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

Method for gene expression specific to cerebellar astrocyte and/or basket cell Download PDF

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WO2007018318A1
WO2007018318A1 PCT/JP2006/316127 JP2006316127W WO2007018318A1 WO 2007018318 A1 WO2007018318 A1 WO 2007018318A1 JP 2006316127 W JP2006316127 W JP 2006316127W WO 2007018318 A1 WO2007018318 A1 WO 2007018318A1
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cells
cerebellar
promoter
synapsin
gene
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PCT/JP2006/316127
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French (fr)
Japanese (ja)
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Hirokazu Hirai
Takashi Torashima
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National University Corporation Kanazawa University
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00Genetically modified animals
    • A01K2217/05Animals comprising random inserted nucleic acids (transgenic)
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • A01K2267/035Animal model for multifactorial diseases
    • A01K2267/0356Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00Reverse Transcribing RNA Viruses
    • C12N2740/00011Reverse Transcribing RNA Viruses
    • C12N2740/10011Retroviridae
    • C12N2740/15011Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00Vector systems having a special element relevant for transcription
    • C12N2830/008Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

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 basket cell-specific gene expression method art

The present invention, cerebellar astrocytes and / or basket cell specific gene expression methods and cerebellar functional studies using the same, a vector for 及 Piso, transgenic animals and the like. 'Akira

Background technology

The cerebellum plays an important role in the cooperative movement, such as walking multiple muscles are involved child

That. 'If there is damage to the cerebellum, can not be a smooth movement not go well regulation of coordinated movement. Command movement from the cerebral cortex through the brainstem, spinal cord, but is transmitted to the muscles, also within the cerebellar cortex via mossy fibers simultaneously. Mossy fiber inputs excitable between granule cells are neurons cerebellar cortex. Granule cells form synapses between the Purukine cells via parallel fibers Ru their axons der. Purukine cells are the only output neurons from cerebellar cortex receives any input is Purukine cells, or outputs it how integrated it can be said that the essence of the function of the cerebellum.

The cerebellar cortex granule cells, three kinds of neurons are present in addition Purukine cells (see Figure 1). The three cell: astrocytes, basket cells, both Golgi is mediated resident neural cells, with the task of adjusting the signal traveling the mossy fibers one granule cells one Purukine cells. Astrocytes exist in a portion called molecular layer close to the surface layer of the cerebellar cortex, it controls the inhibitory Haneda Lucky synaptic connections to Purukine cells 閒 the Purukine dendrites. Basket cells also present in the molecular layer, but unlike. Astrocytes present in the 'near Purukine cell layer, to form the inhibitory synaptic between cell bodies Purukine cells.

This way cerebellar astrocytes and basket cells It is known that Gosei to inhibitory serve the Purukine cells, influence against the Purukine cells of these two cell, how, cerebellar Runoka not contribute to the cooperative movement, or motor learning to control is a completely unknown.

Is reconstituted method for analyzing the functions of cerebellar astrocytes and basket cells, heretofore, the cells were electrically stimulated, stimulation recording, or flat ascending fibers that potential changes in Purukine cells, stellate method for recording a potential change in a cell or basket cells known only. Further, since the promoter that functions specifically in cerebellar astrocytes and basket cells have not been discovered, even unknown These cells specifically transgenic animals plus genetic modification. '

Synapsin I is present in terminal neurons, a protein that 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 in the analysis using the lentiviral vectors, synapsin I promoter one coater will be functional in excitatory and inhibitory of both nerve cells in the cerebral cortex are SaiwaiushitoraTsuge (Dittgen et al., PNAS, Vol. 101, No. 52, pl8207- 211, (2004)). However, for how the function to Luke whether synapsin I promoter is in the cerebellum, no report. In fact, hitherto known cerebellum specific calling expression vector is one in which both using adenovirus and adeno-associated virus or alkylene Chiwirusu, the promoter used is the CMV promoter one, RSV. General promoter ones, and the as a cell-specific is only there is flop Rukine cell-specific L7 promoter (Kohyo 2 0 0 3 - 5 3 4 7 8 7 JP, Hei 9 2 8 3 7 8, JP-T-2 0 0 3 5 1 1 0 8 0 JP). . Shots Ming disclosure - object of the present invention is to a specific gene expression in cerebellar astrocytes and or basket cells were possible, to provide a new tool for the cerebellar function studies or gene therapy . As described above, synapsin I is present in the end of the nerve cells, a protein that plays an important role in the out release of synaptic vesicles. Generally synapsin I is used as a marker of excitable presynaptic terminals. For excitatory neural cerebellar cortex is only granule cells, inventors considered that utilizing the synapsin I promoter as granule cells singular expression vector. Then, the embedded synapsin I promoter gene isolated from rat genome derived vector lentivirus, by connecting the GFP gene downstream thereof, to infect cells of the cerebellar cortex molecular layer of adult mice, the in fluorescence microscopy localization was observed record. Then, the granule cell contrary to expectations not seen the expression of GFP at all, selective expression of GFP in stellate cells and basket cells in the molecular layer was observed instead. Expression of cell nonselective CMV promoter and MSCV under control of a promoter one using lentiviral base Kuta one expressing GFP ¾ and granule cells in GFP is not observed (GFP Other four neurons expression is observed) this and force these, lentiviral vector was considered not have the ability to infect granule cells. These results suggest that wrench viral vector using synapsin I promoter is available as a specific expression vector astrocytes and basket cells.

