JP2002065278A - Gene transfer vehicle containing hvj fusion protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a gene transfer vehicle which is formed by using a HVJ fusion protein, shows safety and stability and exhibits high gene transfer activity. SOLUTION: This gene transfer vehicle is obtained by reconstructing a F- protein derived from HVJ (Sendai virus) to which uv rays have not been irradiated or a recombinant F-protein and a HN protein. Further, the method of preparation for this vehicle is provided. This vehicle is a vector useful in a gene therapy.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a vehicle for gene transfer in vitro and in vivo. In particular, the present invention relates to a gene transfer vehicle for gene transfer, comprising a liposome reconstituted with the HVJ (Sendai virus) F fusion protein and HN fusion protein. Also, the gene transfer vehicle herein can be used for gene therapy.

[0002]

BACKGROUND OF THE INVENTION For gene therapy, a number of viral and non-viral (synthetic) methods for gene transfer have been developed (Mulligan, 1993; Ledle).
y, 1995). Generally, viral methods are more effective than non-viral methods for delivering genes to cells. However, viral vectors require the simultaneous introduction of essential genetic elements from the parent virus, leaky expression of viral genes,
Immunogenicity and alterations in the host genome structure can raise safety concerns. In general, non-viral vectors are less cytotoxic and less immunogenic. However, most non-viral methods have poorer gene transfer efficiency, especially in vivo, than some viral vectors. Thus, both viral and non-viral vectors have advantages with limitations. Therefore, by developing in vivo gene transfer vectors with high efficiency and low toxicity, the limitations of one type of vector system should be compensated for by introducing the advantages of another type of system. It is.

[0003] Under the concept of compensation, the present inventors have developed a novel hybrid gene transfer vector by combining viral and non-viral vectors, and have been able to form a fusion-forming virus derived from Japanese hemagglutinating virus (HVJ; Fusion-forming viral liposomes with an envelope were constructed (Kaneda, 1998; Ka).
neda et al., 1999). In this delivery system, the DN
Fusing the A-loaded liposomes with UV-inactivated HVJ;
Form HVJ liposomes (400-500 nm in diameter), which are fusogenic virus-liposomes. An advantage of fusion-mediated delivery is that transfecting the DNA is protected from endosomal and lysosomal degradation in recipient cells. DNA up to 100 kb
Is taken up in HVJ liposomes and delivered to mammalian cells. RNA, oligonucleotides and drugs are also efficiently introduced into cells in vitro and in vivo. HVJ-liposomes were not shown to induce significant cell damage in vivo. Repeated transfection has been successful in vivo due to the low immunogenicity of HVJ (Hirano et al., 1998). This vector system has been improved and anionic and cationic HVJ-liposomes have been developed for more efficient gene delivery (Saeki et al., 19).
97). Many gene therapy strategies have been successful using this HVJ-liposome system (Dzau et al., 1996;
Kaneda et al., 1999).

[0004]

However, for human gene therapy, the HVJ-liposome system should be improved to achieve complete safety. In terms of safety, the limitations of the current HVJ-liposome system are
Although the J genome has been inactivated by UV irradiation, components other than the fusion protein are contained in the vesicles. HVJ-derived synthetic virosome
Some attempts have been made to construct (Wu et al.
1995; Ramani et al., 1997, 1998).

An object of the present invention is to provide a reconstituted fusion particle containing an HVJ fusion protein, and to provide a vehicle for efficient, stable and safe gene transfer in vitro and in vivo using the reconstituted fusion vesicle. It is to be.

[0006]

According to one aspect of the present invention, there is provided a gene transfer vehicle using a fusion protein of HVJ. The transfection vehicle is a standard HVJ based on whole virions of UV inactivated HVJ.
Oligonucleotides (ODN) and plasmid DNA are introduced into cells as efficiently as liposomes. In addition, gene transfer vehicles containing HVJ fusion proteins also successfully transfer plasmid DNA to cells in tissues such as mouse muscle. The gene transfer vehicle of the present invention has a transfection efficiency comparable to that of HVJ liposomes.

In another aspect of the invention, a standard HV
A gene transfer vehicle excellent in safety and stability is provided for J liposome. Preferably, the gene transfer vehicle of the present invention is HV-based in RT-PCR analysis.
Does not contain J genomic RNA. Therefore, there is no safety issue for virus replication after transfection.

[0008] In certain aspects of the invention, the gene transfer vehicle and the reconstituted fusion particles can be stored more stably than UV-irradiated HVJ. In a further aspect of the invention, the gene transfer vehicle and the reconstituted fusion particles maintain high fusion activity at 4 weeks after preparation, so that
Maintain high gene transfer activity. In another aspect of the invention, the reconstituted fusion particles and the DNA-loaded liposomes can be stored separately after preparation, and then both particles are fused to construct a gene transfer vehicle prior to transfection. obtain.

In yet another embodiment of the present invention, there is provided a vehicle for gene transfer using reconstituted liposomes using recombinant F and HN proteins. Also, in one aspect of the invention, the recombinantly produced fusion protein is processed by a protease in vitro or by an endogenous protease in a mammalian cell host.

[0010] In one aspect of the present invention, there is provided a gene transfer vehicle reconstituted with the HVJ F fusion protein and the HN fusion protein without a detectable amount of HVJ genomic RNA.

In another aspect of the present invention, in vitro gene transfer having preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more gene transfer activity in vitro. Vehicle is provided.

In one aspect of the invention, when the vehicle is formed 4 weeks after the preparation of the reconstituted fusion particles, it is in vitro at least 50%, preferably at least 60%, more preferably at least 70%, most preferably at least 80%. A transfection vehicle is provided that retains at least 100% transfection activity.

In another aspect of the invention, gene transfer into preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, most preferably 60% or more cells for local administration in vivo. An active gene transfer vehicle is provided.

[0014] In yet another aspect of the present invention, 100
A gene transfer vehicle capable of transferring a foreign gene up to kb is provided.

In an aspect of the present invention, there is provided a method for preparing a gene transfer vehicle, wherein the efficiency of enclosing a foreign gene is 20% or more, preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more. Is done.

[0016] In another aspect of the invention, the invention relates to a fusion protein wherein the F fusion protein and the HN protein of the gene transfer vehicle are derived from HVJ that has not been irradiated with UV or are recombinantly expressed fusion proteins. An introgression vehicle is provided. In a further aspect of the present invention, there is provided a gene transfer vehicle comprising a recombinant F protein that has been processed in vivo or in vitro by a protease.

