U.S. patent application number 10/579607 was filed with the patent office on 2007-05-17 for vectors, mutant viruses and methods for generating mutant viruses.
Invention is credited to Susanne Moira Brown, Joe Conner, Paul Dunn.
Application Number | 20070110720 10/579607 |
Document ID | / |
Family ID | 29763982 |
Filed Date | 2007-05-17 |
United States Patent
Application |
20070110720 |
Kind Code |
A1 |
Brown; Susanne Moira ; et
al. |
May 17, 2007 |
Vectors, mutant viruses and methods for generating mutant
viruses
Abstract
A nucleic acid vector comprising first and second nucleotide
sequences corresponding to nucleotide sequences flanking an
insertion site in the genome of a selected herpes simplex virus
strain; and a cassette located between said first and second
nucleotide sequences comprising nucleic acid encoding: (a) one or a
plurality of insertion sites and/or a nucleotide sequence of
interest; and (b) a ribosome binding site or a regulatory
nucleotide sequence; and (c) a marker is disclosed. Herpes simplex
viruses generated using said vector, methods for their generation
and herpes simplex viruses having a genome comprising heterologous
nucleic acid are also disclosed.
Inventors: |
Brown; Susanne Moira;
(Glasgow, GB) ; Conner; Joe; (Glasgow, GB)
; Dunn; Paul; (Glasgow, GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Family ID: |
29763982 |
Appl. No.: |
10/579607 |
Filed: |
November 17, 2004 |
PCT Filed: |
November 17, 2004 |
PCT NO: |
PCT/GB04/04839 |
371 Date: |
May 16, 2006 |
Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 977/802 |
Current CPC
Class: |
C12N 7/00 20130101; C12N
15/1135 20130101; A61K 35/13 20130101; C12N 2710/16643 20130101;
A61P 43/00 20180101; A61P 35/00 20180101; A61K 2039/5256 20130101;
A61K 48/00 20130101; C12N 2840/203 20130101; A61P 35/02 20180101;
C12N 15/86 20130101; A01K 2267/0331 20130101; A61P 35/04
20180101 |
Class at
Publication: |
424/093.2 ;
435/456; 435/235.1; 977/802 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/86 20060101 C12N015/86; C12N 7/00 20060101
C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2003 |
GB |
0326798.6 |
Claims
1. A nucleic acid vector comprising: first and second nucleotide
sequences corresponding to nucleotide sequences flanking a
predetermined insertion site in the RL1 locus of the genome of a
selected herpes simplex virus (HSV); and (I) a cassette located
between said first and second nucleotide sequences comprising
nucleic acid encoding: (a) one or a plurality of insertion sites;
and (b) a ribosome binding site or a regulatory nucleotide
sequence; and (c) a marker, wherein the nucleic acid encoding the
one or plurality of insertion sites is/are arranged upstream (5')
of the ribosome binding site or the regulatory nucleotide sequence
and the nucleic acid encoding the ribosome binding site or the
regulatory nucleotide sequence is arranged upstream (5') of the
marker; or (II) a nucleic acid cassette located between said first
and second nucleotide sequences comprising: (a) a third nucleotide
sequence being of interest: and nucleic acid encoding: (b) a
ribosome binding site or a regulatory nucleotide sequence; and (c)
a marker, wherein the nucleotide sequence of interest is arranged
upstream (5') of the ribosome binding site or the regulatory
nucleotide sequence and the ribosome binding site or the regulatory
nucleotide sequence is arranged upstream (5') of the marker.
2. (canceled)
3. The vector of claim 1 wherein the ribosome binding site
comprises an internal ribosome entry site (IRES).
4-5. (canceled)
6. The vector of claim 1 wherein said regulatory nucleotide
sequence is operably linked to said marker.
7. The vector of claim 1 wherein said regulatory nucleotide
sequence comprises a constitutive or inducible promoter.
8. The vector of claim 1 wherein the nucleotide sequence of
interest encodes an heterologous polypeptide.
9. The vector as claimed in claim 8 wherein the heterologous
polypeptide is selected from the group consisting of: a bacterial
polypeptide; a mammalian polypeptide; a human polypeptide.
10. The vector as claimed in claim 8 wherein the heterologous
polypeptide is selected from the group consisting of: Sodium iodide
symporter (NIS); Nitroreductase (NTR); E. coli NTR; Endothelial
nitric oxide synthase (eNOS); Granulocyte Macrophage
Colony-Stimulating Factor (GM-CSF); a cytokine.
11. The vector of claim 1 wherein the nucleotide sequence of
interest encodes a selected antisense nucleic acid or siRNA.
12. The vector of claim 1 wherein the cassette further comprises a
regulatory nucleotide sequence located upstream (5') of the
nucleotide sequence of interest which has a role in regulating
transcription of the nucleotide sequence of interest.
13. The vector of claim 1 wherein the cassette further comprises a
regulatory nucleotide sequence located upstream (5') of the
insertion site(s).
14. The vector of claim 1 wherein the cassette comprises a
plurality of said insertion sites.
15. The vector of claim 1 wherein each insertion site is formed by
nucleic acid encoding a restriction endonuclease site.
16. The vector of claim 1 wherein the insertion sites comprise one
or more of the ClaI, BglII, NruI and XhoI restriction endonuclease
sites.
17. The vector of claim 1 wherein the first and second nucleotide
sequences each comprise sequence corresponding to nucleotide
sequences in the RL terminal or internal repeat region of the
genome of the selected HSV.
18. The vector of claim 1 wherein said first and second nucleotide
sequences correspond to nucleotide sequences flanking a
predetermined 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.
19. The vector of claim 1 wherein said first and second nucleotide
sequences comprise contiguous portions of nucleotide sequence of
the ICP34.5 gene of a herpes simplex virus.
20. The vector of claim 1 wherein said first and second nucleotide
sequences comprise contiguous portions of nucleotide sequence
encoding the ICP34.5 gene product of a herpes simplex virus.
21. The vector of claim 1 wherein the first and second nucleotide
sequences have at least 60% sequence identity to their
corresponding sequence in the viral genome.
22. The vector of claim 1 wherein said first and second nucleotide
sequences hybridise to their corresponding nucleotide sequence in
the HSV genome, or its complement, under high or very high
stringency conditions.
23. The vector of claim 1 wherein the marker is a defined
nucleotide sequence encoding a polypeptide.
24. The vector of claim 1 wherein the marker comprises the Green
Fluorescent Protein (GFP) protein coding sequence or the enhanced
Green Fluorescent Protein (EGFP) protein coding sequence.
25. The vector of claim 1 wherein the marker comprises a defined
nucleotide sequence detectable by hybridisation under high
stringency conditions with a corresponding labelled nucleic acid
probe.
26. The vector of claim 1 wherein the cassette further comprises
nucleic acid encoding a polyadenylation sequence located downstream
(3') of the nucleic acid encoding the marker.
27. The vector as claimed in claim 26 wherein the polyadenylation
sequence comprises the Simian Virus 40 (SV40) polyadenylation
sequence.
28. The vector of claim 1 wherein the vector further comprises
nucleic acid encoding a second selectable marker.
29. The vector of claim 1 wherein the vector is a DNA vector,
particularly a dsDNA vector.
30. Plasmid RL1.dIRES-GFP (ECACC accession number 03090303).
31. The vector of claim 1 wherein the vector is an expression
vector.
32. A method of generating a herpes simplex virus which expresses a
nucleotide sequence of interest, or polypeptide thereby encoded,
comprising the step of culturing a selected herpes simplex virus
with the vector of claim 1, thereby integrating components (a), (b)
and (c) of said vector at said predetermined insertion site in the
genome of the selected herpes simplex virus.
33. The method of claim 32 wherein said herpes simplex virus is an
HSV-1 or HSV-2.
34. The method of claim 32 wherein the integrated components
disrupt a protein coding sequence resulting in inactivation or lack
of expression of the respective gene product in the generated
virus.
35. The method of claim 32 wherein the generated herpes simplex
virus: is a gene specific null mutant; is an ICP34.5 null mutant;
lacks only one expressible ICP34.5 gene; is non-neurovirulent; or a
combination of two or more thereof.
36.-39. (canceled)
40. A medicament comprising the vector of claim 1.
41.-45. (canceled)
46. A medicament comprising a mutant herpes simplex virus generated
using the vector of claim 1.
47. A kit of parts comprising a first container having a quantity
of the vector of claim 1 and a second container comprising a
quantity of herpes simplex virus genomic DNA.
