WO2007018318A1

WO2007018318A1 - Method for gene expression specific to cerebellar astrocyte and/or basket cell

Info

Publication number: WO2007018318A1
Application number: PCT/JP2006/316127
Authority: WO
Inventors: Hirokazu Hirai; Takashi Torashima
Original assignee: National University Corporation Kanazawa University
Priority date: 2005-08-10
Filing date: 2006-08-10
Publication date: 2007-02-15

Abstract

Disclosed is a method for gene expression specific to a cerebellar astrocyte and/or a basket cell. A method for expressing a gene specifically in a cerebellar astrocyte and/or a basket cell of a target animal, comprising the step of introducing a lentivirus-derived vector carrying the gene into the target animal, wherein the gene is linked to the vector in such a manner that the gene is operable under the control of synapsin I promoter; a study on the function of the cerebellum using the method; and a vector, transgenic animal or the like for use in the method or study.

Description

Cerebellar astrocytes and / X are sputum cell-specific gene expression methods

The present invention relates to a cerebellar stellate cell and / or sputum cell-specific gene expression method, a cerebellar function study using the same, and a vector, a transgenic animal, and the like. 'Ming

Background technology

The cerebellum plays an important role in cooperative movements such as walking involving multiple muscles.

The 'If the cerebellum is damaged, coordinated movements do not work well, and smooth movements cannot be achieved. Movement commands from the cerebral cortex are transmitted to the spinal cord and muscles through the brain stem, but also enter the cerebellar cortex through mossy fibers. Mossy fibers enter excitability between granule cells, which are neurons in the cerebellar cortex. Granule cells form synapses with Purkinje cells via parallel fibers that are axons. Purkinje cells are the only output neurons from the cerebellar cortex, and what input the Purkinje cells receive and how they are integrated and output can be said to be the essence of cerebellar function.

In the cerebellar cortex, there are three types of neurons other than granule cells and Purkinje cells (see Fig. 1). These three types of cells: astrocytes, sputum cells, and Golgi cells are all interneurons, and have the role of regulating signals transmitted to mossy fibers, granule cells, and Purkinje cells. Astrocytes exist in a part of the cerebellar cortex, called a molecular layer, that synapses with the Purkinje cell dendrites and regulates Purkinje cell activity in an inhibitory manner. Vaginal cells are also present in the molecular layer, but unlike astrocytes, they exist near the Purkinje cell layer and form inhibitory synapses with Purkinje cell bodies.

Thus, it is known that cerebellar astrocytes and sputum cells control Purkinje cells in a suppressive manner, but how much these two cells have an effect on Purkinje cells. Contributing to controlled coordination or learning It is completely unknown whether it is.

Until now, cerebellar stellate cells and sputum cells have been analyzed for their functions by electrically stimulating these cells and recording their potential changes with Purkinje cells or stimulating parallel fibers. Only the method of recording potential changes in cells or sputum cells is known. In addition, since a promoter that specifically functions in cerebellar stellate cells and sputum cells has not been found, transgenic animals with these cell-specific genetic modifications are not known. '

Synapsin I is a protein that exists at the end of neurons and plays an important role in the release of synaptic vesicles. In Expression analysis using adenoviral vector, synapsin I promoter is reported to function specifically in nerve Rereru (Kugler et al, Molecular and Cellular Neuroscience, 17, p78 -. 96, (2001)) 0 Analyzes using lentiviral vectors have also been fortunately reported that the synapsin I promoter functions in both excitatory and inhibitory neurons in the cerebral cortex (Dittgen et al., PNAS, Vol. 101, No. 52, pl8207-211, (2004)). However, there is no report on how the synapsin I promoter functions in the cerebellum. In fact, all known cerebellum-specific expression vectors are those using adenovirus, adeno-associated virus, or lentivirus, but the promoters used are general CMV promoters, RSV promoters, etc. As a cell-specific one, there is only a Purkinje cell-specific L7 promoter (Japanese Patent Publication No. 2 0 0 3-5 3 4 7 8 7, Japanese Patent Laid-Open No. 9-2 8 3 7 No. 8 Publication, Special Table 2 0 0 3-5 1 1 0 8 0 Publication). Disclosure of Invention-The object of the present invention is to enable gene expression specific to cerebellar astrocytes and / or sputum cells, and to provide a new means for cerebellar function research and gene therapy. . As mentioned above, synapsin I is a protein that exists at the end of neurons and plays an important role in the release of synaptic vesicles. In general, synapsin I is used as a marker of inspiring presynaptic terminals. Excitatory nerves in the cerebellar cortex are condyles Since it is only granule cells, the inventors considered using the synapsin I promoter as a granule cell-specific expression vector. Then, the synapsin I promoter gene isolated from the rat genome was incorporated into a lentivirus-derived vector, and the GFP gene was ligated downstream of it to infect cells in the cerebellar cortex molecular layer of a living mouse. Localization was observed. Contrary to expectations, no GFP expression was observed in the condylar cells, but instead, selective expression of GFP was confirmed in the astrocytes and sputum cells of the molecular layer. Using a lentiviral vector that expresses GFP under the control of a cell-nonselective CMV promoter or MSCV promoter ¾ and condylar granule cells do not show GFP expression (the other four types of neurons have GFP Therefore, it was considered that the lentiviral vector does not have the ability to infect granule cells. These results suggest that the lentiviral vector using the synapsin I promoter can be used as an expression vector specific for astrocytes and sputum cells.

