U.S. patent application number 12/425952 was filed with the patent office on 2009-09-03 for virus strains.
This patent application is currently assigned to BIOVEX LIMITED. Invention is credited to Robert S. Coffin.
Application Number | 20090220460 12/425952 |
Document ID | / |
Family ID | 27447756 |
Filed Date | 2009-09-03 |
United States Patent
Application |
20090220460 |
Kind Code |
A1 |
Coffin; Robert S. |
September 3, 2009 |
VIRUS STRAINS
Abstract
The present invention relates to clinical herpes simplex virus
(HSV) isolates with improved oncolytic capabilities as compared to
reference laboratory HSV strains. The present invention also
relates to methods of producing HSV strains of the invention and to
pharmaceutical compositions comprising a HSV strain of the
invention.
Inventors: |
Coffin; Robert S.; (London,
GB) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY, SUITE 5100
HOUSTON
TX
77010-3095
US
|
Assignee: |
BIOVEX LIMITED
Oxford
GB
|
Family ID: |
27447756 |
Appl. No.: |
12/425952 |
Filed: |
April 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11372613 |
Mar 10, 2006 |
7537924 |
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12425952 |
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10181692 |
Nov 12, 2002 |
7063835 |
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PCT/GB01/00229 |
Jan 22, 2001 |
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11372613 |
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Current U.S.
Class: |
424/93.6 ;
435/235.1; 435/5 |
Current CPC
Class: |
A61P 25/02 20180101;
A61P 37/04 20180101; A61K 2039/55522 20130101; A61P 25/00 20180101;
C12N 2710/16033 20130101; A61P 35/04 20180101; A61P 31/00 20180101;
A61P 35/00 20180101; C12N 15/869 20130101; C12N 7/00 20130101; A61K
48/00 20130101; C12N 2710/16643 20130101; A61P 25/28 20180101; A61K
35/763 20130101; A61P 43/00 20180101; A61K 2039/55516 20130101;
C12N 15/86 20130101; C12N 2710/16021 20130101; C12N 2710/16632
20130101 |
Class at
Publication: |
424/93.6 ;
435/235.1; 435/5 |
International
Class: |
A61K 35/76 20060101
A61K035/76; C12N 7/00 20060101 C12N007/00; C12Q 1/70 20060101
C12Q001/70 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 21, 2000 |
GB |
0001475.3 |
Feb 8, 2000 |
GB |
0002854.8 |
Jan 5, 2001 |
GB |
0100288.0 |
Jan 6, 2001 |
GB |
0100430.8 |
Claims
1-61. (canceled)
62. A modified, oncolytic herpes simplex virus (HSV) strain
comprising: a modified, oncolytic herpes simplex virus (HSV) strain
wherein said HSV strain is a clinical isolate from a recurrent cold
sore and has a greater ability than a reference laboratory HSV
strain modified in the same manner as the clinical isolate to
replicate in or kill tumor cells, and wherein said reference
laboratory HSV strain is selected from the group consisting of HSV1
strain 17+, HSV1 strain F and HSV1 strain KOS.
63. An HSV strain according to claim 62, which is modified such
that it lacks one or more genes selected from the group consisting
of a functional ICP34.5-encoding gene, a functional ICP6-encoding
gene, a functional glycoprotein H-encoding gene and a functional
thymidine kinase-encoding gene.
64. An HSV strain according to claim 63, which lacks a functional
ICP34.5-encoding gene.
65. An HSV strain according to claim 64, which further lacks a
functional ICP47 gene.
66. An HSV strain according to claim 62, which comprises a
heterologous gene.
67. An HSV strain according to claim 66, wherein said heterologous
gene is a gene capable of modifying immune responses.
68. An HSV strain according to claim 67, wherein said gene capable
of modifying immune responses is selected from the group consisting
of a gene that encodes an immune stimulatory polypeptide, a gene
product capable of modifying immune responses, a prodrug activator,
a tumor suppressor or a pro-apoptotic gene product.
69. An HSV strain according to claim 68, wherein the immune
stimulatory polypeptide is selected from the group consisting of
granulocyte macrophage colony-stimulating factor (GMCSF), a
cytokine, a chemokine, RANTES, B7.1, B7.2 or IL12; wherein the
prodrug activator is nitroreductase or cytochrome p45; or wherein
the tumor suppressor is p53.
70. An HSV1 strain according to claim 62, which is modified such
that it lacks a functional ICP34.5 gene, and optionally a
functional ICP47 gene; and optionally further comprises a
heterologous gene encoding GMCSF.
71. A pharmaceutical composition comprising a HSV strain according
to claim 62 and a pharmaceutically acceptable carrier or
diluent.
72. A method of determining whether a gene enhances the anti-tumor
effects of a herpes simplex virus (HSV) strain comprising: (i)
providing an HSV according to claim 62; (ii) inserting a gene to be
evaluated into said HSV strain to create a modified, oncolytic HSV
strain; and (iii) assessing the ability of said modified, oncolytic
HSV strain to replicate in or kill tumor cells compared to the
ability of the precursor strain provided in step (i).
73. A method of determining the suitability of a herpes simplex
virus (HSV) strain for use in the production of a modified,
oncolytic HSV strain, wherein said HSV strain is a clinical
isolate, said method comprising: (i) providing an HSV virus strain
to be evaluated; (ii) assessing the ability of said HSV to
replicate in or kill tumor cells; and, selecting for modification
HSV strains which have a greater ability than one or more reference
strains selected from the group consisting of HSV1 strain 17+, HSV1
strain F and HSV1 strain KOS, to replicate in or kill tumor
cells.
74. A method of producing a modified oncolytic herpes simplex virus
(HSV) strain, wherein said HSV strain is a clinical isolate, said
method comprising: (i) providing an HSV strain selected by a method
according to claim 73; (ii) modifying said strain by rendering
non-functional one or more genes selected from the genes encoding
ICP34.5, ICP6 and thymidine kinase to create a modified, oncolytic
non-laboratory strain; and (iii) assessing the ability of said
modified, oncolytic non-laboratory strain to kill tumor cells.
75. A method according to claim 74 wherein the tumor cell-killing
capacity of said modified, oncolytic HSV strain is measured by
comparison to the tumor cell-killing capacity of a reference
laboratory strain with the same gene or genes rendered
non-functional, which reference laboratory strain is selected from
HSV1 strain 17+, HSV1 strain F and HSV1 strain KOS.
76. A method according to claim 73, wherein said HSV strain is
isolated from a host.
77. A method according to claim 73, wherein said HSV strain is a
clinical isolate from a recurrent cold sore.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to non-laboratory virus
strains, for example of herpes viruses such as HSV, with improved
oncolytic and/or gene delivery capabilities as compared to
laboratory virus strains.
BACKGROUND OF THE INVENTION
[0002] Viruses have been suggested or demonstrated to have utility
in a variety of applications in biotechnology and medicine on many
occasions. Each is due to the unique ability of viruses to enter
cells at high efficiency. This is followed in such applications by
either virus gene expression and replication and/or expression of
an inserted heterologous gene. Thus viruses can either deliver and
express genes in cells (either viral or other genes) which may be
useful in for example gene therapy or the development of vaccines,
or they may be useful in selectively killing cells by lytic
replication or the action of a delivered gene in for example
cancer.
[0003] Herpes simplex virus (HSV) has been suggested to be of use
both as a gene delivery vector in the nervous system and elsewhere
and for the oncolytic treatment of cancer. In both applications the
virus must however be disabled such that it is no longer pathogenic
but such that it can still enter cells and perform the desired
function. Thus for non-toxic gene delivery to target cells using
HSV it has become apparent that in most cases immediate early gene
expression must be prevented/minimised from the virus. For the
oncolytic treatment of cancer, which may also include the delivery
of gene(s) enhancing the therapeutic effect, a number of mutations
to HSV have been identified which still allow the virus to
replicate in culture or in actively dividing cells in vivo (e.g. in
tumours), but which prevent significant replication in normal
tissue. Such mutations include disruption of the genes encoding
ICP34.5, ICP6 and thymidine kinase. Of these, viruses with
mutations to ICP34.5, or ICP34.5 together with mutations of e.g.
