JP2000302696A - Composition for introducing oligodendrocyte-specific gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specifically allow an oligodendrocyte of normal quiescent state to express a therapeutic gene for a long period. SOLUTION: The objective composition is composed of a recombinant adenovirus vector having CD4 gene on the downstream side of a myelin basic protein(MBP) promoter and a human immunodeficiency virus(HIV) vector containing a therapeutic gene. Accordingly, a recombinant adenovirus vector coding for CD4 gene is introduced into the downstream region of a myelin basic protein(MBP) promoter to effect the transient expression of CD4 molecule by an oligodendrocyte, and a recombinant human immunodeficiency virus(HIV) vector containing the therapeutic gene and recognizing the transiently expressed CD4 molecule is introduced to enable the specific and continuing expression of the therapeutic gene by the oligodendrocyte.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a composition for specifically and for a long term introducing a therapeutic gene into oligodendrocytes. Further, the present invention, using the composition,
The present invention relates to a method for specifically and for a long term introducing a therapeutic gene into oligodendrocytes.

[0002]

2. Description of the Related Art All nervous systems are classified into a central nervous system consisting of the brain and spinal cord, and other peripheral nervous systems. The central nervous system resides in the nerve cells responsible for signal transmission (neurons) and the oligodendrocytes that surround the neurons (oligodendrocytes), astrocytes (astrocytes), and the gaps between them It is composed of microglia (microglia).

[0003] Neurons form a neural network by extending axons from cells, and oligodendrocytes are present so as to surround the axons and form myelin (myelin). Myelin has the role of insulating electrical signals for signal transmission, and it is known that the breakdown (demyelination) of myelin causes severe neuropathy.

[0004] Diseases based on enzyme abnormalities in lipid metabolism in myelin include cerebral white matter degeneration (leukodystrophy: L
D). LD is a collective term for chronic progressive diseases in which myelin in the central nervous system falls off in a wide area. Myelin in the white matter of the cerebral hemisphere and cerebellar hemisphere falls out in a wide range almost symmetrically. LD mainly includes metachromatic brain white matter degeneration (M
LD), adrenoleukodystrophy (ALD), Pelizae
Us-Merzbacher disease (PMD) and the like are considered to be caused by abnormalities in genes encoding allylsulfatase A (ASA), adelenoleukodystrophy protein (ALDP), and proteolipid protein (PLP), respectively.

[0005] The main protein of myelin, myelin basic protein (Myelin basic p
protein (MBP) is a strongly basic protein rich in lysine and arginine, and is present inside the cell membrane of oligodendrocytes. MBP is a major marker of oligodendrocytes, and human or bovine MBP has a molecular weight of 1
It is 8.5 kilodaltons (kD), and two major components are 18.5 kD and 14 kD in mice and rats.

To date, MBP genes have been identified from various animals, and the 1.3 kb promoter region upstream of MBP has been confirmed to be useful as an oligodendrocyte-specific promoter (Ikenaka K.,
et al. , TheNew Biologist,
4, 53-60, 1992).

[0007] Therefore, a method has been attempted in which a gene is introduced into unspecified brain cells using an adenovirus vector to express an oligodendrocyte-specific gene by the MBP promoter (Hashimoto M., et al., Proc.
et al. , Hum. gene Therap
y, 7, 149, 1996), since the adenovirus vector does not allow long-term expression of the introduced gene, oligodendrocyte-specific gene transfer technology has not been established at present.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to express a therapeutic gene specifically for oligodendrocytes which are usually in a stationary phase and for a long period of time.

[0009]

An oligodendrocyte-specific gene transfer composition according to the present invention comprises a CD in the downstream region of a myelin basic protein (MBP) promoter.
A recombinant adenovirus vector having four genes and a recombinant human immunodeficiency virus (HI) containing a therapeutic gene.
V) It consists of a vector. More specifically, the composition for oligodendrocyte-specific gene transfer according to the present invention comprises a CD4 gene downstream of a myelin basic protein (MBP) promoter so as to transiently express a CD4 molecule in an oligodendrocyte. A recombinant adenovirus vector encoding a recombinant human immunodeficiency virus (HIV) vector that recognizes a transiently expressed CD4 molecule and specifically expresses a therapeutic gene in oligodendrocytes. It is.

