U.S. patent application number 11/822373 was filed with the patent office on 2008-01-10 for tetracycline repressor regulated oncolytic viruses.
This patent application is currently assigned to The Brigham and Women's Hospital, Inc.. Invention is credited to Feng Yao.
Application Number | 20080008686 11/822373 |
Document ID | / |
Family ID | 38919348 |
Filed Date | 2008-01-10 |
United States Patent
Application |
20080008686 |
Kind Code |
A1 |
Yao; Feng |
January 10, 2008 |
Tetracycline repressor regulated oncolytic viruses
Abstract
The present invention is directed oncolytic Herpes simplex-l
viruses whose replication is controlled using a tetracycline
operator/repressor system. The invention also includes DNA
sequences used in making the viruses and methods in which these
viruses are used in the treatment of cancer patients with solid
tumors.
Inventors: |
Yao; Feng; (Needham,
MA) |
Correspondence
Address: |
LAW OFFICE OF MICHAEL A. SANZO, LLC
15400 CALHOUN DR.
SUITE 125
ROCKVILLE
MD
20855
US
|
Assignee: |
The Brigham and Women's Hospital,
Inc.
Boston
MA
02115
|
Family ID: |
38919348 |
Appl. No.: |
11/822373 |
Filed: |
July 5, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60819382 |
Jul 10, 2006 |
|
|
|
Current U.S.
Class: |
424/93.2 ;
435/235.1; 536/24.1 |
Current CPC
Class: |
C12N 2710/16632
20130101; C12N 7/00 20130101; C12N 2710/16643 20130101; C12N 15/86
20130101; A61K 35/763 20130101; C12N 2830/003 20130101 |
Class at
Publication: |
424/093.2 ;
435/235.1; 536/024.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/04 20060101 C07H021/04; C12N 7/01 20060101
C12N007/01 |
Goverment Interests
STATEMENT OF GOVERNMENT FUNDING
[0002] The United States Government has a paid-up license in this
invention and the right in limited circumstances to require the
patent owner to license others under reasonable terms as provided
for by the terms of NIH Grant Nos. ROIA105088 and ROIGM51449
awarded by the Department of Health and Human Services.
Claims
1. A Herpes simplex virus 1 or 2 (HSV) recombinant DNA molecule,
comprising: a) a first promoter sequence having a TATA element; b)
a tetracycline operator sequence comprising two op2 repressor
binding sites joined by 2-20 linking nucleotides, wherein the first
nucleotide in said tet operator is between 6 and 24 nucleotides 3'
to the last nucleotide in said TATA element; and c) a gene
necessary for Herpes simplex 1 or 2 (HSV) virus replication lying
3' to said operator and operably linked to said first promoter.
2. The recombinant DNA of claim 1, wherein said first promoter is
an HSV immediate early promoter, early promoter, or late
promoter.
3. The recombinant DNA of claim 2, wherein said first promoter is
an HSV immediate early promoter.
4. The recombinant DNA of claim 2, wherein said first promoter is
selected from the group consisting of: the HSV ICP4 promoter; ICP27
promoter; ICP8 promoter; UL9 promoter; gD promoter; or VP5
promoter.
5. The recombinant DNA of claim 2, wherein said gene necessary for
viral replication is a viral immediate-early, early, or late
gene.
6. The recombinant DNA of claim 5, wherein said gene necessary for
viral replication is ICP27, ICP4, ICP8, UL9, gD, or VP5 and is
under the control of its corresponding promoter.
7. The recombinant DNA of claim 1, wherein said DNA has at least
one sequence encoding the tet repressor under the control of a
second promoter.
8. The recombinant DNA of claim 7, wherein said second promoter is
an HSV immediate early promoter, or the hCMV major immediate-early
promoter.
9. The recombinant DNA of claim 7, wherein said second promoter is
the HSV ICP0 promoter or ICP4 promoter.
10. The recombinant DNA of claim 1, further comprising a ribozyme
sequence or DNA sequence that can reduce translation in the 5'
untranslated region of said gene.
11. The recombinant DNA of claim 1, wherein said recombinant DNA is
part of the HSV genome.
12. The recombinant DNA of claim 11, wherein the HSV ICP6 gene is
deleted.
13. The recombinant DNA of claim 12, wherein the HSV ICP47 gene is
deleted.
14. The recombinant DNA of claim 12, further comprising at least
one therapeutic gene that can augment tumor killing activity or
anti-tumor specific immunity.
15. An oncolytic HSV virus comprising as its genome, the
recombinant DNA of claim 11.
16. The oncolytic virus of claim 15, wherein said recombinant DNA
does not include a functional HSV ICP6 gene.
17. The oncolytic virus of claim 16, wherein said recombinant DNA
does not include a functional HSV ICP47 gene.
18. The oncolytic virus of claim 15, wherein said recombinant DNA
comprises at least one therapeutic gene that can augment tumor
killing activity or anti-tumor specific immunity.