48. An herpes simplex virus (HSV) wherein the herpes simplex virus
comprises a nucleic acid cassette integrated in the RL1 locus of
the HSV genome comprising nucleic acid encoding: (I.) (a) one or a
plurality of insertion sites; and (b) a ribosome binding site or a
regulatory nucleotide sequence, and a (c) marker, wherein the
nucleic acid encoding the one or plurality of insertion sites
is/are arranged upstream (5') of the ribosome binding site or the
regulatory nucleotide sequence and the nucleic acid encoding the
ribosome binding site or the regulatory nucleotide sequence is
arranged upstream (5') of the marker; or (II.) (a) a nucleotide
sequence of interest; and nucleic acid encoding: (b) a ribosome
binding site or a regulatory nucleotide sequence; and (c) a marker,
wherein the nucleotide sequence of interest is arranged upstream
(5') of the ribosome binding site or the regulatory nucleotide
sequence and the ribosome binding site or the regulatory nucleotide
sequence is arranged upstream (5') of the marker.
49. (canceled)
50. The herpes simplex virus of claim 48 wherein the ribosome
binding site comprises an internal ribosome entry site (IRES).
51. The herpes simplex virus of claim 48 wherein a transcription
product of the cassette is a bi- or poly-cistronic transcript
comprising a first cistron encoded by the nucleotide sequence of
interest and a second cistron encoded by the marker nucleic acid
wherein the ribosome binding site is located between said first and
second cistrons.
52.-53. (canceled)
54. The herpes simplex virus of claim 48 wherein said regulatory
nucleotide sequence is operably linked to said marker.
55. The herpes simplex virus of claim 48 wherein said regulatory
nucleotide sequence comprises a constitutive or inducible
promoter.
56. The herpes simplex virus of claim 48 wherein the nucleotide
sequence of interest encodes an heterologous polypeptide.
57. The herpes simplex virus as claimed in claim 56 wherein the
heterologous polypeptide is selected from the group consisting of:
a bacterial polypeptide; a mammalian polypeptide; a human
polypeptide.
58. The herpes simplex virus as claimed in claim 56 wherein the
heterologous polypeptide is selected from the group consisting of:
Sodium iodide symporter (NIS); Nitroreductase (NTR); E. coli NTR;
Endothelial nitric oxide synthase (eNOS); Granulocyte Macrophage
Colony-Stimulating Factor (GM-CSF); a cytokine.
59. The herpes simplex virus of claim 48 wherein the nucleotide
sequence of interest encodes a selected antisense nucleic acid or
siRNA.
60. The herpes simplex virus of claim 48 wherein the cassette
further comprises a regulatory nucleotide sequence located upstream
(5') of the nucleotide sequence of interest which has a role in
regulating transcription of the nucleotide sequence of
interest.
61. The herpes simplex virus of claim 48 wherein the cassette
further comprises a regulatory nucleotide sequence located upstream
(5') of the insertion site(s).
62. The herpes simplex virus of claim 48 wherein the cassette
comprises a plurality of said insertion sites.
63. The herpes simplex virus of claim 48 wherein each insertion
site is formed by nucleic acid encoding a restriction endonuclease
site.
64. The herpes simplex virus of claim 48 wherein the insertion
sites comprise one or more of the ClaI, BglII, NruI and XhoI
restriction endonuclease sites.
65. The herpes simplex virus of claim 48 wherein the nucleic acid
cassette is integrated in the RL terminal or internal repeat region
of the genome of the selected HSV.
66. The herpes simplex virus of claim 48 wherein the nucleic acid
cassette is integrated at a 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.
67. The herpes simplex virus of claim 48 wherein the nucleic acid
cassette is integrated in the genomic nucleotide sequence of the
ICP34.5 gene of a herpes simplex virus.
68. The herpes simplex virus of claim 48 wherein the nucleic acid
cassette is integrated in the genomic nucleotide sequence encoding
the ICP34.5 gene product of a herpes simplex virus.
69. The herpes simplex virus of claim 48 wherein the marker is a
defined nucleotide sequence encoding a polypeptide.
70. The herpes simplex virus of claim 48 wherein the marker
comprises the Green Fluorescent Protein (GFP) protein coding
sequence or the enhanced Green Fluorescent Protein (EGFP) protein
coding sequence.
71. The herpes simplex virus of claim 48 wherein the marker
comprises a defined nucleotide sequence detectable by hybridisation
under high stringency conditions with a corresponding labelled
nucleic acid probe.
72. The herpes simplex virus of claim 48 wherein the cassette
further comprises nucleic acid encoding a polyadenylation sequence
located downstream (3') of the nucleic acid encoding the
marker.
73. The herpes simplex virus as claimed in claim 72 wherein the
polyadenylation sequence comprises the Simian Virus 40 (SV40)
polyadenylation sequence.
74. The herpes simplex virus of claim 48 wherein the cassette
disrupts a protein coding sequence in the HSV genome resulting in
inactivation of the respective gene product.
75. The herpes simplex virus of claim 48 wherein the herpes simplex
virus is a mutant of HSV-1 or HSV-2.
76. The herpes simplex virus of claim 48 wherein the herpes simplex
virus is a mutant of one of HSV-1 strains 17 or F or HSV-2 strain
HG52.
77. The herpes simplex virus of claim 48 which is a gene specific
null mutant.
78. The herpes simplex virus of claim 48 which is an ICP34.5 null
mutant.
79. The herpes simplex virus of claim 48 which lacks at least one
expressible ICP34.5 gene.
80. The herpes simplex virus of claim 48 which lacks only one
expressible ICP34.5 gene.
81. The herpes simplex virus of claim 48 which is
non-neurovirulent
82. The herpes simplex virus of claim 48 for use in a method of
medical treatment.
83. The herpes simplex virus of claim 48 for use in the treatment
of cancer.
84. The herpes simplex virus of claim 48 for use in the oncolytic
treatment of a tumour.
85. (canceled)
86. A method of lysing or killing tumour cells in vitro or in vivo
comprising the step of administering to a patient in need of
treatment a therapeutically effective amount of the herpes simplex
virus of claim 48.
87. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 48.
88. The medicament, pharmaceutical composition or vaccine as
claimed in claim 87 further comprising a pharmaceutically
acceptable carrier, adjuvant or diluent.
89. A method of generating a nucleic acid vector comprising the
steps of: i) providing a first nucleotide sequence comprising a
predetermined second nucleotide sequence corresponding to a
selected nucleotide sequence in the RL1 locus of the genome of a
selected Herpes simplex virus; and ii) inserting nucleotide
sequence(s) in said second nucleotide sequence encoding: a) one or
a plurality of insertion sites and/or a nucleotide sequence of
interest; and b) a ribosome binding site or a regulatory nucleotide
sequence; and c) a marker, wherein the insertion site(s)/nucleotide
sequence of interest is arranged upstream (5') of the ribosome
binding site/regulatory nucleotide sequence and the ribosome
binding site/regulatory nucleotide sequence is arranged upstream
(5') of the marker.
90. The method of claim 89 wherein the inserted nucleotide
sequence(s) separates the second nucleotide sequence into two
vector flanking sequences, the inserted nucleotide sequences
forming a cassette therebetween.
91. The method as claimed in claim 89 wherein the second nucleotide
sequence corresponds to a nucleotide sequence in the RL terminal or
internal repeat region of the genome of the selected herpes simplex
virus.
92. The method of claim 89 wherein the second nucleotide sequence
corresponds to all or a part of the ICP34.5 protein coding sequence
of the genome of the selected herpes simplex virus.
93. The method of claim 89 wherein said second nucleotide sequence
comprises a contiguous portion of nucleotide sequence of the
ICP34.5 gene of the selected herpes simplex virus.
94. The method of claim 91 wherein said second nucleotide sequence
comprises a contiguous portion of nucleotide sequence encoding the
ICP34.5 gene product of the selected herpes simplex virus.
95. The method of claim 89 wherein the second nucleotide sequence
has at least 60% sequence identity to the corresponding sequence in
the viral genome.
96. The method of claim 89 wherein said second nucleotide sequence
hybridises to the corresponding nucleotide sequence in the viral
genome, or its complement, under high or very high stringency
conditions
97. A method of generating a mutant herpes simplex virus (HSV)
comprising inserting a nucleic acid cassette comprising nucleotide
sequence(s) encoding: a) one or a plurality of insertion sites
and/or a nucleotide sequence of interest; and b) a ribosome binding
site or a regulatory nucleotide sequence; and c) a marker into a
predetermined insertion site in the RL1 locus of the genome of a
selected HSV, wherein the insertion site(s)/nucleotide sequence of
interest is arranged upstream (5') of the ribosome binding
site/regulatory nucleotide sequence and the ribosome binding
site/regulatory nucleotide sequence is arranged upstream (5') of
the marker.
