JP2009034029A - Virus vector system having retrograde transport capacity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lentiviral vector system, especially HIV-1 viral vector system, having a high frequency retrograde transport capacity in the brains of mammals including primates. <P>SOLUTION: A kit for preparing a viral vector comprises (1) a packaging plasmid containing the gag and pol genes of HIV-1, (2) a packaging plasmid containing the accessory gene of HIV-1, (3) a transfer plasmid containing a target gene, and (4) an envelope plasmid containing a gene encoding the glycoprotein (RV-G) of rabies virus as an envelope gene. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention particularly relates to a viral vector system having a high degree of retrograde transport ability in the brain, in particular, a lentiviral vector system pseudotyped with a glycoprotein of rabies virus, a gene introduction method using the viral vector system, and It relates to gene therapy methods.

Non-proliferating (non-replicating) recombinant lentiviral vectors are used for gene therapy for various diseases such as a system in which a target gene is transported to non-dividing cells in the central nervous system (CNS) and maintained for a long period of time. It has been used as a vector in many studies (Non-Patent Documents 1 to 4). In particular, primate lentiviral vectors derived from HIV-1 (human immunodeficiency virus type 1) are the most proven vectors for gene therapy (Non-Patent Documents 5 to 8).

On the other hand, for gene therapy for certain types of cranial nerve disease, the target is a cell body that is infected from a nerve terminal site, transported axons retrogradely, and located at a target site located away from the infected site. A viral vector capable of introducing a gene to be transformed is useful.

So far, the recombinant HIV-1 virus has used the V gene (VSV-G) of vesicular stomatitis virus (VSV) as an envelope glycoprotein (envelope gene protein) (pseudotyped) Although a retrograde transport system in the cynomolgus monkey brain has been developed using this, the retrograde transport of this vector has not been efficient (Non-patent Document 9). In the method described in this document, as confirmed by immunostaining, very few cells of the central nervous system were retrogradely infected by recombinant HIV-1 virus injected into monkey striatum. It was.

  On the other hand, rabies virus (RV) is known to be infected from the synaptic terminal and have the activity of transporting axons retrogradely. In fact, it has been reported that the retrograde transport ability of non-primate lentiviral vectors based on equine anemia virus is enhanced by RV-G (Non-patent Documents 10 and 11 and Patent Document 1).

Furthermore, although HIV-1 lentivirus pseudotyped with RV-G has been reported (Non-patent Document 3), no animal experiments (in vivo) using this viral vector have been actually conducted in this report. In addition, gene transfer in the CNS of HIV-1 vector pseudotyped with glycoprotein of mocola lyssavirus, which is a neurotrophic virus causing rabies, or VSV-G has been studied. As a result of the injection of the HIV-1 vector pseudotyped with mocolarissa virus glycoprotein or VSV-G into the nostril of rats, these vectors were comparable to each other in terms of retrograde transport to the olfactory nervous system (non- Patent Document 12). In addition, this document does not describe an example in which a viral vector is administered via the striatum.
Special table 2004-517057 gazette NALDINI, L., BL &Ouml; MER, U., GAGE, FH, TRONO, D., and VERMA, IM (1996) .Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, 11382-11388. REISER, J., HARMISON, G., KLUEPFEL-STAHL, S., BRADY, RO, KARLSSON, S., and SCHUBERT, M. (1996) .Transduction of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proc. Natl. Acad. Sci. USA 93, 15266-15271. MOCHIZUKI, H., SCHWARTZ, JP, TANAKA, K., BRADY, RO, and REISER, J. (1998) .High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells.J. Virol. 72, 8873-8883. MITROPHANOUS, KA, YOON, S., ROHLL, JB, PATIL, D., WILKES, FJ, KIM, VN, KINGSMAN, SM, KINGSMAN, AJ, and MAZARAKIS, ND (1999). Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther. 6, 1808-1818. KORDOWER, JH, EMBORG, ME, BLOCH, J., MA, SY, CHU, Y., LEVENTHAL, L., MCBRIDE, J., CHEN, E.-Y., PALFI, S., ROITBERG, BZ, BROWN , WD, HOLDEN, JE, PYZALSKI, R., TAYLOR, MD, CARVEY, P., LING, Z., TRONO, D., HANTRAYE, P., D &Eacute; GLON, N., and AEBISCHER, P. (2000 ). Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease.Science 290, 767-773. MARR, RA, ROCKENSTEIN, E., MUKHERJEE, A., KINDY, MS, HERSH, LB, GAGE, FH, VERMA, IM, and MASLIAH, E. (2003). Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23, 1992-1996. ROSENBLAD, C., GEORGIEVSKA, B., and KIRIK, D. (2003). Long-term striatal overexpression of GDNF selectively downregulates tyrosine hydroxylase in the intact nigrostriatal dopamine system. Eur. J. Neurosci. 17, 260-270. LO BIANCO, C., SCHNEIDER, BL, BAUER, M., SAJADI, A., BRICE, A., IWATSUBO, T., and AEBISCHER, P. (2004) .Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an α -synuclein rat model of Parkinson's disease.Proc. Natl. Acad. Sci. USA 101, 17510-17515. KITAGAWA, R., MIYACHI, S., HANAWA, H., TAKADA, M., and SHIMADA, T. (2007). Differential characteristics of HIV-based versus SIV-based lentiviral vector systems: gene delivery to neurons and axonal transport of expressed gene. Neurosci. Res. 57, 550-558. MAZARAKIS, ND, AZZOUZ, M., ROHLL, JB, ELLARD, FM, WILKES, FJ, OLSEN, AL, CARTER, EE, BARBER, RD, BABAN, DF, KINGSMAN, SM, KINGSMAN, AJ, O'MALLEY, K , and MITROPHANOUS, KA (2001). Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.Human Mol. Genet. 10, 2109-2121. AZZOUZ, M., RALPH, GS, STORKEBAUM, E., WALMSLEY, LE, MITROPHANOUS, KA, KINGSMAN, SM, CARMELIET, P., and MAZARAKIS, ND (2004) .VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model. Nature 429, 413-417. DESMARIS, N., BOSCH, A., SALA &Uuml; N, C., PETIT, C., PR &Eacute; VOST, M.-C., TORDO, N., PERRIN, P., SCHWARTZ, O., DE ROCQUIGNY, H., and HEARD, JM (2001). Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycoproteins. Mol. Ther. 4, 149-156.

