U.S. patent application number 12/357123 was filed with the patent office on 2009-08-20 for mutant herpes simplex viruses comprising nucleic acid encoding a nitroreductase.
This patent application is currently assigned to CRUSADE LABORATORIES LIMITED. Invention is credited to Susanne Moira Brown, Paul Dunn.
Application Number | 20090208460 12/357123 |
Document ID | / |
Family ID | 29763982 |
Filed Date | 2009-08-20 |
United States Patent
Application |
20090208460 |
Kind Code |
A1 |
Brown; Susanne Moira ; et
al. |
August 20, 2009 |
Mutant Herpes Simplex Viruses Comprising Nucleic Acid Encoding A
Nitroreductase
Abstract
An herpes simplex virus wherein the herpes simplex virus genome
comprises nucleic acid encoding a nitroreductase (NTR) is
disclosed. Disclosed herpes simplex viruses are indicated to be
useful in the treatment of cancer which may involve gene directed
enzyme prodrug therapy.
Inventors: |
Brown; Susanne Moira;
(Glasgow, GB) ; Dunn; Paul; (Glasgow, GB) |
Correspondence
Address: |
SWANSON & BRATSCHUN, L.L.C.
8210 SOUTHPARK TERRACE
LITTLETON
CO
80120
US
|
Assignee: |
CRUSADE LABORATORIES
LIMITED
Glasgow
GB
|
Family ID: |
29763982 |
Appl. No.: |
12/357123 |
Filed: |
January 21, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10579606 |
May 16, 2006 |
7498161 |
|
|
PCT/GB2004/004851 |
Nov 17, 2004 |
|
|
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12357123 |
|
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/69.1 |
Current CPC
Class: |
A61K 35/13 20130101;
C12N 2710/16643 20130101; C12N 15/86 20130101; A61P 35/00 20180101;
C12N 2840/203 20130101; C12N 15/1135 20130101; C12N 7/00 20130101;
A61P 35/04 20180101; A61K 2039/5256 20130101; A61P 35/02 20180101;
A61P 43/00 20180101; A61K 48/00 20130101; A01K 2267/0331
20130101 |
Class at
Publication: |
424/93.2 ;
435/235.1; 435/69.1 |
International
Class: |
A61K 35/76 20060101
A61K035/76; C12N 7/01 20060101 C12N007/01; C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2003 |
GB |
0326798.6 |
Claims
1. An oncolytic herpes simplex virus wherein the herpes simplex
virus genome comprises nucleic acid encoding an heterologous
nitroreductase (NTR), wherein the genome has an inactivating
mutation in the RL1 locus such that the herpes simplex virus does
not produce a functional ICP34.5 gene product, and wherein the
herpes simplex virus is a variant of one of HSV-1 strains 17 or F
or HSV-2 strain HG52.
2. The herpes simplex virus of claim 1, wherein the herpes simplex
virus genome comprises nucleic acid encoding an heterologous NTR,
wherein the genome has an inactivating mutation in the RL1 locus
such that the herpes simplex virus does not produce a functional
ICP34.5 gene product, and wherein the genome of the herpes simplex
virus otherwise is the genome of HSV-1 strain 17 or F or HSV-2
strain HG52.
3. The herpes simplex virus of claim 1, wherein the herpes simplex
virus genome comprises a nucleic acid encoding an heterologous NTR,
wherein the herpes simplex virus genome has an inactivating
mutation in the RL1 locus such that the herpes simplex virus does
not produce a functional ICP34.5 gene product, wherein the herpes
simplex virus genome has a mutation in the ribonucleotide reductase
gene, and wherein the herpes simplex genome otherwise is the genome
of HSV-1 strain 17 or F or HSV-2 strain HG52.
4. The herpes simplex virus of claim 1, wherein the herpes simplex
virus is a variant of one of HSV-1 strains 17 or F.
5. The herpes simplex virus of claim 1 wherein the nitroreductase
encoding nucleic acid comprises SEQ ID NO: 2, or a nucleic acid
encoding the polypeptide of SEQ ID NO: 1, or a nucleic acid having
at least 90% sequence identity to SEQ ID NO: 2 or to a nucleic acid
encoding the polypeptide of SEQ ID NO: 1.
6. A method for the treatment of a tumour involving lysing or
killing tumour cells in a patient in need of treatment, the method
comprising the steps of (i) administering to the patient the herpes
simplex virus of claim 1, and (ii) administering to said patient a
therapeutically effective amount of an NTR prodrug.
7. The herpes simplex virus of claim 1 wherein said herpes simplex
virus genome further comprises a regulatory nucleotide sequence
operably linked to said nucleic acid encoding NTR, wherein said
regulatory nucleotide sequence has a role in controlling
transcription of said NTR.
8. The herpes simplex virus of claim 1 wherein said nucleic acid is
located in at least one RL1 locus of the herpes simplex virus
genome.
9. The herpes simplex virus of claim 1 wherein said nucleic acid is
located in, or overlaps, at least one of the ICP34.5 protein coding
sequences of the herpes simplex virus genome.
10. The herpes simplex virus of claim 1 wherein the herpes simplex
virus is a variant of HSV-1 strain 17 mutant 1716.
11. The herpes simplex virus of claim 1 in which all copies of the
ICP34.5 gene present in the herpes simplex virus genome are
disrupted such that the herpes simplex virus is incapable of
producing a functional ICP34.5 gene product.
12. The herpes simplex virus of claim 1 which lacks only one
expressible ICP34.5 gene.
13. The herpes simplex virus of claim 1 wherein said nucleic acid
encoding the heterologous nitroreductase (NTR) forms part of a
nucleic acid cassette integrated in the genome of said herpes
simplex virus, said cassette encoding: (a) said nucleic acid
encoding NTR; and nucleic acid encoding (b) a ribosome binding
site; and (c) a marker, wherein the nucleic acid encoding NTR is
arranged upstream (5') of the ribosome binding site and the
ribosome binding site is arranged upstream (5') of the marker.
14. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 1.
15. A composition comprising the herpes simplex virus of claim 1
and an NTR prodrug.
16. A composition comprising the herpes simplex virus of claim 1
and CB1954.
17. A kit of parts comprising a first container having a quantity
of the herpes simplex virus of claim 1 and a second container
having a quantity of an NTR prodrug.
18. A method of expressing in vitro or in vivo a nitroreductase,
said method comprising the step of infecting at least one cell or
tissue of interest with the herpes simplex virus of claim 1,
wherein the genome of said virus comprises a nucleic acid sequence
encoding an heterologous nitroreductase in at least one of the long
repeat regions (R.sub.L), said nitroreductase operably linked to a
transcription regulatory sequence.
19. A method for the treatment of a tumour comprising the steps of:
(i) administering to a patient in need of treatment a
therapeutically effective amount of an oncolytic herpes simplex
virus, wherein the genome of said virus comprises a nucleic acid
sequence encoding an heterologous nitroreductase (NTR) in at least
one of the long repeat regions (R.sub.L), and wherein the genome
has an inactivating mutation in the RL1 locus such that the herpes
simplex virus does not produce a functional ICP34.5 gene product,
and wherein the herpes simplex virus is a variant of one of HSV-1
strains 17 or F or HSV-2 strain HG52; and (ii) administering to
said patient a therapeutically effective amount of an NTR
prodrug.
20. An oncolytic herpes simplex virus, wherein the genome of said
virus comprises a nucleic acid sequence encoding an heterologous
nitroreductase (NTR) in at least one of the long repeat regions
(R.sub.L), wherein the genome has an inactivating mutation in the
RL1 locus such that the herpes simplex virus does not produce a
functional ICP34.5 gene product, and wherein the herpes simplex
virus is a variant of one of HSV-1 strains 17.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
10/579,606, filed May 16, 2006, entitled "Mutant Herpes Simplex
Viruses Comprising Nucleic Acid Encoding A Nitroreductase," which
is a '371 national stage patent application of PCT/GB2004/004851
filed Nov. 17, 2004, entitled "Oncolytic Herpes Simplex Virus
Encoding A Heterologous Nitroreductase," which claims priority to
United Kingdom Patent Application No. GB 0326798.6 filed Nov. 13,
2004, entitled "Methods for Generating Mutant Virus," each of which
is incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to mutant herpes simplex
viruses wherein the herpes simplex virus genome comprises nucleic
acid encoding a nitroreductase.
BACKGROUND TO THE INVENTION
Herpes Simplex Virus
[0003] The herpes simplex virus (HSV) genome comprises two
covalently linked segments, designated long (L) and short (S). Each
segment contains a unique sequence flanked by a pair of inverted
terminal repeat sequences. The long repeat (RL or R.sub.L) and the
short repeat (RS or R.sub.S) are distinct.
[0004] The HSV ICP34.5 (also .gamma.34.5) gene, which has been
extensively studied.sup.1,6,7,8, has been sequenced in HSV-1
strains F.sup.9 and syn17+.sup.3 and in HSV-2 strain HG52.sup.4 One
copy of the ICP34.5 gene is located within each of the RL repeat
regions. Mutants inactivating both copies of the ICP34.5 gene (i.e.
null mutants), e.g. HSV-1 strain 17 mutant 17162 (HSV1716) or the
mutants R3616 or R4009 in strain F.sup.5, are known to lack
neurovirulence, i.e. be a virulent, and have utility as both gene
delivery vectors or in the treatment of tumours by oncolysis. HSV-1
strain 17 mutant 1716 has a 759 bp deletion in each copy of the
ICP34.5 gene located within the BamHI s restriction fragment of
each RL repeat.
[0005] ICP34.5 null mutants such as HSV1716 are, in effect,
first-generation oncolytic viruses. Most tumours exhibit individual
characteristics and the ability of a broad spectrum first
generation oncolytic virus to replicate in or provide an effective
treatment for all tumour types is not guaranteed.
[0006] HSV1716 is described in EP 0571410 and WO 92/13943 and has
been deposited on 28 Jan. 1992 at the European Collection of Animal
Cell Cultures, Vaccine Research and Production Laboratories, Public
Health Laboratory Services, Porton Down, Salisbury, Wiltshire, SP4
0JG, United Kingdom under accession number V92012803 in accordance
with the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure (herein referred to as the `Budapest Treaty`).
Nitroreductase Prodrug Activation
[0007] Enzyme prodrug therapy is based on the enzymatic activation
of a non toxic or low toxicity prodrug to a form that is
considerably more cytotoxic. The activation may involve enzymatic
reduction of the prodrug to a cytotoxic reduced form.
[0008] The E. coli nitroreductase enzyme (NTR) has been proposed
for use in gene-directed enzyme prodrug therapy (GDEPT) as an
activating enzyme for nitroaromatic prodrugs of the
dinitrobenzamide class.sup.16. E. coli NTR is a homodimeric enzyme
with two active sites and is the oxygen insensitive enzyme from E.
coli (the nfsB gene product). It has the ability to reduce a wide
range of nitro-containing compounds such as nitrofurazone (to the
hydroxylamines) and quinones such as menadione (to the quinols). It
is specifically inhibited by the irreversible inhibitor
dicoumarol.
[0009] The ability of NTR to reduce aromatic nitro groups to the
corresponding hydroxylamine (and possibly amine) derivatives has
been proposed for cancer chemotherapy mainly with the
dinitrobenzamide class of prodrugs. The
5-aziridin-1-yl-2,4-dinitrobenzamide CB1954 (CAS Registry number
21919-05-1) is one such prodrug which has been studied as a prodrug
for GDEPT with NTR.sup.16.
[0010] Cyclic and acyclic nitroaryl phosphoroamide mustard
analogues have also been shown to be activated by E. coli
NTR.sup.17. The acyclic 4-nitrobenzyl phosphoramide mustard showed
167,500.times. selective cytotoxicity toward
nitroreductase-expressing V79 cells with an IC.sub.50 as low as 0.4
nM which is about 100.times. more active and 27.times. more
selective than CB1954.
[0011] Recombinant adenovirus and recombinant retrovirus.sup.10
expressing nitroreductase have been constructed for use with the
prodrug CB1954 with the intention of providing a treatment for
cancer. The recombinant virus is not oncolytic and relies on gene
directed enzyme-prodrug therapy to achieve tumour cell kill.
SUMMARY OF THE INVENTION
[0012] The inventors have determined that herpes simplex virus
having an inactivating mutation in the RL1 locus, more specifically
a mutation which inactivates the function of the ICP34.5 gene
product, such that the herpes simplex virus does not produce a
functional ICP34.5 gene product and is non-neurovirulent, can be
used in the delivery to a cell of a gene encoding a gene product
useful in targeted tumour therapy.
[0013] The inventors have provided a novel second generation
oncolytic mutant HSV. The genome of this mutant HSV comprises the
heterologous (i.e. non-HSV originating) E. coli nitroreductase
protein coding sequence inserted at one or each ICP34.5 locus,
disrupting the ICP34.5 protein coding sequence such that the
ICP34.5 gene is non-functional and cannot express a functional
ICP34.5 gene product. The generated HSV is capable of expressing
the E. coli nitroreductase gene product under control of the
inserted promoter. This virus thus has the oncolytic activity of
HSV strain 17 mutant 1716 and can be used in gene directed
enzyme-prodrug therapy (GDEPT) and has shown significantly enhanced
tumour cell killing in vitro and in vivo when used with the prodrug
CB1954. The mutant virus is designated HSV1716/CMV-NTR/GFP (also
called HSV1790).
[0014] HSV1716/CMV-NTR/GFP is an engineered herpes simplex virus
ICP34.5 null mutant which expresses the nitroreductase (NTR) gene.
This virus provides for enhanced virus induced tumour cytotoxicity.
It combines NTR transgene delivery and CB 1954 prodrug treatment
with the proliferation-specific, lytic capacity of HSV1716.
[0015] The heterologous nitroreductase polypeptide expressed by an
herpes simplex virus according to the present invention may be
useful in gene directed enzyme-prodrug targeting techniques for
tissue specific delivery of active pharmaceutical agents derived by
nitroreductase dependent activation of the NTR prodrug.
[0016] In vivo, the inventors have demonstrated that the
nitroreductase gene, when introduced by HSV1716/CMV-NTR/GFP into
mouse gliomal xenograft models in combination with the prodrug
CB1954, results in delay in tumour growth and in oncolysis.
Administering both the HSV1716/CMV-NTR/GFP virus and CB1954 prodrug
in combination was observed to produce a greater effect than either
virus or prodrug alone, i.e. the combination exhibits a synergistic
effect.
[0017] The results demonstrate that the combination of oncolytic
HSV therapy with gene therapy directed nitroreductase/prodrug
treatment provides an effective means of tumour cell kill and
tumour growth reduction and thereby a treatment for tumour.
[0018] Combining herpes simplex virus HSV1716-mediated oncolysis
with nitroreductase gene transfer has yielded results exhibiting a
surprising synergy and provides a novel therapeutic strategy for
treatment of tumours of all kinds.
[0019] HSV1716/CMV-NTR/GFP has been deposited in the name of
Crusade Laboratories Limited having an address at Department of
Neurology Southern General Hospital 1345 Govan Road Govan Glasgow
G51 5TF Scotland on 5 Nov. 2003 at the European Collection of Cell
Cultures (ECACC), Health Protection Agency, Porton Down, Salisbury,
Wiltshire, SP4 0JG, United Kingdom under accession number 03110501
in accordance with the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the
Purposes of Patent Procedure (herein referred to as the `Budapest
Treaty`).
