U.S. patent application number 10/579622 was filed with the patent office on 2007-01-04 for mutant viruses.
Invention is credited to Susanne Moira Brown, Paul Dunn, Ian Ganly, Bhuvanesh Singh.
Application Number | 20070003520 10/579622 |
Document ID | / |
Family ID | 34621663 |
Filed Date | 2007-01-04 |
United States Patent
Application |
20070003520 |
Kind Code |
A1 |
Brown; Susanne Moira ; et
al. |
January 4, 2007 |
Mutant viruses
Abstract
An herpes simplex virus wherein the herpes simplex virus genome
comprises nucleic acid encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO); and an herpes simplex virus
wherein the herpes simplex virus genome comprises nucleic acid
encoding a short interfering ribonucleic acid (siRNA) molecule that
is capable of repressing or silencing expression of squamous cell
carcinoma related oncogene (SCCRO) nucleic acid or polypeptide are
disclosed together with methods for generation and applications of
such viruses.
Inventors: |
Brown; Susanne Moira;
(Glasgov, GB) ; Singh; Bhuvanesh; (New York,
NY) ; Ganly; Ian; (New York, NY) ; Dunn;
Paul; (Glasgow, GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Family ID: |
34621663 |
Appl. No.: |
10/579622 |
Filed: |
November 17, 2004 |
PCT Filed: |
November 17, 2004 |
PCT NO: |
PCT/GB04/04908 |
371 Date: |
May 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60541308 |
Feb 3, 2004 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
424/93.6; 435/235.1 |
Current CPC
Class: |
C12N 15/86 20130101;
A61P 35/00 20180101; C12N 15/1135 20130101; C12N 2710/16643
20130101; A61K 38/00 20130101; C12N 2840/203 20130101 |
Class at
Publication: |
424/093.2 ;
424/093.6; 435/235.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 17, 2003 |
GB |
0326798.6 |
Claims
1. An herpes simplex virus wherein the herpes simplex virus genome
comprises nucleic acid encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO).
2. The herpes simplex virus according to claim 1 wherein said
nucleic acid encodes a mammalian asSCCRO.
3. The herpes simplex virus according to claim 1 wherein said
nucleic acid encodes the human asSCCRO.
4. The herpes simplex virus according to claim 1 wherein said
nucleic acid encodes a nucleotide sequence having at least 60%
sequence identity to the nucleotide sequence complementary to: (i)
the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;
(ii) the mRNA transcript of SEQ ID No. 1 or 3; or (iii) to a
fragment of said polynucleotide sequence or mRNA transcript.
5. The herpes simplex virus according to claim 4 wherein said
nucleic acid encodes a nucleotide sequence complementary to: (i)
the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;
(ii) the mRNA transcript of SEQ ID No. 1 or 3; or (iii) to a
fragment of said polynucleotide sequence or mRNA transcript.
6. The herpes simplex virus according to claim 4 wherein said
degree of sequence identity is at least 70%.
7. The herpes simplex virus claim 4 wherein a said fragment
comprises at least 20 nucleotides and no more than 900
nucleotides.
8. The herpes simplex virus according to claim 1 wherein said
nucleic acid hybridises under high stringency conditions to: (i)
the polynucleotide sequence of SEQ ID No. 1 or 3 or its complement;
(ii) the mRNA transcript of SEQ ID No. 1 or 3; or (iii) a fragment
of said polynucleotide sequence or mRNA transcript.
9. The herpes simplex virus of claim 1 wherein said herpes simplex
virus genome further comprises a regulatory sequence operably
linked to said nucleic acid encoding an antisense to the squamous
cell carcinoma related oncogene (as SCCRO), wherein said regulatory
sequence has a role in controlling transcription of said
asSCCRO.
10. The herpes simplex virus of claim 1 wherein said nucleic acid
is located in at least one RL1 locus of the herpes simplex virus
genome.
11. The herpes simplex virus of claim 1 wherein said nucleic acid
is located in, or overlaps, at least one of the ICP34.5 protein
coding sequences of the herpes simplex virus genome.
12. The herpes simplex virus of claim 1 wherein the herpes simplex
virus is a mutant of one of HSV-1 strains 17 or F or HSV-2 strain
HG52.
13. The herpes simplex virus of claim 1 wherein the herpes simplex
virus is a mutant of HSV-1 strain 17 mutant 1716.
14. The herpes simplex virus of claim 1 which is a gene specific
null mutant.
15. The herpes simplex virus of claim 1 which is an ICP34.5 null
mutant.
16. The herpes simplex virus of claim 1 which lacks at least one
expressible ICP34.5 gene.
17. The herpes simplex virus of sclaim 1 which lacks only one
expressible ICP34.5 gene.
18. The herpes simplex virus of claim 1 which is
non-neurovirulent.
19. The herpes simplex virus of claim 1 wherein said nucleic acid
encoding the asSCCRO forms part of a nucleic acid cassette
integrated in the genome of said herpes simplex virus, said
cassette comprising nucleic acid encoding: (a) said asSCCRO; and
nucleic acid encoding: (b) a ribosome binding site; and (c) a
marker, wherein the nucleic acid encoding asSCCRO is arranged
upstream (5') of the ribosome binding site and the ribosome binding
site is arranged upstream (5') of the marker.
20. The herpes simplex virus according to claim 19 wherein a
regulatory nucleotide sequence is located upstream (5') of the
nucleic acid encoding asSCCRO, wherein the regulatory nucleotide
sequence has a role in regulating transcription of said nucleic
acid encoding the asSCCRO.
21. The herpes simplex virus as claimed in claim 19 wherein the
cassette disrupts a protein coding sequence resulting in
inactivation of the respective gene product.
22. The herpes simplex virus of claim 19 wherein a transcription
product of the cassette is a bi- or poly-cistronic transcript
comprising a first cistron encoding the asSCCRO and a second
cistron encoding the marker nucleic acid wherein the ribosome
binding site is located between said first and second cistrons.
23. The herpes simplex virus of claim 19 wherein the ribosome
binding site comprises an internal ribosome entry site (IRES).
24. The herpes simplex virus of claim 19 wherein the marker is a
defined nucleotide sequence encoding a polypeptide.
25. The herpes simplex virus as claimed in claim 24 wherein the
marker comprises the Green Fluorescent Protein (GFP) protein coding
sequence or the enhanced Green Fluorescent Protein (EGFP) protein
coding sequence.
26. The herpes simplex virus of claim 19 wherein the marker
comprises a defined nucleotide sequence detectable by hybridisation
under high stringency conditions with a corresponding labelled
nucleic acid probe.
27. The herpes simplex virus of claim 19 wherein the cassette
further comprises nucleic acid encoding a polyadenylation sequence
located downstream (3') of the nucleic acid encoding the
marker.
28. The herpes simplex virus as claimed in claim 27 wherein the
polyadenylation sequence comprises the Simian Virus 40 (SV40)
polyadenylation sequence.
29.-32. (canceled)
33. 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 the herpes simplex virus claim 1.
34. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 1.
35. The medicament, pharmaceutical composition or vaccine as
claimed in claim 34 further comprising a pharmaceutically
acceptable carrier, adjuvant or diluent.
36. An herpes simplex virus, wherein the genome of said virus
comprises a nucleic acid sequence encoding an antisense to the
squamous cell carcinoma related oncogene (asSCCRO), wherein (1) the
nucleic acid sequence is in at least one of the long repeat regions
(R.sub.L), or (2) the herpes simplex virus is
non-neurovirulent.
37.-41. (canceled)
42. A method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment athe herpes simplex
virus of claim 36.
43. (canceled)
44. The method of claim 42 wherein said herpes simplex virus is
capable of killing tumour cells.
45. A method of expressing in vitro or in vivo an antisense to the
squamous cell carcinoma related oncogene (asSCCRO), said method
comprising the step of infecting at least one cell or tissue of
interest with the herpes simplex virus of claim 36, wherein asSCCRO
is operably linked to a transcription regulatory sequence.
46. (canceled)
47. HSV1716asSCCRO (ECACC accession number 04051901).
48. An herpes simplex virus wherein the herpes simplex virus genome
comprises nucleic acid encoding a short interfering ribonucleic
acid (siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide.
49. The herpes simplex virus according to claim 48 wherein said
siRNA is capable of repressing or silencing expression of a
mammalian SCCRO.
50. The herpes simplex virus according to claim 48 wherein said
siRNA is capable of repressing or silencing expression of human
SCCRO.
51. The herpes simplex virus according to claim 48 wherein said
siRNA comprises a nucleic acid of between 10 and 70 nucleotides in
length and having the sequence of SEQ ID No.5 or the complement
thereof.
52. The herpes simplex virus according to claim 48 wherein said
siRNA comprises a nucleic acid of between 10 and 70 nucleotides in
length and having at least 70% identity to SEQ ID No.5 or the
complement thereof.
53. The herpes simplex virus according to claim 52 wherein said
degree of sequence identity is at least 80%.
54. The herpes simplex virus of claim 48 wherein said herpes
simplex virus genome fuarther comprises a regulatory sequence
operably linked to said siRNA, wherein said regulatory sequence has
a role in controlling transcription of said siRNA.
55. The herpes simplex virus of claim 48 wherein said nucleic acid
is located in at least one RL1 locus of the herpes simplex virus
genome.
56. The herpes simplex virus of claim 48 wherein the said nucleic
acid is located in, or overlaps, at least one of the ICP34.5
protein coding sequences of the herpes simplex virus genome.
57. 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.
58. The herpes simplex virus of claim 48 wherein the herpes simplex
virus is a mutant of HSV-1 strain 17 mutant 1716.
59. The herpes simplex virus of claim 48 which is a gene specific
null mutant.
60. The herpes simplex virus of claim 48 which is an ICP34.5 null
mutant.
61. The herpes simplex virus of claim 48 which lacks at least one
expressible ICP34.5 gene.
62. The herpes simplex virus of claim 48 which lacks only one
expressible ICP34.5 gene.
63. The herpes simplex virus of claim 48 which is
non-neurovirulent.
64. The herpes simplex virus of claim 48 wherein said nucleic acid
encoding said siRNA forms part of a nucleic acid cassette
integrated in the genome of said herpes simplex virus, said
cassette comprising nucleic acid encoding: (a) said siRNA; and
nucleic acid encoding: (b) a first regulatory nucleotide sequence;
and (c) a marker, wherein the nucleic acid encoding said siRNA is
arranged upstream (5') of the first regulatory nucleotide sequence
and the first regulatory nucleotide sequence is arranged upstream
(5') of the marker, wherein said first regulatory sequence has a
role in controlling transcription of said marker.
65. The herpes simplex virus according to claim 64 wherein a second
regulatory nucleotide sequence is located upstream (5') of the
nucleic acid encoding said siRNA, wherein the second regulatory
nucleotide sequence has a role in regulating transcription of said
nucleic acid encoding said siRNA.
66. The herpes simplex virus as claimed in claim 64 wherein the
cassette disrupts a protein coding sequence resulting in
inactivation of the respective gene product.
67. The herpes simplex virus of claim 64 wherein the marker is a
defined nucleotide sequence encoding a polypeptide.
68. The herpes simplex virus as claimed in claim 67 wherein the
marker comprises the Green Fluorescent Protein (GFP) protein coding
sequence or the enhanced Green Fluorescent Protein (EGFP) protein
coding sequence.
69. The herpes simplex virus of claim 64 wherein the marker
comprises a defined nucleotide sequence detectable by hybridisation
under high stringency conditions with a corresponding labelled
nucleic acid probe.
70. The herpes simplex virus of claim 64 wherein the cassette
further comprises nucleic acid encoding a polyadenylation sequence
located downstream (3') of the nucleic acid encoding the
marker.
71. The herpes simplex virus as claimed in claim 70 wherein the
polyadenylation sequence comprises the Simian Virus 40 (SV40)
polyadenylation sequence.