Accordingly, an object of the present invention is to provide a lentiviral vector system having a high frequency of retrograde transport ability (retroportability) in the brain of mammals including primates, particularly an HIV-1 virus vector system, and the virus vector system. It is to provide a gene introduction method and a gene therapy method to be used.

The present inventor uses a glycoprotein (RV-G) of a rabies virus having an infectious property that is transmitted from the synaptic terminal and retrogradely transports axons, and uses a non-proliferating recombinant lentiviral vector system. In the mouse and monkey brain, the viral vector that is actually administered to the striatum is retrogradely transported to the axon of the nerve that projects onto the striatum. The present invention has been completed by demonstrating in vivo that the gene of interest can be introduced and expressed in the target cell area away from the target cell at high frequency and introduced into the neuronal cell body in the target area.

That is, the present invention relates to the following aspects.
[Aspect 1]
(1) a packaging plasmid containing HIV-1 gag and pol genes;
(2) a packaging plasmid containing an accessory gene for HIV-1;
(3) a transfer plasmid containing a target gene (transgene); and (4) an envelope plasmid containing a gene encoding a rabies virus glycoprotein (RV-G) as an envelope gene,
A kit for preparing a retrograde transportable virus vector.
[Aspect 2]
A kit for producing a producer cell, comprising a kit for preparing a viral vector of the present invention and a host cell.
[Aspect 3]
A method for producing a producer cell, comprising co-transfecting an infected cell with a packaging plasmid, a transfer plasmid, and an envelope plasmid, which are contained in the kit for preparing a viral vector of the present invention.
[Aspect 4]
Producer cells obtained by the production method of the present invention.
[Aspect 5]
A method for producing a viral vector, comprising culturing the producer cell of the present invention and recovering virus particles from the culture supernatant.
[Aspect 6]
A viral vector having retrograde transport ability produced by the method for producing a viral vector of the present invention.
[Aspect 7]
Infecting the nerve endings of animals with the viral vector of the present invention, the viral vector is retrogradely transported through the nerve axons, and introduced into the cell body of the nerve in the target region in the brain. A gene transfer method comprising expressing the gene in the cell.
[Aspect 8]
A gene therapy agent comprising the viral vector of the present invention as an active ingredient.
[Aspect 9]
A gene therapy method for brain disease, which comprises incorporating and expressing a target gene introduced by the method of the present invention in a chromosome of a cell in a target region.

According to the present invention, in a mammal such as a monkey and a mouse, a recombinant viral vector having a specific gene to be introduced is injected into a brain region where a nerve terminal (synaptic terminal) exists, and this viral vector reverses axons. The target gene (transgene) can be efficiently introduced and expressed in a region of the cell body of the central nervous system located far from the site of infection (injection) of the viral vector. First demonstrated in vivo. In the present invention, in particular, by using a specific packaging plasmid, transfer plasmid and envelope gene, a viral vector produced using a viral vector preparation kit can obtain a very high virus titer. It has become possible clinically to produce a recombinant virus vector that exhibits a high frequency of retrograde transport ability.

Hereinafter, embodiments for carrying out the present invention will be described in detail. In the kit for preparing a viral vector of the present invention, “gag” is a gene encoding a retrovirus core protein, and “pol” is a gene encoding a reverse transcriptase or the like. The “envelope gene” is a gene encoding an envelope, which is a virus-specific protein present in the envelope, which is the outer membrane of a retrovirus composed of a lipid bilayer membrane. The envelope plays an important role for the virus to adsorb and enter cells. Furthermore, the “accessory gene” means, for example, a rev gene that regulates the expression of a structural gene.

