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Viral Particle-Like Construct and Method of Forming the Same Under Physiological Conditions

a technology of virus-like particles and constructs, which is applied in the field of virus-like structure, can solve the problems of difficult to efficiently reconstitute virus-like particles of uniform size by such methods, and the conditions are not suitable for taking up bioactive substances into particles

Inactive Publication Date: 2008-06-05
TOKYO INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0005]The present invention provides a method which allows a viral particle-like structure to be reconstituted in a test tube to form uniform-sized particles efficiently and under physiological conditions.

Problems solved by technology

Conventional reconstituting methods have been conducted under non-physiological conditions with high salt concentration, but because of problems such as inactivation or poor solvent solubility of bioactive substances included in the particles, the conditions have not been suitable for taking up bioactive substances into particles.
Moreover, it has been difficult to efficiently reconstitute virus-like particles of uniform size by such methods.

Method used

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  • Viral Particle-Like Construct and Method of Forming the Same Under Physiological Conditions
  • Viral Particle-Like Construct and Method of Forming the Same Under Physiological Conditions
  • Viral Particle-Like Construct and Method of Forming the Same Under Physiological Conditions

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Viral Particles

(1) Preparation of Viral Particle (VP1) Pentamers

[0057]After seeding Sf9 cells at 1×107 each into fifty 10 cm-diameter tissue culturing dishes, they were infected with recombinant baculovirus expressing SV40 viral protein (VP1) with a m.o.i. (multiplicity of infection) of 5 to 10.

[0058]At 72 hours after infection, the cells were recovered with medium using a scraper, and rinsed twice with cooled phosphate buffered saline (PBS). To the recovered cells there was added 10 mL of ice-cooled sonication buffer (20 mM Tris-HCl (pH 7.9), 1% (w / v) sodium deoxycholate (DOC), 2 mM PMSF), and then a VP-15S (sonicator) by Taitec was used for 10 minutes of ultrasonic disruption while cooling on ice under conditions with a 50% duty cycle and output at 5. The cell disruptate was centrifuged at 14,000 g, 4° C. for 20 minutes and the supernatant was recovered.

[0059]Cesium chloride solutions with four different densities (50%, 40%, 30%, 20% (w / v)) were gently layered at 1....

example 2

Formation of Virus-Like Particles Incorporating DNA

[0073]Example 1 was repeated. However, in step (3) for in vitro reconstitution of the virus-like particles under physiological conditions, a 3000 bp plasmid was included and the formed virus-like particles comprising DNA incorporated into the virus-like particles were subjected to sucrose density gradient centrifugation, and then fractionation and Southern blotting for detection of DNA. As shown in FIG. 7, the DNA was incorporated into the virus-like particles.

[0074]The prepared SV40-VP1 protein pentamers and SV40-VP2 protein were used for in vitro reconstitution of the virus-like particles under physiological conditions. DNA was added during the procedure. Specifically, at pH 5.0 to 7.0, for example, 2.8 μL of 800 ng / μL VP2 protein was added to 150 μL of 75.7 ng / μL VP1 pentamer protein, and then 21 μL of 5.7 ng / μl 3000 bp circular double-stranded plasmid DNA (pG5vector) was added. The mixture was incubated at 4° C. for 30 minutes a...

example 3

Gene Transfer into Cells Using DNA-Incorporating Virus-Like Particles

[0076]Example 2 was repeated. However, the plasmid used was pEG which can express a fluorescent protein (GFP) in mammalian eukaryotic cells. Formation of virus-like particles containing the pEG plasmid DNA was accomplished by sucrose density gradient centrifugation, and it was confirmed that DNA was contained in the fractions containing the virus-like particles. Specifically, the virus-like particles were reconstituted in a solvent containing 150 mM sodium chloride, 2 mM calcium chloride and 20 mM Tris HCl (pH 7.2) using VP1 and VP2 protein in the presence of the pEG plasmid, and the plasmid DNA-containing virus-like particles were fractionated by sucrose density gradient centrifugation.

[0077]The virus-like particles were detected by Western blotting using anti-VP1 antibody (a-VP1), and pEG was detected by Southern blotting. The results are shown in FIG. 8. The numbers in the image represent the fraction numbers, w...

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Abstract

There is provided a novel method of forming uniform viral particles under physiological conditions. The method of forming uniform-sized viral particle aggregates composed of viral protein is characterized by incubating a viral protein such as SV40 virus VP1 at pH 5.0 to 7.0, room temperature, in the presence of 130 mM to 170 mM sodium chloride and 1.5 mM to 2.5 mM divalent cation, and in the presence of a particle formation acceleration factor such as SV40 VP2. For encapsulation of a substance to be encapsulated in the viral particles, the substance to be encapsulated is included during the incubation.

Description

TECHNICAL FIELD[0001]The present invention relates to a viral particle-like structure comprising viral protein, and to a method of forming it. The viral particle-like structure can encapsulate other substances within it, and therefore has potential use as a carrier for drug delivery and gene therapy.BACKGROUND ART[0002]Conventional formation of viral particle-like structures has involved recovering virus-like-particles formed within cells.[0003]In some methods, the virus-like particles are purified from the cells and are first dissociated into particle structure units (for example, VP1 pentamers in the case of SV40 virus), and then reconstituted into virus-like particles in a test tube.[0004]Conventional reconstituting methods have been conducted under non-physiological conditions with high salt concentration, but because of problems such as inactivation or poor solvent solubility of bioactive substances included in the particles, the conditions have not been suitable for taking up ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12P21/04C12N7/00C12N15/87C07K14/025C12N15/09
CPCA61K39/12A61K2039/5258C07K14/005C12N2710/22022C12N2710/22023C12N7/00A61P43/00Y02A50/30
Inventor HANDA, HIROSHINAKANISHI, AKIRAKAWANO, MASAAKI
Owner TOKYO INST OF TECH
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