U.S. patent application number 10/375700 was filed with the patent office on 2004-01-08 for use of ribozymes in the detection of adventitious agents.
This patent application is currently assigned to Oncolytics Biotech Inc.. Invention is credited to Coffey, Matthew C..
Application Number | 20040005546 10/375700 |
Document ID | / |
Family ID | 30003777 |
Filed Date | 2004-01-08 |
United States Patent
Application |
20040005546 |
Kind Code |
A1 |
Coffey, Matthew C. |
January 8, 2004 |
Use of ribozymes in the detection of adventitious agents
Abstract
The present invention provides a method of detecting
adventitious agents in a composition comprising a microorganism by
using ribozyme-expressing indicator cells, as well as indicator
cells useful in such detection.
Inventors: |
Coffey, Matthew C.;
(Calgary, CA) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Assignee: |
Oncolytics Biotech Inc.
Calgary
CA
|
Family ID: |
30003777 |
Appl. No.: |
10/375700 |
Filed: |
February 26, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60360730 |
Feb 28, 2002 |
|
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60441760 |
Jan 23, 2003 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
C12N 15/1131 20130101;
C12Q 1/701 20130101; C12N 2310/121 20130101; C12Q 1/04 20130101;
C12Q 2521/337 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 001/70 |
Claims
We claim:
1. A method of detecting the presence of an adventitious agent in a
composition comprising a reovirus, comprising: (a) providing a
population of indicator cells that expresses a ribozyme, wherein
the ribozyme is capable of specifically cleaving the genome of the
reovirus; (b) contacting the indicator cells with the composition
under conditions that allow for cleavage of the genome of the
reovirus by the ribozyme; and (c) determining the effect of the
composition on the indicator cells, wherein any pathogenic effect
indicates the presence of an adventitious agent in the composition
in addition to the reovirus.
2. The method of claim 1 wherein the indicator cells express the
ribozyme from a gene that is integrated into the genome of the
cells.
3. The method of claim 1 wherein the ribozyme is capable of
specifically cleaving the s1 RNA of the reovirus.
4. The method of claim 1 wherein the ribozyme is Rz-553 or
Rz-984.
5. The method of claim 1 wherein the reovirus is a mammalian
reovirus.
6. The method of claim 1 wherein the reovirus is a human
reovirus.
7. The method of claim 1 wherein the reovirus is a Dearing strain
reovirus.
8. The method of claim 1 wherein the reovirus is a recombinant
reovirus.
9. The method of claim 8 wherein the recombinant reovirus is
generated by co-infection of mammalian cells with different
subtypes of reovirus.
10. The method of claim 8 wherein the recombinant reovirus is
naturally-occurring.
11. The method of claim 8 wherein the recombinant reovirus is
non-naturally-occurring.
12. The method of claim 8 wherein the recombinant reovirus is from
two or more strains of reovirus.
13. The method of claim 12 wherein the two or more strains of
reovirus are selected from the group consisting of strain Dearing,
strain Abney, strain Jones, and strain Lang.
14. The method of claim 8, wherein the recombinant reovirus results
from reassortment of reoviruses selected from the group consisting
of serotype 1 reovirus, serotype 2 reovirus and serotype 3
reovirus.
15. The method of claim 8 wherein the recombinant reovirus
comprises naturally-occurring variant coat protein coding
sequences.
16. The method of claim 8 wherein the recombinant reovirus
comprises mutated coat protein coding sequences.
17. A method of detecting the presence of an adventitious agent in
a composition comprising a virus wherein the virus contains an RNA
genome or utilizes an RNA transcript to replicate, comprising: (a)
providing a population of indicator cells that expresses a
ribozyme, wherein the ribozyme is capable of specifically cleaving
the RNA genome or RNA transcript of the virus to inhibit
replication or infection of the virus; (b) contacting the indicator
cells with the composition under conditions that allow for cleavage
of the RNA genome or RNA transcript of the virus by the ribozyme;
and (c) determining the effect of the composition on the indicator
cells, wherein any pathogenic effect indicates the presence of an
adventitious agent in the composition in addition to the virus.