In another aspect of the present invention, the present invention provides a gene transfer vehicle used for gene therapy. Further, the present invention provides a method for gene transfer to an isolated cell.

In another aspect of the present invention, the invention relates to
Provided is a method of preparing a gene transfer vehicle containing a fusion protein of VJ, comprising the steps of: isolating the fusion protein from an HVJ virus that has not been UV-irradiated; Reconstituting in the presence to prepare reconstituted particles, preparing a liposome filled with a desired nucleic acid, and reconstituting the particles and the liposome.

In a further aspect of the present invention, the present invention provides a method for preparing a gene transfer vehicle containing the HVJ F and HN proteins, the method comprising the following steps: Expressing, processing F protein with protease, isolating F protein and HN protein, reconstituting F protein and HN protein in the presence of detergent and lipid to prepare reconstituted particles A step of preparing a liposome filled with a nucleic acid, a step of preparing the reconstituted particles and the liposome.

In a further aspect of the present invention, the present invention provides a method for preparing a gene transfer vehicle containing the HVJ F and HN proteins, comprising: a host expressing a protease that processes the F protein. Recombining F protein and HN protein in a cell, isolating F protein and HN protein, reconstituting F protein and HN protein in the presence of a surfactant and a lipid, , A step of preparing a liposome filled with a desired nucleic acid, and a step of fusing the reconstituted particles and the liposome.

[0021]

DETAILED DESCRIPTION OF THE INVENTION (Definition) As used herein, "gene transfer" refers to a natural, synthetic or recombinant desired gene or gene fragment in vivo or in vitro in a target cell. Is introduced so that the introduced gene maintains its function. The gene or gene fragment to be transferred in the present invention includes DNA, RNA or a nucleic acid which is a synthetic analog thereof having a specific sequence.

As used herein, “gene transfer activity” refers to the activity of “gene transfer” by a vehicle, and refers to the function of the transferred gene (eg, in the case of an expression vector, Expression and / or the activity of the protein).

As used herein, a “fusion protein” is involved in HVJ-induced cell fusion.
Refers to a protein derived from HVJ.

The fusion protein of the present invention includes an F protein and an HN protein. The HVJ F and HN proteins are present on the surface of Sendai virus virions and are involved in virus infection and membrane fusion.

Within the context of the present invention, fusion proteins include:
Other variants (including alleles) of wild-type proteins and native protein sequences are included. Briefly, such variants can result from natural polymorphisms, or can be synthesized by recombinant methodology, and
It may differ from the wild-type protein by one or more amino acid substitutions, insertions, deletions, etc., but is intended to retain gene transfer activity when reconstituted in liposomes. For example, if the variant is the result of a synthesis, the amino acid substitutions tend to be conservative, ie, substitutions of amino acids within a group of amino acids such as polar, non-polar, aromatic, charged, and the like. However, it should be understood that variants may contain non-conservative substitutions and other mutations not beyond the scope of the invention, as long as the variant retains the essential function of the native protein or polypeptide. is there.
Variants are the native protein amino acid sequence,
It has at least 70%, preferably 80%, more preferably 90%, even more preferably 92%, even more preferably 95%, and most preferably 97% or more identity.

For the purposes of the present invention, a preferred method of calculating percent identity is using Smith,
-Waterman algorithm. Global DNA sequence identity was determined using the MPSRCH program (Oxford Mole) using an affine gap search using the following search parameters: gap open penalty 12, and gap extension penalty 1.
Smith-W as performed in
aterman homology search algorithm.

The fusion protein herein is HV
Includes both native and recombinantly expressed proteins from the J virus.

As used herein, “vehicle”
Refers to a vehicle for gene transfer containing the fusion protein of HVJ.

As used herein, “reconstituted fusion particles” refers to particles obtained by reconstituting an isolated HVJ fusion protein in the presence of a surfactant and a lipid.
The reconstituted fusion particles fuse with the liposomes containing the nucleic acid to form a vehicle.

In the present specification, "HVJ" and "Sendai virus" can be used interchangeably.

As used herein, "HAU" refers to the activity of a virus capable of aggregating 0.5% of chicken erythrocytes, and 1 HAU corresponds to approximately 30 million virus particles (Okada et al., 1961).

Gene Therapy Therapeutic nucleic acid constructs can be administered either locally or systemically using the gene transfer vehicles of the present invention. Where such a nucleic acid construct includes a coding sequence for a protein, expression of the protein may be induced by use of an endogenous mammalian promoter or a heterologous promoter. Expression of the coding sequence can be constitutive or regulated.

When the gene transfer vehicle of the present invention is used as a composition for gene therapy, administration of the vehicle of the present invention may be performed by suspending the vehicle in PBS (phosphate buffered saline) or physiological saline. This can be done by direct injection of the suspension locally (eg, into cancerous tissue, liver, muscle, brain, etc.), or by intravascular (eg, intra-arterial, intravenous, and intraportal) administration. . The dose varies depending on the site. For example, when a cancer gene vaccine is administered to mouse skeletal muscle, a vehicle containing preferably 1 to 500 μg, more preferably 5 to 150 μg of DNA is injected. In addition, exemplary DNA dosages when administering a 7 kb plasmid using a gene transfer vehicle are: 5-15 μg for mouse liver, blood vessels, skeletal muscle and heart; for rat liver and skeletal muscle:
20-30 μg, 50-60 μm for monkey skeletal muscle
g, as well as 100-150μ for human skeletal muscle
g.

The following examples are illustrative and are not intended to limit the present invention.

[0035]

EXAMPLES Using HVJ (Japanese hemagglutinating virus; Sendai virus) inactivated by UV, efficient in vitro and in vivo gene delivery vehicles have been developed based on the cell fusion properties of Sendai virus.
In vehicles prepared by conventional methods, replication of the viral genome is lethal by prior UV irradiation of HVJ particles, but all proteins and genome of HVJ remain in HVJ liposomes. We have developed a safer, more stable, and more efficient synthetic gene transfer vehicle.

The fusion proteins F1 and HN of HVJ were extracted by mild lysis of the Viruis particles and purified by ion exchange chromatography. The purified virus fusion protein was inserted into the liposome membrane by detergent solubilization and dialysis to construct reconstituted fusion particles.
These particles maintained fusion and gene transfer activity over a storage period of 4 weeks or more.