98. The method of claim 97 wherein said method comprises the steps
of: i) providing the vector of claim 1; ii) where the vector is a
plasmid, linearising the vector; and iii) co-transfecting a cell
culture with the linearised vector and genomic DNA from said
selected HSV.
99. The method of claim 98 wherein said co-transfection is carried
out under conditions effective for homologous recombination of said
cassette into an insertion site in the viral genome.
100. The method of claim 97 wherein said method further comprises
one or more of the steps of: 1) screening said co-transfected cell
culture to detect mutant HSV expressing said marker; and/or 2)
isolating said mutant HSV; and/or 3) screening said mutant HSV for
expression of the nucleotide sequence of interest or the RNA or
polypeptide thereby encoded; and/or 4) screening said mutant HSV
for lack of an active gene product; and/or 5) testing the oncolytic
ability of said mutant HSV to kill tumour cells in vitro.
101. The method of claim 97 wherein the nucleotide sequence of
interest is heterologous to the selected herpes simplex virus.
102. The method of claim 97 wherein the nucleotide sequence of
interest encodes an heterologous polypeptide.
103. The method as claimed in claim 102 wherein the heterologous
polypeptide is selected from the group consisting of: a bacterial
polypeptide; a mammalian polypeptide; a human polypeptide.
104. The method as claimed in claim 102 wherein the heterologous
polypeptide is selected from the group consisting of: Sodium iodide
symporter (NIS); Nitroreductase (NTR); E. coli NTR; Endothelial
nitric oxide synthase (eNOS); Granulocyte Macrophage
Colony-Stimulating Factor (GM-CSF); a cytokine.
105. The method as claimed in claim 101 wherein the nucleotide
sequence of interest encodes a selected antisense nucleic acid or
siRNA.
106. An herpes simplex virus generated by the method of claim
97.
107. An herpes simplex virus gene specific null mutant generated by
the method of claim 97.
108. An herpes simplex virus ICP34.5 null mutant generated by the
method of claim 97.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to nucleic acid vectors for
delivery of a nucleic acid cassette to an insertion site in a
selected viral genome, to methods of generating mutant virus using
said vectors and to the mutant viruses generated, and particularly,
although not exclusively, to mutant herpes simplex viruses and
nucleic acid vectors for use in generating mutant herpes simplex
viruses.
BACKGROUND TO THE INVENTION
[0002] Existing procedure for generating herpes simplex virus (HSV)
mutants requires generation of a unique plasmid by cloning an
entire expression cassette consisting of a promoter, gene of
interest and polyadenylation sequences into a plasmid separately
constructed to contain the relevant flanking sequences and then
co-transfecting BHK cells with the resultant plasmid and HSV-1 DNA.
Homologous recombination drives the formation of recombinant HSV-1
expressing the gene of interest, which is identified by Southern
blotting. The recombinant virus is plaque purified 3-4 times by
Southern blotting. This process takes several months.
[0003] This approach was taken by Liu et al.sup.1 in generating two
distinct plasmids, the first consisting of HSV-1 strain 17+ Sau3A
fragment derived sequences flanking an expression cassette
consisting of a CytoMegalovirus (CMV) promoter, Green Fluorescent
Protein (GFP) gene and bGH polyadenylation (polyA) signal and the
second wherein the GFP gene is replaced with either a human or
mouse Granulocyte Macrophage Colony Stimulating Factor (GM-CSF)
gene.
[0004] Shuttle vectors have been used to generate recombinant
adenoviral vectors, e.g. the pAdEasy.TM. system of vectors
(Stratagene), for use in overexpressing recombinant proteins in
mammalian cells. However, these vectors require the cloning of the
gene of interest into a first shuttle vector which is then
co-transformed into a specially constructed cell line to generate a
recombinant adenoviral plasmid which is transfected into a separate
specially constructed mammalian cell line in which the recombinant
adenoviral plasmid is directly packaged into virus particles.
[0005] The 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.
[0006] 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 1716.sup.2
(HSV1716) or the mutants R3616 or R4009 in strain F.sup.5, are
known to lack neurovirulence, i.e. be avirulent, and have utility
as both gene delivery vectors or in the treatment of tumours by
oncolysis. HSV1716 has a 759 bp deletion in each copy of the
ICP34.5 gene located within the BamHI s restriction fragment of
each RL repeat.
[0007] 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.
[0008] The prior art provides technically challenging, procedurally
slow and inefficient materials and methods for generating
recombinant HSV. In particular the prior art does not provide
methods of, and materials for, generating recombinant HSV which are
easy to detect, may be designed to be specific null mutants and
which may express a selected gene of interest.
[0009] First generation oncolytic viruses such as HSV-1 strain 17
mutant 1716 show significant therapeutic potential in tumour and
gene therapy. Overcoming the existing technical difficulties by
enabling rapid generation and screening of second generation
oncolytic viruses of this kind provides a significant improvement
in the development of novel pharmaceutical compositions, vaccines
and medicaments for the treatment of cancer and disease.
[0010] HSV 1716 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`).
SUMMARY OF THE INVENTION
[0011] The inventors have provided a generic plasmid vector
designated RL1.dIRES-GFP. 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] By using the plasmid RL1.dIRES-GFP to generate a shuttle
vector, designated RL1.dCMV-NTR-GFP, containing the E. coli
nitroreductase gene downstream of a CMV IE promoter, both inserted
at the MCS, the inventors have further provided a novel second
generation oncolytic mutant HSV. The genome of this mutant HSV
comprises the heterologous (i.e. non-HSV) 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 1716 and can be used in gene directed enzyme-prodrug
therapy 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.
[0019] 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.
[0020] 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.
[0021] Thus in some cases a cassette may be provided in which the
sequence of interest and marker are expressed separately from
independent promoters.
[0022] 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 (PGK) promoter. The marker is
thereby expressed under the control of this (the `first`)
regulatory sequence. The nucleotide sequence of interest (e.g.
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. It may also be
useful where the nucleotide sequence of interest has a strong
transcription and/or translation termination signal which may make
it difficult to transcribe and/or translate a single bi- or
poly-cistronic transcript encoding the sequence of interest 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.
[0023] At its most general the present invention relates to: (i) a
nucleic acid vector for delivery of a nucleic acid cassette to an
insertion site in a selected viral genome; (ii) an herpes simplex
virus wherein the herpes simplex virus genome comprises a
nucleotide sequence of interest.
[0024] The present invention further comprises novel HSV mutants
and viral vectors which may be generated using nucleic acid vectors
of the present invention, or vectors derived therefrom, and methods
for the generation of such vectors and HSV mutants.
[0025] According to one aspect of the present invention there is
provided a nucleic acid vector comprising, consisting or consisting
essentially of:
[0026] first and second nucleotide sequences corresponding to
nucleotide sequences flanking a predetermined insertion site in the
genome of a selected herpes simplex virus (HSV); and a cassette
located between said first and second nucleotide sequences
comprising nucleic acid encoding: [0027] (a) one or a plurality of
insertion sites; and [0028] (b) a ribosome binding site; and [0029]
(c) a marker.
[0030] Preferably the nucleic acid encoding the one or plurality of
insertion sites is/are arranged upstream (5') of the ribosome
binding site and the nucleic acid encoding the ribosome binding
site is arranged upstream (5') of the marker.
[0031] According to another aspect of the present invention there
is provided a nucleic acid vector comprising, consisting or
consisting essentially of:
[0032] first and second nucleotide sequences corresponding to
nucleotide sequences flanking a predetermined insertion site in the
genome of a selected herpes simplex virus (HSV); and a nucleic acid
cassette located between said first and second nucleotide sequences
comprising: [0033] (a) a third nucleotide sequence being of
interest; and nucleic acid encoding: [0034] (b) a ribosome binding
site; and [0035] (c) a marker.
[0036] Preferably the nucleotide sequence of interest is arranged
upstream (5') of the ribosome binding site and the ribosome binding
site is arranged upstream (5') of the marker.
[0037] According to another aspect of the present invention there
is provided an herpes simplex virus wherein the herpes simplex
virus (HSV) comprises a nucleic acid cassette integrated in the HSV
genome, the cassette comprising, consisting or consisting
essentially of nucleic acid encoding: [0038] (a) one or a plurality
of insertion sites; and [0039] (b) a ribosome binding site, and a
[0040] (c) marker.