18. The method of claim 17 wherein the indicator cells express the
ribozyme from a gene that is integrated into the genome of the
cells.
19. The method of claim 17 wherein the virus is a DNA virus.
20. The method of claim 17 wherein the virus is capable of
selectively infecting neoplastic cells.
21. The method of claim 17 wherein the virus is selected from the
group consisting of modified adenovirus, modified HSV, modified
vaccinia virus, modified parapoxvirus orf virus, modified influenza
virus, p53-expressing viruses, the ONYX-015 virus, the Delta24
virus, and vesicular stomatitis virus.
22. A method of validating a composition comprising a
microorganism, comprising: (a) providing a population of indicator
cells that expresses a ribozyme, wherein the ribozyme is capable of
specifically cleaving the RNA genome or RNA transcript of the
microorganism to inhibit replication or infection of the
microorganism; (b) contacting the indicator cells with the
composition under conditions that allow for cleavage of the RNA
genome or RNA transcript of the microorganism by the ribozyme; and
(c) determining the effect of the composition on the indicator
cells, wherein the absence of any pathogenic effect validates the
composition as having no detectable adventitious agent.
23. The method of claim 13 wherein the microorganism is a
virus.
24. The method of claim 13 wherein the microorganism is a virus
capable of selectively infecting neoplastic cells.
25. The method of claim 13 wherein the microorganism is a
reovirus.
26. The method of claim 13 wherein the microorganism is selected
from the group consisting of modified adenovirus, modified HSV,
modified vaccinia virus, modified parapoxvirus orf virus, modified
influenza virus, p53-expressing viruses, the ONYX-015 virus, the
Delta24 virus, and vesicular stomatitis virus.
27. An indicator cell useful for detecting an adventitious agent in
a composition of microorganism wherein the indicator cell
permanently expresses a ribozyme that is capable of cleaving the
genome or RNA transcript of the microorganism.
28. The indicator cell of claim 27 wherein a gene coding for the
ribozyme is integrated into the genome of the cell.
29. The indicator cell of claim 27 wherein the microorganism is a
virus.
30. The indicator cell of claim 27 wherein the microorganism is
reovirus.
31. The indicator cell of claim 27 wherein the cell is derived from
human embryonic kidney 293 cells.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Applications Serial No. 60/360,730, filed Feb. 28, 2002; and Serial
No. 60/441,760, filed Jan. 23, 2003. The entire disclosure of these
prior applications is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a method of detecting adventitious
agents in a composition comprising a microorganism, as well as
indicator cells useful in such detection.
REFERENCES
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[0022] Chandron and Nibert, "Protease cleavage of reovirus capsid
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[0027] Duncan et al., "Conformational and functional analysis of
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protein", Virology 182(2):810-9 (1991).
[0028] Farassati, F., et al., "Oncogenes in Ras signalling pathway
dictate host-cell permissiveness to herpes simplex virus 1", Nat.
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Lippincott-Raven (1996).
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the Rb Pathway Produces Anti-Glioma Effect in Vivo", Oncogene
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[0031] Heise, C. et al., "Replication-selective adenoviruses as
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[0050] All of the publications, patents and patent applications
cited above or elsewhere in this application are herein
incorporated by reference in their entirety to the same extent as
if the disclosure of each individual publication, patent
application or patent was specifically and individually indicated
to be incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0051] In the field of medical or biological sciences, there has
always been the demand to produce large quantities of
microorganisms such as viruses, bacteria, yeasts, other fungi,
parasites and prions. The resulting microorganisms can be used to
isolate and purify microbial proteins, generate vaccines, or
provide infectious microorganisms for laboratory or medical
studies. Recently, the new development of virus therapy has further
necessitated the need for efficient production of infectious
viruses.