ICP6 have so far shown the most favourable safety profile. Viruses
deleted for only ICP34.5 have been shown to replicate in many
tumour cell types in vitro and to selectively replicate in
artificially induced brain tumours in mice while sparing
surrounding tissue. Early stage clinical trials have also shown
their safety in man.
[0004] However, while promise has been shown for various viruses
including HSV for gene delivery/therapy or for the oncolytic
treatment of cancer, the majority of this work has used virus
strains which have been maintained in tissue culture cells for many
years. In applications where the virus merely needs enter cells to
deliver genes this may not prove problematical as maintenance in
cell culture also requires the virus to enter cells, albeit often
cells of a different type or species in comparison to the likely
target cells for a vector. However, in applications where other
properties are required, the use of laboratory virus strains may
not allow the full potential of a virus in a particular application
to be utilised.
[0005] HSV has the unique ability amongst viruses currently under
development as vectors in that it has naturally evolved to infect
and remain latent in neurons. HSV has also evolved to be highly
efficiently transported along nerves from the site of infection,
usually at the periphery, to the neuronal cell body, usually in the
spinal ganglia. Such capabilities are not required in cell culture
and as such capabilities require specific evolved properties of
HSV, further adaption to growth in culture may have resulted in
optimally efficient axonal transport capabilities to have been
lost. HSV vectors for gene delivery to the central or peripheral
nervous system are likely to show maximum effectiveness if axonal
transport properties have been retained at maximum efficiency.
Here, inoculation at a peripheral site would then allow maximally
efficient gene delivery to peripheral neuron cell bodies, and
inoculation in the brain would allow maximally efficient gene
delivery to multiple connected sites. Current vectors based on
laboratory strains of HSV may not allow this to occur at the
maximum efficiency possible. Indeed, because of HSV's high capacity
to be transported along nerves, there is potentially a particularly
large discrepancy between the properties which it is desired to
conserve and those likely to be retained in culture.
[0006] HSV and other viruses such as adeno- or rheovirus also have
potential utility in the oncolytic treatment of cancer. However,
again viruses under development for such purposes have previously
been extensively maintained in culture. As the oncolytic treatment
of cancer requires active replication in often relatively slowly
growing human tumour cells, it would be anticipated that adaptation
of laboratory virus strains to growth in particular cultured cells
may have reduced the efficiency with which such lytic replication
in human tumour cells, or infection of human tumour cells, could
optimally occur.
SUMMARY OF THE INVENTION
[0007] The present invention provides the opportunity to develop
viruses with improved in vivo capabilities of gene transfer and/or
lytic destruction of tumour cells. Here, virus strains are
constructed appropriate for these purposes based on recent clinical
isolates of the appropriate virus rather than the serially passaged
laboratory strains which have previously been used. The present
invention therefore has the potential to provide viruses with
improved capabilities of infecting human cells in vivo, improved
replicative/lytic capability in such cells, and (in the case of
HSV) improved abilities of trafficking along nerves from the
inoculation site to the neuronal cell body. The invention is
exemplified using HSV but is equally applicable for other viruses
currently under development as vectors and/or for the oncolytic
destruction of cancer cells.
[0008] We have shown that two clinical isolates of HSV1 (strains
JS1 and BL1) have enhanced replication in some human tumour cell
lines as compared to HSV1 strain 17+ (a standard laboratory
strain).
[0009] We have deleted ICP34.5 from the clinical isolate JS1 strain
and again compared replicative potential in human tumour cell types
in comparison to HSV1 strain 17+ (a standard laboratory strain) in
which ICP34.5 was also deleted. This strain (JS1/ICP34.5-) is a
modified strain derived from a clinical isolate, and is thus a
modified non-laboratory strain of the invention.
[0010] JS1 with ICP34.5 deleted showed enhanced growth in some
human tumour cells tested as compared to HSV1 ICP34.5 deleted
strain 17+, i.e. a laboratory strain virus with the same
modification. However, as compared to the laboratory strain derived
from strain 17+, cell killing capabilities were enhanced with the
JS1/ICP34.5- virus in all tumour cell lines tested.
[0011] Thus, the use of non-laboratory virus strains can be seen to
enhance the anti-tumour capabilities of such viruses and was
evident in all the tumour cell lines tested so far. This will have
applicability for cancer treatment in human patients.
[0012] Further enhanced activity may also be anticipated if these
viruses are then used to deliver genes with anti-tumour activity.
Such genes include those encoding pro-drug activators, tumour
suppressor or pro-apoptotic factors, or immune stimulatory
proteins.
[0013] For this purpose, we have produced an ICP34.5 deleted
clinical isolate of HSV1 which expresses human GMCSF. This virus is
designed to enhance anti-tumour immune responses following
intra-tumoral injection.
[0014] The invention also provides viruses of the invention which
carry a heterologous gene/genes. The term heterologous gene is
intended to embrace any gene not found in the viral genome. The
heterologous gene may be any allelic variant of a wild-type gene,
or it may be a mutant gene. Heterologous genes are preferably
operably linked to a control sequence permitting expression of said
heterologous gene in a cell in vivo. Viruses of the invention may
thus be used to deliver a heterologous gene/genes to a cell in vivo
where it will be expressed. For oncolytic virus therapy, such genes
typically encode proteins capable of enhancing the tumour
destroying properties of the virus. These genes may encode proteins
which are themselves cytotoxic, are pro-drug activating, or which
may be capable of stimulating/enhancing an anti-tumour immune
response. For gene delivery to the peripheral nervous system using
HSV, the heterologous gene/genes may encode a polypeptide capable
of modifying responses to painful stimuli or reducing chronic pain,
for example a protein capable sequestering e.g. nerve growth
factor, other pain modulating neurotrophic factor or neurotrophic
factor-like molecules, or substance P or other neuropeptides. The
heterologous gene/genes may also encode a polypeptide capable of
stimulating the re-growth of damage nerves or preventing the
further degeneration of nerves in degenerative conditions. In the
central nervous system, heterologous genes may include those
potentially beneficial in neurodegenerative disease such as
Parkinson's disease or Alzheimer's disease and might typically
include genes encoding neurotrophic factors and/or enzymes capable
of enhancing the activity of remaining cells in such diseases. In
all cases, single or multiple heterologous genes may be carried by
a single virus.