[0041] Preferably, the nucleic acid encoding the one or plurality
of insertion sites is/are arranged upstream (5') of the ribosome
binding site and the nucleic acid encoding the ribosome binding
site is arranged upstream (5') of the marker.
[0042] According to another aspect of the present invention there
is provided an herpes simplex virus wherein the herpes simplex
virus (HSV) comprises a nucleic acid cassette integrated in the HSV
genome, the cassette comprising, consisting or consisting
essentially of nucleic acid encoding: [0043] (a) a nucleotide
sequence of interest; and nucleic acid encoding: [0044] (b) a
ribosome binding site; and [0045] (c) a marker.
[0046] Preferably, the nucleotide sequence of interest is arranged
upstream (5') of the ribosome binding site and the ribosome binding
site is arranged upstream (5') of the marker.
[0047] In various aspects of the present invention the ribosome
binding site may comprise an internal ribosome entry site (IRES). A
transcription product of the cassette may be a bi- or
poly-cistronic transcript comprising a first cistron encoded by the
nucleotide sequence of interest and a second cistron encoding the
marker nucleic acid wherein the ribosome binding site is located
between said first and second cistrons.
[0048] According to another aspect of the present invention there
is provided a nucleic acid vector comprising, consisting or
consisting essentially of:
[0049] first and second nucleotide sequences corresponding to
nucleotide sequences flanking a predetermined insertion site in the
genome of a selected herpes simplex virus (HSV); and a cassette
located between said first and second nucleotide sequences
comprising nucleic acid encoding: [0050] (a) one or a plurality of
insertion sites; and [0051] (b) a first regulatory nucleotide
sequence; and [0052] (c) a marker.
[0053] Preferably the nucleic acid encoding the one or plurality of
insertion sites is/are arranged upstream (5') of the regulatory
sequence and the nucleic acid encoding the regulatory sequence is
arranged upstream (5') of the marker.
[0054] According to another aspect of the present invention there
is provided a nucleic acid vector comprising, consisting or
consisting essentially of:
[0055] first and second nucleotide sequences corresponding to
nucleotide sequences flanking a predetermined insertion site in the
genome of a selected herpes simplex virus (HSV); and a nucleic acid
cassette located between said first and second nucleotide sequences
comprising: [0056] (a) a third nucleotide sequence being of
interest; and nucleic acid encoding: [0057] (b) a first regulatory
nucleotide sequence; and [0058] (c) a marker.
[0059] Preferably the nucleotide sequence of interest is arranged
upstream (5') of the regulatory sequence and the regulatory
sequence is arranged upstream (5') of the marker.
[0060] According to another aspect of the present invention there
is provided an herpes simplex virus wherein the herpes simplex
virus (HSV) comprises a nucleic acid cassette integrated in the HSV
genome, the cassette comprising, consisting or consisting
essentially of nucleic acid encoding: [0061] (a) one or a plurality
of insertion sites; and [0062] (b) a first regulatory nucleotide
sequence; and [0063] (c) a marker.
[0064] Preferably the nucleic acid encoding the one or plurality of
insertion sites is/are arranged upstream (5') of the regulatory
sequence and the nucleic acid encoding the regulatory sequence is
arranged upstream (5') of the marker.
[0065] According to another aspect of the present invention there
is provided an herpes simplex virus wherein the herpes-simplex
virus (HSV) comprises a nucleic acid cassette integrated in the HSV
genome, the cassette comprising, consisting or consisting
essentially of nucleic acid encoding: [0066] (a) a nucleotide
sequence of interest; and nucleic acid encoding: [0067] (b) a first
regulatory nucleotide sequence; and [0068] (c) a marker.
[0069] Preferably the nucleotide sequence of interest is arranged
upstream (5') of the regulatory sequence and the regulatory
sequence is arranged upstream (5') of the marker.
[0070] In the various aspects the encoded components of the
cassette are preferably arranged in a predetermined order as
described.
[0071] The first regulatory sequence may be operably linked to said
marker and may comprise a constitutive or inducible promoter. The
first regulatory sequence may thus have a role in regulating
transcription of the marker.
[0072] The nucleotide sequence of interest may encode an
heterologous polypeptide, which may be selected from the group
consisting of: a bacterial polypeptide; a mammalian polypeptide; a
human polypeptide or more particularly from the group consisting
of: Sodium iodide symporter (NIS); Nitroreductase (NTR); E. coli
NTR; Endothelial nitric oxide synthase (eNOS); Granulocyte
Macrophage Colony-Stimulating Factor (GM-CSF); a cytokine.
[0073] Alternatively, the nucleotide sequence of interest may
encode a selected antisense nucleic acid or siRNA.
[0074] The cassette may further comprise a regulatory nucleotide
sequence, located upstream (5') of the nucleotide sequence of
interest or insertion site(s), which has a role in regulating
transcription of the nucleotide sequence of interest.
[0075] 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.
[0076] Accordingly, vectors and herpes simplex viruses according to
the present invention may further comprise one or a plurality of
insertion sites, more preferably restriction endonuclease sites
encoded by nucleic acid of the cassette. Each insertion site may be
formed by nucleic acid encoding a restriction endonuclease site.
The insertion sites may comprise one or more of the ClaI, BglII,
NruI and XhoI restriction endonuclease sites.
[0077] The predetermined insertion site is preferably in the RL1
locus of the genome of the selected herpes simplex virus.
[0078] The first and second nucleotide sequences may each comprise
sequence corresponding to: [0079] (a) nucleotide sequences in the
RL terminal or internal repeat region of the genome of the selected
HSV; [0080] (b) nucleotide sequences in the RL1 locus of the genome
of the selected HSV; [0081] (c) nucleotide sequences flanking a
predetermined 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;
[0082] In other arrangements said first and second nucleotide
sequences may comprise contiguous portions of nucleotide sequence
of the ICP34.5 gene of a herpes simplex virus. Or said first and
second nucleotide sequences may comprise contiguous portions of
nucleotide sequence encoding the ICP34.5 gene product of a herpes
simplex virus.
[0083] The first and second nucleotide sequences may have at least
60% sequence identity to their corresponding sequence in the viral
genome. More preferably said identity may be one of at least 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. 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.
[0084] In certain arrangements the first and second nucleotide
sequences may hybridise to their corresponding nucleotide sequence
in the HSV genome, or its complement, under high or very high
stringency conditions.
[0085] The marker may be a defined nucleotide sequence encoding a
polypeptide. In one arrangement the marker may comprise the Green
Fluorescent Protein (GFP) protein coding sequence or the enhanced
Green Fluorescent Protein (EGFP) protein coding sequence.
[0086] In another arrangement the marker may comprise a defined
nucleotide sequence detectable by hybridisation under high or very
high stringency conditions with a corresponding labelled nucleic
acid probe.
[0087] The cassette may further comprise a nucleic acid encoding a
polyadenylation sequence located downstream (3') of the nucleic
acid encoding the marker. The polyadenylation sequence may comprise
the Simian Virus 40 (SV40) polyadenylation sequence.
[0088] Preferably, mutant HSV according to the present invention
are generated by site directed insertion of the cassette into the
viral genome, more preferably by homologous recombination.
[0089] The vector may further comprise nucleic acid encoding a
second selectable marker, e.g. a marker conferring antibiotic
resistance.
[0090] The vector is preferably a DNA vector, particularly a dsDNA
vector and may be an expression vector.
[0091] In one preferred arrangement, the vector 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`).
[0092] The selected herpes simplex virus may be an HSV-1 or HSV-2.
Thus, mutant HSV according to the present invention may be derived
from a strain of either HSV-1 or HSV-2. More preferably the
selected HSV may be one of HSV-1 strains 17 or F or HSV-2 strain
HG52. Thus, herpes simplex viruses of the invention may be mutants
of these preferred `parental` viruses.
[0093] In herpes simplex viruses according to the present invention
the cassette may be integrated in the HSV genome so as to disrupt a
protein coding sequence in the HSV genome resulting in inactivation
of the respective gene product.
[0094] Herpes simplex virus of the invention may be gene specific
null mutants. They may be ICP34.5 null mutants.
[0095] In some aspects of the present invention the herpes simplex
virus may lack at least one expressible ICP34.5 gene. In other
aspects of the present invention the generated herpes simplex virus
may lack only one expressible ICP34.5 gene.
[0096] Herpes simplex virus according to the present invention may
be non-neurovirulent.
[0097] Vectors and herpes simplex viruses according to the present
invention may be used in gene therapy.