[0052] Reovirus therapy (U.S. Pat. No. 6,136,307) is an example of
virus therapy. Reovirus is a double-stranded RNA virus with a
segmented genome. The receptor for the mammalian reovirus is a
ubiquitous molecule, therefore reovirus is capable of binding to a
multitude of cells. However, most cells are not susceptible to
reovirus infection and binding of reovirus to its cellular receptor
results in no viral replication or virus particle production. This
is probably the reason why reovirus is not known to be associated
with any particular disease.
[0053] It was discovered recently that cells transformed with the
ras oncogene become susceptible to reovirus infection, while their
untransformed counterparts are not (Strong et al., 1998). For
example, when reovirus-resistant NIH 3T3 cells were transformed
with activated Ras or Sos, a protein which activates Ras, reovirus
infection was enhanced. Similarly, mouse fibroblasts that are
resistant to reovirus infection became susceptible after
transfection with the EGF receptor gene or the v-erbB oncogene,
both of which activate the ras pathway (Strong et al., 1993; Strong
et al., 1996). Thus, reovirus can selectively infect and kill cells
with an activated ras pathway.
[0054] Ras pathway activation accounts for a large percentage of
mammalian tumors. Activating mutations of the ras gene itself occur
in about 30% of all human tumors (Bos, 1989), primarily in
pancreatic (90%), sporadic colorectal (50%) and lung (40%)
carcinomas, as well as myeloid leukemia (30%). Activation of
factors upstream or downstream of ras in the ras pathway is also
associated with tumors. For example, overexpression of
HER2/Neu/ErbB2 or the epidermal growth factor (EGF) receptor is
common in breast cancer (25-30%), and overexpression of
platelet-derived growth factor (PDGF) receptor or EGF receptor is
prevalent in gliomas and glioblastomas (40-50%). EGF receptor and
PDGF receptor are both known to activate ras upon binding to their
respective ligand, and v-erbB encodes a constitutively activated
receptor lacking the extracellular domain. Accordingly, reovirus
therapy, which is highly selective for ras-associated tumor cells,
can be used to treat a vast variety of tumors.
[0055] Reovirus can be produced and purified in bulk preparations
(U.S. Patent Application Publication Number 2002/0037576 A1). To
ensure that the reovirus preparation does not contain adventitious
agents which may result in undesired side effects, the preparation
is validated by using a susceptible cell line and anti-reovirus
antibodies. Thus, the cell line is exposed to either the virus
preparation alone, or the virus preparation that has been
neutralized by a reovirus-specific neutralizing antibody. If the
antibody neutralized virus preparation is still pathogenic to the
cell line, the virus preparation must contain an adventitious virus
or other organism. The preparation is then discarded or further
purified.
[0056] This validating protocol is expensive, as it requires large
amounts of high affinity, high titer antibodies to neutralize the
virus. This problem is further exacerbated now that we can produce
reovirus very efficiently, and the requirement for antibody is even
higher. Therefore, a more cost effective approach is desirable.
SUMMARY OF THE INVENTION
[0057] The present invention provides a method of validating
microbial preparations using a ribozyme that is specific for the
microorganism being prepared. For example, a plasmid encoding a
ribozyme that specifically cleaves the genome of reovirus can be
introduced into cells that are susceptible to reovirus infection.
The transfected cells, by expressing the ribozyme, are capable of
inactivating reovirus and thus will not be infected by the virus.
The ribozyme-expressing cells are then subjected to a reovirus
preparation, and any pathogenic effects caused by the reovirus
preparation will indicate that an adventitious agent is present in
the reovirus preparation. Conversely, the absence of any pathogenic
effect validates the preparation as having no detectable
adventitious agents.
[0058] This method is also applicable to viruses or other
microorganisms having a DNA genome. Since the DNA genome must be
transcribed into RNA for successful infection by the microorganism,
a ribozyme specific for the RNA transcript will inhibit infection
as well as replication of the microorganism. Again, if the
ribozyme-expressing cells show any pathogenic effects due to the
microbial preparation, an adventitious agent must be present in the
preparation. Similarly, this method can be used to validate
preparations of prions as well.