19. A method of treating a cancer patient having a solid tumor,
comprising: a) locally administering to said solid tumor the
oncolytic virus of claim 15 for a period sufficient to allow
infection of tumor cells b) administering tetracycline either
systemically to said patient or locally to said tumor.
20. The method of claim 19, wherein said oncolytic virus does not
include a functional HSV ICP6 gene within its genome.
21. The method of claim 20, wherein said oncolytic virus does not
include a functional HSV ICP47 gene within its genome.
22. The method of claim 19, wherein said oncolytic virus comprises
at least one therapeutic gene that can augment tumor killing
activity within its genome or anti-tumor specific immunity.
23. The method of claim 19, wherein said tumor is a melanoma or a
tumor of the lung, colon, brain, breast, prostate, pancreas,
kidney, esophagus, liver, ovary, testis or stomach.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to, and the benefit
of, U.S. provisional application 60/819,382, filed on Jul. 10,
2006. This prior application is hereby incorporated by reference in
its entirety.
FIELD OF THE INVENTION
[0003] The present invention is concerned with oncolytic viruses,
especially Herpes Simplex Virus type 1, that rely upon the
tetracycline resistance (tet) operator and repressor to control
their replication after the infection of tumor cells and/or normal
cells. It encompasses DNA sequences used in recombinantly producing
the viruses and methods in which the viruses are used in the
treatment of cancer patients.
BACKGROUND OF THE INVENTION
[0004] Oncolytic viruses are designed to infect and destroy cancer
cells, while leaving normal cells relatively unaffected (MacLean et
al., J. Gen. Virol. 72:630-639 (1991); Robertson et al., J. Gen.
Virol. 73:967-970 (1992); Brown et al., J. Gen. Virol. 75:3767-3686
(1994); Chou et al., Science 250:1262-1265 (1990)). One virus that
has been of particular interest as an oncolytic is Herpes simplex
virus-1 (HSV). HSV can infect a broad range of cell types and has
large genome, which allows multiple therapeutic genes to be
packaged into recombinants. Experimental evidence for the antitumor
effect of HSV against gliomas has existed for over a decade
(Martuza, et al., Science 252:854-856 (1991); see also: Mineta et
al., Nature Med. 1:938-943 (1995); Market et al., J. Neurosurg.
77:590-594 (1992); Randazzo et al., Virology 211:94-101 (1995);
Kesari et al., Lab. Invest. 73:636-648 (1995)) and more recent
studies suggest that it should be effective for the treatment of
tumors of the lung, liver and ovary as well (U.S. Pat. No.
6,428,968; see also Montgomery et al., Cell 87:427-436 (1996)).
Unfortunately, attempts to develop viruses that only target or
replicate in cancer cells while maintaining their cytolytic
effectiveness has met with only limited success. For example, it
has been found that the deletion of genes that impair viral
replication in normal cells also leads to a significant decrease in
the oncolytic activity of the virus in targeted tumor cells (Kramm,
et al. Hum. Gene Ther. 8(17):2057-68 (1997); Advani, et al., Gene
Ther 5(2):160-165 (1998); Chung, et al., J. Virol. 73(9):7556-64
(1999)).
[0005] Thus, it remains a great challenge to construct an oncolytic
HSV recombinant that offers a high degree of safety while its
replication capability in tumor cells remains at levels close to
that of a wild-type virus. In principle, this can be achieved if
one can construct an oncolytic recombinant virus whose de novo
replication can be tightly controlled and adjusted by a
pharmacological agent in the localized tumor microenvironment. This
virus should minimize concerns regarding the potential unwanted
spread of oncolytic virus to other tissues, prevent overloads of
progeny oncolytic viruses at the end of tumor killing and quickly
shut down the oncolytic activity of the virus if unwanted adverse
effects are detected in a patient.
[0006] Recently, a tetracycline-inducible transcription switch for
use in mammalian cells was developed (U.S. Pat. No. 6,444,871; Yao,
et al., Hum. Gene Ther. 9:1939-1950 (1998)) and used to adapt HSV
as a vector for delivering therapeutic genes to cells (US published
application 2005-0266564). However, it has not been clear whether
this degree of regulation is sufficient in controlling de novo
replication of an oncolytic virus or whether it is possible to
develop a safe and effective therapy that does not rely on viruses
being highly specific for cancer cells.
SUMMARY OF THE INVENTION
[0007] The present invention is based upon the use of a tet
operator/repressor system for controlling the replication of
oncolytic viruses. Control is achieved by putting the expression of
a gene needed for viral replication under the control of the tet
operator and expressing large amounts of tet repressor either
before, or soon after, cells are infected. The repressor binds to
the operator, thereby preventing gene transcription and blocking
viral replication. If tetracycline is introduced into the system,
it binds to the repressor and causes it to disassociate from the
operator. As a result, gene transcription is permitted, virus
replication proceeds and, ultimately, this leads to the lysis of
the host cell.
