JP3942362B2 - Viral envelope vector for gene transfer - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a safe and highly efficient vector for gene transfer in vitro and in vivo. In particular, the present invention relates to a gene transfer vector prepared using a virus, an inactivated virus, particularly an inactivated HVJ (Sendai virus). The gene transfer vectors herein can also be used for gene therapy and high throughput screening.
[0002]
[Prior art]
For gene therapy, many viral and non-viral (synthetic) methods for gene transfer have been developed (Mulligan, Science, 260, 926-932 (1993) and Ledley, Human Gene Therapy, Vol. 6, 1129-1144 (1995)). In general, viral methods are more effective than non-viral methods for gene delivery to cells. However, viral vectors can pose safety problems due to co-introduction of essential genetic elements from the parent virus, leaky expression of viral genes, immunogenicity, and alteration of host genome structure. In general, non-viral vectors are less cytotoxic and immunogenic. However, most non-viral methods are particularly poor in gene transfer efficiency into the living body compared to some of the viral vectors.
[0003]
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 by introducing the advantages of another type of system It is.
[0004]
On the other hand, HVJ is highly immunogenic, and is known to induce CTL when NP protein is produced in large quantities (Cole, GA, et al., J. Immunology 158, 4301-4309 (1997)). ). There is also a concern that protein synthesis of the host may be inhibited.
[0005]
For HVJ, centrifugation and column operation of virus fusion protein In Particles made by the method of making and reconstituting a lipid membrane lose the other protein (mainly M protein) possessed by the virus by reconstitution, so that the ratio of Fl to HN protein required for fusion activity Is not maintained in the same manner as the wild-type virus, so that the fusion activity is low. In addition, since the direction in which the fusion protein is inserted into the lipid membrane when reconstituted is not always the same as that of the wild-type virus, an unknown antigen may be presented.
[0006]
In addition, a method for reconstitution by adding a new molecule has also been reported (Uchida, T., et al., J. Cell. Biol. 80, 10-20, 1979). The risk of losing the original virus function is great because it is very different from the virus particles.
[0007]
As represented by conventional HVJ-liposomes, genes and proteins are encapsulated in liposomes and fusion particles prepared by fusing them with inactivated HVJ are non-invasive to cultured cells and living tissues. Gene transfer has become possible, and this technique has been frequently used worldwide at the animal experiment level (Dzau, VJ, et al., Proc. Natl. Acad. Sci. USA, 93, 1412-111425 (1996). And Kaneda, Y. et al., Molecular Medicine Today, 5, 298-303 (1999)). However, it is necessary to prepare two different vesicles, a virus and a liposome, and the method is complicated. A particle whose average diameter is 1.3 times larger than that of a virus particle by fusing with a liposome has a fusion activity of 10% of that of a virus. It has been found that it has some drawbacks such as falling below.
[0008]
Furthermore, with conventional HVJ-based vectors, there were tissues where gene transfer was impossible and tissues with extremely low efficiency. This indicates that the target tissue for gene therapy based on the conventional method can be limited.
[0009]
[Problems to be solved by the invention]
For human gene therapy, it is desired to develop a viral vector that can be prepared safely, highly efficiently and simply, and can be introduced into a wide range of in vivo tissues.
[0010]
Therefore, the object of the present invention is to overcome the drawbacks of the conventional reconstituted HVJ vector method or HVJ-liposome method and develop a safe, highly efficient and simple virus-based gene transfer vector for a wide range of cultured cells and biological tissues. There is to do.
[0011]
[Means for Solving the Problems]
In one aspect of the present invention, a safe and highly efficient gene transfer vector using an inactivated virus is provided. Inactivated viruses in which the viral genome is inactivated are safe and have low cytotoxicity and antigenicity due to the absence of viral protein replication. By enclosing a gene in a virus envelope vector, which is a gene transfer vector using an inactivated virus, a safe, highly efficient and simple gene transfer vector for cultured cells and living tissues is prepared.
[0012]
In a further aspect of the present invention, a viral envelope vector capable of gene transfer into a wide range of in vivo tissues is provided. In one embodiment, the virus used is HVJ. Tissues that are gene-transferred in vivo by the virus envelope vector of the present invention include liver, skeletal muscle, uterus, brain, eye, carotid artery, skin, blood vessel, lung, heart, kidney, spleen, cancer tissue, nerve, B Examples include, but are not limited to, lymphocytes and respiratory tissues.
[0013]
In another aspect of the present invention, a method for easily introducing a gene into a floating cell is provided. A preferable method for introducing a gene into a floating cell using the virus envelope vector of the present invention includes a step of mixing the floating cell and the virus envelope vector in the presence of protamine sulfate, and a step of applying centrifugal force to the mixture. And a gene transfer method.
[0014]
In one aspect of the present invention, high-efficiency and rapid gene transfer into cultured cells and living tissues can be achieved by encapsulating a large amount of genes in a short time using the gene transfer vector of the present invention. Therefore, in a further aspect of the present invention, a high-throughput mass rapid analysis system for genome using the gene transfer vector of the present invention is possible.
[0015]
In a specific aspect of the present invention, the gene transfer vector can be stored for a long period (at least 2 to 3 months or more) in a frozen state at −20 ° C. The gene transfer vector can be sealed, stored and transported in a frozen state, for example.
[0016]
In another aspect of the present invention, a gene transfer vector having gene transfer activity is preferably provided in vitro to 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more of cells. Is done.
[0017]
In one aspect of the present invention, when a virus envelope vector that is a gene transfer vector is formed 2 months after the preparation of an inactivated virus, it is 60% or more, preferably 70% or more, more preferably 80% or more, and most preferably Provides a gene transfer vector having 90% or more gene transfer activity.
[0018]
In another aspect of the present invention, preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, and most preferably 60% or more of cells in the tissue for local administration in vivo. A gene transfer vector having gene transfer activity is provided.
[0019]
In an aspect of the present invention, a gene transfer vector comprising an inactivated virus envelope is provided.
[0020]
In one aspect of the present invention, the virus used for the preparation of the gene transfer vector may be a wild type virus or a recombinant virus.
[0021]
In a further aspect of the invention, the viruses used are retroviridae, togaviridae, coronaviridae, flaviviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, rhabdoviridae, poxviridae, It is a virus belonging to a family selected from the group consisting of Herpesviridae, Baculoviridae, and Hepadnaviridae. In a specific aspect of the present invention, the virus used is HVJ. In a further aspect of the present invention, Hasan, M. et al. K. (Journal of General Virology, 78, 2813-2820 (1997)) or Yonemitsu, Y. et al. (Nature Biotechnology 18, 970 to 973 (2000)), a recombinant Sendai virus is used to prepare a gene transfer vector.
[0022]
In another aspect of the present invention, a gene transfer vector for introducing a gene into a tissue in an animal body is provided.
[0023]
In the method for preparing the gene transfer vector of the present invention, the step of inactivating the virus is not necessarily required. Thus, in one aspect of the invention, without performing the step of inactivating the virus,
1) mixing the virus with a foreign gene, and
2) Freezing and thawing the mixed solution or further mixing the mixed solution with a surfactant;
A gene transfer vector can be prepared by a method comprising
[0024]
In another aspect of the present invention, a method for preparing an inactivated virus envelope vector for gene transfer, comprising:
Inactivating the virus;
Mixing the inactivated virus with a foreign gene; and
Freezing and thawing the mixture,
Are provided.
[0025]
In a further aspect of the invention, a method of preparing an inactivated viral envelope vector for gene transfer comprising:
Inactivating the virus; and
Mixing the inactivated virus with a foreign gene in the presence of a detergent,
Are provided.
[0026]
In a further aspect of the invention, the surfactant used is selected from the group consisting of octyl glucoside, Triton-X100, CHAPS and NP-40.
[0027]
In a specific aspect of the present invention, there is provided a method for preparing an inactivated virus envelope vector further comprising the step of adding protamine sulfate to a foreign gene before mixing with the inactivated virus envelope.
[0028]
In a further aspect of the present invention, a method for introducing a gene into an isolated animal tissue comprising:
Preparing a gene transfer vector containing a desired gene;
A step of introducing a gene into the animal tissue by a gene transfer vector;
Are provided.
[0029]
In another aspect of the present invention, a method for introducing a foreign gene into a floating cell is provided, the method comprising:
A step of mixing the floating cells and the gene transfer vector in the presence of protamine sulfate,
Step of centrifuging the mixture
Is included.