72. The herpes simplex virus of claim 48 for use in a method of
medical treatment.
73. The herpes simplex virus of claim 48 for use in the treatment
of cancer.
74. The herpes simplex virus of claim 48 for use in the oncolytic
treatment of a tumour.
75. (canceled)
76. 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 the herpes simplex virus of claim 48.
77. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 48.
78. The medicament, pharmaceutical composition or vaccine as
claimed in claim 77 further comprising a pharmaceutically
acceptable carrier, adjuvant or diluent.
79. An herpes simplex virus, wherein the genome of said virus
comprises a nucleic acid sequence encoding a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide, wherein (a) the siRNA is in at
least one of the long repeat regions (R.sub.L), or (b) the herpes
simplex virus is non-neurovirulent.
80.-85. (canceled)
86. A method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment a herpes simplex
virus, wherein the genome of said virus comprises a nucleic acid
sequence encoding a short interfering ribonucleic acid (siRNA)
molecule that is capable of repressing or silencing expression of
squamous cell carcinoma related oncogene (SCCRO) nucleic acid or
polypeptide and wherein (a) nucleic acid sequence is in at least
one of the long repeat regions (R.sub.L), or (b) the herpes simplex
virus is non-neurovirulent.
87. The method of claim 86 wherein said herpes simplex virus is
capable of killing tumour cells.
88. A method of expressing in vitro or in vivo a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide, said method comprising the
step of infecting at least one cell or tissue of interest with a
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding said siRNA in at least one of the
long repeat regions (R.sub.L), wherein said nucleic acid sequence
encoding said siRNA is operably linked to a transcription
regulatory sequence.
89. A method of expressing in vitro or in vivo a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide, said method comprising the
step of infecting at least one cell or tissue of interest with a
non-neurovirulent herpes simplex virus, wherein the genome of said
virus comprises a nucleic acid sequence encoding said siRNA,
wherein said nucleic acid sequence encoding said siRNA is operably
linked to a transcription regulatory sequence.
90. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 36.
91. The medicament, pharmaceutical composition or vaccine as
claimed in claim 90 further comprising a pharmaceutically
acceptable carrier, adjuvant or diluent.
92. A medicament, pharmaceutical composition or vaccine comprising
the herpes simplex virus of claim 79.
93. The medicament, pharmaceutical composition or vaccine as
claimed in claim 92 further comprising a pharmaceutically
acceptable carrier, adjuvant or diluent.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to materials and methods
relating to the squamous cell carcinoma related oncogene (SCCRO)
and to mutant herpes simplex viruses.
BACKGROUND OF THE INVENTION
[0002] The herpes simplex virus (HSV) genome comprises two
covalently linked segments, designated long (L) and short (S). Each
segment contains a unique sequence flanked by a pair of inverted
terminal repeat sequences. The long repeat (RL or R.sub.L) and the
short repeat (RS or R.sub.S) are distinct.
[0003] The HSV ICP34.5 (also .gamma.34.5) gene, which has been
extensively studied.sup.1,6,7,8, has been sequenced in HSV-1
strains F.sup.9 and syn17+.sup.3 and in HSV-2 strain HG52.sup.4.
One copy of the ICP34.5 gene is located within each of the RL
repeat regions. Mutants inactivating both copies of the ICP34.5
gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 17162
(HSV1716) or the mutants R3616 or R4009 in strain F.sup.5, are
known to lack neurovirulence, i.e. be avirulent, and have utility
as both gene delivery vectors or in the treatment of tumours by
oncolysis. HSV-1 strain 17 mutant 1716 has a 759 bp deletion in
each copy of the ICP34.5 gene located within the BamHI s
restriction fragment of each RL repeat. ICP34.5 null mutants such
as 1716 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.
[0004] 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
OJG, 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`).
[0005] Squamous cell carcinoma of the head and neck afflicts an
estimated 125,000 patients annually in Europe, North America and
the Far East. Primary therapy for localized disease is surgery and
adjuvant radiotherapy. Tumours recur in approximately one-third of
patients. Once the cancer has recurred and/or metastasized, the
patient is considered incurable. Combination chemotherapy induces
responses in 30-50% of patients but there is no clear impact on
survival. There remains an urgent need for more effective
therapies.sup.12,13.
[0006] There has been much interest in the use of novel therapies
in this disease with particular focus on oncolytic viruses by
direct intratumoural injection. The use of oncolytic viruses to
selectively kill tumours while leaving normal cells unaffected is a
very attractive concept as it has the potential to limit the
toxicity which occurs with conventional modalities. Recent research
has been carried out using intratumoural injections of a
selectively replicating adenovirus (Onyx-015) for the local control
of recurrent disease. Phase I/II studies involving virus alone and
in combination with chemotherapy have produced encouraging
results.sup.14,15,16
[0007] Selectively replicating Herpes simplex viruses HSV may have
better efficacy due to its more potent replication and oncolytic
potential. HSV1716.sup.17 is a deletion mutant of herpes simplex
virus which fails to synthesise the virulence protein ICP34.5. It
has been shown that HSV1716 replicates in actively dividing cells
but not in resting or terminally differentiated cells.sup.18,19. In
vivo, HSV1716 administration has been carried out in mouse models
of a range of cancers including melanoma, teratocarcinoma, glioma,
medulloblastoma and mesothelioma. Animals showed improved survival
and tumour regression following administration of
HSV1716.sup.20,21,22,23,24,25 with no evidence of replication in
normal tissue and no toxicity. HSV1716 has been used in Phase 1
trials in patients with glioblastoma multeforme (GBM).sup.26,
melanoma and head and neck cancer. No toxicity has been experienced
and patients who were seropositive pre HSV1716 seroconverted and
evidence of virus replication contained within tumours has been
obtained.
[0008] It has been shown that the novel oncogene SCCRO (Squamous
cell carcinoma related oncogene (also called Oncoseq and sometimes
called SCRO)) is amplified in 30% of mucosal squamous cell cancers
and that overexpression is associated with poor prognosis in head
and neck cancer patients.
[0009] The Oncoseq nucleic acid sequence was described in U.S. Ser.
No. 10/361,725 having publication number US 2004/0009541, published
on 15 Jan. 2004. This document is incorporated herein in its
entirety by reference. A polynucleotide sequence including an open
reading frame of 780 nucleotides for Oncoseq and the amino acid
sequence of the 259-residue polypeptide encoded thereby was
reported.
[0010] US 2004/0009541 describes Oncoseq alleles to be oncogenes
identified in primary squamous cell carcinoma tissues as being
colocalised with the highest gene duplication peak within the
3q26.3 locus using a positional cloning approach with Oncoseq being
highly duplicated in those carcinomas. Overexpression of Oncoseq is
described to be correlated with gene duplication, aggressive
clinical behaviour and malignant transformation in vitro, making it
a strong candidate as the target for 3q amplification. The gene is
described to be highly oncogenic and to have a basic
region-helix-loop-helix-leucine zipper motif, suggesting it may
function as a transcription factor.
[0011] RNAi
[0012] RNAi utilises small double-stranded RNA molecules (dsRNA) to
target messenger RNA (mRNA), the precursor molecule that cells use
to translate the genetic code into functional proteins. During the
natural process of RNAi, dsRNA is processed into short-interfering
RNA (siRNA) duplexes of 21 nucleotides in length, and it is these
molecules which recognise and target homologous (endogenous) mRNA
sequences for enzymatic degradation (by complementary base-pair
binding), resulting in gene silencing.
[0013] The advantages of RNAi over other gene-targeting strategies
such as anti-sense oligonucleotides include its relative
specificity, its enhanced efficacy (only nanomolar quantities of
siRNA are required for efficient gene-silencing), and the fact that
siRNA treatment feeds into a natural RNAi pathway that is inherent
to all cells.
[0014] The success of gene-silencing by siRNA can be highly
variable depending on the gene target and cell type being
targeted.
SUMMARY OF THE INVENTION
[0015] The inventors have used plasmid RL1.dTRES-GFP to generate a
shuttle vector, designated RL1.dCMV-asSCCRO-GFP, containing the
human antisense squamous cell carcinoma related oncogene (SCCRO)
arranged in an orientation downstream of a CMV IE promoter to
produce antisense RNA transcripts for use in antisense therapeutic
methods. Using this shuttle vector the inventors have provided a
novel second generation mutant HSV, designated
HSV1716/CMV-asSCCRO/GFP (also called HSV1716asSCCRO). The genome of
this mutant HSV comprises the nucleic acid encoding heterologous
(i.e. non-HSV originating) antisense SCCRO 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 an antisense RNA transcript under control of the CMV IE
promoter which is capable of inhibiting the action of the SCCRO
gene by binding to sense SCCRO nucleotide sequences, e.g. SCCRO
mRNA or genomic SCCRO. This virus retains the oncolytic activity of
HSV-1 strain 17 mutant 1716 and can be used in targeted antisense
nucleotide delivery strategies and therapeutic methods.
[0016] In an alternative arrangement, instead of integrating a
nucleic acid encoding an antisense, the inventors have integrated
an siRNA in the genome of a herpes simplex virus. This siRNA is
preferably heterologous to the herpes simplex virus and may be
expressed from the herpes simplex virus genome. In one preferred
embodiment the integrated nucleic acid encodes an siRNA capable of
targeting and repressing or inhibiting expression of the functional
SCCRO gene product. When expressed, the siRNA operates to silence,
wholly or in part, expression of the functional SCCRO gene
product.
[0017] The heterologous asSCCRO expressed by an herpes simplex
virus according to the present invention may be useful in RNA based
antisense therapeutic techniques for repression or silencing of the
SCCRO gene product or of it's expressed function.
[0018] The siRNA expressed by an herpes simplex virus according to
the present invention may be useful in siRNA based therapeutic
techniques for tissue specific repression or silencing of the SCCRO
gene product or of it's expressed function.
[0019] At its most general the present invention relates to (i)
materials and methods relating to the squamous cell carcinoma
related oncogene; and (ii) mutant herpes simplex viruses.
[0020] In one embodiment of the present invention, there is
provided an attenuated replication competent HSV expressing
antisense SCCRO, namely, HSV1716asSCCRO, which may be used in the
treatment of squamous cell cancer, particularly head and neck
squamous cell cancer.
[0021] Accordingly the present invention further provides a
pharmaceutical composition comprising HSV1716asSCCRO and the use of
such virus and/or composition in the treatment of cancer.
[0022] According to one aspect of the present invention there is
provided an herpes simplex virus wherein the herpes simplex virus
genome comprises nucleic acid encoding an antisense to the squamous
cell carcinoma related oncogene (asSCCRO).
[0023] Said nucleic acid may encode an antisense to a mammalian
squamous cell carcinoma related oncogene, more preferably an
antisense to a human squamous cell carcinoma related oncogene.
[0024] In one arrangement said nucleic acid may encode a nucleotide
sequence complementary to: [0025] (i) the polynucleotide sequence
of SEQ ID No.s 1 or 3 or its complement; [0026] (ii) the mRNA
transcript of SEQ ID No.s 1 or 3; or [0027] (iii) to a fragment of
said polynucleotide sequence, complement or mRNA transcript.
[0028] In another arrangement said nucleic acid may encode a
nucleotide sequence having at least 60% sequence identity to the
nucleotide sequence complementary to: [0029] (i) the polynucleotide
sequence of SEQ ID No.s 1 or 3 or its complement; [0030] (ii) the
mRNA transcript of SEQ ID No.s 1 or 3; or [0031] (iii) to a
fragment of said polynucleotide sequence or mRNA transcript.
[0032] More preferably said degree of sequence identity may be at
least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%. The fragment
referred to at (iii) may comprise at least 20 nucleotides and may
be limited to no more than 900 nucleotides. 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.
