U.S. patent application number 10/395287 was filed with the patent office on 2004-02-12 for gene therapy.
This patent application is currently assigned to VICTORIA UNIVERSITY OF MANCHESTER. Invention is credited to Castro, Maria, Lowenstein, Pedro.
Application Number | 20040029227 10/395287 |
Document ID | / |
Family ID | 10863166 |
Filed Date | 2004-02-12 |
United States Patent
Application |
20040029227 |
Kind Code |
A1 |
Lowenstein, Pedro ; et
al. |
February 12, 2004 |
Gene therapy
Abstract
The present invention relates to a method of prolonging the
expression of an exogenous gene in a cell transduced with the
exogenous gene. The method comprises co-administration of the
exogenous gene with a herpes virus gene, whereby such
co-administration prolongs the expression of the exogenous gene in
the transduced cell. The method is particularly useful as a means
of effecting gene therapy.
Inventors: |
Lowenstein, Pedro;
(Manchester, GB) ; Castro, Maria; (Manchester,
GB) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
VICTORIA UNIVERSITY OF
MANCHESTER
|
Family ID: |
10863166 |
Appl. No.: |
10/395287 |
Filed: |
March 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10395287 |
Mar 25, 2003 |
|
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09693970 |
Oct 23, 2000 |
|
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/456 |
Current CPC
Class: |
A61P 37/06 20180101;
A61K 48/0066 20130101; A61P 25/16 20180101; A61P 25/28 20180101;
C12N 2710/10343 20130101; A61P 31/18 20180101; A61K 48/00 20130101;
A61K 38/45 20130101; C12N 15/86 20130101 |
Class at
Publication: |
435/69.1 ;
435/456; 435/320.1; 435/325 |
International
Class: |
C12P 021/02; C12N
015/869 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 1999 |
GB |
9924981.5 |
Claims
1. A method of prolonging the expression of an exogenous gene in a
cell transduced with the exogenous gene, the method comprising
co-administration of the exogenous gene with a herpes virus gene,
whereby such co-administration prolongs the expression of the
exogenous gene in the transduced cell.
2. A method according to claim 1 in which the exogenous gene is
administered to a cell as naked DNA.
3. A method according to claim 1 in which the herpes virus gene is
administered to a cell as naked DNA.
4. A method according to claim 1 in which the exogenous gene and
herpes virus gene are administered to the cell on one piece of
naked DNA.
5. A method according to claim 4 in which the exogenous DNA and
herpes virus gene are provided on a linked piece of DNA.
6. A method according to claim 1 in which the exogenous gene is
administered to a cell in a vector.
7. A method according to claim 1 in which the herpes virus gene is
administered to a cell in a vector.
8. A method according to claim 6 or claim 7 in which the exogenous
gene and herpes virus gene are co-administered to a cell in one
vector.
9. A method according to claim 6 or claim 7 in which the exogenous
gene and herpes virus gene are co-administered to a cell the in two
separate vectors.
10. A method according to any one of claims 6 to 9 in which the
vector comprises a promoter or regulator to control expression of
the genes as required.
11. A method according to any one of claims 6 to 10 in which the
vector further comprises targeting means which targets the vector
to a particular tissue or cell type.
12. A method according to any one of claims 6 to 11 in which the
vector is a viral vector.
13. A method according to claim 12 in which the vector is derived
from a DNA virus, an RNA virus or a retrovirus.
14. A method according to claim 13 in which the vector is an
adenovirus vector.
15. A method according to any preceding claim in which the herpes
virus gene is any gene from the family Herpesviridae, including
Herpes Simplex Virus Type 1, Herpes Simplex Virus Type 2, Varicella
Zoster Virus, Pseudorabies virus, Marek's disease virus,
cercopitecine herpes virus and Epstein Barr virus.
16. A method according to any preceding claim in which the herpes
virus gene is any herpes virus gene is a herpes virus thymidine
kinase gene or variant thereof, which converts ganciclovir to a
toxic agent.
17. A method according to any preceding claim in which the
heterologous gene encodes a therapeutic agent or a marker
protein.
18. A method according to claim 17 in which the gene is selected
from one encoding glial cell derived growth factor (GDNF),
neurotrophic factor (NGF), neurturin, persefin and other family
members, Nurr-1, gli-1, gli-3, brain derived neurotrophic factor,
ciliary derived neurotrophic factor (CNTF), amyloid precursor
protein, .beta. galactosidase, green fluorescent protein(s) (GFP),
transducing growth factors .beta.1, .beta.2, .beta.3, inhibitors of
NF kappaB, anti-apoptotic genes such as bcl-2, bcl-x1,
anti-inflammatory and immune-modulators such as interleukin 1
receptor agonist (IL-1ra), IL-receptor 2, neuropeptide
neurotransmitters such as corticotrophin releasing hormone,
substance P and neurokinins.
19. A method of prolonging the expression of an exogenous gene in a
cell transduced with the exogenous gene, the method comprising
co-administration of the exogenous gene with a conditionally
cytotoxic gene, whereby such co-administration prolongs the
expression of the exogenous gene in the transduced cell.
20. A method according to claim 19 in which the exogenous gene is
administered to a cell as naked DNA.
21. A method according to claim 19 in which the conditionally
cytotoxic gene is administered to a cell as naked DNA.
22. A method according to claim 19 in which the exogenous gene and
conditionally cytotoxic gene are administered to the cell on one
piece of naked DNA.
23. A method according to claim 22 in which the exogenous DNA and
conditionally cytotoxic gene are provided on a linked piece of
DNA.
24. A method according to claim 19 in which the exogenous gene is
administered to a cell in a vector.
25. A method according to claim 19 in which the conditionally
cytotoxic gene is administered to a cell in a vector.
26. A method according to claim 24 or claim 25 in which the
exogenous gene and conditionally cytotoxic gene are co-administered
to a cell in one vector.
27. A method according to claim 24 or claim 25 in which the
exogenous gene and conditionally cytotoxic gene are co-administered
to a cell the in two separate vectors.
28. A method according to any one of claims 24 to 27 in which the
vector comprises a promoter or regulator to control expression of
the genes as required.
29. A method according to any one of claims 24 to 28 in which the
vector further comprises targeting means which targets the vector
to a particular tissue or cell type.
30. A method according to any one of claims 24 to 30 in which the
vector is a viral vector.
31. A method according to claim 30 in which the vector is derived
from a DNA virus, an RNA virus or a retrovirus.
32. A method according to claim 31 in which the vector is an
adenovirus vector.
33. A method according to any one of claims 19 to 32 in which the
conditionally cytotoxic gene encodes thymidine kinase from sources
other than herpes virus, carboxypeptidase G2, alkaline phosphatase,
penicillin--V amidase or cytosine deaminase gene.
34. A method according to any preceding claim further comprising
administration of a pro-drug to the transduced cell.
35. A method according to claim 34 in which the pro-drug is
selected from GCV, aciclovir, trifluorothymidine, 1-[2-deoxy,
2-fluoro, .beta.-D-arabino furanosyl]-5-iodouracil, ara-A, ara-T,
1-.beta.-D-arabinofuranoxyl thymine, 5-ethyl-2'deoxyuridine,
5-iodo-5'-amino-2,5'-dideoxyuridine, idoxuridine, AZT, AIU
(5-iodo-5' amino 2',5'-dideoxyuridine), dideoxycytidine and
Ara-C.
36. Use of the method of any preceding claim in for prophylaxis or
treatment of a disease associated with body tissues.
37. Use of the method of any one of claims 1 to 35 in the treatment
of brain diseases.
38. Use according to claim 36 for treatment of brain tumours,
Alzheimer's disease, Parkinson's disease, Huntington's disease,
lateral amyotrophic sclerosis, neurodegenrative and neurometabolic
disorders, chronic brain infections such as HIV and measles,
pituitary tumours, spinal cord degeneration, spinal cord
regeneration, autoimmune diseases such as multiple sclerosis,
Guillain Barre syndrome and peripheral neuropathies.
39. A method for the treatment of a disorder by suicide gene
therapy comprising more than one cycle of administration of a
cytotoxic pro-drug.
40. A method according to claim 39 in which a cycle of
administration of cytotoxic pro-drug is repeated substantially
every one month, two months, three months, four months, six months,
eight months, ten months or twelve months or fractions thereof.
Description
[0001] The present invention relates to gene therapy.
[0002] Gene therapy is a term used to describe the transfer of one
or more genes to a cell. Gene therapy may be used to introduce a
gene into a cell and provide for subsequent expression of that gene
to alter the phenotype of the cell. For example the gene product
may protect the cell from toxic agents, such as chemotherapeutic
agents, increase the sensitivity of a cell to a cytotoxic drug,
prolong the effect of an agent, either directly or by overcoming
some induced or acquired resistance, correct a genetic defect
within a target cell or to confer a novel function or property on a
target cell.
[0003] Successful gene therapy depends upon the efficient delivery
of a suitable gene to a target cell and expression of the gene at
an adequate level in sufficient target cells to achieve the desired
therapeutic endpoint. The duration of expression required will vary
between different clinical settings, but in most settings continued
therapeutic benefit will depend on targeted, continued, high level,
stable and prolonged expression.
[0004] Several approaches to gene therapy are under investigation
which aim to provide for targeted gene transfer, controlled
expression of the gene transferred and enhanced activity of the
transferred gene product.
[0005] One approach, as described in European Patent Application
90309430.8 in the name of The Wellcome Foundation Limited, is to
use chimeric viral vectors systems to provide for targeted gene
transfer. In this approach a chimera incorporating a tissue
specific transcriptional regulatory sequence (TRS) linked to and
controlling the expression of a heterologous enzyme is packaged in
a synthetic retroviral particle and used for administration to a
patient. The expression of the heterologous gene in the patient is
therefore targeted to the target tissue. This approach has been
used to target expression of cytotoxic agents, for example
thymidine kinase, in cancer cell lines but not in non-cancerous
cell lines.
[0006] Another approach, as described in International Patent
Application PCT/EP98/07380 in the name of Novartis-Erfindungen
Verwaltungsgesellschaft MBH, relates to cell-specific expression
vectors which allow for controlled expression of the gene
transferred. This approach uses an expression vector that contains
at least one gene essential for replication of the vector under the
control of a heterologous transcriptional regulatory system to
produce an expression vector whose replication is controlled by the
presence of an agent which controls the activity of the
transcriptional regulatory system. This allows the gene expression
from the expression vector to be modulated in cells. If, for
example vector replication proceeds at levels that are undesirable
the approach allows the level of replication to be reduced. This
approach has been used to produce an adenovirus vector containing
the herpes simplex virus type 1 thymidine kinase gene (ad HSV-1 TK)
whose replication is modulated in the presence of ganciclovir
(GCV). The vector was administered to subcutaneous tumours induced
in a group of mice and after a period of 5 days GCV given to some
of the group of mice. Immunohistochemistry of all sacrificed mice
showed that HSV1 TK expression was the same between the groups
whereas the mice treated with GCV showed diminished vector
expression.
