# Patent

## Title
Neuron-specific retrograde transport vector

## Abstract

The present invention provides a lentiviral vector system having a higher titer, while sustaining an excellent retrograde transport ability, particularly, in the brain.
The present invention also provides a kit for preparing a retrograde transport viral vector comprising:

    
    
        (1) a packaging plasmid containing the gag gene and the pol gene of HIV-1;
        (2) a packaging plasmid containing an accessory gene of HIV-1;
        (3) a transfer plasmid containing an target gene (a transgene); and
        (4) an envelope plasmid containing, as an envelope gene, a gene encoding a fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G, and an intracellular domain of VSV-G, and the like.

## Background
This application is a National Stage Application of PCT/JP2011/077142, filed Nov. 25, 2011, which claims priority from Japanese Patent Application No. 2010-263148, filed Nov. 26, 2010. The entirety of all of the aforementioned applications is incorporated herein by reference.
FIELD
The present invention relates to a neuron-specific retrograde transport vector (NeuRet) system, having an excellent retrograde transport ability; particularly, in the brain. The vector system has high production efficiency, particular enabling a selective transfer or introduction of a gene into the neuron. More specifically, the present application relates to a lentiviral vector system which is pseudotyped by a fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G and an intracellular domain of VSV-G; and a method of gene transfer and gene therapy using the viral vector system,
BACKGROUND ART
Non-proliferative (non-replicating) recombinant lentiviral vectors are used in a number of studies as a vector for gene therapy to treat various diseases, such as in a system which transports a target gene to a non-dividing cell in the central nervous system (CNS) and maintains its expression over a long period of time (Non-Patent Literatures 1-4). In particular, a primate lentiviral vector from HIV-1 (human immunodeficiency virus type 1) is the most proven vector for gene therapy (Non-Patent Literatures 5-8). However, it is well known that the lentiviral vector has a risk of inducing cancer because it will be integrated into chromosomes. Especially, cases have been reported where leukemia developed in the gene therapy of haematologic diseases. It has been therefore desired to selectively transfer the gene into the neurons in order to reduce the risk of occurrence of cancer and to develop a safer vector system in the gene therapy of nervous diseases.
On the other hand, for gene therapy of a certain cranial nerve disease, useful is a viral vector which can infect a nerve terminal, is retrogradely transported through an axon and introduce a target gene into a cell body in a target site located far from the infected site (FIG. 1).
To date, a retrograde transport system in the brain of cynomolgus monkey was developed using a recombinant HIV-1 virus which uses (is pseudotyped by) a vesicular stomatitis virus (VSV) glycoprotein (VSV-G) as an envelope glycoprotein (an envelope gene protein), but the retrograde transportation of the vector was not efficient (Non-Patent Literature 9). In the method described in the reference, very few cells in the central nervous system were retrogradely infected with the recombinant HIV-1 virus injected into the striatum of the monkey, as indicated by immunostaining.
On the other hand, rabies virus (RV) is known to have an activity that RV infects a synapse terminal, and is retrogradely transported through an axon. Indeed, there is a report that a retrograde transportation ability of a non-primate lentiviral vector based on equine anemia virus was promoted by RV-G (Non-Patent Literatures 10 and 11, and Patent Literature 1).
Further, HIV-1 lentivirus pseudotyped by RV-G has been reported (Non-patent Literature 3), but, in that report, an animal experiment (in vivo) was not actually conducted using that viral vector. In addition, gene transfer in CNS with a HIV-1 vector pseudotyped by a glycoprotein from Mokola lyssavirus, a neurotropic virus causing rabies, or VSV-G, has been studied. As a result of the nasal injection of the HIV-1 vector pseudotyped by the Mokola lyssavirus glycoprotein or VSV-G into a rat, these vectors were mutually comparable with regard to retrograde transportation to the olfactory nerve system (Non-Patent Literature 12). In addition, in that literature, an example in which a viral vector was administered through striatum was not described.
To date, the present inventors have revealed that highly-frequent retrograde gene transfer at various regions in the brain can be feasible by preparing a HIV-1 lentivirus vectors pseudotyped by rabies virus glycoprotein gene (RV-G) (RV-G/HIV-1 vector) (Patent Literature 2, Hum. Gene Ther., 2007). Furthermore, said inventor prepared the fused glycoprotein (FuG-B) wherein an intracellular domain of RV-G was replaced by that of VSV-G, and succeeded in constructing a lentiviral vector system that had a higher titer (functional titer) while sustaining an excellent (highly efficient) retrograde transport ability so as to significantly increase the frequency of retrograde gene transfer or introduction (Hum. Gene Ther., 2010).
REFERENCE LIST
Patent Literature