[0008] Lysis of cancer cells using the viruses described herein
appears to trigger an immune response against tumor-associated
antigens. Thus, in addition to relying upon the direct oncolytic
effects of virus to destroy infected cancer cells, the present
invention also depends upon the subsequent action of the immune
system to induce a specific host immune response against cancer
cells. Damage to non-cancer cells is minimized by applying virus
directly to tumors and then inducing virus replication by applying
tetracycline for a limited time and/or in a localized tumor
microenvironment. Once the tetracycline is removed (or in tissues
that have limited access to tetracycline, i.e., when tetracycline
or its derivatives are introduced into a localized tumor
microenvironment), the virus is no longer able to replicate to a
significant extent and therefore its spread to non-cancerous cells
is minimized.
[0009] In its first aspect, the invention is directed to a
recombinant DNA molecule that includes a promoter sequence with a
TATA element. The promoter is under the control of a tetracycline
operator sequence that has two op2 repressor binding sites joined
together by between two and twenty linking nucleotides. The
positioning of the operator sequence is important to achieve
effective control over the promoter. Specifically, the first
nucleotide in the operator sequence must be located between six and
twenty-four nucleotides 3' to the last nucleotide in the TATA
element. In addition, the recombinant DNA molecule includes a gene
necessary for viral replication that lies 3' to the tet operator
and which is operably linked to the promoter. The term "operably
linked" refers to genetic elements that are joined together in a
manner that enables them to carry out their normal functions. For
example, a gene is operably linked to a promoter when its
transcription is under the control of the promoter and this
transcription results in the production of the product normally
encoded by the gene. The term "recombinant" refers to a nucleic
acid that is formed by experimentally recombining nucleic acid
sequences and sequence elements. A recombinant host cell would be a
cell that has received a recombinant nucleic acid.
[0010] Preferably, the gene necessary for viral replication in the
recombinant DNA described above is a gene essential for the
replication of Herpes simplex virus-1 (HSV) and the gene can be an
essential immediate-early gene, e.g., ICP4 and ICP27, or an
essential early or late gene, e.g., ICP8, UL9, and VP5. In
addition, the recombinant DNA molecule may include a ribozyme
sequence lying in the 5' untranslated region of the essential gene
and a second promoter operably linked to a sequence encoding the
tet repressor. The ribozyme will help to ensure low level of
expression of the essential gene immediately after infection of
cells, especially, if the viral essential immediate-early gene is
used. Preferable promoters for direct expression of tet repressor
in the recombinant DNA molecules are either the HSV immediate-early
promoters or the hCMV major immediate-early promoter.
[0011] In another aspect, the invention is directed to an oncolytic
virus that comprises any of the recombinant DNA molecules described
above. The virus must have a genome in which there is: a) a first
promoter sequence having a TATA element; b) a tetracycline operator
sequence comprising two op2 repressor binding sites joined by 2-20
linking nucleotides, wherein the first nucleotide in the tet
operator is between 6 and 24 nucleotides 3' to the last nucleotide
in the TATA element; and c) a gene necessary for virus replication
lying 3' to the operator and operably linked to the first promoter.
It should preferably also include: d) at least one sequence
encoding a tet repressor and under the control of a second
promoter. The term "second promoter" means that the sequence
encoding the tet repressor is not under the control of the same
promoter as the gene needed for viral replication, although the
same type of promoter may be used in each case. For example there
may be two ICP27 promoters, one operably linked to the gene needed
for viral replication and one to the sequence encoding the tet
promoter, but there should not be a single promoter operably linked
to both.
[0012] The oncolytic virus is preferably HSV and is produced using
standard methodology well-known in the art of virology. Preferred
promoters are the HSV immediate early promoters for directing
expression of tet repressor, and the preferred promoters and genes
needed for virus replication are the HSV essential immediate-early,
early and late genes. It is also preferred that the virus have two
tet repressor sequence elements present, each under the control of
an immediate early promoter. In addition a ribozyme sequence may be
present in the 5' untranslated region of the gene needed for viral
replication. Other viruses with these elements present are also
part of the invention with the next most preferred being
adenovirus.
[0013] The invention also includes methods of treating cancer
patients having a solid tumor by locally administering the
oncolytic viruses described herein. The term "locally
administering" means that the virus is applied directly to the
tumor itself, e.g., by injection, infusion, or, in the case of
certain skin cancers, topical administration. In order for the
therapy to be effective, cancer cells must be treated so that they
have two characteristics. First, they must be infected with a virus
whose replication can be controlled by tetracycline and its
derivatives. This is accomplished by infecting the cancer cells
with a virus, preferably HSV, that has, as described above, at
least the following elements: a) a first promoter sequence having a
TATA sequence; b) a tetracycline operator sequence comprising two
op2 repressor binding sites joined by 2-20 linking nucleotides,
wherein the first nucleotide in the tet operator is between 6 and
24 nucleotides 3' to the last nucleotide in said TATA element; and
c) a gene necessary for virus replication lying 3' to the operator
and operably linked to the first promoter.
