U.S. patent application number 11/075628 was filed with the patent office on 2006-03-09 for transport proteins and their uses.
This patent application is currently assigned to Marie Curie Cancer Care. Invention is credited to Gillian Daphne Elliott, Peter Francis Joseph O'Hare.
Application Number | 20060051869 11/075628 |
Document ID | / |
Family ID | 26307479 |
Filed Date | 2006-03-09 |
United States Patent
Application |
20060051869 |
Kind Code |
A1 |
O'Hare; Peter Francis Joseph ;
et al. |
March 9, 2006 |
Transport proteins and their uses
Abstract
The present invention relates to transport proteins, in
particular VP22 and homologues thereof, and to methods of
delivering these proteins and any associated molecules to a target
population of cells. This transport protein has applications in
gene therapy and methods of targeting agents to cells where
targeting at high efficiency is required.
Inventors: |
O'Hare; Peter Francis Joseph;
(Surrey, GB) ; Elliott; Gillian Daphne; (Surrey,
GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
Marie Curie Cancer Care
|
Family ID: |
26307479 |
Appl. No.: |
11/075628 |
Filed: |
March 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10259198 |
Sep 27, 2002 |
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11075628 |
Mar 8, 2005 |
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09773430 |
Jan 31, 2001 |
6521455 |
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10259198 |
Sep 27, 2002 |
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09011073 |
Jan 26, 1998 |
6184038 |
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PCT/GB96/01831 |
Jul 25, 1996 |
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09773430 |
Jan 31, 2001 |
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Current U.S.
Class: |
435/455 ;
435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 48/00 20130101;
C07K 2319/10 20130101; C12N 2710/16622 20130101; C07K 2319/00
20130101; C12N 15/87 20130101; C07K 14/005 20130101 |
Class at
Publication: |
435/455 ;
435/325; 435/069.1; 530/350; 536/023.5 |
International
Class: |
C12N 15/87 20060101
C12N015/87; C12P 21/06 20060101 C12P021/06; C07H 21/04 20060101
C07H021/04; C07K 14/705 20060101 C07K014/705 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 28, 1995 |
GB |
9515568.5 |
Jan 26, 1996 |
GB |
9601570.6 |
Claims
1-5. (canceled)
6. A transport protein comprising VP22 or an active portion,
fragment, or homologue thereof, non-covalently associated with one
or more other molecules whose transport to a target population of
cells is desired.
7. A transport protein according to claim 6 wherein the one or more
associated molecules are non-peptidyl.
8. A transport protein according to claim 6 wherein the protein is
further capable of being exported from a first part of the target
population of cells in which it is expressed and taken up by a
second part of the target population of cells not directly
producing the protein.
9-15. (canceled)
16. A method of transporting a desired molecule into a population
of cells, the method comprising: i) non-covalently coupling the
desired molecule to a transport protein comprising VP22 or an
active portion, fragment, or homologue thereof to form a complex;
and, ii) exposing the cells to the complex so that cells can take
up the complex.
17. A method according to claim 16 wherein the desired molecule is
non-peptidyl.
18. A method according to claim 16 wherein the desired molecule is
coupled to the transport protein by incorporation into a lipid
based vehicle.
19. A method according to claim 17 wherein the wherein the desired
molecule is coupled to the transport protein by incorporation into
a lipid based vehicle.
20. A transport protein according to claim 7 wherein the protein is
further capable of being exported from a first part of the target
population of cells in which it is expressed and taken up by a
second part of the target population of cells not directly
producing the protein.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to transport proteins, and in
particular to transport proteins based on VP22, homologues of VP22
or fragments thereof, to molecules and compositions including the
transport proteins, and to methods of delivering these proteins and
any associated molecules to a target population of cells, typically
at high efficiency.
BACKGROUND TO THE INVENTION
[0002] The product of the herpes simplex virus type 1 (HSV-1) UL49
gene, the structural protein VP22 (4), is a major component of the
HSV tegument, a compartment of the virion located outside the
capsid and inside the envelope and composed of at least 10 or more
additional virus polypeptides (for a review see (6)). VP22 has a
molecular weight of 32k, is very basic and is modified by
phosphorylation and nucleotidylation (1,4,8,9,11) in the infected
cell.
[0003] Despite being one of the major tegument proteins within the
virion, together with the well characterised transcription
regulatory protein VP16, little is known about the function of VP22
during the virus replicative cycle. It is not yet known if it is an
essential virus protein, but it is possible that, in a manner
similar to VP16, VP22 has two roles to perform during
infection--initially as a functional protein during viral gene
expression, and subsequently as a structural component of the
virion during virus assembly. With regard to the former, some
evidence exists to suggest that VP22 can bind specifically to HSV-1
DNA (2,8,10).
SUMMARY OF THE INVENTION
[0004] We recently demonstrated that there is a stable and specific
interaction between VP16 and VP22, which has implications for the
mechanisms of action of VP16 in assembly and transcriptional
activation. During those studies, we found that when VP22 was in
cells by itself it had an unusual pattern of behaviour. In the work
described here, we extended these studies to investigate the
cellular localisation in detail, and found that VP22 exhibits a
highly unusual property in that it is efficiently transported from
the cell in which it is originally expressed, and in which it
displays cytoplasmic localisation, to adjacent cells within the
monolayer, in which it is taken up into the nucleus. This pattern
of behaviour has not been observed for any other of a range of
proteins we have tested, eg VP16 and to our knowledge these
properties and activities are unprecedented.
[0005] This unexpected property of VP22 was observed when,
approximately 30 hours after introduction of either VP22 or VP16
into a cell monolayer, while VP16 could be detected on average in
about 2-5% of the cells (as is conventional in such experiments),
VP22 could be detected in nearly every cell of the monolayer. We
further found specificity in VP22 intercellular transport and have
demonstrated the involvement of a determinant at the C-terminal end
of the protein. This comes from the result that a variant lacking
the C-terminal 34 amino acids, while being expressed in a
cytoplasmic location in initially expressing cells, was not
transported to adjacent cells.