    Patent Literature 1: National Publication of International Patent Application No. 2004-517057
    Patent Literature 2: Japanese Patent Laid-Open No. 2009-34029


Non Patent Literature


    Non-Patent Literature 1: NALDINI, L., BLÖMER, U., GAGE, F. H., TRONO, D., and VERMA, I. M. (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.
    Non-Patent Literature 2: REISER, J., HARMISON, G., KLUEPFEL-STAHL, S., BRADY, R. O., 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.
    Non-Patent Literature 3: MOCHIZUKI, H., SCHWARTZ, J. P., TANAKA, K., BRADY, R. O., 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.
    Non-Patent Literature 4: MITROPHANOUS, K. A., YOON, S., ROHLL, J. B., PATIL, D., WILKES, F. J., KIM, V. N., KINGSMAN, S. M., KINGSMAN, A. J., and MAZARAKIS, N. D. (1999). Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther. 6, 1808-1818.
    Non-Patent Literature 5: KORDOWER, J. H., EMBORG, M. E., BLOCH, J., MA, S. Y., CHU, Y., LEVENTHAL, L., MCBRIDE, J., CHEN, E.-Y., PALFI, S., ROITBERG, B. Z., BROWN, W. D., HOLDEN, J. E., PYZALSKI, R., TAYLOR, M. D., CARVEY, P., LING, Z., TRONO, D., HANTRAYE, P., DÉGLON, N., and AEBISCHER, P. (2000). Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson's disease. Science 290, 767-773.
    Non-Patent Literature 6: MARR, R. A., ROCKENSTEIN, E., MUKHERJEE, A., KINDY, M. S., HERSH, L. B., GAGE, F. H., VERMA, I. M., and MASLIAH, E. (2003). Neprilysin gene transfer reduces human amyloid pathology in transgenic mice. J. Neurosci. 23, 1992-1996.
    Non-Patent Literature 7: 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.
    Non-Patent Literature 8: LO BIANCO, C., SCHNEIDER, B. L., 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.
    Non-Patent Literature 9: 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.
    Non-Patent Literature 10: MAZARAKIS, N. D., AZZOUZ, M., ROHLL, J. B., ELLARD, F. M., WILKES, F. J., OLSEN, A. L., CARTER, E. E., BARBER, R. D., BABAN, D. F., KINGSMAN, S. M., KINGSMAN, A. J., O'MALLEY, K., and MITROPHANOUS, K. A. (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.
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SUMMARY OF INVENTION
Technical Problem
Since the prior RV-G vector and FuG-B vector have the feature to transfer the gene into both neurons and glia cells at an injected site, the gene would be therefore introduced not only into the neurons but also into dividing cells.
Therefore, an object of the present invention is to provide a much safer lentiviral vector system that enables not only the gene transfer via a retrograde transportation with a higher frequency, but also selective or specific gene transfer into the neurons while reducing the efficiency of gene transfer into the dividing cells such as neural stem cells and glia cells so as to reduce the risk of occurrence of cancer.
Solution to Problems
The present inventor has found that the above problem can be solved by pseudotyping the lentiviral vector by means of a fused polypeptide as an envelope comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G, and an intracellular domain of VSV-G;
That is, the present invention relates to the following aspects.
[Aspect 1]
A kit for preparing a retrograde transport viral vector comprising:
(1) a packaging plasmid containing the gag gene and the pol gene of HIV-1;
(2) a packaging plasmid containing an accessory gene of HIV-1;
(3) a transfer plasmid containing an target gene (a transgene); and
(4) an envelope plasmid containing, as an envelope gene, a gene encoding a fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G, and an intracellular domain of VSV-G.
[Aspect 2]
A kit for preparing a producer cell comprising the kit for preparing a viral vector according to Aspect 1, and a host cell.
[Aspect 3]