The present invention has been made based on the above findings, and can function a transgene under the control of the synapsin I promoter. Cerebellar astrocytes of the target animal

The present invention relates to a method for specifically expressing the transgene in Z or sputum cells. In the above-described method, the transgene is preferably a gene encoding a protein that controls cell survival, a cerebellar development, or a protein that controls physiological functions (for example, a therapeutic protein). For example, selected from diphtheria toxin, diphtheria toxin receptor, simple herpes-derived thymidine kinase, glutamate receptor, GABA receptor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and variants thereof The gene which codes either can be mentioned.

The present invention also includes diphtheria toxin, simple herpes-derived thymidine kinase, glutamate receptor, GABA receptor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor and variants thereof under the control of synapsin I promoter. Lentivirus-derived vectors that contain a gene that encodes a selected gene or a gene that encodes various therapeutic proteins in a manner that can be linked together, or a transgenic animal into which the vector has been introduced To do.

The present invention can also function a transgene under the control of a synapsin I promoter. A lentivirus-derived vector that is ligated and contained in a manner, is inserted into the target animal, and the transgene is specifically expressed in the cerebellar stellate cells and / or sputum cells of the target animal. Also provided are methods for analyzing physiological or pathological functions. .

In particular, in transgenic work products in which a gene encoding one selected from diphtheria toxin, diphtheria toxin receptor, thymidine kinase, Lurcher mutation δ 2 glutamate receptor and its variants is introduced, It is possible to selectively drop cerebellar astrocytes and / or sputum cells, which is useful for elucidating the physiological functions of these two cells and the cerebellum. In addition, a lentiviral-derived vector containing a gene encoding a therapeutic protein such as brain-derived neurotrophic factor and glial cell-derived neurotrophic factor under the control of the synapsin I promoter is linked to the cerebellum. It is useful for gene therapy as a therapeutic protein delivery vector to astrocytes and cells or sputum cells.

The target animals used in the present invention are non-human mammals, particularly rodents such as mice and rats, in the research field. On the other hand, in the field of gene therapy, it is possible to target all mammals including humans. According to the present invention, astrocytes and sputum cells can be selectively dropped, and the role of these two types of neurons can be elucidated at the action level such as coordinated movement and motor learning. It becomes. In addition, gene expression is limited to astrocytes and Z or sputum cells, and functions of these inhibitory nerves can be regulated, providing a useful means for conducting cerebellar neural circuit research. It will be. Brief Description of Drawings

Figure 1 is a schematic diagram of the cerebellar neural circuit. .

FIG. 2 is a schematic diagram of pCL20cMS′CV-GFP.

FIG. 3A is a schematic diagram of pCL20cSynI, which is an example of the expression vector of the present invention. FIG. 3B is a schematic diagram of pCL20cSynI-GFP, which is an example of the expression vector of the present invention.

Figure 3C is a schematic diagram of pCL20cSynI-DTR-HA, which is an example of the expression vector of the present invention. It is. ·

FIG. 4 is a fluorescence micrograph showing an example of expression of a GFP gene specific to cerebellar astrocytes and sputum cells by pCL20cSynI-GFP.

Fig. 5 is a confocal laser micrograph showing an example of expression of GFP gene specific to cerebellar astrocytes and sputum cells by pCL20cSynI-GFP.

FIG. 6A is a fluorescence micrograph showing an example of non-selective expression of GFP gene in cerebellar cells by pCL20cMSCV-GFP.

FIG. 6B is a confocal laser micrograph showing an example of non-selective expression of GFP gene in cerebellar cells by pCL20cMSCV-GFP. , Fig. 7 is a confocal laser micrograph showing an example of expression of GFP gene specific to cerebellar stellate vesicles and sputum cells by pCL20cSynI-DTR-HA. (Upper: Diphtheria toxin not administered) Lower cerebellar cortex of mice, lower cerebellar cortex of mice administered diphtheria toxin twice a day for 1 week). This specification includes the contents described in the specification of Japanese Patent Application No. 2 0 0 5-2 3 1 5 1 4 which is the basis of the priority of the present application. · BEST MODE FOR CARRYING OUT THE INVENTION The vector according to the present invention is a lentivirus-derived vector containing a transgene linked in such a manner that it can function under the control of a synapsin I promoter.

(1) Lentiviral vector

The vector of the present invention is based on a lentiviral vector derived from human immunodeficiency virus. Unlike adenovirus vectors and herpes virus vectors, lentivirus vectors are advantageous in that they have a very weak immune response in the living body and little cytotoxicity. Examples of the vector derived from the lentivirus include pCL20c (provided by St. Jude Children's Research Hospital), pLenti6 / V5-DEST (manufactured by Invitrogen), and the like.