[0010] The method of the present invention for expressing a therapeutic gene in oligodendrocytes specifically and for a long period of time is characterized in that the oligodendrocyte encodes the CD4 gene downstream of the myelin basic protein (MBP) promoter. A human immunodeficiency virus (HIV) vector recognizing the transiently expressed CD4 molecule by introducing a recombinant adenovirus vector into the cell line, which comprises a therapeutic gene. By introducing a recombinant vector, a therapeutic gene is specifically and long-term expressed in oligodendrocytes.

Hereinafter, the present invention will be described in detail with reference to embodiments of the present invention.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION (Composition for Gene Transfer Specific to Oligodendrocytes) The composition for specifically introducing a gene into oligodendrocytes according to the present invention comprises (1) the CD4 gene specifically into oligodendrocytes. It is composed of two vectors: a recombinant adenovirus vector having an MBP promoter and a CD4 gene to be expressed, and an HIV vector having a function of (2) recognizing CD4 and expressing a therapeutic gene. As a method of using the composition,
After administering the recombinant adenovirus vector of (1) to oligodendrocytes and sufficiently expressing CD4,
And (2) administering the HIV vector to express the therapeutic gene.

MBP Promoter According to the present invention, in the step (1), a promoter capable of expressing a CD4 gene introduced by a vector specifically to oligodendrocytes is used.

As such a promoter, any promoter can be used without particular limitation as long as it can specifically express the CD4 gene in oligodendrocytes. Among them, the MBP promoter is definitely used as oligodendrocyte. It can be specifically expressed and is preferred.

[0015] The MBP promoter is present in the upstream region of the MBP gene and has been identified to date from various animals such as humans, chimpanzees, cows, guinea pigs, mice, rats, and chickens. Among them, the promoter region present in the upstream region of the human and chimpanzee MBP gene is preferably used for gene therapy, but the promoter present in the upstream region of the mouse MBP gene also has 90% homology with the human promoter. Yes, it can be used.

Further, the MBP promoter region has a size of 1.3.
If a transcription activity that is the same as or greater than that of the kb promoter region is desired, a partially deleted one may be used.
Or a modified one can be used.

CD4 Gene The CD4 gene used in the present invention is, for example, located downstream of the MBP promoter, incorporated into a cosmid for adenovirus expression (pAdexMBPCD4), and introduced into oligodendrocytes. Furthermore, between the MBP promoter and the CD4 gene, the IRES of picornavirus
Activation of a CD4 protein, such as a sequence, translation of a lentivirus TAR sequence, etc., a transcription promoting sequence, or a Kozak sequence, which is a translation initiation signal, to increase the expression of a CD4 protein;
A regulatory sequence may be included.

The CD4 gene has at least 458
A 1.7 kb region containing an open reading frame encoding an immature CD4 molecule of amino acid residues may be used, and the third region, which is an HIV binding region, may be used.
As long as the region (CDR3) is included, an extracellular region can be used as long as the binding ability to HIV is not reduced.

Recombinant Adenovirus The recombinant adenovirus vector of the present invention and a method for producing the same will be described in detail below. The recombinant adenovirus vector of the present invention can be produced, for example, by the following procedure.

Adenoviruses of animal origin that can be used in the present invention include not only human adenoviruses, but also dogs, cattle,
Adenoviruses of murine, ovine, avian, porcine, or simian origin are mentioned, but human adenoviruses are preferable, and those derived from type 2 or 5 among human adenoviruses are particularly preferable.