A method of producing a producer cell, comprising: co-transfecting an infected cell with the packaging plasmid, the transfer plasmid, and the envelope plasmid, comprised in the kit for preparing a viral vector according to Aspect 1.
[Aspect 4]
A producer cell obtained by the method of producing a producer cell according to Aspect 3.
[Aspect 5]
A method of producing a viral vector, comprising: culturing the producer cell according to Aspect 4 and harvesting virus particles from the supernatant of the culture.
[Aspect 6]
A viral vector possessing a neuron-specific retrograde transportation ability, produced by the method of producing a viral vector according to Aspect 5.
[Aspect 7]
A method of gene transfer, comprising: infecting a nerve terminal of an animal with the viral vector according to Aspect 6; introducing the viral vector specifically into a cell body of the nerve at a target region in the brain by retrograde transportation of the viral vector through an axon of the nerve; and expressing a target gene in the cell body.
[Aspect 8]
An agent for gene therapy containing the viral vector according to Aspect 6 as an active ingredient.
[Aspect 9]
A method of gene therapy for a brain disease, comprising; integrating a target gene introduced by the method according to Aspect to 7 into the chromosome of a cell in a target region to express it.
[Aspect 10]
An envelope for pseudotyping a lentiviral vector, consisting of a fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G and the intracellular domain of vesicular stomatitis virus glycoprotein (VSV-G).
[Aspect 11]
A gene encoding an envelope consisting of the fused polypeptide according to Aspect 10.
[Aspect 12]
A envelope plasmid comprising the gene encoding an envelope consisting of the fused polypeptide according to Aspect 11.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a schematic overview of a HIV-1-pseudotype vector exhibiting highly-frequent retrograde transportation.
FIG. 2 shows the structure of FuG-C wherein the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G) is linked to the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G). It also comprises a transmembrane domain and an intracellular domain of VSV-G. The linking site between the extracellular domains of RV-G and VSV-G is indicated by an arrow, and amino acids in this boundary region (SEQ ID NO: 6) are also shown. “S” means a signal peptide, and “TM” means a transmembrane domain.
FIG. 3 shows photographs of an expression pattern of the transgene: (A) The expression pattern at striatum. (B) The expression pattern of the transgene via the retrograde transportation by RV-G and FuG-C vectors in primary motor cortex (M1), primary somatosensory cortex (S1), parafascicular nucleus of thalamus (PF), substantia nigra pars compacta (SNc), fr (habenulointerpeduncular tract); SNr (substantia nigra rope state par); LV (lateral ventricle). Scale bar: 500 μm.
FIG. 4 shows graphs demonstrating titer-dependency of efficiency of the retrograde gene expression. (Two-way ANOVA: main effect of vector, F(1,30)=223.9 for M1, 231.7 for S1, 46.5 for PF, and 87.7 for SNc, P<0.001 in all regions; and interaction between vector and titer, F(4,30)=20.4 for M1, 21.7 for S1, 3.6 for PF, and 8.4 for SNc, P<0.05 in all regions).
FIG. 5 shows photographs with a confocal microscope of the efficiency of the gene transfer into the neurons and glia cells at an injected site. Scale bar: 50 μm.
FIG. 6 shows photographs with a confocal microscope of the efficiency of the gene transfer into the neural stem cells. (A) Doublestaining with GFAP: (B) Doublestaining with BrdU. Arrows indicate representative double-stained cells. Scale bar: 100 μm (A), 50 μm (B).
FIG. 7 shows the base sequence (nucleotide sequence) encoding the envelope according to the present invention (SEQ ID NO: 1), which is contained in the envelope plasmid pCAG-FuG-C (SEQ ID NO: 5).



