FIELD OF INVENTION
The present invention generally relates to viral nanoparticle (VNP). More particularly,
the present invention relates to viral nanoparticle obtained from capsid protein of Flock
House Virus capable of incorporating and delivering foreign cargo to specific cells5 ,
method of producing the said viral nanoparticle and method of cellular delivery.
BACKGROUND OF THE INVENTION
10 Flock House Virus (FHV), an insect nodavirus, is a structurally simple virus, with a
T=3 icosahedral capsid constructed from 180 copies of a single capsid protein, alpha
(Figure 1A), encapsulating a single stranded, bipartite, positive sense RNA genome
(Fisher, A. J., and Johnson, J. E. (1993). Ordered duplex RNA controls capsid
architecture in an icosahedral animal virus. Nature 361(6408), 176-9) with sequence:
15
MVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNRRRVRG
MNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRKDVLNQ
SISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTSMFGTT
STSRSDQVSSFRYASMNVGIYPTSNLMQAGSITVWKCPVKLSTVQFPVATDP
20 ATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFNDILEGIQTL
PPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVNSAILK
AWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNAS
MWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF
25 having a NCBI Reference Sequence: NC_004144.1. Milligram quantities of FHV
virions, 30 nm in diameter, are usually generated by infection or transfection of DL-1
cells (Gallagher, T. M., and Rueckert, R. R. (1988). Assembly-dependent maturation
cleavage in provirions of a small icosahedral insect ribovirus. J Virol 62(9), 3399-406.
3
Anette Schneemann et.al Specific Encapsidation of Nodavirus RNAs Is Mediated
through the C Terminus of Capsid Precursor Protein Alpha J Virol. 1998 Nov; 72(11):
8738–8746). Virus-like particles (VLPs) of FHV, structurally indistinguishable from
native virus, and containing an equivalent amount of non-genomic, insect cell derived
nucleic acid, are produced by infecting Sf21 cells with recombinant baculovir5 us
expressing alpha (Schneemann A, Dasgupta R, Johnson JE, Rueckert RR. Use of
recombinant baculoviruses in synthesis of morphologically distinct virus like particles
of flock house virus, a nodavirus. Journal of Virology. 1993. 67(5):2756-63.).
However, there is a need for viral nanoparticle for cellular delivery since the isolation
10 of viral nanoparticle and free capsid protein has been challenging.
US 6171591 discloses compositions containing “chimeric proteins in which the
nodavirus capsid protein is present together with a heterologous peptide segment.” The
heterologous peptide segment includes a cell-specific targeting sequence, and the
15 chimeric proteins can be assembled into chimeric virus-like particles. However, in said
invention, the VLPs are assembled in insect cells by over-expressing chimeric proteins
in baculovirus expression system, without any control on assembly.
EP 1736538 A1 provides a “process for the purification recombinantly expressed, self20
assembled VLP from the homogenate of a bacterial host”. While the examples given
are for Qβ VLP, AP205 VLP and HBc VLP the preferred embodiment of the patent
also includes VLP comprising capsid protein of virus selected from various groups,
including Flock House Virus. However, the process consists of a first chromatography
step of anion exchange, followed by a second chromatography step using
25 hydroxyapatite and, optionally, a size exclusion chromatography.
US 7998487 discloses “antitoxin and vaccine compositions based on nodavirus
VLPs”, specifically that of Anthrax antitoxin and vaccine compositions. Here
4
heterologous polypeptides were inserted in place of residues 265-267 of FHV VLP
coat protein.
US 5932426 discloses a molecular presentation system wherein viral proteins, derived
from small insect viruses, primarily from Flock House Virus (FHV), act as carriers 5 for
heterologous amino acid sequences that are inserted in the outward-directed loops of
the viral capsid protein.
US 20020072094 is a method of introducing non-endogenous packageable nucleic
10 acid sequence and non-endogenous capsid protein-encoding sequence in yeast cells
and inducing particle assembly, thereby resulting in the production of infectious
virions (including those of FHV) in yeast Saccharomyces cerevisiae.
US 6700038 discloses gene transfer vectors useful for transmitting genes to plants,
that are based on FHV genome, specifically RNA-1; this invention utilizes the
15 capability of replication in a plant cell in the presence of FHV polymerase.
US20030157130 also provides a nodavirus RNA-1-based gene-delivery vector.