The recombinant adenovirus vector used in the present invention should preferably be defective, that is, the recombinant adenovirus vector cannot be propagated autonomously in the transfected cells. To that end, the autonomous replication ability can be abolished by removing some or all of the regions essential for adenovirus growth, making them non-functional, or modifying them by inserting heterologous DNA sequences. . More specifically,
Cosmids currently used for preparing recombinant adenovirus vectors (pAdex1w, pCMVX, etc.)
To control E1A and / or E1A in order to suppress virus replication in vivo by known genetic engineering methods,
1B and the deletion of the E3 region, E1A and / or E1
It is constructed by inserting a gene construct comprising a promoter, a target gene, and a polyadenylation signal (polyA) so that the target gene is expressed in an arbitrary restriction enzyme site such as SwaI provided in the B deletion region. The gene construct can be inserted in either the forward or reverse transcription of the adenovirus genome, but is preferably inserted in the opposite direction, which results in higher gene expression. Adenovirus vectors can hold and carry foreign DNA sequences of up to about 8.5 kb and can be produced with a titer of 10 ¹¹ pfu / ml or more.

The recombinant adenovirus vector of the present invention is basically produced by co-transfecting genomic DNA of wild-type adenovirus and a cosmid for preparing a vector into, for example, 293 cells, and performing homologous recombination in the cells. Is done. 293 cells are adenovirus E1
It has been transformed by the A and E1B genes, and the recombinant adenovirus vector can grow only in these cells. It is known that the wild-type adenovirus genome is linked to a linear adenovirus genomic DNA, a DNA sequence derived from pBR322, and is functioning as a primer in plasmid adenovirus genomic DNA or as a primer during virus replication. The terminal protein (TP) is a linear adenovirus genome DN
A is supplied by DNA-TCP or the like which is bound to both ends of A.

For the preparation of the recombinant adenovirus vector used in the present invention, a COS using DNA-TCP, which can easily prepare a recombinant adenovirus vector having a high titer and little contamination with wild-type adenovirus, is used. -TCP method (Miyake S., et al., Proc.
Natl. Acad. Sci. U. S. A. , 9
3, 1320-1324, 1996) are particularly preferred.

The linear adenovirus genomic DNA (DNA-TCP) with terminal protein (TP) prepared from the wild-type adenovirus used in the COS-TCP method is as follows:
First, it is desirable to process with EcoT221. Ec
oT221 is E1A of genomic DNA of DNA-TCP,
To cut the region containing E1B at seven sites, EcoT22 was used.
The use of DNA-TCP treated with 1 can reduce the contamination of wild-type adenovirus. E
DNA-TCP treated with coT221 and, for example, cosmid pAdex1 for preparing a recombinant adenovirus vector
PAdexMBPCD4 incorporating a CD4 gene expressed by the MBP promoter and an arbitrary gene expression construct consisting of rabbit β-globin polyA into calcium phosphate method, lipofection method using various cationic lipids, electroporation method, etc. Co-transfect 293 cells by known methods.

After culturing at 37 ° C. for 24 hours, the cells were re-seeded into a 96-well plate and cultured for further 7 to 15 days, and the recombinant adenovirus produced by homologous recombination between viral DNA and cosmid was obtained. The culture solution is collected from the wells in which the vector has proliferated and the cells have died. Each culture solution is repeatedly freeze-thawed several times, and the supernatant liquid obtained by centrifugation to remove cell components is used as a primary virus solution. 3 of this primary virus solution
Since the wild-type adenovirus is mixed, the primary virus solution is added to 293 cells seeded on a 24-well plate, infected and cultured for 3 days to isolate only the recombinant adenovirus. The culture solution is recovered from the wells in which the cells have completely died, and a secondary virus solution is obtained by freeze-thawing and centrifuging several times as in the preparation of the primary virus solution. At this time, cells that have died at this time are collected and all DNA is extracted, and then partially cut with a restriction enzyme and subjected to electrophoresis to confirm the recombinant virus DNA.

The secondary virus solution of the clone in which the recombinant virus DNA was confirmed was used to infect a large amount of 293 cells in order to prepare a recombinant virus to be used in subsequent experiments. Obtain the Ad virus tertiary virus solution. This tertiary virus solution can be used in subsequent experiments as a CD4 / Ad vector virus solution.