In the present specification, the “basic skeleton” means a structural unit containing factors sufficient to be maintained and replicated in a specific cell. Therefore, in the present invention, A product obtained by removing unnecessary portions from a general-purpose lentivirus-derived vector can also be used. From the viewpoint of improving the titer of the viral vector, for example, in the case of pLenti6 / V5-DEST, which is a lentivirus-derived vector, it is preferable that the portion having the base sequence represented by SEQ ID NO: 7 has been removed. Therefore, in the lentiviral vector skeleton containing a synapsin I promoter and a lentiviral vector backbone in which the transgene to be introduced is linked downstream of the promoter in such a manner that it can function, the nucleotide sequence shown in SEQ ID NO: 7 Expression vectors that do not contain are also included in the vector of the present invention. 'The size of an insert that can be incorporated into a lentiviral vector is generally 8 kb, including plug motors. From the experiments conducted by the inventors so far, it is known that ₄ to _{5 kb} is the limit in the high-titer lentil virus vector from which the portion having the base sequence of SEQ ID NO: 6 is removed. Therefore, in order to express a gene of 3 kb or more, it is necessary to use a neuron-specific promoter that does not exceed 1-2 kb.

(2) Synapsin I promoter.

The vector of the present invention contains a synapsin I promoter as a promoter. The synapsin I promoter sequence is available through public databases such as GenBank.For example, the rat synapsin I promoter sequence is Accession No.

As M55300, .T05521, J05630, the human synapsin I promoter sequence is registered as Accession No. M58321, J05431, M57636. Non-registered synapsin I promoter sequences of other animals can be easily obtained according to a well-known method by homology search based on the above synapsin I promoter sequences.

The synapsin I promoter of the present invention includes a modified form thereof (Pariant). The variant may be artificially produced, for example, by a site-directed mutagenesis method according to a conventional method, or may be a naturally occurring variant. As an example, SEQ ID NO: 1 shows the rat synapsin I promoter sequence used in the examples of the present efforts (sequence of positions −225 to 105 of the rat synapsin I promoter:

Sauerwald A, Hoesche C, Osch ald R, Kilimann M., J Biol Chem. 1990 Sep 5; 265 (25): 14932-7. The 5 '-flanking region of the synapsin I gene. A G + C-rich, TATA- and CMT-less, phylogenetically conserved sequence with cell type-specific promoter function: In the above literature, it is reported that neuron-specific expression occurs in the region of -225 to 105). In addition, SEQ ID NOs: 2 and 3 show the human and mouse synapsin I promoter sequences corresponding to the aforementioned positions of rat -225 to 105, respectively. ,

In addition to the 330 bp synapsin I promoter described above, we used the 1046 bp rat synapsin used by Dittgen. Et al. (Dittgen et al., PNAS, Vol. 101, No. 52, pl8207-211, (2004)). The I promoter was also examined. As a result, it was clarified that a foreign gene is expressed specifically in cerebellar stellate cells and / or sputum cells even when a 1046 bp rat synapsin promoter is used. Furthermore, in the cerebellum, the 1046 bp rat synapsin promoter was found to have much higher promoter activity than the 330 bp synapsin I probe motor. SEQ ID NO: 4 shows the 1046 bp rat synapsin I promoter sequence.

However, as described above, the present invention is not limited to this sequence. In the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 4, at least one nucleotide residue (preferably one or several) is a different base sequence. However, as long as it can be expressed specifically in cerebellar stellate cells and / or sputum cells as a synapsin I promoter, it can be expressed in synapsin as well.

It can be used as an I promoter. That is, multiple alignment by the ClustalW method by Higgins et al. (For example, Gappenalty5, Fixed Gap penaltylO, windowssize 5, Floating Gap 10) is aligned to an optimal state, and the calculated sequence identity is at least 80 %, Preferably 85% or more, more preferably 90% or more, and a transgene linked in such a manner that it can function downstream as a synapsin I promoter. Any one having such a base sequence can be used as the synapsin I promoter according to the present invention as long as it can be expressed specifically in cells and cells.

In addition, a sequence that can hybridize with the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4 under stringent conditions is also used as a synapsin I promoter, and a transgene linked in a manner that can function downstream thereof is used as a cerebellar star. Dendritic cells and or It can also be used as a synapsin I promoter as long as it can be expressed specifically in sputum cells. In the present specification, the “stringent condition” means 6xSSC (composition of lxSSC: 0.15M NaCl, 0, 015M sodium quenate, ρΗ7.0), 0.5% SDS, 5χ Den Hart and 100 / ml denatured fragmented salmon sperm DNA and 50. /. In a solution containing formamide, together with the nucleic acid consisting of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, keep it at 55 ° C — low ionic strength, for example, 2xSSC, or more stringent, such as 0.lxSSC Conditions and Z or higher, 37 ° C or higher, stringent 42 ° C or higher, more stringent 50 ° C or higher, even more stringent 60 ° C or higher This can be achieved depending on the conditions under which washing is performed.