Nodavirus RNA-1 molecule modified to include a heterologous protein-coding
insertion which is downstream of its replicase ORF, packaged in a VLP, such as a
papillomavirus VLP. US 6514757 describes nodavirus-like DNA expression vector,
20 based on the RNA-dependent RNA polymerase of nodaviruses like FHV. These
inventions are based on RNA-1 encoding polymerase activity of FHV.
There is a need in the art to explore virus-based nanoparticles, methods to obtain such
nanoparticle in the absence of advanced chromatography columns and FPLC systems.
25
OBJECTIVES OF THE INVENTION
5
It is an object of the present invention to provide Viral nanoparticles from Flock House
virus.
It is another objective of the present invention to provide a method for isolation for
viral nanoparticles from the capsid protein of Flock House 5 virus.
Another objective of the present invention is to engineer the Viral nanoparticles from
the capsid protein of Flock House virus.
10 Another objective of the present invention is to encapsulate small molecules such as
fluorescent dye molecules within the Viral nanoparticles from Flock House virus and
method of cellular delivery.
Yet another objective of the present invention is to genetically engineer a tumor15
homing peptide (LyP-1) onto the external surface of the FHV nanoparticle, capable of
being targeted to tumor cell lines like MDA-MB-435S cells.
Another objective of the present invention is to provide a novel, cheaper, quicker
expression system for FHV particles.
20
SUMMARY OF THE INVENTION
The present invention relates to a viral nanoparticle capsid protein sequence:
MGSSHHHHHHSSGLVPRGSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQ
25 NVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNT
DPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAG
TFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPVATXATSSLVHTLVGLDGVLAVGPDNFSESFIK
GVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVG
30 WGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEV
6
ALQEYRTVARSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGI
SGLSALFEGFGF;
having SEQ ID NO: 1; wherein X is selected from the group consisting of: SEQ ID
NO: 3 HHHHHHAE; SEQ ID NO: 4 CGNKRTRGC; SEQ ID NO: 5 5
CNKRTRGGC; SEQ ID NO: 6 WHSDMEWWYLLG; SEQ ID NO:
7CRGDKGPDC.
In one embodiment, the present invention relates to a method of producing viral
10 nanoparticle capsid protein sequence comprising the steps of:
a) adding a hexahistidine (6x-his) tag “MGSSHHHHHHSSGLVPRGSHM” to
the N-terminal end of the capsid protein alpha of Flock House Virus having a
NCBI Reference Sequence: NC_004144.1, which includes a thrombin
cleavage site downstream of said hexahistidine (6x-his) tag at the N-terminal
15 end;
b) inserting a peptide sequence in the region replacing amino acid residue 207
and amino acid residue 208.
c) protein over-expression by addition of 0.25 – 0.75 mM Isopropyl β-D-1-
thiogalactopyranoside (IPTG) and protein purification;
20 d) thrombin cleavage of the said N-terminal tag in presence of calcium dichloride
in range of 15-25mM to obtain particle assembly.
Further the present invention relates to method of cellular delivery using viral
nanoparticle capsid protein sequence as claimed in claim 1 comprising the steps of:
25 a) thrombin cleavage of the N-terminal tag “MGSSHHHHHHSSGLVPR” in
presence of calcium dichloride in range of 15-25mM to obtain particle
assembly;
b) encapsulation of the cargo molecule to obtain a complex;
c) ultracentrifugation of the assembled particles to obtain purified complex;
30 d) administering the said complex in a concentration range of 0.5 – 1 μg particles
per 104 (or 10000) target cells.
7
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
Figure 1: illustrates A) A representation of Flock House Virus based on its X-ray
crystal structure (PDB ID: 4FTB). B) A ribbon representation of the tertiary structur5 e
of FHV alpha, with the N-terminus (beginning from residue 20 in the crystal structure)
and the loop at residues 207-208 indicated by arrows. C) Schematic showing the
modified version of alpha with two 6x-His-tags, one at the N-terminus and another at
residues 207-208. D) Bacterial expression of modified FHV alpha. Western blot with
10 an anti-His monoclonal antibody, showing soluble and insoluble fractions of protein
induced at 27°C (8 hours) and 37°C (4 hours). E) SDS-PAGE showing FHV alpha
purified using Nickel affinity chromatography. The position of alpha is indicated by
arrows.