The dosage of the tertiary virus solution used is determined according to various parameters, in particular according to the administration method used, the disease, the gene expressed or the time period desired for treatment. Generally speaking, a recombinant adenovirus vector according to the present invention comprises 10 ⁴ to 10 ¹⁴ pfu.
(Plaque forming unit) / ml, preferably 10 ⁶ to 1
It is formulated and administered in a dosage form of 0 ¹⁰ pfu / ml. Techniques for determining the pfu titer of virus solutions are well documented in the literature (Kanegae Y.,
et al. , Jpn. J. Med. Sci.
Biol. , 47, 157-166, 199
4).

HIV Vector Hereinafter, the recombinant HIV vector of the present invention and a method for producing the same will be described in detail. The recombinant HIV vector of the present invention can be produced, for example, by the following procedure.

First, it is necessary to express a functional HIV gene product sufficient for producing a recombinant HIV vector, and to package HIV genomic RNA into HIV virions because it is not itself incorporated into HIV virions. A region (packaging signal) between the downstream of the 5 ′ major splicing donor site and the upstream of the gag gene start codon, which is a sequence required for (1), is deleted. That is, a gene group containing the gag, pol, and env genes of HIV and lacking a packaging signal was prepared, and a gene construct having a cytomegalovirus (CMV) promoter arranged upstream thereof was replaced with an ampicillin resistance gene and SV4.
A helper plasmid inserted into an expression vector containing a 0 origin of replication is constructed by a known gene manipulation method. The above genes may be constructed based on the genome sequences of immunodeficiency virus in mammals, including HIV-1, HIV-2, and simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and the like. it can. By removing the packaging signal from the helper plasmid, RCR (Replication compe
in the sense that it is possible to reduce the probability of the emergence of
The gag, pol, and env genes are, for example, gag, po
2 or 3 plasmids
A recombinant HIV virus vector can be prepared by reconstructing one or more plasmids and co-transfecting them. Furthermore, vif, vpr, vpu, tat, rev, nef other than the gag, pol, and env genes essential for constituting HIV.
The gene or one or more of the corresponding accessory genes can be arbitrarily deleted to enhance safety.

[0030] The promoter used to express the HIV gene is not limited to CMV.
RSV, TK, SV40, SR-α, β-actin,
A promoter such as EF1-α can be used. Also, for example, viral enhancer proteins such as herpes virus and hepatitis B virus or NFκ-
An enhancer sequence that enhances the promoter can be provided by a cellular transcription activating protein such as B or SP-1, and a recombinant HIV vector can be prepared by co-transfection of a plasmid encoding these proteins. .

The cells for preparing the recombinant HIV vector are as follows:
Although any cell line capable of producing any HIV protein can be used, preferably a mammalian cell line, such as CV-1, HeLa, Raji, RD, S
W480, CHO, COS, 293 cells, SV40 l
293T cells and the like, particularly, COS, 293T
Cells are preferred, more preferably 293T cells.

Next, it is packaged in an HIV virion, then reverse transcribed in a target cell, integrated into a chromosome, and sequentially HIV-terminally in the 5 ′ to 3 ′ end necessary for expressing a therapeutic gene. LTR (U3, R, U5
Region), a packaging signal, an optional promoter for expressing the therapeutic gene, the therapeutic gene and the gene construct in which the HIV-LTR is located, for example, was inserted into an expression plasmid containing the ampicillin resistance gene and the SV40 replication origin. A vector plasmid is constructed by a known gene manipulation method. A polyadenylation signal sequence can be added to the therapeutic gene, if necessary. There is no particular limitation on any promoter for expressing the therapeutic gene. For example, CAG, CMV, RS
V, TK, SV40, SR-α, MBP, β-acti
n, EF1-α and the like can be used. In addition, the LTR at the 5 ′ end of the therapeutic gene and the promoter for expressing the same has the U3 region of
AG, CMV, RSV, TK, SV40, SR-α,
A chimeric LTR substituted with a promoter such as MBP, β-actin, EF1-α or the like can be used. The above vector plasmid can be constructed based on the genome sequences of immunodeficiency virus in mammals including HIV-1, HIV-2 and simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV) and the like. it can.