The length of the base sequence of the variant is not particularly limited as long as the desired promoter function is exhibited. However, from the viewpoint of the allowable size of the foreign gene incorporated into the vector, for example, it is 3000 nucleotides or less, preferably 2000 It is desirable that the length be less than or equal to the nucleotide length.

The function as a synapsin I promoter can be evaluated, for example, by detecting the presence or absence of binding between the RNA polymerase contained in the cerebellar astrocytes or sputum cells and the nucleic acid to be tested. The presence or absence of binding can be detected by mobility in gel shift assembly, optical sensorgram or mass sensor in surface plasmon analysis, or footprint method.

The function of the variant as a promoter is, for example, by linking a reporter gene in a manner that allows it to function downstream of the nucleic acid to be tested, obtaining a construct, and then obtaining the construct from mouse or rat cerebellar glial cells + neurons. It can be assessed by infecting mixed cultures or by administering to the subarachnoid surface of the cerebellum of solid mice. When the reporter gene is expressed specifically in cerebellar stellate cells or sputum cells based on the above evaluation, the nucleic acid can be used as a synapsin I promoter suitable for the expression vector of the present invention.

As the synapsin I promoter, it is desirable to use a synapsin I promoter sequence of the same species as that of the target animal to be used or a related species.

Synapsin I is a protein that exists at the end of neurons and plays an important role in the release of synaptic vesicles. Generally, synapsin I is an excitatory presynaptic Used as a terminal marker. Since the excitatory nerves in the cerebellar cortex are only granule cells, it was considered that the synapsin I promoter could be used as a granule cell-specific expression vector. However, the inventors incorporated the rat synapsin I promoter gene (SEQ ID NO: 1 and SEQ ID NO: 4) into a vector derived from a lentivirus and ligated the GFP gene downstream thereof, so that the molecular layer of the cerebellar cortex of a living mouse When the cells were infected, no GFP expression was observed in the granule cells. Instead, selective expression of GFP was confirmed in the astrocytes and sputum cells of the molecular layer. That is, it was confirmed that the lentiviral vector using the synapsin I promoter is useful as an expression vector that can specifically function in S-shaped cells and sputum cells.

(3) Transgene

The vector of the present invention comprises a transgene linked in such a manner that it can function under the control of the synapsin I promoter. Here, “connected in a functional manner” means a state in which it is arranged to exert its original function, and is arranged so that the transgene is appropriately expressed under the control of the synapsin I promoter. It means the state that was done.

The transgene used in the present invention is not particularly limited. For example, a gene that affects cell survival, specifically, a gene encoding a protein that is toxic to the cell, the cerebellum Genes encoding proteins that control the development and physiological functions of the genes, specifically genes encoding cerebellar synapse formation and plasticity control molecules, genes that can express proteins and siRNA that can be used for therapeutic purposes, specifically A gene that codes for an enzyme that breaks down or removes aggregates produced in the cell, a gene that encodes a protein that can capture the function of the cell in the cell, and an excessive function in the cell. Genes that can express siRNA that can suppress development: other genes that code for proteins related to substance production, and degradation of specific substances Such as genes encoding protein is Chi like. More specifically, the gene to be introduced includes, for example, a gene encoding diphtheria toxin, a gene encoding diphtheria toxin receptor, and a simple herpes-derived thymidine kinase as a toxic protein. Genes, cerebellum Sina Encodes NMDA glutamate receptor, AMPA glutamate receptor gene, GABA receptor gene, and brain-derived neurotrophic factor or Dariya cell-derived neurotrophic factor for treatment And a gene encoding a dominant negative for functional inhibition, and the like. .

The expression vector of the present invention only needs to have the structure as described above, and examples thereof include a vector pCL20cSynI (FIG. 3A) having the base sequence shown in SEQ ID NO: 5. In the pCL20cSynI, the nucleic acid to be introduced can be inserted into the EcoRI and Notl recognition sites in the pCL20cSynI. .

2. Functional analysis of the cerebellum

(1) Specific expression of transgenes in cerebellar astrocytes and / or sputum cells

The present invention provides a method for introducing a lentivirus-derived vector comprising a transgene linked in a manner capable of functioning under the control of a synapsin I promoter into a target animal, whereby the cerebellar astrocytes and Z or sputum cells of the target animal A method for specifically expressing a transgene is provided. '

This method enables the expression and delivery of specific genes specific to cerebellar stellate cells and sputum cells, which was not possible before, researching cerebellar function and treating diseases related to abnormalities of cerebellar stellate cells and sputum cells. And research becomes possible.