[0030]
In yet another aspect of the present invention, there is provided a pharmaceutical composition for gene therapy containing the gene transfer vector of the present invention.
[0031]
DETAILED DESCRIPTION OF THE INVENTION
(Definition)
As used herein, “gene transfer” refers to a natural, synthetic or recombinant desired gene or gene fragment in a target cell in vivo or in vitro, and the introduced gene has its function. Introducing to maintain. The gene or gene fragment introduced in the present invention includes a nucleic acid which is DNA, RNA or a synthetic analog thereof having a specific sequence. Also, as used herein, gene transfer, transfection, and transfection are used interchangeably.
[0032]
As used herein, “gene transfer vector”, “gene vector” or “virus envelope vector” refers to a vector in which a foreign gene is encapsulated in a virus envelope. The virus used for the preparation of the gene transfer vector may be a wild type virus or a recombinant virus.
[0033]
In one aspect of the invention, the viruses used are Retroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxviridae A virus belonging to the family selected from the group consisting of Herpesviridae, Baculoviridae, and Hepadnaviridae. In a specific aspect of the present invention, the virus used is HVJ.
[0034]
As used herein, “gene transfer activity” refers to the activity of “gene transfer” by a vector, and functions of the introduced gene (for example, in the case of an expression vector, expression of the encoded protein and / or Alternatively, the activity of the protein can be detected as an index.
[0035]
As used herein, “inactivation” refers to a virus that has inactivated its genome. This inactivated virus is replication defective. Preferably, this inactivation is done by UV treatment or treatment with an alkylating agent.
[0036]
As used herein, “foreign gene” refers to a nucleic acid sequence of a non-viral origin contained within a gene transfer vector. In one aspect of the invention, the foreign gene is a regulatory sequence suitable for expression of the gene introduced by the gene transfer vector (eg, a promoter, enhancer, terminator, and poly A addition signal required for transcription, and A ribosome binding site necessary for translation, start codon, stop codon, etc.). In another aspect of the invention, the foreign gene does not contain regulatory sequences for expression of the foreign gene. In a further aspect of the invention, the foreign gene is an oligonucleotide or a decoy nucleic acid.
[0037]
The foreign gene contained in the gene transfer vector is typically a DNA or RNA nucleic acid molecule, but the nucleic acid molecule to be introduced may include a nucleic acid analog molecule. The molecular species contained in the gene transfer vector may be a single gene molecular species or a plurality of different gene molecular species.
[0038]
As used herein, a “gene library” is a nucleic acid library comprising nucleic acid sequences isolated from nature or synthetic nucleic acid sequences. Sources of nucleic acid sequences isolated from nature include, but are not limited to, genomic and cDNA sequences derived from eukaryotic cells, prokaryotic cells, or viruses. A library obtained by adding an arbitrary sequence (eg, signal, tag, etc.) to a sequence isolated from nature is also included in the gene library of the present invention. In one embodiment, the gene library includes sequences such as promoters that provide for transcription and / or translation of the nucleic acid sequences contained therein.
[0039]
In the present specification, “HVJ” and “Sendai virus” may be used interchangeably.
[0040]
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 24 million virus particles (Okada, Y. et al., Biken Journal 4, 209-213, 1961).
[0041]
(Gene therapy)
Therapeutic nucleic acid constructs can be administered either locally or systemically using the gene transfer vectors of the present invention. Where such a nucleic acid construct includes a coding sequence for a protein, expression of the protein can be induced by use of an endogenous mammalian promoter or a heterologous promoter. The expression of the coding sequence can be constitutive or regulated.
[0042]
When the gene transfer vector of the present invention is used as a composition for gene therapy, the vector of the present invention can be administered by suspending a vector suspension suspended in PBS (phosphate buffered saline) or saline. By local injection (eg, into cancer tissue, liver, intramuscular and brain) or by administration into blood vessels (eg, intraarterial, intravenous and portal vein).
[0043]
In one embodiment, the gene transfer vector is generally used in a unit dose injectable form (solution, suspension or emulsion) of the gene transfer vector (ie, a pharmaceutically acceptable carrier (ie, used). Non-toxic to recipients at different dosages and concentrations, and compatible with the other ingredients of the formulation). For example, the formulation preferably does not include oxidizing agents and other compounds that are known to be deleterious to gene transfer vectors.
[0044]
The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are non-toxic to recipients at the dosages and concentrations used, and buffers such as phosphoric acid, citric acid, succinic acid, acetic acid, and other organic acids or their salts An antioxidant such as ascorbic acid; a low molecular weight (less than about 10 residues) polypeptide (eg, polyarginine or tripeptide); a protein (eg, serum albumin, gelatin, or an immunoglobulin); a hydrophilic polymer ( Amino acids (eg, glycine, glutamic acid, aspartic acid, or arginine); monosaccharides, disaccharides and other carbohydrates (including cellulose or derivatives thereof, glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg mannitol) Other sorbitol); counterions (such as sodium); and / or nonionic surfactants (e.g., may include polysorbates, poloxamers), or PEG.
[0045]
A pharmaceutical composition comprising a gene transfer vector can typically be stored as an aqueous solution in unit or multi-dose containers, such as sealed ampoules or vials.
[0046]
The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical composition of the present invention. Furthermore, the polypeptides of the present invention can be used with other therapeutic compounds.
[0047]
A pharmaceutical composition comprising a gene transfer vector of the present invention comprises a gene transfer vector in a manner consistent with good medical practice, an individual patient's clinical condition (eg, a condition to be prevented or treated). It is formulated and administered taking into account the site of delivery of the composition, the target tissue, the method of administration, the dosing schedule and other factors known to those of skill in the art. Accordingly, the “effective amount” or appropriate dosage of the gene transfer vector herein is determined by such considerations.
[0048]
For example, when the gene vector of the present invention is administered to a mouse, a gene vector equivalent to 20 to 20,000 HAU, preferably equivalent to 60 to 6,000 HAU, more preferably equivalent to 200 to 2,000 HAU per mouse. Be administered. The amount of the foreign gene contained in the administered gene vector is 0.1-100 μg, preferably 0.3-30 μg, more preferably 1-10 μg per mouse.
[0049]
In addition, when the gene vector of the present invention is administered to a human, it corresponds to 400 to 400,000 HAU per subject, preferably 1,200 to 120,000 HAU, more preferably 4,000 to 40,000 HAU. A gene vector is administered. The amount of the foreign gene contained in the administered gene vector is 2 to 2,000 μg, preferably 6 to 600 μg, more preferably 20 to 200 μg per subject.
[0050]
The following examples are illustrative and are not intended to limit the invention.
[0051]
【Example】
(1. Preparation of gene transfer vector using freeze-thaw and its use)
(Example 1: Preparation of HVJ envelope vector by freeze-thawing)
Using a luciferase gene as a foreign gene, the recombinant HVJ virus was freeze-thawed at various times and then introduced into cultured cells.
[0052]
TE500 μl was mixed with 750 μg of luciferase expression vector pcOriPLuc (Saeki and Kaneda et al., Human Gene Therapy, 11, 471-479 (2000)) and various concentrations of HVJ virus. The HVJ virus concentration was adjusted to 10, 25, 50, 100 HAU / μl. This solution was divided into 12 parts, each was stored at 4 ° C. with dry ice, frozen, and thawed repeatedly up to 30 times. A solution having been frozen and thawed a predetermined number of times was added to BHK-21 cells (24-well dish, 4 × 10 4 ^Four Cells / dish, 0.5 ml DMEM, 10% FCS) at 37 ° C., 5% CO ₂ After 20 minutes of reaction, the plate was washed with PBS, 0.5 ml of a new culture solution was newly added, and cultured for 24 hours.
[0053]
After removing the medium and adding 500 μl of 1 × Cell Culture Lys Reagent (Promega) onto the cells to lyse the cells, the cells were transferred to a microtube and centrifuged. From 20 μl of the obtained supernatant, Promega Luciferase Assay System and Lumat Luciferase activity was measured using LB9501 Luminophotometer. The measurement was performed 3 times for each solution, and the average value was obtained.
[0054]
The results are as shown in FIG. Luciferase activity increased as the number of times of freezing and thawing of the recombinant HVJ virus increased, and 10 times or more of luciferase expression was observed in 20 times of freezing and thawing compared to 3 times of freezing and thawing. From this result, it was confirmed that the number of times of freezing and thawing of the recombinant HVJ virus is preferably 5 times or more, more preferably about 15 to 20 times under the conditions used in this example.