[0059] Accordingly, the present invention provides a method of
detecting the presence of an adventitious agent in a composition
comprising a reovirus, comprising:
[0060] (a) providing a population of indicator cells that expresses
a ribozyme, wherein the ribozyme is capable of specifically
cleaving the genome of the reovirus;
[0061] (b) contacting the indicator cells with the composition
under conditions that allow for cleavage of the genome of the
reovirus by the ribozyme; and
[0062] (c) determining the effect of the composition on the
indicator cells, wherein any pathogenic effect indicates the
presence of an adventitious agent in the composition in addition to
the reovirus.
[0063] The cells may express the ribozyme transiently or
permanently. Preferably, the cells comprise a ribozyme-encoding
gene that is integrated into the genome of the cells. The ribozyme
may cleave any part of the reovirus genome that is important for
replication or infection by reovirus. For example, the ribozyme may
cleave the S 1 segment of reovirus, such as Rz-553 or Rz-984.
[0064] This method can be used to detect adventitious agents in any
reovirus. The reovirus is preferably a mammalian reovirus, more
preferably a serotype 3 reovirus, and most preferably a Dearing
strain reovirus. The reovirus may be a recombinant reovirus. The
recombinant reovirus may be generated by co-infection of mammalian
cells with different subtypes of reovirus. The recombinant reovirus
may be naturally-occurring or non-naturally-occurring. The
recombinant reovirus may be from two or more strains of reovirus,
particularly two or more strains of reovirus selected from the
group consisting of strain Dearing, strain Abney, strain Jones, and
strain Lang. The recombinant reovirus may also result from
reassortment of reoviruses from different serotypes, such as
selected from the group consisting of serotype 1 reovirus, serotype
2 reovirus and serotype 3 reovirus. The recombinant reovirus may
comprise naturally-occurring variant coat protein coding sequences
or mutated coat protein coding sequences.
[0065] The present invention can be applied to compositions of any
microorganism, including any virus. Accordingly, the present
invention provides a method of detecting the presence of an
adventitious agent in a composition comprising a virus wherein the
virus contains an RNA genome or utilizes an RNA transcript to
replicate, comprising:
[0066] (a) providing a population of indicator cells that expresses
a ribozyme, wherein the ribozyme is capable of specifically
cleaving the RNA genome or RNA transcript of the virus;
[0067] (b) contacting the indicator cells with the composition
under conditions that allow for cleavage of the RNA genome or RNA
transcript of the virus by the ribozyme; and
[0068] (c) determining the effect of the composition on the
indicator cells, wherein any pathogenic effect indicates the
presence of an adventitious agent in the composition in addition to
the virus.
[0069] The cells may express the ribozyme transiently or
permanently. Preferably, the cells comprise a ribozyme-encoding
gene that is integrated into the genome of the cells.
[0070] The virus may be a DNA virus or RNA virus. Preferably, the
virus is an oncolytic virus, which is capable of selectively
replicating in neoplastic cells. Preferred oncolytic viruses
include, but are not limited to, modified adenovirus, modified HSV,
modified vaccinia virus, modified parapoxvirus orf virus, modified
influenza virus, p53-expressing viruses, the ONYX-015 virus, the
Delta24 virus, and vesicular stomatitis virus.
[0071] Another aspect of the present invention provides a method of
validating a composition comprising a microorganism,
comprising:
[0072] (a) providing a population of indicator cells that expresses
a ribozyme, wherein the ribozyme is capable of specifically
cleaving the RNA genome or RNA transcript of the microorganism to
inhibit replication or infection of the microorganism;
[0073] (b) contacting the indicator cells with the composition
under conditions that allow for cleavage of the RNA genome or RNA
transcript of the microorganism by the ribozyme; and
[0074] (c) determining the effect of the composition on the
indicator cells, wherein the absence of any pathogenic effect
validates the composition as having no detectable adventitious
agent.
[0075] The microorganism is preferably a virus, more preferably a
virus capable of selectively replicating in neoplastic cells, and
most preferably a reovirus.