[0033] In another arrangement said nucleic acid may be selected as
one that hybridises to: [0034] (i) the polynucleotide sequence of
SEQ ID No.s 1 or 3 or its complement; [0035] (ii) the mRNA
transcript of SEQ ID No.s 1 or 3; or [0036] (iii) to a fragment of
said polynucleotide sequence or mRNA transcript under high or very
high stringency conditions.
[0037] The genome of Herpes simplex viruses according to the
present invention may further comprises a regulatory sequence
operably linked to said nucleic acid encoding an antisense to the
squamous cell carcinoma related oncogene, wherein said regulatory
sequence has a role in controlling transcription of said siRNA.
[0038] In a further aspect of the present invention there is
provided an herpes simplex virus wherein the herpes simplex virus
genome comprises nucleic acid encoding a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of the squamous cell carcinoma related
oncogene (SCCRO) nucleic acid or polypeptide.
[0039] Said siRNA may repress or silence expression of a mammalian
SCCRO, more preferably of a human SCCRO.
[0040] Said nucleic acid encoding siRNA may comprise a nucleic acid
of between 10 and 50 nucleotides in length and may have the
sequence of SEQ ID No.5 or the complement thereof.
[0041] In another arrangement said nucleic acid encoding siRNA may
comprise a nucleic acid of between 10 and 50 nucleotides in length
and may have at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%
identity to SEQ ID No.5 or the complement thereof. 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.
[0042] In another arrangement said nucleic acid encoding siRNA may
be selected as one that hybridises to the nucleic acid of SEQ ID
No. 5 or its complement under high or very high stringency
conditions.
[0043] The genome of said herpes simplex virus may further comprise
a regulatory sequence operably linked to said siRNA, wherein said
regulatory sequence has a role in controlling transcription of said
siRNA.
[0044] The nucleic acid encoding asSCCRO or said siRNA may be
located in at least one RL1 locus of the herpes simplex virus
genome. Suitably it may be located in, or overlap, at least one of
the ICP34.5 protein coding sequences of the herpes simplex virus
genome. The nucleic acid may be located in both (usually this is
all) copies of the RL1 locus or ICP34.5 protein coding
sequence.
[0045] The herpes simplex virus is preferably a mutant and may be a
mutant of HSV-1 or HSV-2, more preferably of one of HSV-1 strains
17, F or HSV-2 strain HG52. The herpes simplex virus may be a
further mutant of HSV-1 strain 17 mutant 1716.
[0046] In certain arrangements the herpes simplex virus may be a
gene specific null mutant, such as an ICP34.5 null mutant.
[0047] In other arrangements the herpes simplex virus may lack at
least one expressible ICP34.5 gene.
[0048] In yet another arrangement the herpes simplex virus may lack
only one expressible ICP34.5 gene.
[0049] In yet another arrangement the herpes simplex virus may be
non-neurovirulent.
[0050] In herpes simplex viruses of the present invention the
nucleic acid encoding the asSCCRO or said siRNA may form part of a
nucleic acid cassette permanently integrated in the herpes simplex
virus genome, said cassette comprising nucleic acid encoding:
[0051] (a) said asSCCRO or said siRNA; and nucleic acid encoding:
[0052] (b) a ribosome binding site or a first regulatory nucleotide
sequence; and [0053] (c) a marker, wherein the nucleic acid
encoding said asSCCRO or siRNA is arranged upstream (5') of the
ribosome binding site or first regulatory nucleotide sequence and
the ribosome binding site or first regulatory nucleotide sequence
is arranged upstream (5') of the marker. Said first regulatory
sequence may have a role in controlling transcription of said
marker.
[0054] A second regulatory nucleotide sequence may be located
upstream (5') of the nucleic acid encoding asSCCRO or said siRNA,
wherein the regulatory nucleotide sequence has a role in
controlling and regulating transcription of the nucleic acid
encoding the asSCCRO or siRNA and hence expression of the resulting
transcript. The regulatory sequences may comprise selected promoter
or enhancer elements known to the person skilled in the art, e.g.
the CytoMegalovirus (CMV) or phosphoglycerokinase (PGK)
promoters.
[0055] The components of the cassette are preferably arranged in a
predetermined order.
[0056] In one preferred arrangement, the nucleic acid encoding the
asSCCRO is arranged upstream (i.e. 5') of the ribosome binding site
and the ribosome binding site is arranged upstream (i.e. 5') of the
marker. During transcription a single transcript may be produced
from the cassette comprising a first cistron comprising the asSCCRO
and a second cistron encoding the marker wherein the ribosome
binding site is located between the cistrons.
[0057] A transcription product of this cassette may be a bi- or
poly-cistronic transcript comprising a first cistron encoded by the
nucleic acid encoding the asSCCRO and a second cistron encoding the
marker nucleic acid wherein the ribosome binding site is located
between said first and second cistrons.
[0058] In another preferred arrangement, the nucleic acid encoding
the siRNA is arranged upstream (i.e. 5') of a first regulatory
nucleotide sequence and the first regulatory nucleotide sequence is
arranged upstream (i.e. 5') of the marker.
[0059] The cassette may disrupt a protein coding sequence of the
herpes simplex virus genome resulting in inactivation of the
respective gene product.
[0060] Nucleic acid encoding a selected antisense DNA, that is DNA
corresponding to a gene component (e.g. regulatory sequence, 5'
UTR, 3'UTR or protein coding sequence) or fragment of a gene
component, is inserted in the cassette in an orientation such that
upon transcription an antisense RNA is obtained. Thus the expressed
product of the cassette may ultimately be an antisense nucleic
acid, preferably RNA.
[0061] One suitable ribosome binding site comprises a ribosome
entry site permitting entry of a ribosome to the transcribed mRNA
encoded by the nucleic acid of the cassette such that the ribosome
binds to the translation start signal. Preferably, the ribosome
entry site is an internal ribosome entry site (IRES), more
preferably an encephalomyocarditis virus IRES, permitting
cap-independent initiation of translation. The IRES thus enables
translation of a coding sequence located internally of a bi- or
poly-cistronic mRNA, i.e. of a cistron located downstream of an
adjacent cistron on a single transcript.
[0062] Preferably the marker is a defined nucleotide sequence
coding for a polypeptide which can be expressed in a cell line
(e.g. BHK cells) infected with mutant herpes simplex virus into
which the cassette has been recombined. The function of the marker
is to enable identification of virus plaques containing mutant
virus transformed with the cassette.
[0063] 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.
[0064] In other arrangements the marker may comprise a defined
nucleotide sequence which can be detected by hybridisation under
high stringency conditions with a corresponding labelled nucleic
acid probe, e.g. using a fluorescent- or radio-label.
[0065] The cassette may also comprise nucleic acid encoding a
polyadenylation ("polyA") sequence, which sequence is preferably
located downstream (3') of the nucleic acid encoding the marker.
One preferred polyA sequence is the Simian Virus 40 (SV40)
polyadenylation sequence. The preferred location of the polyA
sequence within the cassette is immediately downstream (i.e. 3') of
the marker.
[0066] By antisense nucleic acid is meant a nucleic acid: [0067]
(i) having substantial sequence identity to the nucleic acid formed
by the sequence of complementary bases to the single strand of a
target nucleic acid; and/or [0068] (ii) a nucleic acid which
hybridises to the target nucleic acid under intermediate, high or
very high stringency conditions.
[0069] In accordance with aspects of the present invention, the
target nucleic acid may be an SCCRO polynucleotide sequence (e.g.
gene sequence), the polynucleotide coding sequence for the SCCRO
polypeptide or protein, or a part/fragment of the gene or
polypeptide coding sequence. Thus, the antisense nucleic acid may
be useful in binding the target nucleic acid (e.g. the SCCRO
genomic coding sequence or mRNA transcript) and may be used as an
inhibitor to prevent or disrupt the normal expression, activity,
folding or binding of the target nucleic acid. The substantial
sequence identity is preferably at least 50% sequence identity,
more preferably one of 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.
[0070] The antisense nucleic acid may comprise all or a fragment of
the antisense to the squamous cell carcinoma related oncogene
(asSCCRO), preferably it is an antisense to the human SCCRO.
[0071] The nucleic acid encoding the asSCCRO which may form part of
the inserted cassette may encode a full length transcript of the
antisense nucleotide sequence to the SCCRO. That full length
antisense transcript may be a sequence complementary to one of the
polynucleotide sequences of SEQ ID No.1 or SEQ ID No.3 or their
complementary sequences. Alternatively, the nucleic acid may encode
one or more fragments of the full length antisense transcript.
[0072] 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, 1500, 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.
[0073] In the case of an antisense to the SCCRO, a full length
transcript comprises a minimum of the contiguous sequence of
nucleotides forming an antisense strand to the corresponding
complete nucleotide sequence encoding the full amino acid sequence
of the SCCRO gene product or to is compliment. The complete
nucleotide sequence of the SCCRO gene product may comprise the
region of SEQ ID No.1 or SEQ ID No.3 respectively encoding the
polypeptide of SEQ ID No. 2 or SEQ ID No.4.
[0074] Preferred antisense nucleic acids may single stranded and
may be DNA or RNA.
[0075] Preferred siRNA may be single or double stranded and may
comprise single stranded nucleic acids capable of forming duplex
structures by stem-loop formation and self-binding of complementary
nucleotides. Preferred siRNA may include RNA molecules having a
sequence encoded by SEQ ID No. 5 or its complement and nucleic
acids having a sequence identity of at least 60% to SEQ ID No. 5 or
a complementary sequence thereof, and more preferably having at
least 70, 80, 85, 90, 95% or 100% sequence 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.
[0076] Preferred siRNA or nucleic acid encoding preferred siRNA may
comprise nucleotide sequences heterologous to the selected HSV
strain being modified, i.e. the siRNA or nucleic acid sequence
encoding the siRNA does not occur in or originate from the
parental, unmodified wild-type, virus.
[0077] Furthermore Herpes simplex viruses according to aspects of
the present invention may contain nucleic acid, encoding siRNA
molecules, which hybridise with SEQ ID No 5 or its complement under
very high, high or intermediate stringency conditions.
[0078] siRNA molecules encoded by nucleic acid molecules integrated
in the genome of Herpes simplex viruses according to the present
invention may be of any length, but preferred siRNA molecules are
small and may comprise at least 10 nucleotides and no more than 50
nucleotides. Herpes simplex virus according to the present
invention may encode an siRNA which is a fragment of the siRNA
encoded by SEQ ID No.5. Particularly suitable siRNA will have a
single strand length in the range 10 to 30 nucleotides and more
suitably in the range 15 to 25 nucleotides. Selected siRNA
molecules may be any of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or
25 nucleotides in length. SiRNA molecules which fold to form their
own duplex structures, e.g. by stem-loop formation will thus have
an unfolded single strand length of about two times the number or
range recited above and siRNA and nucleic acid encoding such siRNA
may be particularly preferred.
[0079] Herpes simplex viruses according to aspects of the present
invention may contain nucleic acid encoding siRNA molecules having
one or more of these sequences.
[0080] Mutant herpes simplex viruses of the present invention may
be generated by site directed insertion of a nucleic acid cassette
into the viral genome, more preferably by homologous recombination.
However, the viruses of the invention are not limited to Herpes
simplex viruses obtained in this way.
[0081] In other aspects of the present invention herpes simplex
viruses according to the present invention are provided for use in
a method of medical treatment. Suitably they are provided for use
in the treatment of disease. Preferably they are provided for use
in the treatment of cancer. Suitably they may be provided for use
in the oncolytic treatment of cancer/a tumour. The use of herpes
simplex viruses according to the present invention in the
manufacture of a medicament for the treatment of cancer is also
provided.
[0082] 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.
[0083] A medicament, pharmaceutical composition or vaccine
comprising an Herpes simplex virus according to the present
invention is also provided. The medicament, pharmaceutical
composition or vaccine may further comprise a pharmaceutically
acceptable carrier, adjuvant or diluent.