In a preferred representative example of the kit for preparing a viral vector of the present invention, (1) a packaging plasmid containing HIV-1 gag and pol genes and (2) a packaging plasmid containing HIV-1 accessory genes, respectively, “PCAGkGP1.1R” and “pCAG4-RTR2” are used, and “pCL20c-MSCV-X (where“ X ”indicates a target gene)” is used as a transfer plasmid. The transfer plasmid encodes the target gene “X” to be introduced downstream of the mouse stem cell virus promoter.

Each of the plasmids contained in the above kit for preparing a viral vector is an HIV-1 vector system “SJ1” developed by Dr. Arthur Nienhuis of St. Jude Children's Research Hospital (HANAWA, H., et al., (2002) Mol. Ther. 5, 242-251; (2004). Blood 103, 4062-4069: provided by St. Jude Children's Research Hospital. This vector system is known to have a titer in HeLa cells of about 10 times that of other vector systems. Therefore, those skilled in the art can easily prepare each of these plasmids by referring to the present specification and the above-mentioned documents. The packaging plasmids (1) and (2) above may be constructed as a single plasmid.

  Examples of the envelope gene contained in the envelope plasmid of the virus vector preparation kit of the present invention include, for example, a rabies virus strain glycoprotein (RV-G), in particular, a specific rabies virus comprising the amino acid sequence shown in SEQ ID NO: 2. An envelope gene encoding the strain RV-G, preferably a nucleic acid having the base sequence shown in SEQ ID NO: 1 can be mentioned. In consideration of codon degeneracy, the base sequence can be appropriately changed so as to be an optimal codon according to other elements in the envelope plasmid. In this envelope plasmid, the envelope gene is linked under the expression control of any expression control sequence known to those skilled in the art.

  “Under expression control” means that a DNA encoding a predetermined amino acid sequence has the ability to express a protein having the amino acid sequence under predetermined conditions. When DNA encoding a predetermined amino acid sequence is bound under the expression control of an expression regulatory sequence, the DNA expresses a predetermined protein under predetermined conditions. Here, “expression control sequence” means a nucleic acid sequence that regulates the expression of other nucleic acid sequences, and also controls and regulates the transcription and preferably translation of other nucleic acid sequences. Expression control sequences include appropriate promoters, enhancers, transcription terminators, initiation codons (ie, ATG) in the gene encoding the protein, splicing signals for introns, polyadenylation sites, and stop codons.

  “Promoter” means a minimal sequence necessary for transcription. The promoter also includes a promoter element that controls the expression of a gene in a promoter-dependent manner by a cell type-specific, tissue-specific, or external signal or regulator. The promoter element is linked to either the 5 ′ region or the 3 ′ region of the expressed DNA. Promoters include both constitutive and inducible promoters. For example, when producing an expression vector in bacteria, promoters such as T7 RNA polymerase promoter, bacteriophage γ pL, lac, ptrp, ptac (ptrp-lac hybrid promoter) can be used as inducible promoters. A promoter known to those skilled in the art can be appropriately selected according to the type of target gene and viral vector to be used, the type of animal and brain disease to be treated, the pathology of the patient, and the like.

  In the envelope plasmid of the present invention, the envelope gene is preferably ligated so as to be expressed under the control of the cytomegalovirus enhancer and the avian β-actin promoter. As a preferred example of such an envelope plasmid, a nucleic acid encoding “VSV-G” in the envelope plasmid “pCAGGS-VSV-G” contained in the vector system “SJ1” has the base sequence described in SEQ ID NO: 1. Nucleic acid (cDNA) encoding the glycoprotein (RV-G) of the rabies virus CVS strain (passed by Prof. Kinjiro Morimoto, National Institute of Infectious Diseases), which has been subcultured in the brains of suckling mice (Morimoto, K. et. al., (1998) Proc Natl. Acad. Sci., USA 95, 3152-3156) and “pCAGGS-RV-G” obtained by a conventional method. Therefore, those skilled in the art can easily prepare these plasmids by referring to the present specification and the above-mentioned documents.

  The target gene contained in the transfer plasmid can be appropriately selected from those known to those skilled in the art according to the intended use of the viral vector, the type of animal and brain disease to be treated, the patient's condition, etc. . Therefore, various genes of mammals such as mice, monkeys and humans, for example, genes necessary for the survival or protection of the nigrostriatal system used for the treatment of cranial nerve diseases or neurodegenerative diseases represented by Parkinson's disease (For example, tyrosine hydroxylase, glial cell line-derived neurotrophic factor) and the like.