[0020] Accordingly, the present invention relates to a herpes
simplex virus, wherein the herpes simplex virus genome comprises a
nucleic acid sequence encoding a nitroreductase. The herpes simplex
virus may also be non-neurovirulent.
[0021] At its most general the present invention relates to an
herpes simplex virus wherein the herpes simplex virus genome
comprises nucleic acid encoding a nitroreductase.
[0022] According to one aspect of the present invention there is
provided an herpes simplex virus wherein the herpes simplex virus
genome comprises nucleic acid encoding an heterologous
nitroreductase (NTR).
[0023] Said nucleic acid may encode an E. coli NTR and may
comprise, consist of or include the nucleic acid sequence of SEQ ID
No. 2. Alternatively the nucleic acid may have at least 60%
sequence identity to SEQ ID No. 2. Said degree of sequence identity
may alternatively be one of at least 70%, 80%, 90%, 95%, 96%, 97%,
98% or 99% provided the polypeptide or protein encoded by such
nucleic acid has a nitroreductase function. Identity of sequences
is determined across the entire length of a given nucleotide
sequence. Where sequences are of different length, sequence
identity of the shorter sequence is determined over the entire
length of the longer sequence.
[0024] Said nucleic acid encoding NTR may be selected by its
ability to hybridise to the nucleic acid of SEQ ID No. 2, or its
complement, under high stringency conditions.
[0025] The genome of said herpes simplex virus may further comprise
a regulatory sequence operably linked to said nucleic acid encoding
NTR, wherein said regulatory sequence has a role in controlling
transcription of said nucleic acid.
[0026] The nucleic acid encoding NTR may be located in at least one
RL1 locus of the herpes simplex virus genome. Suitably it may be
located in, or overlap, at least one of the ICP34.5 protein coding
sequences of the herpes simplex virus genome. The nucleic acid may
be located in both (this will usually be all) copies of the RL1
locus or ICP34.5 protein coding sequence.
[0027] The herpes simplex virus is preferably a mutant and may be a
mutant of HSV-1 or HSV-2, more preferably of one of HSV-1 strains
17, F or HSV-2 strain HG52. The herpes simplex virus may be a
further mutant of HSV-1 strain 17 mutant 1716.
[0028] In certain arrangements the herpes simplex virus may be a
gene specific null mutant, such as an ICP34.5 null mutant.
[0029] In other arrangements the herpes simplex virus may lack at
least one expressible ICP34.5 gene.
[0030] In yet another arrangement the herpes simplex virus may lack
only one expressible ICP34.5 gene.
[0031] In yet another arrangement the herpes simplex virus may be
non-neurovirulent.
[0032] In herpes simplex viruses of the present invention the
nucleic acid encoding the NTR may form part of a nucleic acid
cassette permanently integrated in the herpes simplex virus genome,
said cassette comprising nucleic acid encoding:
[0033] (a) said nucleic acid encoding NTR; and nucleic acid
encoding:
[0034] (a) a ribosome binding site; and
[0035] (b) a marker,
wherein the nucleic acid encoding NTR is arranged upstream (5') of
the ribosome binding site and the ribosome binding site is arranged
upstream (5') of the marker, wherein said ribosome binding site has
a role in controlling transcription of said marker.
[0036] A regulatory nucleotide sequence may be located upstream
(5') of the nucleic acid encoding NTR, wherein the regulatory
nucleotide sequence has a role in controlling and regulating
transcription of the nucleic acid encoding the NTR.sup.X and hence
expression of the resulting transcript and polypeptide. The
regulatory sequence may comprise selected promoter or enhancer
elements known to the person skilled in the art, e.g. the
CytoMegalovirus (CMV) promoter. Suitably the regulatory sequence
may be constitutive or inducible.
[0037] The components of the cassette are preferably arranged in a
predetermined order.
[0038] In one preferred arrangement, the nucleic acid encoding NTR
is arranged upstream (i.e. 5') of the ribosome binding site and the
ribosome binding site is arranged upstream (i.e. 5') of the marker.
During transcription a single transcript may be produced from the
cassette comprising a first cistron comprising nucleic acid
encoding NTR (e.g. an mRNA transcript) and a second cistron
comprising nucleic acid encoding the marker wherein the ribosome
binding site is located between the cistrons.
[0039] A transcription product of this cassette may be a bi- or
poly-cistronic transcript comprising a first cistron encoded by the
nucleic acid encoding NTR and a second cistron encoding the marker
nucleic acid wherein the ribosome binding site is located between
said first and second cistrons.
[0040] In another arrangement, the nucleic acid encoding the NTR
may be arranged upstream (i.e. 5') of a first regulatory nucleotide
sequence and the first regulatory nucleotide sequence is arranged
upstream (i.e. 5') of the marker.
[0041] The cassette may disrupt a protein coding sequence of the
herpes simplex virus genome resulting in inactivation of the
respective gene product.
[0042] One suitable ribosome binding site comprises a ribosome
entry site permitting entry of a ribosome to the transcribed mRNA
encoded by the nucleic acid of the cassette such that the ribosome
binds to the translation start signal. Preferably, the ribosome
entry site is an internal ribosome entry site (IRES), more
preferably an encephalomyocarditis virus IRES, permitting
cap-independent initiation of translation. The IRES thus enables
translation of a coding sequence located internally of a bi- or
poly-cistronic mRNA, i.e. of a cistron located downstream of an
adjacent cistron on a single transcript.
[0043] Preferably the marker is a defined nucleotide sequence
coding for a polypeptide which can be expressed in a cell line
(e.g. BHK cells) infected with mutant herpes simplex virus into
which the cassette has been recombined. The function of the marker
is to enable identification of virus plaques containing mutant
virus transformed with the cassette.
[0044] The marker is preferably a detectable marker, more
preferably an expressible marker polypeptide or protein comprising
at least the coding sequence for the selected polypeptide or
protein. The nucleic acid encoding the marker may further comprise
regulatory sequence upstream and/or downstream of the coding
sequence having a role in control of transcription of the marker
mRNA. Preferred markers include the Green Fluorescent Protein (GFP)
protein coding sequence or gene, preferably the enhanced Green
Fluorescent Protein (EGFP) protein coding sequence or gene.
[0045] In other arrangements the marker may comprise a defined
nucleotide sequence which can be detected by hybridisation under
high stringency conditions with a corresponding labelled nucleic
acid probe, e.g. using a fluorescent- or radio-label.
[0046] The cassette may also comprise nucleic acid encoding a
polyadenylation ("polyA") sequence, which sequence is preferably
located downstream (3') of the nucleic acid encoding the marker.
One preferred polyA sequence is the Simian Virus 40 (SV40)
polyadenylation sequence. The preferred location of the polyA
sequence within the cassette is immediately downstream (i.e. 3') of
the marker.
[0047] Mutant herpes simplex viruses of the present invention may
be generated by site directed insertion of a nucleic acid cassette
into the viral genome, more preferably by homologous recombination.
However, the viruses of the invention are not limited to Herpes
simplex viruses obtained in this way.
[0048] In other aspects of the present invention herpes simplex
viruses according to the present invention are provided for use in
a method of medical treatment. Suitably they are provided for use
in the treatment of disease. Preferably they are provided for use
in the treatment of cancer. Suitably they may be provided for use
in the oncolytic treatment of cancer/a tumour. The use of herpes
simplex viruses according to the present invention in the
manufacture of a medicament for the treatment of cancer is also
provided.
[0049] In another aspect of the present invention medicaments
comprising herpes simplex virus mutants according to the present
invention for use in oncotherapy and methods of treating tumours
comprising administering to a patient in need of treatment an
effective amount of a mutant HSV or a medicament comprising or
derived from such HSV are also provided. Methods of lysing or
killing tumour cells in vitro or in vivo comprising the step of
administering to a patient in need of treatment an amount of an
Herpes simplex virus according to the present invention are also
provided.
[0050] A medicament, pharmaceutical composition or vaccine
comprising an Herpes simplex virus according to the present
invention is also provided. The medicament, pharmaceutical
composition or vaccine may further comprise a pharmaceutically
acceptable carrier, adjuvant or diluent. Pharmaceutical
compositions or vaccines may further comprise an NTR prodrug.
[0051] The present invention may also include the following aspects
which may be provided in combination with any of the other aspects
and features described.
[0052] According to another aspect of the present invention a
herpes simplex virus is provided, wherein the genome of said virus
comprises a nucleic acid sequence encoding an heterologous
nitroreductase (NTR) in at least one of the long repeat regions
(R.sub.L).
[0053] According to another aspect of the present invention a
herpes simplex virus is provided, wherein the genome of said virus
comprises a nucleic acid sequence encoding an heterologous
nitroreductase (NTR) and wherein the herpes simplex virus is
non-neurovirulent.
[0054] A composition comprising a herpes simplex virus of the
invention may be provided in combination with an NTR prodrug. The
NTR prodrug may be CB1954.
[0055] According to another aspect of the present invention a
herpes simplex virus for use in the treatment of a tumour is
provided, wherein the genome of said virus comprises a nucleic acid
sequence encoding an heterologous nitroreductase in at least one of
the long repeat regions (R.sub.L).
[0056] According to another aspect of the present invention a
herpes simplex virus for use in the treatment of a tumour is
provided, wherein the genome of said virus comprises a nucleic acid
sequence encoding an heterologous nitroreductase and wherein the
herpes simplex virus is non-neurovirulent.
[0057] According to another aspect of the present invention a
herpes simplex virus is provided, wherein the genome of said virus
comprises a nucleic acid sequence encoding an heterologous
nitroreductase in at least one of the long repeat regions
(R.sub.L), for use, in combination with an NTR prodrug, in the
treatment of a tumour.
[0058] According to another aspect of the present invention a
herpes simplex virus is provided, wherein the genome of said virus
comprises a nucleic acid sequence encoding an heterologous
nitroreductase and wherein the herpes simplex virus is
non-neurovirulent, for use, in combination with an NTR prodrug, in
the treatment of a tumour.
[0059] According to another aspect of the present invention a kit
of parts is provided comprising a first container having a quantity
of an herpes simplex virus of the invention and a second container
having a quantity of an NTR prodrug.
[0060] In another aspect the use of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase in at least one of the long repeat
regions (R.sub.L), in the manufacture of a medicament for the
treatment of a tumour is also provided.
[0061] In another aspect the use of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase and wherein the herpes simplex virus
is non-neurovirulent, in the manufacture of a medicament for the
treatment of a tumour is also provided.
[0062] In another aspect the use in the manufacture of a medicament
for the treatment of a tumour of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase in at least one of the long repeat
regions (R.sub.L), and an NTR prodrug is also provided.
[0063] In another aspect the use in the manufacture of a medicament
for the treatment of a tumour of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase and wherein the herpes simplex virus
is non-neurovirulent, and an NTR prodrug is also provided.
[0064] In another aspect the use of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase in at least one of the long repeat
regions (R.sub.L) in the manufacture of a first medicament for
administering sequentially or simultaneously with a second
medicament comprising an NTR prodrug in the treatment of a tumour
is also provided
[0065] In another aspect the use of an NTR prodrug in the
manufacture of a first medicament for administering sequentially or
simultaneously with a second medicament comprising a herpes simplex
virus, wherein the genome of said virus comprises a nucleic acid
sequence encoding an heterologous nitroreductase in at least one of
the long repeat regions (R.sub.L), in the treatment of a tumour is
also provided.
[0066] In another aspect the use of an NTR prodrug in the
manufacture of a first medicament for administering sequentially or
simultaneously with a second medicament comprising a herpes simplex
virus, wherein the genome of said virus comprises a nucleic acid
sequence encoding an heterologous nitroreductase and wherein the
herpes simplex virus is non-neurovirulent, in the treatment of a
tumour is also provided.
[0067] In another aspect the use of a herpes simplex virus, wherein
the genome of said virus comprises a nucleic acid sequence encoding
an heterologous nitroreductase and wherein the herpes simplex virus
is non-neurovirulent, in the manufacture of a first medicament for
administering sequentially or simultaneously with a second
medicament comprising an NTR prodrug, in the treatment of a tumour
is also provided.
[0068] The time period between sequential administrations may be
such that the herpes simplex virus and NTR prodrug may interact in
the body to produce an active pharmaceutical agent in situ.
Preferred time periods may be less than 15 minutes, less than one
hour, two hours, three hours, four hours, five hours or six hours,
twelve hours, twenty four hours, forty eight hours, one week or two
weeks. Either the herpes simplex virus or NTR prodrug may be
administered first.
[0069] In another aspect a method of treatment of a tumour is
provided comprising the steps of: [0070] (i) administering to a
patient in need of treatment a herpes simplex virus, wherein the
genome of said virus comprises a nucleic acid sequence encoding a
nitroreductase in at least one of the long repeat regions
(R.sub.L); and [0071] (ii) administering to said patient a
therapeutically effective amount of an NTR prodrug.
[0072] In another aspect a method of treatment of a tumour is
provided comprising the steps of: [0073] (i) administering to a
patient in need of treatment a herpes simplex virus, wherein the
genome of said virus comprises a nucleic acid sequence encoding a
nitroreductase and wherein the herpes simplex virus is
non-neurovirulent; and [0074] (ii) administering to said patient a
therapeutically effective amount of an NTR prodrug.
[0075] In the methods of treatment said herpes simplex virus is
preferably capable of killing tumour cells, e.g. by oncolysis.
[0076] In aspects of the invention involving an NTR prodrug, one
preferred prodrug is CB1954.
[0077] In another aspect a method of expressing in vitro or in vivo
a nitroreductase is provided, said method comprising the step of
infecting at least one cell or tissue of interest with a herpes
simplex virus, wherein the genome of said virus comprises a nucleic
acid sequence encoding a heterologous nitroreductase in at least
one of the long repeat regions (R.sub.L), said nitroreductase
operably linked to a transcription regulatory sequence.
[0078] In another aspect a method of expressing in vitro or in vivo
a nitroreductase is provided, said method comprising the step of
infecting at least one cell or tissue of interest with a
non-neurovirulent herpes simplex virus, wherein the genome of said
virus comprises a nucleic acid sequence encoding a heterologous
nitroreductase, said nitroreductase operably linked to a
transcription regulatory sequence.
[0079] Herpes simplex viruses of the invention having nucleic acid
encoding an heterologous nitroreductase in at least one of the long
repeat regions (R.sub.L) of the HSV genome preferably have said
nucleic acid in each of the long repeat regions of the HSV genome.
Two long repeat regions are usually present in the HSV genome.
[0080] The NTR nucleotide sequence may encode a full length
transcript or polypeptide (i.e. comprise the complete NTR protein
coding sequence). Alternatively, provided the polypeptide product
retains nitroreductase activity, the NTR nucleotide sequence may
comprise one or more fragments of the full length sequence
respectively coding for a fragment of the full length transcript or
a truncated polypeptide.
[0081] A fragment may comprise a nucleotide sequence encoding at
least 10% of the corresponding full length sequence, more
preferably the fragment comprises at least 20, 30, 40, 50, 60, 70,
80, 85, 90, 95, 96, 97, 98 or 99% of the corresponding full length
sequence. Preferably, the fragment comprises at least, i.e. has a
minimum length of, 20 nucleotides, more preferably at least 30, 40,
50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100,
1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 2000, 2100, 2200,
2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300,
3400, 3500, 3600, 3700, 3800, 3900 or 4000 nucleotides. The
fragment may have a maximum length, i.e. be no longer than, 20
nucleotides, more preferably no longer than 30, 40, 50, 100, 150,
200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300,
1400, 1500, 1600, 1700, 1800, 1900 2000, 2100, 2200, 2300, 2400,
2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500,
3600, 3700, 3800, 3900 or 4000. The fragment length may be anywhere
between said minimum and maximum length.