[0055]
(Example 2: Gene transfer efficiency of HVJ envelope vector prepared by freeze-thawing)
The same recombinant HVJ virus as in Example 1 was freeze-thawed 30 times, and then the efficiency of gene transfer into the cells was examined under the same conditions as the number of viruses added to the host cells.
[0056]
The results are as shown in FIG. In FIG. 2, for example, when the X axis is 500 HAU, the addition amount of the solution having a virus concentration of 10 HAU / μl is 50 μl, and the solution of 100 HAU / μl is 5 μl. As shown in FIG. 2, the gene expression efficiency of the solution with the virus concentration of 100 HAU / μl was reduced by about 50% compared to the case of the concentration of 10 to 50 HAU / μl. From this result, it was confirmed that the recombinant virus concentration is preferably in the range of 10 to 50 HAU / μl under the conditions of this example.
[0057]
The recombinant HVJ virus was thawed 29 times and then thawed 30 times, stored in that frozen state for 1 week, thawed and added to the cells. As a result, the recombinant HVJ virus stored frozen for 1 week also showed the same level of luciferase gene expression as that of the virus that had been thawed thirty times continuously.
[0058]
(Example 3: Measurement of gene transfer efficiency using luciferase expression vector)
HVJ envelope vectors were prepared with various amounts of luciferase expression vectors, and the efficiency of gene transfer into host cells was examined.
[0059]
The amount of HVJ virus was 50 HAU per 1 μl TE. The amount of the luciferase expression vector pcOriPLuc was 0.05, 0.1, 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 5.5 μg per 1 μl of TE. After freeze-thawing 20 times with a final solution volume of 100 μl, luciferase activity was measured by the same method as in Example 1.
[0060]
The results are as shown in FIG. The expression level increased in an amount-dependent manner up to 1.5 μg of the addition amount of the expression vector pcOriPLuc (about 9.5 kb), which was a foreign gene, and the expression level was hardly changed thereafter. From the above results, it was confirmed that 1.5 μg / μl or more of exogenous gene DNA is preferably used for gene introduction under the conditions used in this example.
[0061]
(Example 4: Effect of buffer type on gene transfer efficiency)
The efficiency of gene transfer into host cells was examined by changing the type of buffer used for preparing the HVJ envelope vector.
[0062]
The amount of HVJ virus was 50 HAU per 1 μl of buffer, and the amount of luciferase expression vector pcOriPLuc was 15 μg / μl. Buffers are TE, PBS, BSS (137 mM NaCl, 5.4 mM KCl, 10 mM Tris-HCl, pH 7.5), and those obtained by adding sucrose at final concentrations of 0 mM, 20 mM, 40 mM, and 60 mM to these buffers. used. After freeze-thawing 20 times with a final solution volume of 100 μl, luciferase activity was measured by the same method as in Example 1.
[0063]
The results are as shown in FIG. 4. Under the conditions used in this example, it was confirmed that TE alone was preferable as a buffer for the preparation of recombinant HVJ virus.
[0064]
(Example 5: Comparison of AVE (Artificial Viral Envelop) type vector and HVJ envelope vector of the present invention)
Gene transfer using an inactivated HVJ-liposome that is a conventional gene transfer vector (AVE type having the highest gene transfer efficiency (Saeki, Y. et al., Human Gene Therapy, 8, 2133-2141 (1997)), The method of the present invention was compared.
[0065]
The amount of HVJ liposome or HVJ virus was 50 HAU per μl of TE, and the amount of luciferase expression vector pcOriPLuc was 1.5 μg / μl. In addition, the number of times of freezing and thawing of the recombinant HVJ virus was 2 or 15. Other conditions were the same as in Example 1 except that the host cells were human fetal kidney cell line HEK293.
[0066]
The results are as shown in FIG. It was confirmed that the method of the present invention in which the freeze-thawing of the HVJ envelope vector was repeated 15 times was far superior to the conventional method using the HVJ single liposome in terms of gene transfer efficiency.
[0067]
(Example 6: Introduction efficiency of synthetic oligonucleotide)
A synthetic oligonucleotide (20 bp) fluorescently labeled with FITC (fluorescein isothiocyanate) was mixed with an inactivated HVJ virus at a concentration of 1 mg / m1, and this solution was freeze-thawed 20 times, and then BHK-21 cells and 20 Reacted for 1 minute. As a result of observing the fluorescence signal after 17 hours, accumulation of fluorescence was recognized in the nucleus of almost 100% of the cells. From these results, it was confirmed that the method of the present invention is also effective for introducing a synthetic nucleic acid into cells.
[0068]
(Example 7: Introduction efficiency of GFP gene)
After freezing and thawing the mixed solution of GFP (green fluorescent protein) gene and inactivated HVJ virus 20 times, 2ng / 5μ1 Was injected into the rat cerebrum. As a result, a fluorescence signal was observed at the injection site. In addition, the HVJ envelope vector based on the GFP gene was frozen and stored for 3 months and then injected into the rat cerebrum. Similarly, a fluorescence signal due to GFP gene expression was observed at the injection site.
[0069]
From the above results, it was confirmed that the method of the present invention can reliably introduce a gene even in vivo. It was also confirmed that the HVJ envelope vector could be stored frozen.
[0070]
(2. Preparation of gene transfer vector using surfactant and use thereof)
(Example 8: Preparation of inactivated HVJ envelope vector using surfactant)
An outline of a method for preparing an inactivated HVJ envelope vector using a surfactant is shown in FIG.
[0071]
(1: Growth of HVJ)
HVJ can be generally used by inoculating a fertilized egg of a chicken by inoculation with a seed virus. However, cultured cells such as monkeys and humans, and a system for continuously infecting cultured tissues with a virus (cultivating a hydrolase such as trypsin). Any of those that are propagated by using a solution added to the liquid and those that are propagated by infecting a cultured cell with a cloned viral genome and causing persistent infection can be used.
[0072]
In this example, HVJ was propagated as follows.
[0073]
HVJ seeds of HVJ grown using SPF (Specific pathogen free) fertilized eggs, separated and purified, are dispensed into cell storage tubes, and 10% DMSO is added and stored in liquid nitrogen And prepared.
[0074]
Chicken eggs immediately after fertilization were received and placed in an incubator (SHOWA-FURANKI P-03 type; accommodating about 300 chicken eggs) and reared for 10-14 days under conditions of 36.5 ° C. and humidity of 40% or more. In the dark room, use an ophthalmoscope (light bulb light exits through a window with an aperture of about 1.5 cm) to check embryo survival and air chamber and chorioallantoic membrane. About 5 mm above, the location of the virus injection was marked with a pencil (select the location excluding the thick blood vessels). Seed virus (taken from liquid nitrogen) 500 times with polypeptone solution (mixed with 1% polypeptone, 0.2% NaCl, adjusted to pH 7.2 with 1M NaOH, autoclaved and stored at 4 ° C) Diluted to 4 ° C. Eggs were sterilized with isodine and alcohol, and a small hole was made in a thousand holes at the site of virus injection. 1 ml was injected into the chorioallantoic cavity using a 1 ml syringe with a 26 gauge needle. Dissolved paraffin (melting point: 50-52 ° C.) was placed on the hole using a Pasteur pipette to close it. The eggs were placed in an incubator and bred for 3 days under conditions of 36.5 ° C. and humidity of 40% or more. The inoculated eggs were then placed at 4 ° C. overnight. The next day, the air space of the egg was split with tweezers, a 10 ml syringe with an 18 gauge needle was placed in the chorioallantoic membrane, the chorioallantoic fluid was aspirated, collected in a sterile bottle, and stored at 4 ° C.
[0075]
(2: Purification of HVJ)
HVJ can be purified by purification methods by centrifugation, column purification methods, or other purification methods known in the art.
(2.1: Purification method by centrifugation)
Briefly, the grown virus solution was collected, and the tissue and cell debris in the culture solution and chorioallantoic fluid were removed by low speed centrifugation. The supernatant was purified by high-speed centrifugation (27,500 × g, 30 minutes) and ultracentrifugation (62,800 × g, 90 minutes) using a sucrose density gradient (30-60% w / v). It should be noted that the virus should be handled as gently as possible during purification and stored at 4 ° C.
[0076]
In this example, specifically, HVJ was purified by the following method.