[0007] A further approach, as described in International Patent
Application No. PCT/US98/21672 in the name of Danvin Molecular
Corporation relates to expression vectors transferring genes which
encode proteins which are mutated with respect to the wild type
protein to have increased activity. Such vectors provide a gene
product which has enhanced activity within cells transduced with
such vectors.
[0008] It has been previously proposed that gene therapy may be
useful in the treatment of neurological diseases such as
Parkinson's disease, Alzheimer's disease and brain tumours.
Numerous groups are attempting to develop vector systems that will
allow the deliver of potentially therapeutic agents to terminally
differentiated neurones within the intact brain. It is also an aim
to target gene expression to other brain cell types (e.g.
astrocytes and microglia).
[0009] The ability of HSV-1 to establish a lifelong latent
infection within neurones has led to interest in its use as a
neuronal gene delivery vector. However, during HSV-1 latency no
viral proteins are produced and transcription from the viral genome
is limited to a family of nuclear RNAs, the latency-associated
transcripts, whose function is not well understood. Although HSV-1
vectors which express in dorsal root ganglia have been achieved,
whether latency can be achieved in cells of the forebrain is yet to
be determined.
[0010] One viral vector which is particularly used in current gene
therapy techniques under study is the adenovirus vector. The
ability of the adenovirus vector to transduce most cell types
efficiently has resulted in gene therapy trials involving local
administration of adenoviral constructs, including administration
to brain tissues. The complexity of brain function and the
difficulty in non-invasively monitoring and alterations in gene
expression in vivo has meant that the extent and duration of the
therapeutic benefit of these trials has been difficult to
assess.
[0011] Clinical trials of conditional cytotoxic gene therapy of
glioblastoma are currently ongoing using retro- and adenoviral
vectors encoding Herpes simplex virus-I thymidine kinase
(HSV-1-TK), followed by the administration of ganciclovir.sup.1-4.
Much of the efficiency of suicide-gene therapy is thought to be due
to the `bystander effect`, of which inflammation and anti-tumour
immune stimulation appear to be crucial components.sup.5-7. In
spite of many experimental studies examining the efficiency of
suicide-gene therapy-induced glioma regression.sup.8-17, there is
no information on the incidence of subsequent chronic brain
inflammation. It has been reported previously that brain gene
transfer using adenoviral vectors induces acute, short-lived,
inflammatory reactions.sup.18-20, although peripheral
readministration of viral vectors induces a delayed type
hypersensitivity reaction.sup.19, which eliminates transgene
expression, and is accompanied by localised demyelination.
Likewise, most transgenic protein expression is mostly, though not
exclusively, restricted to the injection site. Such experiments
have failed to demonstrate widespread expression of transgenic
proteins in the brain beyond two months.sup.18,19,21-24.
Understanding the long-term consequences of suicide gene therapy of
brain tumours is thus of crucial importance.
[0012] It is an aim of the present invention to obviate or mitigate
a disadvantage of known gene therapy strategies and to provide a
method for prolonged and widespread expression of a gene of
interest. A further aim of the present invention is to provide an
improved gene therapy treatment.
[0013] According to the present invention in a first aspect there
is provided a method of prolonging the expression of an exogenous
gene in a cell transduced with the exogenous gene, the method
comprising co-administration of the exogenous gene with a herpes
virus gene, whereby such co-administration prolongs the expression
of the exogenous gene in the transduced cell.
[0014] According to the present invention in a second aspect there
is provided a method of prolonging the expression of an exogenous
gene in a cell transduced with the exogenous gene, the method
comprising co-administration of the exogenous gene with a
conditionally cytotoxic viral gene, whereby such co-administration
prolongs the expression of the exogenous gene in the transduced
cell.
[0015] The invention is based on the inventors' study of the
long-term outcomes of adenovirus-mediated conditionally cytotoxic
gene therapy in a syngeneic glioblastoma model. CNS-1 cells.sup.25
were implanted into the striatum of Lewis rats, and followed by the
injection of adenovirus expressing HSV1-TK, and systemic
ganciclovir (GCV). The treatment was very efficient, resulting in
the survival of 80-100% of animals for at least 3 months.
Unexpectedly, examination of the brains of long-term survivors
revealed the presence of chronically active brain inflammation, as
well as very strong and widespread HSV1-TK immunoreactivity. These
data have important implications for the design and evaluation of
clinical gene therapy trials of glioblastoma multiforme.
[0016] Based on the results of these experiments the inventors went
on to investigate what gave rise to the prolonged and widespread
HSV1-TK expression and showed that this was not due to the action
of the tumour cells, the adenovirus alone or to the action GCV and
therefore proposed that the sustained expression of HSV1 TK may be
a previously unknown property of HSV-1 TK which may be applicable
to all herpes virus genes or alternatively to all viral
conditionally cytotoxic genes.
[0017] Accordingly, the inventors propose that the expression of
any exogenous gene in a transduced cell may be prolonged if the
exogenous gene is co-administered with a herpes virus gene or a
viral conditionally cytotoxic gene. This proposal should have
enormous impact on the field of gene therapy where it is important
to have prolonged expression of a gene of interest. The method
according to the present invention thus has utility in a wide range
of conditions from cancer to conditions where gene therapy is being
considered, such as Parkinson's disease and muscle degeneration for
example.
[0018] An exogenous gene is defined as a gene or gene fragment
which is provided to a transduced cell. The exogenous gene may be
one that is not normally expressed in the cell which is transduced.
Alternatively, the exogenous gene may be one that is expressed by
the cell that is transduced.
[0019] It is well known that genes may be introduced into cells in
various ways.
[0020] The exogenous gene and/or herpes virus gene may be
transferred to the cells of a subject to be treated by
transfection, infection, microinjection, cell fusion, protoplast
fusion or ballistic bombardment. For example, transfer may be by
ballistic transfection with coated gold particles, liposomes
containing the DNA molecule, viral vectors (e.g. adenovirus) and
means of providing direct DNA uptake (e.g. endocytosis) by
application of plasmid DNA directly to an area topically or by
injection.
[0021] According to one embodiment of the first aspect of the
present invention the exogenous gene and herpes virus gene or
conditionally cytotoxic gene may be introduced into the cell as
"naked" genes by standard physical means including direct
endocytotic uptake.
[0022] The "naked" exogenous gene, herpes virus gene/conditionally
cytotoxic gene or both may be delivered to the cell as separate
pieces of nucleic acid. If the exogenous gene and herpes virus
gene/conditionally cytotoxic gene are delivered to the cell
together they may be provided on one piece of DNA as a chimera
encoding the two full length gene products. The "naked" DNA for the
exogenous gene and/or herpes virus gene/conditionally cytotoxic
gene may be further incorporated within a liposome or virus
particle for delivery to a cell.
[0023] According to an alternative embodiment of the first and
second aspects of the present invention, the exogenous gene, herpes
virus gene/conditionally cytotoxic gene or both may be introduced
into the cell via a recombinant vector delivery system. The vector
may for example be a plasmid, cosmid or phage. Such recombinant
vectors are highly useful according to the first and second aspects
of the present invention for transducing cells with the exogenous
gene and the herpes gene/conditionally cytotoxic gene.
[0024] The exogenous gene and herpes virus gene/conditionally
cytotoxic gene may be co-administered to a cell in one vector or in
two separate vectors. If provided in one vector the two genes may
be arranged to provide a linked transcript under the control of the
same regulatory elements if present or may be provided in separate
regions of the vector. If provided in two separate vectors it is
preferred that the vectors should be arranged to transduce the same
cell population.
[0025] A vector refers to an assembly which is capable of directing
the expression of a gene. Recombinant vectors may also include
functional elements. For instance, recombinant vectors can be
designed such that the vector will autonomously replicate in the
nucleus of the cell. In this case, elements which induce DNA
replication may be required in the recombinant vector.
Alternatively the recombinant vector may be designed such that the
vector and exogenous gene and/or herpes virus gene/conditionally
cytotoxic gene integrate into the genome of a cell. In this case
DNA sequences which favour targeted integration (e.g. by homologous
recombination) are desirable. Recombinant vectors may also have DNA
coding for genes that may be used as selectable markers in the
cloning process.
[0026] The recombinant vector may also further comprise a promoter
or regulator to control expression of the gene as required. The
recombinant vector may further be a virion or a transcriptionally
targeted vector which specifically restricts expression to a
particular tissue or cell type.
[0027] The exogenous gene and or herpes virus gene may (but not
necessarily) be one which becomes incorporated in the DNA of cells
of the subject being treated. Undifferentiated cells may be stably
transduced leading to the production of genetically modified
daughter cells (in which case regulation of expression in the
subject may be required e.g. with specific transcription factors or
gene activators).
[0028] Preferred vector systems for use according to the first
aspect of the present invention are viral vector systems in which
the vector is derived from a DNA virus, for example, parvovirus,
picornavirus, pseudorabies virus, hepatitis virus A, B or C,
papillomavirus, papovavirus (such as polyoma and SV40) or herpes
virus (such as Epstein-Barr Virus, Varicella Zoster Virus,
Cytomegalovirus, Herpes Zoster and Herpes Simplex Virus types 1 and
2), an RNA virus or a retrovirus, such as the Moloney murine
leukemia virus or a lentivirus (i.e. derived from Human
Immunodeficiency Virus, Feline Immunodeficiency Virus, equine
infectious anaemia virus, etc.).
[0029] A particularly preferred vector system for use according to
the first aspect of the present invention is the adenovirus vector
system. A preferred adenovirus vector system is described in
International Application No. PCT/EP98/07380.
[0030] The viral vector particles comprising either the exogenous
gene, the herpes virus gene/conditionally cytotoxic gene or both
may be administered to a host. The host may be an animal host,
including mammalian, non-human primate, rodent and human hosts.
[0031] The viral particles may be administered in combination with
a pharmaceutically acceptable carrier suitable for administration
to a patient. The carrier may be a liquid carrier (for example
saline solution) or a solid carrier, such as for example
microcarrier beads.
[0032] The herpes virus gene which is co-administered to prolong
expression of an exogenous gene in a cell is preferable any gene
encoded by the genome of the family Herpesviridae. Representative
examples of this family include Herpes Simplex Virus Type 1, Herpes
Simplex Virus Type 2, Varicella Zoster Virus, human and murine
cytomegalovirus, Pseudorabies virus, Marek's disease virus,
cercopitecine herpes virus and Epstein Barr virus.