[0082] In one preferred arrangement, the mutant HSV is
HSV1716/CMV-NTR/GFP deposited in the name of Crusade Laboratories
Limited having an address at Department of Neurology Southern
General Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotland on
5 Nov. 2003 at the European Collection of Cell Cultures (ECACC),
Health Protection Agency, Porton Down, Salisbury, Wiltshire, SP4
0JG, United Kingdom under accession number 03110501 in accordance
with the provisions of the Budapest Treaty.
[0083] Suitably, the administration of said herpes simplex virus
and/or said NTR prodrug may comprise parenteral administration.
Preferably administration of the herpes simplex virus is by
injection, more preferably injection to the tumour which is to be
treated. The NTR prodrug may also be administered by injection,
which may also comprise direct injection to the site of the tumour.
Alternatively injections may be intravenous.
[0084] Administration of the herpes simplex virus and NTR prodrug
may be simultaneous, e.g. by combining virus and prodrug in a
single composition, or be substantially simultaneous, e.g. one
being administered immediately after the other. Alternatively, a
predetermined time period may be provided between administration of
the herpes simplex virus and the NTR prodrug. The invention is not
limited by the order of administration.
[0085] In a further aspect of the present invention in vitro or in
vivo methods are provided for delivery of nucleic acid encoding a
nitroreductase to at least one cell or to a tissue of interest said
method comprising the step of infecting said cell(s) or tissue with
a herpes simplex virus according to the invention.
[0086] In another aspect of the invention, a kit of parts is
provided comprising a first container in which a quantity of herpes
simplex virus according to the invention is provided and a second
container in which a quantity of NTR prodrug is provided.
Instructions for the administration, optionally including
information on suitable dosages of herpes simplex virus and/or the
NTR prodrug, may also be provided with the kit.
[0087] In another aspect of the present invention a method of
making or producing a modified herpes simplex virus of the
invention is provided comprising the step of introducing a nucleic
acid sequence encoding a nitroreductase at a selected and/or
predetermined insertion site in the genome of a selected herpes
simplex virus.
[0088] As described, the nucleic acid sequence encoding the
nitroreductase may form part of a nucleic acid cassette which is
inserted in the genome of a selected herpes simplex virus by
homologous recombination. Whether part of a cassette or not, the
site of insertion may be in any genomic location selected. One
preferred insertion site is in one or both of the long repeat
regions (R.sub.L), and one copy of the cassette is preferably
inserted in each copy of the long repeat (R.sub.L). More preferably
the insertion site is in at least one (preferably both) RL1 locus
and most preferably it is inserted in at least one (preferably
both) of the ICP34.5 protein coding sequences of the HSV genomic
DNA. It is preferred that the insertion occurs in identical or
substantially similar positions in each of the two repeat regions,
RL1 loci or ICP34.5 protein coding sequences.
[0089] Insertion may be such as to produce a modified virus which
is a non-neurovirulent mutant capable of expressing the encoded
nitroreductase polypeptide upon transfection into mammalian, more
preferably human, cells in vivo and in vitro in a form which is
functional to facilitate the uptake and/or activation of NTR
prodrug. The non-neurovirulent mutant may be an ICP34.5 null
mutant.
[0090] The nucleic acid cassette may be of any size, e.g. up to 5,
10, 15, 20, 25, 30, 35, 40, 45 or 50 kbp in length.
[0091] Preferably, the herpes simplex virus contains at least one
copy of the nucleic acid encoding the nitroreductase in each long
repeat region (R.sub.L), i.e. in the terminal and internal long
repeat (TR.sub.L and IR.sub.L) regions. In a preferred arrangement
each exogenous sequence or cassette is located in an RL1 locus of
the herpes simplex virus genome, more preferably in the DNA of the
herpes simplex virus genome encoding the ICP34.5 gene or protein
coding sequence. The herpes simplex virus thereby lacks
neurovirulence.
[0092] The parent herpes simplex virus, from which a virus of the
invention is derived may be of any kind, e.g. HSV-1 or HSV-2. In
one preferred arrangement the herpes simplex virus is a variant of
HSV-1 strain 17 and may be obtained by modification of the strain
17 genomic DNA. Suitable modifications include the insertion of the
exogenous nitroreductase nucleic acid sequence or
exogenous/heterologous cassette comprising said sequence into the
herpes simplex virus genomic DNA. The insertion may be performed by
homologous recombination of the exogenous nucleic acid sequence
into the genome of the selected herpes simplex virus.
[0093] Although the non-neurovirulent phenotype of the herpes
simplex virus of the invention may be the result of insertion of
the exogenous nucleic acid sequence in the RL1 locus, herpes
simplex viruses according to the present invention may be obtained
by utilising a non-neurovirulent parent strain, e.g. HSV1716
deposited under the Budapest Treaty at the European Collection of
Animal Cell Cultures (ECACC), Health Protection Agency, Porton
Down, Salisbury, Wiltshire, United Kingdom under accession number
V92012803, and inserting the exogenous nucleic acid sequence at
another location of the genome by standard genetic engineering
techniques, e.g. homologous recombination. In this aspect the
location of the herpes simplex virus genome selected for insertion
of the nitroreductase nucleic acid sequence or cassette containing
said sequence may be a neutral location.
[0094] Herpes simplex viruses of the present invention may be
variants of a known `parent` strain from which the herpes simplex
virus of the invention has been derived. A particularly preferred
parent strain is HSV-1 strain 17. Other parent strains may include
HSV-1 strain F or HSV-2 strain HG52. A variant comprises an HSV in
which the genome substantially resembles that of the parent,
contains the nitroreductase nucleic acid sequence or cassette
containing said sequence and may contain a limited number of other
modifications, e.g. one, two or three other specific mutations,
which may be introduced to disable the pathogenic properties of the
herpes simplex virus, for example a mutation in the ribonucleotide
reductase (RR) gene, the 65K trans inducing factor (TIF) and/or a
small number of mutations resulting from natural variation, which
may be incorporated naturally during replication and selection in
vitro or in vivo. Otherwise the genome of the variant will be that
of the parent strain.
[0095] Herpes simplex viruses of the invention may be used alone,
or in combination with an NTR prodrug in a method of medical
treatment. This may involve treatment of diseases associated with
or involving the proliferation of cells, or cancers or tumours of
any kind. Treatment may involve the selective lysis of dividing
cells. This may be oncolysis, i.e. lysis of tumour cells. Tumours
to be treated may be of any kind, may comprise cancers, neoplasms
or neoplastic tissue and may be in any animal or human patient.
[0096] Cancer/tumour types which may be treated may be primary or
secondary (metastatic) tumours. Tumours to be treated may be
nervous system tumours originating in the central or peripheral
nervous system, e.g. glioma, medulloblastoma, meningioma,
neurofibroma, ependymoma, Schwannoma, neurofibrosarcoma,
astrocytoma and oligodendroglioma, or may be non-nervous system
tumours originating in non-nervous system tissue e.g. melanoma,
mesothelioma, lymphoma, hepatoma, epidermoid carcinoma, prostate
carcinoma, breast cancer cells, lung cancer cells or colon cancer
cells. HSV mutants of the present invention may be used to treat
metastatic tumours of the central or peripheral nervous system
which originated in a non-nervous system tissue.
[0097] Herpes simplex viruses of the invention may be used in `gene
delivery` methods in vitro or in vivo. Non-neurovirulent herpes
simplex viruses of the invention are expression vectors and may be
used to infect selected cells or tissues in order to express the
nitroreductase encoded by the herpes simplex virus genome.
[0098] In one arrangement, cells may be taken from a patient, a
donor or from any other source, infected with a herpes simplex
virus of the invention, optionally screened for expression and/or
function of the encoded nitroreductase, and optionally
returned/introduced to a patient's body, e.g. by injection.
[0099] Delivery of herpes simplex viruses of the invention to the
selected cells may be performed using naked virus or by
encapsulation of the virus in a carrier, e.g. nanoparticles,
liposomes or other vesicles.
[0100] In vitro cultured cells, preferably human or mammalian
cells, transformed with viruses of the present invention and
preferably cells expressing the nitroreductase protein as well as
methods of transforming such cells in vitro with said viruses form
further aspects of the present invention.
[0101] In this specification, a mutant herpes simplex virus is a
non-wild type herpes simplex virus and may be a recombinant herpes
simplex virus. Mutant herpes simplex viruses may comprise a genome
containing modifications relative to the wild type. A modification
may include at least one deletion, insertion, addition or
substitution.
[0102] Medicaments and pharmaceutical compositions according to
aspects of the present invention may be formulated for
administration by a number of routes, including but not limited to,
parenteral, intravenous, intramuscular, intratumoural, oral and
nasal. The medicaments and compositions may be formulated in fluid
or solid (e.g. tablet) form. Fluid formulations may be formulated
for administration by injection to a selected region of the human
or animal body.
[0103] In this specification, non-neurovirulence is defined by the
ability to introduce a high titre of virus (approx 10.sup.6 plaque
forming units (pfu)) to an animal or patient.sup.22,23 without
causing a lethal encephalitis such that the LD.sub.50 in animals,
e.g. mice, or human patients is in the approximate range of
.gtoreq.10.sup.6 pfu.sup.21.
[0104] Where all copies of the ICP34.5 gene present in the herpes
simplex virus genome (two copies are normally present) are
disrupted such that the herpes simplex virus is incapable of
producing a functional ICP34.5 gene product, the virus is
considered to be an ICP34.5 null mutant.
[0105] A regulatory sequence (e.g. promoter) that is operably
linked to a nucleotide sequence may be located adjacent to that
sequence or in close proximity such that the regulatory sequence
can effect and/or control expression of a product of the nucleotide
sequence. The encoded product of the nucleotide sequence may
therefore be expressible from that regulatory sequence.
NTR Prodrug
[0106] In this specification, "NTR prodrug" means any chemical
compound or agent which is not toxic, or exhibits low toxicity, to
a selected human or animal body, or to selected cells or tissues of
the human or animal body, and which may be activated by a
nitroreducase enzyme to a chemical compound or agent which is
cytotoxic to the human or animal body or to those selected
cells.
[0107] "Activation" may involve conversion of a non-toxic (or low
toxicity) prodrug to an active cytotoxic form. That conversion may
involve enzymatic reduction of the prodrug by NTR. The enzymatic
reduction reaction may involve the prodrug as a substrate for NTR
and may involve other co-factors.
[0108] Examples of NTR prodrugs may include compounds from the
following classes of molecules: [0109] 1. dinitirobenzamides;
[0110] 2. dinitroaziridinylbenzamides (e.g. CB1954); [0111] 3.
dinitrobenzamide mustard derivatives (e.g. SN23862); [0112] 4.
4-nitrobenzylcarbamates; [0113] 5. nitroindolines; [0114] 6.
nitroaromatics that are substrates of NTR and are activated to
release a cytotoxic phosphoramide mustard or like-reactive species
upon NTR-reduction (also called nitroaryl phosphoramides).sup.17;
[0115] 7. nitroaromatic prodrugs of the dinitrobenzamide class.
Examples of NTR prodrugs are disclosed in references 16 and 17
which are incorporated herein in their entirety by reference.
Nitroreductase (NTR)
[0116] Nitroreductase enzymes commonly catalyze the reduction of
nitro compounds, quinones, and dyes. The enzymatic reduction may
involve the co-factor NADPH.
[0117] In this specification nitroreductase (NTR) refers to an
enzyme capable of activating an NTR prodrug to an active cytotoxic
form.
[0118] Preferred NTR's may have the ability to reduce a wide range
of nitro-containing compounds such as nitrofurazone (to the
hydroxylamines) and quinones such as menadione (to the
quinols).
[0119] Preferred NTR's may be specifically inhibited by the
irreversible inhibitor dicoumarol.
[0120] One preferred NTR is the E. coli oxygen insensitive
nitroreductase enzyme (the nfsB gene product). Sequence information
for E. coli NTR can be found at the NCBI database
(http://www.ncbi.nlm.nih.gov/) (under accession numbers BA000007
(GI:47118301)--E. coli complete genome sequence--and BAB34039
(GI:13360074)-nitroreductase sequence information.
[0121] The amino acid sequence for the E. coli NTR protein (SEQ ID
No. 1) and polynucleotide sequence for the E. coli NTR gene (SEQ ID
No. 2) are reproduced at FIGS. 32(A) and (B) respectively.
[0122] The nucleotide and amino acid sequences of suitable
nitroreductase enzymes may be derived or obtained from any animal,
insect or microorganism including humans, non-human mammals and
bacteria and may be selected from those sequences which are
publicly available. Many sequences for other nitroreductase genes
are publicly available. Examples of other nitroreductase nucleic
acid sequences which may form part of a herpes simplex virus
according to the present invention include the following which are
referred to by their accession number for the NCBI database
(www.ncbi.nlm.nih.gov): [0123] BAB34039 (GI:13360074)--E. coli
[0124] BAA35218.1 (GI:1651240)--E. coli [0125] AAB72053.1
(GI:2415385)--B. subtilis.
Hybridisation Stringency
[0126] In accordance with the present invention, nucleic acid
sequences may be identified by using hybridization and washing
conditions of appropriate stringency.
[0127] Complementary nucleic acid sequences will hybridise to one
another through Watson-Crick binding interactions. Sequences which
are not 100% complementary may also hybridise but the strength of
the hybridisation usually decreases with the decrease in
complementarity. The strength of hybridisation can therefore be
used to distinguish the degree of complementarity of sequences
capable of binding to each other.
[0128] The "stringency" of a hybridization reaction can be readily
determined by a person skilled in the art.
[0129] The stringency of a given reaction may depend upon factors
such as probe length, washing temperature, and salt concentration.
Higher temperatures are generally required for proper annealing of
long probes, while shorter probes may be annealed at lower
temperatures. The higher the degree of desired complementarity
between the probe and hybridisable sequence, the higher the
relative temperature which can be used. As a result, it follows
that higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so.
[0130] For example, hybridizations may be performed, according to
the method of Sambrook et al., ("Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory Press, 1989) using a
hybridization solution comprising: 5.times.SSC, 5.times.Denhardt's
reagent, 0.5-1.0% SDS, 100 .mu.g/ml denatured, fragmented salmon
sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
Hybridization is carried out at 37-42.degree. C. for at least six
hours. Following hybridization, filters are washed as follows: (1)
5 minutes at room temperature in 2.times.SSC and 1% SDS; (2) 15
minutes at room temperature in 2.times.SSC and 0.1% SDS; (3) 30
minutes-1 hour at 37.degree. C. in 1.times.SSC and 1% SDS; (4) 2
hours at 42-65.degree. C. in 1.times.SSC and 1% SDS, changing the
solution every 30 minutes.
[0131] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules is
to calculate the melting temperature T.sub.m (Sambrook et al.,
1989):
T.sub.m=81.5.degree. C.+16.6 Log [Na+]+0.41(% G+C)-0.63(%
formamide)-600/n
where n is the number of bases in the oligonucleotide.
[0132] As an illustration of the above formula, using [Na+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the T.sub.m is 57.degree. C. The T.sub.m of a DNA
duplex decreases by 1-1.5.degree. C. with every 1% decrease in
sequence complementarity.
[0133] Accordingly, nucleotide sequences can be categorised by an
ability to hybridise to a target sequence under different
hybridisation and washing stringency conditions which can be
selected by using the above equation. The T.sub.m may be used to
provide an indicator of the strength of the hybridisation.