[0014] The second characteristic that the cells must have is that
they must also make the tet repressor. This can be accomplished
using a single virus which, in addition to elements a-c, also
includes a sequence encoding the tet repressor operably linked to a
second promoter. However, other ways of modifying the cancer cells
to express the tet repressor may also be used. For example, the
cells may first be infected with a different virus expressing the
tet repressor or transformed with an expression vector, e.g., a
plasmid, that leads to expression of repressor, and then
subsequently infected with a virus having elements a-c. Many
variations on this scheme will be readily apparent to those of
skill in the art but the ultimate objective of treating cancer
cells so that they have a virus whose replication is controlled by
the tet operator and so that they also produce the tet repressor
will always be the same.
[0015] The amount of virus administered will vary from patient to
patient but it is generally expected that between 1.times.10.sup.6
and 1.times.10.sup.10 PFU (plaque forming units) will be
administered at a time. Tetracycline will be administered either
systemically to the patient or, more preferably, locally to the
tumor either at the time of infection or 1 to 72 h prior to
infection. The administration of tetracycline will have the effect
of allowing de novo viral replication, leading to synthesis of
progeny virus, cell lysis, and subsequent intratumoral spread of
newly synthesized virus within the tumor. This method may be used
for the treatment of any type of solid tumor including tumors of
the lung, colon, brain, breast, prostate, pancreas, kidney,
esophagus, liver, ovary, testis and stomach. Melanomas may also be
treated using the method.
[0016] The procedures for treating cancer by administering
oncolytic viruses described herein may be combined with any other
treatments that are common in the art including surgery, radiation
therapy, and chemotherapy. In addition, the administration of virus
may be repeated based upon the clinical judgment of the attending
physician. Finally, since it is believed that one of the main
actions of the virus will be mediated by an activation of the host
immune system against tumor antigens released as a result of lysis,
other agents designed to boost a patient's immune response may also
be administered or incorporated into the above described
recombinant virus.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is based upon the discovery that the
tet operator/repressor system can be used to tightly regulate the
replication of an HSV oncolytic virus. After infecting tumor cells,
the introduction of tetracycline produced a 1000 to 30,000 fold
increase in virus replication. In addition to directly lysing
infected cells, the virus also inhibits the growth of tumor in
immuno-competent mice at a different site from the site of
infection. This suggests that cell lysis induced by the virus
causes an immune response against tumor specific antigens. While
not being held to any particular theory, it is believed that the
virus contributes to this response both by making antigen more
available due to cell lysis and by somehow inducing a stronger
response, i.e., acting in a manner analogous to an adjuvant. The
methods discussed below are described primarily with reference to
the HSV-1 virus but it will be readily apparent to one of skill in
the art that they may be also applied to other viruses, e.g.,
adenovirus.
[0018] The Tet Operator/Repressor Switch and Recombinant DNA
[0019] The present invention is directed to, inter alia, oncolytic
viruses whose replication is regulated using the tetracycline
operator and repressor protein (for sequences see Postle et al.,
Nucl. Acid Res. 12:4849-4863 (1984); Hillen et al., Ann. Rev.
Microbiol. 48:345-369 (1994); Wissmann et al., J. Mol. Biol.
202:397-406 (1988)). General methods for making recombinant DNA
molecules containing these elements and DNA sequences have been
previously described (see U.S. Pat. No. 6,444,871) and plasmids
which contain the tetracycline-inducible transcription switch are
commercially available (T-REx.TM., Invitrogen, Calif.). An example
of a specific method for viral construction is provided below in
the Examples section but many alternatives will be readily apparent
to one of skill in the art.
[0020] An essential feature of the DNA of the present invention is
the presence of a gene needed for virus replication that is
operably linked to a promoter having a TATA element. A tet operator
sequence is located between 6 and 24 nucleotides 3' to the last
nucleotide in the TATA element of the promoter and 5' to the gene.
The strength with which the tet repressor binds to the operator
sequence is enhanced by using a form of operator which contains two
op2 repressor binding sites (each such site having the nucleotide
sequence: TCCCTATCAGTGATAGAGA (SEQ ID NO:1)) linked by a sequence
of 2-20, preferably 1-3 or 10-13, nucleotides. When repressor is
bound to this operator, very little or no transcription of the
associated gene will occur. If DNA with these characteristics is
present in a cell that also expresses the tetracycline repressor,
transcription of the gene will be blocked by the repressor binding
to the operator and replication of the virus will not occur.
However, if tetracycline is introduced, it will bind to the
repressor, cause it to dissociate from the operator, and virus
replication will proceed.