[0015] Accordingly the invention provides:
[0016] Use of a modified, oncolytic, non-laboratory virus strain in
the manufacture of a medicament for the oncolytic treatment of
cancer;
[0017] Use of a modified, replication incompetent, non-laboratory
virus strain comprising a heterologous gene in the manufacture of a
medicament for the delivery of said gene to a subject;
[0018] A method of determining whether a gene has an effect on a
phenotype associated with a peripheral nervous system disorder or
on a cell of the peripheral nervous system which is relevant to a
peripheral nervous system disorder, which method comprises: [0019]
(i) inoculating a replication incompetent herpes virus of the
invention comprising a heterologous gene into a peripheral nerve;
and [0020] (ii) monitoring a phenotype of said disorder or an
effect of expression of said gene on said cell to determine thereby
whether said gene has an effect relevant to said disorder;
[0021] A method of determining whether a gene has an effect on a
phenotype associated with a central nervous system disorder or on a
cell of the central nervous system which is relevant to a central
nervous system disorder, which method comprises: [0022] (i)
inoculating into a cell of the central nervous system with a
replication incompetent herpes virus of the invention; and [0023]
(ii) monitoring a phenotype of said disorder or an effect of
expression of said gene on said cell to determine thereby whether
said gene has an effect on said cell or said phenotype;
[0024] A method of determining whether a gene encodes an antigen
associated with a pathogenic infection or cancer, which method
comprises infecting a dendritic cell or a macrophage with a
replication incompetent virus of the invention comprising a
heterologous gene encoding an antigen and monitoring antigen
presentation of the polypeptide product of said gene, or an effect
of expression of said gene, or a phenotype of said pathogenic
infection or cancer to determine thereby whether said gene encodes
an antigen associated with said infection or cancer and which
itself has therapeutic potential or is a target for therapeutic
intervention;
[0025] A method of determining the suitability of a non-laboratory
virus strain for modification into a modified strain as defined
herein, comprising: [0026] (i) optionally, isolating a
non-laboratory virus strain from a host; [0027] (ii) providing said
non-laboratory virus strain; [0028] (iii) assessing the properties
of the virus in respect of one or more desirable characteristics;
and, optionally, [0029] (iv) selecting for modification virus
strains with desirable properties;
[0030] A method of determining the suitability of a non-laboratory
virus strain for modification into a modified oncolytic strain of
the invention, comprising: [0031] (i) optionally, isolating a
non-laboratory virus strain from a host; [0032] (ii) assessing the
growth of the virus in one or more types of tumour cell; and
optionally [0033] (iii) selecting for modification virus strains
with a high growth rate or cell killing capability;
[0034] A method of determining the suitability of a non-laboratory
virus strain for modification into a modified oncolytic strain,
comprising: [0035] (i) providing a non-laboratory virus strain,
optionally one selected by a method as just defined; [0036] (ii)
modifying said strain such that it becomes oncolytic; and [0037]
(iii) assessing the ability of said modified, oncolytic
non-laboratory strain to kill tumour cells; and optionally [0038]
(iv) selecting strains that exhibit high tumour cell-killing
capacity for further modifications; and optionally [0039] (v)
carrying out further modifications;
[0040] A method of determining whether a gene enhances the
anti-tumour effects of a virus comprising: [0041] (i) providing a
modified, oncolytic non-laboratory strain of the invention; [0042]
(ii) inserting said gene into said virus as a heterologous gene;
and [0043] (iii) assessing the ability of said modified, oncolytic
non-laboratory strain to kill tumour cells compared to the ability
of the precursor strain provided in step (i);
[0044] A method of producing a modified, oncolytic, non-laboratory
virus strain comprising: [0045] (i) isolating from a host a
non-laboratory strain of a virus; [0046] (ii) optionally
determining its suitability for modification as defined above; and
[0047] (iii) modifying it to render it oncolytic, and optionally
[0048] (iv) carrying out further modifications;
[0049] A method of producing a modified non-laboratory virus strain
comprising: [0050] (i) providing a non-laboratory strain of a
virus; [0051] (ii) modifying it to render it replication
incompetent and, [0052] (iii) inserting a heterologous gene;
[0053] A modified, oncolytic, non-laboratory virus strain as
defined herein;
[0054] A modified non-laboratory virus strain comprising a
heterologous gene, as defined herein
[0055] Use of a gene identified as having an effect on a phenotype
associated with a peripheral nervous system disorder or on a cell
of the peripheral nervous system which is relevant to a peripheral
nervous system disorder by a method as defined above, or of a gene
product encoded by said gene in the manufacture of a medicament for
the treatment of a peripheral nervous system disorder;
[0056] Use of a gene identified as having an effect on a phenotype
associated with a central nervous system disorder or on a cell of
the central nervous system which is relevant to a central nervous
system disorder by a method as defined above, or of a gene product
encoded by said gene in the manufacture of a medicament for the
treatment of a central nervous system disorder;
[0057] Use of a gene identified as encoding an antigen associated
with a pathogenic infection or cancer by a method as defined above,
or of an antigen encoded by said gene in the manufacture of a
medicament for the treatment or prevention of said infection or
cancer;
[0058] A non-laboratory virus strain identified by, or produced in
the course of, a method of the invention;
[0059] Use of a gene identified as enhancing the anti-tumour
effects of a virus by a method as defined above, or of a gene
product encoded by said gene, in the manufacture of a medicament
for the treatment or prevention of cancer;
[0060] A modified non-laboratory virus strain obtained or
obtainable by a method of the invention;
[0061] HSV1 strain JS1 as deposited at the European Collection of
Cell Cultures (ECACC) under provisional accession number 01010209,
or an HSV1 strain derived therefrom, a pharmaceutical composition
comprising such a virus; such a virus for use in the treatment of
the human or animal body;
[0062] A method of treating a tumour in an individual in need
thereof by administering to said individual an effective amount of
an oncolytic virus of the invention;
[0063] A method of delivering a gene to an individual in need
thereof by administering to said individual an effective amount of
a non-oncolytic virus of the invention; and
[0064] A method of treating or preventing a central peripheral
nervous system disorder by administering to a peripheral nerve of
an individual in need thereof an effective amount of a neurotrophic
virus of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1. Viruses
[0066] From top to bottom, diagrams show: laboratory HSV1 strain
17+, clinical strain BL1, clinical strain JS1, 17+/ICP34.5-,
JS1/ICP34.5-, JS1/ICP34.5-/ICP47-/hGMCSF.
[0067] FIG. 2. Clinical isolates show enhanced growth in tumour
cells [0068] (1) Growth of 17+, BL1 and JS1. Left hand diagram: U87
cells. Right-hand diagram: LNCaP cells. [0069] (2) Growth of
ICP34.5-17+ and JS1 on tumour cells. Left-hand diagram: LNCaP
cells. Right-hand diagram: MDA-MB-231 cells. [0070] (3) JS1/34.5-
does not grow on cells non-permissive for HSV ICP34.5 mutants.
Left-hand diagram: 3T6 cells-17+, JS1. Right-hand diagram: 3T6
cells-17+, JS1 ICP34.5-.
[0071] FIG. 3. An ICP34.5 deleted HSV clinical isolate shows
enhanced lysis in all tumour cells tested
[0072] Tumour cell lines were either mock infected, infected with
HSV1 strain 17+/34.5-, or infected with HSV1 strain JS1/34.5- at
the indicated MOI and stained with crystal violet at time points
after infection to allow visualisation of cells. Each block of
photographs relates to a cell type. From top to bottom, these are
HT29 colorectal adenocarcinoma, LNCaP.FGC prostate adenocarcinoma,
MDA-MB-231 breast adenocarcinoma, SK-MEL-28 malignant melanoma and
U-87 MG glioblastoma astrocytoma. Left-hand blocks relate to
results for HSV1 strain 17+/34.5-. Right-hand blocks relate to
results for HSV1 strain JS1/34.5-. Central blocks represent mock
infected cells. Within each block, the top row represents a 24 hour
time-point, the second a 48 hour time-point and the third a 72 hour
time-point within each block, the left-hand column represents
MOI=0.2, the central column MOI=0.1 and the right-hand column
MOI=5.
DETAILED DESCRIPTION OF THE INVENTION
A. Viruses
Virus Strains of the Invention
[0073] The invention is applicable to viruses in general.
Preferably, a virus strain of the invention will be a strain of a
herpes virus, adenovirus, picornavirus, retrovirus or alphavirus.
More preferably, a virus strain of the invention will be a strain
of a herpes virus. Still more preferably, a virus strain of the
invention will be a strain of a herpes simplex virus (HSV),
typically a strain of HSV1 or HSV2.
[0074] When the virus of the invention is a herpes simplex virus,
the virus may be derived from, for example HSV1 or HSV2 strains, or
derivatives thereof, preferably HSV1. Derivatives include
inter-type recombinants containing DNA from HSV1 and HSV2 strains.
Such inter-type recombinants are described in the art, for example
in Thompson et al (1998) and Meignier et al (1988). Derivatives
preferably have at least 70% sequence homology to either the HSV1
or HSV2 genomes, more preferably at least 80%, even more preferably
at least 90 or 95%. More preferably, a derivative has at least 70%
sequence identity to either the HSV1 or HSV2 genome, more
preferably at least 80% identity, even more preferably at least
90%, 95% or 98% identity.