15 Figure 2: illustrates A) Electron micrographs of in vitro assembled particles, stained
with 2% uranyl acetate, with small, intermediate and large particles indicated by white,
black and white dotted arrows, respectively. B) Size distribution (average Feret
diameters) of the particles visualized in A; a total of 300 particles were included.
20 Figure 3: illustrates A)Electron micrograph showing higher order multimers produced
by purified double his-tagged alpha, without thrombin cleavage (white arrow, white
dotted arrow and black arrow indicate oligomers, stacks of oligomer and mis-formed
particles, respectively). B) Pellet obtained after 30% sucrose cushion
ultracentrifugation of sample in A. C) Electron micrographs of particles assembled in
25 vivo from single 6x-his-tag containing alpha (small, intermediate and large particles
are indicated by white, black and white dotted arrows, respectively). D) Size
distribution (average Feret diameters) of particles visualized in C; a total of 300
particles were included.
8
Figure 4: illustrates electron micrograph of negatively stained in vitro assembled LyP-
1 particles encapsulating SulfoB. Scale bar = 100 nm.
Figure 5: illustrates bright-field and fluorescent images of MDA-MB-435S cells (5 top
left panel) and Sf21 cells (top middle panel) treated with Lyp-1-modified, SulfoBencapsulated
particles, and of MDA-MB-435S cells treated with 6x-His-tag modified,
SulfoB-encapsulated particles (top right panel). The untreated controls are shown
(bottom panels). Scale bar = 100 μm.
10
DETAILED DESCRIPTION OF INVENTION
The present invention will be described with respect to preferred embodiments. The
invention is not limited thereto but only by the claims.
15
Identification of Sequences
SEQ ID NO: 1
MGSSHHHHHHSSGLVPRGSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQ
20 NVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNT
DPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAG
TFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPVATXATSSLVHTLVGLDGVLAVGPDNFSESFIK
GVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVG
25 WGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEV
ALQEYRTVARSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGI
SGLSALFEGFGF;
SEQ ID NO: 2
30 GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRK
DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PVATXATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFNDIL
9
EGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAV
NSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIA
AQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF;
5
SEQ ID NO: 3
HHHHHHAE;
SEQ ID NO: 4
CGNKRTRGC10 ;
SEQ ID NO: 5
CNKRTRGGC;
15 SEQ ID NO: 6
WHSDMEWWYLLG;
SEQ ID NO: 7
CRGDKGPDC;
20
SEQ ID NO: 8
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRK
DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
25 MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PVATHHHHHHAEATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEP
DFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIR
VSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVA
RSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGF
30 GF;
SEQ ID NO: 9
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRK
35 DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PATCGNKRTRGCATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEP
DFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIR
VSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVA
40 RSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGF
GF;
10
In one embodiment, the present invention relates a viral nanoparticle (VNP) capsid
protein sequence:
MGSSHHHHHHSSGLVPRGSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQ
NVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFN5 T
DPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAG
TFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPVATXATSSLVHTLVGLDGVLAVGPDNFSESFIK
GVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVG
10 WGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEV
ALQEYRTVARSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGI
SGLSALFEGFGF;
having SEQ ID NO: 1; wherein X is selected from the group consisting of:
15 SEQ ID NO: 3 HHHHAE; SEQ ID NO: 4 CGNKRTRGC; SEQ ID NO: 5
CNKRTRGGC; SEQ ID NO: 6 WHSDMEWWYLLG; SEQ ID NO: 7
CRGDKGPDC.
The present invention teaches the generation and isolation of FHV capsid protein alpha
20 in vitro by addition of a hexahistidine (6x-his)MGSSHHHHHHSSGLVPRGSHMtag
to the N-terminal end of the capsid protein alpha, which sterically blocks assembly. A
thrombin cleavage site downstream of the tag which allows cleavage of the tag is also
present. In addition, to allow the surface expression of a foreign peptide, a 6x-his-tag
is genetically inserted in the surface-exposed loop replacing residues 207-208 of alpha.