[0033] If the herpes virus gene is to be administered on a herpes
viral vector it is preferred that the herpes virus gene is
heterologous to the herpes virus vector. The term "heterologous"
means that the herpes virus gene is not found naturally in the
native herpes vector.
[0034] A preferred herpes gene that is administered to prolong gene
expression of a co-administered exogenous gene is a herpes virus
thymidine kinase gene or variant thereof. The herpes virus TK gene
encodes a viral TK protein which is important in the synthesis of
nucleic acid precursors normally within cells infected with herpes
virus.
[0035] In herpes virus infected cells TK can phosphorylate the
guanosine analogue ganciclovir (GCV) resulting in
GCV-monophosphate, in contrast to uninfected cells which contain a
cellular TK gene which does not act on GCV. GCV monophosphate, if
produced is phosphorylated by intracellular protein kinases
producing a GCV-triphosphate in cells which contain the herpes
virus TK gene. The GCV-triphosphate is preferably incorporated into
the DNA of rapidly dividing cells (e.g. cancer cells) but due to
its chemical structure cannot promote further elongation of nascent
DNA resulting in chain termination and cell death.
[0036] Various mutant forms of herpes virus TK have been proposed
all of which have varying degrees of TK activity. The use of such
mutants, as described in International Patent Application No.
PCT/US98/21672 is envisaged within the scope of the first aspect of
the present invention.
[0037] Other herpes virus genes that are preferred to prolong gene
expression of a co-administered exogenous gene is herpes virus
ribonucleotide reductase, an enzyme involved in purine
metabolism.
[0038] It may be that the addition of a pro-drug to the cell
transduced according to the method of the first or second aspects
of the invention may enhance the prolonged expression of the
exogenous gene. Therefore, preferably, the method according to the
first and second aspects of the invention further comprises
addition of a pro-drug to the transduced cell.
[0039] Examples of suitable pro-drugs include nucleoside analogues
which may be pro-drugs activated by herpes virus TK or other, non
herpes virus conditionally cytotoxic enzymes include purine
arabinosides and substituted pyrimidine compounds, for example as
described in published European Patent Application EP-A-415 731.
Representative examples of nucleoside analogues include GCV,
aciclovir, trifluorothymidine, 1-[2-deoxy, 2-fluoro,
.beta.-D-arabino furanosyl]-5-iodouracil, ara-A, ara-T,
1-.beta.-D-arabinofuranoxyl thymine, 5-ethyl-2'deoxyuridine,
5-iodo-5'-amino-2,5'-dideoxyuridine, idoxuridine, AZT, AIU
(5-iodo-5' amino 2',5'-dideoxyuridine), dideoxycytidine and
Ara-C.
[0040] As described above herpes virus TK is a conditionally
cytotoxic enzyme which can act on a non-toxic compound, GCV (a
nucleoside analogue pro-drug) to produce a compound which is toxic
to a cell, GCV triphosphate (a nucleoside analogue drug).
[0041] Other non-herpes virus genes which encode an enzyme which is
conditionally cytotoxic and act on a pro-drug to produce a drug
will be known to persons skilled in the art, and are included
within the scope of the second aspect of the invention. Such
conditionally cytotoxic enzymes include thymidine kinase from
sources other than herpes virus, carboxypeptidase G2, alkaline
phosphatase, penicillin--V amidase and cytosine deaminase gene.
[0042] Other nucleoside analogues which may be pro-drugs activated
by herpes virus TK or other, non herpes virus conditionally
cytotoxic enzymes include purine arabinosides and substituted
pyrimidine compounds, for example as described in published
European Patent Application EP-A-415 731. Representative examples
of nucleoside analogues include GCV, acyclovir, trifluorothymidine,
1-[2-deoxy, 2-fluoro. .beta.-D-arabino furanosyl]-5-iodouracil,
ara-A, ara-T, 1-.beta.-D-arabinofuranoxyl thymine,
5-ethyl-2'deoxyuridine, 5-iodo-5'-amino-2,5'-dideoxyuridine,
idoxuridine, AZT, AIU (5-iodo-5' amino 2',5'-dideoxyuridine),
dideoxycytidine and Ara-C.
[0043] The nature of the exogenous gene to be co-administered with
the herpes virus gene/conditionally cytotoxic gene for prolonged
expression will depend upon why prolonged expression is desired. If
for example the method is for gene therapy of a particular disease
the exogenous gene will be a therapeutic gene whose expression is
known to be associated with treatment of that disease.
[0044] If the method is used to study the distribution or
expression of a particular gene in a cell, tissue or organ, the
exogenous gene co-administered with the herpes virus
gene/conditionally cytotoxic gene for prolonged expression is a
marker gene.
[0045] Preferred genes to be administered according to the first
aspect and second aspects of the present invention include those
encoding glial cell derived growth factor (GDNF), neurotrophic
factor (NGF), neurturin, persefin and other members of the
Transforming, growth factor .beta. superfamily, Nurr-1, gli-1,
gli-3, brain derived neurotrophic factor, ciliary derived
neurotrophic factor (CNTF), amyloid precursor protein, marker genes
like .beta. galactosidase, green fluorescent protein(s) (GFP)
amongst others, transducing growth factors .beta.1, .beta.2,
.beta.3, inhibitors of NF kappaB, anti-apoptotic genes, e.g. bcl-2,
bcl-x1, anti-inflammatory and immune-modulators such as interleukin
1 receptor agonist (1L-Ira), IL-receptor 2, neuropeptide
neurotransmitters, e.g. corticotrophin releasing hormone, substance
P, neurokinins, etc.
[0046] As the method according to the first and second aspects of
the present invention may provide for widespread distribution of
the herpes gene/conditionally cytotoxic gene and exogenous gene in
brain tissues, the method according to the first and second aspects
of the present invention are proposed to be particularly useful in
gene therapy for brain diseases. In such therapy it is preferred
that the exogenous gene encodes a therapeutic agent associated with
such brain diseases. Such genes may include those provided
above.
[0047] Accordingly a third aspect of the invention provides for the
use of a herpes virus gene co-administered with a heterologous gene
for prophylaxis or treatment of a disease associated with body
tissues.
[0048] Accordingly a fourth aspect of the invention provides for
the use of a conditionally cytotoxic gene co-administered with a
heterologous gene for prophylaxis or treatment of a disease
associated with body tissues.
[0049] Particular brain diseases which the third and fourth aspects
of the invention seek to treat include brain tumours, Alzheimer's
disease, Parkinson's disease, Huntington's disease, lateral
amyotrophic sclerosis, neurodegenerative and neurometabolic
disorders, chronic brain infections (e.g. HIV, measles, etc.),
pituitary tumours, spinal cord degeneration (both inherited and
traumatic), spinal cord regeneration, autoimmune diseases (e.g.
multiple sclerosis, Guillain Barre syndrome, peripheral
neuropathies, etc.) and any other diseases of the brain known to
persons skilled in the art.
[0050] Treatment of diseases associated with tissues of the body
other than the brain are also envisaged within the scope of the
third and fourth aspects of the present invention, such as the
liver, muscle, etc.
[0051] The inventors propose that as well as prolonging gene
expression, the co-administered herpes gene/conditionally cytotoxic
gene provides more widespread distribution of a gene administered
by gene therapy than in the absence of the herpes virus gene.
Histological studies show that a HSV1 TK gene is expressed in the
axons, dendrites and cell bodies of neurones in the contralateral
side to the side of the brain in which a viral vector was injected.
This is in sharp contrast to the observed effects of steroids on
adenoviral vectors where the encoded transgene expression was found
to be local and the majority of the herpes gene was found in
astrocytes and not in neurones. In the treatment of neuronal
diseases such as Parkinson's disease for example, it is essential
that the expression of the exogenous aene, for example in this case
a dopamine receptor or dopamine agonist is in the neurones and not
astroglial cells. Accordingly the third aspect of the invention has
particular utility in the treatment of brain diseases.
[0052] The findings of the inventors have implications in currently
advised treatment regimes involving gene therapy methods. For
example, a trial is underway regarding administering an adenoviral
vector containing a herpes virus TK gene together with GCV
(so-called suicide gene therapy) in a single treatment cycle for
GCV of up to two weeks. The methods underlying this trial are
described in International Patent Application PCT/US98/21672.
[0053] According to current thinking it would be assumed that the
TK administered to a patient in a gene therapy method (e.g. by an
adenovirus vector) would be only transiently expressed and
therefore it would only be worthwhile providing a GCV treatment
cycle for a short period after administration of the TK. According
to the inventors' findings the TK expression would be stable and
could last for up to 12 months from introduction of the TK gene by
gene therapy. Thus, based on these findings the number of cycles of
clinical treatment with GCV should be increased as compared to
currently proposed treatment regimes.
[0054] According to the present invention in a fifth aspect there
is provided a method for the treatment of a disorder by suicide
gene therapy comprising more than one cycle of administration of a
cytotoxic pro-drug.
[0055] As it has been shown that the herpes virus TK gene is still
expressed after one year it is apparent that current treatment
regimes with TK and GCV of one two week cycle of administration of
GCV are curtailed prematurely. Additional cycles of administration
of GCV at one month, two months, three months, up to or more than
12 months, from the administration of the TK gene could still give
therapeutic effect as the TK gene will still be expressed in
vivo.
[0056] The invention will now be described by way of example only,
with reference to the following drawings, in which:
[0057] FIG. 1 shows survival analysis of Lewis rats implanted with
CNS-1 cells, and treated with RAd-128 (TK), RAd-127 (.DELTA.TK),
Rad-35 (.beta.gal), or vehicle, and GCV.
[0058] FIG. 2 shows brain inflammation in long-term suicide-gene
therapy survivors. Abbreviations used in b, d, f, h are: (c)
cortex, (w) white matter, (s) striatum.
[0059] FIG. 3 shows loss of myelinated fibres, HSV1-TK
immunoreactivity and macrophage and lymphocyte infiltration of
perivascular cuffs. The decrease in myelinated fibres in long term
suicide-gene therapy survivors is not due to autoimmune destruction
of oligodendrocytes. Scale bars for a-f is shown in (a) equals 350
.mu.m; scale bar for g-h is shown in (g) equals 35 .mu.m; scale bar
for (i) equals 40 .mu.m; scale bar for (a)=50 .mu.m; and for (k,
1)=30 .mu.m. Abbreviations used in (i) are: (WM) white matter,
(STR) striatum.
[0060] FIG. 4 shows persistence of HSV1-TK within neurones in
long-term survivors of suicide-gene therapy. Small black arrows
indicate the presence of labelling in dendritic spines, while the
thicker arrow indicates labelled immuno-reactive axonal boutons.