[0084] The present invention may also include the following aspects
which may be provided in combination with any of the other aspects
and features described.
[0085] In another aspect of the present invention there is provided
an herpes simplex virus, wherein the genome of said virus comprises
a nucleic acid sequence encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO) in at least one of the long
repeat regions (R.sub.L).
[0086] In another aspect of the present invention there is provided
an herpes simplex virus, wherein the genome of said virus comprises
a nucleic acid sequence encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO) and wherein the herpes simplex
virus is non-neurovirulent.
[0087] In another aspect of the present invention there is provided
an herpes simplex virus for use in the treatment of a tumour,
wherein the genome of said virus comprises a nucleic acid sequence
encoding an antisense to the squamous cell carcinoma related
oncogene (asSCCRO) in at least one of the long repeat regions
(R.sub.L).
[0088] In another aspect of the present invention there is provided
an herpes simplex virus for use in the treatment of a tumour,
wherein the genome of said virus comprises a nucleic acid sequence
encoding an antisense to the squamous cell carcinoma related
oncogene (asSCCRO) and wherein the herpes simplex virus is
non-neurovirulent.
[0089] In another aspect of the present invention the use of an
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO) in at least one of the long
repeat regions (R.sub.L), in the manufacture of a medicament for
the treatment of cancer is provided.
[0090] In another aspect of the present invention the use of an
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding an antisense to the squamous cell
carcinoma related oncogene (asSCCRO) and wherein the herpes simplex
virus is non-neurovirulent, in the manufacture of a medicament for
the treatment of cancer is provided.
[0091] In another aspect of the present invention there is provided
a method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment an effective amount
of an herpes simplex virus, wherein the genome of said virus
comprises a nucleic acid sequence encoding an antisense to the
squamous cell carcinoma related oncogene (asSCCRO) in at least one
of the long repeat regions (R.sub.L).
[0092] In another aspect of the present invention there is provided
a method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment an effective amount
of an herpes simplex virus, wherein the genome of said virus
comprises a nucleic acid sequence encoding an antisense to the
squamous cell carcinoma related oncogene (asSCCRO) and wherein the
herpes simplex virus is non-neurovirulent.
[0093] Suitably, in the methods of treatment of a tumour the herpes
simplex virus is capable of killing tumour cells.
[0094] In another aspect of the present invention there is provided
a method of expressing in vitro or in vivo an antisense to the
squamous cell carcinoma related oncogene (asSCCRO), said method
comprising the step of infecting at least one cell or tissue of
interest with a herpes simplex virus, wherein the genome of said
virus comprises a nucleic acid sequence encoding asSCCRO in at
least one of the long repeat regions (R.sub.L), said asSCCRO
operably linked to a transcription regulatory sequence.
[0095] In another aspect of the present invention there is provided
a method of expressing in vitro or in vivo an antisense to the
squamous cell carcinoma related oncogene (asSCCRO), said method
comprising the step of infecting at least one cell or tissue of
interest with a non-neurovirulent herpes simplex virus, wherein the
genome of said virus comprises a nucleic acid sequence encoding
asSCCRO, said asSCCRO operably linked to a transcription regulatory
sequence.
[0096] In another aspect of the present invention there is provided
an herpes simplex virus, wherein the genome of said virus comprises
a nucleic acid sequence encoding a short interfering ribonucleic
acid (siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide in at least one of the long repeat
regions (R.sub.L).
[0097] In another aspect of the present invention there is provided
an herpes simplex virus, wherein the genome of said virus comprises
a nucleic acid sequence encoding a short interfering ribonucleic
acid (siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide and wherein the herpes simplex virus is
non-neurovirulent.
[0098] In another aspect of the present invention there is provided
an herpes simplex virus for use in the treatment of a tumour,
wherein the genome of said virus comprises a nucleic acid sequence
encoding a short interfering ribonucleic acid (siRNA) molecule that
is capable of repressing or silencing expression of squamous cell
carcinoma related oncogene (SCCRO) nucleic acid or polypeptide in
at least one of the long repeat regions (R.sub.L).
[0099] In another aspect of the present invention there is provided
an herpes simplex virus for use in the treatment of a tumour,
wherein the genome of said virus comprises a nucleic acid sequence
encoding a short interfering ribonucleic acid (siRNA) molecule that
is capable of repressing or silencing expression of squamous cell
carcinoma related oncogene (SCCRO) nucleic acid or polypeptide and
wherein the herpes simplex virus is non-neurovirulent.
[0100] In another aspect of the present invention the use of an
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding a short interfering ribonucleic acid
(siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide in at least one of the long repeat
regions (R.sub.L), in the manufacture of a medicament for the
treatment of cancer is provided.
[0101] In another aspect of the present invention the use of an
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding a short interfering ribonucleic acid
(siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide and wherein the herpes simplex virus is
non-neurovirulent, in the manufacture of a medicament for the
treatment of cancer is provided.
[0102] In another aspect of the present invention there is provided
a method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment an effective amount
of an herpes simplex virus, wherein the genome of said virus
comprises, in at least one of the long repeat regions (R.sub.L), a
nucleic acid sequence encoding a short interfering ribonucleic acid
(siRNA) molecule that is capable of repressing or silencing
expression of squamous cell carcinoma related oncogene (SCCRO)
nucleic acid or polypeptide.
[0103] In another aspect of the present invention there is provided
a method for the treatment of a tumour comprising the step of
administering to a patient in need of treatment an effective amount
of an herpes simplex virus, wherein the genome of said virus
comprises a nucleic acid sequence encoding a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide and wherein the herpes simplex
virus is non-neurovirulent.
[0104] Suitably, in the methods of treatment of a tumour the herpes
simplex virus is capable of killing tumour cells.
[0105] In another aspect of the present invention there is provided
a method of expressing in vitro or in vivo a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide, said method comprising the
step of infecting at least one cell or tissue of interest with a
herpes simplex virus, wherein the genome of said virus comprises a
nucleic acid sequence encoding said siRNA in at least one of the
long repeat regions (R.sub.L), wherein said nucleic acid sequence
encoding said siRNA is operably linked to a transcription
regulatory sequence.
[0106] In another aspect of the present invention there is provided
a method of expressing in vitro or in vivo a short interfering
ribonucleic acid (siRNA) molecule that is capable of repressing or
silencing expression of squamous cell carcinoma related oncogene
(SCCRO) nucleic acid or polypeptide, said method comprising the
step of infecting at least one cell or tissue of interest with a
non-neurovirulent herpes simplex virus, wherein the genome of said
virus comprises a nucleic acid sequence encoding said siRNA,
wherein said nucleic acid sequence encoding said siRNA is operably
linked to a transcription regulatory sequence.
[0107] siRNA according to the invention preferably repress the
function of the squamous cell carcinoma related oncogene (SCCRO)
protein.
[0108] In another aspect of the present invention a method is
provided for repressing the cellular expression of the squamous
cell carcinoma related oncogene (SCCRO) in vitro comprising the
step of: in vitro, contacting a cell with an herpes simplex virus
of the present invention or pharmaceutical composition containing
such virus.
[0109] In one preferred aspect of the invention the herpes simplex
virus is HSV1716/CMV-asSCCRO/GFP, deposited as `HSV1716asSCCRO`, 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 19 May 2004 at the European
Collection of Cell Cultures (ECACC), Health Protection Agency,
Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under
accession number 04051901 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`).
[0110] In yet another aspect of the present invention a cell, in
vitro, in which expression of the squamous cell carcinoma related
oncogene (SCCRO) protein or nucleic acid is repressed or silenced
is provided. The cell may be a mammalian cell, preferably a human
cell.
[0111] Suitably, the administration of said 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. Alternatively
injections may be intravenous.
[0112] In a further aspect of the present invention in vitro or in
vivo methods are provided for delivery of nucleic acid encoding
asSCCRO or siRNA to at least one cell or to a tissue of interest
said method comprising the step of infecting said cell(s) or tissue
with a herpes simplex virus according to the invention.
[0113] In another aspect of the present invention a method of
making or producing a modified herpes simplex virus of the
invention is provided comprising the step of introducing a nucleic
acid sequence encoding asSCCRO or siRNA at a selected or
predetermined insertion site in the genome of a selected herpes
simplex virus.
[0114] As described, the nucleic acid sequence encoding the asSCCRO
or siRNA may form part of a nucleic acid cassette which is inserted
in the genome of a selected herpes simplex virus by homologous
recombination. Whether part of a cassette or not, the site of
insertion may be in any genomic location selected. One preferred
insertion site is in one or both of the long repeat regions
(R.sub.L), and one copy of the cassette is preferably inserted in
each copy of the long repeat (R.sub.L). More preferably the
insertion site is in at least one (preferably both) RL1 locus and
most preferably it is inserted in at least one (preferably both) of
the ICP34.5 protein coding sequences of the HSV genomic DNA. It is
preferred that the insertion occurs in identical or substantially
similar positions in each of the two repeat regions, RL1 loci or
ICP34.5 protein coding sequences.
[0115] Insertion may be such as to produce a modified virus which
is a non-neurovirulent mutant capable of expressing the encoded
asSCCRO or siRNA upon transfection into mammalian, more preferably
human, cells in vivo and in vitro. The non-neurovirulent mutant may
be an ICP34.5 null mutant.
[0116] 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.
[0117] Preferably, the herpes simplex virus contains at least one
copy of the nucleic acid encoding the asSCCRO or siRNA in each long
repeat region (R.sub.L), i.e. in the terminal and internal long
repeat (TR.sub.L and IR.sub.L) regions. In a preferred arrangement
each exogenous sequence or cassette is located in an RL1 locus of
the herpes simplex virus genome, more preferably in the DNA of the
herpes simplex virus genome encoding the ICP34.5 gene or protein
coding sequence. The herpes simplex virus thereby lacks
neurovirulence.
[0118] 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 asSCCRO or siRNA nucleic acid sequences 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.
[0119] Although the non-neurovirulent phenotype of the herpes
simplex virus of the invention may be the result of insertion of
the exogenous nucleic acid sequence in the RL1 locus, herpes
simplex viruses according to the present invention may be obtained
by utilising a non-neurovirulent parent strain, e.g. HSV1716
deposited under the Budapest Treaty at the European Collection of
Animal Cell Cultures (ECACC), Health Protection Agency, Porton
Down, Salisbury, Wiltshire, United Kingdom under accession number
V92012803, and inserting the exogenous nucleic acid sequence at
another location of the genome by standard genetic engineering
techniques, e.g. homologous recombination. In this aspect the
location of the herpes simplex virus genome selected for insertion
of the asSCCRO or siRNA nucleic acid sequence or cassette
containing said sequence may be a neutral location.
[0120] 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 asSCCRO or siRNA encoding 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.
[0121] 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. 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.
[0122] 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
asSCCRO or siRNA encoded by the herpes simplex virus genome.
[0123] 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 asSCCRO or siRNA, and optionally
returned/introduced to a patient's body, e.g. by injection.
[0124] 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.
[0125] In vitro cultured cells, preferably human or mammalian
cells, transformed with viruses of the present invention and
preferably cells expressing the asSCCRO or siRNA as well as methods
of transforming such cells in vitro with said viruses form further
aspects of the present invention.
[0126] Cancer/tumour types to be treated may include primary and/or
secondary (metastatic) tumours. These may be carcinomas of the head
and/or neck. They may be squamous cell carcinomas, which may be of
mucosal origin and may show a predilection for duplication of the
3q locus. Preferred squamous cell carcinomas to be treated may be
those of the head and/or neck. Squamous cell carcinomas to be
treated may include those originating from the lung, head neck,
oesophagus and cervix.
