DEVICE FOR TREATMENT OF VIRAL INFECTION
Technical Field of the Invention
The invention relates
to a device for sequestration of virus from an environment, and for therapy of
viral infection. More specifically the
invention relates to the genetic modification of erythroid cells, to the
production of liposomes, to lymphoid cells, and to the use of the modified
cells, the liposomes, or the lymphoid cells for sequestration of virus from an
environment, and for therapy of viral infection.
Despite the number of
methodologies for treatment of conditions or contamination caused by infectious
agents, few methodologies are available for treatment of conditions or
contamination caused by infectious virus.
There is a need for
alternative methods for treating or at least ameliorating conditions or
contamination caused by viral infection.
The invention seeks to
address the above identified need and in a first aspect provides a device for
sequestering an infective virus from an environment. The device comprises binding means for binding an infective virus
in the environment to the device and container means for containing the
infective virus within the device. The
binding means and container means are arranged for permitting infective virus
bound to the device to be transferred to the container means, for containment
of the infective virus within the device.
The inventors have
surprisingly found that the sequestration of virus from an environment and
containment within the device of the first aspect of the invention is an
effective method for treatment of conditions or contamination caused by
infective virus.
As will be understood
in the art, an “infective virus” is one capable of completing all steps in the
viral life-cycle including adsorption to host cell, production of functionally
competent viral particles capable of infecting further host cells and release
from a host cell.
The binding means of
the device may be any molecule capable of forming a receptor-ligand interaction
with the virus sufficient to bind the virus to the device. An example of a binding means is a receptor
for the virus. The choice of receptor
for the virus will depend on the infective virus that is to be sequestered from
the environment. In one embodiment the
receptor is a receptor for HIV.
Examples of receptors for HIV include, but are not limited to, the
following: CD4, CCR5, CXCR4, CCR2b, CCR3, CCR8, STRL33, AJP, PR1, GPR15, V28
and ChemR23. Preferably, the receptor
for HIV is CD4. Alternatively, the
binding means may be an antibody or antibody fragment capable of interacting
with the virus sufficient to bind the virus to the device. Further, the binding means may be a fusion
protein comprising, for example, a domain derived from a receptor identified
above and a further domain, for example an immunoglobulin domain.
The device may
comprise more than one type of binding means.
The more than one type of binding means may be a receptor, an antibody
or antibody fragment capable of interacting with the virus sufficient to bind
the virus to the device, or a fusion protein sufficient to bind the virus to
the device. In one embodiment, the
device comprises CD4 and at least one other binding means. Typically, the at least one other binding
means is a binding means for binding HIV.
In a preferred embodiment, the at least one other binding means is
selected from the group comprising: CCR5, CXCR4,CCR2b, CCR3, CCR8, STRL33, AJP,
PR1, GPR15, V28 and ChemR23.
The container means
may be any means capable of containing a virus within the device sufficient to
limit the release of virus transferred to the container, to the
environment. The container means is
characterised in that it is deficient in at least one aspect of the molecular
machinery which is necessary for completion of the viral life-cycle. Accordingly, the container means is capable
of containing virus within the device.
In one embodiment, the container means is deficient in one or more of
the following: nucleus, endoplasmic reticulum, golgi apparatus, mitochondria,
functional ribosomes, or metabolic enzymes essential for completion of the
viral life-cycle. Typically, the
container means does not comprise a nucleus. An example of a container means
which does not comprise a nucleus is an erythrocyte. A further example is a liposome.
An example of a container means which does not comprise functional
ribosomes is a lymphoid cell in which the ribosomes have been inactivated by
contacting a lymphoid cell with ricin or diphtheria toxin.
In a second aspect,
the invention provides a device for sequestering a virus from an
environment. The device comprises
binding means for binding a virus in the environment to the device and an
erythroid cell for containing the virus within the device. The binding means and the erythroid cell are
arranged for permitting virus bound to the device to be transferred to the
erythroid cell, for containment of the virus within the device.
An erythroid cell is
particularly advantageous because it provides a means for removal of the device
from the environment, subsequent to containment of virus within the
device. For example, in in vivo applications, the device may be
removed by an organ or tissue capable of eliminating erythrocytes. An example of such a tissue is the spleen or
liver. More importantly, a consequence
of the removal of the device by such tissues or organs is the elimination of
virus contained within the device. In in vitro applications, the device may be
removed by any method capable of isolation of an erythrocyte; for example,
centrifugation, filtration, immunochromatography (panning), etc.