[0075] For example the UWGCG Package provides the BESTFIT program
which can be used to calculate homology (for example used on its
default settings) (Devereux et al. (1984) Nucleic Acids Research
12, p 387-395). The PILEUP and BLAST algorithms can be used to
calculate homology or line up sequences (typically on their default
settings), for example as described in Altschul (1993) J. Mol.
Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.
215:403-10.
[0076] Software for performing BLAST analyses is publicly available
through the National Centre for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pair (HSPs) by identifying short
words of length W in the query sequence that either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., 1990).
These initial neighborhood word hits act as seeds for initiating
searches to find HSPs containing them. The word hits are extended
in both directions along each sequence for as far as the cumulative
alignment score can be increased. Extensions for the word hits in
each direction are halted when: the cumulative alignment score
falls off by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the accumulation of
one or more negative-scoring residue alignments; or the end of
either sequence is reached. The BLAST algorithm parameters W, T and
X determine the sensitivity and speed of the alignment. The BLAST
program uses as defaults a word length (W) of 11, the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad.
Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10, M=5, N=4, and a comparison of both strands.
[0077] The BLAST algorithm performs a statistical analysis of the
similarity between two sequences; see e.g., Karlin and Altschul
(1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a sequence is considered
similar to another sequence if the smallest sum probability in
comparison of the first sequence to the second sequence is less
than about 1, preferably less than about 0.1, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0078] A derivative may have the sequence of a HSV1 or HSV2 genome
modified by nucleotide substitutions, for example from 1, 2 or 3 to
10, 25, 50 or 100 substitutions. The HSV1 or HSV2 genome may
alternatively or additionally be modified by one or more insertions
and/or deletions and/or by an extension at either or both ends.
Properties of Virus Strain of the Invention
[0079] Virus strains of the invention are "non-laboratory" strains.
They can also be referred to as "clinical" strains. A person of
skill in the art will readily be able to distinguish between a
laboratory strain and a non-laboratory, or clinical, one. Further
guidance on the properties likely to be exhibited by virus strains
is given below.
[0080] The key distinction between a laboratory and non-laboratory
strain is that laboratory strains currently in common use have been
maintained for long periods, many years in some cases, in culture.
The culture of viruses such as HSV involves a technique known as
serial passage. To grow and maintain viruses, suitable cells are
infected with the virus, the virus replicates within the cell and
the virus is then harvested; fresh cells are then re-infected. this
process constitutes one cycle of serial passage. Each such cycle
may take, for example, a few days in the case of HSV. As discussed
above, such serial passaging may lead to changes in the properties
of the virus strain, in that selection takes places for properties
that favour growth in culture (e.g. rapid replication), as opposed
to properties useful for practical applications, e.g. maintenance
of the capacity to travel along axons in the case of HSV.
[0081] Virus strains of the invention are non-laboratory strains in
that they are derived from strains recently isolated from infected
individuals. Strains of the invention are modified compared to the
original clinical isolates, and may have spent a time in culture,
but any time spent in culture will be comparatively short. Strains
of the invention are prepared in such a manner as to retain
substantially the desirable properties of the original clinical
isolates from which they are derived.
[0082] A virus of the invention is capable of efficiently infecting
target human cells. Such a virus is recently isolated from an
infected individual and then screened for the desired ability of
enhanced replication in tumour and/or other cells in vitro and/or
in vivo in comparison to standard laboratory strains, or (in the
case of neurotrophic viruses such as HSV) for an enhanced ability
to traffic along nerves as compared to standard laboratory strains
using an in vivo model. Such viruses with improved properties as
compared to laboratory virus strains are viruses of the invention.
Identified viruses with such desired improved properties can then
be engineered such that they can selectively kill tumour cells by
the mutation of appropriate gene(s), or mutated such that they can
deliver a gene(s) to target tissue without toxic effect in
non-oncolytic applications. These modified viruses are also viruses
of the invention. Alternatively, virus strains may be isolated from
an infected individual and mutations anticipated to be appropriate
for oncolytic therapy and/or gene delivery made. These modified
viruses are then screened for the desired improved properties as
compared to laboratory strains, viruses with such improved
properties providing further viruses of the invention.
[0083] Further guidance on the likely properties of the virus
strains of the invention is provided as follows.
[0084] Preferably, a virus strain of the invention has undergone
three years or less in culture since isolation of its unmodified
clinical precursor strain from its host. More preferably, a strain
of the invention has undergone one year or less in culture, for
example nine months or less, six months or less, three months or
less, or two months or less, one month or less, two weeks or less,
or one week or less. By these definitions of time in culture, is
meant time actually spent in culture. Thus, for example, it is a
common practice to freeze virus strains in order to preserve them.
Evidently, preserving by freezing or in an equivalent manner does
not qualify as maintaining the strain in culture. Thus, time spent
frozen or otherwise preserved is not included in the above
definitions of time spent in culture. Time spent in culture is
typically time actually spent undergoing serial passage, i.e. time
during which selection for undesirable characteristics can
occur.
[0085] Preferably, a virus strain of the invention has undergone
1,000 or less cycles of serial passage since isolation of its
unmodified clinical precursor strain from its host. More
preferably, it has undergone 500 or less, 100 or less, 90 or less,
80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or
less, 20 or less, or 10 or less such cycles.
[0086] Preferably, a virus of the invention has a greater ability,
as measured by standard statistical tests, than a reference
laboratory strain with the equivalent modifications to perform
certain functions useful in the application at hand. For example,
in the case of an oncolytic virus for tumour treatment, a virus
strain of the invention will preferably have a greater ability than
a reference laboratory strain with equivalent modifications to
infect or replicate any tumour cell, to kill tumour cells or to
spread between cells in tissue. More preferably, such greater
ability is a statistically significantly greater ability. For
example, according to the invention, a may have up to 1.1 fold, 1.2
fold, 1.5 fold, 2 fold, 5 fold, 10 fold, 20 fold, 50 fold, or 100
fold the capacity of the reference strain in respect of the
property being tested.
[0087] Preferably, a virus of the invention has, i.e. retains,
substantially the ability of its unmodified clinical precursor
strain in respect of one or more of the properties characteristic
of usefulness in the application at hand. For example, in the case
of an oncolytic virus intended for the treatment of tumours, a
virus strain of the invention preferably has substantially the
ability of its unmodified clinical precursor strain to infect or
replicate a tumour cell, kill tumour cells or to spread between
cells in tissue.
[0088] Preferably, according to the invention, a virus retains
substantially the properties of its unmodified clinical precursor
strain if, in a quantitative test, it retains 75%, more preferably
80, 90, 95, 98, 99 or 100% of the capacity of the unmodified
clinical precursor strain in respect of the property being tested.
More preferably, in respect of the property being tested, any
differences between the unmodified clinical precursor strain and
the modified strain of the invention will not be statistically
significant.
[0089] Statistical analysis of the properties described herein may
be carried out by standard tests, for example, t-tests, ANOVA, or
Chi squared tests. Typically, statistical significance will be
measured to a level of p=0.05 (5%), more preferably p=0.01,
p=0.001, p=0.0001, p=0.000001.
Modifications
[0090] Viruses of the invention are typically modified as compared
to their precursor clinical strains. In particular, certain genes
will typically be rendered non-functional, and the viruses may also
comprise a heterologous gene(s). Typically, viruses of the
invention are attenuated.
[0091] Viral regions altered for the purposes described herein may
be either eliminated (completely or partly), or made
non-functional, or substituted by other sequences, in particular by
a heterologous gene sequence. One or more genes may be rendered
non-functional, and one or more heterologous genes inserted.