The DNA-loaded liposomes prepared by vortex-sonication were fused with the reconstituted fusion particles,
NA was delivered to the cells. Using a gene transfer vehicle, fluorescein isothiocyanate (FITC) labeled oligonucleotides were introduced into 100% of the nuclei of target amniotic FL cells. Furthermore, luciferase gene expression upon transfection of human 293 cells with the reconstituted fusion liposomes was almost the same as standard HVJ liposomes. When the LacZ gene was introduced into mouse skeletal muscle using a novel vehicle, it was found that 40-50%
Muscle fibers showed LacZ gene expression.

(Virus) The HVJ, Z strain was purified by differential centrifugation as previously described (Kaneda, 1994). Purified HVJ is equilibrated with a salt solution (BSS: 13
7 mM NaCl, 5.4 mM KCl, 10 mM T
ris-HCl, pH 7.5) and the virus titer was determined by measuring the absorbance at 540 nm. The optical density at 540 nm is
Corresponds to 15,000 hemagglutination units (HAU) and correlates with fusion activity.

(Extraction of F and HN fusion proteins from HVJ) Nonidet dissolved in ethanol
P-40 (NP-40) and phenylmethylsulfonyl fluoride (PMSF) were added to 20 ml of purified HVJ suspension at a final concentration of 0.5% and 2 mM, respectively.
(1,750,000 HAU). The mixture was incubated at 4 ° C. for 30 minutes with rotation. The suspension was then centrifuged at 100,000 g at 4 ° C. for 75 minutes to remove insoluble proteins and viral genome (Uchida et al., 1979). The supernatant was dialyzed against 5 mM phosphate buffer (pH 6.0) for 3 days, and the buffer was changed every day.
-40 and PMSF were washed away. Dialysed solution
Centrifugation was performed at 100,000 g at 4 ° C. for 75 minutes to remove insoluble substances. The supernatant was used as described previously (Yo
CM-Sepharose equilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and 1 mM KCl based on Shima et al., 1981).
CL6B (Pharmacia Fine Chem)
icals, Uppsala, Sweden). Pass through fraction and 0.2M
The NaCl eluate was collected. Both fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) to analyze the protein components. The gel was stained with Coomassie brilliant blue and the ratio of each protein was determined by computerized densitometry (N
IH Image; Apple Computers,
(Cupertino, CA, USA).

(Recombinant Expression) A fusion protein of HVJ can also be prepared by incorporating a gene encoding the fusion protein into an expression vector and expressing it in an appropriate host cell. The amino acid sequences of the F and HN proteins can include, for example, SEQ ID NO: 2 (FIG. 7) and SEQ ID NO: 4 (FIG. 8).

As expression vectors that can be used in various host cells, various commercially available vectors can be used.

[0042] Expression vectors encoding the fusion proteins can be introduced into cells, and may be any of a variety of methods known and described in the art (eg, Sambr).
OK et al., Molecular Cloning: A
Laboratoy Manual, 2nd Ed, V
ols 1 to 3, Cold Spring Ha
rbor Laboratory Press, New
York (1989), and Ausubel et al., C.
current Protocols in Molec
ullar Biology, John Wiley a
nd Sons, Baltimore, MD (199
4), each of which may produce a fusion protein of the invention by virtue of which is incorporated herein by reference)). Methods for introducing a recombinant expression vector into prokaryotic or eukaryotic cells include, for example, transformation or transfection methods such as electroporation.

When the recombinant F protein was expressed in E. coli, it was expressed as an inactive F0 form. In order to convert an inactive F0 form protein expressed in E. coli to an active F1 form, trypsinization at 37 ° C. for 30 minutes using 0.0004-0.001% trypsin was required.

The polypeptide corresponding to the activated trypsin-treated F1 protein can be expressed in E. coli using an expression vector containing a gene encoding the amino acid sequence of the truncated activated F1. The shortened F1 protein must include at least 26 amino acid residues from phenylalanine at 117 to alanine at 142. If the truncated protein forms an inclusion body, one of skill in the art can easily obtain an active protein by refolding the inclusion body (Robert F. Kelley and Marj).
orie E. Winkler, Genetic En
ginering, (1990) vol. 12, 1 ~
See page 19).

Cells capable of replicating HVJ (eg, rodent tracheal epithelial cells; chicken embryos; primary cultured cells of monkey kidney; primary cultured cells of human fetal lung, kidney and amniotic membrane) are used as host cells. When expressing the protein, the expressed full-length F protein is cleaved by the endogenous protease of the host cell and, as a result, is activated.
Activated F protein can be expressed and isolated. Alternatively, Triptase clara (Ki
do, et al., 1999) as endogenous enzymes (eg, rat tracheal epithelial cells) or recombinantly expressing host cells can also be used.

Methods for selecting and constructing these expression vectors, introducing them into host cells, expressing them in host cells, and recovering expressed proteins are well known to those skilled in the art.

(Preparation of Gene Transfer Vehicle) 3.56 m
A lipid mixture of g phosphatidylcholine and 0.44 mg cholesterol was dissolved in chloroform and the lipid solution was evaporated in a rotary evaporator (Uchida et al., 1979). The dried lipid mixture was completely dissolved by vortexing in 2.0 ml of the protein solution (1.6 mg) from the above through-fraction containing 0.85% NP-40. The solution was then diluted with 10 M containing 0.3 M sucrose and 1 mM KCl.
Dialysis against mM phosphate buffer (pH 7.2)
P-40 was removed. Dialysis is performed by changing the buffer every day.
Days. The dialyzed solution was agarose beads (Bio) equilibrated with 10 mM phosphate buffer (pH 5.2) containing 0.3 M sucrose and 1 mM KCl.
-Gel A-50m) (Bio-Rad Labor)
atories, Hercules, CA, USA). Optical density at 540 nm of 1.5
Fractions were collected as reconstituted fusion particles and fused with nucleic acid-loaded liposomes prepared from 10 mg lipid as described below to prepare a gene transfer vehicle.

(Safety) 1) Safety test by RT-PCR Whether the gene transfer vehicle prepared as described above contains HVJ genomic RNA or not is determined by RT-PCR as follows.
Was done.

[0049] Total cell RNA was purified using an ISOGEN ribonucleotide isolation kit (Nippon Gene Co. Ltd.
d. ) Was isolated two days after transfection according to the manufacturer's protocol. The RT-PCR reaction is
0.3 mM dNTP, 1 × reaction buffer, 5 units of r
Gene in a reaction solution (50 μl total volume) containing Tth DNA polymerase, 20 units of RNase inhibitor, 2.5 mM manganese acetate (Toyobo), and 0.4 μM sense and antisense primers
Amp PCR System 2400 (Perkin-El)
mer).