[0076] Another aspect of the present invention provides an
indicator cell useful for detecting an adventitious agent in a
composition of microorganism wherein the indicator cell permanently
expresses a ribozyme that is capable of cleaving the genome or RNA
transcript of the microorganism. Preferably, a gene coding for the
ribozyme is integrated into the genome of the indicator cell. The
microorganism is preferably a virus and more preferably a reovirus.
The cell is preferably derived from the human embryonic kidney 293
cells (HEK 293 cells).
DETAILED DESCRIPTION OF THE INVENTION
[0077] The present invention provides a method of detecting
adventitious agents in a preparation of a microorganism by using a
ribozyme that is specific for the microorganism. Thus, a cell
population susceptible to the microorganism is transfected by an
expressing vector encoding the ribozyme and exposed to the
composition comprising the microorganism. Infection and/or
replication of the microorganism in the cells are inhibited by the
ribozyme. Therefore, the microorganism does not cause any
pathogenic effect on the cells. As a result, any sign of pathogenic
effect is indicative of the presence of an adventitious agent in
the microorganism preparation.
[0078] Prior to describing the invention in further detail, the
terms used in this application are defined as follows unless
otherwise indicated.
[0079] Definitions
[0080] An "adventitious agent" is an agent that is not intended to
be included in a composition. Preferably, an adventitious agent is
an infectious agent, namely an agent capable of infecting a
cell.
[0081] "Infecting" a cell refers to the act of entering into and
replicating in a cell.
[0082] A "ribozyme" is an RNA molecule or RNA derivative that is
capable of catalytically cleaving another RNA (the "target RNA").
The ribozymes of the present invention may have the characteristics
of naturally occurring ribozymes. For example, the ribozymes
isolated from Tetrahymena thermophila has an eight base pair active
site which hybridizes to a target RNA sequence before cleaving the
target (see, for example, Zaug and Cech, 1986). Free guanosine or
guanosine derivatives is required for this reaction, and a
guanosine is added to the 5' end of cleaved RNA. The ribozymes of
the present invention may also be synthetic ribozymes, such as
those described in U.S. Pat. No. 5,254,678. These synthetic
ribozymes have separate hybridizing regions and catalytic regions;
therefore, the hybridizing regions can be designed to recognize any
target sequences. In addition, the cleaved RNA is not modified by
these ribozymes.
[0083] An "indicator cell" is a cell that expresses a ribozyme,
which ribozyme is capable of cleaving and inactivating a
microorganism to be validated. The indicator cell, when not
expressing the ribozyme, is susceptible to infection of the
microorganism.
[0084] A "pathogenic effect" is an adverse effect on the growth or
maintenance of a cell, particularly the effects associated with
microbial infections. Pathogenic effects include, but are not
limited to, cytophathic effect (CPE), cell rupture, inhibition of
growth, inhibition of protein synthesis, and apoptosis.
[0085] "Cytopathic effect" is an observable change in cell
structure. Cytopathic effect may vary with cell types and cause of
death, and can be determined according to established knowledge in
the art. For example, some of the most common effects of viral
infection are morphological changes such as (a) cell rounding and
detachment from the substrate; (b) cell lysis; (c) syncytium
formation; and (d) inclusion body formation. Cytopathic effect
shown by reovirus-infected cells, for instance, is indicated by the
cells becoming swollen and granular in appearance and the cell
clumps breaking up.
[0086] "Validating" a composition, as used herein, means proving
that the composition does not contain an adventitious agent that is
detectable by the method employed.
[0087] "Reovirus" refers to any virus classified in the reovirus
genus, whether naturally occurring, modified or recombinant.
Reoviruses are viruses with a double-stranded, segmented RNA
genome. The virions measure 60-80 nm in diameter and possess two
concentric capsid shells, each of which is icosahedral. The genome
consists of double-stranded RNA in 10-12 discrete segments with a
total genome size of 16-27 kbp. The individual RNA segments vary in
size. Three distinct but related types of reovirus have been
recovered from many species. All three types share a common
complement-fixing antigen.