[0077]
About 100 ml of HVJ-containing chorioallantoic fluid (collecting HVJ-containing chicken egg chorioallantoic fluid and storing at 4 ° C.) was placed in two 50 ml centrifuge tubes with a wide Komagome pipette (Saeki, Y., and Kaneda, Y: Protein modified liposomes (HVJ-1 iposomes) for the delivery of genes, volume of oligonucleotides and proteins, 27th edition, Cell biology; A. laboratory handbook, E. c. ~ 135, 1998), centrifuged in a low speed centrifuge at 3000 rpm for 10 minutes at 4 ° C (brake off) to remove egg tissue debris.
[0078]
After centrifugation, the supernatant was dispensed into four 35 ml centrifuge tubes (for high-speed centrifugation) and centrifuged with an angle rotor at 27,000 g for 30 minutes (accelerator and brake on). The supernatant was removed, and about 5 ml of BSS (10 mM Tris-HCl (pH 7.5), 137 mM NaC1, 5.4 mM KC1; autoclaved and stored at 4 ° C.) (PBS instead of BSS) was added to the precipitate, It left still at 4 degreeC as it was. Gently pipetted with a Komagome pipette, widened to loosen the precipitate, collected in a single tube, and centrifuged at 27,000 g for 30 minutes in the same manner. Excluding the supernatant, about 10 ml of BSS was added to the precipitate, and the mixture was allowed to stand at 4 ° C. overnight. The precipitate was loosened by gently pipetting with a wide-bottom Komagome pipette, centrifuged at 3000 rpm for 10 minutes at 4 ° C. (brake off) with a low-speed centrifuge, and the tissue fragments and virus clumps that could not be removed were removed. The supernatant is placed in a new sterile tube and stored as purified virus at 4 ° C.
[0079]
This virus solution 0. 0.9 ml of BSS was added to 1 ml, the absorption at 540 nm was measured with a spectrophotometer, and the virus titer was converted to hemagglutination activity (HAU). An absorption value 1 at 540 nm corresponded to approximately 15,000 HAU. HAU is considered to be approximately proportional to the fusion activity. In addition, erythrocyte agglutinating activity may be actually measured using chicken erythrocyte fluid (0.5%) (Animal Cell Utilization Manual, REALIZE INC. (Uchida, Oishi, Furuzawa) P259-268, 1984. See
[0080]
Furthermore, purification of HVJ using a sucrose density gradient can be performed as necessary. Specifically, the virus suspension is placed in a centrifuge tube overlaid with 60% and 30% sucrose solutions (autoclaved), and density gradient centrifugation is performed at 62,800 × g for 120 minutes. After centrifugation, the band seen on the 60% sucrose solution layer is collected. The collected virus suspension is dialyzed overnight at 4 ° C. using BSS or PBS as an external solution to remove sucrose. If not used immediately, add glycerol (autoclave sterilization) and 0.5M EDTA solution (autoclave sterilization) to the virus suspension to a final concentration of 10% and 2-10 mM, respectively, and gently at −80 ° C. Frozen and finally stored in liquid nitrogen (cryopreservation is possible with 10 mM DMSO instead of glycerol and 0.5 M EDTA solution).
(2.2: Purification method by column and ultrafiltration)
Instead of the purification method by centrifugation, purification of HVJ using a column is also applicable to the present invention.
[0081]
In brief, purification was performed using concentration by ultrafiltration with a filter having a molecular weight cut-off of 50,000 (about 10 times) and elution by ion exchange chromatography (0.3 M to 1 M NaCl).
[0082]
Specifically, in this example, HVJ was purified by column using the following method.
[0083]
After the chorioallantoic fluid was collected, it was filtered through an 80 μm to 10 μm membrane filter. 0.006-0.008% BPL (final concentration) was added to chorioallantoic fluid (4 ° C., 1 hour) to inactivate HVJ. BPL was inactivated by incubating chorioallantoic fluid at 37 ° C. for 2 hours.
[0084]
The solution was concentrated about 10 times by tangential flow ultrafiltration using 500 KMWCO (A / G Technology, Needham, Massachusetts). As buffer, 50 mM NaCl, 1 mM MgCl ₂ 2% mannitol, 20 mM Tris (pH 7.5) was used. The HAU assay gave excellent results with almost 100% HVJ recovery.
[0085]
HVJ was purified by column chromatography (buffer solution: 20 mM TrisHCl (pH 7.5), 0.2 to 1 M NaCl) by QSepharoseFF (Amersham Pharmacia Biotech KK, Tokyo). The recovery was 40 to 50%, and the purity was 99% or more.
[0086]
The HVJ fraction was concentrated by tangential flow ultrafiltration using 500 KMWCO (A / G Technology).
[0087]
(3: Inactivation of HVJ)
When inactivation of HVJ was required, it was performed by ultraviolet irradiation or alkylating agent treatment as described below.
[0088]
(3.1: UV irradiation method)
Take 1 ml of HVJ suspension in a petri dish with a diameter of 30 mm, 99 or 198 millijoules / cm ² Was irradiated. Gamma irradiation can also be used (5-20 gray), but complete inactivation does not occur.
[0089]
(3.2: Treatment with alkylating agent)
Immediately before use, 10 mM KH ₂ 0.01% β-propiolactone was prepared in PO. During the work, it was kept at a low temperature and worked quickly.
[0090]
Β-propiolactone was added to a suspension of HVJ immediately after purification to a final concentration of 0.01% and incubated on ice for 60 minutes. Thereafter, it was incubated at 37 ° C. for 2 hours. Dispense 10,000 HAU per tube into an Eppendorf tube, centrifuge at 15,000 rpm for 15 minutes, and store the precipitate at -20 ° C. Regardless of the inactivation method described above, the precipitate can be stored at -20 ° C., and the DNA can be directly incorporated by treatment with a surfactant to prepare a vector.
[0091]
(4: Creation of HVJ envelope vector)
92 μl of a solution containing 200 to 800 μg of foreign DNA was added to the preserved HVJ and well suspended by pipetting. This solution can be stored at −20 ° C. for at least 3 months. When protamine sulfate was added to DNA before mixing with HVJ, the expression efficiency was enhanced by a factor of 2 or more.
[0092]
This mixed solution was placed on ice for 1 minute, 8 μl of octyl glucoside (10%) was added, the tube was shaken on ice for 15 seconds, and left on ice for 45 seconds. The treatment time with the surfactant is preferably 1 to 5 minutes. In addition to octylglucoside, Triton-X100 (t-octylphenoxypolyethoxyethanol), CHAPS (3-[(3-colamidopropyl) -dimethylammonio] -1-propanesulfonic acid), NP-40 (nonylphenoxypoly) Surfactants such as ethoxyethanol may also be used. Preferred final concentrations of Triton-X100, NP-40 and CHAPS are 0.24-0.80%, 0.04-0.12% and 1.2-2.0%, respectively.
[0093]
1 ml of cold BSS was added and immediately centrifuged at 15,000 rpm for 15 minutes. 300 μl of PBS or physiological saline was added to the resulting precipitate and suspended by vortexing and pipetting. The suspension can be used directly for gene transfer or can be used for gene transfer after storage at −20 ° C. This HVJ envelope vector maintained comparable gene transfer efficiency after storage for at least 2 months.
[0094]
(Example 9: Ratio of Fl and HN protein in HVJ envelope vector)
(1: Sample preparation)
(I) Purified HVJ 10,000 HAU equivalent was centrifuged at 15,000 rpm for 15 minutes, and the precipitate was suspended in 300 μl of PBS and stored at −20 ° C.
(Ii) Purified HVJ 10,000 HAU equivalent is irradiated with ultraviolet rays (198 mJ / cm ² Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes, and the precipitate was suspended in 300 μl of PBS and stored at −20 ° C.
(Iii) Purified HVJ 10,000 HAU equivalent is irradiated with ultraviolet rays (198 mJ / cm ² After that, the mixture was centrifuged at 15,000 rpm for 15 minutes, and 200 μg of pcLuciDNA (92 μl of the solution) was added to the precipitate and suspended by pipetting. Place on ice, add 8 μl of octylglucoside (10%), shake the tube by hand for 15 seconds, leave on ice for 45 seconds, add 1 ml of cold BSS, immediately centrifuge at 15,000 rpm for 15 minutes, It was suspended in 300 μl of PBS and stored at −20 ° C.
[0095]
(2: Protein electrophoresis)
A 5% Laemli sample buffer was added to 5, 10, and 20 μl of the three types of samples, and 10% SDS-polyacrylamide gel electrophoresis was performed. After the electrophoresis, it was stained with Coomassie blue, destained, pasted on cellophane paper and dried.