[0021] Selection of Promoters and Genes
[0022] During productive infection, HSV gene expression falls into
three major classes based on the temporal order of expression:
immediate-early (.alpha.), early (.beta.), and late (.gamma.), with
late genes being further divided into two groups, .gamma.1 and
.gamma.2. The expression of immediate-early genes does not require
de novo viral protein synthesis and is activated by the
virion-associated protein VP16 together with cellular transcription
factors when the viral DNA enters the nucleus. The protein products
of the immediate-early genes are designated infected cell
polypeptides ICP0, ICP4, ICP22, ICP27, and ICP47 and it is the
promoters of these genes that are preferably used in directing the
expression of tet repressor (tetR). The expression of a gene needed
for virus replication is under the control of the tetO-containing
promoters and these essential genes may be immediate-early, early
or late genes, e.g., ICP4, ICP27, ICP8, UL9, and VP5.
[0023] ICP0 plays a major role in enhancing the reactivation of HSV
from latency and confers a significant growth advantage on the
virus at low multiplicities of infection. ICP4 is the major
transcriptional regulatory protein of HSV-1, which activates the
expression of viral early and late genes. ICP27 is essential for
productive viral infection and is required for efficient viral DNA
replication and the optimal expression of viral .gamma. genes and a
subset of viral .beta. genes. The function of ICP47 during HSV
infection appears to be to down-regulate the expression of the
major histocompatibility complex (MHC) class I on the surface of
infected cells.
[0024] Inclusion of Tet Repressor and Making of Virus
[0025] The recombinant DNA may also include at least one, and
preferably at least two, sequences coding for the tetracycline
repressor with expression of these sequences being under the
control of an immediate early promoter, preferably either an ICP0
or ICP4 promoter. The sequences for the HSV ICP0 and ICP4 promoters
and for the genes whose regulation they endogenously control are
well known in the art (Perry, et al., J. Gen. Virol. 67:2365-2380
(1986); McGeoch et al., J. Gen. Virol. 72:3057-3075 (1991); McGeoch
et al., Nucl. Acid Res. 14:1727-1745 (1986)) and procedures for
making viral vectors containing these elements have been previously
described (see US published application 2005-0266564). These
promoters are not only very active in promoting gene expression,
they are also specifically induced by VP16, a transactivator
released when HSV-1 infects a cell. Thus, transcription from ICP0
or ICP4 is particularly high when repressor is most needed to shut
down virus replication.
[0026] Once appropriate DNA constructs have been produced, they may
be incorporated into HSV-1 virus using methods that are well known
in the art. One appropriate procedure is described in US
2005-0266564 but other methods known in the art may also be
employed.
[0027] Treatment Methods
[0028] The oncolytic viruses described herein will be applied
directly to tumors. In cases where tumors are readily accessible,
e.g., tumors of the skin, mouth or which are accessible as the
result of surgery, virus can be applied topically. In other cases,
it can be administered by injection or infusion. The tetracycline
used prior to infection or at a time of infection can also be
administered in this way or it can be administered
systemically.
[0029] Prior to administration, the oncolytic viruses can be
suspended in any pharmaceutically acceptable solution including
sterile isotonic saline, water, phosphate buffered saline,
1,2-propylene glycol, polyglycols mixed with water, Ringer's
solution, etc. The exact number of viruses to be administered is
not crucial to the invention but should be an "effective amount,"
i.e., an amount sufficient to cause cell lysis extensive enough to
generate an immune response to released tumor antigens. Since virus
is replicated in the cells after infection, the number initially
administered will increase rapidly with time. Thus, widely
different amounts of initially administered virus can give the same
result by varying the time that they are allowed to replicate,
i.e., the time during which cells are exposed to tetracycline. In
general, it is expected that the number of viruses (PFU) initially
administered will be between 1.times.10.sup.6 and
1.times.10.sup.10.
[0030] Tetracycline will be administered either locally or
systemically to induce viral replication at a time of infection or
1-72 h prior to infection. The amount of tetracycline to be
administered will depend upon the route of delivery. In vitro, 1
ug/ml of tetracycline is more than sufficient to allow viral
replication in infected cells. Thus, when delivered locally, a
solution containing anywhere from 0.01 ug/ml to 100 ug/ml may be
administered. However, much higher doses of tetracycline (e.g.,
10-500 mg/ml) can be employed if desired. The total amount given
locally at a single time will depend on the size of the tumor or
tumors undergoing treatment but in general, it is expected that
between 0.5 and 200 ml of tetracycline solution would be used at a
time. When given systemically, higher doses of tetracycline will be
given but it is expected that the total amount needed will be
significantly less than that typically used to treat bacterial
infections (usually 1-2 grams per day in adults divided into 24
equal doses and, in children, 10-20 mg per pound of body weight per
day). It is expected that 100-200 mg per day should be effective in
most cases.
[0031] The effectiveness of a dosage, as well as the effectiveness
of the overall treatment can be assessed by monitoring tumor size
using standard imaging techniques over a period of days, weeks
and/or months. A shrinkage in the size or number of tumors is an
indication that the treatment has been successful. If this does not
occur or continue, then the treatment can be repeated as many times
as desired In addition,.treatment with virus can be combined with
any other therapy typically used for solid tumors, including
surgery, radiation therapy or chemotherapy. In addition, the
procedure can be combined with methods or compositions designed to
help induce an immune response.