[0088] The human reovirus consists of three serotypes: type 1
(strain Lang or T1L), type 2 (strain Jones, T2J) and type 3 (strain
Dearing or strain Abney, T3D). The three serotypes are easily
identifiable on the basis of neutralization and
hemagglutinin-inhibition assays (see, for example, Fields, B. N. et
al., 1996).
[0089] The reovirus may be naturally occurring or modified. The
reovirus is "naturally-occurring" when it can be isolated from a
source in nature and has not been intentionally modified by humans
in the laboratory. For example, the reovirus can be from a "field
source", that is, from a human who has been infected with the
reovirus.
[0090] The reovirus may be modified but still capable of lytically
infecting a mammalian cell having an active ras pathway. The
reovirus may be chemically or biochemically pretreated (e.g., by
treatment with a protease, such as chymotrypsin or trypsin) prior
to administration to the proliferating cells. Pretreatment with a
protease removes the outer coat or capsid of the virus and may
increase the infectivity of the virus. The reovirus may be coated
in a liposome or micelle (Chandron and Nibert, 1998). For example,
the virion may be treated with chymotrypsin in the presence of
micelle forming concentrations of alkyl sulfate detergents to
generate a new infectious subvirion particle.
[0091] The reovirus may be a recombinant reovirus resulting from
the recombination/reassortment of genomic segments from two or more
genetically distinct reoviruses. Recombination/reassortment of
reovirus genomic segments may occur in nature following infection
of a host organism with at least two genetically distinct
reoviruses. Recombinant virions can also be generated in cell
culture, for example, by co-infection of permissive host cells with
genetically distinct reoviruses (Nibert et al. 1995).
[0092] Accordingly, the invention contemplates the recombinant
reovirus resulting from reassortment of genome segments from two or
more genetically distinct reoviruses, including but not limited to,
human reovirus, such as type 1 (e.g., strain Lang), type 2 (e.g.,
strain Jones), and type 3 (e.g., strain Dearing or strain Abney),
non-human mammalian reoviruses, or avian reovirus. The invention
further contemplates recombinant reoviruses resulting from
reassortment of genome segments from two or more genetically
distinct reoviruses wherein at least one parental virus is
genetically engineered, comprises one or more chemically
synthesized genomic segment, has been treated with chemical or
physical mutagens, or is itself the result of a recombination
event. The invention further contemplates the recombinant reovirus
that has undergone recombination in the presence of chemical
mutagens, including but not limited to dimethyl sulfate and
ethidium bromide, or physical mutagens, including but not limited
to ultraviolet light and other forms of radiation.
[0093] The invention further contemplates recombinant reoviruses
that comprise deletions or duplications in one or more genome
segments, that comprise additional genetic information as a result
of recombination with a host cell genome, or that comprise
synthetic genes.
[0094] The reovirus may be modified by incorporation of mutated
coat proteins, such as for example o1, into the virion outer
capsid. The proteins may be mutated by replacement, insertion or
deletion. Replacement includes the insertion of different amino
acids in place of the native amino acids. Insertions include the
insertion of additional amino acid residues into the protein at one
or more locations. Deletions include deletions of one or more amino
acid residues in the protein. Such mutations may be generated by
methods known in the art. For example, oligonucleotide site
directed mutagenesis of the gene encoding for one of the coat
proteins could result in the generation of the desired mutant coat
protein. Expression of the mutated protein in reovirus infected
mammalian cells in vitro such as COS1 cells will result in the
incorporation of the mutated protein into the reovirus virion
particle (Turner and Duncan, 1992; Duncan et al., 1991; Mah et al.,
1990).
[0095] The reovirus is preferably a reovirus modified to reduce or
eliminate an immune reaction to the reovirus. Such modified
reovirus are termed "immunoprotected reovirus". Such modifications
could include packaging of the reovirus in a liposome, a micelle or
other vehicle to mask the reovirus from the mammals immune system.
Alternatively, the outer capsid of the reovirus virion particle may
be removed since the proteins present in the outer capsid are the
major determinant of the host humoral and cellular responses.