The present invention has been made based on the above findings, by introducing a lentiviral derived vector containing linked with can function a transgene under synapsin I promoter data one control. Aspect to a subject animal, the cerebellar astrocytes 及 Pi of the target animal

It relates to a method for specifically expressing the transgene in Z or basket cells. In the above method, the transgene protein to control cell survival, cerebellar development, proteins that regulate physiological functions (e.g., therapeutic protein) genes encoding is preferred. For example, diphtheria toxin, diphtheria toxin receptors, Rupesu derived thymidine kinase into simple, glutamate receptors,. GABA receptors, is selected from brain-derived neurotrophic factor, 及 Pi glial cell-derived neurotrophic factor and their modified variants it can be mentioned a gene encoding any.

The present invention also relates to diphtheria toxin under the control synapsin I promoter, Rupesu derived thymidine kinase into simple, glutamate receptors, GABA receptors, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor and variants thereof selection Bareru les, provide genes encoding Zureka, or 'and lentiviral-derived vectors containing linked in a manner capable of functioning genes encoding various therapeutic protein, also trans diethylene Nick animals introducing the base Kuta one to.

The present invention also provides, synapsin I and ligated in a manner capable of functioning transgene under promoter ruled ^ enter the subject animal lentiviral-derived base compactors including in the Target Animal cerebellar astrocytes and / or basket cells wherein by the transgene was specifically issued manifested ¾, also provides a method of analyzing a physiological or pathological features of the cerebellum in. .

In particular, diphtheria toxin, diphtheria toxin receptor, thymidine kinase, in the trans-di We nick Tsutomubutsu obtained by introducing a gene encoding either Re Izu selected from 及 Pi Lurcher mutagenesis [delta] 2 glutamate receptors and variants thereof, that it becomes possible to selectively shed cerebellar astrocytes and / or basket cells, it is useful in elucidating the this two cell thus physiological function of the cerebellum. . '. Furthermore, lentiviral-derived base restrictor comprising linked with synapsin I promoter under the rule brain-derived neurotrophic factor, it can function a gene encoding a therapeutic protein, such as glial cell-derived neurotrophic factor «cerebellum as therapeutic protein delivery vector into astrocytes 及 Pi Roh or KagoHoso cells, useful for gene therapy.

Subject animal used in the present invention, non-human mammal animal in investigational field, in particular mice, month teeth such as rats. On the other hand, the Te field smell of gene therapy, it is possible to target mammalian general, including humans. According to the present invention, it is possible to selectively shed astrocytes and basket cells, these two neurons Me roles, coordination, can be elucidated Te action level odors such as motor learning to become. Moreover, astrocytes and Z or is localized to gene expression in basket cells, since the functional regulation of these inhibitory nerve becomes possible to provide a useful means for performing cerebellar nerve circuits Research and thus. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of cerebellar circuit. .

Figure 2 is a schematic diagram of pCL20cMS'CV-GFP.

Figure 3 A is a schematic diagram of pCL20cSynI is an example of an expression vector of the present invention. Figure 3 B is Ru schematic der of a is pCL20cSynI- GFP 1 example of the expression vectors of the present invention.

Figure 3 C is a schematic diagram of a is pCL20cSynI- DTR- HA 1 example of the expression vectors of the present invention. -

Figure 4 is a fluorescence micrograph showing the expression example cerebellar astrocytes and basket cells specific GFP gene by PCL20cSynI- GFP.

Figure 5. is a confocal laser microscope photographs showing expression example cerebellar astrocytes and basket cells specific GFP gene by pCL20cSynI-GFP.

Figure 6 A is a fluorescence micrograph showing the expression example of a non-selective GFP gene into cerebellum cells by pCL20cMSCV-GFP.

Figure 6 B is a confocal laser microscope photographs showing nonselective GFP gene Me expressors to cerebellar cells by pCL20cMSCV-GFP. ., 7, PCL20cSynI- DTR-confocal laser microscope photographs showing expression example cerebellar HoshijoHoso '胞及 Pi basket cell-specific GFP gene by HA (upper: not administered diphtheria toxin mouse cerebellar cortex, lower: the diphtheria toxin 1 'twice daily, mouse cerebellar cortex, which was administered between 1 week). The present specification encompasses the contents described in Akira Saisho the No. 2 0 0 5 2 3 1 5 1 4 No. is a priority document of the present application. Vectors according to the best mode invention for carrying out the invention is a lentiviral derived vector containing linked in a manner that can function a transgene under the control synapsin I promoter.

(1) lentivirus-derived vectors

Vectors of the present invention is a basic skeleton lentiviral vectors derived from human immunodeficiency virus. To or adenovirus vector Unlike Le Bae scan viral vectors, lentiviral vectors, the immune response in the organism is very weak, cell failure resistance even in that little advantage. The vector one from the lentivirus, e.g., pCL20c (St. Jude Children s Research Hospital provided), and the like pLenti6 / V5-DEST (Invitrogen Corporation).