25 Said viral nanoparticle (VNP) capsid protein sequence is useful in cellular delivery.
To induce particle assembly for cellular delivery, the N-terminal tag
“MGSSHHHHHHSSGLVPR”is removed from purified alpha by thrombin cleavage
11
in the presence of calcium ions to obtain viral nanoparticle capsid protein
sequencewith SEQ ID NO: 2
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSR5 K
DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PVATXATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFNDIL
EGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAV
10 NSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIA
AQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF;
wherein X is selected from the group consisting of:
SEQ ID NO: 2 HHHHHHAE; SEQ ID NO: 3 CGNKRTRGC; SEQ ID NO: 4
15 CNKRTRGGC; SEQ ID NO: 5 WHSDMEWWYLLG; SEQ ID NO: 6
CRGDKGPDC.
In one embodiment, the present invention relates to viral nanoparticles (VNPs)
obtained from the capsid protein of Flock House Virus (FHV). N-terminal end of the
20 said nanoparticle has a hexahistidine (6x-his) tag and a thrombin cleavage site
downstream of the hexahistidine (6x-his) tag. Further the said sequence includes a
peptide sequence in the region between amino acid residue 207 and amino acid residue
208 as depicted in figure 1 having Sequence ID: 8 and with sequence:
25 GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRK
DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PVATHHHHHHAEATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEP
30 DFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIR
12
VSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVA
RSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGF
GF
In anotherembodiment, the present invention relates toviral nanoparticles obtaine5 d
from the capsid protein of Flock House Virus. N-terminal end of the said nanoparticle
has a hexahistidine (6x-his) tag and a thrombin cleavage site downstream of
thehexahistidine (6x-his) tag. Further the said sequence includes a peptide sequence9-
residue peptide LyP-1 (CGNKRTRGC)in the region between amino acid residue 207
10 and amino acid residue 208 having Sequence ID: 9 and with sequence:
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRTRRNR
RRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRFEGKVVSRK
DVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTSATTFNPVNYPGFTS
15 MFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQF
PATCGNKRTRGCATSSLVHTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEP
DFEFNDILEGIQTLPPANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIR
VSAPEGAVNSAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVA
RSLPVAVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGF
20 GF
Further peptide closely related to LyP-1 (CGNKRTRGC), is LyP-1b (CNKRTRGGC)
also accumulates in the tumor cells as LyP-1. Further examples of such tumor homing
peptides include F56 peptide (WHSDMEWWYLLG), and RGD peptides (e.g.
25 iRGDCRGDKGPDC). Said related sequence can replace LyP-1 (CGNKRTRGC)in
the region between amino acid residue 207 and amino acid residue 208.
In another embodiment, the present invention relates to the method for producing and
isolating viral nanoparticles (VNPs) from the capsid protein of Flock House Virus
30 (FHV), comprising the steps of:
a. adding a hexahistidine (6x-his) tag
“MGSSHHHHHHSSGLVPRGSHM” to the N-terminal end of the
13
capsid protein alpha of Flock House Virus having a NCBI Reference
Sequence: NC_004144.1, which includes a thrombin cleavage site
downstream of said hexahistidine (6x-his) tag at the N-terminal end;
b. inserting a peptide sequence in the region replacing amino acid residue
207 and amino acid residue 5 208.
c. protein over-expression by addition of 0.25 – 0.75 mM Isopropyl β-D-
1-thiogalactopyranosideand protein purification;
d. thrombin cleavage of the said N-terminal tag in presence of calcium
dichloride in range of 15-25mM to obtain particle assembly.
10
The peptide sequence replacing amino acid residue 207 and amino acid residue 208
are selected from the group consisting of:SEQ ID NO: 3 HHHHHHAE; SEQ ID NO:
4 CGNKRTRGC; SEQ ID NO: 5 CNKRTRGGC; SEQ ID NO: 6
WHSDMEWWYLLG; SEQ ID NO: 7 CRGDKGPDC.
15
The said method teaches producing and isolating viral nanoparticles (VNPs) from the
capsid protein of Flock House Virus (FHV), by inducing in vitro assembly of capsid
protein (alpha) expressed in and purified from E. coli. The said VNPs are heterogenous
in size and shape, and retain key biophysical properties as authentic virus and virus20
like particles.