Host cells included in the producer cell preparation kit of the present invention can be infected by the above-described virus vector preparation kit, and as a result, cells that produce retroviral particles called “producer cells” can be prepared. There is no particular limitation as long as it is a cell, and any cell known to those skilled in the art, for example, HEK293T cells (introduced with SV40 large T antigen) and other suitable animal-derived cells should be used. Can do.

The various kits of the present invention include, for example, various reagents, buffers, various auxiliary agents, reaction plates (containers), etc., in addition to the plasmids and / or host cells, depending on the configuration and purpose of use. Other elements or components known to those skilled in the art can be included as appropriate.

Using the producer cell preparation kit of the present invention, a producer cell can be prepared by co-transfecting the packaging plasmid, transfer plasmid, and envelope plasmid contained in the virus vector preparation kit into an infected cell. I can do it. This transfection is transient and can be performed by any method known to those skilled in the art, such as the calcium phosphate method.

Producer cells obtained in this way are cultured by any method / means known to those skilled in the art, and virus particles are recovered from the culture supernatant, thereby having a retrograde transport ability in the brain and exhibiting a high titer. Vectors can be manufactured.

Infecting the nerve endings of animals with the viral vector of the present invention, the viral vector is retrogradely transported through the nerve axons, and introduced into the cell body of the nerve in the target region in the brain. Can be expressed in the cell body. The nerve ending is, for example, in the striatum, and the target area in the brain is, for example, a striatum such as primary motor cortex, primary somatosensory cortex, parathalamic paranucleus, and / or substantia nigra. It is the central region of the brain that projects onto the body.

Therefore, the virus vector of the present invention is effective as an active ingredient of a gene therapy agent. The gene therapy agent can include other ingredients such as any pharmaceutical carrier or diluent known to those of ordinary skill in the art in combination with the active ingredient in combination with the active ingredient.

The effective amount of the active ingredient of the present invention is the type of transgene contained in the viral vector, the type / seriousness of brain disease or neurodegenerative disorder, treatment policy, patient age, weight, sex, general health condition Depending on the (genetic) racial background of the patient, those skilled in the art can select as appropriate. The dose of the active ingredient (virus vector) can be, for example, a total amount of 10 ⁷ to 10 ⁸ TU (Transducing Unit) at several sites of infection (injection) per administration. The virus vector or gene therapy agent can be infected (injected) into a predetermined site of a patient using any administration method / device known to those skilled in the art.

By administering the virus vector of the present invention to a patient, the gene introduced into a predetermined cell in the target region is integrated into the chromosome of the cell, and the target gene is stably expressed. Therefore, gene therapy such as brain disease or neurodegenerative disease (for example, Parkinson's disease) in mammals including primates such as humans can be carried out using this gene transfer method.

  Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples. These examples show a part of the present invention, and the technical scope of the present invention is not limited by these examples. Unless otherwise specified, the experimental conditions in each operation were in accordance with the methods described in the literature cited in this specification or in accordance with standard methods in the technical field.

Virus vector preparation:
The viral vectors of the present invention were prepared utilizing the HIV-1 vector system developed by Dr. Arthur Nienhuis of St. Jude Children's Research Hospital. That is, a packaging plasmid (pCAGkGP1.1R) containing gag and pol genes, a packaging plasmid (pCAG4-RTR2) containing accessory genes, and a transfer plasmid (pCL20c-MSCV-) containing green fluorescent protein (GFP) as a target gene GFP) was used. As the envelope plasmid, a nucleic acid encoding “VSV-G” in the envelope plasmid “pCAGGS-VSV-G” contained in the above vector system is a glycoprotein of a rabies virus strain having the base sequence described in SEQ ID NO: 1 ( “PCAGGS-RV-G” obtained by replacing the nucleic acid (cDNA) encoding RV-G) by a conventional method was used.

Viral vector solutions containing these plasmids were transfected into HEK293T cells using the calcium phosphate method. After culturing for 48 hours, virus particles were collected from the culture supernatant, centrifuged, and filtered through a 0.45 μm cellulose filter. Next, the viral vector contained in this supernatant was concentrated by ultracentrifugation (100,000 × g), suspended in phosphate buffered saline (PBS) containing 0.1% bovine serum albumin, and stored at −80 ° C. . In order to evaluate the virus titer, HEK293T cells were added to a 6-well cell culture plate: MULTIWELL (registered trademark). FALCON), the cultured cells were infected with a virus solution of an appropriate concentration, and titer was measured 3 days after transformation using FACS Calibur (Nippon Becton Dickinson Co., Tokyo, Japan). As a result, it was confirmed that a virus solution of about 5 × 10 ⁸ TU / ml was obtained.