Oncolytic Viruses of the Invention
[0092] In one embodiment, viruses of the invention are modified,
oncolytic, non-laboratory viruses. These will be useful in the
oncolytic treatment of cancer. Such viruses infect and replicate in
tumour cells, subsequently killing the tumour cells. Thus, such
viruses are replication competent. Preferably, they are selectively
replication competent in tumour cells. This means that either they
replicate in tumour cells and not in non-tumour cells, or that they
replicate more effectively in tumour cells than in non-tumour
cells. Measurement of selective replication competence can be
carried out by the tests described herein for measurement of
replication and tumour cell-killing capacity, and also analysed by
the statistical techniques mentioned herein if desired.
[0093] An oncolytic virus of the invention preferably has a greater
ability than a reference laboratory strain with the same
modifications to infect or replicate in a tumour cell, to kill
tumour cells or to spread between cells in tissues. Preferably,
this ability is a statistically significantly greater ability as
described herein. The properties of the virus strain in respect of
tumour cells can be measured in any manner known in the art.
[0094] For example, the capacity of a virus to infect a tumour cell
can be quantified by measuring the dose of virus required to
measure a given percentage of cells, for example 50% or 80% of
cells. The capacity to replicate in a tumour cell can be measured
by growth measurements such as those carried out in the Examples
(see FIG. 2), e.g. by measuring virus growth in cells over a period
of 6, 12, 24, 36, 48 or 72 hours or longer.
[0095] The ability of a virus to kill tumour cells can be roughly
quantitated by eye (see FIG. 3) or more exactly quantitated by
counting the number of live cells that remain over time for a given
time point and MOI for given cell type. For example, comparisons
may be made over 24, 48 or 72 hours and using any known tumour cell
type. In particular, HT29 colorectal adenocarcinoma, LNCaP.FGC
prostate adenocarcinoma, MDA-MB-231 breast adenocarcinoma,
SK-MEL-28 malignant melanoma or U-87 MG glioblastoma astrocytoma
cells can be used. Any one of these cell types or any combination
of these cell types can be used, as may other tumour cell types. It
may be desirable to construct a standard panel of tumour cell types
for this purpose. To count the number of live cells remaining at a
given time point, the number of trypan blue-excluding cells (i.e.
live cells) can be counted. Quantitation may also be carried out by
fluorescence activated cell sorting (FACS) or MTT assay. Tumour
cell-killing ability may also be measured in vivo, e.g. by
measuring the reduction in tumour volume engendered by a particular
virus.
[0096] The ability of a virus to spread in tissue, especially solid
tissue, can be measured by determining the number of cells at sites
connected to the site of the original infection.
[0097] In order to determine the properties of viruses of the
invention, it will generally be desirable to use a standard
laboratory reference strain for comparison. Any suitable standard
laboratory reference strain may be used. In the case of HSV, it is
preferred to use one or more of HSV1 strain 17+, HSV1 strain F or
HSV1 strain KOS. The reference strain will typically have
equivalent modifications to the strain of the invention being
tested. Thus, the reference strain will typically have equivalent
modifications, such as gene deletions and/or heterologous gene
insertions. For example, in the case of an HSV strain, if the
ICP34.5 and ICP47-encoding genes have been rendered non-functional
in the virus of the invention, then they will also be rendered
non-functional in the reference strain. The modifications made to
the reference strain may be identical to those made to the strain
of the invention. By this, it is meant that the gene disruptions in
the reference strain will be in exactly equivalent positions to
those in the strain of the invention, e.g. deletions will be of the
same size and in the same place. Similarly, in these embodiments,
heterologous genes will be inserted in the same place, driven by
the same promoter, etc. However, it is not essential that identical
modifications be made. What is important is that the reference gene
has functionally equivalent modifications, e.g. that the same genes
are rendered non-functional and/or the same heterologous gene or
genes is inserted.
[0098] In an oncolytic virus of the invention, suitable
modifications will be made to the virus to confer oncolytic
activity, if it is not naturally present, and preferably to confer
selective oncolytic activity.
[0099] In the case of HSV, such mutations allowing selective
oncolytic activity include mutation to the genes encoding ICP34.5,
ICP6 and/or thymidine kinase (TK), preferably ICP34.5. Such
mutations to the ICP34.5-encoding gene in laboratory strains of HSV
are described in Chou et al 1990, Maclean et al 1991, although any
mutation in which ICP34.5 is non-functional may be used.
[0100] Accordingly, in an HSV strain, the viruses preferably
modified such that it lacks one or more of a functional
ICP34.5-encoding gene, a functional ICP6-encoding gene, a
functional glycoprotein H-encoding gene, a functional thymidine
kinase-encoding gene; or in a non-HSV strain, the virus lacks a
functional gene equivalent to one of said HSV genes.
[0101] More preferably, the virus lacks a functional
ICP34.5-encoding gene.
[0102] Other modifications may also be made. In particular, in the
case of HSV, the virus may be modified such that it lacks a
functional ICP47 gene. This is because ICP47 usually functions to
block antigen presentation in HSV-infected cells so its disruption
leads to a virus that does not confer on infected tumour cells
particular properties that might protect such HSV infected cells
from the host's immune system.
[0103] Viruses with any other genes deleted/mutated which provide
oncolytic properties (ie selective replication in tumours compared
to surrounding tissue) are also viruses of the invention as those
skilled in the art will recognise that the above list is not
exhaustive and identification of the function of other genes in any
of the viruses above may suggest the construction of new viruses
which are also viruses of the invention.
[0104] Heterologous gene(s) may also be inserted into such viruses
of the invention by techniques known in the art and/or described
herein. In an oncolytic virus, the heterologous gene will typically
be one that enhances the capacity of the virus to counteract
tumours. Any genes conferring on the virus anti-tumour properties
may thus be inserted. In particular, the heterologous gene may be a
gene capable of modifying immune response to the tumour cells in a
beneficial manner, especially an immune stimulatory polypeptide
such as CD40L, granulocyte macrophage-colony-stimulating factor
(GMCSF), another cytokine or chemokine (e.g. RANTES), B7.1 or B7.2
or IL 12. Alternatively, the heterologous gene may encode a
pro-drug activator, such as nitroreductase or cytochrome P450. In
this context, combined treatment of tumours with the pro-drug
activated by the pro-drug activator and a virus of the invention is
envisaged. Alternatively, the heterologous gene may encode a tumour
suppressor, such as p53.
Other Virus Strains of the Invention
[0105] In other embodiments, non-oncolytic viruses are desirable.
These may be replication incompetent viruses. Their function is
typically to deliver heterologous genes to an individual.
[0106] In particular, for use as a vector in non-oncolytic
applications, mutations may be made such that virus regulatory
immediate early gene expression is minimised. Thus, the genes
encoding ICP4, ICP27, ICP22 and/or ICP0 may be inactivated or
deleted either individually or in combination, or mutations in the
virion trans-O activator protein vmw65 included preventing/reducing
its trans-activating ability. In particularly preferred embodiments
for non-oncolytic applications, the genes encoding ICP27, ICP0 and
ICP4 are deleted (with or without additional deletion/inactivation
of ICP22 and/or ICP47), or ICP27 and ICP4 deleted with an
inactivating mutation in vmw65, or ICP4 deleted, again with an
inactivating mutation in vmw65. Examples of such viruses include
viruses reported by Samaniego et al 1998, Krisky et al 1998, or
Thomas et al 1999.
Neurotrophic Virus Strains
[0107] Neurotrophic viruses, and particularly herpes simplex
viruses such as HSV1 and HSV2 may be used according to the
invention in the delivery of heterologous genes to the nervous
system.
[0108] Viruses according to this embodiment of the invention
typically have greater ability than a reference laboratory strain
with equivalent modifications to infect a neuron, spread between
cells in nervous tissue or to be transported within an axon.