The erythroid cell may
be any cell of the erythroid lineage which is deficient in molecular machinery
which is necessary for completion of the viral life-cycle. For example, the erythroid cell may be a
cell which is deficient in at least one aspect of the nucleus. It may comprise part or a fragment of the
nucleus. Alternatively, the erythroid
cell may not have a nucleus. Typically
the erythroid cell is an erythrocyte. A
further example of an erythroid cell is a platelet.
The binding means are
means as described in accordance with the first aspect of the invention.
In one embodiment, the
erythroid cell is derived from a stem cell containing a nucleic acid encoding a
binding means for binding a virus, the nucleic acid being capable of being
expressed during terminal differentiation of stem cell progeny into erythroid
cells. In a preferred embodiment, the
erythrocyte is derived from a haematopoietic stem cell containing a nucleic
acid encoding a binding means for binding a virus, the nucleic acid being
capable of being expressed during terminal differentiation of the
haematopoietic stem cell progeny into erythroid cells. Typically, the nucleic acid comprises a gene
encoding a binding means for binding a virus operably linked to a promoter that
is active during development of the haematopoietic stem cell into an erythroid
cell. Examples of such promoters
include, but are not limited to, promoters for g-globin
gene, e-globin gene, b-globin gene, GATA-1 gene, glycophorin B gene,
ferrochelatase gene, porphobilimogen deaminase gene, 5-aminolevulinate synthase
gene, Kel gene, syndecan-1 gene, ABO blood group genes, RH factor genes, MRS
antigen genes, Duffy antigen genes and Kell antigen genes.
In a third aspect, the
invention provides a device for sequestering a virus from an environment. The device comprises binding means for
binding a virus in the environment to the device and a liposome for containing
the virus within the device. The
binding means and the liposome are arranged for permitting virus bound to the
device to be transferred to the liposome, for containment of the virus within
the device.
Liposomes which are
suitable for use in the device of the third aspect are those which comprise
compounds which lead to accumulation of the device in the lymphatic
system. In one embodiment, the liposome
comprises polyethyleneglycol for targeting the device to the lymphatic
system. The liposome may be produced by
standard techniques, as described in Bestman-Smith et al, Biochimica et Biophysica Acta, 1468, (2000) 161-174.
The binding means of
the device of the third aspect are means as described in accordance with the
first aspect of the invention.
In a fourth aspect,
the invention provides a device for sequestering HIV from an environment. The device comprises CD4 for binding HIV in
the environment to the device and an erythrocyte for containing the virus
within the device. The CD4 and the
erythrocyte are arranged for permitting HIV bound to the device to be
transferred to the erythrocyte, for containment of the HIV within the device.
The device of the fourth aspect may further comprise one or more binding
means for binding HIV, including for example any of the following binding
means: CXCR4, CCR5, CCR2b, CCR3, CCR8, STRL33, AJP, PR1, GPR15, V28 and
ChemR23.
In a fifth aspect, the invention provides a device for sequestering HIV
from an environment. The device
comprises CD4 for binding HIV in the environment to the device, and a lymphoid
cell for containing the virus within the device. The CD4 and the lymphoid cell
are arranged for permitting HIV bound to the device to be transferred to the
lymphoid cell, for containment of the HIV within the device.
The lymphoid cell is characterised in that it is capable of containing a
virus within the device sufficient to limit the release of virus transferred to
the lymphoid cell, to the environment.
The lymphoid cell is deficient in at least one aspect of the molecular
machinery which is necessary for completion of the viral life-cyle. Accordingly, the lymphoid cell is capable of
containing virus within the device. The
lymphoid cell may be any cell of the lymphoid lineage including for example,
thymocytes. Typically, the lymphoid
cell is a CD4+ cell. In one embodiment,
the lymphoid cell is deficient in ribosome function. Preferably, ribosome function of the lymphoid cell is inactivated
by contacting the lymphoid cell with an agent such as ricin or diphtheria toxin.
The container means,
erythroid cell, liposome, erythrocyte or lymphoid cell of the devices of the
first, second, third, fourth and fifth aspects of the invention may further
contain antiviral agents for inhibiting or interfering with viral life-cycle. Examples of antiviral agents include
restriction endonuclease, DNase, RNase, protease specific to viral protein,
reverse-transcriptase inhibitor, and antibody or antibody fragments capable of
binding viral components.