[0127] Other tumour types which may be treated may be primary or
secondary (metastatic) tumours. Tumours to be treated may be
nervous or non-nervous system tumours. Nervous system tumours may
originate either in the central or peripheral nervous system, e.g.
glioma, medulloblastoma, meningioma, neurofibroma, ependymoma,
Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma.
Non-nervous system tumours may originate in any other non-nervous
tissue, examples include melanoma, mesothelioma, lymphoma,
hepatoma, epidermoid carcinoma, prostate carcinoma, breast cancer
cells, lung cancer cells or colon cancer cells. HSV mutants of the
present invention may be used to treat metastatic tumours of the
central or peripheral nervous system which originated in a
non-nervous system tissue.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
SCCRO
[0133] The polynucleotide sequence of SEQ ID No.1, positions 43-918
and the polynucleotide of SEQ ID No.2 are disclosed in GenBank
Accession No. AF456425 (GI:18700655) released to the public as of
19 February 2002.
[0134] A second Oncoseq (Oncoseq2) polypeptide is encoded by the
polynucleotide sequence of SEQ ID No.3, which together with the
polypeptide thereby encoded (SEQ ID No.4) are disclosed in GenBank
Accession No. AF456426 (GI:18700657) released to the public as of
19 February 2002.
[0135] The GenBank database may be accessed at
http://www.ncbi.nlm.nih.gov/.
Therapeutic Strategies
[0136] The following therapeutic strategies are provided by way of
example only. The invention is not limited to a theory of operation
of a given antisense or siRNA.
Antisense
[0137] Herpes simplex viruses according to the present invention
may express an antisense nucleic acid, e.g. single stranded RNA
that targets and binds, by complementary sequence binding, to the
target mRNA thereby blocking translation of that mRNA and
expression of the gene product.
[0138] Expressed antisense nucleic acid may also be arranged to
bind sense genomic nucleic acid and inhibit transcription of a
target nucleotide sequence.
siRMA
[0139] Herpes simplex viruses according to the present invention
may encode nucleic acid designed such that on transcription an RNA
having internal complementary sequence is provided and which may
bind to form a short hairpin siRNA duplex having a stem-loop
structure. Preferably, the hairpin siRNA mediates specific
repression and/or silencing of gene expression by RNA
interference.
[0140] Alternatively, two siRNA molecules may be encoded which are
designed to bind by complementary sequence binding and form a
functionally active duplex molecule.
Repression and Silencing
[0141] siRNA and antisense molecules provided under the invention
are designed to repress or silence the expression of a target
nucleic acid, peptide, polypeptide or protein or to repress a
function of such nucleic acid, peptide, polypeptide or protein.
[0142] A repression of expression results in a decrease in the
quantity or expressed function of the target. For example, in a
given cell the repression of SCCRO by expression of an siRNA or
antisense may result in a decrease in either the quantity of the
SCCRO gene product or the expressed function of the SCCRO gene
product relative to an untreated cell.
[0143] Repression of a function may involve the decrease in
transcription of an mRNA, or translation of a peptide or
polypeptide.
[0144] Repression may be partial. Preferred degrees of repression
are at least 50%, more preferably one of at least 60, 70, 80, 85 or
90%. A level of repression between 90% and 100% is considered a
`silencing` of expression or function.
Hybridisation Stringency
[0145] In accordance with the present invention, nucleic acid
sequences may be identified by using hybridization and washing
conditions of appropriate stringency.
[0146] 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.
[0147] The "stringency" of a hybridization reaction can be readily
determined by a person skilled in the art.
[0148] 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.
[0149] 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-l 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.
[0150] 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.
[0151] 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.
[0152] Accordingly, nucleotide sequences can be categorised by an
ability to hybridise to a target sequence under different
lybridisation 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.
[0153] 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.
[0154] 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.
[0155] The invention includes the combination of the aspects and
preferred features described except where such a combination is
clearly impermissible or expressly avoided.
[0156] 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
[0157] FIG. 1. Generation of plasmid RL1.dIRES-GFP from plasmids
pNAT-IRES-GFP and RL1.del.
[0158] 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.
[0159] 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.
[0160] 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 (Ampr)
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.
[0161] *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.
*N.B. Inserts could have been cloned in two orientations, both of
which were acceptable.
[0162] FIG. 5. Determination of the orientation of
pCMV-NAT-IRES-GFP-PolyA in clone 5 (RL1.dCMV-NAT-GFPb).
pCMV-NAT-IRES-GFP-PolyA (blunt ended) could have been cloned into
the HpaI site of RL1.del in two orientations. To determine the
orientation of the insert in clone 5, the plasmid was digested with
XhoI and the digested DNA electrophoresed, beside a 1 Kbp DNA
ladder (Promega), on a 1% agarose gel. If the insert had been
cloned in the orientation shown in A, two fragments of 10.2 Kbp and
3.8 Kbp would be generated from XhoI digestion. If it had been
cloned in the opposite orientation (B), two fragments of 12.4 Kbp
and 1.6 Kbp would be generated. The presence of two fragments of
10.2 Kbp and 3.8 Kbp in the gel confirmed that the insert had been
cloned in the orientation shown in A.
*This XhoI site was present in the initial cloning vector
(RL1.del), upstream of the HpaI site into which
pCMV-NAT-IRES-GFP-PolyA was cloned.
[0163] FIG. 6. Removal of pCMV-NAT from clone 5 (A) and large scale
plasmid preparation of RL1.dIRES-GFP (B). Four samples of clone 5
were digested with XhoI and electrophoresed, beside a 1 Kbp DNA
ladder (L) (Promega), on a 1% agarose gel (A). The larger fragment
of DNA generated from this digestion (10.2 Kbp) was purified from
the gel and ligated back together, at the XhoI sites, to form a
single XhoI site in a new plasmid, designated RL1.dIRES-GFP. A
large-scale plasmid preparation was grown up and the preparation
checked by digesting with XhoI. 1 .mu.l and 4 .mu.l of the digested
DNA was electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega),
on a 1% agarose gel (B). The DNA should produce a single fragment
of 10.2 Kbp when digested with XhoI. The ClaI, BglII, NruI and XhoI
sites of RL1.dIRES-GFP are all unique.
*Clone 5 is the RL1.del plasmid into which has been cloned the 5.4
Kbp pCMV-NAT-IRES-GFP-PolyA fragment from pNAT-IRES-GFP.
[0164] FIG. 7. Generation, detection and purification of ICP34.5
null HSV-1 expressing a gene product of interest.
[0165] FIG. 8. Strategy used to clone pCMV-asSCCRO, from
pUSEamp-asSCCRO, into RL1.dIRES-GFP. (1) Digest pUSEamp-asSCCRO
with Ssp1 and XhoI and purify the 1.96 Kbp PCMV-asSCCRO fragment;
(2) Digest RL1.dIRES-GFP with BglII, blunt end using Klenow
polymerase and treat with Calf Intestinal Phosphatase (CIP). (3)
Clone the blunt ended pCMV-asSCCRO fragment (1.96 Kbp) into BglII
digested/blunt ended/CIP treated RL1.dIRES-GFP. (*pUSEamp-asSCCRO
was provided by Memorial Sloan-Kettering Cancer Centre, New
York.)
[0166] FIG. 9. Agarose gel electrophoresis of BglII digested, blunt
ended, CIP treated RL1.dIRES-GFP. RL1.dIRES.GFP was digested with
BglII. The digested plasmid was then blunt ended using Klenow
polymerase and treated with Calf Intestinal Phosphatase (CIP) to
prevent the vector re-annealing to itself in subsequent ligation
reactions. A sample of the digested/blunt ended/CIP treated DNA was
electrophoresed, beside a 1 Kbp DNA ladder (Promega), on a 1%
agarose gel to check its concentration. pCMV-asSCCRO was
subsequently cloned into this digested/CIP treated vector.
[0167] FIG. 10. Agarose gel electrophoresis of SspI/XhoI digested
pUSEamp-asSCCRO (A) and the purified pCMV-asSCCRO fragment (B).
Four samples of pUSEamp-asSCCRO were digested with SspI and XhoI
then electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega), on a
1% agarose gel. The 1.96 Kbp fragments, consisting of DNA antisense
to the squamous cell carcinoma related oncogene (asSCCRO)
downstream of the CMV IE promoter (pCMV), were purified from the
gel, blunt ended using Klenow polymerase, purified again and a
sample of the purified DNA electrophoresed on an agarose gel to
check its concentration.
[0168] FIG. 11. Identification of RL1.dIRES-GFP clones containing
the pCMV-asSCCRO insert. Ligation reactions were set up with the
purified, blunt ended pCMV-asSCCRO fragment and BglII digested,
blunt ended, CIP treated RL1.dIRES-GFP. Bacteria were transformed
with samples from the ligation reactions and plated onto LBA
(Amp.sup.r) plates. Colonies were picked and plasmid DNA was
extracted and digested with BglII. Digested samples were
electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega), on a 1%
agarose gel
*Clone 11 contained the pCMV-asSCCRO insert as two fragments of the
predicted size--1.4 Kbp and 10.8 Kbp were generated from BglII
digestion. Clones without the insert would not produce a fragment
of 1.4 Kbp when digested with BglII.
[0169] FIG. 12. Determination of the orientation of pCMV-asSCCRO in
clone 11. The presence of an NruI site, .about.320 bp into the
cloned pCMV-asSCCRO fragment, was utilized to determine the
orientation of pCMV-asSCCRO. Clone 11 was digested with NruI and
electrophoresed, beside a 1 Kbp DNA ladder (L) (Promega), on a 1%
agarose gel. If pCMV-asSCCRO was in the desired orientation (A),
NruI digestion would produce a fragment of 1.64 Kbp. If in the
opposite orientation (B), no 1.64 Kbp fragment would be generated
from this digestion. The presence of a fragment at 1.64 Kbp in the
gel confirmed that pCMV-asSCCRO was in the desired orientation.
(*This NruI site was already present in the initial cloning vector
(i.e. RL1.dIRES-GFP))
[0170] FIG. 13. Agarose gel electrophoresis of ScaI digested clone
11 (A) and HSV1716/CMV-asSCCRO/GFP virus titre (B). Clone 11
(RL1.dCMV-asSCCRO-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 11. 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-asSCCRO/GFP, grown up. HSV1716/CMV-asSCCRO/GFP was
titrated on BHK cells.
[0171] FIG. 14. Cytotoxicity assay for cell lines SCC15 and 584
after infection with HSV1716 or HSV1716asSCCRO at MOI of 1 pfu/cell
and 5 pfu/cell.
[0172] FIG. 15. Cytotoxicity assay for cell lines 1483 and 1986
after infection with HSV1716 or HSV1716asSCCRO at MOI of 1 pfu/cell
and 5 pfu/cell.
[0173] FIG. 16. Cytotoxicity assay for cell line 1186 and 1386
after infection with HSV1716 or HSV1716asSCCRO at MOI of 1 pfu/cell
and Spfu/cell.
[0174] FIG. 17. Viral proliferation assays for head and neck
squamous cell carcinoma cell lines after infection with HSV1716 or
HSV1716asSCCRO at MOI 1 pfu/cell.
[0175] FIG. 18. Infectivity assay--gfp expression 6 hours post
infection with 1716gfp virus.
[0176] FIG. 19. Western blot results of the cell line SCC15 showing
downregulation of SCCRO protein at 12 hours with HSV1716asSCCRO but
not in 584.
[0177] FIG. 20. Nude mice xenograft growth curves in SCC15 and 584
following single intratumoural injection of HSV1716 or
HSV1716asSCCRO.
[0178] FIG. 21. Nude mice xenograft growth curves in SCC15
following single intratumoural injection of PBS, HSV1716 or
HSV1716asSCCRO.