The constructed plasmid is co-transfected into, for example, COS cells by a known method such as a calcium phosphate method, a lipofection method using various cationic lipids, or an electroporation method. In order to efficiently produce a recombinant HIV vector,
The cells are cultured at the optimum temperature for various cells for 24 to 72 hours, preferably 24 hours or more, and more preferably 48 hours or more. The recombinant HIV vector released into the culture supernatant was obtained by
It can be prepared as a recombinant HIV vector solution by centrifuging at 0 rpm for 10 minutes or by using a filter having a pore size of 0.45 μm to remove contaminants derived from cells. Furthermore, impurities can be removed by a density gradient centrifugation method, a PEG precipitation method, or a column method such as a cellulose sulfate column or an affinity column.

Therapeutic gene The HIV vector contains a therapeutic gene.
Target diseases for which gene therapy is performed in cerebral white matter degeneration (leukodystrophy: LD) include metachromatic cerebral white matter degeneration (MLD), adrenoleukodystrophy (ALD), and Peli
There are zaeus-Merzbacher disease (PMD) and the like, and the therapeutic genes for each include arylsulfatase A (ASA), adrenoleukodystrophy protein (ALDP), and proteolipid protein (PLP) gene.

[0035] Oligodendroma, an oligodendroglioma, which is a type of brain tumor, is a brain tumor derived from oligodendrocytes and is a subject disease of the present invention. Therapeutic genes include, for example, IL-2 gene, GM-CSF
Genes such as cytokine genes, herpes virus thymidine kinase (hsv-tk) gene, suicide genes such as cytosine deaminase (CD) gene, p53 gene,
Examples include cancer suppressor genes such as the Rb gene. Also, ra
s gene, CRIPTO gene, c-fos gene, c
-Myc gene, oncogene, VEGF gene, MM
Antisense, ribozyme, or triplex-forming oligonucleotide (TFO) targeting a gene involved in tumor angiogenesis such as the P gene can also be used as the therapeutic gene of the present invention.

In order to efficiently express a therapeutic gene, a promoter can be introduced upstream of the therapeutic gene. Such a promoter used for expressing a therapeutic gene is not limited, and CAG,
CMV, RSV, TK, SV40, SR-α, MB
Promoters such as P, β-actin and EF1-α are mentioned, and CAG, CMV and SV40 promoters are preferably used. In particular, the CAG promoter is
It is constructed by incorporating the enhancer region of the CMV promoter (CMV-IE enhancer) upstream of the hybrid promoter in which the splicing acceptor of the rabbit β-globin gene is connected downstream of the intron region of the in β-actin promoter, and is used in a wide variety of cell types. It is known that gene expression is possible and gene expression level is high (Ni
wa H. , Et al. , Gene, 108,
193-200, 1991), which is particularly desirable as a promoter used in the practice of the present invention. Further, as a promoter for expressing a relatively short RNA such as ribozyme, antisense, triple-stranded RNA, and RNA decoy, tR is used.
Promoters such as NAmet, tRNAval, U6, and VA1 can be used. (Method of Administering Recombinant Adenovirus and Recombinant HIV Vector to Oligodendrocytes) When administering the recombinant adenovirus and recombinant HIV vector of the present invention, adsorption to cells or gene transfer into cells is performed. Compositions for enhancing, for example, liposomes such as cationic liposomes and immunoliposomes, polycations such as DMRIE, polyethyleneimine and polybrene, or polypeptides such as fibronectin and polylysine can be added.