As used herein, "basic skeleton", within a particular cell, maintenance, refers to a structural unit that contains a sufficient factor to be replicated. Accordingly, in the present invention may also be used those obtained by removing unnecessary portions of vectors derived from generic lentiviral. From the viewpoint of improving the titer of viral vectors, for example, in the case of pLenti6 / V5- DEST is a base compactors from lentivirus, it is preferable that the portion having the nucleotide sequence represented by SEQ ID NO: 7 is removed. Therefore, synapsin a vector of lentivirus-derived containing the I promoter, and the lentiviral vector backbone and operably linking a transgene introduced interest downstream of the promoter, the nucleotide sequence shown in SEQ ID NO: 7 expression vectors containing no also included in the vectors of the present invention. 'The size of the insert that can be incorporated into the lentiviral vector is said to generally 8kb, including flop opening motor. From the inventors' previous experiments, the high titer lentivirus Virus vector removed a portion having a nucleotide sequence of the SEQ ID NO: 6, it has been found that 4 ~ 5 kb is the limit. Therefore, in order to express more genes 3kb, it is necessary to use a neural-specific promoter one not exceeding L~2kb.

(2) synapsin I promoter.

Vectors of the invention include synapsin I promoter as a promoter. Sequence of synapsin I promoter is available through public databases such as GenBank, for example, rat synapsin I promoter sequence, Accession No.

M55300, .T05521, as J05630, human Toshinapushin I promoter sequence as Accession No. M58321, J05431, M57636, mouse synapsin I promoter sequences are registered. Not registered, synapsin I promoter sequence in other animals, by homology search based on the synapsin I promoter sequence, it is possible to obtain accordance connexion readily to known methods.

The synapsin I promoter of the present invention, a variant thereof (Parianto) is also included. Variants, artificially, for example be those made Ri by the site-specific mutagenesis method and the like by a conventional method, or may be a variant that occur spontaneously. As an example, in SEQ ID NO: 1 shows the rat synapsin I promoter sequence used in the examples of MotoTsutomuAkira (rat synapsin I promoter - 225-105 of SEQ:

. 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: the document, - the part of the two hundred and twenty-five to one hundred and five neuron - to be expressed in specific has been reported). Further, in SEQ ID NO: 2 及 Pi 3 shows human and murine synapsin I promoter sequences corresponding to each of the rats -225~105 position. ,

In addition to the synapsin I promoter of the above 330 bp, it Dittgen. Et al (Dittgen et al., PNAS ,. Vol. 101, No. 52, pl8207-211, (2004)) is that not using 1046bp rat synapsin I was also examined I promoter. As a result, to express specific foreign gene cerebellar astrocytes and / or basket cells even with rat synapsin promoter 1046bp and has decreased obviously. In addition Oite in the cerebellum, rat synapsin promoter of 1046bp was found to be much higher promoter activity than the synapsin I-flops opening motor of 330bp. In SEQ ID NO: 4 shows the rat synapsin I promoter sequence of 1046bp.

However, as described above, is not limited to this arrangement, in the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 4, (is rather preferably, one or several) of at least one nucleotide residue had a different nucleotide sequence even, as long as it is as synapsin I promoter one coater, which can specifically expressed the transgene ligated in a manner that can function downstream thereof in cerebellar astrocytes and / or basket cells, similarly synapsin

It can be used as I promoter. That is, multiple § line e n t by ClustalW method by Higgins et al. (E.g., Gappenalty5, Fixed Gap penaltylO, windowssize 5, Floating Gap 10), the optimum condition is Arainme cement, calculated sequence less identity even 80 %, preferably 85% or more, good Ri preferably a nucleotide sequence at least 90%, it as synapsin I promoter, like the cerebellum star transgenes were linked in a manner that can function downstream thereof as long as such may specifically expressed in fine 胞及 Pi or basket cells, can also be used as synapsin I promoter according to the present invention having such a nucleotide sequence.

The arrangement also may Haiburidizu in sequence under stringent conditions shown in SEQ ID NO: 1 and SEQ ID NO: 4, it as synapsin I promoter, cerebellar Review a transgene ligated in a manner that can function downstream thereof as long as capable of specifically expressed in Jo cells and Roh or basket cells, can be used as well as synapsin I promoter. In the present specification, the "stringent conditions", 6xSSC. (Composition of lxSSC: 0. 15M NaCl, 0, sodium 015M Kuen acid, ρΗ7 · 0) and 0. 5% SDS and 5χ den Halt and 100 / ml denatured fragmented salmon sperm DNA and 50. /. Solution containing Horumuamido, SEQ ID NO: 1 and SEQ ID NO: 55 ° C with a nucleic acid consisting of the nucleotide sequence shown in 4 - 晚保 raised, low ionic strength, 'for example, 2 × SSC, more stringently, such as 0. LxSSC conditions and Z or higher temperatures, 37 ° C or more, the stringent, 42 ° C or higher, more stringent, 50 ° C or higher, more and more stringent, under conditions such as 60 ° C or higher It can be achieved by the condition for cleaning.

Note that the length of the nucleotide sequence of variants, but the desired promoter function is not particularly limited as long exerted, in terms of allowed size 'of a foreign gene incorporated into a vector, for example, 3000 nucleotides in length or less, preferably, 2000 nucleotides in length or less der Rukoto is desirable.