Capacity to infect neurons can be determined as set above as for
cells in general. Ability to be transported within an axon or to
spread between cells in nervous tissue can be determined by
measuring the infection of cells in the nervous system at sites
connected to the site of the original infection. Results may be
analysed statistically as described above.
[0109] In these embodiments, it is preferred that, in an HSV
strain, the virus is modified such that it lacks one, two, three or
all of a functional ICP27-encoding gene, a functional ICP4-encoding
gene, a functional ICP0-encoding gene, or a functional
ICP22-encoding gene; or, in an non-HSV strain, the virus lacks a
functional gene equivalent to one of said HSV genes; and/or, in an
HSV strain, the virus lacks a functional vmw65 gene due to a
mutation in said gene which abolishes its
transcriptional-activation activity; or in a non-HSV strain, the
virus lacks a functional gene equivalent to vmw65 due to a mutation
in said gene which abolishes its transcriptional-activation
activity.
[0110] Preferably, two or more of the genes encoding ICP27, ICP4,
ICP0 and ICP22 are rendered non-functional, more preferably three
and, still more preferably all four. Preferably, according to these
embodiments a virus of the invention lacks both a functional gene
encoding ICP4 and a functional gene encoding ICP27 and which has an
inactivating mutation in the gene encoding vmw65 abolishing its
transcriptional activation activity.
[0111] Such viruses may be used for the treatment of either
peripheral nervous system disorders or central nervous system
disorders. In the context of central nervous system disorders, it
is particularly preferred that at least two immediate early genes
selected from ICP0, ICP4, ICP22 and ICP27 are rendered
non-functional.
Immunotherapeutic Viruses
[0112] In immunotherapeutic applications, herpes viruses of the
invention are used to infect dendritic cells or other cells of the
immune system, for example macrophages. Normally, herpes virus
infection of dendritic cells reduces the capacity of the dendritic
cells to stimulate an immune response. Accordingly, viruses of the
invention are modified so that they are capable of efficiently
infecting dendritic cells without preventing the dendritic cells
from stimulating the immune system (WO00/08191). In such
applications, the virus of the invention typically comprises a
heterologous gene(s) which encodes an antigen gene product. This
may be an antigen associated with a pathogenic infection or cancer.
The gene is expressed in the dendritic cell and the gene produce
presented as an antigen on the dendritic cell's surface. This
stimulates an immune response to the antigen by activating T cells,
which seek out cells displaying that antigen on their surface, i.e.
tumours or pathogen cells or cells infected with a pathogen and
destroy them.
[0113] Preferably, in an HSV strain, said virus lacks a functional
UL43 gene, and/or a functional vhs gene, or, in a non-HSV strain,
lacks a functional equivalent of UL43 and/or vhs; and optionally,
in an HSV strain, lacks a functional vmw65 gene due to a mutation
in said gene which abolishes its transcriptional-activation
activity, or, in a non-HSV strain, lacks a functional gene
equivalent to vmw65 due to a mutation in said gene which abolishes
its transcriptional-activation activity; and optionally lacks at
least one functional immediate early gene selected from ICP0, ICP4,
ICP22 and ICP27.
[0114] In this context, it is preferred that a virus of the
invention has greater ability than a reference laboratory strain
with equivalent modifications to infect a dendritic cell or to
stimulate an immune response. Infection of dendritic cells can be
assessed as described above for cells in general. In particular, a
non-laboratory virus strain of the invention will typically infect
a greater percentage of dendritic cells than the reference
laboratory strain when the dose of the two strains is the same.
Statistical analysis can be conducted by the methods described
above.
B. Complementing Cell Lines
[0115] When the virus of the invention is a herpes simplex virus
which lacks a particular functional essential gene, for example a
gene encoding ICP4 or ICP27, the virus of the invention is
propagated on a cell line expressing that essential gene. For
example, when the virus lacks a functional ICP27 gene, the virus
may be propagated on V27 cells (Rice and Knipe, 1990), 2-2 cells
(Smith et al, 1992) or B130/2 cells (Howard et al, 1998). When the
virus lacks a functional ICP4 gene the virus may be propagated on a
cell line expressing ICP4, for example E5 cells (DeLuca et al,
1985). When the virus lacks a functional ICP4 gene and a functional
ICP27 gene the virus is propagated on a cell line expressing both
ICP4 and ICP27 (such as E26 cells; Samaniego et al, 1995), and when
the virus additionally lacks a functional vmw65 gene the virus may
be propagated on a cell line also containing a non-HSV homologue of
vmw65 (e.g. from equine herpes virus as in Thomas et al, 1999).
Mutations to vmw65 may also be partially compensated for by
inclusion of hexamethylene bisacetamide (HMBA) in the media used
for virus growth (MacFarlane et al, 1992).
[0116] ICP27-expressing cell lines can be produced by
co-transfecting mammalian cells, for example the Vero or BHK cells,
with a vector, preferably a plasmid vector, comprising a functional
HSV ICP27 gene capable of being expressed in said cells, and a
vector, preferably a plasmid vector, encoding a selectable marker,
for example neomycin resistance. Clones possessing the selectable
marker are then screened further to determine which clones also
express functional ICP27, for example on the basis of their ability
to support the growth of ICP27--HSV strains, using methods known to
those skilled in the art (for example, as described in Rice and
Knipe, 1990).
[0117] Cell lines which do not allow reversion of an ICP27-mutant
HSV strain to a strain with functional ICP27 are produced as
described above, ensuring that the vector comprising a functional
ICP27 gene does not contain sequences that overlap with (i.e. are
homologous to) sequences remaining in the ICP27-mutant virus.
[0118] Where HSV strains of the invention comprise inactivating
modifications in other essential genes, for example ICP4,
complementing cell lines will comprise a functional HSV gene which
complements the modified essential gene in the same manner as
described for ICP27. For example, in the case of HSV strains
comprising mutations in both ICP27 and ICP4, a cell line expressing
both ICP27 and ICP4 is used (such as described in Samaniego et al,
1995 or in Thomas et al, 1999). HSV strains expressing other
essential genes can be constructed in a similar manner to that
described for ICP27. Here again, if it is ensured there is no
sequence overlap between the remaining virus DNA and that inserted
into the cell line for virus growth, the possibility of reversion
of the virus to a less disabled form during growth will be
minimised.
C. Methods of Mutation
[0119] The various viral genes referred to may be rendered
functionally inactive by several techniques well known in the art.
For example, they may be rendered functionally inactive by
deletion(s), substitution(s) or insertion(s), preferably by
deletion. A deletion may remove a portion of the genes or the
entire gene. For example, deletion of only one nucleotide may be
made, resulting in a frame shift. However, preferably a larger
deletion is made, for example at least 25%, more preferably at
least 50% of the total coding and non-coding sequence (or
alternatively, in absolute terms, at least 10 nucleotides, more
preferably at least 100 nucleotides, most preferably at least 1000
nucleotides). It is particularly preferred to remove the entire
gene and some of the flanking sequences. An inserted sequence may
include one or more of the heterologous genes described below. In
the case of the vmw65 gene, the entire gene is not deleted since it
encodes an essential structural protein, but a small inactivating
mutation is made which abolishes the ability of vmw65 to activate
transcriptionally IE genes (e.g. as in Ace et al, 1989 or Smiley et
al, 1997).
[0120] Mutations are made in the herpes viruses by homologous
recombination methods well known to those skilled in the art. For
example, HSV genomic DNA is transfected together with a vector,
preferably a plasmid vector, comprising the mutated sequence
flanked by homologous HSV sequences. The mutated sequence may
comprise a deletion(s), insertion(s) or substitution(s), all of
which may be constructed by routine techniques. Insertions may
include selectable marker genes, for example lacZ or GFP, for
screening recombinant viruses by, for example .beta.-galactosidase
activity or fluorescence.