[0006] Protein secretion or export normally occur via specific
pathways requiring well characterised signal sequences for sorting
into the compartments and vesicles involved in export pathways for
a review see (12). VP22 does not possess any conventional signal
sequences and its route and mechanism of transport is highly
unusual and is previously uncharacterised. Further studies on the
determinants required within VP22 and of the physiological
requirements within the cell should help clarify the pathway
involved. Thus, VP22, or more particularly the determinants
involved in VP22 transport, could be transferred to other proteins
to enable transport and efficient expression or uptake within a
target cell population. Widespread utility and applications of this
property can easily be envisaged.
[0007] Accordingly, the present invention is based on the above
finding that it is possible to introduce into a first part of a
target population of cells nucleic acid encoding a transport
protein, and optionally with nucleic acid encoding proteins
associated with the transport protein (eg. as fusion partners), to
express the nucleic acid, optionally from tissue specific
promoters, to produce the protein(s), after which the transport
protein is exported from the cells, together with any associated
protein(s), to be taken up by a second part of the target
population of cells not directly producing the protein(s).
Typically, the protein(s) are found to be taken up by the second
part of the population of cells at high efficiency, and tend to
localise in the nuclei of the second part of the population of
cells. Thus, the combination of initial introduction and subsequent
transport allows the transport protein and any associated proteins
to be delivered at high efficiency to the target population of
cells.
[0008] Accordingly, in one aspect, the present invention provides a
protein that is capable of being exported from the cells in which
it is expressed and is capable of being taken up in other cells,
for example those not directly producing the protein. Preferably,
the transport protein is associated with one or more other proteins
whose delivery to the populations of cells is desired.
[0009] Further experiments have confirmed that VP22 is imported
into cells when it is added as an extract to the extracellular
medium. This confirms that it is not necessary for the VP22 to be
expressed in at least a part of the population of cells for the
observed intercellular transport to occur.
[0010] In one alternative aspect, the VP22 transport protein can be
coupled to the associated molecules, eg covalently, or incorporated
with associated molecules, and used in that form, as opposed to the
use of an expression vector to produce the protein and/or the
associated molecule. In particular, this could allow non-peptidyl
molecules, such as nucleic acid, drugs or markers (in addition to
or as alternatives to proteins) to be associated to the transport
protein, and be taken up into a population of cells, without the
need to express the VP22 and the associated molecule in at least a
part of the population of cells to which delivery of the VP22
and/or the associated molecule is desired.
[0011] In a preferred embodiment, the present invention provides a
transport protein which is: [0012] (i) VP22 or an active portion
thereof; [0013] (ii) a fragment or homologue of VP22 including one
or more of the determinants providing the transport property; or,
[0014] (iii) a fragment from the C-terminal 34 amino acids of VP22;
[0015] the transport protein being optionally associated with one
or more other molecules whose transport to the target population of
cells is desired.
[0016] In this invention, "an active portion" means a peptide which
is less than full length VP22, but which retains the property of
being secreted by the cells producing it and of being taken up
other cells.
[0017] In a further aspect, the present invention provides a
composition comprising one or more of the above transport proteins,
the proteins being optionally associated with one or more molecules
whose delivery to a population of cells is desired.
[0018] In a further aspect, the present invention provides a method
of transporting a desired molecule into a population of cells by
exposing the cells to the desired molecule and the transport
protein as set out above.
[0019] Thus, in this aspect, the invention provides a method which
avoids the need initially to transfect the population of cells with
nucleic acid encoding the transport protein and optionally the
desired molecule. Thus, this can also allow the transport of
molecules which are non-peptidyl, which could not be expressed in a
cell, as the desired molecule and the transport protein can be
added to the the medium surrounding the cells.
[0020] In some embodiments, the desired molecule can be coupled
covalently to the transport protein and these entities exposed to
the target population of cells. Alternatively, the desired molecule
and the transport protein can be non-covalently associated, eg
using lipid based vehicles incorporating a desired molecule such as
nucleic acid and the transport protein.
[0021] Accordingly, in a further aspect, the present invention
provides a method of preparing a composition including a desired
molecule for transport to a target population of cells comprising
covalently or non-covalently associating the desired molecule and
transport protein.
[0022] In a further aspect, the present invention provides nucleic
acid encoding the transport protein and (optionally) the associated
molecules. For example, in embodiments in which the transport
protein is expressed as a fusion construct, the nucleic acid can be
provided which encodes the transport protein and its fusion
partner(s).
[0023] In a further aspect, the present invention provides
expression vectors incorporating nucleic acid encoding the
transport protein or an active portion or fragment thereof, and
optionally nucleic acid encoding the associated molecules.
[0024] In a further aspect, the present invention provides host
cells transfected with the above expression vectors.
[0025] In a further aspect, the present invention provides a method
of transporting a desired protein or peptide to a target population
of cells, the method comprising: [0026] (i) transfecting, infecting
or otherwise introducing into a first part of a target population
of cells with nucleic acid encoding a transport protein, and
optionally with nucleic acid encoding proteins associated with the
transport protein; [0027] (ii) expressing the nucleic acid to
produce the protein(s), after which the transport protein is
exported from the cells, together with any other proteins
associated with it, to be taken up by a second part of the target
population of cells not directly producing the protein(s).
[0028] It will be appreciated that introduction into transfection
of a first part of a target population of cells could be achieved
by infecting with a recombinant virus.