DETAILED DESCRIPTION
Advantages of the Invention
The present invention demonstrates, in vivo, that in an animal including mammals such as mice, by injecting a recombinant viral vector containing a specific gene for transfection at a region of the brain where a nerve terminal (a synapse terminal) is present, and by allowing the viral vector to be retrogradely transported through an axon, the target gene (the transgene) can be efficiently and specifically (selectively) introduced for expression into the neuron that is a non-dividing cell in the central nervous system distant from the infected (injected) site of the viral vector. In particular, by using a kit for preparing a viral vector which utilizes a specific packaging plasmid, transfer plasmid and envelope gene, a viral vector having an unexpectedly higher virus titer can be obtained, and a recombinant viral vector showing a highly-frequent and neuron-specific retrograde transportation ability in the brain can be advantageously produced.
The viral vector possessing a neuron-specific retrograde transportation ability according to the present invention suppresses the frequency of gene transfer into the dividing cells such as neural stem cells and glia cells. As a result, the risk of occurrence of cancer can be reduced, and any side effects caused by the gene transfer and expression in other cells than the neurons can be decreased.
DESCRIPTION OF EMBODIMENTS
The viral vector according to the present invention is the neuron-specific retrograde transport vector (NeuRet), which is characterized by having a high titer. The vector may be prepared by means of a kit comprising:
(1) a packaging plasmid containing the gag gene and the pol gene of HIV-1;
(2) a packaging plasmid containing an accessory gene of HIV-1;
(3) a transfer plasmid containing an target gene (a transgene); and
(4) an envelope plasmid containing, as an envelope gene, a gene encoding a fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of an extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), a transmembrane domain of RV-G or VSV-G, and an intracellular domain of VSV-G.
In the kit for preparing the viral vector according to the present invention, “gag” is a gene which encodes retroviral core proteins, and “pol” is a gene which encodes reverse transcriptase and the like. In addition, an “envelope gene” is a gene which encodes an envelope, a virus specific protein which is located in an envelope that is an outer membrane of a retrovirus comprised of a lipid bilayer membrane. The envelope plays an important role for a virus to adhere to and invade into a cell. Further, an “accessories gene” means, for example, the rev gene which regulates the expression of structural genes.
A preferred and representative example of the kit for preparing a viral vector according to the present invention is characterized by the use of “pCAGkGP1.1R” and “pCAG4-RTR2” as (1) a packaging plasmid containing the gag and the pol gene of HIV-1 and (2) a packaging plasmid containing an accessories gene of HIV-1 respectively, and further by the use of “pCL20 c-MSCV-X as a transfer plasmid where “X” represents a target gene. The target gene “X” to be transfected is encoded downstream of a mouse stem cell virus promoter in the above transfer plasmid.
Each plasmid contained in the above kit for preparing a viral vector is constructed based on a HIV-1 vector system “SJ1” developed by Dr. Arthur Nienhuis at St. Jude Children's Research Hospital (HANAWA, H., et al., (2002) Mol. Ther. 5, 242-251; (2004). Blood 103, 4062-4069. Supplied by St. Jude Children's Research Hospital). This vector system is known to show an about 10-fold greater titer in HeLa cells than other vector systems. Therefore, those skilled in the art can readily produce each of these plasmids by referring to the specification of the present application and the above references. Note that the above (1) and (2) of the packaging plasmids may be constructed as one plasmid.
The envelope gene contained in the envelope plasmid of the kit for preparing the viral vector according to the present invention encodes the fused polypeptide comprising a fused extracellular domain consisting of the N-terminal region of the extracellular domain of rabies virus glycoprotein (RV-G) and the C-terminal region of an extracellular domain of vesicular stomatitis virus glycoprotein (VSV-G), the transmembrane domain of RV-G or VSV-G, and the intracellular domain of VSV-G. Note that at the fusion boundaries of each domain, one or more amino acids can be optionally altered by deletion, insertion, substitution, or the like, and all of the amino acids constituting each domain are not necessarily included.
Examples of the envelope gene can include an envelope gene encoding a polypeptide consisting of, for example, about 420 amino acids or more, or 430 amino acids or more of the N terminal region of the extracellular domain of RV-G originally consisting of 458 amino acids as the N terminal region constituting the fused extracellular domain.
As a preferred example, there may be mentioned an envelope gene encoding the amino acid sequence shown in SEQ ID NO: 2 (FuG-C), and preferably a nucleic acid molecule having the base sequence of 1 ̃1,365 bases from N terminal shown in SEQ ID NO: 1 (FIG. 7) wherein the 1 ̃1,317 bases (including start codon) at the 5′ side are originated from RV-G and the 1,318 ̃1,365 bases at the 3′ side are originated from VSV-G. Given the codon degeneracy, the above base sequences can be optionally altered to optimize the codon along with other elements in the envelope plasmid. SEQ ID NOs: 4 and 3 show examples of the amino acid sequence of rabies virus glycoprotein (RV-G) and the base sequence encoding thereof.