D. Heterologous Genes and Promoters
[0121] The viruses of the invention may be modified to carry a
heterologous gene/genes. The term "heterologous gene" encompasses
any gene. Although a heterologous gene is typically a gene not
present in the genome of a herpes virus, herpes gene/genes may be
used provided that the coding sequence is not operably linked to
the viral control sequences with which it is naturally associated.
The heterologous gene may be any allelic variant of a wild-type
gene, or it may be a mutant gene. The term "gene" is intended to
cover nucleic acid sequences which are capable of being at least
transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are
included within this definition. However, the present invention is
concerned with the expression of polypeptides rather than tRNA and
rRNA. Sequences encoding mRNA will optionally include some or all
of 5' and or 3' transcribed but untranslated flanking sequences
naturally, or otherwise, associated with the translated coding
sequence. It may optionally further include the associated
transcriptional control sequences normally associated with the
transcribed sequences, for example transcriptional stop signals,
polyadenylation sites and downstream enhancer elements.
[0122] The heterologous gene/genes may be inserted into the viral
genome by homologous recombination of HSV strains with, for example
plasmid vectors carrying the heterologous gene/genes flanked by HSV
sequences. The heterologous gene/genes may be introduced into a
suitable plasmid vector comprising herpes viral sequences using
cloning techniques well-known in the art. The heterologous
gene/genes may be inserted into the viral genome at any location
provided that the virus can still be propagated. It is preferred
that the heterologous gene/genes is inserted into an essential
gene. Heterologous genes may be inserted at multiple sites within
the virus genome.
[0123] The transcribed sequence of the heterologous gene/genes is
preferably operably linked to a control sequence permitting
expression of the heterologous gene/genes in mammalian cells,
preferably a tumour cell or a cell of the nervous system. The term
"operably linked" refers to a juxtaposition wherein the components
described are in a relationship permitting them to function in
their intended manner. A control sequence "operably linked" to a
coding sequence is ligated in such a way that expression of the
coding sequence is achieved under conditions compatible with the
control sequence.
[0124] The control sequence comprises a promoter allowing
expression of the heterologous gene/genes and a signal for
termination of transcription. The promoter is selected from
promoters which are functional in mammalian, preferably human,
cells of the nervous system or in tumours or in cells of the immune
system. The promoter/promoters may be derived from promoter
sequences of eukaryotic genes. For example, promoters may be
derived from the genome of a cell in which expression of the
heterologous gene is to occur, preferably a mammalian, preferably
human cell. With respect to eukaryotic promoters, they may be
promoters that function in a ubiquitous manner (such as promoters
of P-actin, tubulin) or, alternatively, a tissue-specific manner,
such as the neuron-specific enolase (NSE) promoter. They may also
be promoters that respond to specific stimuli, for example
promoters that bind steroid hormone receptors. Viral promoters may
also be used, for example the Moloney murine leukaemia virus long
terminal repeat (MMLV) LTR promoter or other retroviral promoters,
the human or mouse cytomegalovirus (CMV) IE promoter, or promoters
of herpes virus genes including those driving expression of the
latency associated transcripts.
[0125] Expression cassettes and other suitable constructs
comprising the heterologous gene/genes and control sequences can be
made using routine cloning techniques known to persons skilled in
the art (see, for example Sambrook et al, 1989, Molecular
Cloning--A laboratory manual: Cold Spring Harbor Press).
[0126] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the heterologous gene
can be regulated during the life-time of the cell. Inducible means
that the levels of expression obtained using the promoter can be
regulated. For example, in a preferred embodiment where more than
one heterologous gene is inserted into the HSV genome, one promoter
would comprise a promoter responsive to the tet repressor/VP16
transcriptional activator fusion protein previously reported
(Gossen and Bujard, 1992, Grossen et al, 1995), and driving the
heterologous gene the expression of which is to be regulated. The
second promoter would comprise a strong promoter (e.g. the CMV IE
promoter) driving the expression of the tet repressor/VP 16 fusion
protein. Thus, in this example, expression of the first
heterologous gene would depend on the presence or absence of
tetracycline.
[0127] Heterologous genes will typically encode polypeptides of
therapeutic use. For example, in the nervous system in
non-oncolytic applications, genes which may modulate pain,
stimulate nerve re-growth or prevent nerve degeneration. In
oncolytic applications, heterologous genes may encode proteins
which are themselves cytotoxic, encode pro-drug activating enzymes
or which are capable of stimulating or enhancing an anti-tumour
immune response.
[0128] Heterologous genes may also include marker genes (for
example, encoding .beta.-galactosidase or green fluorescent protein
or other fluorescent proteins) or genes whose products regulate the
expression of other genes (for example, transcriptional regulatory
factors including the tet repressor/vmw65 transcriptional activator
fusion protein described above).
[0129] Gene therapy and other therapeutic applications may well
require the administration of multiple genes. The expression of
multiple genes may be advantageous for the treatment of a variety
of conditions. Herpes viruses are uniquely appropriate as they do
not have the limited packaging capabilities of other viral vector
systems. Thus, multiple heterologous genes can be accommodated
within its genome. For example, from 2 to 5 genes may be inserted
into the genome.
[0130] There are, for example, at least two ways in which this
could be achieved. For example, more than one heterologous gene and
associated control sequences could be introduced into a particular
HSV strain either at a single site or at multiple sites in the
virus genome. It would also be possible to use pairs of promoters
(the same or different promoters) facing in opposite orientations
away from each other, these promoters each driving the expression
of a heterologous gene (the same or different heterologous gene) as
described above.
E. Therapeutic Uses
[0131] Viruses of the invention may be used in methods of therapy.
In particular, oncolytic viruses of the invention may be used in
applications including the oncolytic treatment of cancer, e.g. by
direct intra-tumour injection. Where the virus comprises a
heterologous gene encoding a prodrug activator, additional pro-drug
therapy may be carried out. Additionally, treatment may be combined
with or stimulation of an immune response by any means known in the
art. Viruses of the invention may be used in the therapeutic
treatment of any solid tumour in a mammal, preferably in a human.
For example viruses of the invention may be administered to a
subject with prostate, breast, lung, liver, endometrial, bladder,
colon or cervical carcinoma; adenocarcinoma; melanoma; lymphoma;
glioma; or sarcomas such as soft tissue and bone sarcomas.
[0132] Replication incompetent viruses of the invention may be used
in the delivery of genes to individuals requiring gene therapy. In
particular, neurotrophic viruses of the invention may be used in
the treatment of disorders of the central or peripheral nervous
system. Preferred central nervous system disorders for treatment or
prevention include neurodegenerative disorders. Particularly
preferred central nervous system disorders for treatment or
prevention are stroke, Parkinson's disease, Alzheimer's disease,
Tay Sachs disease and mucopolysaccaride diseases. Preferred
peripheral nervous system disorders for treatment or prevention
include motor neuron disease, chronic pain and peripheral nerve
damage.
[0133] Immunotherapeutic viruses of the invention may be used in
the prevention or treatment of the pathogenic infection or cancer
with which the antigen their inserted heterologous coding gene is
associated.
F. Administration
[0134] The viruses of the invention may thus be used in a patient,
preferably a human patient, in need of treatment. Viruses of the
invention may be used for the oncolytic treatment of cancer, and
herpes viruses of the invention (in addition to oncolytic
applications) for the treatment of, for example pain, degenerative
conditions of the nervous system, or to stimulate nerve re-growth.
The aim of therapeutic treatment is to improve the condition of the
patient. Typically therapeutic treatment using a virus of the
invention will alleviate the symptoms of the disease or condition
of the patient being treated. A method of treatment according to
the invention therefore comprises administering a therapeutically
effective amount of a virus of the invention to a patient suffering
from cancer, pain, a neurodegenerative condition or nerve
damage.