[0029] In a further aspect, the present invention provides a method
of transporting a desired molecule into a population of cells
comprising: [0030] (I) coupling the desired molecule to a transport
protein to form a complex; and, [0031] (ii) exposing the cells to
the complex so that the cells can take up the complex.
[0032] In this method, the desired molecule, which may be
non-peptidyl, can be coupled to, e.g. covalently or incorporated
with, the transport protein.
[0033] In a further aspect, the present invention provides the
above transport proteins and vectors for use in methods of
transporting molecules, both in research methods and in methods of
therapy. The present invention is particularly applicable in
methods of targeting agents to populations of cells where targeting
at high efficiency is required, eg in gene therapy techniques, the
treatment of cancer, eg by the delivery of tumour suppressing
agents to cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows the immunofluorescence of COS-1 cells
expressing VP22. COS-1 cells grown on coverslips were transfected
with the VP22 expression vector. Forty hours after transfection the
cells were fixed in 100% methanol for 15 mins at room temperature
and incubated with the anti-VP22 monoclonal antibody P43, followed
by an FITC-conjugated secondary antibody. The cells were then
analysed using confocal microscopy. (a) Typical nuclear
localisation exhibited by VP22 expressing cells. (b) Cytoplasmic
pattern of VP22 expression present in about 5-10% of expressing
cells. (c) Pattern of VP22 localisation observed when the levels of
expression are increased. (d) VP22 expression resulting in the
protein being localised in every cell within the monolayer.
[0035] FIG. 2 shows VP22 transport in Vero cells. Transfection and
detection were performed as for FIG. 1 except that the experiment
was performed in Vero cells. The field shows a foci of VP22 in a
central cell where it is cytoplasmic and in a gradient of intensity
in surrounding cells where it is localised to the nucleus.
[0036] FIG. 3 shows a transfection time-course of VP22 expression
demonstrates that VP22 is transported between cells. COS-1 cells
were transfected and fixed at 14, 20, 26, and 38 hrs
post-transfection. The confocal images shown are for the 20 hr time
point at 20.times. magnification (panel a) and 38 hr at 100.times.
magnification (panel b)
[0037] FIG. 4 shows the protein coding sequence of VP22, the
product of the HSV-1 UL49 gene showing the truncation point for the
.DELTA.267 mutant;
[0038] FIGS. 5(a) and (b) show the results of immunofluorescence
carried out after full length VP22 and .DELTA.267 VP22 containing
extracts were added to the extracellular medium; and,
[0039] FIG. 5(c) shows a Western blot of the full length VP22, and
.DELTA.267 VP22 cellular extracts;
[0040] FIG. 6 shows the import of VP22 or modifications thereof
when applied to medium.
[0041] FIG. 7a shows a typical picture of a field with one
microinjected cell stained with an antibody to .beta.-gal.
[0042] FIG. 7b shows the same field of cells where VP22 is seen not
only in the microinjected cells, but in numerous surrounding
cells;
[0043] FIG. 8a shows typical fields of mixed cells marked by
T-antigen positive COS cell. The surrounding BHK cells are not
detected by the T-antigen antibody;
[0044] FIG. 8b shows the VP22 positive cells, i.e. the central COS
cell plus the surrounding non-transfected BHK cells are seen;
[0045] FIG. 9a shows Cos-1 cells expressing full-length VP22 were
stained for both VP22 and DNA, and cells at different stages of
mitosis analysed. To stain DNA propidium iodide was added to the
glycerol mountant at a final concentration of 3 .mu.g/ml. Cells
representing prophase, prometaphase and anaphase are shown;
[0046] FIG. 9b shows increasing amounts of GST and GST-VP22 fusion
protein were incubated with an end-labelled 40 bp oligonucleotide
(T24), and resulting complexes analysed by gel-shift assay. The
VP22-specific complexes are labelled 1 to 3; and
[0047] FIG. 10 shows vero cells infected with HSV-1 [gH-ve]. a
shows a field 24 hour after infection stained for
beta-galactosidase. b shows the same field stained for VP22. c
shows a field 66 hour after infection stained for
beta-galactosidase. d shows the same field stained for VP22.
DETAILED DESCRIPTION
Materials and Methods
Cells and Viruses.
[0048] HeLa cells (human epithelial carcinoma cells), COS-1 cells
(monkey kidney fibroblasts transformed by SV40 T-antigen), Vero
cells (monkey kidney fibroblasts) and BHK (baby hamster cells) were
grown in Dulbecco's modified minimal essential medium containing
10% newborn calf serum.
Plasmids.
[0049] The eukaryotic expression vector pGE109 contains the VP22
open reading frames, under the control of the hCMV IE promoter, and
has been described previously (4). Briefly, the expression
construct was made by using the polymerase chain reaction to
amplify the VP22 open reading frame with linkers containing BgIII
or BamHI restriction enzyme sites, to facilitate subsequent
introduction into a vector under the control of the hCMV
enhancer/promoter region.
Antibodies.
[0050] These experiments used anti-VP16 monoclonal antibody LP1 and
anti-VP22 monoclonal antibody p43. The p43 antibody resulted from
immunisation of mice with a preparation of virion proteins from
herpes simplex virus. A polyclonal antibody (AGV30) reactive
against VP22 was produced by immunisation of rabbits with a fusion
protein consisting of glutathione-s-transferase linked to the VP22
open reading frame. The fusion protein was created by in frame
insertion of a Bgl II-BamHI fragment from pGE109 into the BamHI
site of the commercially available (Pharmacia) bacterial expression
vector pGEX2T to create the vector pGST-VP22. E. coli strain HB101
containing pGST-VP22 were induced by addition of IPTG (0.1 mM) to a
logarithmic phase culture, and the cells harvested 3 hours later.