In a sixth aspect, the
invention provides a method for sequestering a virus from an environment
comprising contacting the virus with a binding means of a device of any one of
the first, second, third, fourth or fifth aspects of the invention, to permit
the virus to bind to the device. Typically,
the virus is contacted with the binding means in conditions for permitting
virus bound to the binding means to be transferred to the container means,
erythroid cell, liposome, erythrocyte or lymphoid cell, for containment of the
virus within the device.
In one embodiment, the
method comprises the further step of removing the device from the
environment. In in vivo applications, the environment is typically blood, lymphatic
fluid or cerebrospinal fluid (CSF). In in vitro applications, the environment
may be blood, lymphatic fluid, CSF or a fractioned product of blood, lymphatic
fluid, CSF or tissue culture medium, buffered solution, or the like.
In a seventh aspect
the invention provides a method of treating or ameliorating a viral infection
in a mammal, the method comprising administering a device of any one of the
first, second, third, fourth and fifth aspects of the invention to the mammal.
The inventors
recognise that in in vivo
applications, the blood type of the erythroid cell for use in the device may be
dependent on the blood type of the individual in which the virus is
present. The determination of the
appropriate erythroid cell can be made using standard techniques known in the
art.
In an eighth aspect,
the invention provides a method of treating or ameliorating a HIV infection in
a mammal, the method comprising administering a device of the fourth or fifth
aspect of the invention to the mammal.
In one embodiment, the
method of the seventh and eighth aspects of the invention comprises the further
step of administering at least one antiviral drug to the mammal. The at least one antiviral drug may be
administered simultaneously with the devices of the invention.
The method of the
seventh or eighth aspect of the invention may therefore be used in conventional
therapies based on antiviral drugs to reduce the number of virus that are
resistant to antiviral drugs.
Alternatively, by eliminating virus sensitive to antiviral drugs, a
lesser amount of a device according to the invention may be required to contain
virus resistant to the antiviral drugs.
The method of the
seventh aspect of the invention may be used in gene therapies which employ
viral delivery of genes, the method being used to reduce the number of virus
that are present following the gene therapy.
In a further
embodiment, the device of the fourth or fifth aspect of the invention may be
administered in combination with an immunosuppressive agent. The immunosuppressive agent permits
proliferation of latent HIV in the patient.
The latent HIV may then be reduced by administration of the device,
thereby providing a means of also reducing latent virus numbers.
Advances in
leukapheresis and cell sorting technology have given rise to the ability to
extract precursor cells from which CD4 expressing erythrocytes may be
derived. Human bone marrow and foetal
umbilical cord blood are examples of sources of suitable cells.
Following extraction
of blood cells from a serologically similar donor (may be the intended
recipient), CD34+ cells are extracted by leukapheresis and suitable aliquots
selected using the method of Freyssinier et
al, British Journal of Haematology 1999, 06, 912-922; Fukuda, Kaneko,
Egashira and Oshimi. See: Stem Cells
1998 : 16 : 294-300. An enriched
population of cells exhibiting desirable erythropoietic stem cell behaviour are
further isolated as described in Ratajczak, et
al, Leukemia (1998) 12, 942-950.
The exact nature of the extraction procedure is not critical provided it
makes available a supply of viable, undamaged and undifferentiated CD34+ cells
for subsequent transfection, selection and later serum-free culture with
Interleukin 3, Interleukin 6 and Stem Cell Factor in order to promote the
appearance of CD36+ cells, which are precursor cells to erythrocytes.
Given a harvest of
appropriate nucleated CD34+ precursor stem cells, exogenous DNA encoding the
CD4 receptor protein under the control of a promoter that is expressed during
erythroid development is incorporated into the DNA of the CD34+ cells.
Numerous ways exist to
make this exogenous DNA. The commonest
is to use PCR employing a high fidelity polymerase in a PCR reaction. The desired sequence of DNA can be amplified
from nuclear DNA of cells expressing the desired form of CD4 receptor.
Different alleles for
the CD4 protein are known to exist. The
commonest allele of CD4 protein is exhibited on what are called CD4+
T-lymphocytes, and this allele of CD4 represents a significant adherent
receptor which interacts with a glycoprotein (gp120) found on the surface of
HIV.