More specifically, a viral vector was produced by the following procedure.
(1) antibiotic 293T cells (3x10 ⁶ cells / 10 cm of material containing DMEM 10% FBS (10 ml) dish: CELL STAR ( TM) gleiner bio-one) was seeded approximately 24 hours prior to transfection, or confluent 293T cells were split 1: 7 and incubated at 5% CO ₂ , 37 ° C.
(2) 2xHBSP, CaCl ₂ and sterile milli-Q water were warmed to room temperature.
(3) In a 15 ml conical tube, the following DNA mixture:
pCAGkGP1.1R 6 μg;
pCAG4-RTR2 2 μg;
pCAGGS-VSV-G or pCAGGS-RV-G 2 μg; and
pCL20c MSCV-GFP 10 μg,
Sterilized milli-Q water was added to 450 μl, and 2.5 M CaCl ₂ 50 μl was added thereto.
(4) 500 μl of 2 × HBSP was added dropwise with stirring.
(5) The DNA-CaCl ₂ -HBSP mixture obtained above was dropped onto 293T cells and mixed well. The incubation time of the DNA-CaCl ₂ -HBSP mixture varies depending on the lot of HBSP. When adding the DNA-CaCl ₂ -HBSP mixture to the culture dish, it was added slowly and evenly and then the dish was slowly mixed. After dropping, it was examined whether or not particles were formed. I saw trash like garbage. After incubation at 37 ° C. for 1 h, it was examined again. As a result, DNA particles were clearly observed at the bottom of the dish.
(6) After about 18 hours of incubation, the medium was aspirated, washed well twice with warm PBS (5 ml), and antibiotic-containing DMEM 10% FBS (7 ml) was added thereon.
(7) After about 12 hours, the conditioned medium was collected, centrifuged at a low speed (1,200 rpm, 10 min, 4 ° C.), and this was added to a 0.45 μm cellulose filter (“Millex” (registered trademark): PVDF membrane, Millipore Co., Ltd.). cat No. SLHV033RB). Furthermore, antibiotic-containing DMEM 10% FBS (7 ml) was added, and after about 12 hours, a second collection operation was performed.
(8) The virus vector contained in the supernatant thus recovered was concentrated by ultracentrifugation (25,000 rpm 90 min 4 ° C., SW28) or ultrafiltration (Centricon (registered trademark) Plus-80, Amicon). However, when performing ultrafiltration, a serum-free medium is required. After ultracentrifugation, the virus ppt was clearly visible at the bottom of the tube.
(9) The virus ppt was suspended in PBS / 0.1% BSA solution, frozen at a constant rate with dry ice, and stored at −80 ° C.

In addition, the preparation of the reagent used by said method is as follows.
CaCl ₂ 2.5M
Water was added to 2.5M CaCl ₂ 2H ₂ O (36.76 g) to make 100 ml, sterilized by filtration through a 0.45 μm filter, and aliquoted (10 ml) and stored at −20 ° C.
10x HBSP stock solution
Add 1.4M NaCl (40.9g), 50mM HEPES (29.8g), 7.5mM Na ₂ HPO ₄ · 12H ₂ O (1.35g), 50mM KCl (1.9g) and 60mM glucose (5.4g) to 500ml And sterilized by filtration through a 0.45 μm filter and stored at 4 ° C.
2x HBSP stock solution
Mix 100ml 10x HBSP stock solution and water (300ml), adjust to pH 7.05 (7.05-7.20) with 1N NaOH, add water to 500ml, filter through 0.45 μm filter, sterilize, and add a certain amount ( 50 ml) and stored at -20 ° C. After thawing, it was stored at 4 ° C.

Introduction of viral vectors into the mouse brain:
Animal breeding and handling operations were performed based on guidelines established by the Animal Experiment Committee of Fukushima Medical University and the Animal Experiment Ethics Committee of the Tokyo Metropolitan Institute for Neuroscience.
A 12-week-old mouse (C57BL / 6J) was anesthetized with sodium pentobarbital (50 mg / kg, ip), and the solution containing the viral vector prepared above (3.0 x 10 ⁸ TU / ml) was placed in the mouse brain ( In accordance with the mouse brain atlas (PAXINOS, G., and FRANKLIN, KBJ (2001). The Mouse Brain in Stereotaxic Coordinates, 2nd edn. (Academic Press, San Diego)) Then, 0.5 μl of the solution was injected into the dorsal region of the striatum through the glass microinjection capillary connected to the microinjection pump at two locations on the track (0.1 μl / min). The coordinates of the anteroposterior, mediolateral, and dorsoventral directions from the bregma were 0.50, 2.00, and 2.50 / 3.25 (mm), respectively.