After pelleting, the cells were resuspended in phosphate buffered
saline containing 0.5% NP-40, sonicated using a Branson Ultrasonic
cell homogeniser and clarified by centrifugation at 12000 rpm for
20 minutes at 4 degrees C. The GST-VP22 fusion protein was then
purified by affinity chromatography on glutathione Sepharose beads
(Pharmacia), washing of unbound protein in PBS 0.5% NP40 and
elution in 10 mM Tris-HCl buffer (pH 8.0) containing 5 mM
glutathione. The fusion protein was injected (approx. 50 .mu.g per
injection) in adjuvant at 4-weekly intervals into each of three
rabbits and sera harvested after a total of 17 weeks.
SDS-PAGE and Western Blotting.
[0051] Proteins were separated by electrophoresis through
SDS-polyacrylamide gels crosslinked with bis-acrylamide. Gels
containing radiolabelled samples were dried and exposed to X-ray
film. Gels for Western blotting were transferred to nitrocellulose
filters and reacted with the appropriate antibody. A horseradish
peroxidase linked secondary conjugate was used, with reactive bands
being visualised by development with 3, 3' diaminobenzidine
tetrahydrochloride dihydrate (DAB) or with enhanced
chemiluminescence.
Transfections and Immunofluorescence.
[0052] The day before transfection cells were plated into 6 well
trays (6.times.35 mm) containing one coverslip per well, at a
density of 2.times.10.sup.5- cells per well. DNA transfections were
carried out with 500 ng expression plasmid made up to 2 .mu.g with
pUC19 DNA, using the calcium phosphate precipitation technique
modified with BES [N,N-bis(2-hydroxethyl)-2-aminoethanesulfonic
acid] buffered saline in place of HEPES-buffered saline previously
described (3,5). Cells were fixed 40 hrs after transfection in 100%
methanol for 15 mins at RT, then washed in phosphate buffered
saline (PBS) For transfections, the cells on coverslips were
blocked by incubation with PBS/10% calf serum for 15 mins at RT.
Primary antibody was added in the same solution (p43 at a dilution
of 1:100,) and incubated for 20 mins at RT. Following extensive
washing with PBS, secondary antibodies (FITC-conjugated anti-mouse
IgM and/or Texas Red-conjugated anti-rabbit IgG) were added in
PES/10% calf serum and incubated for 10 mins. Extensive washing was
again carried out in PBS before the coverslips were mounted in
glycerol and examined in dual channels using a Biorad MRC600
confocal microscope. Phase contrast pictures were photographed
using standard light microscope.
Results
VP22 Localises in Two Distinct Patterns.
[0053] The cellular localisation of VP22 was investigated by
immunofluorescence of VP22 expressing cells using the anti-VP22
monoclonal P43, followed by confocal microscopy. COS cells were
transfected as described above, and the cells fixed 30 to 40 hours
later in methanol and processed for detection of VP22. Initial
results demonstrated that, in the majority of VP22 containing
cells, the protein was present in a distinctive nuclear pattern,
frequently showing enrichment around the nuclear rim. FIG. 1a
illustrates typical examples of the nuclear patterns of VP22.
However in a number of cells VP22 was present in a very different
cytoplasmic-filamentous pattern (FIG. 1b). In this case, the
protein was detected in a meshwork or cage like pattern within the
cytoplasm and was excluded from the nucleus. This heterogeneity in
distribution, although not by itself unusual, was not observed with
the other HSV tegument protein under study (VP16), which exhibited
a pattern differing little from cell to cell and consisted mainly
of a diffuse cytoplasmic pattern with minor, amounts present in the
nuclei.
[0054] Further examination revealed that the cytoplasmic versus
nuclear distribution of VP22 was not random with respect to each
other, ie one in which a central cell containing cytoplasmic VP22
was surrounded by a halo of cells containing nuclear VP22, where
the protein was clearly enriched in the region of these latter
cells adjacent to the central cell containing the cytoplasmic VP22
(FIG. 1c). Finally, the areas of the monolayer in which VP22 could
be detected in every cell again displayed this distribution with
the cytoplasmic pattern being observed in a minority of cells, with
a nuclear pattern in the surrounding cells (FIG. 1d).
[0055] Initially, these experiments well performed in COS cells
which by virtue of the SV40T-antigen allow replication of the VP22
expression plasmid used here. Although we did not detect this type
of distribution with other proteins (e.g. VP16 see above) which
were expressed from identical vectors, we wished to rule out the
possibility that the pattern of VP22 distribution was related to
the use of COS cells. Similar experiments were therefore performed
with Vero cells and HeLa cells and identical results were obtained.
FIG. 2 shows expression of VP22 in Vero cells in the pattern
described above in which a central cell containing cytoplasmic VP22
is surrounded by cells containing nuclear VP22. In this case, VP22
is seen in a gradient of decreasing intensity, from the central
cell to the adjacent cells with intense nuclear VP22, to cells
further away with less intense nuclear VP22.
[0056] These surprising results indicate that VP22 was being
transported from the cells in which it was initially being
synthesised and was present in a cytoplasmic pattern, to adjacent
cells and also to cells further removed again, where it localised
to the nucleus. To demonstrate VP22 moving between cells, a time
course of transfection was carried out. By 14 hours after
transfection, it was possible to detect VP22 expression in
individual cells. At 20 hrs, foci of cells containing VP22 could be
seen wherein a central cell containing intense VP22 was surrounded
by several cells containing less intense VP22 (FIG. 3a, lower
magnification to facilitate point), while by 38 hrs virtually every
single cell contained the protein (FIG. 3b)
[0057] As an additional piece of evidence that VP22 was being
transported between cells, two separate dishes of COS-1 cells were
transfected with either the VP22 expression vector, and pRG50, a
VP16 expression vector. Twenty-four hours later, each dish was
trypsinised and the two populations mixed and plated onto
coverslips. Twenty-four hours later, it was possible to detect VP16
expressing cells which also contained VP22 within the nucleus,
demonstrating that cells which had never been exposed to the VP22
expression vector had taken up the VP22 protein.