As an approach for introducing a gene into the central nervous system in which oligodendrocytes are present, for example, the method of the present invention may be applied to a site of fibrotic gliosis due to myelin shedding in leukodystrophy or a brain tumor site containing oligodendrocyte glioma. An in vivo method of directly introducing the recombinant virus vector is possible. Specifically, after fixing the head with a stereotaxic apparatus, three-dimensional coordinate values of the target site are determined using CT and MRI, and by using a surgical microscope and a neuroendoscope together. It is possible to administer the recombinant viral vector directly to the target site under visible conditions.

It is preferable to administer the HIV virus vector 12 to 72 hours after administration of the recombinant adenovirus vector, since the CD4 protein can be sufficiently expressed in an oligodendrocyte-specific manner.
More preferably, it is administered 24 to 48 hours later.

As described above, the present invention provides (1) an MB that specifically expresses the CD4 gene in oligodendrocytes.
The recombinant adenovirus vector comprises (1) a recombinant adenovirus vector having a P promoter and a CD4 gene, and (2) an HIV vector having a function of recognizing CD4 and expressing a therapeutic gene. Is administered to an oligodendrocyte to sufficiently express CD4, and further, the method of (2) administering an HIV vector enables a therapeutic gene to be expressed in an oligodendrocyte-specific manner.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0041]

EXAMPLES (Example 1) Recombinant adenovirus vector
Preparation of Recombinant Adenovirus Vector (CD4 / Ad Vector) Used for Expressing CD4 Molecule on Cell Surface, Expression of CXCR4 Molecule (2nd HIV Receptor, One of Chemokine Receptors) on Cell Surface Adenovirus vector (CXCR4 / A) used for
d vector) by a known method (Proc. Natl. Ac).
ad. Sci. U. S. A. , 93, 1320-
1234, 1996) by the COS-TCP method.

An expression cosmid pAdexMBPC into which genes encoding the MBP promoter and the CD4 molecule have been inserted
D4 (Fig. 1) or CAG promoter and CXC
Expression cosmid p into which gene encoding R4 molecule is inserted
AdexCAGCXCR4 (FIG. 2) was constructed by a known method and treated with EcoT221 for parental virus DNA-
Each 293 cells were co-transfected with TCP (FIG. 3). After overnight cultivation, re-seed in a 96-well dish and cultivation for 7 to 15 days.
The culture was recovered from the wells where the virus grew and the cells died. Each culture was repeatedly freeze-thawed several times and centrifuged to remove the cell components, and the supernatant was used as the primary virus solution.

The primary virus solution was added to 293 cells seeded on a 24-well plate, infected, and cultured for 3 days. The culture solution was recovered from the wells in which the cells had completely died, and a secondary virus solution was obtained by freeze-thawing and centrifuging several times as in the preparation of the primary virus solution. At this time, the cells that had died at this time were collected, and after extracting the total DNA, the DNA was partially cut with a restriction enzyme and subjected to electrophoresis to confirm the recombinant virus DNA.

The secondary virus solution of the clone in which the recombinant virus DNA was confirmed was used to infect a large amount of 293 cells in order to prepare a recombinant virus to be used in subsequent experiments. Ad vector, CXC
A tertiary virus solution of the R4 / Ad vector was obtained. This tertiary virus solution is a CD4 / Ad vector solution, CXCR4 / A
It was used as a d vector solution in the subsequent experiments.

(Example 2) Preparation of recombinant HIV vector
A recombinant HIV vector capable of specifically introducing a gene into cells expressing the CD4 molecule produced on the cell surface is obtained by the method of Shimada T., e.
t al. , J. et al. Clin. Invest. ,
88: 1043, 1991).

A gene construct in which the cytomegalovirus promoter was located upstream of a group of genes containing the gag, pol, and env genes of HIV and lacking a packaging signal was inserted into an expression vector containing the ampicillin resistance gene and the replication origin of SV40. Plasmid pCGPE
(-) (FIG. 4) and HI sequentially from the 5 'end to the 3' end
A vector plasmid HXCAGEGFP (FIG. 5) obtained by inserting a gene construct in which a V-LTR, a CAG promoter, a green fluorescent protein (EGFP) gene and an HIV-LTR were placed in an expression vector containing an ampicillin resistance gene and an SV40 replication origin was used as a calcium phosphate. After cotransfection into COS cells by the method, the cells were cultured for 2 days. The obtained culture supernatant was centrifuged at 2000 rpm for 10 minutes, and the precipitate was removed to obtain a GFP / HIV vector solution.