[0098] According to another aspect of the present invention there
is provided a method of generating a herpes simplex virus which
expresses a nucleotide sequence of interest, or polypeptide thereby
encoded, comprising the step of culturing a selected herpes simplex
virus with a vector according to the present invention, thereby
integrating components (a), (b) and (c) of said vector at said
predetermined insertion site in the genome of the selected herpes
simplex virus.
[0099] Said integration at the predetermined insertion site is
preferably a permanent integration.
[0100] According to another aspect of the present invention there
is provided a method of generating a nucleic acid vector comprising
the steps of: [0101] i) providing a first nucleotide sequence
comprising a predetermined second nucleotide sequence corresponding
to a selected nucleotide sequence in the genome of a selected
Herpes simplex virus; and [0102] ii) inserting nucleotide
sequence(s) in said second nucleotide sequence encoding: [0103] a)
one or a plurality of insertion sites and/or a nucleotide sequence
of interest; and [0104] b) a ribosome binding site or a regulatory
nucleotide sequence; and [0105] c) a marker.
[0106] Preferably the insertion site(s)/nucleotide sequence of
interest is arranged upstream (5') of the ribosome binding
site/regulatory nucleotide sequence and the ribosome binding
site/regulatory nucleotide sequence is arranged upstream (5') of
the marker.
[0107] Said predetermined second nucleotide sequence may preferably
correspond to a selected nucleotide sequence in the RL1 locus of
the genome of the selected HSV.
[0108] The inserted nucleotide sequence(s) may separate the second
nucleotide sequence into two vector flanking sequences, the
inserted nucleotide sequences forming a cassette therebetween.
[0109] The second nucleotide sequence may correspond to a: [0110]
(a) nucleotide sequence in the RL terminal or internal repeat
region of the genome of the selected herpes simplex virus; [0111]
(b) nucleotide sequence in the RL1 locus of the genome of the
selected herpes simplex virus; [0112] (c) a nucleotide sequence
formed in, or comprising all or a part of, the ICP34.5 protein
coding sequence of the genome of a selected herpes simplex
virus.
[0113] The second nucleotide sequence may comprise a contiguous
portion of nucleotide sequence of the ICP34.5 gene of a herpes
simplex virus. Alternatively the second nucleotide sequence may
comprise a contiguous portion of nucleotide sequence encoding the
ICP34.5 gene product of a herpes simplex virus.
[0114] The second nucleotide sequence may have at least 60%
sequence identity to the corresponding sequence in the viral
genome. More preferably said identity may be one of at least 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%. 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.
[0115] Said second nucleotide sequence may hybridise to the
corresponding nucleotide sequence in the viral genome, or its
complement, under high or very high stringency conditions.
[0116] According to another aspect of the present invention there
is provided a method of generating a mutant herpes simplex virus
(HSV) comprising inserting a nucleic acid cassette comprising
nucleotide sequence(s) encoding: [0117] a) one or a plurality of
insertion sites and/or a nucleotide sequence of interest; and
[0118] b) a ribosome binding site or a regulatory nucleotide
sequence; and [0119] d) a marker into the genome of a selected
HSV.
[0120] Preferably the insertion site(s)/nucleotide sequence of
interest is arranged upstream (5') of the ribosome binding
site/regulatory nucleotide sequence and the ribosome binding
site/regulatory nucleotide sequence is arranged upstream (5') of
the marker.
[0121] The site of insertion may preferably be a predetermined
insertion site in the RL1 locus of the selected HSV.
[0122] The method of generating a mutant herpes simplex virus may
comprise the steps of: [0123] i) providing a vector according to
the present invention; [0124] ii) where the vector is a plasmid,
linearising the vector; and [0125] iii) co-transfecting a cell
culture with the linearised vector and genomic DNA from said
HSV.
[0126] The co-transfection may be carried out under conditions
effective for homologous recombination of said cassette into an
insertion site in the viral genome.
[0127] The method of generating a mutant herpes simplex virus may
further comprise one or more of the steps of: [0128] 1) screening
said co-transfected cell culture to detect mutant HSV expressing
said marker; and/or [0129] 2) isolating said mutant HSV; and/or
[0130] 3) screening said mutant HSV for expression of the
nucleotide sequence of interest or the RNA or polypeptide thereby
encoded; and/or [0131] 4) screening said mutant HSV for lack of an
active gene product; and/or [0132] 5) testing the oncolytic ability
of said mutant HSV to kill tumour cells in vitro.
[0133] In the method of generating a mutant herpes simplex virus
the nucleotide sequence of interest may be heterologous to the
selected herpes simplex virus. It may encode an heterologous
polypeptide which may be selected from the group consisting of: a
bacterial polypeptide; a mammalian polypeptide; a human polypeptide
or from the group consisting of: Sodium iodide symporter (NIS);
Nitroreductase (NTR); E. coli NTR; Endothelial nitric oxide
synthase (eNOS); Granulocyte Macrophage Colony-Stimulating Factor
(GM-CSF); a cytokine.
[0134] Alternatively the nucleotide sequence of interest may encode
a selected antisense nucleic acid.
[0135] In another aspect of the present invention there is provided
a mutant herpes simplex virus generated by the method of generation
disclosed herein. Herpes simplex viruses generated by such methods
may be gene specific null mutants, e.g. ICP34.5 null mutants.
[0136] The present invention may also include the following aspects
and features which may be provided in combination with any of the
other aspects and features described.
[0137] In aspects of the present invention the nucleotide sequence
of interest contained in the cassette may encode a polypeptide of
interest, or fragment thereof, or comprise selected antisense DNA,
that is DNA corresponding to at least one gene component (e.g.
regulatory sequence 5' UTR, 3'UTR or protein coding sequence) or
fragment of a gene component, which is inserted in the cassette in
an orientation such that upon transcription an antisense RNA is
obtained. Thus the expressed product of the nucleotide sequence of
interest may ultimately be a polypeptide, complete or truncated
(e.g. a polypeptide fragment), or an antisense nucleic acid,
preferably RNA.
[0138] By antisense nucleic acid is meant a nucleic acid having
substantial sequence identity to the nucleic acid formed by the
sequence of complementary bases to the single strand of a target
nucleic acid. Thus, the antisense nucleic acid is useful in binding
the target nucleic acid and may be used as an inhibitor to prevent
or disrupt the normal activity, folding or binding of the target
nucleic acid. The substantial sequence identity is preferably at
least 50% sequence identity, 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.
[0139] Where the nucleotide sequence of interest encodes a
polypeptide of interest the polypeptide may be any selected
polypeptide. Preferably, the polypeptide of interest is an
heterologous or exogenous polypeptide (i.e. a non-HSV originating
polypeptide), preferably a bacterial polypeptide, alternatively a
mammalian polypeptide or a human polypeptide. The heterologous
polypeptide may be useful in gene directed enzyme-prodrug targeting
techniques for tissue specific delivery of active pharmaceutical
agents. For example, the polypeptide of interest may be the Sodium
iodide symporter (NIS), Nitroreductase (NTR), preferably E. coli
NTR, Endothelial nitric oxide synthase (eNOS), Granulocyte
Macrophage Colony-Stimulating Factor (GM-CSF) or a cytokine.
[0140] The cassette preferably further comprises at least one
regulatory nucleotide sequence such as one or more selected
promoter or enhancer elements known to the person skilled in the
art, e.g. the CytoMegalovirus (CMV) promoter or
Phosphoglycerokinase (PGK) promoter. A regulatory nucleotide
sequence may be located upstream (i.e. 5') of the nucleotide
sequence of interest and have a role in controlling and regulating
transcription of the nucleotide sequence of interest and hence
expression of the resulting transcript or polypeptide. In certain
arrangements another regulatory sequence may be located upstream
(5') of the marker sequence and downstream (3') of the nucleotide
sequence of interest and have a role in controlling and regulating
transcription of the marker and hence expression of the resulting
transcript or polypeptide. In some arrangements this latter
regulatory sequence may take the place of the ribosome binding
site.
[0141] The components of the cassette are preferably arranged in a
predetermined order. In certain aspects, the nucleotide sequence of
interest 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. Thus during transcription a single transcript may be
produced from the cassette comprising a first cistron comprising
the nucleotide sequence of interest and a second cistron encoding
the marker wherein the ribosome binding site is located between the
cistrons.
[0142] A suitable ribosome binding site may comprise 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.
[0143] 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. One function of the marker
is to enable identification of virus plaques containing mutant
virus transformed with the cassette.
[0144] Alternatively, 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.