Functions as synapsin I promoter, e.g., a RNA polymerase contained in cerebellar astrocytes or KagoHoso alveoli can be assessed by Rukoto detecting the presence or absence of binding of the nucleic acid to be tested. Detection of the presence or absence of binding may be performed mobility that put the gel shift Atsu Si, optical sensorgram or mass sensor-grams of the surface plasmon analysis, the footprinting like.

Also functions as a promoter variants, for example, downstream of the nucleic acid to be tested is operably linking a reporter gene to give the construct, resulting construct of mouse or rat cerebellar glial cells + neurons infecting mixed culture, there have can be assessed by administering the cerebellum surface subarachnoid space of the solid mouse. The pre-Symbol evaluation, the reporter gene is. Cerebellar astrocytes or basket cells specifically when expressed, the nucleic acid can be used as synapsin I promoter suitable for expression vectors of the present invention.

The synapsin I promoter, it is desirable to use the same or, alternatively synapsin I promoter sequence related species subject animal used.

Synapsin I is present in terminal neurons, a protein that plays an important role in the release of synaptic vesicles. Generally synapsin I is used as a marker of excitatory presynaptic terminals. Der excitatory neural cerebellar cortex only granule cells because, Toto the synapsin I promoter can be utilized as a granule cell-specific expression vector is considered. However, we have built a rat Sina god of military arts I promoter gene (SEQ ID NO: 1 and SEQ ID NO: 4) to base click terpolymer derived from a lentivirus, by connecting the GFP gene downstream thereof, cerebellar cortical molecular layer of adult mice was infected with the cell, not observed GFP expression at all in the granule cells, expression of selective GFP in astrocytes and basket cells in the molecular layer was observed instead. Ie, lentiviral vectors using the synapsin I promoter, it was confirmed to be useful as an expression vector that can specifically function in S JoHoso cells and basket cells.

(3) transgene

Vector of the invention comprises linked in a manner that can function a transgene under the control of the synapsin I promoter. Here, "linked in a manner that can function" is a state arranged to perform the function of the years, arranged so as in one dominant under synapsin I promoter, transgene expression properly It refers to the state.

Although not particularly limited transgene used in the present invention, if example embodiment, genes that affect the survival of cells, specifically, genes encoding lifting one protein toxic to. Cells, cerebellar development, genes encoding proteins that control the physiological functions, specifically, genes encoding cerebellum synapse formation and plasticity control molecule, a gene capable of expressing proteins or siRNA that can be used in therapeutic applications, specifically , gene enzyme code to decompose or remove the aggregates produced intracellularly, in the cell, the gene encoding the protein or the like that may Toi functions are 俾下, in the cell, excessive functions gene and the like capable of expressing the siRNA capable of suppressing the origination other genes encoding proteins associated with substance production, degrades certain substances Such as genes encoding protein is Chi like. As the gene introduction target, and more specifically, for example, as a toxic protein, encoded genes encoding diphtheria toxin, a gene that encoding a diphtheria toxin receptor, the Rupesu derived thymidine kinase to simple gene, NMDA-type glutamate receptors as cerebellar synaptic formation and plasticity control molecule, AMPA-type grayed gene encoding glutamic acid receptor, a gene encoding a GABA receptor, brain-derived neurotrophic factor or Daria cell-derived neural for therapeutic gene that encoding a trophic factor, further variants of the protein, Yadenko like encoding a dominant negative Restorative material for functional inhibition. .

As expression vectors of the present invention, Re as it has a structure as described above Bayoku, for example, vectors pCL20cSynI having the nucleotide sequence shown in SEQ ID NO: 5 (Figure 3 A), and the like. In the above PCL20cSynI, nucleic acid introduced target EcoRI 及 Pi Notl recognition site in the PCL20cSynI can be 揷入. ..

2. Functional analysis of the cerebellum

(1) cerebellar astrocytes transgene and or basket cell-specific expression

The present invention, by introducing a lentiviral derived vector containing linked in a manner that can function a transgene under the control synapsin I promoter in the target animal, the cerebellum astrocytes and Z or basket cells of the target animal It provides a method of specifically expressing a transgene. '

In this way, hitherto it is possible to express and deliver cerebellar astrocytes and basket cells specific specific gene was impossible, therapeutic research and cerebellar astrocytes and basket cells abnormality relating diseases of cerebellar function and research is made possible.

In particular, by using a vector of the present invention, cerebellar astrocytes and basket cells specifically diphtheria § toxin, Le Bae scan virus-derived thymidine kinase into simple, if express Lurcher mutagenesis δ.2 glutamate receptors, thereby dropping these cells specifically: is possible. By observing the change by dropping, it is possible to know the function in the cerebellum cerebellar astrocytes and basket cells. . For example, if shed small brain astrocytes and basket cells using the method in immature mice, leading to the elucidation of the role in the cerebellum 祌経 circuit formation. In addition, the use in adult mice, whether the two kinds of ^ standing nerve to contribute to throat in cerebellar function is clear. Other, it is possible to specifically express a variety of genes in cerebellar astrocytes and basket cells, it is possible to widely research on synaptic transmission between cerebellar astrocytes and basket cells and flop Rukine cells. (2) trans-diethyl Nick animal

The vectors of the present invention by injecting into the subarachnoid space, it is possible to express a specific transgene to a wide range of small brain astrocytes and / or basket cells of the target animal. Such trans Jie Nick animals, for example, is available as cerebellar function research model animals and cerebellar disease model animal. Target trans di We nick animal is not particularly limited, a mouse, rat, Usagi, © Ma, can be used Hijji, I j, cats, baboons small mammals typified monkeys', in particular the mouse month teeth such as rat are preferred. '3. Gene therapy

Lentiviral derived base restrictor comprising linked with synapsin I manner can function a gene encoding a therapeutic protein under promoter one coater dominance, as a delivery vector into cerebellar astrocytes and Z or basket cells, gene therapy it is useful.