[0096]
(3: Protein identification)
The electrophoresed and stained sample was incorporated into LAS2000 (Fuji Film, Tokyo) (FIG. 7), and the concentration of the protein band corresponding to F1 and HN was measured. Three different amounts were run per sample, the F1 / HN density of each was calculated, and the average and standard deviation were determined for each sample.
[0097]
(4: Result)
In the samples (i), (ii), and (iii), the F1 / HN was almost equal to 1.7. Considering the molecular weights of F1 51 kDa and HN 68 kDa, the molar ratio is about 2.3. The optimal value of fusion with reconstituted liposomes using F1 and HN is consistent with the report (Exp. Cell Res. 142, 95-101, 1985) that F1 / HN is given at about 2. This ratio is different from that of the wild-type virus in the rearranged form reported by other researchers (J. Virol. 67, 3312-3318, 1993). Other protein compositions were almost the same for the HVJ and HVJ envelope vectors.
[0098]
(Example 10: Encapsulation of DNA in HVJ envelope vector and encapsulation rate)
(1: electron microscope image of HVJ envelope vector (DNA encapsulated, unencapsulated))
As described above, 130 μg of pSFV-LacZ (14.5 kb) was enclosed in 10,000 HAU HVJ (UV-inactivated) to prepare an HVJ envelope vector.
[0099]
The encapsulated HVJ envelope vector was suspended in 300 μl of PBS and stored at −20 ° C. Ten days later, 1 μl of the suspension was placed on a grid and observed with a negative staining method using an electron microscope. As a control, an HVJ envelope vector without DNA encapsulation was used.
[0100]
(result)
The results are shown in FIG. Many HVJ envelope vectors were of the same type as the HVJ virus itself observed in the past. Compared with DNA-unencapsulated ones, those using DNA showed structures with a higher electron density in the HVJ envelope vector. On the other hand, the unencapsulated one had high internal permeability, and it was estimated that the viral genome was destroyed or lost.
[0101]
(2: DNA encapsulation rate in HVJ envelope vector)
As described above, 157 μg of pcLuci (7.4 kb) was encapsulated in HVJ (UV-inactivated) of 6,700 HAU to prepare an HVJ envelope vector. The HVJ envelope vector is suspended in 300 μl of BSS, 15 units of micrococcal nuclease, CaCl ₂ 2 mM, RNase A 20 μg / ml, treated at 20 ° C. for 30 minutes and dialyzed against 1 liter of PBS (4 ° C., overnight). Treated with 1% SDS at 37 ° C. for 1 minute. Treatment with 500 μl phenol, 500 μl chloroform-isoamyl alcohol, followed by ethanol precipitation. The sample was suspended in 100 μl of BSS, and 260 nm and 280 nm were measured with a spectrophotometer.
[0102]
(result)
Since the recovery rate was 85.7%, the conversion efficiency of DNA into the HVJ envelope vector was 3.8%. The uptake efficiency when 279 μg of pcLuci was taken into 10,000 HAU HVJ was 7.2%.
[0103]
From the above, the DNA uptake efficiency of 10,000 HAU into HVJ is estimated to be about 6-7% when octyl glucoside is used, but it may vary somewhat depending on the amount of DNA used. In addition, it has been known that the introduction efficiency increases when protamine sulfate coexists with DNA and HVJ envelope vector, but this is considered to be due to the increased efficiency of DNA encapsulation into the HVJ envelope vector. With Triton-X100 and NP-40, the efficiency is further increased, and it is estimated that it is about 10 to 40%.
[0104]
(Example 11: Gene transfer into cells using HVJ envelope vector)
(1: Gene transfer method)
1,000 HAU was placed in an Eppendorf tube (30 μl), and 5 μl of protamine sulfate (1 mg / ml) was added. The medium of BHK-21 cells (200,000 per well and spread in 6 wells the day before) was changed, and 0.5 ml of medium (10% FCS-DMEM) was added per well. To each well, a mixture of the above-mentioned vector (equivalent to 1,000 HAU) and protamine sulfate is added, and the vector and cells are mixed well by shaking the plate back and forth and left and right. ₂ It was left in the incubator for 10 minutes.
[0105]
Change the medium, 5% CO at 37 ° C ₂ The gene expression was examined by standing overnight (16-24 hours) in an incubator. In the case of luciferase (pcLuci; luciferase gene having a CMV promoter), cells were lysed with 0.5 ml of Cell Lysis Buffer (Promega), and the activity in a 20 μl solution was measured using a luciferase assay kit (Promega). In the case of green fluorescent protein (pCMV-GFPE; Promega), it was directly observed with a fluorescence microscope, 5 to 8 fields were observed at 400 magnifications, and the ratio of cells that emitted fluorescence was calculated.
[0106]
(2: Examination of conditions for introduction efficiency in cultured cells)
BHK-21 cells were used as cultured cells.
[0107]
(2.1: Examination of octylglucoside (OG) concentration in HVJ envelope vector preparation)
The effect of octyl glucoside (OG) at each concentration (final concentration of OG used when creating the HVJ envelope vector) on gene transfer using the HVJ envelope vector was investigated by making the following changes to the gene transfer method of (1). Was:
(A) Octyl glucoside concentration: 1, 2, 3%;
Time when inactivated HVJ was treated with OG when creating the HVJ envelope vector: 1 minute, 5 minutes, 10 minutes;
Sonication was performed or not performed.
(B) Octyl glucoside concentration: 0.125-1.25%;
Volume of vector used for transfection: 10 μl, 100 μl.
(C) Octyl glucoside: 0.55 to 0.8%;
Transfection time: 30 minutes, overnight (O / N).
The results are shown in FIG.
[0108]
(2.2: Conditions for gene introduction into cells, concentration of protamine sulfate (PS) and treatment time)
The effect of protamine sulfate on gene transfer using the HVJ envelope vector was examined by making the following changes to the gene transfer method of (1):
(A) Protamine sulfate: 0-100 μg / ml medium;
Transfection time: 20, 40, 60 minutes.
(B) Protamine sulfate: 0 to 40 μg / ml medium;
Transfection time: 5, 10, 20 minutes.
The results are shown in FIG.
[0109]
(2.3: Effect of DNA concentration enclosed in HVJ envelope vector on gene expression level)
The effect of the amount of DNA used in the experiment on the gene expression level by the HVJ envelope vector was investigated by making the following changes to the gene introduction method of (1):
(A) DNA amount: 20-200 μg;
The HVJ envelope vector was stored at -20 ° C or -80 ° C for 5 days.
(B) DNA amount: 180-360 μg / HVJ 10,000 HAU.
The results are shown in FIG.
[0110]
(2.4: Effect of HVJ titer used for gene transfer on gene expression level)
The effect of the HVJ titer used for gene introduction on the gene expression level by the HVJ envelope vector was examined by making the following changes to the gene introduction method of (1):
HVJ envelope vectors were prepared using HVJ with titers of 5,000, 10,000, and 20,000 HAU, and amounts corresponding to 250, 500, 1,000, and 2,000 HAU of each were used for BHK. -21 cells were transfected.
The results are shown in FIG.
[0111]
(2.5: Effect of HVJ inactivation conditions on HVJ envelope vector gene transfer efficiency)
The effect of the HVJ inactivation method (UV or β-propiolactone) on luciferase gene expression in BHK-21 cells was examined by making the following changes to the gene introduction method of (1).
(A) Irradiation dose for UV inactivation: 0 to 231 millijoules / cm ² .
(B) β-propiolactone (BPL) concentration used for HVJ treatment: 0 to 0.025%.
The results are shown in FIG.
[0112]
(Example 12: Gene transfer of HVJ envelope vector into various cells)
The gene was transferred to squamous cell carcinoma (SAS) of the human tongue by the method described in Example 11. The results are shown in FIG. The concentration of protamine sulfate and the incubation time at the time of transfection during gene introduction were changed as shown in FIG. 14, and the efficiency of gene introduction was measured by the expression of the luciferase gene. Within the range of transfection conditions, 200 μg / ml of protamine sulfate was used, and when transfection was performed for 60 minutes, the gene transfer efficiency was maximum, but if the protamine sulfate concentration was further increased, Further increase in gene transfer efficiency is expected.
[0113]
Gene transfer was performed on human aortic endothelial cells (HAEC) by the method described in Example 11. The results are shown in FIG. The concentration of protamine sulfate and the incubation time at the time of transfection during gene introduction were varied as shown in FIG. 15, and the efficiency of gene introduction was measured by the expression of the luciferase gene. Within the range of the transfection conditions, 100 μg / ml protamine sulfate was used and the transfection treatment was performed for 60 minutes. The gene transfer efficiency was maximum, but if the protamine sulfate concentration was further increased, Further increase in gene transfer efficiency is expected.