EXAMPLES
[0032] The current example describes the creation and testing of an
oncolytic HSV-1 recombinant, KTR-27, which encodes two copies of
the tetr gene controlled by the HSV-1 immediate-early ICP0 promoter
in the ICP0 locus and the essential ICP27 gene under the control of
the tetO-bearing ICP27 promoter. To reduce the levels of ICP27
expression immediately after HSV infection, a riboenzyme sequence
was inserted at the 5' untranslated region of ICP27. Alternative
designs and methods will be readily apparent to those of skill in
the art.
[0033] Materials and Methods
[0034] Plasmids
[0035] p27BS is an ICP27-expressing plasmid with flanking sequences
406 bp upstream of the ICP27 open-reading frame to 461 bp
downstream of ICP27 poly A signal ptetO27, derived from p27BS,
contains two tandem tet operators (5'-TCCCTATC
AGTGATAGAGATCTCCCTATCAGTGATAGAGATCGCTGCA-3' (SEQ ID NO:2)) at 20 bp
downstream of the last nucleotide of the TATAAGG element of the
ICP27 promoter. Note that the tet operator consists of two op2
repressor binding sites joined by the 2 nucleotide sequence TC.
Insertion of a DNA sequence containing the mentioned tetO elements
(underlined text) at the Eag I site downstream of the ICP27 TATA
element in the described orientation generates a unique Eag I site
at the 5' end of the insert and a unique Pst I site at the 3' end
of the insert. Using lacZ as a reporter, we demonstrate that, like
the teto-bearing hCMV major immediate-early promoter, the
tetO-bearing ICP27 promoter can be sensitively regulated by
tetracycline in the presence of tetR.
[0036] ptetORZ27, derived from ptetO27, contains a DNA sequence
encoding a self-cleavage ribozyme N107 (Yen, et al., Nature
431:471476 (2004)), flanked with 5' Pst I and 3' Age I sequences,
into the 5' untranslated region of ICP27 coding sequence of ptetO27
at the Pst I and Age I sites. Presence of tetO and N107 sequences
in ptetORZ27 was verified by DNA sequencing. Western blot analysis
of cell extracts prepared from ptetO27 and ptetORZ27 transfected
U2OS cells demonstrate that the presence of N107 in ptetORZ27
significantly reduced levels of ICP27 expression in
ptetORZ27-transfected cells.
[0037] Cells and Viruses
[0038] Osteosarcoma line U2OS, African green monkey kidney (Vero)
cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) (Yao, et al., J.
Virol 69(10):6249-6258 (1995)). U2OS cells encode a cellular
function that can complement the function of the HSV-1
immediate-early regulatory protein ICP0. Human embryonic lung
cells, HEL638 were grown in DMEM in the presence of 10% FBS plus
1.times. non-essential amino acid (Sigma). R27 is an ICP27
expressing cell line derived from tetr-expressing U2OS cells
(Augustinova, et al., J. Virol. 78(11):5756-65 (2004)). R27 cells
were grown in DMEM growth medium in the presence of G418 (400
ug/ml) and hygromycin B (50 ug/ml).
[0039] Human Non-Small cell lung cancer cells H1299, human breast
cancer cells MCF7, human prostate cancer cells PC1435, and
pancreatic cancer cells Pan 1 were cultured in DMEM containing 10%
FBS. Mouse melanoma cells M3 were grown in Kaighan's modification
of Ham's F12 medium supplemented with 15% horse serum and 2.5% FBS.
7134 is an ICP0 null mutant, in which both copies of the ICP0
coding sequence are replaced by the Lac Z gene of Escherichia coli
(Cai, et al., J. Virol. 63(11):4579-(1989)). 7134 was propagated
and assayed in U2OS cells ((Yao, et al., J. Virol. 69(10):6249-6258
(1995)). KOR is an HSV-1 recombinant that was generated by
recombinational replacement of the Lac Z genes of 7134 with DNA
sequence-encoding tetR (Yao et al., Mol. Ther. 13(6):113341
(2006)). KOR27-lacZ was derived from KOR in which the ICP27 coding
sequence was replaced by the lacZ gene by homologous recombination.
KOR27-lacZ is replication-defective in U2OS cells, and was
propagated and assayed in R27 cells.
[0040] Construction of KTR27
[0041] KTR27 was constructed by replacing the Lac Z gene of
KOR27-lacZ with the ribozyme-containing ICP27 gene under the
control of the tetO-bearing ICP27 promoter. In brief, we
transfected U2OS cells with linearized ptetORZ27 DNA followed by
KOR27-lacZ super-infection. Progeny viruses were harvested at 24 h
post-infection and plaque assayed on U2OS cell monolayers in the
presence of tetracycline. Plaques, indicating replacement of the
lacZ gene by the ICP27 gene, were isolated and plaque purified four
times on U2OS cell monolayer in the presence of tetracycline. KTR27
is a viral recombinant that is replication-competent in the
presence of tetracycline and replication impaired in the absence of
tetracycline in both Vero and U2OS cells.