The cDNA encoding FHV capsid protein alpha are cloned into the BamHI and NdeI
sites of the bacterial expression vector pET28b(+) (Novagen), which allows the
inclusion of an N-terminal overhang consisting of a 6x-his-tag and a thrombin
25 cleavage site (MGSSHHHHHHSSGLVPRGSHM). In addition, residues 207-208 of
alpha are replaced by the coding sequence for LyP-1 (CGNKRTRGC) by overlap
extension PCR. Further said protein purification is done through Ni-affinity
chromatography
14
In one embodiment, the present invention relates to method of cellular delivery using
viral nanoparticle (VNP) capsid protein of Flock House Virus comprising the steps of:
a. thrombin cleavage of the N-terminal tag
“MGSSHHHHHHSSGLVPR” in presence of calcium dichloride 5 in
range of 15-25mM to obtain particle assembly;
b. encapsulation of the cargo molecule to obtain a complex;
c. ultracentrifugation of the assembled particles to obtain purified
complex;
10 d. administering the said complex in a concentration range of 0.5 – 1 μg
particles per 104 target cells.
The cargo selected from a group consisting of a peptide, a polypeptide, or a protein, a
polysaccharide, a lipid, a lipoprotein, a glyco lipid, a nucleic acid, a small molecule
drug or a toxin, a nanoparticle and an imaging or contrast agent.
15
FHV alpha containing the N-terminal tag, upon thrombin cleavage and inclusion of 50
mM SulfoB during subsequent in vitro assembly reactions results in the fluorescent
dye being packaged in the particles (~ 25 molecules dye/particle, assuming an average
of 180 protein molecules per particle) (Figure 4).
20
The present invention involves genetically engineering a tumor-homing peptide (LyP-
1) onto the external surface of the FHV nanoparticle, capable of being targeted to
tumor cell lines like MDA-MB-435S cells. The engineered FHV particles of the
present invention hold utility as they possess tumor binding and cargo carrying
25 capabilities.
The present invention is further explained by way of non-limiting examples:
15
Example 1:In order to generate and purify FHV capsid protein alpha for in vitro
assembly, hexahistidine (6x-his) tag was added to the N-terminal end of the capsid
protein alpha, which sterically blocked assembly. A thrombin cleavage site
downstream of the tag which would allow cleavage of the tag was also present. In
addition, to allow the surface expression of a foreign peptide, a 6x-his-tag wa5 s
genetically inserted in the surface-exposed loop replacing residues 207-208 of alpha.
The locations of N-terminus and loop 207-208 in the capsid protein alpha are shown
in Figure 1B. This site has been previously used to accommodate large insertions,
without interfering with capsid protein association and particle assembly. The 6x-his10
tag is hydrophilic and flexible in nature, and is known to rarely interfere with the
conformation or functionality of proteins. This dual his-tagged construct (shown
schematically in Figure 1C) was expressed in E. coli Rosetta (DE3) pLysS cells, with
protein production induced at low temperature (27˚C) to improve solubility of alpha
(Figure 1D). Modified alpha was purified, almost to homogeneity, using nickel affinity
15 chromatography (Figure 1E).
Further to induce particle assembly, the N-terminal tag was removed from purified
alpha by thrombin cleavage in the presence of calcium ions. The inclusion of calcium
ions was expected to promote assembly, since divalent metal cations have been shown
20 to stabilize FHV particles. Following removal of the N-terminal tag by thrombin
cleavage, the protein remaining in solution was subsequently pelleted by
ultracentrifugation through a 30% sucrose cushion to pellet any particles thus formed.
Negative stain (uranyl acetate) electron microscopy showed presence of closed
particles (Figure 2A), mostly in the size range of 29-35 nm (Figure 2B). No particles
25 were formed without thrombin cleavage (Figures 3A, 3B). In vivo assembly of FHV
particles was also done, using a clone that did not contain the 6x-His-tag at the Nterminus.
Such particles formed looked morphologically similar to in vitro assembled
particles, but had a slightly different size distribution (Figures 3C, 3D).
16
Example2: For investigation of the effect of foreign insertions in engineered
assembly, a 9-residue peptide LyP-1 (CGNKRTRGC) was introduced in the loop at
position 207-208. LyP-1 specifically associates with the endothelium of tumor
lymphatics and cells, and has previously been utilized for specialized targeting as 5 s a
“tumor-homing” peptide. Insertion of this peptide in a construct of FHV alpha
containing the N-terminal tag, followed by thrombin cleavage and inclusion of 50 mM
SulfoB during subsequent in vitro assembly reactions resulted in the fluorescent dye
being packaged in the particles (~ 25 molecules dye/particle, assuming an average of
10 180 protein molecules per particle) (Figure 4). Dye-loaded LyP-1-functionalized
particles were targeted to the human cancer cell line MDA-MB-435S cells, and red
fluorescence corresponding to SulfoB was detected inside the cells (Figure 5, left
panel). A similar experiment with insect Sf21 cells did not result in fluorescent dye
being deposited in the cells (Figure 5, middle panel). Dye-loaded particles, containing
15 a 6x-His-tag in the surface loop, were unable to deposit significant amounts of the dye
into MDA-MB-435S cells (Figure 5C, right panel), indicating that specific binding of
the LyP-1 peptide to target cells is essential for entry of particles inside cells.