In particular, if the vector of the present invention is used to express diphtheria toxin, herpes simplex virus-derived thymidine kinase, Lurcher mutation δ.2 glutamate receptor specifically in cerebellar astrocytes and sputum cells, It is possible to specifically shed these cells: By observing changes due to dropout, it is possible to know the functions of cerebellar astrocytes and sputum cells in the cerebellum. For example, dropping cerebellar stellate cells and sputum cells using this method in young mice leads to the elucidation of the role in cerebellar transduction circuit formation. When used in mature mice, it is clear how these two types of nerves contribute to cerebellar function. In addition, various genes can be specifically expressed in cerebellar stellate cells and sputum cells, and it becomes possible to conduct extensive research on synaptic transmission between cerebellar stellate cells and sputum cells and Purkinje cells. (2) Transgenic animals

By injecting the vector of the present invention into the subarachnoid space, a transgene can be expressed specifically in a wide range of cerebellar astrocytes and / or sputum cells of the subject animal. Such a transgenic animal can be used as, for example, a cerebellar function research model animal or a cerebellar disease model animal. The target of the transgenic animal is not particularly limited, but non-small mammals represented by mice, rats, rabbits, horses, hidges, inu, cats, monkeys, etc. can be used, especially mice. Rodents such as rats are preferred. '3. Gene therapy

Lentiviral-derived vectors containing a gene encoding a therapeutic protein linked in a functional manner under the control of the synapsin I promoter are used as gene delivery vectors for cerebellar astrocytes and Z or sputum cells. Useful.

That is, the vector of the present invention is dissolved and suspended in a suitable buffer together with a pharmacologically acceptable carrier, injected into the subarachnoid space on the surface of the cerebellum, cerebellar astrocytes and Z or sputum cells. It can be used as a therapeutic agent for diseases caused by disorders in Since the vector of the present invention can efficiently deliver and express a gene encoding a therapeutic protein to cerebellar stellate cells and / or sputum cells, diseases caused by disorders in these cells (as opposed to It is possible to achieve a therapeutic effect with efficiency.

So far, there has been no known disease that specifically damages cerebellar astrocytes and cells. Typical examples of secondary astrocyte and sputum cell damage following Purkinje cell degeneration / dropping include spinocerebellar degeneration. Purkinje cell degeneration is prominent in spinocerebellar degeneration type 1, type 2 and type 6. Since astrocytes and sputum cells regulate Purkinje cell activity in a suppressive manner, it may be possible to suppress Purkinje cell degeneration by regulating these cell activities. The pharmacological evaluation of gene therapy varies depending on the type of disease to be treated, but in the case of spinocerebellar degeneration, the vector is administered to the subarachnoid space on the surface of the cerebellum and then cytologically Purkinje cells, stars By preventing degeneration of neurons in the cerebellum, including dendritic cells and sputum cells, clinically (behaviorally in animals) to the extent of improvement in cerebellar ataxia More can be done.

The transgene to be incorporated into the vector can be appropriately selected according to the type of disease to be treated. For example, a nucleic acid encoding a brain-derived or glial cell-derived neurotrophic factor can be mentioned.

The vector can preferably be administered by injection into the subarachnoid space on the surface of the cerebellum. The injected vector contains a small number of cerebellar astrocytes around the injection site and

Without being limited to Z or sputum cells alone, the active ingredient can be delivered over a wide range with high affinity to cerebellar stellate cells and / or whole sputum cells. Moreover, injection of the vector of the present invention into the subarachnoid space on the surface of the cerebellum is superior in clinical application because it can suppress damage to the brain parenchyma compared to injection into the lower olive nucleus and the cerebellar nucleus.

In addition, for the administration of the vector of the present invention, from the viewpoint of stably maintaining the brain pressure during the injection, it is preferable to attach a means capable of injection at a constant rate, for example, a Norton syringe and the like. It is desirable to use a micromanipulator that can be used and a microinjection pump for injection. The injection rate is not particularly limited as long as the brain pressure can be stably maintained, and can be appropriately set according to the age, weight, disease state, etc. of the individual. For example, it is desirable to be lOnl / min to 800 nl / min, preferably 50 nl / min to 400 nl / min, and more preferably lOOnl to 200 nl / min.

The dose of the vector of the present invention is not particularly limited as long as it is an amount suitable for exerting a therapeutic effect, and can be appropriately set according to the age, weight, disease state, etc. of the individual. In addition, the number of administrations of the therapeutic agent of the present invention is sufficient as long as the therapeutic effect is exhibited. This vector, which uses lentivirus as the basic skeleton, can be administered once because the gene is integrated into the chromosome, but in order to increase the copy number of the integrated gene and introduce it into a wider range of cells. In some cases, the surface of the cerebellum may be changed (right, midline, left, etc.) about 3 times. Example

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the examples.

[Example 1] Preparation of cerebellar stellate cell and sputum cell-specific expression vector (1) Virus production

The following operations were performed in the P2 laboratory.

HEK293T cells in logarithmic growth phase were dispersed in PB $ (—), and then seeded at ⁵ × 10 ⁵ cells per l (km dish (manufactured by Falcon)). In a 10 cm dish after seeding, 10 and添Ka卩weight ./. © Shi calf serum containing DMEMlOml, after which the cells, 5 vol% C0 _2, 37 ° were cultured in C. 24 hours later, the medium in the Deitsushu, new, medium ( was replaced with 10-fold bulk% © shea fetal serum-containing DMEM) 10 ml. cells then 5 volumes ./. C0 _2, 37 ° and 0.5 hours at C.