[0042] Regulation of De Novo Viral Replication in Vero Cells
[0043] Vero cells were seeded at 7.5.times.10.sup.5 cells per 60-mm
dish. At about 24 h post-seeding, cells in triplicate dishes were
infected with KTR-27 at a multiplicity of infection (MOI) of 1
PFU/cell in a volume of 0.5 ml. After 1.5 h incubation at
37.degree. C., virus-containing inoculation medium was removed, and
cells were washed twice with acid-glycine saline followed by two
more washes with DMEM. Infections were then carried out either in
the absence or presence of 2.5 .mu.g/ml of tetracycline. Infected
cells were harvested at 24, 48, and 72 h post-infection,
respectively. Viral titers were determined by standard plaque assay
on U2OS cell monolayer in the presence of tetracycline.
[0044] Regulation of Viral Replication in Human Cancer Cells
[0045] Viral replication was examined in the human cancer cells
PC1435 (prostate), H1299 (lung), MCF7 (breast), and Panc 1
(pancreas). PC 1435, HI 299, MCF7, and Panc 1 cells were seeded at
5.times.10.sup.5 cells per 60-mm dish. At 48 h post-seeding, cells
in triplicate dishes were infected with KTR-27 at an MOI of 1
PFU/cell in a volume of 0.5 ml. After 1.5 h incubation at
37.degree. C., inoculation medium was removed followed by two
washes with acid-glycine saline, then with DMEM. Infections were
carried out in the absence or presence of tetracycline at 2.5
.mu.g/ml. Infected cells were harvested at 72 h post-infection and
viral titers were determined by standard plaque assay on U2OS cell
monolayer in the presence of tetracycline.
[0046] Regulation o Viral Replication in Human Embryonic Lung
Cells
[0047] Tetracycline-dependent de novo viral synthesis of KTR-27 was
examined in primary human embryonic lung cells (HEL638). HEL cells
were seeded at 7.5.times.10.sup.5 cells per 60-mm dish. At about 48
h post-seeding, cells in triplicate dishes were infected with
KTR-27 at an MOI of 1 or 3 PFU/cell in the absence or presence of
2.5 .mu.g/ml of tetracycline as described above. Infected cells
were harvested at 48 h post-infection for cells that were infected
at an MOI of 3 PFU/cell and at 72 h post-infection for cells
infected at an MOI of 1 PFU/cell. Viral titers were determined on
U2OS cell monolayer in the presence of tetracycline.
[0048] Dose Dependent Regulation
[0049] Tetracycline dose-dependent regulation of de novo viral
synthesis of KTR27 in Vero cells. Vero cells were seeded at
5.times.10.sup.5 cells per 60-mm dish. At 48 h post-seeding, cells
in triplicate dishes were infected with KTR27 at an MOI of 1
PFU/cell in the absence or presence of tetracycline at
concentrations of 0.01, 0.05, 0.5 and 2.5 .mu.g/ml, respectively.
Infected cells were harvested at 72 h post-infection and viral
titers were determined on U2OS cell monolayer in the presence of
tetracycline.
[0050] Test of Effectiveness in an In Vivo Model of Non-Small Cell
Lung Cancer
[0051] Female BALB/c (nu/nu), 6 to 8-weeks-old, were implanted s.c.
with 7.5.times.10.sup.6 human lung cancer cells H1299 in a volume
of 100 ul at the left and right flanks. Once mice developed
palpable tumors, they were randomly divided intro three groups, and
one group was started on doxycycline-containing diet. Three days
later, each tumor with a maximum diameter of 4-5 mm was injected
with 50 .mu.l of DMEM or KTR27 at a dose of 1.times.10.sup.7 PFU.
The inoculated tumors received identical treatment 3 and/or 7 days
later. For the group of mice treated with doxycycline, doxycycline
special diet was discontinued 6 days after the first virus
inoculation. Tumor volumes were quantified every third day using
the formula V=(L.times.(W)2)/2 for 24 days.
[0052] Effectiveness in a Synergeneic M3 Melanoma Model in
Immuno-Competent Mice
[0053] Female DBA/2 mice, 6 to 8-weeks-old, were implanted s.c.
with 1.times.10.sup.5 syngeneic M3 Cloudman melanoma cells in a
volume of 100 .mu.l at both the left and right flanks. When
subcutaneous tumors reached a palpable size, mice were randomly
divided intro three groups of 6 mice each and one group was started
on Doxycycline special diet. Three days later (maximum diameter of
tumor size: 4-5 mm), mice were anesthetized and received a single
intratumoral inoculation of KTR-27 at 5.times.10.sup.7 PFU or DMEM
in a volume of 50 ul unilaterally. Mice were fed ad libitum either
a standard diet or a doxycycline-containing diet. Doxycyline
special diet was discontinued 5 days after virus inoculation and
henceforth all mice received a normal diet throughout the
experiment. Volumes of injected and contralateral tumors were
quantified every third day by a caliper using the formula
V=(L.times.(W).sup.2)/2 until 21 days after treatment. Mean tumor
volumes +SEM were calculated.