Experiment 3: Cloning: The cDNA encoding FHV alpha was cloned into the BamHI
20 and NdeI sites of the bacterial expression vector pET28b (+) (Novagen), allowing the
inclusion of an N-terminal overhang consisting of a 6x-his-tag and a thrombin
cleavage site(MGSSHHHHHHSSGLVPRGSHM). In addition, residues 207-208 of
alpha were replaced by a 6x his-tag and a two-amino acid linker (Alanine-Glutamic
acid) (HHHHHHAE) by overlap extension PCR. A second construct, containing the
25 N-terminal extension (MGSSHHHHHHSSGLVPRGSHM), and with residues 207-
208 replaced by the coding sequence for LyP-1 (CGNKRTRGC), was similarly
generated. A third construct, which did not contain the N-terminal extension, was
generated by introducing a stop codon in the 5’ end of the forward primer (Primer
17
sequence: 5’-AAACATATGTAGAAAATGGTTAATAACAACAGACCAAG-3’),
to prevent the expression of the N-terminal 6x-his-tag. This construct, however,
contained a 6x-his-tag in place of residues 207-208, which was introduced by overlap
extension PCR. All constructs were confirmed by sequencing.
Protein overexpression: Recombinant expression vector pET28b (+) containing 5 the
gene of interest was transformed into E.coli Rosetta (DE3) pLysS cells. One colony
was inoculated into a 50 ml flask containing 10 ml LB broth and grown overnight,
shaking at 37°C. This primary culture was used to set up multiple 1L secondary
cultures, which were incubated at 37°C with shaking until the OD600 reached 0.5-0.6.
10 Protein overexpression was induced by addition of 0.5mM IPTG at 27°C, and cells
were grown for a further 8 hours at this temperature. Post-induction, cells were
harvested by centrifugation at 4500g for 15 minutes and were frozen at -20°C until
further processing.
15 Protein purification: The cell pellets were resuspended in TN buffer (50mM Tris pH
7.4, 100mM NaCl) supplemented with 0.5 mM PMSF (Phenylmethanesulfonyl
fluoride). Resuspended pellets were ruptured by treatment with 1 mg/ml lysozyme at
room temperature for 30 minutes, followed by sonication at 4°C. The supernatant
containing soluble proteins was collected by centrifugation at 12900g at 4°C for 30
20 minutes. The soluble supernatant was allowed to bind nickel NTA-agarose beads
(Qiagen) for 90 minutes. The unbound and nonspecifically bound proteins were
removed by washing with TN buffer containing increasing concentrations of imidazole
(20 mM and 50 mM). Protein bound to the column was eluted using TN
buffer containing 250 mM imidazole. The eluate was dialyzed in TN buffer using
25 Amicon Ultra-4 centrifugal filter (10 kDa cut-off, Merck Millipore) to remove
imidazole. All purification steps were carried out at 4˚C. The N-terminal 6x-his-tag
was removed by cleavage with thrombin (~10 units per mg of protein) in TN buffer
supplemented with 20mM CaCl2, for 2 hours at room temperature. Specifically, during
18
purification of the LyP-1-modified alpha protein, 5 mM 2-Mercaptoethanol was
included in the buffer. Any precipitated protein was removed through centrifugation
at 8000xg for 10 minutes at 4°C and the soluble fraction used for further processing.
For packaging fluorescent dye within particles, fluorescent dye Sulforhodamine 5 B
(SulfoB), at a final concentration of 50 mM, was included in the buffer during
thrombin cleavage, and particle pelleting performed as below.