—PcAG-KGPIR (St. Jude Children's Research Hospital) 3 μg, pCAG-RTR2 (St. Jude Children's Research Hospital) 1 μg, pCAGGS-VSVG (St. Jude

Chil dren's Research Hospital (1 μg) and pCL20cSynI-GFP (5 g) were each dissolved in 450 to 1 sterile water to obtain a plasmid solution. The pCL20cSynI-GFP is a part of the MSCV promoter of pCL20c MSCV-GFP (St. Jude Children's Research Hospital: SEQ ID NO: 8) shown in FIG. It is replaced with a synapsin I promoter having a base sequence.

• pCAG-KGPIR: gag (encodes the structural protein of the virus) pol (codes reverse transcriptase)

• pCAG- RTR2: tat (transcriptional regulatory gene)

• pCAGS-VSVG: VSVG stands for Vesicular somatitis virus, glycoprotein. The original envelope of lentivirus can only infect CD4 positive cells. By substituting this with a VSV envelope that targets phospholipids, it becomes possible to infect various cells including nerves.

• pCL20c: Lentiviral main vector ", the region between the two LTRs is integrated into the host genome.

In this way, when the gene essential for virus production is divided into four plasmids, the virus produced is infectious, but after infection there is a lack of self-propagating ability, increasing safety.

To the obtained plasmid solution, 2.5 M CaCl 2 O ^ ul was added and stirred. The obtained mixture was allowed to stand for 5 minutes, and then the mixture was mixed with 2 × BBS [composition: 50 mM N, N-bis (2_hydroxychetyl) -2-aminoethanesulfonic acid (BES), 280 mM NaCl, 1. 5mM Na ₂ HP0 ₄ (pH 6.95)] 500 μ 1 was added and stirred rapidly. Thereafter, the obtained mixture was allowed to stand at room temperature for 30 minutes. The plasmid solution was evenly dropped onto the dish and gently mixed with the medium in the dish. Then cells, 3 volumes. /. The cells were cultured at C0 ₂ and 35 ° C. The following operation was performed in a safety cabinet for biohazard measures.

After 16 hours, the medium in the dish was replaced with 10 ml of fresh medium (DMEM containing 10% by weight urchin fetal serum). 'Then cells, 5 volumes. /. They were cultured for 24 hours in C0 _2, 37 ° C.

(2) Concentration

The medium was collected from the dish obtained in (1) above, transferred to a 50 ml centrifuge tube, and centrifuged at 1000 rpm (l20 × g) for 4 minutes to obtain a supernatant. The obtained supernatant was passed through a filter (Millipore, 0.22 / z m diameter). The obtained filtrate was subjected to ultracentrifugation (25,000 rpm, 2 hours, 4 ° C) using Beckman's mouthpiece SW28.1 to precipitate virus particles, and the supernatant was removed.

The resulting virus particle precipitate is suspended in phosphate buffered saline (not containing Mg ²⁺ and Ca ²⁺ ) [hereinafter PBS (-)] to a final volume of 200 μ1, for infection. A virus solution was obtained. In addition, the virus solution that was not used immediately was dispensed 20 μl at a time and stored at −80 ° C.

(3) Measurement of virus titer

HeLa cells are cultured in a 10cm dish and infected 24 hours after seeding. Sensation. Three days later, 5.0 × 10 ⁶ cells were obtained. In parallel, the same number of LAV-8E5 cells (ATCC, Manassas, VA, USA) were obtained. LAV-8E5 cells have one copy of HIV type 1 provirus per cell, and this is the standard. Genomic DNA was extracted from these cells and finally dissolved in 100 1 TE buffer. Of these, 1 and 1 were used to amplify a 290 bp region contained in the HIV provirus RRE (nev responsive element) using the following primers:

5, -ATGAGGGACAATTGGAGAAGTGAATTA-3 '(SEQ ID NO: 1 1),

5'-CAGACTGTGAGTTGCAACAGATGCTGT-3 '(SEQ ID NO: 1 2).

The copy number of provirus integrated into the genome was determined by serial dilution of genomic DNA. That is, by determining the dilution ratio immediately before the panda disappeared and comparing it with the standard using LAV-8E5 cells, it was determined how many cells per 5.0 X 10 ⁶ HeLa cells. It was calculated whether the provirus was integrated (genome copy number / virus solution lml).

[Example 2] Inoculation of vector into mouse cerebellar subarachnoid space

The following operations are performed in the P2 laboratory. In addition, the mouse after inoculation with the virus vector is a rack for infected animals equipped with a HEPA filter (trade name: Pioclean Gap Cellulite T- BCC-M4).

Mice (SLC supply, 4-10 weeks old) were anesthetized by intraperitoneal administration of pentobarbital (trade name: Nembutal) at 200/1 per 30g body weight. ·, In addition, the following operations were performed in the safety cabinet for biohazard countermeasures.

After anesthesia, the mouse was fixed using a small animal fixing device [trade name: SG-4, manufactured by Narishige Co., Ltd.]. In addition, the body temperature of the mouse was maintained at 37 ° C with a body temperature controller [F 'S' T, product name: body temperature control system (for mouse) FST-HPSM]. After shaving the head of the mouse, the skin was incised from the rostral side of several millimeters to just above the cerebellum. Next, under a stereomicroscope (Nikon Corporation, trade name: 1SMZ645), a micro drill (Urawa Kogyo, trade name: power controller) UC100 + HB1 (drill)] was used to drill holes with an inner diameter of 2 to 3 mm. In addition, a hole was made in the dura mater and spider under the bone using an injection needle.