That is, the vector of the present invention is dissolved or suspended together with pharmaceutically acceptable carriers in a suitable puffer one, is injected into the subarachnoid space on the surface of the cerebellum, cerebellum HoshijoHoso 胞及 Pi Z or basket cells It may be utilized as a therapeutic agent for diseases pathogenesis faults in. The vectors of the present invention, a gene encoding a therapeutic protein efficiently delivered to cerebellar astrocytes and / or basket cells, since it is possible to express, against disease (this to pathogenesis of disorders in these cells, efficiency by Les, as possible out is possible to achieve a therapeutic effect.,

Previously diseases cerebellar astrocytes and Roh or basket cells are specifically impaired at the not disclosed. As Purukine cytopathic & representative of secondary HoshijoHoso 胞及 Pi basket cytotoxicity following dropping of Ru can be exemplified spinocerebellar degeneration. Spinocerebellar ataxia type 1, Type 2 and type 6 Ru degeneration significantly der of Purukine cells. Astrocytes 及 Pi basket cells to adjust suppressively the Purukine cellular activity, may be able to suppress Purukine cytopathic by adjusting the these cellular activities. Pharmacological evaluation of gene therapy varies depending on the type of disease to be treated, in the case of spinal cerebellum degeneration, after the vector was administered into the subarachnoid space on the surface of the cerebellum, Purukine cells in cytological, stars the prevention of degeneration separation of cerebellar neurons including Jo cells and basket cells, may (in the case of animal behavioral) further clinically to be more done about improvement of cerebellar ataxia.

Transgene incorporated into the vector, according to the type of disease to be treated, 'suitable Yibin can be selected. For example, a nucleic acid, and the like encoding the brain-derived or glial cell-derived neurotrophic factor.

Vector preferably by injecting into the subarachnoid space on the surface of the cerebellum can be dosing. Injected vector few cerebellar astrocytes near the injection site and

Without localized only in the Z or basket cells, with high affinity to the entire cerebellar astrocytes and / or basket cells, and can be delivered extensively active ingredient. Also, injection of the vectors of the present invention to subarachnoid space of the cerebellum table surface, it is possible to suppress damage to the brain parenchyma in comparison to the Note-entry into the inferior olive Ya cerebellar nuclei, excellent clinical applications.

Further, the administration of the vector of the present invention, upon implantation, from the viewpoint of maintaining the intracranial pressure stable, preferably means capable injected at a constant rate, for example, Nono Milton syringe and take it. Put it it is desirable to use Michael injection pump for micromanipulator and injection can. Injection speed is not limited in particular as long as it can maintain the intracranial pressure stable, age of the individual, weight, depending on the disease state or the like may appropriately be set. For example, LOnl / min ~ 800 NL / min, preferably 50 nl / min ~ 400 nl / min, more preferably, it is desirable that lOOnl min ~ 200 nl min.

The dose of the vector of the present invention, 'is not limited especially as long as it is an amount suitable to exert a therapeutic effect, the individual's age, body weight, depending on the disease state or the like, may be appropriately set. Further, the number of doses of the therapeutic agent of the present invention may be a sufficient number of times to exert a therapeutic effect. The vectors of the present MizunotoAkira that lentiviral as a basic skeleton, since the gene is integrated into the chromosome, may be administered once, but increases the number of copies of the incorporated Ru gene, for introducing a wider range of cell to, change the location of the small brain surface may be administered (right, median, left, etc.) about 3 times. Example

The present invention will hereinafter be described in detail by referring to Examples, but the present invention is not limited to these examples.

Example 1 Preparation of cerebellar astrocytes, basket cell-specific expression vector (1) Production of virus

The following operations were performed in a P2 laboratory.

$ The HEK293T cells in the logarithmic growth phase PB (-). Dispersed in, then, were seeded in such a way that l (km dish (made off Alcon) per 5 X 10 5 cells in 10cm Disshi Interview 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 ( It 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.

- How, pCAG-KGPIR (St. Jude Chi ldren s Research Hospital) 3 μ g, pCAG - RTR2 (St. Jude Chi ldren 's Research Hospital) 1 μ g, pCAGGS-VSVG (St. Jude

Chi l dren 's Research Hospi tal) 1 μ g, dissolved in 450〃 1 sterile water pCL20cSynI- GFP 5 g each, to obtain a plasmid solution. Incidentally, the PCL20cSynI- GFP is, pCL20c MSCV-GFP shown in Figure 2: shown in the MSCV flop port motor portion of (St. Jude Chi ldren 's Research Hospital SEQ ID NO: 8), SEQ ID NO: 1 or SEQ ID NO: 4 it is replaced with synapsin I promoter having the nucleotide sequence.