Particle pelleting: The supernatant containing particles generated through in vitro
10 assembly was added to a polyallomer ultracentrifuge tube (Beckman Coulter) with a
30% sucrose cushion at the bottom of the tube, followed by ultracentrifugation in a
SW41 Ti rotor at 40,000 rpm at 11°C for 150 minutes in Beckman Coulter Optima L-
100K ultracentrifuge. The pellet was resuspended in phosphate buffered saline (PBS),
pH 7.4, and analyzed by negative stain-electron microscopy.
15
Electron microscopy:5 μl of sample was applied to a 300-mesh carbon-formvar coated
copper grid (Electron Microscopy Sciences) and allowed to adsorb onto the surface.
After removal of excess solution, the grids were stained with 2% uranyl acetate, airdried
and viewed using a Tecnai TF20 transmission electron microscope (FEI
20 Company), at an accelerating voltage of 200 keV.
Particle size analysis: The feret diameters (Feretmax and Feretmin) of 300 particles were
measured from the electron micrographs using ImageJ (Schneider et al., 2012), and
the average feret diameter calculated for each particle. The distribution of the average
25 feret diameter was plotted.
Protein estimation/particle quantitation: Concentration of the protein was determined
by measuring absorbance at 280 nm (A280) and calculating concentration using an
19
extinction coefficient 38180 M-1 cm-1 corresponding to FHV alpha (ExpasyProtparam
tool; assuming all pairs of Cys residues form cystines). This concentration was
confirmed semi-quantitatively by SDS-PAGE densitometry analysis (comparing
protein band intensity with standard BSA quantities). The concentration of in vitroassembled
particles was calculated by assuming that on an average, 180 copies 5 s of
alpha make up one particle.
Cell culture and localization studies: MDA-MB-435S cells were maintained in
Leibovitz’s L-15 medium (Gibco) supplemented with 10% heat-inactivated Fetal
10 Bovine Serum (Gibco) and Penicillin-Streptomycin (100 U/ml) at 37°C. Sf21 insect
cells were maintained in TC-100 medium, supplemented with 10% FBS and
Penicillin-Streptomycin, at 27°C.
To study qualitative cellular uptake of sulfoB-encapsulating, LyP-1-modified particles
15 by fluorescence microscopy, MDA-MB-435S cells were seeded in 96-well flat-bottom
tissue culture plates at a density of 1x104 cells per well in complete growth medium
and allowed to grow overnight at 37°C. The SulfoB-LyP-1-particles were added to the
cell culture medium at a particle concentration of 10 μg/ml, and 100 μl added per well.
After 12 hours of incubation at 37°C, the cells were washed three times with PBS and
20 viewed under fluorescence microscope (Olympus, Japan). Cellular uptake of the above
particles in Sf21 insect cells was similarly done, at 27°C. SulfoB-encapsulating, 6x-
His-modified particles were studied for cellular uptake in MDA-MB-435S cells as
described above for the LyP-1-modified particles.

We Claim:
1. A viral nanoparticle capsid protein sequence:
MGSSHHHHHHSSGLVPRGSHMMVNNNRPRRQRAQRVVVTTTQTAP
VPQQNVPRNGRRRRNRTRRNRRRVRGMNMAALTRLSQPGLAFLK5 C
AFAPPDFNTDPGKGIPDRFEGKVVSRKDVLNQSISFTAGQDTFILIAPT
PGVAYWSASVPAGTFPTSATTFNPVNYPGFTSMFGTTSTSRSDQVSSF
RYASMNVGIYPTSNLMQFAGSITVWKCPVKLSTVQFPVATXATSSLV
HTLVGLDGVLAVGPDNFSESFIKGVFSQSACNEPDFEFNDILEGIQTLP
10 PANVSLGSTGQPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVN
SAILKAWSCIEYRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPV
AVIAAQNASMWERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGF
GF;
15 having SEQ ID NO: 1; wherein X is selected from the group consisting of:
SEQ ID NO: 3 HHHHHHAE; SEQ ID NO: 4 CGNKRTRGC; SEQ ID NO: 5
CNKRTRGGC; SEQ ID NO: 6 WHSDMEWWYLLG; SEQ ID NO: 7
CRGDKGPDC.