Thus, the above fused polypeptide is effective as an envelope to pseudotype various kinds of lentiviral vectors, in particular a HIV-1 lentiviral vector. Therefore, the present invention also relates to an envelope for pseudotyping a lentiviral vector comprising the above fused polypeptide, a gene encoding an envelope comprising the above fused polypeptide, and an envelope plasmid itself containing the above gene. In each of the plasmids contained in the kit for preparing a viral vector according to the present invention, each gene is linked under expression control of any expression regulatory sequences known to persons skilled in the art.
The phrase “under expression control of” means DNA encoding a given amino acid sequence has the ability to express a protein having that amino acid sequence under given conditions. In case that DNA encoding a given amino acid sequence is linked under expression control of an expression regulatory sequence, that DNA will express a given protein under given conditions. The term “an expression regulatory sequence” herein means a nucleic acid sequence that regulates expression of other nucleic acid sequences, and it regulates and modulates transcription and preferably even translation of other nucleic acid sequences. Expression regulatory sequences include an appropriate promoter, an enhancer, a transcription terminator, the start codon (namely, ATG) in a gene encoding a protein, a splicing signal for intron, a polyadenylation site and the stop codons.
The term “a promoter” means an essential sequence for transcription. Promoters also include promoter elements that regulate gene expression cell-type specifically, tissue specifically, or promoter-dependently via a signal or a modulator from the outside. A promoter element is linked at either the 5′ or 3′ region of DNA to be expressed. In addition, promoters include any of those constitutive or inducible. Promoters known for persons skilled in the art can be selected accordingly, depending on the classes of target genes and viral vectors to be used, the kinds of animals and brain diseases to be treated, pathological conditions of patients, and so on.
For example, in the envelope plasmid according to the present invention, an envelope gene is preferably linked such that it will be expressed under control of the cytomegalovirus enhancer and the avian β actin promoter. Such an envelope plasmid can be obtained by replacing, in accordance with the standard method, the base sequence encoding an extracellular domain and a transmembrane domain of vesicular stomatitis virus glycoprotein (VSV-G) with the base sequence encoding an extracellular domain and a transmembrane domain in the nucleic acid (cDNA) encoding a glycoprotein from a CVS strain of rabies virus (RV-G) which is passed in the brain of an infected infant mouse (provided by Dr. Kinjiro Morimoto at National Institute of Infectious Diseases) (Morimoto, K. et al., (1998) Proc Natl. Acad. Sci., USA 95, 3152-3156: SEQ ID NO 3) in the envelope plasmid “pCAGGS-VSV-G” included in the above vector system “SJ1”. Therefore, persons skilled in the art can readily produce these plasmids described above by referring to the specification of the present application and the above references. Note that the glycoprotein (RV-G) of the rabies virus CVS strain is not limited to those having the base sequence shown in SEQ ID NO 3 above, but glycoproteins (RV-G) from any strains of any known rabies viruses can be used.
A target gene contained in a transfer plasmid known for persons skilled in the art can selected accordingly, depending on the intended use of a viral vector, the kinds of animals and brain diseases to be treated, pathological conditions of patients, and so on. Therefore, they include various genes of mammal, such as mouse, monkey and human, for example, a gene required for survival or protection of nigrostriatal system, which is used to treat cranial nerve diseases or neurodegenerative diseases represented by Parkinson's disease, etc. (for example, tyrosine hydroxylase, a neurotrophic factor from an glial cell line), or genes such as the interleukin-2 receptor α subunit (a target molecule of a recombinant immunotoxin) for research on cranial nerve systems and a light dependent ion channel, etc.
Host cells contained in the kit for producing a producer cell according to the present invention have no particular limitation as long as they can be infected by the above kit for preparing a viral vector so that they can produce a cell called “a producer cell” which can produce a retroviral particle. Any cells known to persons skilled in the art, for example, commercially available appropriate animal cells such as HEK293 T-cells (SV40 large T antigen is introduced) can be used.
Depending on their composition, intended use, etc., in addition to each of the above plasmids and/or host cells, the various kits according to the present invention can optionally contain other elements or ingredients known to persons skilled in the art, such as various reagents, buffers, various adjuvants, reaction plates (containers) and the like.
Using the kit for preparing a producer cell according to the present invention, a producer cell can be produced by co-transfecting an infected cell with a packaging plasmid, a transfer plasmid and an envelope plasmid contained in the kit for preparing a viral vector. This transfection is transient and can be performed by any methods known to persons skilled in the art, such as the calcium phosphate method.
A viral vector having a neuron-specific or neuron-selective retrograde transport ability and a high titer in the brain can be produced by culturing the resultant producer cells using any methods or means known to persons skilled in the art, and harvesting virus particles from the culture supernatant.