Primers were designed to PCR amplify a 715 bp fragment of the HVJ F protein coding sequence (Hokkaido System S).
science Co. Ltd. ). The sense strand 5′GTGATTGGTACTATCGCACTT 3 ′ antisense strand 5′CTGGCTGTCAGGTATCAGTTG 3 ′ The reaction mixture was subjected to one cycle of reverse transcription at 60 ° C. for 30 minutes;
One cycle of PCR amplification at 94 ° C for 3 minutes and 50 ° C for 1.5 minutes; 38 cycles of PCR amplification at 94 ° C for 1 minute and 50 ° C for 1.5 minutes; 94 ° C for 1 minute and 50 ° C
One cycle of PCR amplification for 8.5 minutes. All assays were performed in triplicate. RT-PCR products were subjected to agarose gel electrophoresis in the presence of ethidium bromide and analyzed with a UV illuminator. A 715 bp band indicating the presence of the sequence encoding the F protein was not observed in the sample after transfection.

From the above results, it was revealed that the above-mentioned gene transfer vehicle does not contain HVJ genomic RNA. Thus, the gene transfer vehicle is safe because it does not cause viral replication and is very weakly stimulating the immune system.

2) Safety test for immunogenicity Furthermore, this gene transfer vehicle was used for the NP protein of HVJ (Col.
e et al., 1997; Chen et al., 1998). Therefore, this gene vehicle is considered to be less immunogenic than HVJ liposomes containing NP protein. The immunogenicity of this gene transfer vehicle can be analyzed by repeated in vivo injections of the reconstituted vehicle reported for the HVJ liposome system (Hirano et al., 1998).

(Preparation of Gene Transfer Vehicle Containing Fluorescein Isothiocyanate-Labeled Oligonucleotide)
Fluorescein isothiocyanate (FITC) -oligonucleotide (ODN) was prepared as previously described (Mo
Rishita, 1994). Briefly, 10 mg of the dry lipid mixture (phosphatidylserine, phosphatidylcholine and cholesterol) was added to 20 nmole FITC-conjugated ODN.
(5′-GAT-CCG-CGG-GAA-ATF-
3 '; Clontech Laboratories,
Inc. (Palo Alto, CA, USA). FITC-ODN loaded liposomes were prepared by vortexing and sonication. This 20 nmole FITC-OD
N-containing liposomes (containing 10 mg lipid)
Incubated with 4 g of the reconstituted fusion particles containing lipid for 10 minutes at 4 ° C. and then for 60 minutes at 37 ° C. This FITC
The gene transfer vehicle containing -ODN was purified by sucrose gradient ultracentrifugation as previously described (Kaneda, 1994).

(Transfer of FITC-ODN to FL cells)
Human amniotic FL cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 1/10 of the transfection vehicle with FITC-ODN prepared as described above at 37 ° C. Incubated for 30 minutes. The cells were then washed with fresh medium to remove the vehicle and fixed with cold methanol containing 1% acetic acid at 4 ° C. for 5 minutes. After washing with phosphate buffered saline, nuclei were removed at 1 μg / ml Hoech.
st 33258 (Sigma Chemicals)
Inc. , St. Louis, MO, USA) for 5 minutes. Next, cells that received FITC-ODN were observed with a fluorescence microscope.

(Luciferase gene expression in transfected cells derived from human HEK293 strain) pCMV
-Luciferase (7.4 kb) was converted to pGEM-luc
(Promega Corp., Madison, W
I, USA) from the pcDN
A3 (5.4 kb) (Invitrogen, San
(Diego, CA, USA) by cloning at the HindIII and BamHI sites. A transfection vehicle containing about 40 μg of pCMV-luciferase was constructed as described above, and the transfection vehicle (about 1.5 × 10 ¹¹ particles / ml,
The DNA concentration is about 1/10 of the amount (100 μg / ml) (100
μl) was transferred to a human 293 cell line (human fetal kidney: HEK).
Incubated with 2 × 10 5 cells from the origin. The same amount of luciferase DNA was transferred to 2 × 10 5 HEK293 cells using HVJ liposomes. Twenty-four hours after transfection, cells are harvested and described (Saek
i et al., 1997), luciferase activity assay.

(LacZ Gene Transfer into Mouse Muscle) The expression vector pAct-LacZ-NII was constructed as described previously (Saeki et al., 1997). Eight week old male C57BL / 6 mice were anesthetized by intraperitoneal injection of diluted pentobarbital (1 mg). One-third of the transfection vehicle containing 40 μg of pAct-LacZ-NII prepared as described above (DNA concentration of about 40
μg / ml) was injected directly into the quadriceps using a 1 ml syringe equipped with a 26G needle. On day 3 of transfer, muscles at the injection site were isolated, fixed with 1% glutaraldehyde, immersed in 20% sucrose overnight, and frozen sections (10 μm) were cryostated (Miles-San).
(Kyo, Kanagawa, Japan).
LacZ gene expression was determined as described elsewhere (Sanes
1986).

(Example 1) (Purification of fusion protein from HVJ) For purification of fusion protein, a lysate of NPJ-treated HVJ was clarified by ultracentrifugation. Supernatant protein,
Before further purification, it was analyzed by SDS-PAGE (FIG. 1a). This supernatant contained many proteins from HVJ. Next, the supernatant was applied to ion exchange chromatography. 52 kDa and 72
The kDa protein was mainly eluted in the flow-through fraction (FIG. 1b, lane 1). Depending on the mobility on SDS-PAGE, these two proteins were separated into F1 and H, respectively.
N (Okada, 1993). 52k
The faint band below the Da protein was considered to be a degradation product of the fusion proteins (F1 and HN).
This is because these proteins were not reproducible between different experiments. 0.2% more protein
Eluted with M NaCl. However, the fusion protein was not obtained efficiently. And NP of HVJ
An additional 60 kDa protein appeared to be a protein (FIG. 1b, lane 2). as a result,
Only the flow through fraction was used as a source of fusion protein for further experiments. Densitometry showed that the F1 to HN concentration ratio of the flow through fraction was 2.3: 1. This was consistent with the ratio of both proteins in the virus envelope. Previous paper (Nakanishi
Et al., 1982) report that this ratio is required for efficient fusion of HVJ.

(Example 2) (Preparation of reconstituted fusion particles)
Was added to the liquid mixture solubilized and the liposomes were prepared by dialysis. This liposome contained F1 and HN (FIG. 1b, lane 3). However, the inventors have failed to capture DNA in the same liposome by dialysis. DNA-containing fusion particles were constructed by incubating empty fusion particles with DNA-loaded liposomes prepared by vortex sonication. A schematic diagram of the construction is shown in FIG.