[0058] Intercellular transport requires a C-technical determinant
within VP22.
[0059] To begin characterisation of the requirements within VP22,
we tested a series of variants containing short (3-4 amino acids)
in frame insertions and a deletion mutant lacking residues 267 to
the end (residue 301) of the protein. The insertion mutants
contained insertion at position 60, 159, 192 and 267. None of the
insertion mutants Ins 60, Ins 159 or Ins 267 had any effect on the
pattern of localisation of VP22 or its ability to undergo
intercellular transport (data not shown, all the same as wild
type). The insertion at position 192 did however have a significant
effect. This mutant exhibited a pattern of distribution similar to
the cytoplasmic distribution seen with the wild type; thus, nuclear
staining in adjacent cells was not observed, fewer positive cells
were observed overall and in VP22 positive cells, the pattern was
exclusively cytoplasmic (FIG. 3c)
[0060] An identical phenotype was observed for the deletion mutant
lacking the C-terminal 34 residues 267-301 (FIG. 3d). Note that the
insertion mutant at position 267 was normal. The pattern of
distribution showing exclusively cytoplasmic VP22 and lack of
transport to surrounding cells for deletion mutant 267-301 and
insertion mutant 192 was not due to inefficient synthesis. When
total synthesis was compared by Western blotting, similar amounts
of the w/t and mutant proteins were observed.
[0061] To begin to address the possibility that this property of
intercellular transport could be utilised for the transport of
additional peptides or proteins, we tested whether a variant of
VP22 (VP22ep) which contained a 12 residue extension on its
C-terminal end (containing a recognition site for a monoclonal
antibody), would also be transported. The results demonstrated
efficient transport of the extended protein. In fact, FIG. 3b,
which demonstrates transport in Vero cells, utilised this
C-terminal extended variant. As indicated above, a foci of cells
containing central cytoplasmic VP22ep was surrounded by cells
containing nuclear VP22ep and, as for w/t protein, expression of
VP22 could be detected in virtually every cell in the monolayer.
Moreover, the protein which was being detected was indeed the
fusion protein, and not some fortuitous deletion product, since it
could be detected using either the anti-VP22 antibody p43 or the
antibody to the C-terminal peptide extension. This important result
demonstrates that it is indeed feasible to promote the delivery of
peptides or proteins to cells by linking them to a suitable
transport protein such as VP22.
[0062] Those skilled in the art could routinely employ these or
other site-directed mutagenesis methodologies to map or refine the
determinants in VP22 that are critical and/or important for the
transport property, thereby allowing the use of fragments of VP22,
rather than the full length protein.
VP22 Uptake from Medium.
[0063] Although VP22 was readily detected on the cell surface, we
have as yet been unable to detect VP22 ir the medium of transfected
cells. However to examine the ability of VP22 to be imported from
the extracellular medium, a soluble extract made from
VP22-expressing cells was added to the media of untransfected
cells. VP22 was imported and was localised to the nucleus with high
efficiency and rapidly (see FIG. 5(a)), uptake occurring within 5
mins after addition to media (possibly explaining our inability to
detect it in the medium). The uptake mechanism was not affected by
incubation at 4.degree. C. suggesting that internalisation was not
via normal endocytosis. In control experiments uptake from the
medium was not observed for any other protein we have tested.
[0064] Soluble extracts containing either full-length (WT), or
truncated (4267) VP22 were added to the media covering COS-1 cells
grown on coverslips, and the cells fixed 1 hr later. Equal amounts
of the extracts were also analysed by SDS-PAGE and Western
blotting.
[0065] To make the soluble extract, one million COS-1 cells were
transfected with either full-length or 6267 VP22 expression
vectors, harvested 36 hrs later, and the extract made in 10 mM
HEPES (pH 7.9), 400 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT and 5%
glycerol. Half of the extracts were then added to the media
covering 5.times.10.sup.5 COS-1 cells grown on coverslips, the
cells fixed 1 hr later, and immunofluorescence carried out using
the polyclonal anti-VP22 antibody (see FIGS. 5(a) and 5(b). One
tenth of the extract described was analysed by electrophoresis on a
10% acrylamide gel followed by Western blotting using monoclonal
antibody P43 (see FIG. 5(c)).
[0066] Furthermore, the VP22 deletion mutant .DELTA.267, while
present in the soluble extract in equivalent amounts to full-length
(see FIG. 5(c), .DELTA.267), was not detected intercellularly
indicating a requirement for the C-terminal 34 residues for this
process (FIG. 5(b)).
[0067] To investigate this further, we have made a peptide
corresponding to the C-terminal 34 residues and added a 12 residue
tag to allow immunological detection. This peptide, when applied to
cell media, gains entry to the cell and is transported to the
nucleus (FIG. 6b). These experiment shows that these C-terminal 34
residues are a) required and b) sufficient to give transport.
Transport of Fusion Proteins
[0068] Although, as indicated above, we have demonstrated that
peptides can be transported by fusion to VP22 it was unknown how
big a protein could be fused to VP22 to facilitate transport.
[0069] COS-1 cells in 6 mm dishes were transfected with plasmids
pGE109, .DELTA.267 or VP22-GFP which had been constructed by
insertion of the UL49 open frame reading frame into the BamHI site
of the plasmid pGFP-N1 (Clontech), resulting in a fusion of VP22 to
the N-terminus of green fluorescent protein (GFP). 40 hrs after
transfection the cells were harvested and high salt extracts were
prepared. Western blotting of these extracts demonstrated that
while there were equivalent amounts of the full length and
.DELTA.267 variants of VP22 present in the extracts, the level of
22-GFP protein was about 5-fold less. Equal volumes of each extract
was added to the media covering the COS-1 cells on coverslips, left
for 1 hour, then fixed in 100% methanol. Immunofluorescence was
carried out using AGV30. The results showed (FIG. 6a) that the
VP22-GFP fusion protein could be detected in the recipient cells
and that it had accumulated in the nuclei.