A nerve terminal can be infected with the viral vector according to the invention, and the retrograde transportation of the viral vector through an axon of that nerve can specifically or selectively introduce the viral vector into a cell body of that nerve at the target region in the brain, and a target gene can be expressed in the cell body. Target regions in the brain include primary motor cortex, primary somatosensory cortex, parafascicular nucleus of thalamus and substantia nigra pars compacta, which are projecting to striatum, and the brain center such as piriform cortex, subiculum, amygdala basolateral nucleus, anterior paraventricular nucleus, mediodorsal nucleus of thalamus and lateral hypothalamus, which are projecting to ventral striatum (nucleus accumbens). Further, the viral vector according to the present invention is retrogradely transported through an axon of motor neuron in the spinal cord.
Therefore, the viral vector according to the present invention is effective as an active ingredient of an agent for gene therapy. The agent for gene therapy can contain, in combination with the active ingredient, any pharmaceutically acceptable careers or diluents or other components known to those skilled in the art.
The effective amount of the active ingredient according to the present invention can be selected accordingly by persons skilled in the art, depending on the classes of the transgene contained in the viral vector; the kinds and seriousness of brain diseases or neurodegenerative disorders; therapeutic strategy; age, body weight, sex, general health of patient; and racial (genetic) background of patient. A dose of the active ingredient (the viral vector) can be, for example, a total amount of 108 to 109 TU (Transducing Unit) per administration for several infection (injection) sites. Note that the viral vector or the agent for gene therapy can be infected (injected) at a predetermined site in a patient using any administration methods or devices known to those skilled in the art.
By administering the viral vector according to the present invention to a patient, a gene introduced into a predetermined cell in a target region will be integrated into the chromosome of that cell, and the target gene will be stably expressed. Therefore, the present method of gene transfer can be used to perform gene therapy for brain diseases, neurodegenerative diseases (for example, Parkinson's disease) or the like of mammals including primate such as human.
Now, the present invention will be described in detail by Examples and test examples. These examples represent a part of the present invention, and the technical scope of the present invention is not limited at all by these Examples. Unless otherwise stated, experimental conditions and the like in each procedure were according to the methods described in the references cited herein, or the standard methods in the art.
Example 1
Preparing a Viral Vector
A viral vector according to the present invention was prepared using a HIV-1 vector system developed by Dr. Arthur Nienhuis at St. Jude Children's Research Hospital. Namely, a packaging plasmid containing the gag and pol gene (pCAGkGP1.1R), a packaging plasmid containing an accessories gene (pCAG4-RTR2) and a transfer plasmid containing a green fluorescence protein (GFP) as a target gene (pCL20 c-MSCV-GFP) were used.
As an envelope plasmid (pCAG-FuG-C), a vector containing a base sequence (SEQ ID NO 5) encoding an envelope was produced according to the conventional method, wherein the envelope was a fused glycoprotein (FuG-C) consisting of the C-terminal region of the extracellular domain of VSV-G linked to the N-terminal region of the extracellular domain of RV-G that had been provided by Dr. Kinjiro Morimoto at National Institute of Infectious Diseases, and the transmembrane domain and the intracellular domain of VSV-G. (FIG. 2). The extracellular domain of FuG-C consisted of 439 amino acids from the N-terminal region of the extracellular domain of RV-G and 16 amino acids from the C-terminal region of the extracellular domain of VSV-G. Similarly, viral vectors comprising VSV-G and RV-G, respectively, were prepared for the purpose of comparison.
Determination of Viral Titer:
HEK293 T-cells (eighteen 10-cm dish) were transfected with a viral vector solution containing these plasmids using the calcium phosphate method. After cultured for 48 hours, virus particles were harvested from the culture supernatant and centrifuged, which was filtered with a 0.45-μm cellulose filter. Then, the vector particles were collected by centrifugation (10,000×g, 16 to 18 hours), and suspended in PBS (1 ml). The suspension was subjected to Sepharose Q FF ion exchange column chromatography, which was washed with PBS and then eluted using a linear gradient from 0 to 1.5 M NaCl. Fractionations were monitored by the absorbance at 260/280 nm. Fractionations containing the vector particles were collected, concentrated using an ultrafiltration filter, and stored at −80° C.
In order to evaluate a viral titer, the following culture cells that were easily available from the public depositories and the like were plated to a 6-well cell culture plate (MULTIWELL (R), FALCON) to infect the cultured cells with an appropriate concentration of the virus solution: Human renal cell:HEK293 T (available from a cell bank of RIKEN, Accession No: RCB2202), mouse neuroblast: Neuro2A (available from ATCC, ID No: CCL-131TM), mouse neuroblast: N1E-115 (available from ATCC, ID No: CRL-2263TM).
Titers were measured using FACS Calibur (Nippon Becton Dickinson Co., Tokyo, Japan) (FIG. 2B). Then, the amount of RNA contained in vector stock was measured using a quantitative RT-PCR method.
The results are shown in Table 1 below. Although the functional titer of FuG-C vector was detected only in Neuro2A, its value was significantly decreased compared to that of VSV-G or RV-G vector (ANOVA, Tukey HSD, p<0.01), and the RNA titer of FuG-C was comparable to that of VSV-G or RV-G vector.