Introduction of viral vectors into the monkey brain:
Cynomolgus monkeys (Macaca fascicularis, 25-3.5 kg) were sedated with ketamine hydrochloride (5 mg / kg, im) and xylazine hydrochloride (0.5 mg / kg, im), then pentobarbital sodium (20 mg / kg, iv The solution containing the viral vector prepared above using a Hamilton microsyringe (50 μl) (3.0 × 10 ⁸ TU / ml) was added to a monkey brain atlas (SZABO, J. and COWAN, WM (1984). A stereotaxic atlas of the brain of the cynomolgus monkey (Macaca fascicularis). According to J. Comp. Neurol. 222, 265-300), 2 μl of solution on each track and 5 μl of solution per place in the putamen Injected. The coordinates (mm) in the front, back, medial and lateral, and dorsal and ventral directions from the interauralline, midline and dura connecting the ear canals are 22, 10, 14/16, respectively. (Track 1); 20, 10, 14/16 (Track 2); 20, 8, 13/17 (Track 3); 18, 11, 14/17 (Track 4); 18, 9, 14/18 (Track) 5); 16, 12, 13/17 (Track 6); 16, 10, 13/17 Track 7); 14, 13, 13/17 (Track 8); 14, 11, 13/17 (Track 9); 12, 12, 13/16 (Track 10) and 10, 12, 16/13 (Track 11).

Four weeks after injection, the animals were deeply anesthetized with sodium pentobarbital (50 mg / kg, ip) and via the heart, in the case of mice, 4% formalin and 0.1 M phosphate buffer (PB: pH 7.4). In the case of monkeys, 10% formalin and 15% picric acid in 0.1 M PB were perfused.

Histological operation For immunostaining by avidin-biotin-peroxidase method, cross sections (for mice: 30 μm thick, for monkeys: 60 μm) were prepared using a cryostat, and rabbit anti-GFP polyclonal Incubated with antibody (Molecular Probes, Eugene, OR: 1: 2,000 dilution) and further incubated with biotinylated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA: 1: 1,000 dilution). Immune response signals were visualized with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA).

For double immunofluorescence histochemical staining, sections were incubated with the above rabbit anti-GFP polyclonal antibody and one of the following mouse monoclonal antibodies: anti-NueN antibody (1: 400 dilution, Chemicon, Temecula, CA), Anti-glial fibrillary acidic protein (GFAP) antibody (1: 400 dilution, Sigma, St, Louis, MO) or anti-tyrosine hydroxylase (TH) antibody (1: 400 dilution, Chemicon). The sections were then incubated with FITC-conjugated goat anti-rabbit IgG and Cy3-conjugated donkey anti-mouse antibody (1: 500 dilution, Jackson, ImmunoResearch Laboratories, West Groove, PA). Fluorescence images were captured under a confocal laser scanning microscope (LSM510, Zeiss, Thornwood, NY) with appropriate filter cube specifications for FITC and Cy3 fluorescence channels. These fluorescence images were taken with a high-quality CCD camera system adjusted by the Zeiss Axiovision software package.

Cell counts A series of sections through the forebrain and midbrain were used to perform immunostaining with the avidin-biotin-peroxidase method described above. The number of immunostained cells in each brain region was counted with a computerized image program (NIH Image 1.62, National Institutes of Health, Bethesda, MD). For identification of striatal cells at the site of vector injection, double immunofluorescent histochemical staining was performed using representative sections. In each animal, the number of immunostained cells in the target area (0.2 x 0.2 mm) was counted using an image program. Measurements were made using 8-10 sections from each animal and the average per section was calculated.

Statistical analysis Statistical comparison was performed using ANOVA, post hoc Tukey's test and Student's t-test. Values are expressed as mean of data + SEM.

Results: Mouse (1) As a result of immunostaining with rabbit anti-GFP polyclonal antibody, when a viral vector carrying VSV-G or RV-G was injected into the striatum of the mouse, both vectors were Transgene expression was induced in many neurons (FIG. 1A).

(2) Next, the primary motor cortex (primary motor cortex :) represents that the injected viral vector is transported axon retrogradely and the target gene is expressed as a representative of the brain region projected onto the striatum. M1), primary somatosensory cortex (S1), parafascicular nucleus of the thalamus (PF), and substantia nigra pars compacta (SNc) . As a result, when a viral vector pseudotyped with VSV-G was injected, there were very few or no GFP-positive cells in these brain regions, whereas RV-G When a pseudotyped viral vector was injected, significant expression of GFP was observed in the PF region, moderate expression was observed in the M1 and S1 regions, and the expression in the SNc region was slight ( FIG. 1B). As summarized in Table 1 below, when a viral vector pseudotyped with RV-G was injected, each of the viral vectors pseudotyped with VSV-G was compared to when injected with a viral vector pseudotyped with VSV-G. The number of GFP positive cells in the brain region was significantly increased (n- = 4, p <0.001 for M1 and S1, p <0.01 for PF and SNc, Student's t-test). In Table 1, “ND” indicates that it was not detected.

In addition, axonal endings of striatal nerves showing GFP immune responses were observed in the substantia nigra pars reticulata (SNr) due to the antegrade transport of GFP. is there. The intensity of the GFP immunoreaction in the substantia nigra was lower for the virus vector pseudotyped with RV-G (FIG. 1B).