(Example 3) Rat primary oligode
Selective Residue to Recombinant HIV Vector to Ndrocyte
Gene introduction rat primary brain cells were prepared according to the method of Bottenstein (Bottenstein J.
E. FIG. , Proc. Natl. Acad. Sc
i. U. S. A. , 83, 1955-1959,
1986).

CD4 / Ad prepared according to Example 1
Rat primary brain cells (5 ul) in which 1 ul of the vector solution and the CXCR4 / Ad vector solution were seeded on a
x10 ⁴ cells / well). 2 days C
After culturing in an O ₂ incubator, an EGFP / HIV vector solution was added at 0.1 ml / well. After culturing in a CO ₂ incubator for another 2 days,
Cells were fixed with 1% paraformaldehyde and oligodendrocyte-specific antibodies (anti-Carbonic A
nhydrorase II antibody) (FIG. 6,
7, 8).

As a result, cells transfected with the recombinant HIV vector emit green fluorescence (GFP positive).
This was confirmed (FIG. 7). Furthermore, the GFP-positive cells were oligodendrocytes (FIGS. 6 and 8), and it was confirmed that the gene was introduced specifically to oligodendrocytes.

[0050]

Industrial Applicability The composition for oligodendrocyte-specific gene transfer of the present invention comprises a myelin basic protein (MB).
P) Consists of a recombinant adenovirus vector having the CD4 gene downstream of the promoter and a recombinant human immunodeficiency virus (HIV) vector containing a therapeutic gene.
Therefore, a recombinant adenovirus vector encoding the CD4 gene was introduced into the oligodendrocyte downstream of the myelin basic protein (MBP) promoter to transiently express the CD4 molecule. By introducing a recombinant human immunodeficiency virus (HIV) vector containing a therapeutic gene by recognizing the CD4 molecule, the therapeutic gene can be expressed in oligodendrocytes specifically and for a long time.

In addition, it is possible to introduce a therapeutic gene specifically for oligodendrocytes, which cannot be achieved by conventional gene therapy or DDS, thereby providing an effective therapeutic method for many diseases.

[Brief description of the drawings]

FIG. 1 shows the MBP promoter and CD4 used in Example 1.
Expression cosmid pAd into which gene encoding molecule is inserted
It is a figure which shows exMBPCD4.

FIG. 2 shows the CAG promoter and CXC used in Example 1.
Expression cosmid p into which gene encoding R4 molecule is inserted
It is a figure which shows AdexCAGCXCR4.

FIG. 3 is a view showing parental virus DNA-TCP used in Example 1.

FIG. 4 shows a plasmid pCGPE (−) used in Example 2.
FIG.

FIG. 5: Vector plasmid HXCA used in Example 2
It is a figure showing GEGFP.

FIG. 6 is a micrograph showing the staining of the brain cells obtained in Example 3.

FIG. 7 is a fluorescence micrograph of the brain cells obtained in Example 3.

8 is a photograph obtained by superimposing the micrographs of FIGS. 6 and 7. FIG.

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Claims (2)

[Claims] 1. An oligodendrocyte-specific recombinant adenovirus vector comprising a recombinant adenovirus vector having a CD4 gene downstream of a myelin basic protein (MBP) promoter and a recombinant human immunodeficiency virus (HIV) vector containing a therapeutic gene. For gene transfer. 2. A recombinant adenovirus vector encoding the CD4 gene is introduced into the oligodendrocyte downstream of the myelin basic protein (MBP) promoter to transiently express the CD4 molecule, A method for specifically and for a long term expressing a therapeutic gene in oligodendrocytes by introducing a recombinant human immunodeficiency virus (HIV) vector containing a therapeutic gene by recognizing the CD4 molecule expressed in the gene.