TABLE 1







Titer of lentiviral vectors










Functional titer













HEK293T
Neuro2A
N1E-115
RNA titer















VSV-G
1.0 ± 0.02 ×
5.0 ± 0.1 ×
2.8 ± 0.1 ×
6.8 ± 0.6 ×



109
108
108
109


RV-G
5.2 ± 1.0 ×
1.9 ± 0.04 ×
3.4 ± 0.2 ×
8.6 ± 0.7 ×



107
107
106
109


FuG-C
ND
1.2 ± 0.01 ×
ND
8.9 ± 0.8 ×




106 *

109





ND, Not detected.


* P < 0.01 vs VSV-G or RV-G (ANOVA, Tukey HSD)






Example 2
Introduction of the Viral Vector into the Brain of Mouse
Animal care and use was performed according to a guideline by the animal care and use committee of Fukushima Medical University. A 12-week old mouse (C57BL/6J) was anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and a solution containing a vector produced as described above (4.8×1010 copies/ml) was injected in the brain (striatum) of the mouse using a brain stereotaxic apparatus. The injection was performed according to the mouse brain atlas (PAXINOS, G., and FRANKLIN, K. B. J. (2001). The Mouse Brain in Stereotaxic Coordinates, 2nd edn. (Academic Press, San Diego). 2 μl of the solution was injected (0.1 μl/min) at two points along the track respectively in the dorsal region of striatum through a glass microinjection capillary connected to a microinjection pump. Anteroposterior, mediolateral and dorsoventral coordinates from bregma were 0.50, 2.00 and 2.50/3.25 (mm), respectively.
Four weeks after the injection, a mouse was deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and then the brain was perfused and fixed with 4% formalin and 0.1 M phosphate buffer (PB: pH 7.4) via the heart before extirpating the brain. Sections were prepared using a cryostat and analyzed using the immunostaining method.
Although immuno-positive signals were observed in a wide range of the striatum of the mouse that received the injection of the FuG-C vector, their intensity had been significantly decreased compared to those of RV-G vectors (FIG. 3A). In addition, the expression of the transgene was analyzed by the immunostaining method in primary motor cortex (M1), primary somatosensory cortex (S1), parafascicular nucleus of thalamus (PF) and substantia nigra pars compacta (SNc), which are representative brain regions projecting to striatum. Both the same and opposite regions of the brain cortex as the injected site, and in the same region of PF and SNc in the brain as the injected side were observed (FIG. 3B), and the number of the positive cells in each region was counted (Table 2). The efficiency of transfer of the gene with the FuG-C vector was significantly increased in all of the regions in the brain when compared to that with the RV-G vector (Student t-test, p<0.01 or 0.05). These data indicate that the FuG-C vector has improved the efficiency of transfer of the gene to the neurons via the retrograde transport.







TABLE 2







Efficiency of the retrograde transfer of gene with lentiviral vectors









Brain region











M1
S1















Ipsi
Contra
Ipsi
Contra
PF
SNc

















RV-G
61.2 ± 9.0 
10.4 ± 2.3 
59.6 ± 8.6 
9.1 ± 1.8
121.2 ± 13.6 
2.0 ± 0.4 


FuG-C
246.6 ± 20.5b
67.1 ± 3.6 *
248.8 ± 21.7 *
50.8 ± 4.4 *
191.6 ± 24.5**
7.2 ± 0.7 *