[0134] The concept of distinguishing sequences based on the
stringency of the conditions is well understood by the person
skilled in the art and may be readily applied.
[0135] Sequences exhibiting 95-100% sequence complementarity may be
considered to hybridise under very high stringency conditions,
sequences exhibiting 85-95% complementarity may be considered to
hybridise under high stringency conditions, sequences exhibiting
70-85% complementarity may be considered to hybridise under
intermediate stringency conditions, sequences exhibiting 60-70%
complementarity may be considered to hybridise under low stringency
conditions and sequences exhibiting 50-60%% complementarity may be
considered to hybridise under very low stringency conditions.
[0136] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0137] Aspects and embodiments of the present invention will now be
illustrated, by way of example, with reference to the accompanying
figures. Further aspects and embodiments will be apparent to those
skilled in the art. All documents mentioned in this text are
incorporated herein by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0138] FIG. 1. Generation of plasmid RL1.dIRES-GFP from plasmids
pNAT-IRES-GFP and RL1.del.
[0139] FIG. 2. Agarose gel electrophoresis of HpaI digested, CIP
treated, RL1.del. RL1.del was digested with HpaI. The digested DNA
was then treated with Calf Intestinal Phosphatase (CIP) to prevent
the vector re-annealing to itself in subsequent ligation reactions.
A sample of the digested/CIP treated DNA was electrophoresed,
beside a 1 Kbp DNA ladder (Promega), on a 1% agarose gel. HpaI
linearises the vector at 8.6 Kbp.
[0140] FIG. 3. Agarose gel electrophoresis of NsiI/SspI digested
pNAT-IRES-GFP (A) and purified/blunt-ended pCMV-NAT-IRES-GFP-PolyA
(B). Four NsiI/SspI digestions of pNAT-IRES-GFP were
electrophoresed, beside a 1 Kbp DNA Ladder (Promega) on a 1%
agarose gel. The 5.4 Kbp fragments (pCMV-NAT-IRES-GFP-PolyA) were
purified from the gel. The purified DNA was blunt ended using
Klenow polymerase and a sample electrophoresed on an agarose gel to
check its concentration.
[0141] FIG. 4. Identification of RL1.del clones containing the
pCMV-NAT-IRES-GFP-PolyA insert. Ligation reactions were set up with
the purified, blunt ended pCMV-NAT-IRES-GFP-PolyA fragment and HpaI
digested, CIP treated RL1.del. Bacteria were transformed with
samples from the ligation reactions and plated out onto LBA
(Amp.sup.r) plates. Colonies were picked and plasmid DNA was
extracted and digested with AflII. Digested samples were
electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega), on a 1%
agarose gel.
*Clones 5 and 8 contained the pCMV-NAT-IRES-GFP-PolyA insert as two
fragments of the predicted size--4.8 Kbp and 9.2 Kbp--were
generated from AflII digestion. Clones without inserts would not be
digested with AflII as there is no AflII site in RL 1.del. N.B.
Inserts could have been cloned in two orientations, both of which
were acceptable.
[0142] FIG. 5. Determination of the orientation of
pCMV-NAT-IRES-GFP-PolyA in clone 5 (RL1.dCMV-NAT-GFPb).
pCMV-NAT-IRES-GFP-PolyA (blunt ended) could have been cloned into
the HpaI site of RL1.del in two orientations. To determine the
orientation of the insert in clone 5, the plasmid was digested with
XhoI and the digested DNA electrophoresed, beside a 1 Kbp DNA
ladder (Promega), on a 1% agarose gel. If the insert had been
cloned in the orientation shown in A, two fragments of 10.2 Kbp and
3.8 Kbp would be generated from XhoI digestion. If it had been
cloned in the opposite orientation (B), two fragments of 12.4 Kbp
and 1.6 Kbp would be generated. The presence of two fragments of
10.2 Kbp and 3.8 Kbp in the gel confirmed that the insert had been
cloned in the orientation shown in A.
*This XhoI site was present in the initial cloning vector
(RL1.del), upstream of the HpaI site into which
pCMV-NAT-IRES-GFP-PolyA was cloned.
[0143] FIG. 6. Removal of pCMV-NAT from clone 5 (A) and large scale
plasmid preparation of RL1.dIRES-GFP (B). Four samples of clone 5
were digested with XhoI and electrophoresed, beside a 1 Kbp DNA
ladder (L) (Promega), on a 1% agarose gel (A). The larger fragment
of DNA generated from this digestion (10.2 Kbp) was purified from
the gel and ligated back together, at the XhoI sites, to form a
single XhoI site in a new plasmid, designated RL1.dIRES-GFP. A
large-scale plasmid preparation was grown up and the preparation
checked by digesting with XhoI. 1 .mu.l and 4 .mu.l of the digested
DNA was electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega),
on a 1% agarose gel (B). The DNA should produce a single fragment
of 10.2 Kbp when digested with XhoI. The ClaI, BglII, NruI and XhoI
sites of RL1 dIRES-GFP are all unique.
*Clone 5 is the RL1.del plasmid into which has been cloned the 5.4
Kbp pCMV-NAT-IRES-GFP-PolyA fragment from pNAT-IRES-GFP.
[0144] FIG. 7. Generation, detection and purification of ICP34.5
null HSV-1 expressing a gene product of interest.
[0145] FIG. 8. Strategy used to clone pCMV-NTR from pPS949 into
RL1.dIRES-GFP. (1) Digest pPS949 with BamHI and purify the 1.6 Kbp
pCMV-NTR fragment; (2) Digest RL1 dIRES-GFP with BglII and treat
with Calf Intestinal Phosphatase (CIP); (3) Clone the pCMV-NTR
fragment (BamHI ends) into the BglII site of RL1.dIRES-GFP.
*The pPS949 plasmid was a kind gift from Professor Lawrence Young
(University of Birmingham) and contains the E. coli nitroreductase
(NTR) gene downstream of the CMV-IE promoter (pCMV) in pLNCX
(Clontech).
[0146] FIG. 9. Agarose gel electrophoresis of BamHI digested pPS949
(A) and the purified pCMV-NTR fragment (B). Four samples of pPS949
were digested with BamHI and electrophoresed, beside a 1 Kbp DNA
ladder (L) (New England Biolabs), on a 1% agarose gel. The 1.6 Kbp
fragments, consisting of the E. coli nitroreductase (NTR) gene
downstream of the CMV IE promoter (pCMV), were purified from the
gel and a sample of the purified DNA was electrophoresed on an
agarose gel to check its concentration.
[0147] FIG. 10. Agarose gel electrophoresis of BglII digested, CIP
treated RL1.dIRES-GFP.RL1.dIRES.GFP was digested with BglII. The
digested plasmid was then treated with Calf Intestinal Phosphatase
(CIP) to prevent the vector re-annealing to itself in subsequent
ligation reactions. A sample of the digested/CIP treated DNA was
electrophoresed, beside a 1 Kbp DNA ladder (Promega), on a 1%
agarose gel to check its concentration. pCMV-NTR from pPS949 was
subsequently cloned into this digested/CIP treated vector.
[0148] FIG. 11. Determination of the orientation of pCMV-NTR in
clone 4. pCMV-NTR (BamHI ends) could have been cloned into the
BglII site of RL1.dIRES-GFP in two orientations. To determine the
orientation, clone 4 was digested with BglII and XhoI and the
digested DNA electrophoresed, beside a 1 Kbp DNA ladder (Promega),
on a 1% agarose gel. If the insert was in the desired orientation
(A), two fragments (11.5 Kbp and 300 bp) would be generated. If in
the opposite orientation, two fragments of 10.5 Kbp and 1.3 Kbp
would be generated. The presence of a band at 300 bp (and the
absence of a band at 1.3 Kbp) confirmed that the pCMV-NTR fragment
had been cloned into the vector in the desired orientation.
[0149] FIG. 12. Agarose gel electrophoresis of ScaI digested clone
4 (A) and HSV1716/CMV-NTR/GFP viral titres (B). Clone 4
(RL1.dCMV-NTR-GFP) was digested with ScaI, the digested DNA
purified and 5 .mu.l electrophoresed, beside a 1 Kbp DNA ladder
(Promega), on a 1% agarose gel, to check its concentration. 80%
confluent BHK cells were then co-transfected with 10 .mu.l
HSV17.sup.+ DNA and an appropriate volume of the remaining digested
clone 4. The cells were incubated at 37.degree. C. for 3 days until
cpe was evident. Recombinant viral plaques were picked under the
fluorescent microscope, purified and a virus stock, named
HSV1716/CMV-NTR/GFP, grown up. The cell-associated and
cell-released fraction of the virus stock was titrated on BHK
cells.
[0150] FIG. 13. Growth kinetics of HSV17, HSV1716 and
HSV1716/CMV-NTR/GFP in confluent BHK and 3T6 cells. Confluent BHK
and 3T6 cells were infected at a MOI of 0.1 pfu/cell. Infected
cells were harvested at 0, 4, 24, 48 and 72 hrs post infection,
sonicated and progeny virus titrated on BHK cell monolayers. All
viruses replicated with similar kinetics in BHK cells (A); HSV1716
and HSV1716/CMV-NTR/GFP both failed to replicate efficiently in
confluent 3T6 cells (B).
[0151] FIG. 14. Western blot analysis of ICP34.5 expression in
HSV17.sup.+ and HSV1716/CMV-NTR/GFP infected BHK cells. BHK cells
were infected with HSV17.sup.+ and HSV1716/CMV-NTR/GFP at a MOI of
10 pfu/cell. 16 hrs post infection, the cells were harvested and
protein extracts analysed using 10% SDS-PAGE in a Western blot
using a polyclonal anti-ICP34.5 antibody. ICP34.5 was strongly
expressed in HSV17.sup.+ infected cells but was not expressed in
HSV1716/CMV-NTR/GFP infected cells.
[0152] FIG. 15. Western blot analysis of NTR expression in
HSV1716/CMV-NTR/GFP infected cell lines. BHK, C8161, VM and 3T6
cells were infected with 10 pfu/cell HSV1716/CMV-NTR/GFP, HSV17 or
mock infected. 16 hrs post infection, the cells were harvested and
protein extracts analysed in a Western blot using a polyclonal
NTR-specific antibody. Significant NTR expression was detected in
all the HSV1716/CMV-NTR/GFP infected cells. No NTR expression was
detected in the mock or HSV17.sup.+ infected cells.
[0153] FIG. 16. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with
or without CB1954 (50M) on confluent 3T6 cells. Confluent 3T6 cells
in three wells of a 96-well plate were mock infected, infected with
1 or 10 pfu/cell HSV1716/CMV-NTR/GFP or infected with 1 pfu/cell of
HSV1716-GFP. 45 minutes later, infected cells were overlaid with
media containing 50M CB1954 or with media alone and incubated at
37.degree. C. 24, 48, 72, 96, and 120 hrs later, % cell survival
was determined relative to that of mock infected cells without
prodrug using CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (Promega). Figures shown represent the mean of 3
values+/-standard error of the mean.
[0154] FIG. 17. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with
or without CB1954 (50M) on confluent C8161 cells. Confluent C8161
cells in three wells of a 96-well plate were mock infected,
infected with 1 or 10 pfu/cell HSV1716/CMV-NTR/GFP or infected with
1 pfu/cell of HSV1716-GFP. 45 minutes later, infected cells were
overlaid with media containing 50M CB1954 or with media alone and
incubated at 37.degree. C. 24, 48 and 72 hrs later, % cell survival
was determined relative to that of mock infected cells without
prodrug using CellTiter 96 Aqueous One Solution Cell Proliferation
Assay (Promega). Figures shown represent the mean of 3
values+/-standard error of the mean.
[0155] FIG. 18. Confluent 3T6 cells 72 hrs post treatment with 10
pfu/cell HSV1716/CMV-NTR/GFP (A), or 10 pfu/cell
HSV1716/CMV-NTR/GFP with 50 .mu.M CB1954 (B). The extent of cell
death is significantly more pronounced in HSV1716/CMV-NTR/GFP
infected cells overlaid with media containing 50M CB1954 than in
HSV1716/CMV-NTR/GFP infected cells overlaid with normal media. The
extent of cell death following infection of these cells with 10
pfu/cell HSV1716, with or without CB1954, is comparable to that
seen in A (data not shown). 50M CB1954 alone has no effect on these
cells.
[0156] FIG. 19. Confluent C8161 cells 72 hrs post treatment with 10
pfu/cell HSV1716/CMV-NTR/GFP (A), or 10 pfu/cell
HSV1716/CMV-NTR/GFP with 50 .mu.M CB1954 (B). The extent of cell
death is significantly more pronounced in HSV1716/CMV-NTR/GFP
infected cells overlaid with media containing 50 .mu.M CB1954 than
in HSV1716/CMV-NTR/GFP infected cells overlaid with normal media.
The extent of cell death following infection of these cells with 10
pfu/cell HSV1716, with or without CB1954, is comparable to that
seen in A (data not shown). 50 .mu.M CB1954 alone has no effect on
these cells.
[0157] FIG. 20. Weight change (as a guide to health) in athymic
nude mice with subcutaneous A2780 (xenograft) tumours injected
intratumourally with HSV1790. Group size=3 mice per dose. A2780
xenografts at date of intratumoural injection (Day 0) are between
0.5-1 mm in diameter. The xenografts have reached this size 12 days
after injection with 10 million A2780 cells subcutaneously on the
flank of female athymic nude mice.
[0158] FIG. 21. Change in tumour volume over time in athymic nude
mice with A2780 xenografts after intratumoural injection of
HSV1790.
[0159] FIG. 22. Starting tumour sizes of mice.
[0160] FIG. 23. Alterations in weight after treatment with CMV-ntr,
CB1954 or a combination of both.
[0161] FIG. 24. Change in tumour volume after treatment with
CMV-ntr, CB 1954 or a combination of both.
[0162] FIG. 25. Starting tumour volume of each treatment group (see
Table 2).
[0163] FIG. 26. Weight (as a measurement of health) in athymic nude
mice with A2780 xenograft treated with either HSV1790, HSV1716,
CB1954 or a combination of them.
[0164] FIG. 27. Change in tumour volume of xenografts treated with
the prodrug CB1954.
[0165] FIG. 28. Changes in tumour volume in xenograft treated with
10.sup.5 PFU HSV1790 and CB1954.
[0166] FIG. 29. Changes in tumour volume in xenografts treated with
10.sup.6 PFU HSV1790 and CB1954.
[0167] FIG. 30. Changes in tumour volume in xenografts treated with
10.sup.5 PFU HSV1716 and CB1954.
[0168] FIG. 31. Comparison of 10.sup.5 PFU, 10.sup.6 PFU HSV1790
and 10.sup.5 PFU HSV1716.
[0169] FIG. 32. Sequence information for E. coli NTR. (A) Amino
acid sequence of NTR polypeptide (SEQ ID No. 1); (B) polynucleotide
sequence for NTR gene (SEQ ID No.2).
[0170] FIG. 33. Structure of two NTR prodrugs. (A) CB1954; (B)
SN23862.
DETAILED DESCRIPTION OF THE BEST MODE OF THE INVENTION
[0171] Specific details of the best mode contemplated by the
inventors for carrying out the invention are set forth below, by
way of example. It will be apparent to one skilled in the art that
the present invention may be practiced without limitation to these
specific details.
Vectors Useful for Generation of Herpes Simplex Virus Mutants
[0172] Mutant herpes simplex viruses of the invention may be
generated by use of nucleic acid vectors.