[0135] Administration of an oncolytic virus of the invention to a
patient suffering from a tumour will typically kill the cells of
the tumour thus decreasing the size of the tumour and/or preventing
the spread of malignant cells from the tumour. Administration of a
virus of the invention to a patient suffering from other diseases
such as pain, degenerative conditions or nerve damage will
typically improve the condition of the patient. For example by
lessening the severity of the pain, slowing the degeneration of
nervous tissue or promoting nerve regrowth.
[0136] One method of administering therapy involves combining the
virus with a pharmaceutically acceptable carrier or diluent to
produce a pharmaceutical composition. Suitable carriers and
diluents include isotonic saline solutions, for example
phosphate-buffered saline.
[0137] Oncolytic treatment and/or gene delivery to cells for
therapeutic purposes may then be carried out following direct
injection of the vector composition into target tissue. The amount
of virus administered is in the case of HSV in the range of from
10.sup.4 to 10.sup.10 pfu, preferably from 10.sup.5 to 10.sup.8
pfu, more preferably about 10.sup.6 to 10.sup.8 pfu. When injected
for oncolytic or non-oncolytic treatment, typically up to 5001,
typically from 1-200 .mu.l, preferably from 1-10 .mu.l of a
pharmaceutical composition consisting essentially of the virus and
a pharmaceutically acceptable suitable carrier or diluent, would be
used for injection. However, for some oncolytic therapy
applications larger volumes up to 10 ml may also be used, depending
on the tumour and inoculation site.
[0138] The routes of administration and dosages described are
intended only as a guide since a skilled practitioner will be able
to determine readily the optimum route of administration and
dosage. The dosage may be determined according to various
parameters, especially according to the age, weight and condition
of the patient to be treated, the severity of the disease or
condition and the route of administration.
[0139] The preferred route of administration to a patient suffering
from cancer is by direct injection into the tumour. The virus may
also be administered systemically or by injection into a blood
vessel supplying the tumour. The optimum route of administration
will depend on the location and size of the tumour. The dosage may
be determined according to various parameters, especially according
to the location of the tumour, the size of the tumour, the age,
weight and condition of the patient to be treated and the route of
administration.
G. Non-Therapeutic Aspects
[0140] Also provided are methods of identifying suitable clinical
strains for modification according to the invention. In addition,
methods of target validation are provided. These concern the
identification of genes suitable for use in the therapeutic
applications of the invention as described above.
[0141] Methods of production of viruses of the invention are also
provided.
[0142] The following Examples illustrate the invention.
[0143] Herpes simplex type-1 virus (HSV1) in which the
neurovirulence factor ICP34.5 is inactivated has previously been
shown to direct tumour specific cell lysis in tumour models both in
vitro and in vivo. Such viruses have also been shown to be safe in
Phase I clinical trials by direct intra-cerebral injection in late
stage glioma patients.
[0144] Previous work has used serially passaged laboratory isolates
of HSV1 (viruses derived from HSV1 strain 17+ or HSV1 strain F)
which might be anticipated to be attenuated in their lytic
capability in human tumour cells as compared to more recent
clinical isolates.
[0145] In work aimed at producing ICP34.5 deleted HSV with enhanced
oncolytic and anti-tumour potential, we have deleted ICP34.5 from
an HSV1 clinical isolate and compared replicative and lytic
potential in a number of human tumour cell types in comparison to
HSV1 strain 17+(a standard laboratory strain).
Virus Construction (see FIG. 1)
[0146] The viruses used were either based on HSV1 strain 17+(a
standard laboratory strain) or two clinical isolates derived from
cold sores from frequent re-activators of HSV1. These strains were
named BL1 and JS1. ICP34.5 was completely deleted from strain 17+
and JS1 together with the insertion of a CMV-GFP cassette. JS1 was
also further engineered by the insertion of human or mouse GM-CSF
so as to replace the ICP34.5 gene. BL1 and JS1 are thus clinical
isolates, or "non-laboratory" strains. The derivatives of JS1
discussed herein are also non-laboratory strains, i.e. modified
non-laboratory strains of the invention.
Virus Growth in Tumour Cells (see FIG. 2)
[0147] JS1 and BL1 showed enhanced growth in some human tumour
cells tested as compared to HSV1 ICP34.5 deleted strain 17+ when
tested over a 72 hour period (FIG. 2). JS1 was selected for further
study and the modifications described above (see FIG. 1, and above)
were made to it.
Lytic Capabilities of Viruses (see FIG. 3)
[0148] Lytic (cell killing) capabilities were enhanced with the
JS1-derived non-laboratory strains derived virus in all tumour cell
lines tested. More particularly, with reference to FIG. 3, the
JS1/34.5- virus, i.e. JS1 with ICP34.5 removed by deletion, showed
enhanced lytic capabilities in HT29 colorectal adenocarcinoma,
LNCaP.FGC prostate adenocarcinoma, MDA-MB-231 breast
adenocarcinoma, SK-MEL-28 malignant melanoma and U-87 MG
glioblastoma astrocytoma cells.
[0149] Lytic capabilities were also assessed in SK-MEL-28,
MDA-MB-231 and HT29 cells by trypan blue exclusion assay of
infected cells at various doses and times after infection with BL1,
JS1 as compared to strain 17+. Trypan blue is excluded from live
cells and so numbers of live cells remaining in a culture can be
assessed by this means. Tumour cell lines cultured in duplicate
wells of six well dishes were infected for 24, 48 or 72 hrs at an
MOI of 0.1 or 1 with either 17+, BL1 or JS1 and numbers of live
cells were counted. The percentage of the number of live cells in
equivalent uninfected control wells are shown in Table 1.
[0150] Thus, as in all cases more tumour cells are killed with the
clinical isolate viruses BL1 and JS1 than the laboratory isolate
17+, to provide increased oncolytic activity, the use of recent
clinical virus strains is likely to enhance the anti-tumour
capabilities of such viruses modified to give tumour selective
replication (e.g. by the deletion of ICP34.5) when used in human
patients for cancer treatment.
TABLE-US-00001 TABLE 1 JS1 17 BL1 time after MOI = 0.1 MOI = 1 MOI
= 0.1 MOI = 1 MOI = 0.1 MOI = 1 Cell line infection percentage of
number of live cells in uninfected control wells SK-MEL-28 24 h 41
8 57.3 19 43.7 6.67 duplicate 33.7 7 62.6 19.3 39 6.33 samples 48 h
5.51 1.9 7.4 3.7 4.5 0.8 5.05 0.8 7.1 2.6 4.8 1.1 72 h 0 0 0 0 0 0
0 0 0 0 0 0 MDA-MB-231 24 h 44.91 16.7 69.37 36.34 55.63 26.79
44.02 16.96 65.8 34.55 60.45 25.27 48 h 14.1 4.7 27.9 8.3 18 6.7
13.5 3.8 27 8.5 20 8.3 72 h 0 0 2.91 0.73 1.46 0 0 0 2.91 1.27 1.64
0 HT-29 24 h 37.53 15 47.28 23.61 42.22 22.15 39.24 15 45.76 24.24
43.04 21.33 48 h 13.2 2.3 29.4 4.2 18.4 4.4 14 3 27.7 4.7 21.2 3.7
72 h 0 0 1.57 0 1.64 0 0 0 1.89 0 1.57 0
Further Enhanced Anti-Tumour Activity
[0151] Further enhanced activity may also be anticipated if these
viruses are then used to deliver genes with anti-tumour activity.
Such genes include those encoding pro-drug activators or immune
stimulatory proteins.
[0152] For this purpose, we have produced from JS1 an ICP34.5
deleted clinical isolated of HSV1 which expresses human or mouse
GM-CSF GM-CSF is a potent immune stimulator. This virus is designed
to enhance anti-tumour immune responses following intra-tumoral
injection.
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Deposit Information
[0170] HSV1 Strain JS1 has been deposited at the European
Collection of Cell Cultures (ECACC), CAMR, Salisbury, Wiltshire SP4
0JG, United Kingdom, on 2 Jan. 2001 under provisional accession
number 01010209.
* * * * *
References