Scale bars: (a)=235 .mu.m, (b)=50 .mu.m, and (c)=10 .mu.m.
[0061] FIG. 5 shows PCR analysis of brain sections from long term
suicide-gene therapy survivors. Immunohistochemical staining for
HSV-1 TK in a 3 months survivor is shown in a coronal section in
(a). The results of the PCR detection of vector genome (IVa2),
transgene (TK), or replication competent genome (E1B), in brain
sections of Rad128 and Rad127 treated animals, and which survived
for 90 days is shown in (b). A schematic representation of the
regions amplified by PCR is shown in (c). The sites of
amplification for primer pairs a/b, c/d and e/f are indicated.
Scale bar (a)=2 mm.
[0062] FIG. 6 shows immune-mediated elimination of CNS-1 cells does
not lead to chronic persistent infiltration of CD4+ or CD8+
T-cells. Sections were photographed at a low magnification (a, e,
i, c, g, k), and at higher magnification (b, f, j, d, h, l). Scale
bars for a, e, i, c, g, k shown in (i,k)=1 mm and for b, f, j, d,
h, l shown in (j, l)=0.5 mm.
[0063] FIG. 7 shows that injection of RAd-128 followed by
ganciclovir leads to chronic sustained infiltration of CD8+
T-cells. Arrows indicate the boundaries of the white matter showing
T-cell infiltration. Abbreviations used in a-d are: (c) cortex, (w)
white matter, (s) striatum. Scale bar for a-d, shown in (d)=450
.mu.M.
[0064] FIG. 8 shows persistence of HSV1-TK within neurons 1, 5 and
12 months following injection of adenovirus TK.
[0065] FIG. 9 shows widespread distribution HSV1-TK
immunoreactivity throughout the anterior frontal and cingulate
neocortex at 30 days post-vector injection. a: right side of this
panel illustrates the hemisphere ipsilateral to the injection side.
b shows boxed area of cingulate cortex at higher power. d shows
boxed area of the piriform cortex at higher power. c and e are
boxes enlarged from b and d respectively; notice the strong
immunolabeling of pyramidal neurons, their dendrites as well as
afferent and efferent axonal processes. Scale bars are shown in
each of the panels.
[0066] FIG. 10 shows widespread distribution HSV1-TK
immunoreactivity at the level of the anterior striatum at 30 days
post-vector injection. Arrows indicate the exact location of the
original injection site. * indicates the enlarged lateral
ventricle, ipsilateral to viral vector injection. Boxed areas in
panel (a) are shown enlarged in (b) (insular cortex), (d) (anterior
commissure) and (e) (corpus callosum). Box in (b), is shown
enlarged in (c) to illustrate neuronal morphology. Box in (e), is
shown enlarged in (f) to illustrate the detailed axonal morphology
of axons coursing along white matter and entering or exiting the
overlying cerebral cortex. Scale bars are shown in each of the
panels.
[0067] FIG. 11 shows a time-course of HSV-1 TK expression in rat
neocortex at 1 month (a,d), 3 months (b,e) and 5 months (c,f). Rats
illustrated in a-c received i.p. GCV and rats d-f received i.p.
saline. Boxes to the right of each lettered panel show higher power
views to illustrate neuronal morphology. Scale bar for the lettered
(low power) panels is shown in (a), and the scale bar for all
higher power views is shown to the panel to the right of (a).
[0068] FIG. 12 shows sections from the neocortex of all 6 rats 1
year following the injection of adenovirus encoding HSV-1 TK.
Panels shown in (a-c) illustrate the neocortex of rats treated with
i.p. GCV; panels (d-e) illustrate the neocortex of rats treated
with i.p. saline. Notice that there is individual variability in
the HSV1-TK immunoreactivity detected. The highest level of
immunoreactivity was detected in the animal illustrated in (d).
[0069] FIG. 13 shows co-injection of RAd HSV-TK with RAd
.beta.-Galactosidase. Panels shown in a-f illustrate sections taken
from the same animal; panels a-c illustrate the forebrain and
substantia nigra immunoreacted with .beta.-galactosidase
antibodies. Panels illustrated in d-f, are all serial sections to
those shown in panels a-c, and were immunoreacted with antibodies
against HSV-1 TK.
[0070] FIG. 14 shows inflammatory brain responses to the injection
of RAd128 expressing HSV1-TK. ED1 staining of
macrophages/microglial cells at 1 month (a,d), 3 months (b,e) and 5
months (c,f) post-vector injection is shown. Brains illustrated in
a-c were obtained from animals which received i.p. GCV, while those
shown in d-f received i.p.saline. CD8 staining is shown at 1 month
(g,j), 3 months (h,k) and 5 months (i,l) post-vector injection;
panels shown in g-i were obtained from animals which received i.p.
GCV, and those illustrated in j-l received i.p.saline. Notice that
inflammatory responses appear somewhat increased in those animals
injected with ganciclovir.
[0071] FIG. 15 shows immune response to RAd HSV1-TK in the brains
of all 6 rats injected 1 year earlier with RAd128. Panels shown in
a-f illustrate ED1 staining of macrophages/microglial cells; those
in a-c received i.p. GCV, and those in d-f are from animals which
received i.p.saline. Panels g-1 illustrate CD8 staining; panels g-i
are from animals that received i.p. GCV, and those shown in j-l are
from animals which received i.p.saline. Arrow in g show mainly
brownish coloured haemosiderin deposition.
EXAMPLES
[0072] Methods
[0073] Cell Culture. The rat glioma cell line CNS-1 was kindly
provided by Prof. W. Hickey (Dartmouth Medical Center, Department
of Pathology, Lebanon, N.H., USA).sup.25. The kidney embryonic cell
line 293 was obtained from Microbix Biosystems Inc. (Toronto,
Ontario, Canada). The maintenance of the cell lines was described
previously.sup.37,38.
[0074] Adenovirus vectors expressing HSV1-TK, HSV1-.DELTA.TK, or
.beta.-galactosidase RAd vectors encoding HSV1-TK (RAd-128) and
HSV1-.DELTA.TK (RAd-127) under the short immediate/early human
cytomegalovirus (sMEhCMV) promoter.sup.37, 38 were generated and
characterised as described previously.sup.27-28. Viruses were
purified using double caesium chloride gradient and titres of
1.times.10.sup.10-1.times.10.sup.11 infectious units (U)/ml, and
particle/pfu ratios of 30 were obtained as described in detail
elsewhere.sup.17,20,27,28,37,38. As control virus, a double caesium
chloride gradient purified adenovirus, RAd-35, expressing the E.
coli .beta.-galactosidase gene driven by the sMIEHCMV promoter, was
used.sup.38. We have reported elsewhere that in vitro, CNS-1 cells
did express the transgene HSV1-TK following infection with
adenoviral vectors, and were sensitive to apoptosis induced
following the addition of ganciclovir to infected
cultures.sup.27,28.
[0075] Lipopolysaccharide endotoxin (LPS) assay and replication
competent adenovirus (RCA) assays LPS contamination in each RAd
stock was assessed by using the amoebocyte horseshoe crab lysate
method (E-Toxate assay, Sigma, Poole, Dorset, UK).sup.39. RAd-117
and RAd-128 showed levels of LPS below 5 milli-endotoxin units
(mEU)/.mu.l; thus, in the 4 .mu.l injected total LPS was below 20
mEU. .beta.-galactosidase expressing RAd-35, showed levels of
LPS.ltoreq.2 mEU/.mu.l; thus, in the 4 .mu.l injected total LPS was
.ltoreq.8 mEU. The amount of LPS needed to produce inflammatory
responses in the brain is several fold above the upper limit of
bioactive LPS activity value obtained in our bio-assays.sup.40. Our
viral volumes injected were essentially LPS free. RCA presence was
tested by the supernatant rescue assay.sup.41. No RCA was detected
in 2.6.times.10.sup.8 IU of either of the RAd-127 and RAd-128
vector stocks used in our experiments, showing the absence of RCA
in an amount of vector three times higher than the total amount of
infectious units that were injected in vivo.
[0076] In vivo treatment of gliomas. Male Lewis rats (250-300 g)
were anesthetized with halothane (Zeneca Ltd., Macclesfield,
Cheshire, UK) and placed in a stereotaxic frame. A burr hole in the
skull was made with a drill 3 mm to the right and 1 mm anterior to
bregma. A 5 .mu.l syringe fitted with a 26 gauge needle was
connected to the manipulating arm of a stereotaxic frame, and 5 or
10.times.10.sup.3 CNS-1 cells (in 3 .mu.l of phosphate buffered
saline (PBS) were injected over a 3 min period into the striatum at
the following location: bregma+1 mm; lateral+3 mm; ventral-4 mm.
The needle was left in place for another 5 min before removing.
[0077] Viruses were injected into the tumour site three days after
tumour implantation. Using the same anterior and lateral
coordinates, 1 .mu.l of PBS or 1 .mu.l (2.times.10.sup.7 IU/.mu.l)
of RAd127, RAd-128 or RAd-35 were injected at each of the following
ventral coordinates: -5 mm; -4.5 mm; -4 mm; -3.5 mm. Starting 12
hours after the injection of the viral vector, 25 mg/kg of
ganciclovir (GCV) (Cymevene, Roche Products Ltd., Welwyn Garden
City, UK) was injected intra-peritoneally twice daily for 7 days.
Animals injected with RAd-127, RAd-128, RAd-35 or PBS (n=5 per
group) were monitored daily. Any animal showing any sign of
morbidity, was perfusion-fixed and brains were removed for
histological analysis.
[0078] In other groups of animals not implanted with CNS-1 tumours,
the same amount of RAd-128 was injected into the brain, and was
followed by ganciclovir or saline administration for 7 days (n=3
per group). Animals were perfused either at 1 or 3 months
post-adenoviral vector injection.
[0079] Histological Analysis, Fixation, Paraffin and Plastic
Sections
[0080] Rats were anaesthetised, and fixed by cardiac perfusion.
First, animals were perfused with approximately 100 ml of Tyrode
solution (0.14 M NaCl, 2.7 mM KCl, 1.8 mM CaCl.sub.2, 0.32 mM
NaH.sub.2PO.sub.4, 5.6 mM glucose and 11.6 mM NaHCO.sub.3),
containing heparin (10 units/ml) (CP Pharmaceuticals Ltd., Wrexham,
UK), and this was followed by 250 ml of 4% paraformaldehyde in PBS,
pH 7.4. Brains were removed and placed in 4% paraformaldehyde for
24 h. Serial Vibratome sections (70 .mu.m) were maintained at
4.degree. C. in PBS. Sections were stained with hematoxylin and
eosin, or Luxol fast blue, or processed by immunohistochemistry.