[0173] One such vector useful for generation of mutant herpes
simplex viruses according to the present invention is a nucleic
acid vector comprising, consisting or consisting essentially
of:
first and second nucleotide sequences corresponding to nucleotide
sequences flanking an insertion site in the genome of a selected
herpes simplex virus; and a cassette located between said first and
second nucleotide sequences comprising nucleic acid encoding:
[0174] a) one or a plurality of insertion sites; and
[0175] b) a ribosome binding site; and
[0176] c) a marker.
[0177] Another vector useful for generation of mutant herpes
simplex viruses according to the present invention is a nucleic
acid vector comprising, consisting or consisting essentially
of:
first and second nucleotide sequences corresponding to nucleotide
sequences flanking an insertion site in the genome of a selected
herpes simplex virus; and a cassette located between said first and
second nucleotide sequences comprising nucleic acid encoding:
[0178] a) one or a plurality of insertion sites; and
[0179] b) a first regulatory nucleotide sequence; and
[0180] c) a marker.
[0181] The first and second nucleotide sequences may correspond to
nucleotide sequences flanking an insertion site formed in, or
comprising all or a part of, the ICP34.5 protein coding sequence of
the genome of a selected herpes simplex virus.
[0182] The cassette may comprise a plurality of insertion sites,
each insertion site preferably formed by nucleic acid encoding a
specific restriction endonuclease site (`restriction site`).
Together the restriction sites may form a multiple cloning site
(MCS) comprising a series of overlapping or distinct restriction
sites, preferably a series of distinct restriction sites comprising
one or more of the ClaI, BglII, NruI, XhoI restriction sites.
[0183] The encoded components of the cassette may be arranged in a
predetermined order. In one arrangement, the one or plurality of
insertion sites is/are arranged upstream (i.e. 5') of the ribosome
binding site/first regulatory sequence and the ribosome binding
site/first regulatory sequence is arranged upstream (i.e. 5') of
the marker.
[0184] The first and second nucleotide sequences may comprise
nucleotide sequences having identity to regions of the genome
surrounding the insertion site in the selected herpes simplex virus
(the `viral insertion site`). These sequences enable the cassette
to be incorporated at the viral insertion site by homologous
recombination between the first and second nucleotide sequences and
their respective corresponding sequences in the viral genome.
[0185] Thus the first and second nucleotide sequences are flanking
sequences for homologous recombination with corresponding sequences
of a selected viral genome, such homologous recombination resulting
in insertion of the cassette at the viral insertion site.
[0186] The first and second nucleotide sequences may correspond to
nucleotide sequences flanking an insertion site in the RL1 locus of
the HSV genome, more preferably in the ICP34.5 protein coding
sequence of the HSV genome.
[0187] The first and second nucleotide sequences may each be at
least 50 bp in length, more preferably at least 100, 150, 200, 300,
400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500,
1600, 1700, 1800, 1900 2000, 2100, 2200, 2300, 2400, 2500, 2600,
2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700,
3800, 3900 or 4000 bp in length. Each of the first and second
nucleotide sequences may have at least 50% sequence identity to
their corresponding sequence in the viral genome, more preferably
at least 60%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%
99% or 100% identity. Identity of sequences is determined across
the entire length of a given nucleotide sequence. Where sequences
are of different length, sequence identity of the shorter sequence
is determined over the entire length of the longer sequence.
[0188] The first and second nucleotide sequences may be
characterised by the ability of one strand of a given sequence to
hybridise with the corresponding single-stranded complement of the
HSV genome under varying hybridisation stringency conditions.
Suitably, the first and second nucleotide sequences will hybridise
with their corresponding complement under very low, low or
intermediate stringency conditions, more preferably at high or very
high stringency conditions.
[0189] The viral insertion site is the position between the genomic
nucleotide sequences corresponding to the first and second
nucleotide sequences of the vector (the `genomic` and `vector
flanking sequences` respectively) at which homologous recombination
will occur and may be predetermined by selection of the vector
flanking sequences. Where the genomic flanking sequences are
immediately adjacent, the insertion site is the position between
the peripheral and immediately adjacent bases of the two genomic
flanking sequences, such that insertion of the cassette separates
the genomic flanking sequences. Where the genomic flanking
sequences are separated by one or a plurality of bases in the viral
genome, the insertion site is formed by said one or a plurality of
bases which are excised from the genome by the homologous
recombination event.
[0190] The position of the viral insertion site may be accurately
selected by careful selection and construction of the vector
flanking sequences. Accordingly, the vector may be constructed such
that homologous insertion of the cassette results in disruption of
a chosen protein coding sequence and inactivation of the respective
gene product or such that the cassette is inserted at a non-protein
coding region of the viral genome. The complete genome sequences of
several herpes simplex virus strains have been reported and are
publicly available. The complete genome sequence for HSV-1 strain
17syn+ was reported by Dolan et al.sup.3 (incorporated herein by
reference) and the complete genome sequence of HSV-2 strain HG52
was reported by Dolan et al.sup.4 (incorporated herein by
reference) and is available from the EMBL database under accession
code Z86099. Using this information, the vector of the present
invention may preferably be designed for use in generating mutant
HSV-1 (e.g. in strain 17 or F) or mutant HSV-2 (e.g. in strain
HG52).
[0191] The first and second nucleotide sequences (vector flanking
sequences) may each comprise sequence corresponding to the RL
terminal repeat region of the genome of the selected HSV (e.g.
HSV-1 strains 17 or F or HSV-2 strain HG52). The vector flanking
sequences may comprise, consist or consist essentially of
nucleotide sequences of the RL repeat region which flank the
ICP34.5 protein coding sequence. In flanking the ICP34.5 coding
sequence, one or both of the selected sequences may, in the
corresponding HSV genome, overlap, i.e. extend into, the ICP34.5
protein coding sequence or one or both sequences may be selected so
as to not overlap the ICP34.5 protein coding sequence. In a similar
manner, the selected sequences may be chosen to overlap completely
or partially other important encoded signals, e.g. transcription
initiation site, polyadenylation site, defined promoters or
enhancers. In this preferred arrangement the insertion site will
thus comprise all or a part of the ICP34.5 protein coding sequence
and/or be such that the inserted cassette disrupts the ICP34.5
protein coding sequence.
[0192] The vectors described, comprising first and second
nucleotide sequences corresponding to regions of the RL repeat
region flanking and/or overlapping the ICP34.5 protein coding
sequence, may be used in the generation of ICP34.5 null mutants
wherein all or a portion of the ICP34.5 protein coding sequence is
excised and replaced during the homologous recombination event such
that both copies of the ICP34.5 coding sequence are disrupted.
[0193] The recombination may result in an insertion of nucleic acid
within the ICP34.5 protein coding sequence thereby disrupting that
sequence. In that case, successfully transformed virus are thus
mutants incapable of generating the ICP34.5 active gene product
from at least one copy, and preferably from both copies, of the
ICP34.5 gene.
[0194] Successfully transformed viruses are thus mutants incapable
of generating the ICP34.5 active gene product.
[0195] Each component of the cassette may be positioned
substantially adjacent the neighbouring component such that a
single bicistronic transcript comprising or consisting essentially
of the mRNA encoding the nucleotide sequence of interest, ribosome
binding site and marker is obtainable.
[0196] The vectors described may further comprise, consist, or
consist essentially of a nucleic acid encoding a selectable marker
such as a polypeptide or protein conferring antibiotic resistance
e.g. kanamycin resistance or ampicillin resistance.
[0197] The vectors described are preferably DNA vectors,
particularly dsDNA vectors. The vector may be provided as a linear
or circular (plasmid) DNA vector. The vector preferably contains
nucleotide sequences, e.g. restriction endonuclease site(s),
permitting transition between the two forms by use of DNA ligation
and restriction materials (e.g. enzymes) and techniques known to
the person skilled in the art. To achieve homologous recombination
with a selected HSV, the vector is preferably provided in linear
form.
[0198] One such vector provided by the inventors is plasmid
RL1.dIRES-GFP deposited in the name of Crusade Laboratories Limited
having an address at Department of Neurology Southern General
Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotland on 3 Sep.
2003 at the European Collection of Cell Cultures (ECACC), Health
Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG,
United Kingdom under accession number 03090303 in accordance with
the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purposes of
Patent Procedure (herein referred to as the `Budapest Treaty`).
[0199] RL1.dIRES-GFP provides a platform for generating a plurality
of `shuttle vectors` which can exploit the process of homologous
recombination to transfer a nucleotide sequence of interest
(downstream of a selected promoter) into the disabling RL1 locus of
HSV-1, generating easily identifiable, oncolytic, ICP34.5 null
HSV-1 mutants expressing the products of the nucleotide sequence of
interest, e.g. an RNA transcript or a polypeptide, and GFP.
RL1.dIRES-GFP thus provides for ease of generation and purification
of ICP34.5 null HSV.
[0200] RL1.dIRES-GFP is a useful vector for making
second-generation oncolytic viruses having enhanced cytotoxic
potential and which may express the product(s) of selected gene(s)
to enhance the oncolytic and/or therapeutic effect of the
administered virus.
[0201] The RL1.dIRES-GFP plasmid incorporates a multi-cloning
sequence (MCS), upstream of an internal ribosome entry site (IRES),
the GFP gene and SV40 polyadenylation sequences flanked by HSV-1
RL1 sequences. Incorporation of the encephalomyocarditis virus IRES
(EMCV IRES) permits translation of two open reading frames from a
single transcribed mRNA.
[0202] Following generation of a specific shuttle vector by cloning
of the nucleotide sequence of interest (and the selected promoter)
into RL1.dIRES-GFP, recombinant HSV-1 expressing the desired
nucleic acid transcript or protein, can be generated and purified
within 2 weeks. This compares with 2-3 months using prior art
protocols.
[0203] In the ICP34.5 null HSV generated using the RL1.dIRES-GFP
plasmid provided by the inventors transcription of both the
nucleotide sequence of interest and GFP as a single transcript is
controlled by the same promoter upstream of the nucleotide sequence
of interest, the transcribed IRES directing cap-independent
translation of GFP. The generated ICP34.5 null HSV are
non-neurovirulent. By modifying the RL1.dIRES-GFP plasmid to
incorporate appropriate flanking sequences surrounding the cassette
other gene-specific HSV null mutants expressing GFP can be
generated.
[0204] RL1.dIRES-GFP is promoterless, thus enabling a promoter of
choice to be incorporated in the homologously recombined shuttle
vector for controlling expression of the nucleotide sequence of
interest from the inserted cassette.
[0205] Plasmid RL1.dIRES-GFP or modified plasmid shuttle vectors
thereof further comprising nucleotide sequence encoding a nucleic
acid transcript or polypeptide of interest may be provided in
isolated or purified form.
[0206] The vector may be a variant of plasmid RL1.dIRES-GFP.
[0207] As the plasmid RL1.dIRES-GFP is designed for tandem
expression of a sequence of interest and the marker gene encoding
green fluorescent protein (GFP). The sequence of interest is cloned
into RL1.dIRES-GFP along with its promoter (e.g. CMV) such that the
promoter drives transcription of an mRNA for the sequence of
interest along with the IRES-GFP. Translation results in expression
of the GFP from the internal ribosomal entry site and the gene of
interest and promoter must be cloned into RL1.dIRES-GFP in the
correct orientation to achieve this. There are a number of
instances where this tandem expression arrangement may be
unsuitable and a variation of the cassette design is
favourable.
[0208] One example is the expression of siRNAs as short hairpin
RNAs using RNA polIII promoters such as H1 or U6. These promoters
are unable to drive the additional tandem expression of the
IRES-GFP as the RNApolIII expression cassette is designed only to
produce short transcripts. Additionally, sequences of interest
derived from genomic DNA with strong mRNA shut-off signals in their
3' untranslated regions may not support IRES-GFP expression.
[0209] Thus in some cases a cassette may be provided in which the
sequence of interest and marker are expressed separately from
independent promoters.
[0210] One variant contains a cassette in which the ribosome
binding site of plasmid RL1.dIRES-GFP is replaced with a regulatory
nucleotide sequence, preferably a strong, constitutive promoter
such as the Phosphoglycerokinase promoter. The marker is thereby
expressed under the control of this (the `first`) regulatory
sequence. The nucleotide sequence of interest (e.g. NTR, an
antisense or siRNA) is expressed under the control of a second
regulatory sequence upstream (5') of the nucleotide sequence of
interest, e.g. the CMV promoter. This vector variant is
particularly suitable for expression of siRNA where a weak promoter
may be used for expression of the siRNA molecule or where the
nucleic acid encoding the NTR may have a strong termination signal
making it difficult to transcribe or translate a single bi- or
poly-cistronic transcript encoding the NTR and marker sequence. In
this arrangement the transformed virus containing the cassette
integrated in the viral genome produces two separate transcripts
under the control of the first and second promoters.
[0211] One such cassette was constructed in the following manner.
The 1.3 kbp blunt-ended EcoRI/AfIII fragment that contains the PGK
promoter/GFP gene was obtained by restriction digestion followed by
Klenow treatment from the vector pSNRG and cloned into the RL1-del
vector cut with the restriction enzyme NruI that generates blunt
ends. Successful insertion of the PGK/GFP DNA was confirmed by
BamHI digestion and the orientation of the inserted DNA identified
using the unique XhoI site in RL1-del and the BsrGI site at the 3'
end of PGK/GFP. Plasmids with PGK/GFP in both forward and reverse
orientation were obtained and the plasmids were designated
RL1-dPGK/GFP for and RL1-dPGK/GFPrev. Expression of GFP was
confirmed in BHK cells transfected with the forward and reverse
orientation plasmids.
[0212] Thus, sequences of interest along with their own promoters
(although it is preferred that the PGK promoter is not also used
for this purpose) can then be cloned into either RL1-dPGK/GFP for
or RL1-dPGK/GFPrev in either orientation using the remaining unique
BglII, XhoI or HpaI unique restriction enzyme sites. The resulting
plasmid can be used to derive recombinant HSV in which the marker
GFP gene and the gene of interest are expressed independently from
their own promoters.
[0213] The vectors described may be constructed for use in
generating engineered HSV-1 or HSV-2 by insertion of a nucleic acid
cassette through a mechanism of homologous recombination between
nucleotide sequences flanking the cassette and corresponding
sequences in the selected herpes simplex virus genome.
[0214] The vectors described may comprise and have use as: [0215]
i) gene delivery (gene therapy) vectors for delivery of a selected
nucleotide sequence, e.g. NTR, to a specific locus of the HSV
genome; and/or [0216] ii) expression vectors for expression of the
delivered nucleotide sequence of i) from the HSV genome under the
control of a selected regulatory element; and/or [0217] iii)
vectors for the generation of HSV gene-specific null mutants
wherein the cassette is inserted at a selected genomic location to
disrupt the protein coding sequence of a selected HSV gene such
that the gene product is inactive in the resultant mutant
virus.
[0218] The vectors described may be used in the manufacture of
engineered gene specific HSV null mutants, i.e. HSV mutants
incapable of expressing an active gene product of a selected gene.
They may be used in the manufacture of engineered viruses which
express a selected protein from only one gene copy the other gene
copy being disrupted or modified such that it cannot express a
functional gene product. Such vectors may also be used in the
manufacture of a medicament, preferably comprising said gene
specific HSV null mutant, for use in treating cancer and tumours,
preferably by the oncolytic treatment of the tumour.