[0114]
(Example 13: Gene transfer of HVJ envelope vector into various living tissues)
In this example, gene transfer into various in vivo tissues using the HVJ envelope vector described in Example 11 is exemplified.
[0115]
(13.1: Mouse liver)
The HVJ envelope vector was prepared by leaving 0.8% octyl glucoside and 200 μg pcLuci on ice for 1 minute and suspending in 300 μl PBS. 30 μl of 1/10 volume of the prepared suspension (corresponding to 1,000 HAU) was diluted with 70 μl PBS (total volume 100 μl) and injected into one of the lobes of the mouse (C57BL / 6) liver.
[0116]
HVJ-AVE (Artificial Viral Envelop) liposomes were prepared by vortexing / extrusion followed by sucrose gradient centrifugation (62,000 g, 90 min) with 200 μg pcLuci. The preparation is then centrifuged by centrifugation (27,000 g, 30 minutes) pellet And suspended in 500 μl of PBS. 100 μl of this sample was injected into one of the mouse (C57BL / 6) liver lobes.
[0117]
After 24 hours, injected liver lobes were isolated and the luciferase activity of the leaves was assayed using the Promega Luciferase Assay System. The results are shown in FIG. 16A. As is clear from this result, the HVJ envelope vector of the present invention showed a gene transfer efficiency that was significantly higher than that of the conventional HVJ-AVE liposome by about 2 times.
[0118]
(13.2: Mouse uterus)
An HVJ envelope vector was prepared in the same manner as in 13.1. 50 μl and 100 μl samples were diluted with PBS to 500 μl, injected into the mouse fallopian tube, and the cervix was ligated for 10 minutes. Twenty-four hours later, mouse uterus was isolated and leaf and uterine luciferase activity was assayed using the Promega Luciferase Assay System. The results are shown in FIG. 16B. Although gene transfer into the mouse uterus was possible with the HVJ envelope vector of the present invention, no detectable gene transfer into the uterine tissue was found by the method using HVJ-AVE liposomes.
[0119]
An HVJ envelope vector containing pcLuci was prepared as in 13.1. For LacZ expression, an HVJ envelope vector containing pEB-CMV-LacZ (13 kb) was prepared using 200 μg of plasmid. HVJ envelope vectors containing each of these vectors were injected into the uterus as described above. The results are shown in FIG. 16C. LacZ staining detected the expression of the LacZ gene mainly in the endometrial glandular epithelium.
[0120]
(13.3: Rat brain)
PEGFP-1 (the jellyfish green fluorescent protein gene (approximately 0.7 kb) was incorporated into an expression vector having a cytomegalovirus promoter by a method similar to the preparation of the HVJ envelope vector containing pcLuci described above. HVJ envelope vector containing vector (available from Clontech, Palo Alto, CA) was prepared. 30 μl of vector (1/10 of the preparation, equivalent to 1,000 HAU) was injected into SD rats (Sprague-Dawley rats) via carotid artery injection or intrathecal injection from the cisterna magna. Three to four days after gene transfer, rats were sacrificed, brain sections were prepared without fixation, and fluorescence was observed under a fluorescence microscope. As in the results shown in FIG. 16D, expression of green fluorescent protein (GFP) in the brain was observed in both cases of carotid artery injection and intrathecal injection from the cisterna. In contrast, when HVJ-AVE liposomes were used to introduce the same gene from the rat carotid artery, GFP expression in the brain was not observed.
[0121]
(13.4: Rabbit eyes)
pCMV-NK4 constructed by cloning NK4 cDNA (1.4 kb), a mutant of human HGF (hepatocyte growth factor) gene, at the HindIII / XbaI site of pCDNA3 (In Vitrogen, San Diego, Calif.) Produced by Prof. Toshikazu Nakamura, Graduate School of Medicine.
[0122]
The HVJ envelope was prepared in the same manner as the HVJ envelope vector containing pcLuci described above, except that 10,000 HAU inactivated HVJ was used with either 400 μg or 800 μg pCMV-NK4. Vector was prepared. pCMV-NK4 expresses mutant HGF that inhibits HGF function Plasmid It is. 50 μl of vector (1/6 of the preparation) was injected into rabbit corneal tissue treated with a pellet of recombinant VEGF to induce neovascularization. Seven days after treatment, the rabbits were sacrificed and observed for neovascularization in the eyes. The result is shown in FIG. 16E. pCMV-NK4 suppressed angiogenesis induced by VEGF in a dose-dependent manner.
[0123]
(13.5: Rat pulmonary artery)
An HVJ envelope vector containing pSV-LacZ (Promega, Madison, WI) having a LacZ gene under the control of the SV40 promoter was prepared in the same manner as the above-described HVJ envelope vector containing pcLuci was prepared. did. 50 μl of vector (1/6 of the preparation) was injected into the rat trachea. Three days after gene transfer, the rats were sacrificed, the tissues were fixed with 1% glutaraldehyde, and the expression of LacZ in the arteries was visualized with X-gal. The results are shown in FIG. 16F. LacZ staining was observed in the bronchial epithelium. In addition, gene expression was also observed in bronchial epithelium when HVJ envelope vector was introduced from pulmonary artery (data not shown).
[0124]
(Example 14: Function of virus envelope vector as drug delivery system (DDS))
The gene transfer vector of the present invention is also useful as a drug delivery system for oligonucleotide or decoy nucleic acid therapy.
[0125]
(14.1: Introduction of fluorescent oligonucleotide)
Using the virus envelope vector of the present invention, fluorescently labeled oligonucleotides were introduced into cells.
[0126]
20-mer oligonucleotide labeled 5 ′ with FITC (5′-CCTTgAAGGGATTCCCTCC-3 ′) 194 μg / 92 μ1 BSS in 10,000 HAU HVJ (UV 198 mJ / cm ² Inactive), add Triton X-100 (0.24%, final concentration), treat on ice for 1 minute, add 1 ml BSS, and centrifuge (15,000 rpm, 15 minutes, 4 ° C.). 100 μl of PBS was added to the precipitate and stored at −20 ° C. One month later, thawed, 10 μl aliquots were mixed with 5 μg protamine sulfate and incubated (10 min, 60 min) with 5 million BHK-21 cells (in 0.5 ml medium). When the fluorescence of the cells was observed on the next day after the introduction with a fluorescence microscope, the oligonucleotide introduction efficiency was about 10% in 10 minutes as shown in FIG. 17B, but it was 80% or more in 60 minutes as shown in FIG. 17A. Oligonucleotides were introduced into the cells.
[0127]
(14.2: Treatment of contact dermatitis with Stat6 decoy nucleic acid)
The viral envelope vector of the present invention was used to introduce decoy nucleic acid into cells.
[0128]
Double-stranded nucleic acid (5′-GATCAAGACCTTTTCCCAAGAATCTAT-3 ′ and 3′-CATGTTCTGGAAAAGGGTTCTTAGATA-5 ′, (Wang, LH et al., Blood 95, 1249 to 1257, 2000)) 250 μg / 300 μl with Stat6 DNA binding sequence 30,000 HAU HVJ (UV 99 mJ / cm ² Inactive), add Triton X-100 (final concentration 0.24%), treat on ice for 1 min, add 1 ml BSS, centrifuge (15,000 rpm, 15 min, 4 min). ° C). 300 μl of PBS was added to the precipitate and stored at −20 ° C. When this HVJ envelope vector was used for subcutaneous injection in mice, it suppressed IgE-induced allergies and delayed skin reactions.
[0129]
(Example 15: Gene transfer to suspension cells)
Gene transfer experiments were conducted on human leukemia cell-like CCRF-CEM, NALM-6, and K-562.
[0130]
200 μg (92 μl) of pCMV-Luciferase was used to inactivate 10,000 HAU of HVJ (UV 99 mJ / cm ² ), And Triton X-100 (0.24%, final concentration) was added, treated on ice for 1 minute, 1 ml of BSS was added, and centrifuged (15,000 rpm, 15 minutes, 4 ° C.). HVJ envelope vector was prepared by adding 300 μl of PBS to the precipitate. Vector 60 μl (equivalent to 2,000 HAU), protamine sulfate, and 4 million suspension cells were mixed in a 1.5 ml Eppendorf tube, and centrifuged at 10,000 to 15,000 rpm for 10 minutes at 20 ° C. It was. Then, the culture solution was added to the precipitate, transferred to a culture dish, and the luciferase activity of the cells was measured after 1 day.