Alternatively, some animals were perfused with 1% glutaraldehyde,
2% paraformaldehyde in 0.1M phosphate buffer, pH 7.4, and
post-fixed for 2-3 days. Some of the paraformaldehyde fixed brains
were dissected, and tissue blocks containing the needle track were
embedded on paraffin. Serial sections were performed on each block
to define precisely the location of the injection area. Five micron
thick paraffin sections were stained with hematoxylin and eosin,
Luxol fast blue myelin stain, and Bielschowski silver impregnation
for axons. Immunocytochemistry was performed using the
avidin-biotin technique, using the following primary antibodies:
W3/13 (leucosyalin, mouse monoclonal staining of rat T cells,
Serotec); OX8 (mouse monoclonal antibodies recognising CD8+
T-lymphocytes, Serotec); and cyclic nucleotide phosphodiesterase
(CNPase; mouse monoclonal SMI 91). Glutaraldehyde fixed tissue was
dissected into small tissue blocks containing the injection site.
Material was then further fixed/stained in 1% osmic acid in
phosphate buffered saline, and embedded into Epon; 0.5 .mu.m thick
plastic sections were stained with toluidine blue.
[0081] Immunohistochemistry on Vibratome Sections
[0082] Immunohistochemistry was performed on free floating
sections, as described before.sup.36,42. Anti-GFAP (Boehringer
Mannheim Ltd., Lewes, East Sussex, UK) and anti-vimentin antibodies
(Sigma, Poole, Dorset, UK), specific for astrocyte-specific
intermediate filaments, were used to identify astrocytes, and
.beta.-tubulin III (Sigma, Poole, Dorset, UK), to detect neurons
and their axons. Anti-ED1 antibodies, to identify
monocytes/macrophages/microglial cells, anti-CD3 antibodies to
detect total lymphocytes, and anti-CD8 antibodies, which detect CD8
positive lymphocytes and NK cells, were from Serotec Ltd.,
Kidlington, Oxford, UK. HSV1-TK proteins were detected using an
anti-HSV1-TK polyclonal antibody (kindly provided by M. Janicot,
Rhone Poulenc Rorer, Paris, France). Sections were washed twice
with Tris buffered saline (TBS) (50 mM Tris; 0.9% NaCl; 0.5%
Triton; pH 7.4), incubated for 15 min with 0.3% H2O2, washed three
times with 2 ml of TBS, incubated with 10% horse normal serum (HNS,
Life Technologies Ltd., Inchinnan Business Park, Paisley, UK) in
TBS for 45 min, and washed briefly for 10 min with 1% NHS in TBS.
Sections were then incubated overnight at room temperature with
primary antibodies at the following concentration: anti-GFAP
dilution {fraction (1/200)}, monoclonal anti-vimentin clone V9
({fraction (1/1000)}), anti-ED-1 ({fraction (1/1000)}), anti-CD3
({fraction (1/500)}), anti-CD8 ({fraction (1/500)}), anti-_-tubulin
III ({fraction (1/2000)}), and polyclonal anti-HSV1-TK ({fraction
(1/1000)}). Antibodies were diluted in 1% NHS in TBS. The following
day, sections were washed three times with TBS before incubation
with a {fraction (1/200)} dilution of the secondary antibody
(rabbit anti-mouse immunoglobulins biotinylated, Dako Ltd., High
Wycombe, Bucks, UK) for 4 h at room temperature. Sections were then
washed three times with TBS before incubation with Avidin/Biotin
complex (Vectastain ABC Kit, Vector Laboratories, Bretton,
Peterborough, UK) for 3 h at room temperature. Subsequently,
sections were washed three times with PBS and two additional times
with 0.1 M acetate buffer, pH 6.
[0083] Staining was developed by incubating the sections for 5 min
at room temperature with a solution containing equal volumes of:
(i) 0.2 M acetate buffer pH 6 containing 48 g/l ammonium nickel
sulphate, 4 g/l glucose, 0.8 g/l ammonium chloride, and, (ii) 1 g/l
3,3'-diaminobenzidine and 50 mg/l glucose oxidase in distilled
water. The staining reaction was stopped by washing the sections
two times in 0.1 M acetate buffer pH 6 and two additional times in
PBS. Sections were placed on gelatin coated slides, dehydrated,
coverslipped and mounted.
[0084] Statistical Analysis
[0085] Survival data were analysed by Kaplan-Meier estimator
analysis, and compared using the generalised Wilcoxon test
(Prentice-Peto).
[0086] Magnetic Resonance Imaging
[0087] Proton magnetic resonance imaging of the rat brain was
performed with a 4.7T, 15 cm horizontal bore Biospec (Bruker/Oxford
Instruments) system, using a 2.5 cm surface coil. During imaging,
animals were maintained under general anaesthesia by means of a
Halothane (Zeneca Ltd., Macclesfield, Cheshire, UK)/oxygen gas
mixture. In order to detect the presence of turnouts in the brain,
axial images, of 2 mm slice thickness, were acquired at
approximately 2 mm intervals. The position of the slice of
interest, relative to the plane of coil, was selected by applying a
90.degree. "hard" pulse of between 35 and 90 .mu.s duration. In
preliminary studies, T.sub.1, T.sub.2, magnetisation transfer
contrast (MTC) and diffusion-weighted pulse sequences were
compared, in order to determine the conditions required to give
optimum contrast between tumour and normal brain tissue. While the
tumours were detectable in T.sub.2-weighted images, MTC.sup.43,44
provided greater contrast and was used in all subsequent
experiments. The MTC sequence involved the application of a pulse
of radiation on 1 s duration, with an offset of 5 kHz and an
amplitude of 1.5.times.10.sup.-5 T. In-plane resolution was
240.times.480 .mu.m for a field of view of 6.2 cm. The superiority
of this imaging technique in the detection of gliomas has been
demonstrated previously.sup.45.
[0088] Detection of Adenoviral Genome in Brain Sections Using
PCR
[0089] Adenoviral sequences were detected in free-floating
vibratome-cut brain sections using the polymerase chain reaction
(PCR). Briefly, sections were digested for 24 hours at 37.degree.
C. in 10 mM Tris-HCl (pH 8), 10 mM NaCl, 25 mM EDTA, 1% SDS and 4
mg/ml proteinase K. The proteinase K was heat inactivated at
95.degree. C. for 10 minutes after which two rounds of
phenol:chloroform:isoamyl alcohol (25:24:1) extraction were carried
out. The genomic DNA was then ethanol precipitated with 3M sodium
acetate (pH 5.2), washed with 70% ethanol and then re-suspended in
sterile water contaning 20 .mu.g/ml DNase-free RNase.
[0090] Ad 5 transcription unit IVa2, Ad 5 E1B, HSV-1 TK and
.beta.-actin sequences were detected using four different primer
sets. Primers A and B (FIG. 5c) are specific to the IVa2
transcription unit of the Ad 5 genome and produce a PCR product of
686 bp. Primers C and D (FIG. 5c) are specific to the EIB
transcription unit of the Ad 5 genome and produce a PCR product of
560 bp.sup.35. Primers E and F (FIG. 5c) are specific to HSV-1 TK
and produce a PCR product of 365 bp from TK and .DELTA.TK. Primers
G and H have been modified from a method for detecting chicken
.beta.-actin.sup.46 and produce a PCR product of 340 bp from exon 4
of rat cytoplasmic .beta.-actin. In a 50 .mu.l PCR reaction, 5-10
.mu.l of genomic DNA was used in a solution containing 1.times.PCR
buffer (Promega, Southampton, UK), 200 .mu.M dATP, 200 .mu.M dTTP,
200 .mu.M dCTP, 200 .mu.M dGTP, 2 mM MgCl.sub.2, 2 ng/ml each
primer and 1U Taq polymerase (Promega, Southampton, UK). PCR
conditions were 35 cycles of: 30 seconds denature, 30 seconds
anneal, and 1 minute extension followed by a further 10 minutes
extension. The annealing temperatures for primer pairs a/b, c/d,
e/f and g/h were 56.degree. C., 57.degree. C., 63.degree. C. and
63.degree. C. respectively. The PCR products were separated on a 2%
agarose gel and visualised on a UV transilluminator using ethidium
bromide staining.
1 Sequences were as follows; A: 5'-AAGCAAGTGTCTTGCTGTCT-3'; (SEQ ID
NO. 1) B: 5'-GGATGGAACCATTATACCGC-3'; (SEQ ID NO. 2) C:
5'-CAAGAATCGCCTGCTACTGTTGTC-3'; (SEQ ID NO. 3) D:
5'-CCTATCCTCCGTATCTATCTCCACC-3'; (SEQ ID NO. 4) E:
5'-AAAACCACCACCACGCAACT-3'; (SEQ ID NO. 5) F:
5'-GTCATGCTGCCCATAAGGTA-3'; (SEQ ID NO. 6) G:
5'-CCAGCCATGTACGTAGCCATCC-3'; (SEQ ID NO. 7) H:
5'-GCAGCTCATAGCTCTTCTCCAGG-3'. (SEQ ID NO. 8)
[0091] Peripheral Priming with CNS-1 Cells
[0092] CNS-1 cells were treated with 2 .mu.g/ml mitomycin C
overnight to arrest cell division. Twenty-five thousand mitomycin C
treated CNS-1 cells (primed rats), or PBS (controls), was injected
subcutaneously into the flank of Lewis rats (n=4, per each group).
Thirty days later all animals were rechallenged by implanting 5,000
CNS-1 cells into the striatum, unilaterally. All animals primed
peripherally with mitomycin C treated CNS-1 cells survived, while
those primed with PBS died by day 30 post-tumour implantation.
Surviving animals were perfused 90 days after intracerebral
challenge.
[0093] Results
[0094] Adenovirus Encoding HSV-1 TK Plus Ganciclovir, Inhibits the
Growth of CNS-1 Gliomas Implanted into the Brains of Syngeneic
Lewis Rats
[0095] Implantation of 5000 CNS-1 cells unilaterally into the
striatum of Lewis rats killed animals within 30 days (FIG. 1).
Injection of 8.times.10 infectious units (IU) of a
replication-defective recombinant adenovirus (RAd) expressing
either the full length HSV1-TK gene (RAd-128), or a truncated,
biologically active HSVI-TK gene of reduced intrinsic
toxicity.sup.26-28--HSV1-(TK(RAd-127), into the same site at 3 days
post-implantation, followed by ganciclovir treatment for seven
days, almost completely inhibited CNS-1 glioma growth. Animals were
monitored by weekly magnetic resonance imaging (MRI) brain scans to
assess treatment effectiveness. Tumour growth was only seen in a
single animal treated with RAd-117. No MRI, clinical, or anatomical
evidence of tumour growth was observed in any other animals.