Example 3 (Introduction of FITC-ODN into Cultured Cells by Reconstituted Fusion Particles) 30 minutes after transfection, FIT
C-ODN was found in all nuclei of FL cells (FIGS. 3a and 4). Without the use of fusion particles, very faint signals were found in FL cells (FIG. 3b).
FITC-OD achieved by gene transfer vehicle
The efficiency of N introduction was comparable to that of standard HVJ liposomes based on UV irradiation of whole HVJ virions.

The empty reconstituted fusion particles were stored at 4 ° C. for 1-4 weeks, and reconstituted fusion particles containing FITC-ODN were assembled at weekly intervals and the FITC-ODN was FL
The cells were introduced. Using reconstituted fusion particles stored for 4 weeks, FITC-ODN was introduced into more than 80% of the cells (FIG. 4).

For comparison, 20 nmole of FITC-O was added to a liposome containing 10 mg lipid according to the conventional method.
The DNC was incorporated by vortex-sonication, the liposomes were fused with HVJ (equivalent to 15000 HAU) inactivated by ultraviolet irradiation at 198 mjoule / cm ² , and free HVJ was purified by sucrose gradient ultracentrifugation to obtain FITC. -HVJ liposomes filled with ODN were prepared and stored at 4 ° C for 1-4 weeks. When a gene vehicle was prepared using this HVJ liposome, the efficiency of introduction of FITC-ODN into the cell nucleus was dramatically reduced. The use of HVJ liposomes prepared with inactivated HVJ stored at 4 ° C. for only one week reduced the efficiency of gene transfer to less than 10%.

Example 4 In Vitro Gene Expression Using Reconstituted Fusion Protein As shown in FIG. 2, a luciferase gene-loaded transfection vehicle was prepared and added to HEK293 cells. One day after transfection, luciferase activity in the cells was determined as previously reported (Sa
eki et al., 1997). As shown in FIG.
Luciferase gene expression obtained using the gene transfer vehicle was almost identical to expression obtained using HVJ liposomes. Both F and HN proteins are required for virus-cell fusion. In practice, as previously reported (Bagai and S
anker, 1993) Luciferase gene transfer was dramatically reduced when the HN protein was inactivated with 3 mM dithiothreitol (DTT) before being incorporated into reconstituted vesicles.

Briefly, the fusion protein was purified and HN
Exposed to inactivated DTT treatment and used to prepare reconstituted liposomes. The resulting vesicles could transfer the luciferase gene into HEK293 cells, but with low efficiency. Under these conditions, luciferase activity was less than 10% of the level obtained with intact reconstituted fusion particles.

Example 5 In Vivo Gene Expression Using Reconstituted Fusion Protein The LacZ gene was directly transferred in vivo into mouse skeletal muscle using a gene transfer vehicle. FIG.
As shown in the figure, LacZ expression is 40-50% of the muscle fiber.
Was found in No significant toxicity was found in mouse muscle.

From the foregoing, it is apparent that while certain embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. It is. Accordingly, the invention is not limited except as by the appended claims.

[0066]

The present invention provides a gene transfer vehicle using an HVJ fusion protein. The gene transfer vehicle of the present invention introduces ODN and plasmid DNA into cells as efficiently as standard HVJ liposomes based on whole virions of UV-inactivated HVJ. In addition, the gene transfer vehicle of the present invention is safe and can be stably stored.

(References)

[0068]

[Table 1]

[0069]