* P < 0.01,


**P < 0.05 vs RV-G (Student t-test)






Next, in order to confirm the improvement in the retrograde transfer of gene with the FuG-C vector, vector solution with various concentration of the vector (3.0×109 ̃4.8×1010 copies/ml) was injected into the mouse striatum, and the number of the positive cells was counted in M1, S1, PF and SNc (FIG. 4). The number of the positive cells clearly showed a concentration-dependent increase in every region in the case of the injection of the FuG-C vector. Although the concentration-dependent increase was also observed in the animals with the injection of the RV-G vector, its tendency was lower than that of the FuG-C vector (two-way ANOVA). These results show that the FuG-C vector enables the transfer of gene via the retrograde transport with a higher efficiency than the RV-G vector.
Example 3
Selective Transfer of Gene into Neurons
The efficiency of the gene transfer into the neurons and glia cells at the injected site of the FuG-C vector was analyzed. After each vector of VSV-G, RV-G and FuG-C (1.2×1010 copies/ml) was injected into the striatum of the mouse, the sections of the striatum were prepared in the same way as in Example 2. Double immunostaining was done with the neuron marker of NeuN and GFP, or with the glia cell marker of GFAP and GFP (FIG. 5). First, a ratio of the number of GFP+/NeuN+ double positive cells to the number of the total NeuN+ positive cells, and a ratio of the number of GFP+/GFAP+ double positive cells to the number of the total GFAP+ positive cells were measured, respectively. The ratio of the number of GFP+/NeuN+ double positive cells was 81.7±2.9%, 21.4±1.8%, and 6.2±1.4% for the vectors of VSV-G, RV-G and FuG-C, respectively (n=4). The efficiency of the gene transfer with the FuG-C vector into the neurons in the striatum was significantly decreased compared to those with the other vectors (ANOVA, Tukey HSD, p<0.001 vs VSV-G, p<0.01 vs RV-G). On the other hand, the ratio of the number of GFP+/GFAP+ double positive cells was 5.9±0.7%, 71.5±3.6%, and 0.3±0.03% for the vectors of VSV-G, RV-G and FuG-C, respectively (n=4). Thus, almost no gene transfer into the glia cells was observed in the case of the FuG-C vector, showing that the FuG-C vector can transfer the gene selectively into the neurons mainly via the retrograde transportation.
Example 4
Properties of the Transfer of Gene with Vectors into Neural Stem Cells
It is known that the VSV-G vector has a high gene-transfer efficiency into neural stem cells. The properties of the vectors for the gene transfer into the neural stem cells localized in circumventricular region (SVZ) were analyzed. The viral vector solution (1.2×1010 copies/ml) was injected into the SVZ of the mouse, the brain sections were prepared, followed by double immunostaining using the neural stem cell marker of GFAP and GFP (FIG. 6A). Many GFAP positive cells were observed when the gene transfer was done with the injection of the VSV-G or the RV-G vector. On the other hand, almost no positive cell was observed in the case of the FuG-C vector. Furthermore, BrdU was administered to label dividing cells and the brain sections were prepared one week after in order to carry out the double immunostaining with BrdU and GFP (FIG. 6B). While the expression of the transgene was observed in many dividing cells in SVZ in the case of the injection of the VSV-G and the RV-G vectors, almost no gene transfer into the dividing cells was observed in the case of the injection of the FuG-C vector. These results show that the frequency of the gene transfer into the neural stem cells and dividing cells with the FuG-C vector is very low.
Histological Procedures
For immunostaining by the avidin-biotin-peroxidase method, transverse sections (for mice: thickness of 30 μm) were prepared using a cryostat. The sections were then incubated with rabbit anti-GFP polyclonal antibody (Molecular Probes, Eugene, Oreg.: 1:2,000 dilution), and further incubated with a biotinylated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, Calif.: 1:1,000 dilution). Immunoreaction signals were visualized by the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif.).
For double immunofluorescence histochemical staining, the sections were incubated with either one of the above rabbit anti-GFP polyclonal antibody or anti-choline acetyltransferase mouse antibody (Chemicon, Temecula, Calif.: 1:100 dilution). Then, the sections were 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, N.Y.) equipped with a filter cube having a suitable specification for FITC and Cy3 fluorescence channels. These fluorescence images were taken with an advanced CCD camera system controlled by the Zeiss Axiovision software package.
Cell Count
Immunostaining by the above avidin-biotin-peroxidase method was performed using a series of sections passing through the forebrain and the midbrain. The number of immunostained cells in each brain region was counted by a computer-controlled graphics program (NIH Image 1.62, National Institutes of Health, Bethesda, Md.). In order to identify striatum cells at the injection site of the vector, double immunofluorescence histochemical staining was performed using representative sections. In each animal, the number of immunostained cells within the target region was counted by the graphics program. Using 8 to 10 sections from each animal, a mean value per section was calculated.
Contents described in the references cited herein constitute the contents of the disclosure of the present specification as a part of the present specification.
INDUSTRIAL APPLICABILITY
The retrograde transport vector according to the present invention allows the selective gene transfer into the neurons mainly via the retrograde transport, while significantly suppressing the introduction of gene into the dividing cells such as glia cells and neural stem cells. Thus, the vector according to the present invention can reduce the risk of occurrence of cancer so as to alleviate any side effects caused by non-specific gene expression in other cells than the neurons. Therefore, the present invention provides an effective technology for gene therapy of cranial nerve diseases such as Parkinson's disease.
In addition, the present invention is to provide a novel and effective technology for experiments for gene therapy of cranial nerve diseases and for creating a disease model.

## Claims

1. A chimeric envelope protein capable of pseudotyping an HIV-1 lentivirus vector system to produce pseudotyped particles comprising said chimeric envelope such that the pseudotyped virus particles can facilitate selective retrograde gene transfer into neurons, wherein the chimeric envelope protein comprises the amino acid sequence of SEQ ID NO: 2.

2. A polynucleotide encoding the chimeric envelope protein of SEQ ID NO: 2.

3. An envelope plasmid, comprising the polynucleotide of claim 2.