Survival at 3 months post tumour implantation was 100% in animals
injected with RAd-128 and 83-100% in animals injected with RAd-127
(survival of animals injected with RAd-127 shown in FIG. 1 was
83%). The survival rates of animals injected with either RAd-128 or
RAd-127 were significantly better than of those animals treated
with either RAd-35, an adenovirus vector expressing
(-galactosidase, or vehicle alone (p=0.0079). No significant
differences were observed in the survival either between vehicle
and RAd-35 treated groups (p=0.1232), or between groups of animals
treated with either RAd-128 or RAd-127 (p=0.3613). In two identical
repeat experiments, no tumour growth was detected in animals
treated with either RAd128 or RAd127.
[0096] Chronic Active Inflammation Following the Complete
Inhibition of CNS-1 Tumour Growth by Gene Therapy: Astrocytosis,
Microglia/Macrophage and Lymphocyte Infiltration, and Loss of
Myelinated Fibres.
[0097] General Histopathological Analysis Long-term (ninety days)
survivors in our experimental syngeneic glioma trials were
perfusion-fixed, and their brains analysed histopathologically for
the distribution of glial, inflammatory, and immune cell markers,
as well as for the integrity of myelin fibres and oligodendrocytes.
Examination of haematoxylin and eosin stained sections revealed the
presence of inflammatory infiltrates (i.e. diffuse hypercellularity
within the white matter, striatum and perivascular cuffs), and
lateral ventricle enlargement (also detected by MRI), ipsilateral
to tumour and viral vector injection (FIGS. 2a, b).
[0098] Astrocytosis Immunohistochemical staining for the astrocyte
markers vimentin (FIGS. 2c, d) and glial fibrillary acidic protein
(GFAP) (FIGS. 2e, f) indicated a widespread activation of
astrocytes. GFAP is expressed by astrocytes, and is upregulated
upon activation. Vimentin is undetectable in resting adult rodent
astrocytes, but is also upregulated upon activation. Astrocyte
activation was bilateral, but was strongest in the ipsilateral
subcortical white matter. Vimentin-positive cells displayed typical
astrocytic morphology, with perivascular end-feet. The distribution
of activated GFAP immunoreactive astrocytes was much wider than the
area occupied by vimentin-immunopositive cells. Astrocyte
activation was seen in all animals.
[0099] Microglial/macrophage activation and lymphocyte infiltration
Activated ED1 immunoreactive macrophages/microglia, displaying
ongoing phagocytosis (i.e. containing tissue debris), were found
mainly ipsilaterally, over a more restricted area than that
occupied by activated astrocytes (illustrated in FIGS. 2g, h).
Within the ipsilateral subcortical white matter, the area occupied
by ED1+, CD3+, leucosyalin+, or CD8+ cells overlapped with the
hypercellularity detected in the hematoxylin and eosin, GFAP or
vimentin stained sections (FIG. 2). In the striatum, activated
microglia/macrophages were found surrounding the needle track, and
infiltrating trans-striatal white matter tracts. Activated
microglia/macrophages were distributed throughout the dorsal and
ventral subcortical white matter, the corpus callosum, and the
ipsilateral anterior commissure. Only very few could be detected in
the contralateral subcortical white matter (see FIGS. 3e, f).
Activated microglial/macrophages were also found within
perivascular cuffs (FIG. 3g), together with, leucosyalin+, CD3+and
CD8+lymphocytes (FIG. 3h). Lymphocytes were also found within the
ipsilateral subcortical white matter, as well as infiltrating
striatal tissue (FIG. 3i, l).
[0100] Loss of Myelinated Fibres
[0101] The loss of Luxol fast blue staining strongly suggested a
substantial reduction of myelinated fibres in the ipsilateral
subcortical white matter (FIGS. 3a-b), which spread into its
ventral extension. Luxol fast blue staining in the injected
striatum was weaker than in the contralateral side, suggesting
actual loss of myelinated fibres also within the striatum.
Examination of semithin Epon-embedded sections (FIG. 3j) stained
with osmium and toluidine blue to highlight myelinated fibres,
confirmed the loss of myelinated fibres within the subcortical
white matter. This also indicated the presence of increased
extracellular space (due to fibre loss and edema), and an increase
in cellularity, composed mostly of astrocytes and oligodendrocytes
(FIG. 3j-l). The reduced density of myelinated fibres, the presence
of oligodendrocytes (identified using specific CNPase antibodies;
illustrated in FIG. 3k), together with the absence of primary
demyelinated axons, strongly sugoests that the loss of myelin
fibres is secondary to tissue degeneration, rather than due to
primary immunemediated demvelination. The presence of axonal
spheroids (FIG. 3j) further suggests on going axonal
degeneration.
[0102] Long-Term Presence of Immunoreactive HSV1-TK Transgene in
the Brains of Rats
[0103] We assessed the presence of HSV1-TK immunoreactivity in the
brains of animals surviving tumour gene therapy for 3 months.
Surprisingly, very strong and widespread immunoreactivity was
detected (FIGS. 3c-d, FIG. 4, FIG. 5a). In the ipsilateral
hemisphere, strong immunoreactivity was encountered in an area
overlapping with the distribution of ED1+ microglia/macrophages
within the subcortical white matter. Further, we detected strong
HSV1-TK immunoreactivity throughout the ipsilateral striatum, both
in neurons and axonal processes, as well as throughout the
contralateral hemisphere (FIGS. 4, 5a).
[0104] Large numbers of HSV1-TK immunoreactive neurons, axons, and
synaptic boutons, were distributed throughout significant areas of
the ipsilateral and contralateral cortex (FIGS. 3c, d; 4a-c; 5a).
Immunoreactive neurons were mainly of pyramidal morphology, and
were concentrated in layers II/III and V (FIG. 4a-c). This strongly
suggests that cortico-cortically projecting neurons contain high
levels of HSV-1 TK protein. Importantly, brain areas displaying
large numbers of strongly immunoreactive HSV1-TK neurons throughout
the contralateral hemisphere (outside of the subcortical white
matter), proved to be completely devoid of any ED1+ activated
macrophages, or CD3/CD8 positive lymphocytes (see FIGS. 2g, 3e-f).
Contralateral striata, only contained a large number of HSV1-TK
immunoreactive axons (FIG. 3d, FIG. 5a). These most likely
represent axons of cortical neurons projecting to lower levels of
the neuraxis. Although strong labelling was found in all animals
examined, the distribution of labelled cells varied between
animals.
[0105] To exclude any non-specific immunoreactivity, the following
controls were performed:
[0106] (i) Sections from animals injected with RAd-127 or RAd-128
and ganciclovir were immunoreacted with secondary antibodies in the
absence of primary antibodies. No positive staining was observed,
indicating that the secondary antibodies were not cross-reacting
with non-specific tissue components.
[0107] (ii) Sections from animals treated with RAd35 and
ganciclovir, or vehicle and ganciclovir were immunoreacted with
primary anti-HSV1-TK and secondary antibodies. No immunopositivity
was observed. This excludes the possibility that virus and/or
ganciclovir injection might induce the production of an endogenous
protein detected by anti-HSV1-TK antibodies.
[0108] Also, viral stocks were tested prior to injection using the
supernatant rescue assay and were shown to be devoid of replication
competent adenovirus. To confirm that no very low level
contamination could have been amplified in the brain during the
three months of the experiment, a PCR based method was devised to
detect the presence of any replication competent virus in the
brain. Three regions of the viral genome were amplified by PCR from
the same brain sections used for immunohistochemistry (FIGS. 5b,
c). The IVa2 region is present in the genome of vectors, and in the
genome of any replication competent virus. The E1B region is
present in the genome of a replication competent virus, and in 293
cells, but not in the E1 deleted vectors. TK sequences will be
present only in RAd127 and RAd128, and a .beta.-actin sequence was
used as a control for DNA extraction.
[0109] We amplified the TK and the IVa2 region, but not the E1B
fragment, from sections of brains injected with either viral vector
3 months earlier (FIG. 5b). This demonstrates that vector genomes,
but not replication competent viral genomes, were present. To
confirm that the E1B fragment, if present, could be amplified from
brain tissue, a preparation of an unrelated viral vector
contaminated with replication competent adenovirus (as assessed
using the supernatant rescue assay) was injected into the brain.
From sections taken from such brains we amplified both the IVa2 and
the E1B region, as expected (FIG. 5b).
[0110] Persistent Lymphocyte Infiltration is Not Seen Following
Immune-Mediated Elimination of CNS-1 Glioma Cells, but Does Occur
Following the Injection of RAd128 and Ganciclovir Treatment.
[0111] To determine whether the chronic lymphocyte infiltration and
inflammation was caused by (a) the elimination of CNS-1 cells, (b)
the administration of viral vectors expressing HSV1-TK, or (c) the
subsequent administration of GCV, these variables were tested
independently. Peripheral priming of Lewis rats with 25,000
mitomycin-C treated CNS-1 cells, protected rats from a lethal
intracerebral challenge with 5,000 CNS-1 cells. Surviving rats were
perfusion-fixed 90 days following the intracerebral challenge. No
tumour, CD8+, or CD4+, cells could be detected (FIG. 6). Only a
modest increase of ED1+ macrophages/microglial cells was detected,
compared to that following the inhibition of tumour growth by gene
therapy (compare FIGS. 6a, b with FIGS. 2g, h). Thus,
immune-mediated elimination of CNS-1 cells does not lead to a
prolonged infiltration of lymphocytes into the brain. Intracerebral
injection of 8.times.10.sup.7 IU of RAd-128 in the absence of CNS-1
cells, followed by administration of ganciclovir or saline for 7
days, and perfusion-fixation 1 or 3 months later, led to a chronic
brain inflammatory infiltration, with higher numbers of CD8+
lymphocytes in animals treated with ganciclovir (FIG. 7).
[0112] Long Term Transgene Expression using RAd-128 Encoding the
Herpes Simplex Virus Type 1 Thymidine Kinase Gene Under the Control
of a Short Powerful Immediate Early CMV Promoter.
[0113] Animals were injected with 1.times.10.sup.8 infectious units
(iu) of RAd-128, and either injected with ganciclovir or saline
twice daily for 7 days. Groups of animals were then perfused 1
month, 3 months, 5 months, or 12 months later. Animals were
perfusion-fixed and brains were cut in serial sections.
[0114] Immunostaining with antibodies against HSV1-TK showed very
widespread distribution of immunoreactive neurons, which were found
throughout the striatum, cortex, and even distant sites such as the
substantia nigra (FIG. 8). It is important to notice that not only
were neurons labelled on the ipsilateral side, but also on the
contralateral side of the brain. This wide distribution has never
been reported with any other transgene encoded by either adenoviral
vectors, or any other viral vector.