[0179] FIG. 22. [0180] (A) SEQ ID No. 1--a Human SCCRO nucleic acid
sequence. Also showing the amino acid sequence of the encoded
polypeptide (an SCCRO gene product); [0181] (B) SEQ ID No. 2--Amino
acid sequence of the polypeptide encoded by SEQ ID No.1; [0182] (C)
SEQ ID No. 3--a Human SCCRO nucleic acid sequence. Also showing the
amino acid sequence of the encoded polypeptide (an SCCRO gene
product); [0183] (D) SEQ ID No. 4--Amino acid sequence of the
polypeptide encoded by SEQ ID No.3.
[0184] FIG. 23. (A) DNA nucleotide sequence encoding the siRNA
construct designed to target expression of the SCCRO gene (SEQ ID
No. 5); and (B) nucleotide sequence encoding control siRNA (SEQ ID
No 6). Sequences either side of the central nucleotides are
respectively complimentary enabling the transcribed RNA to form a
hairpin structure (stem-loop) by binding of complementary
nucleotides.
DETAILED DESCRIPTION OF THE BEST MODE OF THE INVENTION
[0185] Specific details of the best mode contemplated by the
inventors for carrying out the invention are set forth below, by
way of example. It will be apparent to one skilled in the art that
the present invention may be practiced without limitation to these
specific details.
Vectors Useful for Generation of Herpes Simplex Virus Mutants
[0186] Mutant herpes simplex viruses of the invention may be
generated by use of nucleic acid vectors.
[0187] One such vector useful for generation of mutant herpes
simplex viruses according to the present invention is a nucleic
acid vector comprising, consisting or consisting essentially
of:
[0188] first and second nucleotide sequences corresponding to
nucleotide sequences flanking an insertion site in the genome of a
selected herpes simplex virus; and
[0189] a cassette located between said first and second nucleotide
sequences comprising nucleic acid encoding: [0190] a) one or a
plurality of insertion sites; and [0191] b) a ribosome binding
site; and [0192] c) a marker.
[0193] Another vector useful for generation of mutant herpes
simplex viruses according to the present invention is a nucleic
acid vector comprising, consisting or consisting essentially
of:
[0194] first and second nucleotide sequences corresponding to
nucleotide sequences flanking an insertion site in the genome of a
selected herpes simplex virus; and
[0195] a cassette located between said first and second nucleotide
sequences comprising nucleic acid encoding: [0196] a) one or a
plurality of insertion sites; and [0197] b) a first regulatory
nucleotide sequence; and [0198] c) a marker.
[0199] The first and second nucleotide sequences may correspond to
nucleotide sequences flanking an insertion site formed in, or
comprising all or a part of, the ICP34.5 protein coding sequence of
the genome of a selected herpes simplex virus.
[0200] 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.
[0201] The encoded components of the cassette may be arranged in a
predetermined order. In one arrangement, the one or plurality of
insertion sites is/are arranged upstream (i.e. 5') of the ribosome
binding site/first regulatory sequence and the ribosome binding
site/first regulatory sequence is arranged upstream (i.e. 5') of
the marker.
[0202] The first and second nucleotide sequences may comprise
nucleotide sequences having identity to regions of the genome
surrounding the insertion site in the selected herpes simplex virus
(the `viral insertion site`). These sequences enable the cassette
to be incorporated at the viral insertion site by homologous
recombination between the first and second nucleotide sequences and
their respective corresponding sequences in the viral genome.
[0203] 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.
[0204] The first and second nucleotide sequences may correspond to
nucleotide sequences flanking an insertion site in the RL1 locus of
the HSV genome, more preferably in the ICP34.5 protein coding
sequence of the HSV genome.
[0205] The first and second nucleotide sequences may each be at
least 50bp 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 4000bp 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.
[0206] 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.
[0207] 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.
[0208] 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).
[0209] The first and second nucleotide sequences (vector flanking
sequences) may each comprise sequence corresponding to the RL
terminal repeat region of the genome of the selected HSV (e.g.
HSV-1 strains 17 or F or HSV-2 strain HG52). The vector flanking
sequences may comprise, consist or consist essentially of
nucleotide sequences of the RL repeat region which flank the
ICP34.5 protein coding sequence. In flanking the ICP34.5 coding
sequence, one or both of the selected sequences may, in the
corresponding HSV genome, overlap, i.e. extend into, the ICP34.5
protein coding sequence or one or both sequences may be selected so
as to not overlap the ICP34.5 protein coding sequence. In a similar
manner, the selected sequences may be chosen to overlap completely
or partially other important encoded signals, e.g. transcription
initiation site, polyadenylation site, defined promoters or
enhancers. In this preferred arrangement the insertion site will
thus comprise all or a part of the ICP34.5 protein coding sequence
and/or be such that the inserted cassette disrupts the ICP34.5
protein coding sequence.
[0210] The vectors described, comprising first and second
nucleotide sequences corresponding to regions of the RL repeat
region flanking and/or overlapping the ICP34.5 protein coding
sequence, may be used in the generation of ICP34.5 null mutants
wherein all or a portion of the ICP34.5 protein coding sequence is
excised and replaced during the homologous recombination event such
that both copies of the ICP34.5 coding sequence are disrupted. 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.
[0211] Successfully transformed virus are thus mutants incapable of
generating the ICP34.5 active gene product.
[0212] Each component of the cassette may be positioned
substantially adjacent the neighbouring component such that a
single bicistronic transcript comprising or consisting essentially
of the mRNA encoding the nucleotide sequence of interest, ribosome
binding site and marker is obtainable.
[0213] The vectors described may further comprise, consist, or
consist essentially of a nucleic acid encoding a selectable marker
such as a polypeptide or protein conferring antibiotic resistance
e.g. kanamycin resistance or ampicillin resistance.
[0214] The vectors described are preferably DNA vectors,
particularly dsDNA vectors. The vector may be provided as a linear
or circular (plasmid) DNA vector. The vector preferably contains
nucleotide sequences, e.g. restriction endonuclease site(s),
permitting transition between the two forms by use of DNA ligation
and restriction materials (e.g. enzymes) and techniques known to
the person skilled in the art. To achieve homologous recombination
with a selected HSV, the vector is preferably provided in linear
form.
[0215] One such vector provided by the inventors is plasmid
RL1.dIRES-GFP deposited in the name of Crusade Laboratories Limited
having an address at Department of Neurology Southern General
Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotland on 03 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`).
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] The vector may be a variant of plasmid RL1.dIRES-GFP.
[0224] 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.
[0225] One example is the expression of siRNAs as short hairpirn
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.
[0226] Thus in some cases a cassette may be provided in which the
sequence of interest and marker are expressed separately from
independent promoters.
[0227] One variant contains a cassette in which the ribosome
binding site of plasmid RL1.dIRES-GFP is replaced with a regulatory
nucleotide sequence, preferably a strong, constitutive promoter
such as the Phosphoglycerokinase promoter. The marker is thereby
expressed under the control of this (the `first`) regulatory
sequence. The nucleotide sequence of interest (e.g. antisense or
siRNA) is expressed under the control of a second regulatory
sequence upstream (5') of the nucleotide sequence of interest, e.g.
the CMV promoter. This vector variant is particularly suitable for
expression of siRNA where a weak promoter may be used for
expression of the siRNA molecule or the nucleic acid encoding the
siRNA may have a strong termination signal making it difficult to
produce a single bi- or poly-cistronic transcript containing the
transcribed siRNA 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.
[0228] This 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.
[0229] Thus, sequences of interest along with their own promoters
(although it is preferred that the PGK promoter is not also used
for this purpose) can then be cloned into either RL1-dPGK/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.
[0230] The vectors described may be constructed for use in
generating engineered HSV-1 or HSV-2 by insertion of a nucleic acid
cassette through a mechanism of homologous recombination between
nucleotide sequences flanking the cassette and corresponding
sequences in the selected herpes simplex virus genome.
[0231] The vectors described may comprise and have use as: [0232]
i) gene delivery (gene therapy) vectors for delivery of a selected
nucleotide sequence, e.g. antisense nucleic acid or siRNA, to a
specific locus of the HSV genome; and/or [0233] ii) expression
vectors for expression of the delivered nucleotide sequence of i)
from the HSV genome under the control of a selected regulatory
element; and/or [0234] iii) vectors for the generation of HSV
gene-specific null mutants wherein the cassette is inserted at a
selected genomic location to disrupt the protein coding sequence of
a selected HSV gene such that the gene product is inactive in the
resultant mutant virus.
[0235] The vectors described may be used in the manufacture of
engineered gene specific HSV null mutants, i.e. HSV mutants
incapable of expressing an active gene product of a selected gene.
They may be used in the manufacture of engineered viruses which
express a selected protein from only one gene copy the other gene
copy being disrupted or modified such that it cannot express a
functional gene product. Such vectors may also be used in the
manufacture of a medicament, preferably comprising said gene
specific HSV null mutant, for use in treating cancer and tumours,
preferably by the oncolytic treatment of the tumour.
[0236] The vectors described may also be used in the manufacture of
engineered HSV mutants wherein the genome of the mutant HSV
comprises a nucleotide sequence which has 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 or siRNA. 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.
[0237] The vectors described may also be used in the manufacture of
an engineered HSV mutant wherein the genome of the mutant HSV
comprises a nucleotide sequence which has been inserted in a
protein coding sequence of the HSV genome by homologous
recombination of the cassette such that the mutant HSV is incapable
of expressing the active gene encoded by said protein coding
sequence and wherein the inserted nucleotide sequence is expressed
under the control of a regulatory element to produce an antisense
transcript or siRNA. 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.
[0238] The vectors described may be used to generate mutant HSV by
inserting the cassette into the genome of a selected HSV, the
method of generation may comprise providing a vector described
above, where the vector is a plasmid, linearising the vector; and
co-transfecting a cell culture with the linearised vector and
genomic DNA from said HSV.
[0239] The co-transfection may be carried out under conditions
effective for homologous recombination of said cassette into an
insertion site of the viral genome.
[0240] The method may further comprise one or more of the steps of:
[0241] 1) screening said co-transfected cell culture to detect
mutant HSV expressing said marker; and/or [0242] 2) isolating said
mutant HSV; and/or [0243] 3) screening said mutant HSV for
expression of the nucleotide sequence of interest or the RNA or
polypeptide thereby encoded; and/or [0244] 4) screening said mutant
HSV for lack of an active gene product; and/or [0245] 5) testing
the oncolytic ability of said mutant HSV to kill tumour cells in
vitro.
EXAMPLE 1
[0245] Construction of Plasmid RL1.dIRES-GFP
General Approach
[0246] Plasmid RL1.dIRES-GFP was generated in three stages,
illustrated in FIG. 1.
[0247] 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.
[0248] 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.
[0249] 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.
[0250] 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
[0251] 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).
[0252] 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 lhr 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).
[0253] Ligation reactions were carried out in small eppendorf tubes
containing 5 units T4 DNA Ligase (Promega), a suitable volume of
10.times. DNA Ligase Buffer (Promega), nuclease free water
(Promega) and various volumes of the HpaI digested/CIP treated
RL1.del and blunt ended pCMV-NAT-IRES-GFP-SV40 Poly A DNA, at
16.degree. C. overnight. Competent JM109 bacterial cells (Promega)
were then transformed with various aliqouts of the ligation
reactions***. Colonies formed on the plates were picked, had their
plasmid DNA extracted using a Qiagen Plasmid Mini kit and screened
for inserts using AflII (New England Biolabs) restriction enzyme
analysis. Plasmid DNA containing the insert would produce two
fragments of 4.8 Kbp and 9.2 Kbp following digestion with AflII.
Two clones (clone 5 and 8) contained the insert (FIG. 4). The
orientation of the insert in clone 5 (RL1.dCMV-NAT-GFP) was
determined using XhoI restriction enzyme analysis (FIG. 5).
[0254] 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 lhr (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.