[0070] In summary, we have shown that we can make extracts of cells
expressing a fusion protein where the attached protein was 32 Kda,
and after applying the extract to cell medium, we can detect the
fusion protein in the nuclei.
Microinjection of VP22
[0071] Although transport by transfection is valuable for tissue
culture uses, it may not be a really a valid route for delivery in
tissues or patients.
[0072] Microinjection (and also liposome delivery) are utilised in
vivo and it is additionally useful to demonstrate VP22 transport by
a route of delivery which could have in vivo utility.
[0073] Cos-1 cells plated onto coverslips were microinjected with a
mixture of 100 ng/.mu.l pGE109 and 100 ng/.mu.l pBAG as a marker
for the injected cell(PNAS 84: 156-160), using a Carl Zeiss
semi-automatic microinjector in manual mode. The cells were
incubated for 24 hrs, fixed in 100% methanol, and double
immunofluorescence using the polyclonal anti-VP22 antibody AGV30
(1:500) and monoclonal anti-s galactosidase. (1:50) (Promega). FIG.
7a shows a typical picture of a field with one microinjected cell,
stained by antibody to .beta.-gal. In the same field of cells, VP22
(FIG. 7b) is seen not only in the microinjected cells, but in
numerous non-injected cells. Thus, this route of delivery could be
used in vivo by microinjection into e.g. the centre of tumours, to
deliver VP22 or a variant thereof. Other routes of delivery would,
of course, be equally viable, this just demonstrates one route
directly. Currently two main routes of delivery of genes are
utilised, namely liposome-mediated and virus infection. Therefore,
the gene for VP22 or various modifications including fusion
proteins, could be incorporated into liposome vesicles for in vivo
delivery. Likewise, liposome delivery is also utilised in tissue
culture systems. Alternatively, the gene for VP22 or varients could
be inserted into the genomes of e.g. adenovirus or retroviruses for
delivery of infection. In both cases expression would be in the
initial population of transfected or infected cells, but VP22 would
be delivered to surrounding cells. This is particularly useful
where delivery is by disabled virus vectors which are designed not
to replicate. Delivery in this case would be enhanced. Direct
microinjection of the genes or direct application of the naked DNA
are also routes currently being explored in the field.
[0074] Transport of VP22 into Different Cell Types
[0075] We have further demonstrated that VP22 can be transported
into a different cell type from the one in which it is initially
expressed.
[0076] COS-1 cells in a 60 mm plate were transfected with 1 .mu.g
pGE109 made up to 4 .mu.g with PUC19 DNA. 24 hrs after transfection
the cells were removed by trypsinization and mixed with previously
trypsinized BHK-21 cells (hamster) at a ratio of 1:20. The mixture
of cells was plated onto coverslips in 6 well plates and incubated
for a further 20 hrs, following which they were fixed in 100%
methanol. Double immunofluorescence was carried out using AGV30
(1:500) and the monoclonal anti-T antigen pAB419 (neat) to identify
the COS-1 cells (J. Virol. 39:861-869).
[0077] FIG. 8 shows a typical field of mixed cells where the
T-antigen positive COS cells are seen (FIG. 6a, left hand panel),
(the surrounding BHK cells of course are not detected by the
T-antigen antibody), and in FIG. 8b, right hand panel the VP22
positive cells, i.e. the central COS cell plus the surrounding
non-transfected BHK cells are seen.
DNA Binding Properties of VP22
[0078] We have shown that in cells where VP22 is nuclear, it binds
to the condensing chromosomes during mitosis. This shows a is
strong association with the chromosomes during mitosis and
demonstrates that once VP22, or variants is taken up into the cell,
they will also be passed onto the daughter cells after division.
This property makes VP22 additionally useful for delivery. Moreover
we have shown that in vitro, VP22 has non-specific DNA binding
properties probably explaining (at least in part) the in vivo
chromatin association. These two observations combined add to the
potential of VP22 to bind DNA and to deliver DNA/genes to
nuclei.
[0079] During the analysis we observed that a proportion of the
cells with nuclear VP22 contained the protein in a pattern similar
to that of mitotic chromatin. Double staining of cells for both
VP22 and DNA revealed that cells at all stages of mitosis could be
detected with VP22 localised around the condensed chromosomes (FIG.
9a). To determine if VP22 could interact directly with DNA, we
tested the ability of a GST-VP22 fusion protein to bind a 40 bp
oligonucleotide probe, by gel retardation assay. Dose-dependent
increase in the appearance of 3 complexes (FIG. 9b), not present in
the GST controls, was observed for GST-VP22. The complexes of
increasing size may represent multimerization of the protein, since
the largest one formed less efficiently on shorter probes (not
shown). Moreover VP22 was capable of binding to a range of DNA
probes suggesting that the protein interacts with DNA in a
non-specific manner.
VP22 Transport in Virus Infected Cells.
[0080] One of the main routes of gene delivery for gene therapy is
via the use of viruses. Typically such viruses are disabled in some
way so that they will replicate inefficiently or not at all. The
desire is that limiting replication will prevent complications due
to pathogenesis. Safety of virus vectors is a major issue in gene
therapy and much effort is going into developing stringently
disabled viruses. But the disadvantage of using disabled viruses is
that the genes of interest are then delivered only to those
initially infected cells.