2TABLE 1 Expression and distribution of transgenes acutely and long
term following the injection of RAd-HSV1-TK or RAd-.beta.gal into
the brains of rodents RAd-HSV1-TK RAd-.beta.gal Striatum ++++ ++
Distant cortical sites ++++ - Distant subcortical sites ++++ -
Longevity of expression ++++ +
[0115] The results shown in the above table indicate that following
the injection of RAd-HSV1-TK expression of transgene is very
widespread and long term. Following injection of RAd-.beta.gal,
transgene expression is only seen in the striatum, and only very
little can be detected long term.
[0116] Discussion
[0117] Three main findings were made in the course of our
conditional cytotoxic gene therapy studies in a syngeneic rat
glioblastoma model: (i) complete tumour growth inhibition in the
majority of animals; (ii) a chronic, ongoing, inflammatory process,
characterised by T-cell and macrophage/microglial infiltration and
activation, and loss of myelinated fibres and axons in long-term
survivors; and, (iii) transgenic HSV1-TK was still being expressed
at very high levels in neurons throughout the brains of survivors
ninety days post vector administration.
[0118] This is the first report of: (i) chronic active inflammation
in response to a single, successful, brain glioma gene therapy
regime; and, (ii) the long term presence of the therapeutic enzyme,
HSV1-TK. Furthermore, we demonstrate that the chronic inflammatory
process does not impair long-term transgene expression in the
brain. As the presence of HSV1-TK throughout the contralateral
cortex and striatum did not result in overt local inflammatory
responses, additional mechanisms will have to be invoked to explain
the usual short lived transgene expression following adenovirus
vector-mediated gene transfer to the brain.sup.18-21,23-24.
[0119] Long-Term Striatal and Peri-Striatal Inflammatory Responses
to the Treatment of Syngeneic Gliomas
[0120] Most previous experimental models of glioblastoma gene
therapy have used C6, 9L, or F98 glioma cells8-17, and failed to
report any chronic inflammatory responses. Following a single
administration of adenovirus encoding the marker gene
.beta.-galactosidase or HSV1-TK to either rats, mice, non-human
primates, or human glioma, mainly acute, short-lived, and
dose-dependent, inflammatory responses have so far been
described.sup.18-21, 23-24, 29-31. The ongoing nature of the
inflammatory process detected in our model is supported by our
finding of perivascular cuffs, composed of both T-cells and
activated microglia/macrophages. Importantly, injection volumes,
viral and ganciclovir doses, and our experimental paradigm, are
within the range described in the literature.sup.8-17. However,
this is the first report of a syngeneic glioma gene therapy model
in Lewis rats, which are highly susceptible to experimental
allergic encephalomyelitis.sup.32.
[0121] Previously, localised demyelination has only been described
following the peripheral readministration of adenovirus
vectors.sup.19. Moreover, in our experiments, the chronic
inflammatory response was also limited to the hemisphere originally
injected with tumour cells and viruses. Importantly, no
inflammatory responses were detected at any distant sites
expressing high levels of immunoreactive HSV1-TK. Further, our data
uncovered a loss of myelinated fibres, edema, and indices of
ongoing axonal degeneration, while oligodendrocytes were preserved
and primary demyelinated axons absent. This demonstrates that the
loss of myelinated fibres, is not primary, or immune-mediated, but
secondary to tissue injury and axonal loss.
[0122] Adenovirus injection into brain parenchyma stimulates the
secretion of L-1 and IL-6, while injection into the lateral
ventricle induces secretion of IL-1, L-6, and TNF-.alpha..sup.20.
Thus, the immune-suppressive microenvironment of the brain and
gliomas (which express TGF-.beta..sup.33 and Fas-Ligand.sup.34)
could be modified by viral-mediated gene therapy, through the
secretion of pro-inflammatory cytokines, coupled to inflammation
elicited through HSV1-TK and ganciclovir mediated cell killing.
This could enhance tumour immunogenicity and improve gene therapy's
anti-tumour activity.
[0123] The persistent inflammation is not exclusively due to the
elimination of CNS-1 cells, since the subcutaneous priming with
growth arrested CNS-1 cells completely protected animals from an
intracerebral challenge, without leading to a chronic inflammatory
response. Thus, immune system-mediated elimination of glioma cells
(as opposed to adenovirus mediated gene therapy) does not lead to
chronic inflammation and lymphocyte infiltration. Importantly
however, injection of RAd-128 followed by ganciclovir
administration did cause an important influx of CD8+ cells, which
was much reduced in the absence of ganciclovir treatment. Hence,
the persistent inflammation is a result of the combined effect of
HSV1-TK and ganciclovir, but is not a direct result of the
elimination of the tumour cells per se. Whether this effect will be
shown to be specific to adenovirally-encoded HSV1-TK, or whether it
will be seen when HSV1-TK is expressed by other viral vectors
remains to be determined.
[0124] Long-Term Presence of the Adenovirally Encoded HSV1-TK
Transgene
[0125] Another significant finding was the widespread presence of
immunoreactive HSV1-TK within ipsilateral and contralateral
neocortex and striatum, as well as within ipsilateral glia and
inflammatory cells 3 months following vector injection and
ganciclovir administration. So, HSV1-TK immunoreactive cells either
became infected following the administration of ganciclovir, or are
resistant to HSV1-TK plus ganciclovir dependent cytotoxicity. HSV-1
immunoreactive neurons displayed normal morphologies, suggesting
that the long-tern presence of HSV1-TK and ganciclovir
administration did not compromise neuronal survival. This contrasts
with sympathetic neurons in culture, in which infection with
adenovirus vectors led to neuronal death in a few days.sup.35. Our
findings also contrast with previously published experiments
describing much more anatomically restricted adenoviral encoded
neuronal protein expression.sup.18, 19, 21-24.
[0126] The presence of HSV1-TK throughout the ipsilateral and
contralateral neocortex was restricted to pyramidal neurons, mainly
located in layers II-III and V, which contain callosally and
cortico-cortically projecting pyramidal cells. Such neurons may
have taken up vectors through axonal varicosities present on axons
coursing throughout the subcortical white matter overlying the
injected striatum.sup.36. Alternatively, HSV1-TK protein could have
been released by dying cells (if spared from intracellular
degradation) and taken up by axonal terminals to be transported
retrogradely to parent neuronal cell bodies. This is unlikely,
however, given the widespread distribution of HSV1-TK protein and
viral vector genomes, in distant cells, including neurons,
throughout the brain. The presence of vector genomes, but the
absence of replication competent virus from brains of long term
survivors, indicates the apparent stability of, and long term
expression from, adenoviral vectors injected into the rodent
brain.
[0127] Implications for Clinical Gene Therapy Trials of
Glioblastoma Multiforme
[0128] In spite of aggressive surgery, chemo- and radiotherapy,
median survival of glioblastoma patients is below 12-15 months, and
has not improved during the last 30 years. This calls for novel
treatments, such as gene therapy. All current treatments have
significant side effects. Surgery can damage vital brain areas,
chemotherapy has very high toxicity, and widespread demyelination
is a long-term consequence of radiotherapy.
[0129] The above experiments appear to show the operation of two
phenomena. One, is the enhanced distribution of transgene, which is
not seen with any other transgene. The second one, is the very long
term and distributed expression of the transgene throughout large
areas of the brain. The basis underlying this phenomenon could be
an intrinsic characteristic of the HSV1-TK gene, or the mRNA
encoding HSV1-TK, or the protein itself. This intrinsic
characteristic may be one found in all herpes virus genes. Further,
it could be a characteristic of the gene that is dependent on the
viral vector environment. It is possible that the widespread
distribution and longevity of expression will be conferred onto any
second gene co-expressed with a herpes virus gene, for example
HSV1-TK, either in the same vector, or an associated vector that
can infect the exact same cells simultaneously. These hypothesis
are now being tested. It is further possible that the
administration of ganciclovir confers an advantage, but how big
this is, also remains to be determined.
[0130] Several clinical trials of glioblastoma suicide gene therapy
using retro- and adenoviruses encoding HSV1-TK, in combination with
ganciclovir, are currently ongoing.sup.1-4. Our work has several
important implications for clinical trials of glioblastoma gene
therapy using adenoviruses expressing HSV1-TK: (i) effective gene
transfer may occur to so far unexpected widespread areas of the
human brain; (ii) the development of a chronic inflammatory
response in humans could lead to a loss of myelinated fibres; (iii)
long term persistence of HSV1-TK could lead to improvements in the
clinical trial's schedule of ganciclovir administration. Extending
the administration of ganciclovir could improve the anti-tumour
effect by allowing killing of transduced glioma cells that have not
yet entered the cell cycle during short periods of post-surgical
ganciclovir administration in current use. The severity of gene
therapy's untoward effects will have to be balanced with its
increased anti-glioblastoma efficiency, vis-a-vis the limitations
of currently used therapies.
[0131] Further Examples
[0132] Further studies to evaluate the spread, level, and longevity
of RAd mediated HSV-1 derived-TK expression, driven by the MIE-hCMV
promoter in the Lewis rat brain were carried out and described
below. In particular the aim was to determine whether the high
level and widespread expression of HSV-1-TK following the
administration of an adenoviral vector into the brain seen in the
earlier experiments described above was due to: (i) the ganciclovir
treatment. (ii) tumour presence, or (iii) whether it was transgene
dependent. These factors could not be dissected in the original
experimental design.
[0133] In summary the results of these additional experiments
demonstrate that, using the HSV-1 derived TK as a transgene:
[0134] 1) Despite an inflammatory immune response, intra-striatal
RAd mediated delivery leads to widespread, high-level and long
lived neuronal transgene expression that is transgene
dependent.
[0135] 2) This effect does not depend on ganciclovir treatment of
animals injected with the vector.
[0136] 3) This effect is not dependent upon the co-implantation of
potentially immune-suppressive glioma cells in the brain.
[0137] 4) The effects detected are restricted to the RAd expressing
HSV1-TK, since neither the spread, high level expression, nor
widespread distribution are conferred upon a co-injected vector
expressing a different transgene.
[0138] Harnessing this transgene dependent property could increase
the spread and expression levels of therapeutic transgenes, thus
improving their efficacy in the treatment of neurological
disorders.
[0139] Materials and Methods
[0140] Vectors:
[0141] Experiment 1: Adenoviral vectors used were E1A deleted
recombinant adenoviral vectors encoding the full length Herpes
Simplex Virus-derived Thymidine Kinase gene.sup.47 driven by the
MIE-hCMV promoter, as described above.