[0145] 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.
[0146] In another preferred arrangement, the cassette further
comprises a polyadenylation sequence (`polyA sequence`). Preferably
the polyA sequence comprises the Simian Virus 40 (SV40) polyA
sequence. The preferred location of the polyA sequence within the
cassette is immediately downstream (i.e. 3') of the marker.
[0147] The first and second nucleotide sequences preferably
comprise nucleotide sequences having identity to regions of the
genome surrounding the insertion site in the selected herpes
simplex virus strain (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.
[0148] 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.
[0149] Preferably, the first and second nucleotide sequences
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.
[0150] 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.
[0151] 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.
[0152] The nucleotide sequence of interest which forms part of the
inserted cassette may encode a full length transcript or
polypeptide (i.e. comprise the complete protein coding sequence).
Alternatively, the nucleotide sequence of interest 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 or antigenic peptide respectively. 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.
[0153] 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.
[0154] 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).
[0155] Preferably the first and second nucleotide sequences (vector
flanking sequences) 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). More preferably,
vector flanking sequences 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 or so as to
be within it entirely. 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 may 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.
[0156] Thus, vectors according to the present invention comprising
first and second nucleotide sequences corresponding to regions of
the RL repeat region flanking and/or overlapping or being entirely
within the ICP34.5 protein coding sequence may be used in the
generation of ICP34.5 null mutants. In such mutants all or a
portion of the ICP34.5 protein coding sequence may be excised and
replaced during the homologous recombination event such that both
copies of the ICP34.5 coding sequence are disrupted. Alternatively,
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.
[0157] 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.
[0158] Preferably, each component of the cassette is 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.
[0159] Preferably, the vector further comprises, consists, or
consists 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.
[0160] A vector of the present invention preferably comprises a DNA
vector, particularly a dsDNA vector. 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 strain, the vector is preferably
provided in linear form.
[0161] In one preferred arrangement, the vector 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`).
[0162] In another preferred arrangement, the vector is a variant of
plasmid RL1.dIRES-GFP.
[0163] Variant vectors may incorporate a regulatory nucleotide
sequence, e.g. promoter, in place of the ribosome binding site of
RL1.dIRES-GFP to control transcription of the nucleic acid encoding
the marker.
[0164] Vectors according to the present invention are preferably
constructed for use in generating engineered HSV-1 or HSV-2 by
insertion of a nucleic acid cassette, which insertion may be
through a mechanism of homologous recombination between nucleotide
sequences flanking the cassette and corresponding sequences in the
selected herpes simplex virus genome. The cassette may thereby be
permanently integrated at a selected site of the viral genome.
[0165] Thus, vectors according to the present invention may
comprise and have use as: [0166] i) gene delivery (gene therapy)
vectors for delivery of a selected protein coding sequence or
antisense nucleic acid to a specific locus of the HSV genome;
and/or [0167] ii) expression vectors for expression of the
delivered protein coding sequence or antisense nucleic acid of i)
from the HSV genome under the control of a selected regulatory
element; and/or [0168] iii) vectors for the generation of HSV
gene-specific mutants (optionally 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.
[0169] Vectors according to the present invention 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. Alternatively 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 tumours,
preferably by the oncolytic treatment of the tumour. Preferably,
such tumours may be primary or secondary (metastatic) tumours
originating either in the central or peripheral nervous system,
e.g. glioma, medulloblastoma, meningioma, neurofibroma, ependymoma,
Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma,
or 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 generated using vectors 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.
[0170] Vectors according to the present invention 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.
[0171] Vectors according to the present invention 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.
[0172] Vectors according to the present invention may also be used
in the manufacture of engineered HSV mutants wherein the genome of
the mutant HSV comprises a nucleotide sequence which may have been
inserted in the HSV genome by homologous recombination of the
cassette such that the nucleotide sequence is arranged to be
transcribed from the HSV genome under the control of a regulatory
element e.g. promoter, preferably a regulatory element forming part
of the inserted cassette, to produce an antisense transcript.
Preferably the antisense nucleotide sequence is an
exogenous/heterologous (i.e. non-HSV originating) sequence. 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.
[0173] Vectors according to the present invention may also be used
in the manufacture of an engineered HSV mutant wherein the genome
of the mutant HSV comprises a nucleotide sequence 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 inserted nucleotide sequence is
expressed under the control of a regulatory element to produce an
antisense 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.
[0174] In another aspect of the present invention there is provided
a method of lysing or killing tumour cells in vitro or in vivo
comprising the step of administering mutant HSV, having a mutation
in each ICP34.5 protein coding sequence and generated by a method
according to an aspect of the present invention, to tumour
cells.
[0175] In another aspect of the present invention there is provided
a method of treating a tumour comprising administering to a subject
mutant HSV having a mutation in each ICP34.5 protein coding
sequence and generated by a method according to an aspect of the
present invention.
[0176] In another aspect of the present invention there is provided
a medicament, pharmaceutical composition or vaccine comprising a
mutant HSV generated by a method according to an aspect of the
present invention. The medicament, pharmaceutical composition or
vaccine is preferably for use in the oncolytic treatment of tumours
and may further comprise a pharmaceutically acceptable carrier,
adjuvant or diluent.
[0177] In another aspect of the present invention there is provided
a kit of parts comprising a first container having a quantity of a
vector according to an aspect of the present invention and a second
container comprising a quantity of HSV genomic DNA.
[0178] In another aspect of the present invention there is provided
a mutant HSV generated using the vector of, or vectors derived
from, an aspect of the present invention. Preferably, the mutant is
a gene specific null mutant, more preferably an HSV ICP34.5 null
mutant, wherein the HSV genome comprises an inserted nucleotide
sequence of interest encoding a selected antisense RNA or an
heterologous polypeptide. Preferably the nucleotide sequence of
interest has been inserted in each RL region of the HSV genome,
more preferably at both of the ICP34.5 loci, still more preferably
the inserted heterologous nucleic acid disrupts the ICP34.5 protein
coding sequence such that both ICP34.5 genes are non-functional and
the mutant HSV is incapable of expressing an active ICP34.5 gene
product from the disrupted ICP34.5 protein coding sequences.
Preferably, the inserted heterologous nucleotide sequence is
non-endogenous to HSV and encodes a polypeptide of interest
selected from the group comprising or consisting of Sodium iodide
symporter (NIS), Nitroreductase (NTR), preferably E. coli NTR,
Endothelial nitric oxide synthase (eNOS), Granulocyte Macrophage
Colony-stimulating Factor (GM-CSF) or a cytokine.
[0179] The inserted nucleotide sequence of interest is preferably
expressed or capable of expression under the control of an inserted
regulatory element, preferably the CMV IE promoter. The mutant HSV
genome preferably encodes the GFP gene product. More preferably the
GFP coding sequence and nucleotide sequence of interest are
arranged to be transcribed on a single bi- or poly-cistronic
transcript such that expression of GFP is an indicator of HSV gene
specific null mutants transformed with the nucleotide sequence of
interest.
[0180] 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.
[0181] 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.
[0182] Medicaments comprising HSV 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 according to an aspect of the
present invention or a medicament comprising or derived from such
HSV are also provided.
[0183] The use of such mutant HSV in the treatment of disease,
including methods for the treatment of tumours/cancer, preferably
by oncolysis is provided. Use of such mutant HSV in the manufacture
of a medicament for use in these treatments is also provided.
[0184] Suitably, the administration of herpes simplex virus 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.
[0185] Alternatively injections may be intravenous. Alternative
administration routes may comprise oral or nasal
administration.
[0186] Herpes simplex viruses according to the present invention
may contain at least one copy of a nucleotide sequence of interest
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/heterologous 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.
[0187] 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 nucleotide sequence of interest 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.
[0188] 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.
[0189] In other aspects of the present invention vectors and/or
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/tumour of
any kind. The use of vectors and/or herpes simplex viruses
according to the present invention in the manufacture of a
medicament for the treatment of cancer is also provided.
[0190] 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.
[0191] A medicament, pharmaceutical composition or vaccine
comprising a vector or 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.
[0192] Herpes simplex viruses of the invention may be used 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.
[0193] 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. Cancer/tumour types which may be treated may be
primary or secondary (metastatic) tumours of any kind.
[0194] Treatable tumour types may include primary or secondary
(metastatic) tumours originating either in the central or
peripheral nervous system, e.g. glioma, medulloblastoma,
meningioma, neurofibroma, ependymoma, Schwannoma,
neurofibrosarcoma, astrocytoma and oligodendroglioma, or
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. Treatable metastatic tumours may be those of the central or
peripheral nervous system which originated in a non-nervous system
tissue.