DNA encoding CD4 is
isolated from CD4+ T-lymphocytes using reverse-transcriptase Polymerase Chain
Reaction (RT-PCR). It is anticipated
that DNA encoding for other viral pathogen receptors of interest could be
amplified from mRNA (or if required, nuclear DNA) in the same way, given
appropriate primer design. Lee et al (see: Blood, Vol. 93 No. 4
pp1145-1156, 1999) described primers and methods that are sufficient to produce
the required DNA for subsequent incorporation into erythrocyte precursor
nuclear DNA of genes encoding the CD4 receptor.
Once amplified, the
CD4 DNA fragment is cloned downstream of a promoter which is active during
erythroid development of the CD34+ cells and therefore results in expression of
CD4 on the surface of erythrocyte derived from the transfected CD34+ precursor
cells.
The CD4 clone may be
introduced into the CD34+ stem cells using any methods which exist for the
incorporation of exogenous DNA transfection.
Such methods include calcium phosphate / DNA method, DNA-ligand methods,
electroporation, glass-bead, gene guns (Proc Natl Acad Sci USA 87 : 9568-72),
and cationic lipids (Proc Natl Acad Sci USA 87 : 3655-59).
Electroporation has
been shown to have a 2.7% efficiency when used in transferring genes into human
haematopoietic stem cell precursor cells (for methods see: Toneguzzo, Keating,
Proc. Nat. Acad. Sci. USA 83: 3496-99).
Other more recently developed methods may have higher efficiency but
this has no real significance on the engineering protocol, which necessarily
incorporates processes to select only those cells which have undergone
successful gene transfer.
Individual candidate
CD4 transfected CD 34+ precursor cells are first proliferated in an appropriate
atmosphere, temperature regime and growth medium containing human stem cell
factor (see Muta, et al Blood, 86, No
2, p572-580 1995), and other colony-stimulating factors such as interleukin-1
and interleukin-3, see Lemoli et al,
Exper. Hematol. 20: 569-575, 1992) in small batches. These small batches may be stored frozen for subsequent bulk
culture.
This also enables some
proliferated transfected cells from known small batches derived from the aforementioned
individual cells, to themselves be proliferated, terminally differentiated (a
process triggered by, for example, granulocyte/macrophage colony stimulating
factor, stem cell factor and a glycoprotein hormone, erythropoietin, see Wu et al, cell, Vol 83, 59-67, 1995) and
analysed for terminally differentiated erythrocytes.
Erythrocytes which
express the CD4 receptor are then matched back to the specific batch of
precursor cells. These precursor cells
are then used as a source of CD4+ erythrocytes, and (see sieving, below) can be
maintained in culture and proliferated by interleukin-3 mediated self-renewal
for many generations (see Lewis, et al,
Cytokine, Vol 10, No 1, p49-54 Jan 1998).
From this culture, occasional aliquots of these precursors can be taken
for bulk-culture and subsequent differentiation into receptor-expressing
erythrocytes.
Batches of precursor
cells with progeny not exhibiting desired traits or failing to exhibit traits
typical of fully functional erythrocytes (for example, a cell which doesn’t
exhibit correct haemoglobin behaviour) are discarded.
It is envisaged that
progeny of cells which have successfully taken up the gene expressing the CD4
receptor can be expected to express the receptor on their surface. These cells can thus be expected to adhere
to a surface plated with (immunoglobulin or the like) antibody specific to the
receptor, when washed over such a surface.
Cells failing to take up and express the receptor would not bind to such
a surface. Adherent cells would, by
their adherence, designate the small-batch from which they originated as
suitable for subsequent bulk culture, proliferation and differentiation.
If desired, the
location of newly inserted sequence can be determined using comparison against
online human genome sequences. For
example, sequences flanking the newly inserted DNA can be sequenced and used to
search the human genome sequence to determine the position of the inserted DNA. If the inserted DNA is found to be located
in a sequence of endogenous non-coding DNA, then the clone is less likely to
have its usual behaviour disrupted at the DNA level.
In order to make enough CD4 receptor expressing erythrocytes to
effectively mop-up viral particles, a considerable number of such erythrocytes
is needed in the bloodstream, given that the daily turnover is in the order of
2x1011/day and half life is approximately 120 days. This therefore requires bulk culture
proliferation and subsequent differentiation.