[0081] We wished to demonstrate directly that VP22 could be
transported from a virally infected cell to a non-infected cell. To
this end we infected cells with a disabled herpes simplex virus
mutant HSV-1 [gH-ve]. This virus lacks the gene for an essential
virus glycoprotein, gH, and must be propagated on a specialised
complementing cell line containing gH. But when this virus infects
non-complementing cells (ie any cell type not containing the virus
gE), the virus can penetrate an initial population of cells,
synthesis virus proteins (except gH), and assemble virus particles,
but these are non-infectious. No second round or infection occurs.
We wished to demonstrate that VP22 could be transported form a cell
initially infected with the HSV-1 [gH-ve] virus mutant (Desai, P.
J. et al. J Gen Viral 6.9 (Pt 6): 1147-56 (1988); Forrester, A. et
al. J Virol 66: 341-8 (1992) U. Gompels & A. Minson Virology
153: 230-47 (1986)).
[0082] Vero cells were infected with HSV-1 [gH-ve] at a
multiplicity of approximately 0.2 pfu/cell. Therefore roughly 1 in
5 cells would be initially infected. Twenty four or sixty six hours
after infection the cells were fixed in methanol and assessed for
the presence of VP22, or beta galactosidase. Note that this virus
contains the gene for beta-galactosidase and the presence of
beta-galactosidase serves as a marker for the primary infected
cell. The results are shown in FIG. 10.
[0083] FIG. 10a shows a field 24 hour after infection stained for
beta-galactosidase.
[0084] FIG. 10b shows the same field stained for VP22.
[0085] FIG. 10c shows a field 0.66 hour after infection stained for
beta-galactosidase.
[0086] FIG. 10d shows the same field stained for VP22.
[0087] In FIG. 10a two cells expressing beta-galactosidase are
arrowed. Note the surrounding cells lacking beta-galactosidase. In
the same field stained for VP22 (FIG. 10), these two cells are also
VP22 positive (large arrowheads), but in addition several
surrounding cells are detested containing VP22 (small arrowheads),
which were beta-galactosidase negative.
[0088] Again in FIG. 10c (66 hours post infection) three cells are
shown which are beta-galactosidase positive (large arrowheads). In
the same field stained for VP22 (FIG. 10d), these same three cells
are positive but in addition surrounding cells, beta-galactosidase
negative and therefore non-infected, nonetheless contain VP22 which
has accumulated in the nuclei (small arrowheads). Note these latter
cells completely lack detectable beta-galactosidase, despite the
intense staining in the three central positive cells.
[0089] The results demonstrate that VP22 can be transported from
virally infected cells. This is not property of only herpes virus
infection since VP22 can be transported from transfected or
microinjected cells. Thus transport does not require any other
herpes virus encoded protein and will almost certainly occur
utilising any virus vector such as adenovirus or retroviruses.
[0090] Therefore we have directly demonstrated proof of principle
for VP22 transport from virus infected cells. It is easily
envisioned that fusion proteins containing a gene of interest could
be constructed linked to the gene for VP22, or the active portion
thereof, and inserted into virus vectors under the control of a
constitutively efficient or tissue-specific promoter. The fusion
protein will then be delivered to a much greater population than
those initially infected.
Discussion
[0091] The above results demonstrate that VP22 exhibits a highly
unusual property in that it is efficiently transported from the
cells in which it is originally expressed, and in which it displays
cytoplasmic localisation, to adjacent cells within a cell the
monolayer, where it is taken up into the nucleus. This pattern of
behaviour has not been observed for any other of a range of
proteins we have tested and we can find no previous demonstration
of this activity. Thus, for example, VP16 is also expressed in the
cytoplasm of cells, but its pattern of expression is completely
homogeneous within the population of expressing cells and it is not
transported to adjacent cells. This difference and the unexpected
property of VP22 is shown from the result that approximately 30
hours after introduction of either VP22 or VP16 into a cell
monolayer, while VP16 can be detected on average in about 2-5% of
the cells, VP22 can be detected in every cell of the monolayer.
[0092] This work also demonstrates the specificity in VP22
intercellular transport and demonstrates the involvement of a
determinant at the C-terminal end of the protein since a variant
lacking the C-terminal 34 amino acids, while being expressed in a
cytoplasmic location in initially expressing cells, is not
transported to adjacent cells. The experiments show intercellular
transport of VP22 in a number of different cell types including
COS-1 cells, Vero cells, and HeLa cells, ie the phenomenon does not
appear to be specific to the COS cells in which it was initially
observed.
[0093] Protein secretion or export normally occur via specific
pathways requiring well characterised signal sequences for sorting
into the compartments and vesicles involved in export pathways. In
one of the main mechanisms of protein secretion, signal sequences
usually residing in the extreme N-terminus of a protein, are
recognised by a complex of a 7S RNA and at least 6 polypeptides
which together comprise the signal recognition particle (SRP).
Recognition by the SRP is followed by docking and transfer of the
signal peptide to a receptor within the endoplasmic membrane. The
emerging polypeptide is then discharged across the ER membrane and
subsequently processed through a complex network of interactions
and vesicle assembly in the Golgi network. VP22 does not possess
any conventional signal sequence and its pattern of distribution
within the cytoplasm does not resemble ER or Golgi
distributions.
[0094] There have been a small number of previous examples of
mammalian protein secretion by non-classical pathway(s), including
for example the cytokines interleukin 1alpha and 1beta, and the
basic and acidic fibroblast growth factors (7). One possibility for
the export of such components is via ABC-transporter systems. These
proteins comprise ATP-dependent and membrane bound proteins which
contain membrane spanning helices. They are involved in protein
specific binding and transport across membranes and are best
characterised in bacterial systems. However, to date there is no
direct evidence for a role of any mammalian ABC-transporters in
proteins secretion.
