4. Construstion of bionanocapsule and its application
Due to the progression of genetic engineering and molecular
virology, several viruses have been developed as useful viral vectors for
gene therapy, mass production of useful recombinant proteins and so forth,
although infectious viruses are hazardous to human health. Intrinsic features
of viruses which come from selective recognition of their target cells
and their high efficiency of gene delivery have fully been utilized for
these viral vectors yet developed. However, these recombinant viral vectors
have still some problems on their pathogeny, immunological response, integration
site of viral genome into cellular chromosome and so on. To solve these,
many artificial vectors or carriers, constituted of liposomes, cationic
polymers, sugar-based polymers, and ceramic materials have been developed.
But their delivering efficiency is quite low as compared with that of viral
vectors. These artificial carriers, therefore, have not yet reached the
practical use.
To develop another
possible carriers that possess selective recognition to target cells and high
delivering efficiency, we had an idea to make use of nanocapsules composed of
viral capsid protein, a component of virus particle, instead of whole virus.
Viral capsid protein has multiple functions, (1) selective recognition of target
cells through binding membrane receptors, (2) self-assembly in vitro and in vivo
into virus-like particles (VLP), that are nanocapsules, (3) encapsulation of
viral genome into VLP, (4) intrusion into target cells and release of viral
genome therein. On the virtue of these properties, viral capsid proteins would
be utilized for developing nanocapsules used as carriers of drug delivery system
(DDS).
We have selected simian virus 40 (SV40) major capsid protein,
VP1. SV40 with an icosahedoral structure 45nm in diameter belongs in the
Papovaviridae, small animal viruses. Its capsid, composed of VP1 only, consists
of 360 VP1 molecules, or 72 VP1 pentamers, each of which consists of 5 VP1
molecules. SV40 was first isolated as a contaminating virus in rhesus macaque
monkey cells which were used to grow the early versions of the active polio
vaccine developed in the late 1950s. Afterward SV40 has been found to induce
tumors in rodents such as hamsters or mice while not in humans.
We
have demonstrated that recombinant VP1 forms VLP in the nuclei when expressed in
insect Sf-9 cells using recombinant baculovirus vectors, (Kosukegawa, A., et
al., Biochim. Biophys. Acta., 1996). Moreover we have developed a technology for
preparing VLPs with high-purity from recombinant baculovirus-infected cells. We
have also shown that purified VLPs are dissociated into VP1 pentamers by
treatment with EGTA, a calcium chelating agent, and DTT, a reducing agent, and
vice versa (Ishizu, K. I., et al., J. Virol., 2001).
We have
subsequently found that VP1 pentamers self-assemble and form structures of
various shapes and sizes in different reaction conditions. They form mixed
structures, including 20nm to 45nm spherical capsules and tubular structures, in
a high salt buffer condition with 1M NaCl and 2mM CaCl2, but they only form 20nm
tiny capsules in the same condition without CaCl2. On the other hand, they only
form tubular structures in a physiological condition with 150mM NaCl and 2mM
CaCl2 at pH5.0, whereas VP1 pentamers do not form any three-dimentional
structure in the same condition except at pH 7.2 (Kanesashi, S. N., et al., J.
Gen. Virol. 2003).
We have then searched for factors which induce or
direct VP1 pentamer's assembly in vitro in the physiological condition
containing 150mM and 2mM CaCl2 at pH 7.2. We have found that minor capsid
proteins, VP2 or VP3, induce a formation of 20nm to 45nm spherical nanocapsules
like VLPs in shape. The sphelical nanocapsules are somewhat smaller compared
with VLPs formed in a baculovirus expression system and are dissimilar in that
the inside of the minor capsid protein-induced spherical nanoparticules was not
stained well with uranium acetate, suggesting empty nanocapsules (Kawano, M. A.,
et al., J. Biol. Chem., 2006). Minor capsid proteins also induce nanocasule
formation even at pH5.0. These suggest that minor capsid proteins direct VP1
pentamers toward forming viral particles. We have, recently, found that a
chimeric protein containing a part of VP2 and GFP can be encapsulated into
nanocapsules and successfully introduced into target cells through infection
like viruses.
We have also developed a technology with which exogenous
plasmid DNA is packaged in nanocapsules formed by VP1 pentamer's assembly. This
nano- capsule is able to deliver and express exogenous genes into target cells
through infection like viruses (Tsukamoto et al., submitted). This indicates
that the in vitro reconstituted nanocapules have the same physiogical property
as wild type SV40 virions.
These findings obtained from the study of
in vitro VP1 pentamer's assembly are important not only for understanding the
control of viral particle formation by interactions of VP1 pentamers with other
viral components, but also for applying the in vitro assembling and packaging
system to novel DDS development.
We are also interested in virus
tropism on infection. SV40 selectively binds target cell membrane receptors
through VP1. This selective binding fixes tropism. The membrane receptors for
SV40 has been known to be MHC class I and GM1. Both are necessary for intrusion
of SV40 into target cells via endocytosis. We have recently developed a
technology for changing tropism by altering VP1 chemically or genetically or
both. We are now constructing various functional nanocapsules with altered VP1
to suit each individual purpose. We will also try to loose or reduce
antigenisity originated from SV40 VP1.
On the basis of our results
obtained so far, we are now constructing new types of bionanocapsules composed
of SV40 VP1 pentamers or its variant pentamers for developing novel drug
delivery system, which enables us to deliver optimum amounts of DNA, drug,
protein, ferrite, etc and their mixture to target cells. We are collaborating
with many scientists at home and abroad to achieve this projects. We have
described bionanocapsules constructed from SV40 VP1 pentamers in details in gThe
Present Situation and Future Prospect of DDS Researchh published by Japan
Society of Drug Delivery System as the 20th year anniversary (PHARM TECH JAPAN,
21, 168-171, 2005).