Unoletra micropump in which the lentiviral vector obtained in Example 1 was filled in a trade name: Rexfil microsyringe (manufactured by WPI, 10 / zl capacity), and the flexfil microsyringe was attached to the micromanipulator.

2 (made by WPI).

Insert the needle of the microsyringe through the hole No. 5 for about 0.5 mm and place it in the subarachnoid space, and then place the lentiviral vector for expressing the foreign gene product into the ultra micro pump 2 digital controller Micro4 (trade name) For 4 minutes at a rate of lOOnl / min for a total of 4 μΙ.

After injection, the skin of the incised mouse was sutured with an ophthalmic microneedle with a suture thread (trade name. Ophthalmic weak needle C67-0, manufactured by Natsume Seisakusho Co., Ltd.). After that, remove the mouse from the fixing device, and on the heating pad (made by Showa Seiki Kogyo Co., Ltd., trade name: rubber mat heater 1 SG-15) And observed in a cage placed in the safety cabinet. After the mice woke up from anesthesia, the mouse cage was returned to the infected animal rack and maintained for 7-: 14 days.

As a control, mice were similarly inoculated using a lentiviral vector carrying the MSCV promoter instead of the synapsin I promoter.

For mice 7 to 14 days after the inoculation, the brain was removed after perfusion fixation with 4% formaldehyde-PB. After taking a fluorescent photograph of the whole brain using a fluorescent stereomicroscope, it was treated with 30% sucrose for 24 hours, and then a frozen section was prepared. The prepared brain sections were incubated with the primary antibody (Rat Anti-GFPAb: Nacalai) for 24 hours at room temperature, and then with the secondary antibody (Goat Anti- Rat IgGAb; manufactured by Molecular Probe) for 2 hours. Incubation at room temperature yielded a microscope specimen. Then, the localization of the expressed protein was investigated by observing the specimen with a fluorescence microscope (trade name: CKX41, manufactured by Olympus) and a confocal microscope (trade name: 'LSM5 Pascal, manufactured by Carl Zeiss).

As a result, as shown in Figs. 4 and 5 (when using a lentiviral vector that retains the synapsin I promoter) and Fig. 6 (when using a lentiviral vector that retains the MSCV promoter), the arachnoid membrane just above the cerebellum. GFP expression localized to the cerebellum was observed by administration of the virus into the lower space. In frozen sections, when a lentiviral vector carrying the synapsin I promoter (invention) was used, gene expression was restricted to cerebellar astrocytes and / or silkworm cysts (Figs. 4 and 5). In addition to cerebellar stellate cells and sputum cells, expression in Purkinje cells and Pergman glia was also observed when using a lentiviral vector that retained MSCV and mouth motor (Fig. 6). In stereomicrographs, one reason for the low GFP fluorescence intensity of cerebellum inoculated with a lentiviral vector carrying the synapsin I promoter is that gene expression is confined to cerebellar astrocytes and sputum cells. In addition, by this method, gene expression was observed in a wide range of cerebellar astrocytes and sputum cells, and it was found that transgenic animals that express transgenes specifically for these cells could be prepared.

[Example 3] Selective elimination of astrocytes and sputum cells by diphtheria toxin GFP of lentiviral vector pCL20cSynI (SEQ ID NO: 5) prepared in Example 1 A vector pCL20cSynI-DTR-HA (FIG. 7, SEQ ID NO: 10) was prepared by substituting the cDNA sequence of the diphtheria toxin receptor (DTR) (SEQ ID NO: 9) '. An HA tag sequence for antibody recognition was added to the carboxyl terminus of DTR. According to Example 1, a lentiviral vector for expression of diphtheria toxin receptor was obtained.

According to Example 2, a lentiviral vector for diphtheria toxin receptor expression was administered into the subarachnoid space of an adult mouse (after 4 weeks of age). 7 days after inoculation 20 (day 9 am and 8 pm) Diphtheria toxin dissolved in PBS at a concentration of 1 μg / ml (manufactured by Rubiochem) 150 / zl (diphtheria toxin 150 ng) Inoculated intraperitoneally for a total of 7 days. Thereafter, perfusion fixation was performed with 4% formaldehyde-PB, and the brain was removed. Using an electric slicer (DTK-1000, manufactured by Dosaka EM), a piece with a thickness of 100 / m was produced at room temperature. The prepared brain sections were incubated with the primary antibody mouse monoclonal anti-parvalbumin antibody (manufactured by Sigma) and rat monoclonal monoclonal anti-HA antibody (manufactured by Kuchish) for 24 hours, and then secondary antibody (568 Goat Anti-mouse IgG antibody; Opi 488 Goat Anti-rat IgG antibody manufactured by Molecular Probes) was added and incubated at room temperature for 2 hours to obtain a specimen for microscope. After that, the specimen was observed with a confocal laser microscope. The results are shown in FIG.