[0195] 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
nucleotide sequence of interest encoded by the herpes simplex virus
genome.
[0196] Mutant HSV according to aspects of the present invention may
be useful in the treatment of disease by gene directed
enzyme-prodrug therapy and/or the treatment of disease, including
tumours, by the use of antisense RNA or siRNA technology.
[0197] 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 nucleotide sequence of interest, and
optionally returned/introduced to a patient's body, e.g. by
injection.
[0198] 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.
[0199] In vitro cultured cells, preferably human or mammalian
cells, transformed with viruses of the present invention and
preferably cells expressing the nucleotide sequence of interest as
well as methods of transforming such cells in vitro with said
viruses form further aspects of the present invention.
[0200] A nucleotide sequence of interest may be an
exogenous/heterologous sequence, i.e. one not originating in the
parent (wild-type) herpes simplex virus from which the herpes
simplex virus of the invention is derived.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
Hybridisation Stringency
[0206] In accordance with the present invention, nucleic acid
sequences may be identified by using hybridization and washing
conditions of appropriate stringency.
[0207] 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.
[0208] The "stringency" of a hybridization reaction can be readily
determined by a person skilled in the art.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0217] 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
[0218] FIG. 1. Generation of plasmid RL1.dIRES-GFP from plasmids
pNAT-IRES-GFP and RL1.del.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] *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 RL1.del.
[0223] N.B. Inserts could have been cloned in two orientations,
both of which were acceptable.
[0224] 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.
[0225] *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.
[0226] 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.
[0227] *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.
[0228] FIG. 7. Generation, detection and purification of ICP34.5
null HSV-1 expressing a gene product of interest.
[0229] FIG. 8. Strategy used to clone pCMV-NTR from pPS949 into
RL1.dIRES-GFP. (1) Digest pPS949 with BamH1 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.
[0230] * 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).
[0231] 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.
[0232] 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.
[0233] 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 .about.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.
[0234] 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.
[0235] FIG. 13. Growth kinetics of HSV17.sup.+, 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).
[0236] 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.
[0237] 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.sup.+ 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.
[0238] FIG. 16. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with
or without CB1954 (50 .mu.M) 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 50 .mu.M 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.
[0239] FIG. 17. Effect of HSV1716/CMV-NTR/GFP and HSV1716-GFP with
or without CB1954 (50 .mu.M) 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 50 .mu.M 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.
[0240] 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 C1954 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.
[0241] 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.
[0242] FIG. 20. Weight change (as a guide to health) in athymic
nude mice with subcutaneous A2780 (xenograft) tumours injected
intratumourally with HSV 1790. 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.
[0243] FIG. 21. Change in tumour volume over time in athymic nude
mice with A2780 xenografts after intratumoural injection of HSV
1790.
[0244] FIG. 22. Starting tumour sizes of mice.
[0245] FIG. 23. Alterations in weight after treatment with CMV-ntr,
CB1954 or a combination of both.
[0246] FIG. 24. Change in tumour volume after treatment with
CMV-ntr, CB1954 or a combination of both.
[0247] FIG. 25. Starting tumour volume of each treatment group (see
Table 2).
[0248] FIG. 26. Weight (as a measurement of health) in athymic nude
mice with A2780 xenograft treated with either HSV 1790, HSV 1716,
CB 1954 or a combination of them.
[0249] FIG. 27. Change in tumour volume of xenografts treated with
the prodrug CB1954.
[0250] FIG. 28. Changes in tumour volume in xenograft treated with
10.sup.5 PFU HSV 1790 and CB1954.
[0251] FIG. 29. Changes in tumour volume in xenografts treated with
10.sup.6 PFU HSV 1790 and CB1954.
[0252] FIG. 30. Changes in tumour volume in xenografts treated with
10.sup.5 PFU HSV 1716 and CB1954.
[0253] FIG. 31. Comparison of 10.sup.5 PFU, 10.sup.6 PFU HSV 1790
and 10.sup.5 PFU HSV 1716.
DETAILED DESCRIPTION OF THE BEST MODE OF THE INVENTION
[0254] 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.
Example 1
Construction of Plasmid RL1.dIRES-GFP
General Approach
[0255] Plasmid RL1.dIRES-GFP was generated in three stages,
illustrated in FIG. 1.
[0256] 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.
[0257] 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.
[0258] 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.
[0259] 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.
Materials and Methods
[0260] 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).
[0261] 4.times.1 .mu.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).
[0262] 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 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 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).
[0263] 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.
[0264] Ligation reactions were performed in small eppendorf tubes
containing 10 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`.
[0265] 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.
RL1.del
[0266] *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 PflMI-BstEII fragment of the RL1 gene
(125292-125769) has been removed and replaced with a multi-cloning
site (MCS) to form RL1.del.
pNAT-IRES-GFP
[0267] ** 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.
***Transformation of Bacterial Cells
[0268] 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 1/10.sup.th 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.
[0269] 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
General Approach
[0270] 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 RL1.dIRES.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.
[0271] 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.
[0272] 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.
[0273] 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.
[0274] 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).
[0275] 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.
Materials and Methods
[0276] 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.
[0277] 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.
Co-Transfection of Virus and Plasmid DNA by CaPO.sub.4 and DMSO
Boost
[0278] 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).
[0279] 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.
Plaque Purification
[0280] Sonicated samples from co-transfection plates are thawed and
serially diluted 10 fold in ETC10. 100 .mu.l from neat to the
10.sup.5 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.
Tissue Culture Media
[0281] 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
General Approach
[0282] 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.
Materials and Methods
[0283] 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).
[0284] 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).
[0285] 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 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 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`.
[0286] 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).
[0287] 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.
HSV1716/CMV-NTR/GFP Cell Killing
[0288] 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.
[0289] 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.
[0290] 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.
[0291] 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).
[0292] 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.
[0293] 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.
[0294] 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
In Vivo Evaluation of the Anti-Tumour Activity of a Selectively
Replication Competent Herpes Simplex Virus in Combination with
Enzyme Pro-Drug Therapy
[0295] 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.
[0296] The parental virus, HSV 1716 is a selectively replication
competent mutant of the herpes simplex virus 1 (HSV 1) 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.
[0297] It has already been shown that HSV 1716 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.
[0298] 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.
[0299] In order to enhance the efficacy of the tumour cell
killing--hence kill the entire tumour--the inventors have
constructed a derivative HSV1790 of HSV1716 that expresses the E.
Coli nitroreductase gene (ntr) under the control of a CMV early
promoter (see example 3 above). 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.
[0300] 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.
[0301] 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.
[0302] This example further evaluates this combination approach in
vivo in appropriate animal models.
Results
Months 1-3
[0303] 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.
[0304] 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).
[0305] 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.
[0306] 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
xenograft 28 No of cells days after injected per Number of cell
Cell Line mice mice injection LN 18 5 million 5 0/5 U373 MG 5
million 5 0/5
A2780 Tumour Take
[0307] 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%.
[0308] Thus increasing the number of gliomal cells injected may
increase the take rate of these cell lines
Dose Response to the HSV 1790 Virus
[0309] 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.
[0310] 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.
[0311] 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.
[0312] 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.
[0313] A dose of 10.sup.9 PFU of the HSV 1790 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.
[0314] 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.
Response of the Tumour to HSV 1790 Treatment
[0315] Tumour volume was measured daily after intratumoural
injection of the HSV 1790 virus to look for any growth delay or
regression of the tumours.
[0316] 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.
[0317] 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.
[0318] 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.
Naked DNA Experiments
[0319] 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 HSV 1716 virus, an experiment was
set up looking at the effect of the CMV-ntr plasmid DNA alone and
in combination with the prodrug CB1954.
[0320] 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).
Scheduling Experiment
[0321] 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.
[0322] 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.
[0323] 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.
[0324] 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.
[0325] 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.
[0326] 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.
[0327] 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.
[0328] 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
(Day10) 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)
[0329] 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.
[0330] 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.
[0331] 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.
[0332] 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.
[0333] 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.
S No Virus+CB1954 Prodrug
[0334] 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.
10.sup.5 PFU Virus+/-CB1954 Prodrug
[0335] 10.sup.5 PFU virus was given at Day 0 followed by either
prodrug or vehicle at Days 2 and 10.
[0336] 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.
[0337] Treatment with virus and prodrug resulted in tumours which
grew to only approx 2-3 mm.sup.3 in volume. This is significantly
less than the untreated tumours which grew in size to approx 20
mm.sup.3.