20
2. The viral nanoparticle capsid protein sequence as claimed in claim 1, wherein
the sequence is:
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRT
RRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRF
25 EGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTS
ATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPVATXATSSLVHTLVGLDGVLAVGPDNFS
ESFIKGVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQPFTMDSG
AEATSGVVGWGNMDTIVIRVSAPEGAVNSAILKAWSCIEYRPNPNAM
30 LYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASMWERVKSIIK
SSLAAASNIPGPIGVAASGISGLSALFEGFGF;
having SEQ ID NO: 2
3. The viral nanoparticle capsid protein sequence as claimed in claim 2, wherein
35 the sequence is:
21
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRT
RRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRF
EGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTS
ATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPATCGNKRTRGCATSSLVHTLVGLDGVL5 A
VGPDNFSESFIKGVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTGQ
PFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVNSAILKAWSCIEY
RPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASMW
ERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF; having SEQ ID
10 NO:9.
4. The viral nanoparticle capsid protein sequence as claimed in claim 2, wherein
the sequence is:
GSHMMVNNNRPRRQRAQRVVVTTTQTAPVPQQNVPRNGRRRRNRT
15 RRNRRRVRGMNMAALTRLSQPGLAFLKCAFAPPDFNTDPGKGIPDRF
EGKVVSRKDVLNQSISFTAGQDTFILIAPTPGVAYWSASVPAGTFPTS
ATTFNPVNYPGFTSMFGTTSTSRSDQVSSFRYASMNVGIYPTSNLMQF
AGSITVWKCPVKLSTVQFPVATHHHHHHAEATSSLVHTLVGLDGVL
AVGPDNFSESFIKGVFSQSACNEPDFEFNDILEGIQTLPPANVSLGSTG
20 QPFTMDSGAEATSGVVGWGNMDTIVIRVSAPEGAVNSAILKAWSCIE
YRPNPNAMLYQFGHDSPPLDEVALQEYRTVARSLPVAVIAAQNASM
WERVKSIIKSSLAAASNIPGPIGVAASGISGLSALFEGFGF; having SEQ
ID NO:8.
25 5. The viral nanoparticle capsid protein sequence as claimed in claim 1, wherein
nanoparticle assembly, is induced by removing N-terminal
tagMGSSHHHHHHSSGLVPR by thrombin cleavage in the presence of
calcium ions.
30 6. A method of producing viral nanoparticle capsid protein sequence comprising
the steps of:
a. adding a hexahistidine (6x-his) tag
“MGSSHHHHHHSSGLVPRGSHM” to the N-terminal end of the
capsid protein alpha of Flock House Virus having a NCBI Reference
22
Sequence: NC_004144.1, which includes a thrombin cleavage site
downstream of said hexahistidine (6x-his) tag at the N-terminal end;
b. inserting a peptide sequence in the region replacing amino acid residue
207 and amino acid residue 208.
c. protein over-expression by addition of 0.25 – 0.75 mM Isopropyl β-5 D-
1-thiogalactopyranoside and protein purification;
d. thrombin cleavage of the said N-terminal tag in presence of calcium
dichloride in range of 15-25 mM to obtain particle assembly.
10 7. The method of producing viral nanoparticle capsid protein sequence as claimed
in claim 6, wherein said peptide sequence is selected from the group consisting
of: SEQ ID NO: 3 HHHHHHAE; SEQ ID NO: 4 CGNKRTRGC; SEQ ID NO:
5 CNKRTRGGC; SEQ ID NO: 6 WHSDMEWWYLLG; SEQ ID NO: 7
CRGDKGPDC.
15
8. The method of producing viral nanoparticle capsid protein sequence as claimed
in claim 6, wherein said protein purification is done through Ni-affinity
chromatography.
20 9. The method of cellular delivery using viral nanoparticle capsid protein
sequence as claimed in claim 1 comprising the steps of:
e. thrombin cleavage of the N-terminal tag
“MGSSHHHHHHSSGLVPR” in presence of calcium dichloride in
range of 15-25mM to obtain particle assembly;
25 f. encapsulation of the cargo molecule to obtain a complex;
g. ultracentrifugation of the assembled particles to obtain purified
complex;
23
h. administering the said complex in a concentration range of 0.5 – 1 μg
particles per 104 target cells.
10. The method as claimed in claim 6, wherein said cargo selected from a group
consisting of a peptide, a polypeptide, or a protein, a polysaccharide, a lipid, 5 d, a
lipoprotein, a glyco lipid, a nucleic acid, a small molecule drug or a toxin, a
nanoparticle and an imaging or contrast agent.