The method of Fibach and Rachmilewitz (see: Stem cells, 1993, 11
(suppl.1) 6-41) may be used for erythrocyte production on a large scale.
Cells selected using the methods above as expressing the CD4 receptor
can be proliferated into large quantities, then signalled to commence
differentiation into erythrocytes using the growth factors indicated above.
In order to mount a successful infection, HIV need only successfully
infect enough susceptible cells as is required to provide slightly more than a
maintenance population of HIV. Thus, in
order to treat HIV infection in the patient, a concentration of CD4+ red blood
cells is maintained in the bloodstream such that the maintenance population of
virus gradually decreases to levels undetectable by sensitive assays such as
PCR. This concentration needs to be
experimentally determined and will vary from patient to patient. The engineered cells still perform their
usual task of oxygen transport and are not anticipated to exert any additional
effects outside of their usual roles.
However, this may require experimental confirmation prior to a full
scale treatment of each patient.
Problematic viral latency can be dealt with by immunosuppressive therapy
and other immune system compromising agents (starvation, or other means to
stimulate latently infected cells into viral production), at the same time as
infusion with CD4+ erythrocytes, which can be expected to enable latently
infected cells to become active and spill their load of virus particles such
that the particles are accessible to the CD4+ erythrocytes. This would typically be done at times of low
or no detectable viral load.
THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
1.
A device for
sequestering an infective virus from an environment, the device comprising
binding means for binding an infective virus in the environment to the device,
and container means for containing the infective virus within the device, the
binding means and container means being arranged for permitting infective virus
bound to the device to be transferred to the container means, for containment
of the infective virus within the device.
2.
The device of
claim 1 wherein the binding means is a receptor for the virus.
3.
The device of
claim 1 or 2 wherein the binding means is a receptor for HIV.
4.
The device of any
one of claims 1 to 3 wherein the binding means is CD4.
5.
The device of any
one of claims 1 to 4 wherein the container means does not comprise a
nucleus.
6.
The device of any
one of claims 1 to 5 wherein the container means is an erythroid cell.
7.
The device of any
one of claims 1 to 6 wherein the container means is an erythrocyte.
8.
The device of any
one of claims 1 to 6 wherein the container means is a platelet.
9.
The device of any
one of claims 1 to 5 wherein the container means is a liposome.
10.
The device of any
one of claims 1 to 4 wherein the container means is a lymphoid cell.
11.
The device of
claim 6 wherein the erythroid cell is derived from a stem cell containing a
nucleic acid encoding a binding means for binding a virus, the nucleic acid
being capable of expressing the binding means during terminal differentiation
of stem cell progeny into erythroid cells.
12.
The device of
claim 6 wherein the erythroid cell is derived from a haematopoietic stem cell
containing a nucleic acid encoding a binding means for binding a virus, the
nucleic acid being capable of being expressed during terminal differentiation
of the haematopoietic stem cell progeny into erythroid cells.
13.
The device of
claim 11 or 12 wherein the nucleic acid comprises a gene encoding a binding
means for binding a virus operably linked to a promoter that is active during
development of the haematopoietic stem cell into an erythroid cell.
14.
The device of
claim 13 wherein the promoter is selected from the group comprising g-globin gene, e-globin
gene, b-globin gene,
GATA-1 gene, glycophorin B gene, ferrochelatase gene, porphobilimogen deaminase
gene, 5-aminolevulinate synthase gene, Kel gene, syndecan-1 gene, ABO blood
group genes, RH factor genes, MRS antigen genes, Duffy antigen genes and Kell
antigen genes.
15.
A method for
sequestering a virus from an environment comprising contacting the virus with a
binding means of a device of any one of claims 1 to 14, to permit the virus to
bind to the device wherein the virus is contacted with the binding means in
conditions for permitting virus bound to the binding means to be transferred to
the container means for containment of the virus within the device.
16.
The method of
claim 15 comprising the further step of removing the device from the
environment.
17.
The method of
claim 15 wherein the environment is blood, lymph, cerebrospinal fluid or a
fractioned product of blood, lymph or cerebrospinal fluid or tissue culture
medium, buffered solution, or the like.
18.
A method of
treating or ameliorating a viral infection in a mammal, the method comprising
administering a device of any one of claims 1 to 14 to the mammal.
19.
A method of
treating or ameliorating a HIV infection in a mammal, the method comprising
administering a device of claim 3 or 4 to the mammal.