[0255] Ligation reactions were performed in small eppendorf tubes
containing 100 ng-500 ng purified DNA, 3 units T4 DNA Ligase
(Promega), a suitable volume of 10.times. DNA Ligase Buffer
(Promega) and nuclease free water (Promega) overnight at 16.degree.
C. Competent JM109 bacterial cells (Promega) were then transformed
with various aliquots of the ligation reactions***. Colonies formed
on the plates were picked, had their plasmid DNA extracted using a
Qiagen Plasmid Mini kit and screened using XhoI (Promega)
restriction enzyme analysis. Colonies containing plasmid DNA from
which CMV-NAT had been removed would produce one fragment of 10.2
Kbp when digested with XhoI. Several positive clones were found,
one was isolated, and a large-scale plasmid preparation undertaken
using Promega's Wizard Plus Maxipreps kit. The large-scale plasmid
preparation was checked by digesting with XhoI (FIG. 6B). This
plasmid DNA was subsequently named `RL1.dIRES-GFP`.
[0256] 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 03 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.
[0257] RL1.del
[0258] *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.
[0259] pNAT-IRES-GFP
[0260] **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.
[0261] ***Transformation of Bacterial Cells
[0262] 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 3hrs. 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.
[0263] 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
[0264] 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 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.
[0265] 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.
[0266] 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.
[0267] 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.
[0268] 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).
[0269] 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
[0270] To generate recombinant ICP34.5 null HSV-1 expressing a gene
of interest and GFP, requires the gene of interest and 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.
[0271] To generate replication restricted HSV-1, expressing the
gene of interest and GFP, the gene of interest must be cloned into
RL1dIRES-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
[0272] 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).
[0273] 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
[0274] 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 48hrs. 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's in roller bottles, in
50 ml ETC10, and a virus stock grown.
Tissue Culture Media
[0275] 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-asSCCRO/GFP
General Approach
[0276] HSV1716/CMV-asSCCRO/GFP was generated by first digesting
pUSEamp-asSCCRO with SspI and XhoI and purifying the 1.96 Kbp
fragment generated from the digestion. The 1.96 kbp SspI/XhoI
fragment comprises DNA antisense to squamous cell carcinoma related
antigen (asSCCRO), downstream of the CMV IE promoter (pCMV). This
fragment was cloned 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-asSCCRO-GFP, was then linearised and recombinant virus
generated and purified as described in Example 2. The plasmid
pUSEamp-asSCCRO was obtained from Bhuvanesh Singh, Memorial Sloan
Kettering Cancer Center, New York.
Materials and Methods
[0277] 2 .mu.g of the RL1.dIRES-GFP plasmid 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 5 units of
Klenow polymerase (Promega) for 20 minutes at room temperature,
then purified again. The purified DNA was then added to 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. 9).
[0278] 4.times.1 .mu.g of pUSEamp-asSCCRO was digested with 10
units of SspI and 10 units of XhoI (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 1.96 Kbp DNA
fragment, consisting essentially of the CMV promoter upstream of
DNA antisense to SCCRO (PCMV-asSCCRO), was excised using a sterile
scalpel and the DNA purified from the gel using a QIAquick Gel
Extraction kit (Qiagen). The purified DNA was blunt ended using 5
units of Klenow polymerase (Promega) for 20 minutes at room
temperature, then purified again. 5 .mu.l of the purified DNA
fragment was electrophoresed on a 1% agarose gel to check its
concentration (FIG. 10).
[0279] 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/blunt ended/CIP
treated RL1.dIRES-GFP plasmid and blunt ended pCMV-asSCCRO, at
16.degree. C. overnight. Competent JM109 bacterial cells (Promega)
were then transformed with various aliqouts of the ligation
reactions. Colonies formed on the plates were picked, had their
plasmid DNA extracted using a Qiagen Plasmid Mini kit and screened
for inserts using BglII (Promega) restriction enzyme analysis.
RL1.dIRES-GFP plasmid DNA containing the pCMV-asSCCRO insert would
produce two fragments of 10.8 Kbp and 1.4 Kbp following digestion
with BglII. One clone (clone 11) was found to contain the insert
(FIG. 11). The pCMV-asSCCRO insert could have been cloned into
RL1.dIRES-GFP in two orientations. To confirm that the pCMV-asSCCRO
fragment had been cloned into RL1.dIRES-GFP in the desired
orientation, clone 11 was digested with 10 units of NruI (Promega),
in a suitable volume of 10.times. buffer (Promega) and nuclease
free water (Promega), at 37.degree. C. for 16 hrs. If the insert
was in the correct orientation, a fragment of 1.64 Kbp would be
generated. As a 1.64 Kbp fragment was generated following digestion
with NruI (FIG. 12), it was confirmed that pCMV-asSCCRO had been
cloned in the desired orientation. This plasmid (clone 11) was
named `RL1.dCMV-asSCCRO-GFP`.
[0280] 0.1-1 .mu.g of RL1.dCMV-asSCCRO-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 asSCCRO), was identified and purified using a
fluorescent microscope and a virus stock, named
HSV1716/CMV-asSCCRO/GFP, was grown and titrated on BHK cells (FIG.
13).
[0281] HSV1716/CMV-asSCCRO/GFP has been deposited as
`HSV1716asSCCRO` 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 19 May 2004 at
the European Collection of Cell Cultures (ECACC), Health Protection
Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom
under accession number 04051901 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`).
EXAMPLE 4
The Use of HSV1716asSCCRO as a Novel Therapeutic Agent for Head and
Neck Squamous Cell Cancer.
[0282] The inventors believe that insertion of the antisense to
SCCRO into the herpes simplex virus HSV1716 may provide a virus
with a dual hit mechanism of cell kill. This would involve virus
induced cell death via cytolysis in addition to cell death via
downregulation of endogenous SCCRO expression.
[0283] The HSV1716asSCCRO virus was constructed, amplified and
purified in accordance with the present invention. Following this,
in vitro and in vivo analysis was carried out on a series of head
and neck squamous cell cancer (HNSCC) cell lines. HNSCC cell lines
studied were SCC15, 1483, 1186, 1386, 1986 and 584. The relative
expression of SCCRO protein expression in these cell lines was
initially determined by western blotting. This showed the cell
lines SCC15, 1483 and 1186 had high levels of expression of SCCRO,
1386 intermediate expression and 1986, 584 low expression. All cell
lines were then infected with HSV1716 or HSV1716asSCCRO viruses and
cytotoxicity determined by LDH release cytotoxicity assay at MOI
(multiplicity of infection) of 1 and 5 pfu/cell (FIGS. 14, 15 and
16). Viral proliferation was determined by serial plaque assays at
an MOI of 1 pfu/cell (FIG. 17) and infectivity determined by green
fluorescent protein (gfp) using HSV1716gtp virus (FIG. 18). In the
cell lines with low or intermediate expression (1986,584,1386)
cytotoxicity increased in a dose dependent fashion with both
viruses but there was no significant difference in cytotoxicity
between the 2 viruses. Viral proliferation assays (FIG. 17) showed
an increase in viral production over a range of 10.sup.2 to
10.sup.4 with equivalent proliferation with both viruses. In the
cell lines with high expression of SCCRO the inventors found that
the cell line SCC15 showed enhanced cytotoxicity with the
HSV1716asSCCRO virus. This observation occurred 12 hours post viral
infection which is premature for virus induced cell death by a
cytolytic mechanism. In addition, virus proliferation of the 2
viruses was equivalent with an increase in virus production of
10.sup.4 for both viruses. These results suggested that the
enhanced cell kill at 12 hours was by an alternative mechanism
possibly by downregulation of the endogenous high expression of
SCCRO by antisenseSCCRO expression. To investigate this hypothesis
the inventors analysed the cell lines SCC15 (high expression) and
584 (low expression) post virus infection by serial protein
expression over a 36 hour period. Cells were infected at an MOI of
1 pfu/cell with HSV1716 or HSV1716asSCCRO and cells harvested and
lysed for protein at 12, 24 and 36 hours post infection. Western
blotting of the cell line SCC15 showed downregulation of SCCRO
protein at 12 hours with HSV1716asSCCRO but not in 584 (see FIG.
19). This suggested that this was the mechanism by which
HSV1716asSCCRO had enhanced efficacy in cell line SCC15.
[0284] In vivo studies were then carried out in the cell lines
SCC15 and 584. Subcutaneous tumour were grown in athymic nude mice
and injected intratumorally with a single injection of HSV1716,
HSV1716asSCCRO or PBS control and tumour sizes monitored at serial
time points (FIG. 20 and 21). In SCC15, efficacy was enhanced with
HSV1716asSCCRO compared to HSV1716. All 6 mice injected with
HSV1716asSCCRO showed complete responses by 21 days post infection.
Inhibition of tumour growth occurred with HSV1716 with only 3/6
mice showing a complete response over a 48 day follow up period. In
the cell line 584, both viruses were able to inhibit tumour growth
but neither virus produced a complete response in any mouse
xenograft injected. This in vivo data was further evidence that
HSV1716asSCCRO was a more potent antitumour agent than HSV1716 in
the cell line SCC15 with high SCCRO expression.
[0285] These results suggest that HSV1716 and HSV1716asSCCRO has
great potential as useful therapeutic agents in the treatment of
recurrent or locally advanced head and neck cancer by direct
intratumoral injection. However, this data also suggests that
HSV1716asSCCRO may augment anti-tumour activity in SCCRO
over-expressing tumours. Since SCCRO is overexpressed in a
significant number of squamous cell cancers of the head and neck
this modified virus may be particularly efficacious in this
disease. Therefore, the inventors believe that HSV1716asSCCRO will
be an important therapeutic agent in head and neck cancer patients
with locally advanced or recurrent head and neck cancer,
particularly as these cancers are amenable to direct intratumoural
injection.
EXAMPLE 5
Construction of HSV1716 Variants Expressing siRNA
General Strategy
[0286] A plasmid that contains the siRNA construct designed to
target expression of the SCCRO-gene (SEQ ID No. 5) and designated
339i was provided by Dr Bhuv Singh, MSKCC, New York. A plasmid
encoding a control siRNA (SEQ ID No 6), designated Coni, was also
provided.
[0287] 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.
[0288] 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.
[0289] 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
[0290] 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.
[0291] 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.
[0292] 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.
[0293] 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, 501 of HSV-1
strain 17+ DNA was added along with 20 .mu.l lipofectamine 2000 in
a final volume of 500 .mu.l 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)
[0294] 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.