• pCAG-KGPIR: gag (encoding the structural proteins of the virus) throat pol (reverse transcriptase co once)

• pCAG- RTR2: tat (transcriptional regulatory gene)

• pCAGS - VSVG: VSVG is Vesicular somatitis virus, it stands for glycoprotein. Not able to infect only to the original Envelope in CD4-positive cells of the lentivirus. This By substituting Envelope of VSV that phosphorus lipid targets, it is possible to infect various cells including neurons.

• pCL20c: is a lentivirus of the main base Kuta ", two of the LTR in Is the Murrell area in this is integrated into the host genome.

While thus Dividing the essential gene in virus production in the four plasmid is produced virus with infectivity, infected after safety for self-propagating ability is lacking is increased.

To the resulting plasmid solution 2. 添Ka卩 a 5M CaCl O ^ ul, it was 撹拝. The resulting mixture was allowed to stand for 5 minutes, to the mixture, '2XBBS [Composition: 50 mM N, N-bis (2_ heat Dorokishechiru) - 2-aminoethanesulfonic acid (BES), 280 mM NaCl, 1. 5mM Na 2 HP0 4 (pH6. 95)] the 500 mu 1 was added Caro, was quickly stirred. Thereafter, the resulting mixture was allowed to stand at room temperature for 30 minutes. .. Uniformly dropwise plasmid solution to the dish and mixed with medium gently in the dish. Then, the cell, 3 volume. /. They were cultured in C0 2, 35 ° C. Below, it was carried out the operation in the safety Kiyabinetto for bio 'hazard measures.

After 16 hours, the medium in the dish was replaced with fresh medium (10 wt% © shea calf serum containing organic DMEM) 10 ml. 'Then, the cells, 5 vol. /. They were cultured for 24 hours in C0 2, 37 ° C.

(2) concentrated

Wherein the resulting dish (1), and the medium was recovered, transferred to a 50ml centrifuge tube, and centrifuged 1000 rpm (L20 X g) 4 minutes to obtain a supernatant. The resulting supernatant, filter (Millipore, 0. 22 / zm diameter) was passed through. The resulting filtrate, Beckman port one coater SW28. 1 ultracentrifuge using (25,000 rpm, 2 hours, 4 ° C) was subjected to precipitate the virus particles child, and the supernatant was removed.

The resulting virus particles precipitate phosphate buffered saline (not containing the Mg 2+ and Ca 2+) [hereinafter, PBS (-)] were suspended in a final amount is 200 mu 1, for infection to obtain a virus solution. Incidentally, immediately without using a virus solution, dispensed by 20 mu 1 minute, - stored at 80 ° C.

(3) measurement of virus titer

HeLa cells were cultured in a 10cm dish, Ru were infected with the virus solution 24 hours after seeding. Sensitive. Was obtained 5. 0 X 10 6 cells in dyeing 3 days. The same number of LAV In parallel - 8E5 cells were obtained (ATCC Inc., Manassas, VA, USA) and. LAV- 8E5 cells are to have one copy of the HIV1 type of professional virus in 1 cells, it was used as a standard. Extracting the cells or et genomic DNA, it was finally dissolved in 100 1 of TE buffer. Among used 1 1, the region of 290bp which contained in the HIV provirus RRE (nev responsive element), was amplified 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 is serially diluted genomic DNA determined meth. Namely, a dilution ratio was determined immediately before Pando no longer appear, LAV- 8E5 cells and compared with the standard using, 5.0 or X 10 6 cells of HeLa how many proviral per cell is incorporated ( genome copy number / virus solution lml) was calculated.

Inoculation of vector into Example 2 Mouse cerebellar subarachnoid space

The following operation is carried out in the P2 laboratory, also, mice after viral vector inoculation rack for infected animals equipped with HEPA filter one (Tokiwa Scientific Instruments Co., Ltd., trade name: pie O clean moth-flop cells Interview knit T - BCC- M4) was housed in.

The mice (SLC supply, 4-10 weeks old), pentobarbital (trade name: Nembutal), per body weight 30g 200/1, were anesthetized by intraperitoneal administration. -, It should be noted that the following operations, was row summer in a biological safety cabinet.

After anesthesia, a mouse, a small animal fixing device [NARISHIGE Co., Ltd., trade name: SG-4] were fixed with. Further, temperature controllers: at [F 'S' T, trade name body temperature control system (for mice) FST- HPSM], mice were maintained body temperature so that the 37 ° C. After the cut the hair of the mouse's head, were dissected the skin over the cerebellum above from Bregma by. Ri few millimeters rostral. Next, a stereoscopic microscope (Nikon Corporation, trade name 1SMZ645) below, in from 5-5~7- 5 Yuzuruo side of the midline of the head 蓥骨 Bregma, micro drill [Urawa Industry Co., Ltd., trade name: Power Controller UC100 + HB1 using (drill)], a hole having an inner diameter of 2 to 3 mm. Also, using an injection needle, a hole in the dura 及 Pi arachnoid under the bone.

The lentiviral vectors obtained in Example 1, trade name: Rex fill microsyringe (WPI Inc., 10 / zl volume) was filled, Uno Retora micropump attached flex fill microsyringe micromanipulator

It was set to 2 (WPI Co., Ltd.).