[Sequence List] SEQUENCE LISTING <110> MedGene Bioscience, Inc. <120> A Gene Transfer Vehicle Comprising HVJ Fusion Proteins <130> J100083473 <140> <141> 2000-08-31 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 1813 <212> DNA <213> Sendai virus <220> cDNA to viral ss-RNA <221> CDS <222> (54) .. (1751) <223> F protein <223> mature peptide F2 <223> (54) .. (401) <223> mature peptide F1 <223> (402) .. (1748) <400> 1 agggataaag tcccttgtga gtgcttgatt gcaaaactct ccccttggga aacatgacag 60 catatatcca gagatcacag tgcatctcagt catcactcgt gtca tccatc gt gattcccagg gataggctct ctaacatagg ggtcatagtc gatgaaggga 180 aatcactgaa gatagctgga tcccacgaat cgaggtacat agtactgagt ctagttccgg 240 gggtagactt tgagaatggg tgcggaacag cccaggttat ccagtacaag agcctactga 300 acaggctgtt aatcccattg agggatgcct tagatcttca ggaggctctg ataactgtca 360 ccaatgatac gacacaaaat gccggtgctc cacagtcgag attcttcggt gctgtgattg 420 gtactatcgc acttggagtg gcgacatcag cacaaatcac cgcagggatt gcactagccg 480 aagcgaggga ggccaaaaga gacatag cgc tcatcaaaga atcgatgaca aaaacacaca 540 agtctataga actgctgcaa aacgctgtgg gggaacaaat tcttgctcta aagacactcc 600 aggatttcgt gaatgatgag atcaaacccg caataagcga attaggctgt gagactgctg 660 ccttaagact gggtataaaa ttgacacagc attactccga gctgttaact gcgttcggct 720 cgaatttcgg aaccatcgga gagaagagcc tcacgctgca ggcgctgtct tcactttact 780 ctgctaacat tactgagatt atgaccacaa tcaagacagg gcagtctaac atctatgatg 840 tcatttatac agaacagatc aaaggaacgg tgatagatgt ggatctagag agatacatgg 900 tcaccctgtc tgtgaagatc cctattcttt ctgaagtccc aggtgtgctc atacacaagg 960 catcatctat ttcttacaac atagacgggg aggaatggta tgtgactgtc cccagccata 1020 tactcagtcg tgcttctttc ttagggggtg cagacataac cgattgtgtt gagtccagat 1080 tgacctatat atgccccagg gatcccgcac aactgatacc tgacagccag caaaagtgta 1140 tcctggggga cacaacaagg tgtcctgtca caaaagttgt ggacagcctt atccccaagt 1200 ttgcttttgt gaatgggggc gttgttgcta actgcatagc atccacatgt acctgcggga 1260 caggccgaag accaatcagt caggatcgct ctaaaggtgt agtattccta acccatgaca 1320 actgtggtct tataggtgtc aatggggtag aattgtatgc taaccggaga gggcacgatg 1380 ccacttgggg ggtccagaac ttgacagtcg gtcctgcaat tgctatcaga cccattgata 1440 tttctctcaa ccttgctgat gctacgaatt tcttgcaaga ctctaaggct gagcttgaga 1500 aagcacggaa aatcctctcg gaggtaggta gatggtacaa ctcaagagag actgtgatta 1560 cgatcatagt agttatggtc gtaatattgg tggtcattat agtgatcatc atcgtgcttt 1620 atagactcag aaggtcaatg ctaatgggta atccagatga ccgtataccg agggacacat 1680 acacattaga gccgaagatc agacatatgt acacaaacgg tgggtttgat gcaatggctg 1740 agaaaagatg atcacgacca ttatcagatg tcttgtaaag caggcatggt atccgttgag 1800 atctgtatat aat 1813 <210> 2 <211> 565 <212> PRT <213> Sendai virus <223> F protein <400> 2 Met Thr Ala Tyr Ile Gln Arg Ser Gln Cys Ile Ser Thr Ser Leu Leu 1 5 10 15 Val Val Leu Thr Thr Leu Val Ser Cys Gln Ile Pro Arg Asp Arg Leu 20 25 30 Ser Asn Ile Gly Val Ile Val Asp Glu Gly Lys Ser Leu Lys Ile Ala 35 40 45 Gly Ser His Glu Ser Arg Tyr Ile Val Leu Ser Leu Val Pro Gly Val 50 55 60 Asp Phe Glu Asn Gly Cys Gly Thr Ala Gln Val Ile Gln Tyr Lys Ser 65 70 75 80 Leu Leu Asn Arg Leu Leu Ile Pro Leu Arg Asp Ala Leu Asp Leu Gln 85 90 95 Glu Ala Leu Ile Thr Val Thr Asn Asp Thr Thr Gln Asn Ala Gly Ala 100 105 110 Pro Gln Ser Arg Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly 115 120 125 Val Ala Thr Ser Ala Gln Ile Thr Ala Gly Ile Ala Leu Ala Glu Ala 130 135 140 Arg Glu Ala Lys Arg Asp Ile Ala Leu Ile Lys Glu Ser Met Thr Lys 145 150 155 160 Thr His Lys Ser Ile Glu Leu Leu Gln Asn Ala Val Gly Glu Gln Ile 165 170 175 Leu Ala Leu Lys Thr Leu Gln Asp Phe Val Asn Asp Glu Ile Lys Pro 180 185 190 Ala Ile Ser Glu Leu Gly Cys Glu Thr Ala Ala Leu Arg Leu Gly Ile 195 200 205 Lys Leu Thr Gln His Tyr Ser Glu Leu Leu Thr Ala Phe Gly Ser Asn 210 215 220 Phe Gly Thr Ile Gly Glu Lys Ser Leu Thr Leu Gln Ala Leu Ser Ser 225 230 235 240 Leu Tyr Ser Ala Asn Ile Thr Glu Ile Met Thr Thr Ile Lys Thr Gly 245 250 255 Gln Ser Asn Ile Tyr Asp Val Ile Tyr Thr Glu Gln Ile Lys Gly Thr 260 265 270 Val Ile Asp Val Asp Leu Glu Arg Tyr Met Val Thr Leu Ser Val Lys 275 280 285 Ile Pro Ile Leu Ser Gl u Val Pro Gly Val Leu Ile His Lys Ala Ser 290 295 300 300 Ser Ile Ser Tyr Asn Ile Asp Gly Glu Glu Trp Tyr Val Thr Val Pro 305 310 315 320 Ser His Ile Leu Ser Arg Ala Ser Phe Leu Gly Gly Ala Asp Ile Thr 325 330 335 Asp Cys Val Glu Ser Arg Leu Thr Tyr Ile Cys Pro Arg Asp Pro Ala 340 345 350 Gln Leu Ile Pro Asp Ser Gln Gln Lys Cys Ile Leu Gly Asp Thr Thr 355 360 365 Arg Cys Pro Val Thr Lys Val Val Asp Ser Leu Ile Pro Lys Phe Ala 370 375 380 Phe Val Asn Gly Gly Val Val Ala Asn Cys Ile Ala Ser Thr Cys Thr 385 390 395 400 Cys Gly Thr Gly Arg Arg Pro Ile Ser Gln Asp Arg Ser Lys Gly Val 405 410 415 Val Phe Leu Thr His Asp Asn Cys Gly Leu Ile Gly Val Asn Gly Val 420 425 430 Glu Leu Tyr Ala Asn Arg Arg Gly His Asp Ala Thr Trp Gly Val Gln 435 440 445 Asn Leu Thr Val Gly Pro Ala Ile Ala Ile Arg Pro Ile Asp Ile Ser 450 455 460 Leu Asn Leu Ala Asp Ala Thr Asn Phe Leu Gln Asp Ser Lys Ala Glu 465 470 475 480 Leu Glu Lys Ala Arg Lys Ile Leu Ser Glu Val Gly Arg Trp Tyr Asn 485 490 495 495 Ser Arg Glu Thr Val I le Thr Ile Ile Val Val Met Val Val Ile Leu 500 505 510 Val Val Ile Ile Val Ile Ile Ile Val Leu Tyr Arg Leu Arg Arg Ser 515 520 525 Met Leu Met Gly Asn Pro Asp Asp Arg Ile Pro Arg