[0131]
The cell line used is a cell line (especially CCRF-CEM, NALM-6) with very low introduction efficiency when HVJ-liposomes or existing liposome reagents (Gibc BRL Lipofectin, Lipofectamine, etc.) are used. As shown in FIG. 18, highly efficient gene transfer into these cell lines was observed.
[0132]
The preferable conditions for gene transfer were conditions in which 600 to 1,000 μg / ml of protamine sulfate was added and the centrifugation was performed at 10,000 rpm or 15,000 rpm for 10 minutes at 20 ° C. No significant cytotoxicity was observed with the HVJ envelope vector. Moreover, both centrifugation and addition of protamine sulfate were necessary for gene transfer.
[0133]
(Example 16: Gene transfer to cancer tissue)
354 μg (92 μl) of pCMV-Luciferase was used to inactivate 34,000 HAU of HVJ (UV 99 mJ / cm ² ), Add TritonX-100 (0.24%, final concentration), treat on ice for 1 minute, add 1 ml BSS, and centrifuge (10,000-15,000 rpm, 10 minutes) , 20 ° C.). 300 μl of PBS was added to the precipitate and stored at −20 ° C. One day later, protamine sulfate was mixed with 500 μg / ml or 1000 μg / ml, and 100 μl thereof was injected into a tumor mass (diameter: 7 to 8 mm) of mouse melanoma B16-Fl, and luciferase activity was measured one day later. As shown in FIG. 19, gene expression was observed in the tumor mass. A preferred protamine sulfate concentration was 500 μg / ml. However, gene expression could not be detected at lower protamine sulfate concentrations.
[0134]
(Example 17: Preparation of herpesvirus envelope vector)
(17.1: Preparation of inactivated virus)
Herpes simplex virus type 1 (HSV-1) (10 ^Ten Plaque formation unit / m1) was provided by Prof. Yamanishi of the Department of Bacteriology, Osaka University Graduate School of Medicine. The virus inactivation conditions were examined by virus plaque formation in cultured monkey cells (Vero cells). In inactivation of the virus with β-propiolactone (BPL) 0.05%, the frequency of plaque appearance in Vero cells is 9. l × l0 ^-Four (Plaque / Vero cells). On the other hand, ultraviolet rays 200 and 400 millijoules / cm ² Inactivation of the virus by irradiation is 4.3 × 10 respectively. ^-Four 2.2 × 10 ^-6 (Plaque / Vero cells).
[0135]
(17.2: Gene transfer using inactivated virus)
100 μl of HSV-1 (10 ⁹ Particles) diluted with 620 μl of PBS, and ultraviolet rays of 400 millijoules / cm ² Irradiate, mix 10% amount (72 μl) with DNA (pCMV-Luciferase 8.83 μg / μl), add 8 μl of 3% Triton-X100 on ice (final concentration, 0.24%), After 3, 4, 5, 6 minutes, 1 ml of PBS was added for dilution. 100 μl of each sample was directly introduced into BHK-21 cells (in a 6-well plate). The cells were cultured in Dulbecco's minimal essential medium (DME) 0.5 ml / well containing 10% fetal calf serum (FCS). In another experiment, 100 μl of each sample was mixed with 5 μg of protamine sulfate and then introduced into BHK-21. 37 ° C, 5% CO ₂ After standing for 60 minutes in the incubator, the culture medium was replaced with 10% FCS-DME. The luciferase activity was measured after 22 hours. As shown in FIG. 20, high-efficiency gene transfer was confirmed using a herpesvirus envelope vector prepared using the method of the present invention. In each sample, no cytotoxicity was observed by morphological observation.
[0136]
Next, the herpesvirus envelope vector treated with Triton-X100 for 5 minutes was stored at −80 ° C. for 2 days, thawed, added again to BHK-21 cells, and the introduction efficiency was measured. In this case, 60 minutes after introduction, 2.5 ml / well of 10% FCS-DME was added and cultured overnight to measure the activity. The influence of serum and the amount of vector were examined.
[0137]
As shown in FIG. 21, highly efficient gene transfer was still confirmed even after storage at −80 ° C. for 2 days. The one using a medium supplemented with 10% serum has higher introduction efficiency than the serum-free medium, and the vector solution 200 μ1 (estimated amount: 2.8 × 10 ⁷ Virus particles / well) 100 μ1 solution (estimated amount: 1.4 × 10 ⁷ The gene transfer activity was higher than that of virus particles / well.
[0138]
(17.3: Gene transfer using inactivated virus)
From the above disclosure, it will be apparent to those skilled in the art that the technique for producing an envelope vector using this surfactant is applicable not only to HVJ but also to envelope viruses having a wide lipid membrane. Therefore, it is clear that those skilled in the art can easily prepare an envelope vector for gene transfer using other enveloped viruses according to the disclosure of the present invention. Therefore, Retroviridae, Togaviridae, Coronaviridae, Flaviviridae, Paramyxoviridae, Orthomyxoviridae, Bunyaviridae, Rhabdoviridae, Poxviridae, Herpesviridae, Baculoviridae, Hepadnavirus Envelope vectors for viruses such as families can be created. Thus, it is considered possible to introduce a target into a specific organ using the tissue directivity of the virus. For example, the herpes simplex virus envelope vector can be applied as a neurotrophic vector, the Epstein-Barr virus envelope vector as a B lymphocyte-directed vector, and the influenza envelope vector as a respiratory directional vector.
[0139]
From the foregoing, it will be apparent that while particular embodiments of the invention are described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Specifically, although the examples of the present specification have been described for gene transfer vectors using inactivated HVJ, viruses other than HVJ can be inactivated by using a similar preparation method. It will be apparent to those skilled in the art from the disclosure herein that the gene transfer vector is prepared and the gene transfer vector of the present invention is prepared without performing an inactivation step. Accordingly, the invention is not limited except as by the appended claims.
[0140]
【The invention's effect】
Provided is a new gene introduction method that is simple in operation and excellent in gene introduction efficiency. This will enable rapid screening of gene libraries. A kit for high throughput screening comprising the viral envelope vector of the present invention is also provided. In addition, the virus envelope vector provided by the present application can be cryopreserved for a long period of time and does not require preparation. Introduction becomes possible. Furthermore, the gene transfer vector of the present invention enables higher-efficiency gene transfer than conventional vectors prepared based on HVJ, and enables gene transfer to a wider range of in vivo tissues than conventional methods. To do.
[0141]
Drug delivery systems for drug administration, drug screening systems, and gene therapy vectors comprising the gene transfer vector of the present invention are also provided.
[Brief description of the drawings]
FIG. 1 shows the results of measuring the degree of expression of a foreign gene (luciferase gene) (luciferase activity) by freezing and thawing an HVJ envelope vector at various times and transfecting the cultured cells.
FIG. 2 shows the results of measurement of luciferase activity when transfected into cultured cells with the same number of viruses used to prepare the HVJ envelope vector added to the cultured cells.
FIG. 3 shows the results of measurement of luciferase activity when the amount of a foreign gene (luciferase expression vector) of the HVJ envelope vector is variously changed.
FIG. 4 shows the results of measurement of luciferase activity when the type of buffer for preparing the HVJ envelope vector was changed.
FIG. 5 shows the result of comparison between gene transfer using a conventional gene transfer vector-inactivated HVJ-liposome and the method of the present invention.
FIG. 6 is a diagram showing an outline of a method for preparing an inactivated HVJ envelope vector using a surfactant.
FIG. 7 is a diagram showing an SDS-PAGE pattern of a protein contained in an HVJ envelope vector prepared using a surfactant.
FIG. 8 is an electron micrograph using a negative staining method for the HVJ envelope vector. (1) Untreated HVJ; (2) octylglucoside-treated HVJ not containing DNA; (3) octylglucoside-treated HVJ containing DNA.
FIGS. 9A-C show gene transfer at each octyl glucoside concentration, HVJ treatment time with octyl glucoside, sonicated or not sonicated, and sonicated sonication, and vector volume used, respectively. It is the graph which showed efficiency by luciferase activity.
FIGS. 9A-C show gene transfer at each octyl glucoside concentration, HVJ treatment time with octyl glucoside, sonication or sonication, and sonication, and each vector volume used, as shown in the figure. It is the graph which showed efficiency by luciferase activity.
