco) and basic fibroblast growth factor at 10 ng / mL and epidermal growth factor at 20 ng / mL. When the TAT-VHL (157-171) peptide was added at a concentration of 100 ng / mL to the cells after 10 days of the primary culture, differentiation into neurons (neurons) was observed after 8 hours, and clearly after 24 hours. Differentiation into cells (neurons) was observed. For bone marrow stromal cells, first, bone marrow cells were isolated from femur bone marrow of 6-week-old rats, cultured in 10% FCS in RPMI medium, and then passed through 2 or 3 passages. After only treating adherent cells with β-mercaptoethanol added to the medium overnight, and further treating with β-mercaptoethanol and DMSO for 3 hours, TAT-VHL (157-171) peptide was added at a concentration of 100 ng / mL. When the culture was continued in the culture dish coated with lysine and coated with lysine, differentiation into neurons was observed after 8 hours, and differentiation into neurons (neurons) was clearly observed after 24 hours.

  The present invention enables neural differentiation of tissue stem cells by introducing a peptide (protein) without introducing a gene. Gene transfer involves various risks with current technology. When using a viral vector, virus infection and cytotoxicity due to the virus must be considered. Moreover, in the case of a plasmid vector, it is necessary to consider that the gene transfer efficiency is low and that there is cytotoxicity depending on the transfer method. In contrast, when a peptide (protein) was introduced and a fusion protein that facilitated cell membrane penetration was bound using TAT or ANT, little cytotoxicity was observed. In addition, there is no risk associated with infection as seen in a viral vector when a peptide (protein) is introduced. Therefore, the synthesized peptide (protein) can be used in the same manner as a normal drug. Accordingly, when the peptide of the present invention is considered as a drug, administration allows specific differentiation into nerve cells (neurons) not only in vitro but also in vivo. By utilizing this, the tissue stem cells of the self are taken out and differentiated into neural stem cells in vitro first, and then the nerves are regenerated by returning them (self-transplantation), Parkinson's disease, cerebral infarction, Alzheimer's disease, spinal cord Intractable neurological diseases such as injury can be treated. The present invention provides a VHL peptide as a drug necessary for this purpose. Furthermore, by administering a VHL peptide having nerve differentiation-inducing activity as a drug in vivo, it is possible to induce differentiation into nerve cells (neurons) in vivo without taking out its own endogenous neural stem cells. . This makes it possible to regenerate nerves and treat intractable neurological diseases such as Parkinson's disease, cerebral infarction, Alzheimer's disease, and spinal cord injury.

It is a fluorescence micrograph showing that skin stem cells are induced to differentiate into neurofilament positive neurons. It is explanatory drawing of the peptide synthesis | combination of this invention by a peptide solid-phase synthesis method. When a VHL peptide is introduced into a neural stem cell using a BioPORTER reagent, the morphological change is noticeable compared to the control group 8 hours after introduction and is a micrograph showing a tendency to differentiate into a neuron. When the VHL peptide was introduced into neural stem cells using BioPORTER reagent, a fluorescence microscope of cells immunofluorescently stained showing that 72% were differentiated into neurons and 12% into glia 24 hours after introduction. It is a photograph. Every week after transplantation of neural stem cells into which a VHL peptide has been introduced into a Parkinson's disease model rat as a neurological disease animal depleted of dopaminergic cells on one side prepared by injecting a small amount of 6-hydroxydopamine into the rat brain It is a figure which shows the result of having measured the apomorphine induction rotation speed. The results of the control group-only transplantation group and the neuronal liver cell-only transplantation group are also shown. It is a figure which shows the result of the Paw reaching test with respect to the said Parkinson's disease model rat. The horizontal axis shows the pre- and post-transplant (week) VHL peptide transplantation, and the vertical axis shows the percentage of pellets taken and eaten on the impaired leg. Parkinson's disease model rats transplanted with neural stem cells showed a significant improvement in this behavioral analysis compared to the control group. It is a microscope picture of the structure | tissue of the brain of the said Parkinson's disease model rat which transplanted VHL peptide. It can be seen that about 70% of the transplanted cells are engrafted in the brain and most of them are neurofilaments. In the figure, Brd-U represents bromodeoxyuridine, GFAP represents a glial cell marker, and O4 represents an oligodendrocyte marker.

Claims (5)

A peptide agent for inducing differentiation of stem cells into neural cells,
A peptide drug comprising a synthetic peptide consisting of the amino acid sequence of SEQ ID NO: 1, or a synthetic peptide in which the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 3 is fused to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1. A method for inducing differentiation of stem cells to produce neurofilament positive neurons,
Using neural stem cells, skin stem cells, bone marrow stromal cells or adipose stem cells as the stem cells,
A synthetic peptide consisting of the amino acid sequence of SEQ ID NO: 1 or a synthetic peptide in which the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 3 is fused to the N-terminal or C-terminal of the amino acid sequence of SEQ ID NO: 1; A method characterized by introducing. The method according to claim 2, wherein primary stem cells of skin stem cells are used as the stem cells. 3. The method according to claim 2, wherein subcultured cells of bone marrow stromal cells are used as the stem cells. The method according to any one of claims 2 to 4, wherein any one of the synthetic peptides is added to a medium for culturing the stem cells so as to have a concentration of 10 ng / mL or more.

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