Asp Thr Tyr Thr 530 535 540 Leu Glu Pro Lys Ile Arg His Met Tyr Thr Asn Gly Gly Phe Asp Ala 545 550 555 560 Met Ala Glu Lys Arg 565 <210> 3 <211> 1883 <212> DNA <213> Sendai virus <220> cDNA to viral ss-RNA <221> CDS <222> (57) .. (1784) <223> HN protein <400> 3 agggtgaaag tgaggtcgcg cggtacttta gctttccacct caaacaagca cagatcatgg 60 atggtgatag gggcaaacgt gactc gagcag tagtaggag aggtagcat aggtagct aggtagct agg ggctttgtca attgccacag tgatcatctg tatcataatt tctgctagac 240 aagggtatag tatgaaagag tactcaatga ctgtagaggc attgaacatg agcagcaggg 300 aggtgaaaga gtcacttacc agtctaataa ggcaagaggt tatagcaagg gctgtcaaca 360 ttcagagctc tgtgcaaacc ggaatcccag tcttgttgaa caaaaacagc agggatgtca 420 tccagatgat tgataagtcg tgcagcagac aagagctcac tcagcactgt gagagtacga 480 t cgcagtcca ccatgccgag ggaattgccc cacttgagcc acatagtttc tggagatgcc 540 ctgtcggaga accgtatctt agctcagatc ctgaaatctc attgctgcct ggtccgagct 600 tgttatctgg ttctacaacg atctctggat gtgttaggct cccttcactc tcaattggcg 660 aggcaatcta tgcctattca tcaaatctca ttacacaagg ttgtgctgac atagggaaat 720 catatcaggt cctgcagcta gggtacatat cactcaattc agatatgatc cctgatctta 780 accccgtagt gtcccacact tatgacatca acgacaatcg gaaatcatgc tctgtggtgg 840 caaccgggac taggggttat cagctttgct ccatgccgac tgtagacgaa agaaccgact 900 actctagtga tggtatcgag gatctggtcc ttgatgtcct ggatctcaaa gggagaacta 960 agtctcaccg gtatcgcaac agcgaggtag atcttgatca cccgttctct gcactatacc 1020 ccagtgtagg caacggcatt gcaacagaag gctcattgat atttcttggg tatggtggac 1080 taaccacccc tctgcagggt gatacaaaat gtaggaccca aggatgccaa caggtgtcgc 1140 aagacacatg caatgaggct ctgaaaatta catggctagg agggaaacag gtggtcagcg 1200 tgatcatcca ggtcaatgac tatctctcag agaggccaaa gataagagtc acaaccattc 1260 caatcactca aaactatctc ggggcggaag gtagattatt aaaattgggt gatcgggtgt 1320 acatctatac aag atcatca ggctggcact ctcaactgca gataggagta cttgatgtca 1380 gccacccttt gactatcaac tggacacctc atgaagcctt gtctagacca ggaaataaag 1440 agtgcaattg gtacaataag tgtccgaagg aatgcatatc aggcgtatac actgatgctt 1500 atccattgtc ccctgatgca gctaacgtcg ctaccgtcac gctatatgcc aatacatcgc 1560 gtgtcaaccc aacaatcatg tattctaaca ctactaacat tataaatatg ttaaggataa 1620 aggatgttca attagaggct gcatatacca cgacatcgtg tatcacgcat tttggtaaag 1680 gctactgctt tcacatcatc gagatcaatc agaagagcct gaatacctta cagccgatgc 1740 tctttaagac tagcatccct aaattatgca aggccgagtc ttaaatttaa ctgactagca 1800 ggcttgtcgg ccttgctgac actagagtca tctccgaaca tccacaatat ctctcagtct 1860 cttacgtctc tcacagtatt aag 1883 <210> 4 <211> 575 <212> PRT <223> GRT <213> GRT virus Asp Ser Tyr Trp Ser Thr Ser Pro 1 5 10 15 Ser Gly Ser Thr Thr Lys Leu Ala Ser Gly Trp Glu Arg Ser Ser Lys 20 25 30 Val Asp Thr Trp Leu Leu Ile Leu Ser Phe Thr Gln Trp Ala Leu Ser 35 40 45 Ile Ala Thr Val Ile Ile Cys Ile Ile Ile Ser Ala Arg Gln Gly Tyr 50 55 60 Ser Met Lys Glu Tyr Ser Met Thr Val Glu Ala Leu Asn Met Ser Ser 65 70 75 80 Arg Glu Val Lys Glu Ser Leu Thr Ser Leu Ile Arg Gln Glu Val Ile 85 90 95 Ala Arg Ala Val Asn Ile Gln Ser Ser Val Gln Thr Gly Ile Pro Val 100 105 110 Leu Leu Asn Lys Asn Ser Arg Asp Val Ile Gln Met Ile Asp Lys Ser 115 120 125 Cys Ser Arg Gln Glu Leu Thr Gln His Cys Glu Ser Thr Ile Ala Val 130 135 140 His His Ala Glu Gly Ile Ala Pro Leu Glu Pro His Ser Phe Trp Arg 145 150 155 160 Cys Pro Val Gly Glu Pro Tyr Leu Ser Ser Asp Pro Glu Ile Ser Leu 165 170 175 Leu Pro Gly Pro Ser Leu Leu Ser Gly Ser Thr Thr Ile Ser Gly Cys 180 185 190 Val Arg Leu Pro Ser Leu Ser Ile Gly Glu Ala Ile Tyr Ala Tyr Ser 195 200 205 Ser Asn Leu Ile Thr Gln Gly Cys Ala Asp Ile Gly Lys Ser Tyr Gln 210 215 220 Val Leu Gln Leu Gly Tyr Ile Ser Leu Asn Ser Asp Met Ile Pro Asp 225 230 235 240 Leu Asn Pro Val Val Ser His Thr Tyr Asp Ile Asn Asp Asn Arg Lys 245 250 255 Ser Cys Ser Val Val Ala Thr Gly Thr Arg Gly Tyr Gln Leu Cys S er 260 265 270 Met Pro Thr Val Asp Glu Arg Thr Asp Tyr Ser Ser Asp Gly Ile Glu 275 280 285 Asp Leu Val Leu Asp Val Leu Asp Leu Lys Gly Arg Thr Lys Ser His 290 295 300 Arg Tyr Arg Asn Ser Glu Val Asp Leu Asp His Pro Phe Ser Ala Leu 305 310 315 320 Tyr Pro Ser Val Gly Asn Gly Ile Ala Thr Glu Gly Ser Leu Ile Phe 325 330 335 Leu Gly Tyr Gly Gly Leu Thr Thr Pro Leu Gln Gly Asp Thr Lys Cys 340 345 350 Arg Thr Gln Gly Cys Gln Gln Val Ser Gln Asp Thr Cys Asn Glu Ala 355 360 365 Leu Lys Ile Thr Trp Leu Gly Gly Lys Gln Val Val Ser Val Ile Ile 370 375 380 Gln Val Asn Asp Tyr Leu Ser Glu Arg Pro Lys Ile Arg Val Thr Thr 385 390 395 400 Ile Pro Ile Thr Gln Asn Tyr Leu Gly Ala Glu Gly Arg Leu Leu Lys 405 410 415 Leu Gly Asp Arg Val Tyr Ile Tyr Thr Arg Ser Ser Gly Trp His Ser 420 425 430 Gln Leu Gln Ile Gly Val Leu Asp Val Ser His Pro Leu Thr Ile Asn 435 440 445 Trp Thr Pro His Glu Ala Leu Ser Arg Pro Gly Asn Lys Glu Cys Asn 450 455 460 Trp Tyr Asn Lys Cys Pro Lys Glu Cys Ile Ser Gly Val Tyr Thr Asp Four 65 470 475 480 Ala Tyr Pro Leu Ser Pro Asp Ala Ala Asn Val Ala Thr Val Thr Leu 485 490 495 Tyr Ala Asn Thr Ser Arg Val Asn Pro Thr Ile Met Tyr Ser Asn Thr 500 505 510 Thr Asn Ile Ile Asn Met Leu Arg Ile Lys Asp Val Gln Leu Glu Ala 515 520 525 525 Ala Tyr Thr Thr Thr Ser Cys Ile Thr His Phe Gly Lys Gly Tyr Cys 530 535 540 540 Phe His Ile Ile Glu Ile Asn Gln Lys Ser Leu Asn Thr Leu Gln Pro 545 550 555 555 560 Met Leu Phe Lys Thr Ser Ile Pro Lys Leu Cys Lys Ala Glu Ser 565 570 575