About 0.5 瞧刺 incoming city than the hole of the No. 5 needle tip of a microsyringe, placed in the subarachnoid space, the lentiviral vectors for foreign gene product expression, Ultra 2-specific digital controller Micro Micro4 (trade name , using manufactured WPI Inc.), lOOnl / min velocity at 40 minutes was injected in total 4 mu iota.

After the injection, the skin of the dissected mouse, suture with ophthalmic micro-needle (Natsume Seisakusho Co., Ltd., product name. Ophthalmic weak Ishiyumihari C67-0) was sutured in. Thereafter, the mice were removed from the stereotaxic apparatus, heating pad (Showa Seiki Kogyo Co., Ltd., trade name: Rubber mat heaters one SG - 15) were observed on the (in the safety cabinet) placed. Mouse, after awakening from the 'anesthesia, return the mouse cage to infected animals rack, 7: were maintained for 14 days.

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

The mice in 7-14 days after inoculation, perfused with 4% formaldehyde over PB solid funnel, and the brains were excised. After taking fluorescence photograph of the whole brain using fluorescence stereoscopic microscope after 24 hours at 30% Shiyukurosu, frozen sections were prepared. The prepared brain sections, the primary antibody (Rat Anti-GFPAb: Nacalai Inc.) with 24 hours at room temperature Inkyubeshi Yonshi, then the secondary antibody; 2 hours (Goat Anti- Rat IgGAb Molecular Probe Inc.), incubation at room temperature, to obtain a microscope specimen. Thereafter, the fluorescence microscope (Orinpasu trade name: CKX41) 及 Pi confocal microscope (Carl Zeiss, Inc., trade name: 'LSM5 Pascal) at localized tone base 7 This expressed protein by performing the sample to be observed.

As a result, directly above the cerebellum subarachnoid as shown in figures and (if using lentiviral vector carrying the synapsin I promoter) 4 及 Pi 5 6 (when using a wrench Virus vector carrying the MSCV promoter) the virus administration to the lower cavity, the expression of GFP was localized to the cerebellum was observed. In frozen sections, when using a lentiviral vector (invention) which holds the synapsin I promoter, confined to cerebellar astrocytes and or basket Itoda cells expression of genes was observed (Fig. 4 及 Pi 5) Power MSCV-flop, when using a lentiviral vector carrying the mouth motor, in addition to the cerebellar astrocytes and basket cells, Purukine cells, was also observed expression into Pagumanguria (Figure 6). In stereomicroscope photograph, as the GFP fluorescence intensity One reason weak cerebellar inoculated with lentiviral vector carrying the synapsin I promoter, the gene expression was localized to cerebellar astrocytes and basket cells. Further, by such a method, from the gene expression in a wide range of cerebellar astrocytes and basket cells are found, these cells specifically transgenic animals expressing the transgene it was found that it is possible to produce.

Example 3 cDNA sequence of Jifuteria toxin receptor GFP portion of lentiviral vector pCL20cSynI prepared in selective E successful example 1 astrocytes and basket cells by diphtheria toxin (SEQ ID NO: 5) (DTR) (SEQ solid terpolymer pCL20cSynI- DTR- HA (Fig. 7 substituted on No. 9) ', SEQ ID NO: 1 0) was prepared. The carboxyl terminus of the DTR added with HA tag sequence for antibody recognition. In accordance with Example 1, was obtained Renchiui ^^ scan vectors for Jifuteria toxin receptor expression.

In accordance with Example 2, was administered lentiviral vector for diphtheria toxin receptor expression in the subarachnoid space of mature mice (age 4 weeks later). . After inoculation 7 days 1 2 0 (9 am and 8:00 pm) Jifuteria toxin (manufactured by force Rubiokemu Co.) was dissolved at a concentration of 1 mu g / ml in PBS with 0.99 / zl (the diphtheria toxin 150 ng) It was inoculated with a total of 7 days in the abdominal cavity. Thereafter, perfusion fixed with 4% formaldehyde over PB, and brains removed. Electric slicer (DTK-1000, DoBan Iemu Inc.) was used to generate a switching piece of the thickness of 100 / m at room temperature. Brain sections were prepared is incubated at room temperature for 24 hours with the primary antibody mouse monoclonal anti-parvalbumin antibody (Sigma) and rat monochrome one null anti-HA antibody (manufactured by mouth Mesh Co.), followed by secondary antibody (568 Goat Anti-mouse IgG antibody; molecular probes, Inc. Contact Yopi 488 Goat Anti-rat IgG antibody) was added 2 h, fin-incubated over Chillon at room temperature to obtain a microscope specimen. Later, were specimens observed with a confocal laser microscope. The results are shown in Figure 7.

In the mice that were not administered the diphtheria toxin mainly in molecular layer and Punorekine cell layer, expression of the DTR recognized by anti-HA antibody (green) were observed (Figure 7 top left). At high magnification, DTR (green) was observed mainly along the cell membrane of the cell bodies of astrocytes and basket cells. (In Figure 7 the upper, right). Although finer granular dyed was observed in the molecular layer (Figure 7 top right), which was measured dendrites or axons within the estimated of astrocytes and basket cells. Twice daily diphtheria toxin, the fluorescent stained specimen cerebellar cortex of mice administered 1 week, DTR (green) recognized by anti-HA antibody was not observed at all (FIG. 7 lower left). The same fluorescence photograph, in two different cerebellar small force leaves are reflected S, left cerebellar cortex, compared to the cerebellar cortex of mice with no added diphtheria toxin (Fig. 7 upper left), astrocytes Oyo the number of Pi basket cells it can be seen that has decreased. Furthermore the two cells was hardly observed in the right cerebellar cortex (astrocytes, the position appears to KagoHoso vesicles missing black. Figure 7 the lower middle is an enlarged).