[0095] Further while certain proteins are secreted by a
nonclassical pathway, the pattern of VP22 secretion is extremely
unusual. While not wishing to be bound by any particular theory,
the simplest interpretation of our results is that VP22 is
secreted, but then taken up and concentrates in the nucleus of
target cells. Again, there are no obvious nuclear localisation
signals in VP22 although the protein has a predicted molecular
weight of 32k and may enter the nucleus without a requirement for a
specific signal. Further studies on the determinants required
within VP22 and of the physiological requirements within the cell
should help clarify the pathway involved, and understanding how
VP22 is secreted may contribute to an understanding of how other
proteins are transported in non-classical export pathways.
[0096] These results open up the possibility of using the
determinants involved in VP22 transport to enable the transport and
efficient expression or uptake within a target cell population of
other proteins or other molecules associated with the VP22
determinants. We have already shown that a short peptide (12
residues) can be linked to VP22 and the fusion protein still be
transported to every cell in the monolayer. This result
demonstrates that it is indeed feasible to promote the delivery of
peptides by linking them to VP22.
[0097] Further, we have shown that a large protein (32 Kd), linked
to VP22 can be imported into cells from the cell medium and is seen
to accumulate in the nuclei.
[0098] Widespread utility of such ability can easily be
envisaged.
[0099] For example it may be possible to link DNA binding proteins
to the VP22 determinant(s), such that when delivered to a target
cell population they will be efficiently expressed in a much larger
population.
Other Utilities Include:
Costimulatory Molecules and Cytokines are Recruited to Boost
Vaccine Response by Host
[0100] Recent data from the field of vaccine development highlights
the requirment for costimulatory molecules and cytokines for
vaccines to work effectively to boost the host immune response at
the cellular level. Tumour cells do not express costimulatory
molecules on their cell surface and this is the reason why there is
no clonal expansion of CTL (cytotoxic T-lymphocytes) in the host,
resulting in the patient's inability to effect tumour shrinkage
without medical intervention.
Direct Stimulation of Cytotoxic T-Cell Response in Host
[0101] Delivery of foreign protein with anti-cancer properties to
the nucleus. The foreign protein may be a tumour-specific antigen
known to induce an immune (T-cell) response to the tumour.
[0102] The foreign protein may be delivered directly to the nucleus
with VP22 molecules (this would also emulate a virus infection--see
Note). The foreign protein would then be broken down to peptides in
the proteosome of the cell. If the classical T-cell induction
pathway is followed, the peptides are targeted through the ER and
Golgi apparatus of the cell and presented on the cell surface in
conjunction with MHC class-I molecules.
[0103] Boosting the CTL response in patients has obvious
applications to non-cancer therapy, particularly where diseases are
caused by viral infection. The belief that inducing a strong CTL
response in the host may lead to viral clearance from the body has
recently gained scientific credibility.
Boost Immune Response with Delivery of MHC Molecule with
Protein/Antigen to be Targeted
[0104] It would be possible to solve the problem of MHC restriction
in the population and boost cellular responses by targeting antigen
plus HLA molecule. This application would also have non-cancer
applications.
Modulation of Signal Transduction Pathways
[0105] Many signal transduction pathways operate by transferring an
environmental signal, hormones, binding of ligands to membranes,
stress (DNA damage, heat osmotic etc) to the nucleus to effect
alterations in gene expression. It may be possible to use VP22 to
mediate such signals by coupling the protein or peptide to
signalling molecules. Delivery of the fusion protein, or the
corresponding gene, will enhance signal because to fusion is also
exported to neighboring cells. For example, fusing VP22 to a
dominant mutant of a plasma membrane associated signal effector (eg
small GTPases and their effectors or apoptotic regulatory
molecules) may effect the desired pathway not only in the recipient
cell with the gene, but also in surrounding cells despite the
absence of the gene in them. Many scenarios can be envisaged that
are variations on the theme of modulation of signal transduction
pathways.
[0106] The ability of VP22 to bind DNA in vitro and the ability for
it to bind condensing chromosomes during mitosis provides for an
application with regard to gene/DNA therapy. This application can
take several forms: [0107] a) virus infection where the gene for
VP22 or variants thereof is inserted into the genomes of
adenoviruses or retroviruses for delivery of infection. Likewise, a
gene of a desired protein could also be inserted such that a fusion
protein is expressed comprising VP22 and the protein of interest.
VP22 would then serve to deliver the protein of interest to the
surrounding population of cells. In this way the amount and
therefore risk of such virus infection is reduced. [0108] b)
microinjection of nucleic acid coding for VP22 (or a variant
thereof) and the protein of interest directly into a cell such that
once expressed VP22 can transport the protein of interest into the
surrounding population of cells. [0109] c) liposome mediated
infection where the gene for VP22 or various modifications
including fusion proteins are incorporated into liposome vesicles
for in vivo delivery.
[0110] Other techniques for introducing DNA into cells will be
apparent to the skilled man, for example, direct scarification
where the DNA is taken up directly by the tissue; receptor mediated
DNA transfer; calcium phosphate mediated transfection; and
ballistic delivery.
[0111] Efficient delivery of functional molecules is a desired aim
in gene/protein therapy. This mechanism will have the additional
advantage that the gene expressing VP22 will only be present in a
small population of cells while the protein is present in a much
wider population. This will be a useful factor where potentiality
harmful effects of the delivered gene are a consideration. Lower
delivery of genes while maintaining high delivery of functional
protein may be desirable. Virtually any application requiring or
desiring efficient expression of test proteins can be envisaged in
finding application for a VP22 delivery mechanism. Delivery of
tumour suppressor proteins, enzymes and so on. We have also shown
that it is possible to bind nucleic acids to VP22, and therefore in
addition to dna it may be possible to deliver antisense RNA
ribozymes etc.
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Sequence CWU 1
1
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