[0219] The vectors described may also be used in the manufacture of
engineered HSV mutants wherein the genome of the mutant HSV
comprises an exogenous or heterologous gene which may have been
inserted in the HSV genome by homologous recombination of the
cassette. Preferably, the exogenous/heterologous gene is expressed
in the mutant HSV, which expression may be regulated by a
regulatory element, e.g. promoter, forming part of the inserted
cassette. Such vectors may be used in the manufacture of a
medicament, preferably comprising the engineered HSV mutant, for
use in the treatment of disease, including the oncolytic treatment
of tumours.
[0220] The vectors described may also be used in the manufacture of
an engineered HSV mutant wherein the genome of the mutant HSV
comprises an exogenous/heterologous gene (i.e. a non-HSV
originating gene) which may have been inserted in a protein coding
sequence of the HSV genome by homologous recombination of the
cassette such that the mutant HSV is incapable of expressing the
active gene encoded by said protein coding sequence and wherein the
exogenous/heterologous gene product is expressed under the control
of a regulatory element. Preferably, the regulatory element forms
part of the cassette. Such vectors may be used in the manufacture
of a medicament, preferably comprising the engineered HSV mutant,
for use in the treatment of disease, including the oncolytic
treatment of tumours.
[0221] The vectors described may also be used in the manufacture of
an engineered HSV mutant wherein the genome of the mutant HSV
comprises a nucleotide sequence which has been inserted in a
protein coding sequence of the HSV genome by homologous
recombination of the cassette such that the mutant HSV is incapable
of expressing the active gene encoded by said protein coding
sequence and wherein the inserted nucleotide sequence is expressed
under the control of a regulatory element to produce a desired
transcript. Preferably, the regulatory element forms part of the
cassette. Such vectors may be used in the manufacture of a
medicament, preferably comprising the engineered HSV mutant, for
use in the treatment of disease, including the oncolytic treatment
of tumours.
[0222] The vectors described may be used to generate mutant HSV by
inserting the cassette into the genome of a selected HSV, the
method of generation may comprise providing a vector described
above, where the vector is a plasmid, linearising the vector; and
co-transfecting a cell culture with the linearised vector and
genomic DNA from said HSV.
[0223] The co-transfection may be carried out under conditions
effective for homologous recombination of said cassette into an
insertion site of the viral genome.
[0224] The method may further comprise one or more of the steps of:
[0225] 1) screening said co-transfected cell culture to detect
mutant HSV expressing said marker; and/or [0226] 2) isolating said
mutant HSV; and/or [0227] 3) screening said mutant HSV for
expression of the nucleotide sequence of interest or the RNA or
polypeptide thereby encoded; and/or [0228] 4) screening said mutant
HSV for lack of an active gene product; and/or [0229] 5) testing
the oncolytic ability of said mutant HSV to kill tumour cells in
vitro.
Example 1
Construction of Plasmid RL1.dIRES-GFP
General Approach
[0230] Plasmid RL1.dIRES-GFP was generated in three stages,
illustrated in FIG. 1.
[0231] 1. The DNA sequences containing the CMV IE promoter (pCMV),
the NAT gene, the internal ribosome entry site (IRES), the GFP
reporter gene and the SV40 polyadenylation sequences were excised
from pNAT-IRES-GFP using NsiI and SspI and purified.
[0232] 2. The purified pCMV-NAT-IRES-GFP-PolyA DNA fragment was
cloned into RL1.del to form a new plasmid designated
RL1.dCMV-NAT-GFP.
[0233] 3. The pCMV-NAT DNA sequences of RL1.dCMV-NAT-GFP were
excised using XhoI and the remainder of the plasmid re-ligated to
form a novel plasmid designated RL1.dIRES-GFP. This novel plasmid
contained a multi-cloning site (all sites shown are unique)
upstream of an IRES, the GFP gene and the SV40 polyA sequences all
within the HSV-1 RL1 flanking sequences. Recombinant ICP34.5 null
HSV-1, expressing a gene of interest in the RL1 locus, can be
generated by cloning the gene of interest (downstream of a suitable
promoter) into the multi-cloning site and co-transfecting BHK cells
with the plasmid and HSV-1 DNA. Recombinant virus expressing the
target gene can be identified using GFP fluorescence.
[0234] Removal of the CMV promoter and noradrenaline transporter
gene (pCMV-NAT) from RL1.dCMV-NAT-GFP, followed by re-ligation of
the remainder of the plasmid, resulted in a novel plasmid
(RL1.dIRES-GFP) containing a multi-cloning site (MCS), upstream of
the encephalomyocarditis virus internal ribosome entry site (EMCV
IRES), the GFP reporter gene and the SV40 PolyA sequences, all
within RL1 flanking sequences. This novel arrangement of DNA
sequences or `smart cassette` allows ICP34.5 null HSV-1, expressing
a gene of interest in the RL1 locus, to be easily generated by
simply inserting the desired transgene (downstream of a suitable
promoter) into the MCS and co-transfecting BHK cells with the
plasmid and HSV-1 DNA. The IRES situated between the GFP gene and
the MCS permits expression of two genes from the same promoter and
so recombinant virus expressing the gene of interest also expresses
GFP and can therefore be easily identified under a fluorescence
microscope and purified.
[0235] Materials and Methods
[0236] 1 .mu.g of RL1 del* was digested with 10 units HpaI
(Promega) in a suitable volume of 10.times. buffer (Promega) and
nuclease free water (Promega) at 37.degree. C. for 16 hrs. The
digested plasmid was then purified using the QIAquick PCR
purification kit (Qiagen), treated with 10 units of Calf Intestinal
Phosphatase (Promega), in a suitable volume of 10.times.CIP buffer
and nuclease free water for 4 hrs at 37.degree. C., before being
purified again using a Qiaquick PCR purification kit. 5 .mu.l of
the purified DNA was electrophoresed on a 1% agarose gel to check
its concentration (FIG. 2).
[0237] 4.times.1 g of pNAT-IRES-GFP** was digested with 10 units of
NsiI and 10 units of SspI in a suitable volume of 10.times. buffer
(Promega) and nuclease free water (Promega) at 37.degree. C. for 16
hrs. The reaction mixture was electrophoresed in a 1% agarose gel
for 1 hr at 110 volts. The 5.4 Kbp DNA fragment consisting of the
CMV IE promoter (pCMV), upstream of the noradrenaline transporter
gene (NAT), the encephalomyocarditis virus internal ribosome entry
site (IRES), the gene for green fluorescent protein (GFP) and the
SV40 polyadenylation sequences (SV40 Poly A), was excised using a
sterile scalpel and the DNA purified from the gel using a QIAquick
Gel Extraction kit (Qiagen). The eluted DNA was blunt ended using 3
units Klenow Polymerase (Promega) in accordance with the
manufacturers instructions and the DNA purified using a QIAquick
PCR purification kit (Qiagen). 5 .mu.l of the purified DNA fragment
was electrophoresed on a 1% agarose gel to check its concentration
(FIG. 3).
[0238] Ligation reactions were carried out in small eppendorf tubes
containing 5 units T4 DNA Ligase (Promega), a suitable volume of
10.times.DNA Ligase Buffer (Promega), nuclease free water (Promega)
and various volumes of the HpaI digested/CIP treated RL1.del and
blunt ended pCMV-NAT-IRES-GFP-SV40 Poly A DNA, at 16.degree. C.
overnight. Competent JM109 bacterial cells (Promega) were then
transformed with various aliqouts of the ligation reactions***.
Colonies formed on the plates were picked, had their plasmid DNA
extracted using a Qiagen Plasmid Mini kit and screened for inserts
using AflII (New England Biolabs) restriction enzyme analysis.
Plasmid DNA containing the insert would produce two fragments of
4.8 Kbp and 9.2 Kbp following digestion with AflII. Two clones
(clone 5 and 8) contained the insert (FIG. 4). The orientation of
the insert in clone 5 (RL1.dCMV-NAT-GFP) was determined using XhoI
restriction enzyme analysis (FIG. 5).
[0239] To generate RL1.dIRES-GFP from clone 5, the CMV-NAT portion
of the CMV-NAT-IRES-GFP-SV40 PolyA insert was removed by digesting
4.times.500 ng of clone 5 with 10 units of XhoI in a suitable
volume of buffer and water (Promega), overnight at 37.degree. C.
The digested DNA was electrophoresed on a 1% agarose gel at 110
volts for 1 hr (FIG. 6A). The 10.2 Kbp fragment consisting of the
IRES, the GFP gene, the SV40 PolyA sequences and RL1 flanking
sequences in a pGEM3Zf(-) (Promega) backbone, was excised using a
sterile scalpel and the DNA purified from the gel using a QIAquick
Gel Extraction kit.
[0240] Ligation reactions were performed in small eppendorf tubes
containing 100 ng-500 ng purified DNA, 3 units T4 DNA Ligase
(Promega), a suitable volume of 10.times.DNA Ligase Buffer
(Promega) and nuclease free water (Promega) overnight at 16.degree.
C. Competent JM109 bacterial cells (Promega) were then transformed
with various aliquots of the ligation reactions***. Colonies formed
on the plates were picked, had their plasmid DNA extracted using a
Qiagen Plasmid Mini kit and screened using XhoI (Promega)
restriction enzyme analysis. Colonies containing plasmid DNA from
which CMV-NAT had been removed would produce one fragment of 10.2
Kbp when digested with XhoI. Several positive clones were found,
one was isolated, and a large-scale plasmid preparation undertaken
using Promega's Wizard Plus Maxipreps kit. The large-scale plasmid
preparation was checked by digesting with XhoI (FIG. 6B). This
plasmid DNA was subsequently named `RL1.dIRES-GFP`.
[0241] Plasmid RL1.dIRES-GFP has been deposited in the name of
Crusade Laboratories Limited having an address at Department of
Neurology Southern General Hospital 1345 Govan Road Govan Glasgow
G51 5TF Scotland on 3 Sep. 2003 at the European Collection of Cell
Cultures (ECACC), Health Protection Agency, Porton Down, Salisbury,
Wiltshire, SP4 0JG, United Kingdom under accession number 03090303
in accordance with the provisions of the Budapest Treaty.
[0242] RL1.del
*RL1.del was provided by Dr. E. McKie and is the pGEM-3Zf(-)
plasmid (Promega) into which has been cloned an HSV-1 fragment
(123459-129403) consisting of the RL1 gene and its flanking
sequences. The 477 bp Pf7MI-BstEII fragment of the RL1 gene
(125292-125769) has been removed and replaced with a multi-cloning
site (MCS) to form RL1.del.
[0243] pNAT-IRES-GFP
** pNAT-IRES-GFP was supplied by Dr. Marie Boyd (CRUK Beatson
Laboratories) and is the pIRES2-EGFP plasmid (BD Biosciences
Clontech) into which she has cloned the bovine noradrenaline
transporter (NAT) gene (3.2 Kbp), at the NheI and XhoI sites.
[0244] ***Transformation of Bacterial Cells
10 .mu.l of a glycerol E. coli stock was added to 10 ml 2YT medium
in a 20 ml griener tube. This was placed in a 37.degree. C. shaking
incubator for 16-24 hrs until a saturated culture was obtained. 1
ml of this culture was then added to 100 ml of 2YT in a 500 ml
sterile glass bottle and placed in the 37.degree. C. shaking
incubator for 3 hrs. The bacterial cells were pelleted by
centrifugation at 2,000 rpm for 10 minutes (Beckman). The cells
were then resuspended in 1/10th volume of transformation and
storage buffer (10 mM MgCl.sub.2, 10 mM Mg(SO).sub.4, 10% (w/v) PEG
3,500, 5% (v/v) DMSO). The cells were placed on ice for between 10
minutes and 2 hrs, after which time they were considered competent
for transformation.
[0245] 1-10 .mu.l of DNA was mixed with 100 .mu.l of competent
bacteria in eppendorf tubes, and the tubes placed on ice for 30
minutes. After this, the samples were `heat shocked` by incubating
the tubes in a 42.degree. C. water bath for exactly 45 seconds
before placing them on ice for a further 2 minutes. 1 ml of L-Broth
was added, the tube inverted 2-3 times, and the bacteria incubated
for 1 hr at 37.degree. C. 100 .mu.l of the transformed bacteria was
plated out onto L-broth agar plates containing 100 .mu.g/ml of the
appropriate antibiotic (usually ampicillin or kanamycin). Plates
were allowed to dry at room temperature, before incubating in an
inverted position at 37.degree. C. overnight.
Example 2
Generation of ICP34.5 null HSV-1 expressing a gene product of
interest and GFP using plasmid RL1.dIRES-GFP.
[0246] General Approach
Generation of ICP34.5 null HSV-1 expressing a gene product of
interest requires insertion of nucleotide sequence encoding the
gene product (polypeptide) of interest, and often a desired
promoter, at the MCS of RL1dIRES.GFP followed by co-transfection of
BHK cells with the linearised plasmid, containing the gene of
interest, and HSV DNA. Following homologous recombination viral
plaques expressing GFP are identified. FIG. 7 illustrates the
method steps involved. Referring to FIG. 7A plasmid DNA, containing
the gene of interest and the desired promoter (X), is digested with
restriction endonucleases to release the promoter/gene
fragment.
[0247] The promoter/gene fragment is purified and cloned into the
multi-cloning site (MCS) of RL1.dIRES.GFP forming a shuttle vector
suitable for generating oncolytic HSV-1 (FIG. 7B). This vector
contains HSV-1 sequences that flank the essential RL1 gene but does
not contain the RL1 gene. The plasmid also contains the gene for
Green Fluorescent Protein (GFP) downstream of an internal ribosome
entry site (IRES). The IRES permits expression of both the gene of
interest and the GFP gene from the same upstream promoter.
[0248] BHK cells are then co-transfected with linearised
RL1.dIRES.GFP, now containing the gene of interest, and HSV-1 DNA
(FIG. 7C). Following homologous recombination, designer virus,
expressing the gene of interest and GFP, is generated and can be
distinguished from wild type virus (also generated but not
expressing GFP) under a fluorescence microscope.
[0249] Viral plaques, expressing GFP (and hence the gene of
interest), are picked under the fluorescence microscope and
purified until all wild-type HSV-1 has been removed. The
recombinant HSV-1 is considered 100% pure when all the viral
plaques are expressing GFP (FIG. 7D).
[0250] Once the recombinant virus is completely pure, an isolated
plaque is picked and a highly concentrated stock is grown and
titrated (FIG. 7E). Oncolytic HSV-1, expressing a gene product of
interest from a selected promoter, is then ready for
characterisation and in vitro examination of its tumour killing
potential.
[0251] Materials and Methods
To generate recombinant ICP34.5 null HSV-1 expressing a gene of
interest and GFP, requires the gene of interest, and often a
suitable promoter, to be cloned into the MCS of RL1.dIRES-GFP in
the forward orientation with respect to the GFP gene in this
plasmid. Once this has been achieved the plasmid is linearised
(i.e. digested with a restriction enzyme that cuts only once,
usually SspI or ScaI) in an irrelevant region. 80% confluent BHK
cells in 60 mm petri dishes are then co-transfected with HSV-1 DNA
and linearised plasmid DNA as described below.
[0252] To generate replication restricted HSV-1, expressing the
gene of interest and GFP, the gene of interest must be cloned into
RL1.dIRES-GFP downstream of a suitable promoter (e.g. CMV IE). The
promoter is required upstream of the gene of interest for the
production of a bicistronic mRNA transcript. The IRES sequence
between the two open reading frames in the transcript functions as
a ribosome binding site for efficient cap-independent internal
initiation of translation. The design enables coupled transcription
of both the gene of interest and GFP, followed by cap-dependent
initiation of translation of the first gene (gene of interest) and
IRES-directed, cap-independent translation of GFP. Co-ordinate gene
expression is thus ensured in this configuration.