In mice that did not receive diphtheria toxin, DTR expression (green) recognized by anti-HA antibody was observed centered on the molecular layer and the Punoreckin cell layer (Figure 7, upper left). At high magnification, DTR (green) was observed mainly along the cell membranes of astrocytes and sputum cell bodies (right in the upper row of Fig. 7). A finer granular stain was observed in the molecular layer (Fig. 7, upper right), which was presumed to be in the dendrites or axons of astrocytes and sputum cells. DTR (green) recognized by anti-HA antibody was not observed at all in the cerebellar cortex fluorescently stained in mice administered with diphtheria toxin twice a day for 1 week (lower left in Fig. 7). In the same fluorescent photograph, the force S shows two different cerebellar lobules, and the left cerebellar cortex has astrocytes compared to the cerebellar cortex of the mouse without the addition of diphtheria toxin (upper left in Fig. 7). It can be seen that the number of pith cells is decreasing. In addition, these two cells were hardly observed in the cerebellar cortex on the right side (the positions that appear to be astrocytes and sputum cells are blackened out. The center in the lower part of Fig. 7 is an enlargement).

Based on the above results, by using this viral vector, the diphtheria toxin receptor is selectively expressed in astrocytes and sputum cells, and this can be achieved by continuous administration of diphtheria toxin. It was thought that two types of cells had dropped out.

By conducting similar experiments using young mice (0 to 14 days after birth) in which neural circuit formation has not been completed instead of mature mice, the cerebellar neural circuit in which astrocytes and cells are developing The role played in formation can be examined. As a result, the formation of neural circuits can be examined morphologically or electrophysiologically. All publications, patents and patent references cited in this specification are incorporated herein by reference. '' Possibility of industrial use

According to the present invention, gene expression limited to astrocytes and sputum cells can be studied, and a useful means for studying the function of the cerebellum will be provided. In addition, by selectively dropping out astrocytes and sputum cells, it is possible to elucidate the role of these two types of neurons at the behavioral level such as coordinated movement and motor learning. Furthermore, the present invention can be used for gene therapy targeting astrocytes and sputum cells. Sequence listing free text

SEQ ID NO: 1—rat synapsin I promoter (Synlp) _ ² 25 ~: part of L05 position SEQ ID NO: 2—rat of rat synapsin I promoter (Synlp) 2 ² 5 to position 105 to 105 part, '

SEQ ID NO: 3—Rat of mouse synapsin I promoter (Synlp) -225 ~: position 105

SEQ ID NO: 4—rat synapsin I promoter (Synlp)-941-105 position SEQ ID NO: 5—artificial sequence description: pCL20cSynI (Synlp is rat 1046 bp) SEQ ID NO: 6—artificial sequence description: pCL20cSynI-GFP (Synlp is 1046bp of rat)

SEQ ID NO: 7—Description of artificial sequence: Sequence containing SV40 promoter and Blasticidine resistance gene

SEQ ID NO: 8—Description of artificial sequence: pCL20cMSCV-GFP

SEQ ID NO: 9 Description of one artificial sequence: diphtheria toxin receptor (DTR) cDNA sequence SEQ ID NO: 10—Description of artificial sequence: pCL20cSynI DTR-HA (Synlp is rat 1046 bp)

SEQ ID NO: 1 1 Description of one artificial sequence: Primer

SEQ ID NO: 1 '2—Description of artificial sequence: primer

Claims

The scope of the claims

1. By introducing a lentivirus-derived vector containing a transgene linked in a manner capable of functioning under the control of the synapsin I promoter into the target animal, the introduction is performed in the cerebellar astrocytes and Z or sputum cells of the target animals. A method of specifically expressing a gene.

2. The method according to claim 1, wherein the transgene is a gene encoding a protein that controls development or physiological function or life of cerebellar neurons. .

3. A gene encoding a protein that controls the development or physiological function or survival of the cerebellar neurons is diphtheria toxin, diphtheria toxin receptor, thymidine kinase, glutamate receptor, and GABA receptor, brain-derived neurotrophic factor, 3. The method according to claim 2, which is a gene encoding any one selected from glia cell-derived neurotrophic factor and variants thereof.

4. Selected from diphtheria toxin, diphtheria toxin receptor, thymidine kinase, glutamate receptor, GABA receptor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor and variants thereof under the control of synapsin I promoter A vector derived from a lentivirus comprising a gene encoding any of the above linked in a functional manner.

5. A transgenic animal into which the lentivirus-derived vector according to claim 4 is introduced. .

6. A gene encoding any one selected from diphtheria toxin, diphtheria toxin receptor, thymidine kinase, Lurcher mutagenesis δ2 glutamate receptor and its variants is linked under the control of the synapsin I promoter in a manner capable of functioning. A method for selectively dropping off cerebellar astrocytes and cells of the subject animal by introducing a vector derived from lentivirus into the subject animal.

7. A method for analyzing the function of the cerebellum using the method according to any one of claims 1 to 3 or the method according to claim 6.