(3) The above-mentioned rabbit anti-GFP polyclonal antibody and anti-NueN (neuron marker) antibody for sections of M1, S1 and PF, and the above-mentioned rabbit anti-GFP polyclonal antibody and anti-TH (dopamine neuron for sections of SNc region) Double immunofluorescent histochemical staining using a marker antibody was performed. As a result, in mice injected with a viral vector pseudotyped with RV-G, almost all GFP positive cells in the M1, S1 and PF regions were double-stained for NueN, and in the SNc region GFP positive cells were double stained for TH (Figure 2). These results indicate that the efficiency of gene transfer via retrograde axonal transport was increased in the mouse brain by pseudotyping the HIV-1 viral vector with RV-G.

(4) Double immunofluorescent histochemical staining using the above rabbit anti-GFP polyclonal antibody and anti-NueN antibody or anti-GFAP (glial cell marker) antibody in order to examine the gene transfer pattern into mouse striatal cells (FIG. 3). As a result, in mice injected with VSV-G pseudotyped viral vectors, the number of cells double-stained for NueN and GFP was 90.2 + 6.8% of total GFP-positive cells, The number of cells double-stained for NueN and GFAP was 6.9 + 0.8% of all GFP positive cells (n = 4). In contrast, in mice injected with viral vectors pseudotyped with RV-G, the number of cells double-stained for NueN and GFP was 25.8 + 2.5% of all GFP-positive cells. On the other hand, the number of cells double-stained for NueN and GFAP was 70.6 + 4.1% of all GFP positive cells (n = 4). That is, the efficiency of gene transfer into striatal neurons was significantly lower with the virus vector pseudotyped with RV-G (p <0.001, Turkey's test). In contrast, the efficiency of gene transfer into striatal astroglia was significantly higher with the virus vector pseudotyped with RV-G (p <0.001, Turkey's test).

  As a result of the analysis using the mouse brain as described above, the virus vector of the present invention pseudotyped with RV-G has a low efficiency of transfer to the striatum itself, but it projects to the neuronal cells that project to the striatum of the mouse. It was shown to have a high frequency of gene transfer through retrograde transport to the population.

Results: The axonal retrograde transport ability of viral vectors to nigrostriatal dopamine neurons by administration into monkey (1) striatum is very important for model experiments aimed at gene therapy of Parkinson's disease. Viral vectors were injected into the striatum (putamen) of cynomolgus monkeys. As a result, even when a viral vector pseudotyped with VSV-G was injected, only a small number (15.8 cells / intercept, n = 2) of cells were introduced into monkey SNc (FIG. 4A). This result was in agreement with the recently reported result of the retrograde transport system in monkey nigrostriatal (Non-Patent Document 9). In sharp contrast, when the viral vector of the present invention pseudotyped with RV-G was injected, the gene was introduced into a large number of cells in SNc (376.9 cells / section, n = 2). This number was about 24 times that in the case of using a viral vector pseudotyped with VSV-G. In addition, the strength of the GFP immune reaction at the striatonigral axon terminals of the striatum nigra system is greater in the pseudotype of RV-G than in the case of using the viral vector pseudotyped with VSV-G. There was a decrease in the case of injecting the modified virus vector of the present invention (FIG. 4A).

(2) Next, a series of SNc sections prepared from monkeys injected with the viral vector of the present invention pseudotyped with RV-G were examined by double immunofluorescent histochemical staining for GFP and TH. Virtually all GFP-positive neurons in SNc were double-stained for TH, and many of the TH immunoreactive neurons (68.0%, n = 2) expressed the GFP transgene (FIG. 4B). These data indicate that the viral vector of the present invention pseudotyped with RV-G has a high frequency of gene transfer ability by retrograde transport to the nigrostriatal dopamine system in monkey brain. It was.

(3) Double immunofluorescent histochemical staining for GFP and NueN was performed in order to examine gene transfer efficiency into monkey striatal neurons. For the viral vector pseudotyped with VSV-G and the viral vector of the present invention pseudotyped with RV-G, the percentage of cells double-stained for NueN and GFP with respect to the total GFP-positive cells is Respectively, 51.9% and 21.4% (n = 2) (FIG. 4C). As in the case of the mouse brain, the efficiency of introduction of a viral vector pseudotyped with RV-G into striatal neurons has decreased, and for this reason, the present invention pseudotyped with RV-G. In this virus vector, it seems that the GFP immunoreaction intensity at the axonal terminal of the striatum nigra system decreased. However, in the case of monkeys compared to mice (comparison between FIG. 4A and FIG. 1B), the viral vector of the present invention pseudotyped with RV-G is converted to the nigrostriatal system by retrograde transport. It was shown that gene transfer can be performed to a higher degree.

  The contents described in the documents cited in the present specification constitute the disclosure of the present specification as a part of the present specification.