10.sup.6 PFU Virus+/-CB1954 Prodrug
[0338] 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.
HSV 1716 Virus in Combination with CB1954 Prodrug
[0339] 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.
[0340] The combination of the virus and the prodrug appeared to
produce some reduction in tumour growth compared to the untreated
control tumours.
[0341] 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 HSV 1716 virus.
Comparison of HSV 1790 (at 10.sup.5 and 10.sup.6) and HSV 1716 in
Combination with CB1954 Prodrug
[0342] FIG. 31 shows a comparison between the two doses of the HSV
1790 virus in combination with the prodrug and the HSV 1716 prodrug
combination. The parental virus HSV 1716 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.
[0343] 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.
[0344] In conclusion it would appear from these results that indeed
the HSV 1790 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.
[0345] 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.
[0346] 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.
Example 5
Construction of HSV1716 Variants Expressing siRNA
General Strategy
[0347] A plasmid that contains the siRNA construct designed to
target expression of the SCCRO gene (SEQ ID No. 1) and designated
339i was provided by Dr Bhuv Singh, MSKCC, New York. A plasmid
encoding a control siRNA (SEQ ID No 2), designated Coni, was also
provided.
[0348] DNA sequences for the two constructs are as follows:
TABLE-US-00003 339isiRNA (SEQ ID No.1):
gatcCCCGTTCAGAGCAGCAACACAGTTCAAGAGACTGTGTTGCTGCTCT
GAACTTTTTGGAAA
[0349] TABLE-US-00004 ConisiRNA (SEQ ID No.2):
gatcCCCCGTCTACCTACACTCCCTCTTCAAGAGAGAGGGAGTGTAGGTA GACGTTTTTA
[0350] Both siRNA constructs were in the vector pSNRG and their
expression is driven by the RNA polIII H1 promoter. RNA polIII only
transcribes short RNA molecules and the H1 promoter would be
insufficient to drive expression of IRES-gfp from the normal
recombinant virus producing shuttle vector RL1-del.IRES.gfp so an
alternative cloning strategy was adopted.
[0351] A cassette was constructed in the following manner. The 1.3
kbp blunt-ended EcoRI/AflII 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/GFPfor and RL1-dPGK/GFPrev. Expression of GFP was
confirmed in BHK cells transfected with the forward and reverse
orientation plasmids.
[0352] Thus, sequences of interest along with their own promoters
(in this arrangement it is preferred that a different promoter is
used to drive transcription of the nucleotide sequence of interest
and marker) can then be cloned into either RL1-dPGK/GFPfor 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
Materials and Methods
[0353] In the pSNRG plasmid and adjacent to the H1/siRNA coding
sequence is a green fluorescent protein (gfp) expression cassette
comprising the gfp gene with a Phosphoglycerokinase (PGK) promoter.
Using the restriction enzymes HindIII and AflII sequentially, the
1.6 kbp DNA fragment that contains the H1/siRNA and PGK/EGFP
expression cassettes were excised from their Coni and 339i
plasmids. The 1.6 kbp DNA fragment was purified from a 1% agarose
gel and blunt-ended by incubation with Klenow DNA polymerase for 30
minutes at 30.degree. C. The blunt-ended fragment was ligated into
the RL1-del shuttle vector which had been digested with the
restriction enzyme Nru1 that produces a blunt-ended cut. Before
ligation the Nru1-cut RL1-del was gel purified and
phosphatase-treated using Calf Intestinal Alkaline Phosphatase.
After an overnight ligation with either the blunt-ended 339i or
Coni DNA fragments with the blunt-ended RL1-del plasmid, the
reaction mix was used to transform DH5alpha cells and these were
plated-out on LB amp plates. After overnight incubation at
37.degree. C., individual clones from each of the LB amp plates
were grown overnight in 3 ml of LB broth and plasmid DNA
extracted.
[0354] To screen for recombinants, plasmids were initially digested
with BamHI, as insertion of the H1/siRNA and PGK/gfp cassette
increases the size of the RL1 BamHI fragment in the plasmid from
5.4 kbp to 7.0 kbp. For both Coni and 339i ligations 1/24 clones
screened demonstrated a 7.0 kbp BamHI fragment and the presence of
the H1/siRNA and PGK/EGFP cassette in these plasmids was confirmed
by EcoR1, EcoR1/HindIII and EcoR1/SalI digests, the inserted
H1/siRNA and PGK/EGFP cassette introduces a novel EcoR1 site into
the RL1-del vector.
[0355] From a glycerol stock of the positive 339i and Coni clones,
additional plasmid was prepared and used to transfect BHK cells.
Fifty microlitres (50 .mu.l) of plasmid was mixed with 6 .mu.l
lipofectamine 2000 in a final volume of 100 .mu.l serum free medium
and used to transfect BHK cells plated out on a 13 mm glass
coverslip in a 24-well plate. After 48 hrs of transfection the
cells were washed once in PBS, incubated for 2 hrs in 4%
paraformaldehyde, washed once more in PBS and mounted on microscope
slides using Vectashield. The presence of c5% gfp-positive cells
following transfection with the RL1-del/339i and RL1-del/Coni
plasmids confirmed the presence of the PGK/GFP cassette.
[0356] The RL1-del/339i and RL1-del/Coni plasmids were linearized
using either of the restriction enzymes ScaI and XmnI and the
linearized plasmid was used along with viral DNA to transfect BHK
cells plated out to c80% confluency in 60 mm dishes. To 100 .mu.l
of linearized plasmid or undigested circular plasmid, 50 .mu.l of
HSV-1 strain 17+ DNA was added along with 20 .mu.l lipofectamine
2000 in a final volume of 5001 serum free medium and the mix added
to the BHK cells. After 4 hrs of transfection, the cells were
shocked with 25% DMSO in HBSS for exactly 4 minutes, washed
.times.3 with medium and returned to 37.degree. C. incubation in 5
ml of medium for 48 hrs. Viral cpe was evident after 48 hrs and the
cells and medium were harvested together, sonicated and stored at
-80.degree. C. Undiluted medium/cells and 4.times.10-fold dilutions
were plated out on BHK cells and, after 48 hrs, viral plaques were
examined by fluorescent microscopy for gfp expression. On the
undiluted plate from cells transfected with XmnI-linearized plasmid
>100 gfp-positive plaques were observed for both Coni and 339i
indicating a high degree of recombination. Interestingly,
recombination, but at a lower frequency (c50 plaques/plate), was
observed for the transfected circular plasmid but recombination
with the ScaI-linearized plasmid was very low (<5
plaques/plate).
[0357] Using the highest dilution at which gfp-positive plaques
were clearly visible (the PGK/GFP cassette gave a very strong
fluorescent signal), two plaques each of Coni and 339i viruses were
picked using a sterile pipette tip, placed in 1 ml medium,
sonicated for 1 minute and stored at -80.degree. C. Plaques were
then subjected to 6 rounds of plaque purification, after, the
6.sup.th round no wild type, non-gfp expressing plaques were
visible and 6 plaques each of Coni or 339i virus were picked for
Southern blotting.
[0358] Each of the six plaques of Coni and 339i virus was used to
infect a T175 flask of Vero cells, after 72 hrs of infection virus
was harvested and titred. For 3 each of the Coni and 339i viruses
that gave the highest titres 0.5 ml was used to infect a second
T175 flask for 24 hrs. Viral DNA was then harvested from each of
the 6 flasks. The BamHI-digested viral DNA was Southern blotted
with the Alu/Rsa RL1 probe and the band pattern compared to wild
type and HSV1716 DNA digested also with BamHI. A novel c6 kbp band,
consistent with the insertion of the 1.6 kbp H1/siRNA and PGK/GFP
cassette in the RL1 locus, was clearly visible in all six viral
isolates and no wild type bands were detected. Stocks of the Coni
and 339i viruses that gave the strongest signal on Southern
blotting were produced.
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Papanastassiou V et al. Toxicity evaluation of
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1716) in patients with recurrent malignant glioma. Gene Ther 2000;
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Sequence CWU 1
1
2 1 64 DNA Artificial sequence 339isiRNA; siRNA construct designed
to target expression of the SCCRO gene 1 gatccccgtt cagagcagca
acacagttca agagactgtg ttgctgctct gaactttttg 60 gaaa 64 2 60 DNA
Artificial sequence ConisiRNA; control siRNA 2 gatcccccgt
ctacctacac tccctcttca agagagaggg agtgtaggta gacgttttta 60
* * * * *