[0054] Results
[0055] Regulation of Viral Replication in Cultured Cells
[0056] A greater than 150,000-fold of tetracycline-dependent de
novo viral production was detected in African green monkey kidney
(Vero) cells. Infection of human tumor cell lines, such as breast,
lung, prostate, and pancreatic tumors, with KTR-27 in the presence
of tetracycline led to 1000- to 30,000-fold higher progeny viral
production than that produced in cells infected in the absence of
tetracycline. Similarly effective regulation of KTR-27 viral
production by tetracycline was also seen in proliferating primary
human embryonic lung cells. Moreover, it was found that the degree
of de novo replication of KTR-27 can be adjusted by tetracycline in
a dose-dependent fashion.
[0057] Safety of In Vivo Injection
[0058] In order to examine the safety of KTR27 in a potential
clinical application, the neurovirulence of KTR-27 after
intracerebral inoculation was tested. Female CD-1 mice, 6 to
8-weeks-old, were randomly assigned to three groups and
anesthetized with sodium pentobarbital. Groups of mice were fed
either a normal diet (n=7) or a diet containing doxycycline (n=8).
After 3 days of feeding, mice were inoculated with KTR27 through
intracerebral injection into the left frontal lobe of the brain in
a volume of 10 .mu.l (1.times.10.sup.7 PFU) at a depth of 4.5 mm.
As a negative control, a group of mice received intracerebral
injection of DMEM (n=4) in a similar fashion. Mice were examined
for signs and symptoms of illness for 35 days after KTR27
inoculation. It was found that all mice behaved normally and did
not display any symptoms of disease over this period.
[0059] Effectiveness in an In Vivo Model of Non-Small Cell Lung
Cancer
[0060] We examined the direct oncolytic effect of KTR27 in killing
human tumors in a pre-established mouse tumor xenograft model of
pre-established subcutaneous human non-small-cell lung cancer in
nude mice in either the presence or absence of tetracycline
treatment. It was found that intratumoral inoculation of KTR27
significantly inhibited the growth of established lung tumor
compared to DMEM treated control. After 24 days, tumor volume in
mice receiving a standard rodent diet and inoculated with KTR27 was
less than half of the tumor volume in the DMEM control mice. For
mice receiving the doxycycline diet, tumor volume after 24 days was
less than ten percent the tumor volume in the DMEM control mice. No
toxic effects or signs of herpetic infection were observed in
surrounding normal tissue following intratumoral injection of KTR27
in the tetracycline-fed mice.
[0061] Effectiveness in a Syngeneic M3 Melanoma Model in
Immuno-Competent Mice
[0062] The therapeutic treatment of established bilateral tumors in
DBA/2 mice was examined. It was found that intratumoral inoculation
of KTR-27 into pre-established melanoma lead to little measurable
growth in KTR-27 treated tumors, while tumors that received a DMEM
injection grew more than 100-fold in size. It was found that KTR-27
can also provide a potent anti-tumor effect on contralateral tumors
that received no viruses, suggesting that intratumoral inoculation
of KTR27 can elicit a strong effective anti-tumor specific
immunity. No difference in tumor killing efficiency was observed in
mice that received doxycycline diet or standard rodent diet, which
can likely be explained by the inefficient replication of KTR27 in
M3 cells in the presence of tetracycline and rapid cytotoxicity
induced by KTR27 infection of M3 cells in the absence of
tetracycline. We observed that infection of M3 cells with KTR27 at
an MOI of 3 PFU/cell led to about 100% cells detached from dishes
in both the presence and absence of tetracycline, and virus yield
at 24 h post-infection in the presence of tetracycline was only
0.21 PFU/cell. Again, no signs of herpetic infection were observed
on surrounding normal tissue following intratumoral injection of
KTR-27.
[0063] Conclusions
[0064] In summary, we have developed an oncolytic HSV recombinant
whose de novo viral replication can be sensitively regulated by
tetracycline in normal replicating cells and various tumor cells.
KTR-27 is highly effective and safe against pre-established
non-small cell lung cancer in nude mice and can prevent the growth
of pre-established M3 mouse melanoma in immuno-competent mice.
Intratumoral inoculation of KTR-27 can elicit a systemic immune
response that can effectively prevent the growth of a distant tumor
in immuno-competent mice.
[0065] All references cited herein are fully incorporated by
reference. Having now fully described the invention, it will be
understood by those of skill in the art that the invention may be
practiced within a wide and equivalent range of conditions,
parameters and the like, without affecting the spirit or scope of
the invention or any embodiment thereof.
Sequence CWU 1
1
2 1 19 DNA Escherichia coli 1 tccctatcag tgatagaga 19 2 48 DNA
Escherichia coli 2 tccctatcag tgatagagat ctccctatca gtgatagaga
tcgctgca 48
* * * * *