FIGS. 9A-C show gene transfer at each octyl glucoside concentration, HVJ treatment time with octyl glucoside, sonicated or not sonicated, and sonicated sonication, and each vector volume used. It is the graph which showed efficiency by luciferase activity.
FIGS. 10A and 10B are graphs showing luciferase activity of the concentration of each protamine sulfate (PS) shown in the drawings and the gene transfer efficiency at each transfection time.
FIGS. 10A and 10B are graphs showing luciferase activity with the concentration of each protamine sulfate (PS) shown in the drawings and the gene transfer efficiency at each transfection time.
11A and 11B are graphs showing the amount of each DNA shown in the drawings (the amount used in the experiment) and the gene transfer efficiency at each storage temperature in terms of luciferase activity.
11A and 11B are graphs showing the amount of each DNA shown in the drawings (the amount used in the experiment) and the gene transfer efficiency at each storage temperature in terms of luciferase activity.
FIG. 12 is a graph showing the gene transfer efficiency in terms of luciferase activity when an HVJ envelope vector was prepared using HVJ having the titer of each HAU shown in the drawing and gene transfer was performed.
FIG. 13A is a graph showing the gene transfer efficiency at each UV irradiation dose shown in the drawing in terms of luciferase activity.
FIG. 13B is a graph showing the gene transfer efficiency at each β-propiolactone (BPL) concentration shown in the drawing in terms of luciferase activity.
FIG. 14 is a graph showing luciferase activity of gene transduction efficiency at each protamine sulfate concentration and incubation time of each transfection shown in the figure for squamous cell carcinoma (SAS) of human tongue.
FIG. 15 is a graph showing luciferase activity of the gene transfer efficiency in human aortic endothelial cells (HAEC) at each protamine sulfate concentration and incubation time of each transfection shown in the drawing.
FIG. 16A is a graph showing the gene transfer efficiency using HVJ envelope vector of the present invention and HVJ-AVE (Artificial Viral Envelop) liposome to mouse liver in terms of luciferase activity.
FIG. 16B is a graph showing the gene transfer efficiency using the HVJ envelope vector of the present invention and the HVJ-AVE (Artificial Viral Envelop) liposome to the mouse uterus as luciferase activity.
FIG. 16C shows the result of LacZ staining of uterine tissue after gene transfer of pEB-CMV-LacZ using the HVJ envelope vector of the present invention to the mouse uterus. LacZ staining detected the expression of the LacZ gene mainly in the endometrial glandular epithelium.
FIG. 16D shows the results of administration of an HVJ envelope vector containing 10,000 HAU of pEGFP-1 via the cisterna and the carotid artery to SD rats (male, body weight 300 to 400 g). Three to four days after administration, the mice were sacrificed, and raw sections were prepared and observed under a fluorescence microscope. (1) Administration via cisterna
Gene transfer to the surface of the brain was confirmed. On the other hand, gene transfer into the deep brain was not confirmed. No gene transfer to the choroid plexus was confirmed.
(2), (3) Administration via the carotid artery
Significantly higher expression was confirmed on the left side of administration. Expression was confirmed not only in the brain surface but also in the basal ganglia. It was also confirmed on the brain surface of the brain. The expression on the brain surface of the antibrain was thought to have flowed to the contralateral side due to collateral flow.
FIG. 16E is a diagram showing the results of pCMV-NK4, a vector expressing mutant HGF that inhibits HGF function, suppressing angiogenesis induced by VEGF in a dose-dependent manner.
FIG. 16F is a diagram showing the results of gene transfer performed by injecting an HVJ envelope vector into the trachea.
FIGS. 17A and 17B show the results of observing fluorescence of cells with a fluorescence microscope on the day after introduction of oligonucleotides into cells. FIGS. The oligonucleotide introduction efficiency was about 10% after 10 minutes of incubation (FIG. 17B). On the other hand, in the incubation for 60 minutes, the oligonucleotide was introduced into 80% or more of the cells (FIG. 17A).
FIG. 18 shows the results of an introduction experiment on CCRF-CEM, NALM-6, and K-562, which are human leukemia cell lines. These cell lines are cell lines (especially CCRF-CEM, NALM-6) in which introduction efficiency is extremely low with HVJ-liposomes and existing liposome reagents (Gibc BRL Lipofectin, Lipofectamine, etc.). High luciferase activity was obtained under the conditions of adding 600 to 1000 μg / ml of protamine sulfate and centrifuging at 10000 rpm or 15000 rpm for 10 minutes at 20 ° C. There was no significant cytotoxicity. Both centrifugation and protamine sulfate were necessary for introduction.
FIG. 18 shows the results of introduction experiments on human leukemia cell lines CCRF-CEM, NALM-6, and K-562. These cell lines are cell lines (especially CCRF-CEM, NALM-6) in which introduction efficiency is extremely low in HVJ-liposomes and existing liposome reagents (Gibc BRL Lipofectin, Lipofectamine, etc.). High luciferase activity was obtained under the conditions of adding 600 to 1000 μg / ml of protamine sulfate and centrifuging at 10000 rpm or 15000 rpm for 10 minutes at 20 ° C. There was no significant cytotoxicity. Both centrifugation and protamine sulfate were necessary for introduction.
FIG. 18 shows the results of an introduction experiment on CCRF-CEM, NALM-6, and K-562, which are human leukemia cell lines. These cell lines are cell lines (especially CCRF-CEM, NALM-6) in which introduction efficiency is extremely low with HVJ-liposomes and existing liposome reagents (Gibc BRL Lipofectin, Lipofectamine, etc.). High luciferase activity was obtained under the conditions of adding 600 to 1000 μg / ml of protamine sulfate and centrifuging at 10000 rpm or 15000 rpm for 10 minutes at 20 ° C. There was no significant cytotoxicity. Both centrifugation and protamine sulfate were necessary for introduction.
FIG. 19 shows the results of gene transfer into cancer tissue. Gene expression was observed in a tumor mass which is a cancer tissue, and in particular, high gene transfer activity was obtained when protamine sulfate was 500 μg / ml. When a lower concentration of protamine sulfate was used, gene expression could not be detected.
FIG. 20 shows the results of gene transfer into cells using a herpesvirus envelope vector. Although the total luciferase activity was low, it was judged that a vector with the highest introduction efficiency could be prepared by treatment with Triton-X100 for 5 minutes. In addition, the preparation method without using protamine sulfate had high introduction efficiency. In each sample, no cytotoxicity was observed by morphological observation.
FIG. 21 shows the results of gene transfer into cells using a herpesvirus envelope vector after storage at −80 ° C. When a medium supplemented with 10% serum was used, the introduction efficiency was higher than that of the serum-free medium, and the vector solution 200 μ1 (estimated amount: 2.8 × 10 6 ⁷ Virus particles / well) 100 μ1 solution (estimated amount: 1.4 × 10 ⁷ More active than virus particles / well).

Claims (12)

An inactivated virus envelope of Sendai virus (HVJ), which is purified and separated from the virus body and has the same ratio of virus proteins required for fusion activity as wild-type virus. The inactivated viral envelope according to claim 1 which is not fused to a liposome. The inactivated virus envelope according to claim 1 or 2, wherein the HVJ is wild-type or recombinant. The inactivated virus envelope according to any one of claims 1 to 3, wherein the inactivation is achieved by ultraviolet irradiation or treatment with an alkylating agent. After purification of the HVJ, 33 to 198 mJ / cm ² The inactivated virus envelope according to any one of claims 1 to 4, which is prepared by centrifuging after irradiating the ultraviolet ray. After refining the HVJ, 33-165 mJ / cm ² The inactivated virus envelope according to any one of claims 1 to 5, which is prepared by centrifuging after irradiating the ultraviolet ray. The inactivated virus envelope according to any one of claims 1 to 4, wherein the purified HVJ is subjected to an alkylating agent treatment and then centrifuged. The inactivated virus envelope according to claim 7, wherein the alkylating agent is β-propiolactone. The inactivated virus envelope according to claim 8, wherein the β-propiolactone treatment concentration is 0.0025 to 0.025%. The inactivated virus envelope according to claim 8, wherein the β-propiolactone treatment concentration is 0.0075 to 0.025%. The inactivated virus envelope according to any one of claims 1 to 10, wherein the viral proteins are F1 protein and HN protein. The inactivated virus envelope according to any one of claims 1 to 10, wherein F1 protein / HN protein is contained in the same ratio as that of the wild type.

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