[0253] Co-Transfection of Virus and Plasmid DNA by CaPO.sub.4 and
DMSO Boost HSV-1 (17.sup.+) DNA and 0.1-1 .mu.g linearized SMART
cassette containing the gene and promoter of interest is pipetted
into 1.5 ml eppendorf tubes containing 1 .mu.l of calf thymus DNA
(10 .mu.g/ml) and an appropriate volume of distilled water to give
a final volume of 165 .mu.l. The solutions are very gently mixed
using a 200 .mu.l pipette tip. 388 .mu.l of HEBS, pH 7.5, (130 mM
NaCl, 4.9 mM KCl, 1.6 mM Na.sub.2HPO.sub.4, 5.5 mM D-glucose, 21 mM
HEPES) is then added, the solution mixed, before adding 26.5 .mu.l
of 2M CaCl.sub.2 dropwise and flicking the eppendorf tube two or
three times. The samples are left at room temperature for 10-15
minutes then added dropwise to 80% confluent BHK's in 60 mm petri
dishes from which the medium has been removed. Following incubation
at 37.degree. C. for 45 minutes, the cells are overlaid with 5 ml
of ETC10 and incubated at 37.degree. C. Three to four hours later,
the media is removed and the plates washed with ETC10. For exactly
4 minutes, the cells are overlaid with 1 ml 25% (v/v) DMSO in HEBS
at room temperature. After the 4 minutes, the cells are immediately
washed three times with 5 ml ETC10 before overlaying with 5 ml of
ETC10 and returning to the incubator. The following day, fresh
medium is added to the cells. Two days later, when cpe is evident,
cells are scraped into the medium, transferred to small bijoux and
sonicated thoroughly. The sample is then stored at -70.degree. C.
until required (see section below on plaque purification).
[0254] N.B. The volume of virus DNA to add is determined by
undertaking the above procedure without plasmid DNA, using a range
of virus DNA volumes and choosing the volume that gives the
greatest number of viral plaques on the BHK monolayer after 2 or 3
days.
[0255] Plaque Purification
Sonicated samples from co-transfection plates are thawed and
serially diluted 10 fold in ETC10. 100 .mu.l from neat to the 105
dilution is plated out on confluent BHK's in 60 mm petri dishes
from which the media has been removed. After 45 minutes incubation
at 37.degree. C., the cells are overlaid with 5 ml EMC10 and
incubated at 37.degree. C. for 48 hrs. The plates are then checked
for the presence of viral plaques and those dishes with the fewest,
most separated plaques are placed under a fluorescent
stereomicroscope. Recombinant virus, designed to express the green
fluorescent protein (GFP) in addition to the gene of interest, can
clearly be distinguished from wild type virus using a GFP filter.
Fluorescent plaques are picked using a 20 .mu.l pipette and placed
(including the tip) into an eppendorf tube containing 1 ml ETC10.
The sample is thoroughly sonicated before making serial 10 fold
dilutions in ETC10 and repeating the above purification procedure.
The process is repeated typically 3-4 times until every plaque on
the BHK monolayer is fluorescent. Once this has been achieved, 50
.mu.l of this sample is used to infect BHK cells in roller bottles,
in 50 ml ETC10, and a virus stock grown.
[0256] Tissue Culture Media
BHK21/C13 cells are grown in Eagle's medium (Gibco) supplemented
with 10% newborn calf serum (Gibco) and 10% (v/v) tryptose
phosphate broth. This is referred to as ETC10. For virus titrations
and plaque purification, EMC10 (Eagles medium containing 1.5%
methylcellulose and 10% newborn calf serum) is used to overlay the
cells.
Example 3
Construction of HSV1716/CMV-NTR/GFP
[0257] General Approach
HSV1716/CMV-NTR/GFP was generated by cloning a 1.6 Kbp BamHI
fragment from pPS949.sup.10, consisting of the E. coli
nitroreductase (NTR) gene downstream of the CMV IE promoter (pCMV),
into the MCS of the RL1.dIRES-GFP smart cassette, in the forward
orientation with respect to the GFP gene in RL1.dIRES-GFP (FIG. 8).
The resultant plasmid, named RL1.dCMV-NTR-GFP, was then linearised
and recombinant virus generated and purified as described above.
The plasmid pPS949 (referred to as `pxLNC-ntr` in Ref 10)
containing the NTR gene downstream of the CMV IE promoter
(pCMV-NTR) in a pLNCX (Clontech) backbone, was a kind gift from
Professor Lawrence Young, University of Birmingham, UK.
[0258] Materials and Methods
4.times.1 .mu.g of pPS949 was digested with 10 units of BamHI
(Promega), in a suitable volume of 10.times. buffer (Promega) and
nuclease free water (Promega), at 37.degree. C. for 16 hrs. The
reaction mixture was electrophoresed in a 1% agarose gel for 1 hr
at 110 volts. The 1.6 Kbp DNA fragment consisting of the CMV
promoter upstream of the NTR gene (pCMV-NTR), was excised using a
sterile scalpel and the DNA purified from the gel using a QIAquick
Gel Extraction kit (Qiagen). 5 .mu.l of the purified DNA fragment
was electrophoresed on a 1% agarose gel to check its concentration
(FIG. 9).
[0259] 2 .mu.g of the RL1.dIRES-GFP smart cassette was then
digested with 15 units of BglII (Promega), in a suitable volume of
10.times. buffer (Promega) and nuclease free water (Promega), at
37.degree. C. for 16 hrs. The digested plasmid was then purified
using the QIAquick PCR purification kit (Qiagen), treated with 10
units of Calf Intestinal Phosphatase (Promega), in a suitable
volume of 10.times.CIP buffer and nuclease free water for 4 hrs at
37.degree. C., before being purified again using the Qiaquick PCR
purification kit. 5 .mu.l of the purified DNA was electrophoresed
on a 1% agarose gel to check its concentration (FIG. 10).
[0260] Ligation reactions were carried out in small eppendorf tubes
containing 5 units T4 DNA Ligase (Promega), a suitable volume of
10.times.DNA Ligase Buffer (Promega), nuclease free water (Promega)
and various volumes of the BglII digested/CIP treated RL1.dIRES-GFP
smart cassette and pCMV-NTR (BamHI ends), at 16.degree. C.
overnight. Competent JM109 bacterial cells (Promega) were then
transformed with various aliqouts of the ligation reactions.
Colonies formed on the plates were picked, had their plasmid DNA
extracted using a Qiagen Plasmid Mini kit and screened for inserts
using BglII/XhoI (Promega) restriction enzyme analysis.
RL1.dIRES-GFP plasmid DNA containing the pCMV-NTR insert in the
correct orientation would produce two fragments of 11.5 Kbp and 300
bp following digestion with BglII and XhoI. One clone (clone 4) was
found to contain the insert in the correct orientation (FIG. 11).
This plasmid was named `RL1.dCMV-NTR-GFP`.
[0261] 0.1-1 .mu.g of RL1.dCMV-NTR-GFP was linearized by digesting
with 10 units of ScaI (Promega), in a suitable volume of 10.times.
buffer (Promega) and nuclease free water (Promega), at 37.degree.
C. for 16 hrs. A sample (5 .mu.l) of the digested DNA was
electrophoresed on a 1% agarose gel for 1 hr at 110 volts to check
that it had been linearized. 80% confluent BHK cells were then
co-transfected with a suitable volume of the remaining linearised
DNA and HSV-1 DNA. Recombinant HSV-1, expressing GFP (and hence
NTR), was identified and purified using a fluorescent microscope
and a virus stock, named HSV1716/CMV-NTR/GFP, was grown and
titrated on BHK cells (FIG. 12).
[0262] HSV1716/CMV-NTR/GFP has been deposited in the name of
Crusade Laboratories Limited having an address at Department of
Neurology Southern General Hospital 1345 Govan Road Govan Glasgow
G51 5TF Scotland on 5 Nov. 2003 at the European Collection of Cell
Cultures (ECACC), Health Protection Agency, Porton Down, Salisbury,
Wiltshire, SP4 0JG, United Kingdom under accession number 03110501
in accordance with the provisions of the Budapest Treaty.
[0263] HSV1716/CMV-NTR/GFP Cell Killing
[0264] HSV1716/CMV-NTR/GFP replicates with almost identical
kinetics to HSV1716 in BHK cells and 3T6 cells. BHK cells support
the replication of ICP34.5 null HSV while confluent 3T6 cells do
not. FIG. 13 shows that HSV1716/CMV-NTR/GFP will replicate as well
as HSV1716 in permissive cell lines and that the introduction of
exogenous genes, e.g. NTR and GFP, has not reduced the oncolytic
potential of the ICP34.5 null HSV. The fact that
HSV1716/CMV-NTR/GFP fails to replicate in 3T6 cells also indicates
that this recombinant HSV is an ICP34.5 null mutant.
[0265] FIG. 14 is a Western blot demonstrating that no ICP34.5
polypeptide is expressed from HSV1716/CMV-NTR/GFP, and that the
virus is thus useful as a gene therapy vector.
[0266] FIG. 15 is another Western blot demonstrating expression of
NTR in a variety of cell lines infected with HSV1716/CMV-NTR/GFP,
including a human malignant melanoma cell line (C8161) and
confluent 3T6 cells in which ICP34.5 null HSV does not replicate.
Expression of NTR in confluent 3T6 cells, following infection with
HSV1716/CMV-NTR/GFP, is encouraging as it demonstrates that
replication of this ICP34.5 null mutant is not required for
expression of the prodrug-activating gene (i.e. NTR). Some tumour
cells in vivo will not support the replication of ICP34.5 null HSV
and as such, will not be killed with HSV1716.
[0267] FIG. 16 shows the results from a cytotoxicity assay
performed in confluent 3T6 cells. Infecting confluent 3T6 cells
with an ICP34.5 null mutant (HSV1716/CMV-NTR/GFP), at a
multiplicity of infection (MOI) of 1 plaque forming units
(pfu)/cell, does not result in any significant cell death, neither
does separate incubation of the cells with 50 .mu.M CB1954.
However, significant cell death is evident 72 hrs post infection
with 1 pfu/cell HSV1716/CMV-NTR/GFP when 50 .mu.M CB1954 is
included in the growth medium. This clearly demonstrates that when
there is no replication of the virus, substantial cell death is
still possible from virus directed enzyme prodrug therapy
(VDEPT).
[0268] Infecting confluent 3T6 cells with an ICP34.5 null mutant at
a MOI of 10 pfu/cell will result in cell death, by a mechanism
known as `viral antigen overload`. However, the level of cell
killing is even more pronounced (approximately 20% more), when 50
.mu.M CB1954 is included in the growth medium.
[0269] A similar cytotoxicity assay was performed in human C8161
melanoma cells, the results are set out in FIG. 17. Unlike
confluent 3T6 cells, C8161 cells do support the replication of
ICP34.5 null HSV. Therefore, cell death will occur following
infection of the cells with ICP34.5 null HSV, at 1 pfu/cell.
However, when CB1954 is included in the overlay of
HSV1716/CMV-NTR/GFP infected cells, the cells are killed more
efficiently and more quickly. No enhanced cell killing is evident
when CB1954 is included in the overlay of cells infected with
HSV1716-GFP. These results demonstrate that enhanced cell killing
is possible in human tumour cells.
[0270] Cell culture images for the cytotoxicity assays performed in
confluent 3T6 and human C8161 melanoma cells are shown in FIGS. 18
and 19.
Example 4
[0271] In vivo evaluation of the anti-tumour activity of a
selectively replication competent herpes simplex virus in
combination with enzyme pro-drug therapy. The anti-tumour activity
of a selectively replication competent herpes simplex virus in
combination with an enzyme prodrug therapy approach in appropriate
animal models in vivo was investigated.
[0272] The parental virus, HSV1716 is a selectively replication
competent mutant of the herpes simplex virus 1 (HSV1) which lacks
both copies of the RL1 gene that encodes the protein ICP 34.5. This
protein is a specific determinant of virulence. The function of
this protein has been described at length elsewhere.sup.12. The
virus can grow only in cells that have a high level of functional
PCNA. High levels of PCNA are found only in cells that are dividing
such as tumour cells and not normal differentiated cells.
[0273] It has already been shown that HSV1716 can achieve selective
tumour cell killing with minimal toxicity and improved survival
times in a number of animal models.sup.13. Initial phase 1 clinical
trials using HSV1716 virus in patients has also meet with some
success.sup.14,15.
[0274] Although HSV1716 selectivity replicates in tumour reducing
the tumour bulk by cell lysis the inventors did not anticipate
HSV1716 to lytically replicate in all cells in the tumour due to
the heterogeneity of the cell type and growth state.
[0275] In order to enhance the efficacy of the tumour cell
killing--hence kill the entire tumour--the inventors have
constructed a derivative of HSV1716 designated HSV1716/CMV-NTR/GFP
that expresses the E. Coli nitroreductase gene (ntr) under the
control of a CMV early promoter (see example 3 above). In this
example and the figures referred to HSV1716/CMV-NTR/GFP is called
HSV1790.
[0276] The enzyme ntr converts the inactive prodrug CB1954 to a
functional cytotoxic alkylating agent that kills both dividing and
non-dividing cells by apoptosis. This active drug is diffusible and
membrane permeable resulting in an efficient bystander effect, i.e.
wherein the activated drug may have an effect on surrounding
cells.
[0277] As the prodrug will only be converted to its active form in
the tumour which has been infected with ntr expressing virus,
toxicity to normal cells is avoided hence improving the therapeutic
index following systemic delivery of this compound.
[0278] Initial in vitro experiments using this combination have
already shown enhanced cell kill using this virus in combination
with CB1954 in a number of cell lines.
[0279] This example further evaluates this combination approach in
vivo in appropriate animal models.
[0280] Results
Months 1-3
[0281] Months 1-3 were taken up mainly by in vitro work. During
this time period high titre, sterile virus stock was generated for
use in the xenograft models.
[0282] Xenograft models were also generated in athymic nude mice
using the cell line A2780, a human ovarian epithelial carcinoma
line initially derived from a tumour sample from an untreated
patient (European Collection of Cell Cultures (ECACC) CAMR, Porton
Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom, accession
number 93112520).
[0283] Generation of a gliomal xenograft model was attempted using
2 gliomal lines that were available in house, LN-18 and U373MG.
There are reports in the literature of both being successfully
grown as xenografts in athymic mice. However, as shown in the table
below the inventors failed to see any xenograft growth up to 28
days after injection with 5 million cells subcutaneously.
TABLE-US-00001 TABLE 1 Presence of No of cells injected xenograft
28 days Cell Line per mice Number of mice after cell injection LN
18 5 million 5 0/5 U373 MG 5 million 5 0/5
[0284] A2780 Tumour Take
As reported previously A2780 have a Take Rate of approximately
50%--that is 50% of mice that are injected with 5 million cells per
flank subcutaneously will develop xenografts. When the number of
cells injected was increased to 10 million or more an increase in
take rate of approximately 15-25% was seen, giving an overall take
rate of 65-75%.
[0285] Thus increasing the number of gliomal cells injected may
increase the take rate of these cell lines
[0286] Dose Response to the HSV1790 Virus
Before the mice can be treated with a combination of virus and
prodrug, first one must carry out experiments to make informed
decisions about how much of the virus, and the prodrug to give.