[Brief description of the drawings]

FIG. 1 shows purification of HVJ fusion proteins (HN and F1). (A) The protein component of the supernatant of HVJ solubilized with detergent was analyzed on SDS-PAGE after ultracentrifugation. By mobility on SDS-PAGE, these proteins were identified as H
It was estimated to be N, NP, F1 and M proteins. (B) subjecting the supernatant to ion exchange chromatography,
The protein components of the flow-through fraction (lane 1) and the eluate at 0.2 M NaCl (lane 2) were analyzed by SDS-PAGE.
Analyzed on E. The fusion protein was mixed with lipids and reconstituted fusion particles were constructed by dialysis.
Both F1 and HN were detected in the reconstituted liposomes (lane 3). Molecular weight markers are indicated by arrows on the left.

FIG. 2 is a schematic diagram showing the construction of a gene transfer vehicle. HVJ is solubilized and the fusion protein is isolated. This protein is inserted into the liposome by dialysis to form reconstituted fusion particles. Plasmid DN
Liposomes containing A or FITC-ODN are constructed, fused with reconstituted fusion particles, and prepared as a gene transfer vehicle.

FIG. 3. FITC by gene transfer vehicle.
-Shows the transfer of ODN into FL cells. 30 minutes after the transfer,
Fluorescence in all FL cells was detected (a), while FI
No fluorescence was observed when using a gene transfer vehicle without TC-ODN (b). The left figure is F
The detection of ITC is shown, and the figure on the right shows Ho for nuclear detection.
3 shows echst staining.

FIG. 4 shows the stability of the reconstituted fused particles. Reconstituted fused particles (squares) and UV-irradiated HVJ (circles)
Stored for 1 to 4 weeks at ° C. Each week, liposomes containing FITC-ODN were fused with either reconstituted fusion particles or UV-inactivated HVJ. FIT for FL cells
The efficiency of C-ODN transfer was measured by counting the fluorescent nuclei of approximately 500 cells. The means and standard deviations of triplicate experiments are shown.

FIG. 5 shows luciferase gene expression in HEK239 cells. The luciferase gene was replaced with HEK2
39 cells were transfected with (1) HVJ liposomes, (2) gene transfer vehicle, or (3) DTT treated gene transfer vehicle.

FIG. 6 shows LacZ expression in mouse skeletal muscle. (A) The transfection vehicle containing pAct-LacZ-NII was injected into C57BL / 6 mice.
(B) As a negative control, an empty gene transfer vehicle was injected into the same muscle. 3 days after transfer, L
The acZ gene expression was determined by X-g
Tested by al staining. 1 shows a cross section of a quadriceps.

FIG. 7 shows the nucleic acid sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) of the F protein derived from the HVJ Z strain.

FIG. 8 shows the nucleic acid sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 4) of the HN protein derived from the HVJ Z strain.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 AA01 BA80 CA02 DA03 EA04 FA20 GA13 HA17 4B064 CA02 CA10 CA19 CB06 CC24 DA01 4C084 AA06 AA13 BA03 CA01 DC50 NA14 ZB332 4H045 AA10 AA20 AA30 BA10 BA55 CA01 GA20 GA72 HA06

Claims (14)

[Claims] 1. A gene transfer vehicle reconstituted from the HVJ F fusion protein and HN fusion protein, wherein the gene transfer vehicle does not contain an amount of HVJ genomic RNA detectable by RT-PCR. 2. The gene transfer vehicle according to claim 1, which has gene transfer activity for 40% or more of cells in a living body. 3. When the gene transfer vehicle is formed 4 weeks after the preparation of the reconstituted fusion particles, retains 70% or more gene transfer activity to cells in vitro.
The gene transfer vehicle of claim 1. 4. The gene transfer vehicle according to claim 1, wherein the gene transfer vehicle can transfer a foreign gene up to 100 kb. 5. The gene transfer vehicle according to claim 1, wherein the F fusion protein and the HN protein are derived from HVJ that has not been irradiated with UV. 6. The gene transfer vehicle according to claim 1, wherein the F fusion protein and the HN protein are recombinantly expressed fusion proteins. 7. The gene transfer vehicle according to claim 6, wherein the F protein is a protein processed by a protease. 8. A pharmaceutical composition comprising the gene transfer vehicle according to claim 1 for gene therapy. 9. A method for transferring a gene to an isolated cell, comprising the steps of:
Preparing a gene transfer vehicle according to the above, and transferring the gene to the cells by the gene transfer vehicle. 10. A method for preparing a gene transfer vehicle containing a fusion protein of HVJ, comprising the steps of: isolating the fusion protein from HVJ virus not subjected to UV irradiation; And reconstituting in the presence of a lipid to prepare reconstituted particles, preparing a liposome filled with a desired nucleic acid, and fusing the reconstituted particles and the liposome. 11. A method for preparing a gene transfer vehicle containing an HVJ F protein and an HN protein, comprising: a step of recombinantly expressing the F protein and the HN protein; a step of processing the F protein with a protease; Isolating the F protein and the HN protein; reconstituting the F protein and the HN protein in the presence of a surfactant and a lipid to prepare reconstituted particles; preparing a liposome filled with a desired nucleic acid And fusing the reconstituted particles and the liposome. 12. A method for preparing a gene transfer vehicle containing an HVJ F protein and an HN protein, comprising: combining the F protein and the HN protein in a host cell that expresses a protease that processes the F protein. A step of expressing the F protein and the HN protein, a step of reconstituting the F protein and the HN protein in the presence of a surfactant and a lipid to prepare reconstituted particles, and a step of preparing a desired nucleic acid. Preparing a loaded liposome, fusing the reconstituted particles and the liposome. 13. A gene transfer vehicle prepared by the method according to any one of claims 10 to 12. 14. A pharmaceutical composition comprising the gene transfer vehicle of claim 13 for gene therapy.