[0142] Experiment 2: As experiment 1, in addition to a similar
vector encoding lacZ (RAd 35) and a RAd encoding HPRT (RAd HPRT).
Vectors were titrated by end point dilution, characterised by
Southern blot or PCR and were negative for replication competent
virus, as analysed by supernatant rescue assay, and
lipopolysaccharide.
[0143] Experiment 1:
[0144] 18 adult male Lewis rats (weight 250-300 g) had
5.times.10.sup.7 infectious units (iu) of RAd 128 injected
stereotactically into the mid striatum (co-ordinates: anterior to
bregma+1 mm; lateral+3 mm; ventral-4 mm) whilst under halothane
anaesthesia, using a 25 gauge needle on a 10 .mu.l Hamilton
syringe. 12 hours later, 9 rats received intraperitoneal injections
of ganciclovir 25 mg/kg twice daily and 9 received i.p. saline
injections for 7 days. Four rats, 2 treated with GCV, and 2
injected with saline, were sacrificed by perfusion-fixation with
Tyrode's solution containing 10 units/ml heparin (approx. 200 ml)
followed by 250 ml of 4% paraformaldehyde in phosphate buffered
saline (PBS) at 1 month, 3 months and 5 months. The remaining 6
rats (3 treated with ganciclovir, and three injected with saline)
were sacrificed at 1 year.
[0145] Experiment 2:
[0146] To assess the effects of HSV-1 TK on the expression another
transgene, lacZ, a combination of either RAd TK+RAd HPRT or RAd
35+RAd HPRT or RAd 35+RAd TK was injected into the mid striatum of
9 rats (3 rats per group). The total vector dose was
8.times.10.sup.7 iu (i.e. 4.times.10.sup.7 iu per vector).
[0147] Tissue Processing
[0148] Rat brains were post fixed overnight in 4% paraformaldeheyde
in PBS, then sectioned, using a Leica VT 1000S vibrating blade
microtome at 50 .mu.m. Sections were stained by free-floating
immunohistochemistry for HSV1-TK (rabbit polyclonal anti-HSV1-TK,
courtesy of M. Janicot, Rhone Poulenc Rorer, Paris, France.
Dilution 1:400), .beta.-galactosidase (mouse monoclonal, Promega,
dilution 1:1000), the macrophage marker ED1 (mouse monoclonal,
Serotec, 1:1000 dilution) and cytotoxic T cell and NK cell marker
CD8 (mouse monoclonal, Serotec, 1:500 dilution). Secondary
antibodies used were biotinylated rabbit anti-mouse immunoglobulin
or swine anti-rabbit immunglobulin (Dako, Carpinteria, Calif.,
dilution 1:200).
[0149] RESULTS
[0150] Spread of HSV1-TK
[0151] FIGS. 9 and 10 show coronal brain sections at different
anterior-posterior levels along the neuraxis, 1 month following a
single intra-striatal injection of 5.times.10.sup.7 i.u. of RAd
HSV-1 TK. We detected a diffuse, high level HSV1-TK protein
immunoreactivity throughout the striatum and many areas of the
neocortex, both ipsilateral and contralateral to the injection
site. In addition there was immunoreactivity in the anterior
commissure, nucleus accumbens, ipsilateral nucleus of the
horizontal limb of the diagonal band, magnocellular preoptic
nucleus and several thalamic nuclei e.g. the paracentral,
anteroventral and anteromedial thalamic nuclei (not shown), among
others. FIG. 9 illustrates a difference in the distribution of
HSV-1 TK immunoreactivity between the two hemispheres, and anterior
levels of the neuraxis. Notice the lack of immunoreactivity in the
olfactory cortical areas contralateral to the injection side. As
opposed to the cingulate, frontal and parietal cortices, this area
does not have axonal connections with the contralateral cortex or
striatum, so vector could not reach this area through axonal
pathways. Neurons, as well as their dendritic processes, axons, and
axonal terminals could be clearly detected throughout both
hemispheres. The decussating fibres of cortico-cortical axons,
coursing through the corpus callosum could be clearly identified,
and were seen branching off and entering the neocortex [FIG.
10].
[0152] Longevity of HSV1-TK Immunoreactivity
[0153] Cortical HSV1-TK immunoreactivity was maximal at 1 month
post-vector administration and subsequently declined. FIGS. 11 and
12 show the brains displaying the strongest levels of
immunoreactivity for each time point. Importantly, however,
staining persisted even at 1 year post-vector injection in all 6
brains. HSV1-TK immunoreactivity in the brains of all animals
sacrificed at 1 year are illustrated in FIG. 12. In one brain (FIG.
12d), cortical staining at 1 year was almost as high as at 1 month
(FIG. 11). Striatal staining showed a similar maximum at 1 month
and a gradual decline with persistence at 1 year (results not
illustrated).
[0154] Although at 3 and 5 months there appeared to be more
neocortical HSV1-TK immunoreactivity in the GCV treated group, this
was not the case at either 1 month or 1 year. Indeed, at 1 year the
highest level of immunoreactivity was detected in the brain of an
animal injected with saline. Thus, we conclude that GCV had no
effect on the maintenance of HSV1-TK immunoreactivity in the rodent
brain. Furthermore, we did not detect any major differences in the
degree of spread between the GCV and saline treated groups at any
time point.
[0155] Co-Injection of RAd HSV-1 TK with RAd 35
[0156] To investigate whether the above results were due to a
property of the HSV1-TK protein potentiating transgene expression,
RAd HSV1-TK was co-injected with RAd 35, a first generation
adenoviral vector expressing the transcene lacZ, under the control
of the exact same MIEhCMV promoter [FIG. 13]. The striatal HSV1-TK
immunoreactivity indicates that, from a technical point of view,
the injection in these animals was successful in accuracy and
delivery. Retrograde transport and expression is illustrated by the
numerous HSV1-TK immunopositive neurons in the substantia nigra
pars compacta, known to project to the corpus striatum. However,
serial vibratome sections taken from the same site and
immunoreacted for .beta.-galactosidase, showed very little
expression, limited to the area immediately around the needle tract
of the corpus striatum with negligible retrograde expression in the
substantia nigra pars compacta. A control with RAd 35 co-injected
with RAd HPRT showed similar .beta.-galactosidase expression.
[0157] Inflammatory Response
[0158] There was a strong initial immune response to the vector as
illustrated by the ED1 (a macrophage and activated microglial
marker) [FIG. 14, a-f] and CD8 (NK cell and cytotoxic T cell
marker) [FIGS. 14, g-h] immunoreactivity. Apart from the 1-year
time point [illustrated in FIG. 15], all brains showed similar
levels of inflammation, which gradually declined over the year.
Despite widespread transgene expression, the ED1 and CD8 response
was limited to the area of brain tissue surrounding the injection
site. At 1 year, 5 of the brains showed similar levels of
inflammation. Interestingly, one brain showed widespread ED1
staining [FIG. 15c] associated with enlargement of the ventricles
and reduced striatal volume bilaterally. However, this was not
accompanied by comparable CDS persistence. Levels of inflammation
detected at 1 and 3 months post-injection were similar to those
reported previously.
[0159] DISCUSSION
[0160] This paper illustrates the remarkable phenomenon of
widespread, high level and long term HSV-1 TK expression in the
brain when delivered by a first generation recombinant adenoviral
vector. This phenomenon is not dependent upon the promoter or
vector, but unexpectedly, the HSV1-TK transgene itself. It also
illustrates the shortcomings of relying on gene expression as an
indicator of the efficiency of adenovirus transduction of the
brain: RAd-mediated expression of intracellular proteins would be
judged much less efficient on the basis of most results described
in the current literature.
[0161] This phenomenon can be explained by a combination of
factors. Firstly, since RAds are known to undergo retrograde axonal
transport, the pattern of staining is likely to due to this, as
opposed to diffusion. A recent paper by Gerdes et al g48 showed
that using the MIEmCMV promoter, much higher transgene expression
levels could be achieved in the brain, following the injection of
very low doses of viral vectors. However, although spread of
transgene expression increased, it was still limited to the
ipsilateral striatum. Since the MIEmCMV is a glial specific
promoter, this was not totally unexpected. Even if virus spread to
other parts of the brain by axonal transport, this could possibly
not be detected using MIEmCMV driven transgene expression as a
marker for viral spread. These data show, however, that adenovirus
can diffuse in the brain over much larger distances than previously
thought.
[0162] The most likely explanation that retrograde transport lead
to the spread of RAd-encoded HSV1-TK throughout the brain is
illustrated by the immunopositive axons seen in the corpus
callosum, the main `decussating highway` of the brain. Furthermore,
the piriform cortex contralateral to the injection site was devoid
of HSV1-TK immunoreactivity, while the ipsilateral piriform cortex
did display strong HSV1-TK staining. Importantly, the piriform
cortex is not connected by strong contralateral cortico-cortical or
cortico-striatal connections. Retrograde transport of adenovirus
has been described previously, but the extent detected in our
experiments. was unexpected. Thus, there is either more expression
and/or less clearance of the HSV1-TK protein or HSV1-mRNA. Apart
from the transgene, the vector and expression cassette are the same
as in our vector RAd35, which expresses .beta.-gal. The difference
between .beta.-gal and HSV1-TK expression when co-injected excludes
the possibility of altered anti-vector immune responses leading to
such high levels and widespread HSV1-TK expression. However, it
does not exclude the possibility that the HSV1-TK protein could be
less immunogenic. However, this in unlikely since when tested side
by side HSV1-TK was shown to be more inflammatory than
.beta.-gal.
[0163] The most likely explanation for the high levels of
expression is that the HSV1-TK transgene is potentiating or
stabilising its own expression levels above that dependent on the
MIE-hCMV promoter. This is supported by two studies, which showed
that the HSV-1 TK gene contains several short nucleotide sequences
or sub-elements within its translated region that facilitate
pre-mRNA transport from the nucleus to the cytoplasm. These
sub-elements may also act by enhancing transcriptional activation,
stabilising mRNA and enhancing translation. The mechanisms by which
they function have yet to be fully elucidated. However, at least
one of these `RNA processing enhancers`, an 119 nucleotide sequence
isolated by Liu and Mertz, binds in a sequence specific manner to
heterogeneous nuclear ribonucleoprotein (hnRNP) L. hnRNPs are a
group of ribonucleoproteins which are known to be involved in the
regulation of mRNA transport, turnover and translation.
[0164] The widespread transgene expression with HSV1-TK illustrates
how important either vector and/or transcriptional targeting will
be when using potentially cytotoxic transgenes in the CNS. On the
other hand, harnessing this phenomenon will allow more widespread
transgene expression in the treatment of global brain
disorders.
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