The present invention is useful for gene therapy of Parkinson's disease caused by, for example, selective degeneration of the substantia nigra-striatal system. In this case, a recombinant virus is injected into the striatum and is deeply localized in the deep brain. It is possible to efficiently introduce a gene into a dense substantia nigra. This technology provides a new and effective experimental means for gene therapy of cranial nerve diseases.

It is the photograph of the immuno-staining which shows the expression of the transgene in the brain area | region after inject | pouring a lentiviral vector in a mouse | mouth striatum. The right column of B is an enlargement of the square frame of the left column. The dotted line in the lowermost photograph (four) shows the boundary between SNc and SNr. The scale bar is 400 μm. It is a photograph which shows the result of double immunofluorescence histochemical staining about the section | slice of each area | region of M1, S1, PF, and SNc. GFP positive signal (green), NeuN or TH positive signal (red), and merged image signal (yellow), scale bar is 50 μm. It is a photograph which shows the result of the double immunofluorescence histochemical dyeing | staining which shows gene transfer in a mouse | mouth striatal cell. GFP positive signal (green), NeuN or GFAP positive signal (red), and merged image signal (yellow), scale bar is 50 μm. A: Photograph of an immunostaining showing transgene expression in the brain region after injecting a lentiviral virus vector into the striatum of the cynomolgus monkey (the fruit nucleus: putamen). The right column is an enlargement of the square frame of the left column. The scale bar is 1 mm. B: Photograph showing the results of double immunofluorescent histochemical staining for a section of the SNc region. GFP positive signal (green), TH positive signal (red), and merged image signal (yellow), scale bar is 50 μm. C: Photograph showing the results of double immunofluorescent histochemical staining in the striatum. GFP positive signal (green), NeuN positive signal (red), and merged image signal (yellow), scale bar is 50 μm.

Claims (25)

(1) a packaging plasmid containing HIV-1 gag and pol genes;
(2) a packaging plasmid containing an accessory gene for HIV-1;
(3) a transfer plasmid containing a target gene; and (4) an envelope plasmid containing a gene encoding a rabies virus glycoprotein (RV-G) as an envelope gene. RV-G has the amino acid sequence shown in SEQ ID NO: 2, the packaging plasmid (1) is pCAGkGP1.1R, the packaging plasmid (2) is pCAG4-RTR2, and the transfer plasmid is pCL20c- The kit for preparing a viral vector according to claim 1, wherein the kit is MSCV-X (where "X" represents a target gene). The kit for preparing a viral vector according to claim 1 or 2, wherein in the envelope plasmid, the envelope gene is expressed under the control of a cytomegalovirus enhancer and an avian β-actin promoter. The kit for preparing a virus vector according to claim 3, wherein the base sequence of the gene encoding the rabies virus glycoprotein (RV-G) is shown in SEQ ID NO: 1. The kit for preparing a viral vector according to claim 4, wherein the envelope plasmid is pCAGGS-RV-G. The kit for preparing a viral vector according to any one of claims 1 to 5, wherein the target gene is a human gene. The kit for preparing a viral vector according to claim 6, wherein the target gene is a gene for treating cranial nerve diseases. The kit for producer cell preparation containing the kit for virus vector preparation as described in any one of Claims 1-7, and a host cell. The kit according to claim 8, wherein the infected cells are HEK293T cells. A method for producing a producer cell, comprising co-transfecting an infected cell with a packaging plasmid, a transfer plasmid, and an envelope plasmid, which are included in the virus vector preparation kit according to any one of claims 1 to 7. . The production method according to claim 10, wherein the infected cells are HEK293T cells. The production method according to claim 10 or 11, wherein the transfection is performed using a calcium phosphate method. Producer cell obtained in claims 10-12. A method for producing a virus vector, comprising culturing the producer cell according to claim 13 and recovering virus particles from the culture supernatant. A viral vector having retrograde transport ability produced by the method according to claim 14. The virus vector according to claim 15 is introduced into the nerve ending of an animal, and the virus vector is retrogradely transported through the nerve axon to be introduced into the cell body of the nerve in the target region in the brain. A gene transfer method comprising expressing a target gene in the cell body. The gene introduction method according to claim 16, wherein the nerve ending is in the striatum, and the target region in the brain is a brain central region projected onto the striatum. 18. The method according to claim 17, wherein the brain central region projected onto the striatum is primary motor cortex, primary somatosensory cortex, parathalamic paranucleus, and / or substantia nigra. The method according to any one of claims 16 to 19, wherein the animal is a mammal. 20. The method of claim 19, wherein the mammal is a primate. 21. The method of claim 20, wherein the mammal is a human. A gene therapy agent comprising the viral vector according to claim 15 as an active ingredient. A gene therapy method for brain disease, which comprises incorporating and expressing a target gene introduced by the method according to claim 16 to 21 in a chromosome of a cell in a target region. 24. A treatment method according to claim 23, comprising administering the gene therapy agent according to claim 22 to a patient. The treatment method according to claim 23 or 24, wherein the brain disease is Parkinson's disease.