[0287] A dose response experiment will allow one to find both the
most appropriate does of the virus to use in the experiments and
the maximum tolerated dose (MTD) of the virus, that is, the largest
amount of the virus that can be given to a single mouse without
adverse side effects. Small groups of tumour bearing mice are given
a small dose of virus. Assuming they do not have any adverse
effects another group is given a larger dose of virus. This
continues until either the mice start to suffer ill effects or we
reach a maximum dose.
[0288] The maximum amount of virus that can be intratumourally
injected is 100 .mu.l, hence the maximum dose from our current
stock is 10.sup.9 PFU per injection.
[0289] FIG. 20 shows the weight change in the mice after injection
with a variety of doses of virus. Weight is a good indicator of the
animals overall health. Any loss of weight signifies that the
treatment is not being well tolerated. Where an animal loses more
than 20% of its initial body weight it was sacrificed
immediately.
[0290] A dose of 10.sup.9 PFU of the HSV1790 virus is not tolerated
by these mice, they rapidly lost body weight and were sacrificed at
Day 3 post injection. Doses of 10.sup.8 PFU or less were better
tolerated, the mice initially lost weight in the days following
injection but quickly recovered to approximately their initial body
weights.
[0291] It should be pointed out that as the experiment progresses
the animals appear to be increasing in weight. This is almost
certainly due to the fact that it is total body weight that is
measured, which includes the weight of any tumour that is
forming.
[0292] Response of the Tumour to HSV1790 Treatment
Tumour volume was measured daily after intratumoural injection of
the HSV1790 virus to look for any growth delay or regression of the
tumours.
[0293] FIG. 21 shows the change in tumour volume as measured over a
period of 100 days. If the tumour was injected with PBS only as a
control the tumour increased in size rapidly and by approximately
Day 13 post injection the tumours had become too large and the
animals had to be sacrificed.
[0294] Treatment with all doses of virus appeared to delay the
growth of the tumour to some degree. Doses of 10.sup.5 PFU
increased the longevity of the mice by approximately 12 days while
mice injected with 10.sup.6 PFU virus tumours survived for an extra
23 days compared to the control group before the tumours became
prohibitively large. Perhaps surprisingly the group of mice
injected with 10.sup.8 PFU of virus survived only slightly longer
than the control group. It is possible there were a large number of
non infectious particles or that sheer number of particles caused
the cells which the virus would have grown in to be killed.
[0295] The group of mice treated with 10.sup.7 PFU of virus
survived the longest and indeed two out of three of the mice did
not have any visible signs of tumour when sacrificed at day
100.
[0296] Naked DNA Experiments
In order to check the alterations in tumour growth are due to the
virus itself and not a result of the CMV-ntr plasmid DNA that had
been introduced to the HSV1716 virus, an experiment was set up
looking at the effect of the CMV-ntr plasmid DNA alone and in
combination with the prodrug CB1954.
[0297] Mice were randomised into treatment groups of 6 animals each
when tumour diameters are approximately 5 mm (this is Day 0). FIG.
22 shows the starting tumour diameters for the mice used in this
experiment. Two groups of mice were administered CMV-ntr plasmid by
direct intratumoural injection at a dose of 0.2 mg DNA per
injection. One of these groups was then administered with a single
dose of 80 mg/kg of CB1954 on Day 2 by intra-peritoneal injection.
The third group of mice had a single administration of CB1954 (80
mg/kg) by intra-peritoneal injection on Day 2 following
intratumoural injection of saline control at Day 0. Animals were
weighed daily (FIG. 23) and daily caliper measurements performed
until the tumour sizes were in the region of 20 mm by 20 mm. Tumour
volumes were estimated from these measurements (volume=d3.times.6)
(FIG. 24). In addition any toxicity from these administered agents
was determined. On the basis of these experiments the inventors
determined that neither the CMV-ntr alone, CB1954 alone or the
combination of both CMV-ntr and CB1954, has any anti-tumour
activity as determined by tumour regression in this model system
(FIG. 24).
[0298] Scheduling Experiment
Previous dose response experiments have shown that doses of less
than 10.sup.8 PFU virus per mouse do not appear to have any adverse
effect of the animals health.
[0299] A dose of 10.sup.7 PFU virus per mouse resulted in a great
reduction in tumour growth, indeed after 100 days two out of three
of the mice in the group had no visible tumour. This is very
encouraging--the virus only at high doses may be enough to delay
growth or cause tumour regression.
[0300] However to look at the effect of a combination of the virus
and the prodrug CB 1954 a lower dose of the virus was studied--if
the treatment of the virus alone results in growth delay for such a
long period one would be unable to ascertain the addition or
synergistic effects of the prodrug.
[0301] The inventors proceeded to investigate two doses of the
virus in combination with CB1954. The doses selected were 10.sup.5
PFU and 10.sup.6 PFU. Both these doses caused some tumour growth
delay in earlier experiments.
[0302] The prodrug is given as an 80 mg/kg intra-peritoneal
injection, after dissolving the powdered form in 10% acetone and
then making up the volume with peanut oil.
[0303] Another factor that determines how well the drug will
work--hence how much tumour growth delay or regression is seen--is
when the drug is actually given. As the prodrug will only be
converted to an active substrate in the presence of NTR it was
considered that the virus containing the NTR will have to been
given first. It was also considered that if the virus is given time
to replicate and produce more NTR then the prodrug may have a more
pronounced effect.
[0304] To discover the optimal doses of both the virus and the drug
and the optimal times of these treatments a scheduling experiment
was set up.
[0305] Mice were randomized into treatment groups (treatment
regimes shown in Table 2) of 3 animals when tumour diameters were
approximately 5 mm (tumour volume 0.5-1.5 mm.sup.3). FIG. 25 shows
the starting tumour volumes of each of the groups.
TABLE-US-00002 TABLE 2 Treatment groups 1 10.sup.5 HSV 1790 + drug
(Day 2) + Drug (Day 10) 2 10.sup.5 HSV 1790 + drug (Day 2) +
vehicle (Day 10) 3 10.sup.5 HSV 1790 + vehicle (Day 2) + drug (Day
10) 4 10.sup.5 HSV 1790 + vehicle (Day 2) + vehicle (Day 10) 5 No
virus + drug (Day 2) + drug (Day 10) 6 No virus + drug (Day 2) +
vehicle (Day 10) 7 No virus + vehicle (Day 2) + drug (Day 10) 8 No
virus + vehicle (Day 2) + vehicle (Day 10) 9 10.sup.6 HSV 1790 +
vehicle (Day 2) + vehicle (Day 10) 10 10.sup.6 HSV 1790 + drug (Day
2) + vehicle (Day 10) 11 10.sup.6 HSV 1790 + drug (Day 2) + drug
(Day 10) 12 10.sup.6 HSV 1790 + drug (Day 2) + drug (Day 10) + drug
(Day 15) 13 No virus + drug (Day 2) + drug (Day 10) 14 No virus +
vehicle (Day 2) + vehicle (Day 10) 15 10.sup.5 HSV 1716 + drug (Day
2) + drug (Day 10) 16 10.sup.5 HSV 1716 + vehicle (Day 2) + vehicle
(Day 10) 17 10.sup.5 HSV 1716 + drug (Day 2) + vehicle (Day 10) 18
10.sup.5 HSV 1716 + vehicle (Day 2) + drug (Day 10)
[0306] The treatment groups were administered with a single direct
intratumoural injection of the virus and dose determined for that
group. The virus was diluted PBS+10% serum. `No virus` control
groups received an intratumoural injection of 100 .mu.l of PBS+10%
serum. This day was designated as Experimental Day 0.
[0307] The intratumoural injections did not appear to have any
adverse effects on the mice. Some tumours bleed slightly after
injection but not to a great degree. The animals did not lose body
weight (FIG. 26) and their behaviour did not appear to alter. In
all the tumours that bleed slightly, the following day the healing
process had begun and within 3-5 days there was little evidence of
any puncture wound on any tumour.
[0308] Injections of CB1954 were given to the appropriate groups at
days 2, 10 and 15. A dose of 80 mg/kg--the equivalent of approx. 2
mg per mouse--was given. The powdered form of the CB1954 drug (from
Sigma) was dissolved in acetone to 10% of the final volume (10
.mu.l per 2 mg). The volume was then made up to 2 mg CB1954 in 100
.mu.l using peanut oil. A syringe was used to mix the drug as
peanut oil is thick and viscous. The drug was made up fresh every
time.
[0309] The appropriate groups were then injected intra-peritoneal
with this solution. Control groups which were not receiving drug
were injected intra-peritoneal with a 100 .mu.l solution of 10%
acetone in peanut oil.
[0310] No swelling or irritation at the site of injection was noted
on any of the mice either at time of injection or at any later time
point. The mice appeared slightly lethargic for a short period
after the injection but did not lose any body weight (FIG. 26) or
show signs of lethargy the following day.
[0311] No virus+CB1954 prodrug
Groups 5, 6, 7, 8, 13 & 14 looked at the effect of prodrug
alone on tumour growth. FIG. 27 shows that there is little effect
on tumour growth when CB1954 is given alone.
[0312] 10.sup.5 PFU Virus+/-CB1954 Prodrug
10.sup.5 PFU virus was given at Day 0 followed by either prodrug or
vehicle at Days 2 and 10. As can be seen from the graph in FIG. 28
tumours treated with either virus only or virus and prodrug did not
grow as large as the untreated tumour. The tumour treated with the
virus grew only to approximately half the size of the untreated
control.
[0313] Treatment with virus and prodrug resulted in tumours which
grew to only approx 2-3 mm in volume. This is significantly less
than the untreated tumours which grew in size to approx 20
mm.sup.3
[0314] 10.sup.6 PFU Virus+/-CB1954 Prodrug
FIG. 29 shows the changes in tumour volume over time after
treatment with a higher dose of virus, 10.sup.6 PFU per injection,
in combination with the prodrug, given as described in Table 2. As
with the lower virus dose, treatment with either virus only, or in
combination with CB1954, results in significantly smaller tumours
compared to the untreated controls.
[0315] HSV1716 Virus in Combination with CB1954 Prodrug
The parental strain of the virus, which has not been engineered to
contain the CMV-ntr DNA was examined for its effects on tumour
growth delay. This virus does have an oncolytic effect, however it
doesn't contain the NTR gene needed to convert the inactive prodrug
into its active metabolite. Therefore one would not expect any
additional or synergistic effects when the prodrug is added in
combination with the virus. FIG. 30 shows the results of this
experiment.
[0316] The combination of the virus and the prodrug appeared to
produce some reduction in tumour growth compared to the untreated
control tumours.
[0317] The groups used in these results contained only 2 or 3
animals. The animals used were also older and their tumours had
taken longer to grow than those used in previous experiments. Hence
it is possible that repeating the experiment with a larger number,
with younger mice or quicker forming tumours may result in a more
marked growth delay after treatment with the HSV1716 virus.
[0318] Comparison of HSV1790 (at 10.sup.5 and 10.sup.6) and HSV1716
in Combination with CB1954 Prodrug
FIG. 31 shows a comparison between the two doses of the HSV1790
virus in combination with the prodrug and the HSV1716 prodrug
combination. The parental virus HSV1716 shows some growth delay in
comparison with the untreated control. We would assume that this
growth delay is due to the oncolytic effect of the virus as the NTR
gene is not present to alter the inactive prodrug into its active
form.
[0319] Tumour growth is reduced further when the tumour is treated
with the HSV 1790 virus containing the NTR gene. This appears to be
dose dependent--the higher dose of the virus results in a greater
growth delay than the lower dose.
[0320] In conclusion it would appear from these results that indeed
the HSV1790 virus used in combination with the prodrug CB1954
results in growth delay in the model system examined. Giving both
virus and drug in combination has a greater effect than given
either alone.
[0321] It appears that the timing at which the prodrug is given
after virus treatment is important. When CB1954 was given soon
after viral injection (Day 2 post viral injection) tumour growth
was not delayed as much as if the drug was given at a later date
(Day 10). It may be that given at Day 2 the drug killed the cells
that were supporting viral growth and replication and actually
reduced the oncolytic effect of the virus.
[0322] By day 10 the virus may have replicated and killed as many
cells by oncolysis as possible. It is anticipated that due to
heterogeneity of the cell type and growth state that all the cells
within a tumour would not be susceptible to lysis by the virus. The
drug then comes in and `mops up` by killing any cells that are
supporting viral growth (hence containing the NTR gene) but were
not susceptible to oncolysis. As the active drug is diffusible and
membrane permeable it may have a bystander effect--killing not only
the cells infected with the virus but also its near neighbours.
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Sequence CWU 1
1
21217PRTEscherichia coli 1Met Asp Ile Ile Ser Val Ala Leu Lys Arg
His Ser Thr Lys Ala Phe1 5 10 15Asp Ala Ser Lys Lys Leu Thr Pro Glu
Gln Ala Glu Gln Ile Lys Thr 20 25 30Leu Leu Gln Tyr Ser Pro Ser Ser
Thr Asn Ser Gln Pro Trp His Phe 35 40 45Ile Val Ala Ser Thr Glu Glu
Gly Lys Ala Arg Val Ala Lys Ser Ala 50 55 60Ala Gly Asn Tyr Val Phe
Asn Glu Arg Lys Ile Leu Asp Ala Ser His65 70 75 80Val Val Val Phe
Cys Ala Lys Thr Ala Met Asp Asp Ala Trp Leu Lys 85 90 95Leu Val Val
Asp Gln Glu Asp Ala Asp Gly Arg Phe Ala Thr Pro Glu 100 105 110Ala
Lys Ala Ala Asn Asp Lys Gly Arg Lys Phe Phe Ala Asp Met His 115 120
125Arg Lys Asp Leu His Asp Asp Ala Glu Trp Met Ala Lys Gln Val Tyr
130 135 140Leu Asn Val Gly Asn Phe Leu Leu Gly Val Ala Ala Leu Gly
Leu Asp145 150 155 160Ala Val Pro Ile Glu Gly Phe Asp Ala Ala Ile
Leu Asp Ala Glu Phe 165 170 175Gly Leu Lys Glu Lys Gly Tyr Thr Ser
Leu Val Val Val Pro Val Gly 180 185 190His His Ser Val Glu Asp Phe
Asn Ala Thr Leu Pro Lys Ser Arg Leu 195 200 205Pro Gln Asn Ile Thr
Leu Thr Glu Val 210 2152654DNAEscherichia coli 2atggatatca
tttctgtcgc cttaaagcgt cattccacta aggcatttga tgccagcaaa 60aaacttaccc
cggaacaggc cgagcagatc aaaactctcc tgcaatacag cccatccagc
120accaactccc agccgtggca ttttattgtt gccagcacgg aagaaggtaa
agcgcgtgtt 180gccaaatccg ctgccggtaa ttatgtgttc aacgaacgta
aaatacttga tgcctcgcac 240gtcgtggtgt tctgtgcaaa aaccgcgatg
gacgatgcct ggctgaagct ggttgttgac 300caggaagatg ctgatggccg
ctttgccacg ccggaagcga aagccgcgaa cgataaaggt 360cgcaagttct
tcgccgatat gcaccgtaaa gatctgcatg atgatgcaga gtggatggca
420aaacaggttt atctcaacgt cggtaatttc ctgctcggcg tggcggctct
gggtctggac 480gcggtaccca tcgaaggttt tgacgccgcc atcctcgatg
cagaatttgg tctgaaagag 540aaaggctaca ccagtctggt ggtagttccg
gtgggtcatc acagcgttga agattttaac 600gctacgctgc cgaaatctcg
tctgccgcaa aacattacct taaccgaagt gtaa 654
* * * * *
References