[0295] 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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Sequence CWU 1
1
6 1 918 DNA Homo sapiens CDS (58)..(837) 1 cgccgtccat tcgctgcgga
gccggaggag gaggggagag gcctggagga caccaac 57 atg aac aag ttg aaa tca
tcg cag aag gat aaa gtt cgt cag ttt atg 105 Met Asn Lys Leu Lys Ser
Ser Gln Lys Asp Lys Val Arg Gln Phe Met 1 5 10 15 atc ttc aca caa
tct agt gaa aaa aca gca gta agt tgt ctt tct caa 153 Ile Phe Thr Gln
Ser Ser Glu Lys Thr Ala Val Ser Cys Leu Ser Gln 20 25 30 aat gac
tgg aag tta gat gtt gca aca gat aat ttt ttc caa aat cct 201 Asn Asp
Trp Lys Leu Asp Val Ala Thr Asp Asn Phe Phe Gln Asn Pro 35 40 45
gaa ctt tat ata cga gag agt gta aaa gga tca ttg gac agg aag aag 249
Glu Leu Tyr Ile Arg Glu Ser Val Lys Gly Ser Leu Asp Arg Lys Lys 50
55 60 tta gaa cag ctg tac aat aga tac aaa gac cct caa gat gag aat
aaa 297 Leu Glu Gln Leu Tyr Asn Arg Tyr Lys Asp Pro Gln Asp Glu Asn
Lys 65 70 75 80 att gga ata gat ggc ata cag cag ttc tgt gat gac ctg
gca ctc gat 345 Ile Gly Ile Asp Gly Ile Gln Gln Phe Cys Asp Asp Leu
Ala Leu Asp 85 90 95 cca gcc agc att agt gtg ttg att att gca tgg
aag ttc aga gca gca 393 Pro Ala Ser Ile Ser Val Leu Ile Ile Ala Trp
Lys Phe Arg Ala Ala 100 105 110 aca cag tgc gag ttc tcc aaa cag gag
ttc atg gat ggc atg aca gaa 441 Thr Gln Cys Glu Phe Ser Lys Gln Glu
Phe Met Asp Gly Met Thr Glu 115 120 125 tta gga tgt gac agc ata gaa
aaa cta aag gcc cag ata ccc aag atg 489 Leu Gly Cys Asp Ser Ile Glu
Lys Leu Lys Ala Gln Ile Pro Lys Met 130 135 140 gaa caa gaa ttg aaa
gaa cca gga cga ttt aag gat ttt tac cag ttt 537 Glu Gln Glu Leu Lys
Glu Pro Gly Arg Phe Lys Asp Phe Tyr Gln Phe 145 150 155 160 act ttt
aat ttt gca aag aat cca gga caa aaa gga tta gat cta gaa 585 Thr Phe
Asn Phe Ala Lys Asn Pro Gly Gln Lys Gly Leu Asp Leu Glu 165 170 175
atg gcc att gcc tac tgg aac tta gtg ctt aat gga aga ttt aaa ttc 633
Met Ala Ile Ala Tyr Trp Asn Leu Val Leu Asn Gly Arg Phe Lys Phe 180
185 190 tta gac tta tgg aat aaa ttt ttg ttg gaa cat cat aaa cga tca
ata 681 Leu Asp Leu Trp Asn Lys Phe Leu Leu Glu His His Lys Arg Ser
Ile 195 200 205 cca aaa gac act tgg aat ctt ctt tta gac ttc agt acg
atg att gca 729 Pro Lys Asp Thr Trp Asn Leu Leu Leu Asp Phe Ser Thr
Met Ile Ala 210 215 220 gat gac atg tct aat tat gat gaa gaa gga gca
tgg cct gtt ctt att 777 Asp Asp Met Ser Asn Tyr Asp Glu Glu Gly Ala
Trp Pro Val Leu Ile 225 230 235 240 gat gac ttt gtg gaa ttt gca cgc
cct caa att gct ggg aca aaa agt 825 Asp Asp Phe Val Glu Phe Ala Arg
Pro Gln Ile Ala Gly Thr Lys Ser 245 250 255 aca aca gtg tag
cactaaagga accttctaga atgtacatag tctgtacaat 877 Thr Thr Val
aaatacaaca gaaaattgca cagtcaattt ctgctggctg g 918 2 259 PRT Homo
sapiens 2 Met Asn Lys Leu Lys Ser Ser Gln Lys Asp Lys Val Arg Gln
Phe Met 1 5 10 15 Ile Phe Thr Gln Ser Ser Glu Lys Thr Ala Val Ser
Cys Leu Ser Gln 20 25 30 Asn Asp Trp Lys Leu Asp Val Ala Thr Asp
Asn Phe Phe Gln Asn Pro 35 40 45 Glu Leu Tyr Ile Arg Glu Ser Val
Lys Gly Ser Leu Asp Arg Lys Lys 50 55 60 Leu Glu Gln Leu Tyr Asn
Arg Tyr Lys Asp Pro Gln Asp Glu Asn Lys 65 70 75 80 Ile Gly Ile Asp
Gly Ile Gln Gln Phe Cys Asp Asp Leu Ala Leu Asp 85 90 95 Pro Ala
Ser Ile Ser Val Leu Ile Ile Ala Trp Lys Phe Arg Ala Ala 100 105 110
Thr Gln Cys Glu Phe Ser Lys Gln Glu Phe Met Asp Gly Met Thr Glu 115
120 125 Leu Gly Cys Asp Ser Ile Glu Lys Leu Lys Ala Gln Ile Pro Lys
Met 130 135 140 Glu Gln Glu Leu Lys Glu Pro Gly Arg Phe Lys Asp Phe
Tyr Gln Phe 145 150 155 160 Thr Phe Asn Phe Ala Lys Asn Pro Gly Gln
Lys Gly Leu Asp Leu Glu 165 170 175 Met Ala Ile Ala Tyr Trp Asn Leu
Val Leu Asn Gly Arg Phe Lys Phe 180 185 190 Leu Asp Leu Trp Asn Lys
Phe Leu Leu Glu His His Lys Arg Ser Ile 195 200 205 Pro Lys Asp Thr
Trp Asn Leu Leu Leu Asp Phe Ser Thr Met Ile Ala 210 215 220 Asp Asp
Met Ser Asn Tyr Asp Glu Glu Gly Ala Trp Pro Val Leu Ile 225 230 235
240 Asp Asp Phe Val Glu Phe Ala Arg Pro Gln Ile Ala Gly Thr Lys Ser
245 250 255 Thr Thr Val 3 876 DNA Homo sapiens CDS (16)..(795) 3
ctggaggaca ccaac atg aac aag ttg aaa tca tcg cag aag gat aaa gtt 51
Met Asn Lys Leu Lys Ser Ser Gln Lys Asp Lys Val 1 5 10 cgt cag ttt
atg atc ttc aca caa tct agt gaa aaa aca gca gta agt 99 Arg Gln Phe
Met Ile Phe Thr Gln Ser Ser Glu Lys Thr Ala Val Ser 15 20 25 tgt
ctt tct caa aat gac tgg aag tta gat gtt gca aca gat aat ttt 147 Cys
Leu Ser Gln Asn Asp Trp Lys Leu Asp Val Ala Thr Asp Asn Phe 30 35
40 ttc caa aat cct gaa ctt tat ata cga gag agt gta aaa gga tca ttg
195 Phe Gln Asn Pro Glu Leu Tyr Ile Arg Glu Ser Val Lys Gly Ser Leu
45 50 55 60 gac agg aag aag tta gaa cag ctg tac aat aga tac aaa gac
cct caa 243 Asp Arg Lys Lys Leu Glu Gln Leu Tyr Asn Arg Tyr Lys Asp
Pro Gln 65 70 75 gat gag aat aaa att gga ata gat ggc ata cag cag
ttc tgt gat gac 291 Asp Glu Asn Lys Ile Gly Ile Asp Gly Ile Gln Gln
Phe Cys Asp Asp 80 85 90 ctg gca ctc gat cca gcc agc att agt gtg
ttg att att gcg tgg aag 339 Leu Ala Leu Asp Pro Ala Ser Ile Ser Val
Leu Ile Ile Ala Trp Lys 95 100 105 ttc aga gca gca aca cag tgc gag
ttc tcc aaa cag gag ttc atg gat 387 Phe Arg Ala Ala Thr Gln Cys Glu
Phe Ser Lys Gln Glu Phe Met Asp 110 115 120 ggc atg aca gaa tta gga
tgt gac agc aca gaa aaa cta aag gcc cag 435 Gly Met Thr Glu Leu Gly
Cys Asp Ser Thr Glu Lys Leu Lys Ala Gln 125 130 135 140 ata ccc aag
atg gaa caa gaa ttg aaa gaa cca gga cga ttt aag gat 483 Ile Pro Lys
Met Glu Gln Glu Leu Lys Glu Pro Gly Arg Phe Lys Asp 145 150 155 ttt
tac cag ttt act ttt aat ttt gca aag aat cca gga caa aaa gga 531 Phe
Tyr Gln Phe Thr Phe Asn Phe Ala Lys Asn Pro Gly Gln Lys Gly 160 165
170 tta gat cta gaa atg gcc att gcc tac tgg aac tta gtg ctt aat gga
579 Leu Asp Leu Glu Met Ala Ile Ala Tyr Trp Asn Leu Val Leu Asn Gly
175 180 185 aga ttt aga ctc tta gac tta tgg aat aaa ttt ttg ttg gaa
cat cat 627 Arg Phe Arg Leu Leu Asp Leu Trp Asn Lys Phe Leu Leu Glu
His His 190 195 200 aaa cga tca ata cca aaa gac act tgg aat ctt ctt
tta gac ttc agt 675 Lys Arg Ser Ile Pro Lys Asp Thr Trp Asn Leu Leu
Leu Asp Phe Ser 205 210 215 220 acg atg att gca gat gac atg tct aat
tat gat gaa gaa gga gca tgg 723 Thr Met Ile Ala Asp Asp Met Ser Asn
Tyr Asp Glu Glu Gly Ala Trp 225 230 235 cct gtt ctt att gat gac ttt
gtg gaa ttt gca cgc cct caa att gct 771 Pro Val Leu Ile Asp Asp Phe
Val Glu Phe Ala Arg Pro Gln Ile Ala 240 245 250 ggg aca aaa agt aca
aca gtg tag cactaaagga accttctaga atgtacatag 825 Gly Thr Lys Ser
Thr Thr Val 255 tctgtacaat aaatacaaca gaaaattgca cagtcaattt
ctgctggctg g 876 4 259 PRT Homo sapiens 4 Met Asn Lys Leu Lys Ser
Ser Gln Lys Asp Lys Val Arg Gln Phe Met 1 5 10 15 Ile Phe Thr Gln
Ser Ser Glu Lys Thr Ala Val Ser Cys Leu Ser Gln 20 25 30 Asn Asp
Trp Lys Leu Asp Val Ala Thr Asp Asn Phe Phe Gln Asn Pro 35 40 45
Glu Leu Tyr Ile Arg Glu Ser Val Lys Gly Ser Leu Asp Arg Lys Lys 50
55 60 Leu Glu Gln Leu Tyr Asn Arg Tyr Lys Asp Pro Gln Asp Glu Asn
Lys 65 70 75 80 Ile Gly Ile Asp Gly Ile Gln Gln Phe Cys Asp Asp Leu
Ala Leu Asp 85 90 95 Pro Ala Ser Ile Ser Val Leu Ile Ile Ala Trp
Lys Phe Arg Ala Ala 100 105 110 Thr Gln Cys Glu Phe Ser Lys Gln Glu
Phe Met Asp Gly Met Thr Glu 115 120 125 Leu Gly Cys Asp Ser Thr Glu
Lys Leu Lys Ala Gln Ile Pro Lys Met 130 135 140 Glu Gln Glu Leu Lys
Glu Pro Gly Arg Phe Lys Asp Phe Tyr Gln Phe 145 150 155 160 Thr Phe
Asn Phe Ala Lys Asn Pro Gly Gln Lys Gly Leu Asp Leu Glu 165 170 175
Met Ala Ile Ala Tyr Trp Asn Leu Val Leu Asn Gly Arg Phe Arg Leu 180
185 190 Leu Asp Leu Trp Asn Lys Phe Leu Leu Glu His His Lys Arg Ser
Ile 195 200 205 Pro Lys Asp Thr Trp Asn Leu Leu Leu Asp Phe Ser Thr
Met Ile Ala 210 215 220 Asp Asp Met Ser Asn Tyr Asp Glu Glu Gly Ala
Trp Pro Val Leu Ile 225 230 235 240 Asp Asp Phe Val Glu Phe Ala Arg
Pro Gln Ile Ala Gly Thr Lys Ser 245 250 255 Thr Thr Val 5 64 DNA
Artificial sequence DNA nucleotide sequence encoding the siRNA
construct designed to target expression of the SCCRO gene 5
gatccccgtt cagagcagca acacagttca agagactgtg ttgctgctct gaactttttg
60 gaaa 64 6 60 DNA Artificial sequence Nucleotide sequence
encoding control siRNA 6 gatcccccgt ctacctacac tccctcttca
agagagaggg agtgtaggta gacgttttta 60
* * * * *
References