From the above results, by using the present viral vector, diphtheria toxin receptors selectively expressed in astrocytes and basket cells, it was considered two cell this was eliminated by sequential administration of Jifuteria toxin .

Juvenile mice neural formed instead is not completed in adult mice by performing the same experiment using a (0 day after birth until about 1 day 4), astrocytes and KagoHoso is cerebellar circuit during development it is possible to examine the role of the formation. Thus, formation of neural circuits can be examined morphologically or electrophysiological. All of the publications cited herein, it is assumed that incorporate herein and as it is reference to patents and patent out ¾. 'The possibility of use on the industry

The present invention, studies of gene was localized to astrocytes and basket cells expressing becomes possible, and to provide a useful means to cerebellar functional studies. Further, by selectively dropping the astrocytes and basket cells, the role of these two neurons, coordination, it is possible to elucidate the behavior level, such as' motor learning. In addition, the present onset Ming the stellate cells and basket cells can also be used in gene therapy to target. Sequence Listing Free Text

2 2 5-105 of the person portion, '- rats L05 position of a portion SEQ ID NO: 2 human Toshinapushin I promoter (Synlp): SEQ ID NO: 1 over rat synapsin I promoter (Synlp) _ 2 25~

105 of the relevant part: rat -225~ of SEQ ID NO: 3 mouse synapsin I promoter (Synlp)

SEQ ID NO: 4 over rat synapsin I promoter (Synlp) - Description of 941-105 position partial SEQ ID NO: 5 Artificial Sequence: Description of pCL20cSynI (Synlp those of 1046bp Rat) SEQ ID NO: 6 Artificial Sequence: PCL20cSynI- GFP (Synlp is also the 1046bp of rat)

Description of SEQ ID NO: 7 Artificial Sequence: sequence comprising the SV40 promoter and Blasticidine resistance gene

Description of SEQ ID NO: 8 Artificial Sequence: pCL20cMSCV- GFP

Description of SEQ ID NO: 9 one Artificial Sequence: Jifuteria toxin receptor (DTR) Description of the cDNA sequence SEQ ID NO: 1 0- Artificial Sequence: pCL20cSynI DTR-HA (Synlp those of 1046bp Rat)

Description of SEQ ID NO: 1 1 one Artificial Sequence:. Primer

Description of SEQ ID NO: 1 '2 Artificial Sequence: Primer

Claims

The scope of the claims
1. Synapsin by introducing into the subject animal lentiviral-derived vectors containing linked in a manner that can function a transgene under I promoter dominant, the introduction in the cerebellum astrocytes and Z or basket cells of the Target animals how to specifically issued revealed the gene.
2. The transgene is a gene encoding the development or physiology or raw braking Gosuru protein cerebellar neurons The method of claim 1. ..
3. The gene encodes a protein that controls the development or physiology or survival of cerebellar cells, diphtheria toxin, diphtheria toxin receptor, thymidine kinase, glutamate receptors, and GABA receptors, brain-derived neurotrophic factor, '及 Pi glial cell-derived neurotrophic factor and a gene encoding any selected from these variants, the method of claim 2.
4. Synapsin I promoter governed under the diphtheria toxin, diphtheria toxin receptor, thymidine kinase, glutamate receptors, 及 Pi GABA receptors, selected from brain-derived nerve trophic factor, and glial cell-derived neurotrophic factor and variants thereof Renchiui Luz derived vector containing linked in a manner that can function a gene encoding any of the.
5. Transgenic click animals introduced lentiviral-derived vector according to claim 4. .
6. Synapsin I promoter governed under the diphtheria toxin, diphtheria toxin receptors, linked in a manner that thymidine kinase, can function a gene encoding any selected from 及 Pi Lurcher mutagenesis [delta] 2 glutamate receptors and variants thereof how it makes to selectively shed cerebellar astrocytes and Roh or basket cells of the target animal to be introduced into the subject animal lentiviral-derived vectors containing Te.
7. Claim 1 The method according to any one of 3, or functional analysis method cerebellum using a method according to claim 6.
PCT/JP2006/316127 2005-08-10 2006-08-10 Method for gene expression specific to cerebellar astrocyte and/or basket cell WO2007018318A1 (en)

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WO2015164739A1 (en) * 2014-04-25 2015-10-29 Bluebird Bio, Inc. Kappa/lambda chimeric antigen receptors

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EP2318537A1 (en) * 2008-08-07 2011-05-11 Her Majesty The Queen In Right of Canada as represented by The Minister of Health Optimized promoter sequence
EP2318537A4 (en) * 2008-08-07 2012-01-18 Ca Minister Health & Welfare Optimized promoter sequence
WO2015164739A1 (en) * 2014-04-25 2015-10-29 Bluebird Bio, Inc. Kappa/lambda chimeric antigen receptors

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