[0096] A "neoplastic cell", also known as a "cell with a
proliferative disorder", refers to a cell which proliferates at an
abnormally high rate. A new growth comprising neoplastic cells is a
neoplasm, also known as a tumor. A neoplasm is an abnormal tissue
growth, generally forming a distinct mass, that grows by cellular
proliferation more rapidly than normal tissue growth. A neoplasm
may show partial or total lack of structural organization and
functional coordination with normal tissue. As used herein, a
neoplasm is intended to encompass hematopoietic tumors as well as
solid tumors.
[0097] A neoplasm may be benign (benign tumor) or malignant
(malignant tumor or cancer). Malignant tumors can be broadly
classified into three major types. Malignant neoplasms arising from
epithelial structures are called carcinomas, malignant neoplasms
that originate from connective tissues such as muscle, cartilage,
fat or bone are called sarcomas and malignant tumors affecting
hematopoietic structures (structures pertaining to the formation of
blood cells) including components of the immune system, are called
leukemias and lymphomas. Other neoplasms include, but are not
limited to neurofibromatosis.
[0098] "Ras-activated neoplastic cells" or "ras-mediated neoplastic
cells" refer to cells which proliferate at an abnormally high rate
due to, at least in part, activation of the ras pathway. The ras
pathway may be activated by way of ras gene structural mutation,
elevated level of ras gene expression, elevated stability of the
ras gene message, or any mutation or other mechanism which leads to
the activation of ras or a factor or factors downstream or upstream
from ras in the ras pathway, thereby increasing the ras pathway
activity. For example, activation of EGF receptor, PDGF receptor or
sos results in activation of the ras pathway. Ras-mediated
neoplastic cells include, but are not limited to, ras-mediated
cancer cells, which are cells proliferating in a malignant manner
due to activation of the ras pathway.
[0099] An "oncolytic" virus is a virus that is capable of
selectively infecting and killing neoplastic cells. In particular,
the oncolytic virus is capable of selectively replicating in and
lysing neoplastic cells. Examples of oncolytic viruses include, but
are not limited to, modified adenovirus, modified HSV, modified
vaccinia virus, modified parapoxvirus orf virus, modified influenza
virus, p53-expressing viruses, the ONYX-015 virus, the Delta24
virus, vesicular stomatitis virus, the herpes simplex virus 1
mutant which is defective in hrR3, Newcastle disease virus,
encephalitis virus, herpes zoster virus, hepatitis virus, influenza
virus, varicella virus, and measles virus.
[0100] The term "attenuated adenovirus" or "modified adenovirus"
means an adenovirus in which the gene product or products which
prevents the activation of PKR is lacking, inhibited or mutated
such that PKR activation is not blocked. Preferably, the VAI RNA's
are not transcribed. Such attenuated or modified adenovirus would
not be able to replicate in normal cells that do not have an
activated ras pathway, however, it would be able to infect and
replicate in cells having an activated ras pathway.
[0101] The term "attenuated HSV" or "modified HSV" means a herpes
simplex virus (HSV) in which the gene product or products that
prevents the activation of PKR is lacking, inhibited or mutated
such that PKR activation is not blocked. Preferably, the HSV gene
.sub..gamma.134.5 is not transcribed. Such attenuated or modified
HSV would not be able to replicate in normal cells that do not have
an activated ras pathway, however, it would be able to infect and
replicate in cells having an activated ras pathway.
[0102] "Parapoxvirus orf virus" is a poxvirus. It is a virus that
induces acute cutaneous lesions in different mammalian species,
including humans. Parapoxvirus orf virus naturally infects sheep,
goats and humans through broken or damaged skin, replicates in
regenerating epidermal cells and induces pustular leasions that
turn to scabs (Haig et al., 1998). The term "attenuated
parapoxvirus orf virus" or "modified parapoxvirus orf virus" means
a parapoxvirus orf virus in which the gene product or products
which prevents the activation of PKR is lacking, inhibited or
mutated such that PKR activation is not blocked. Preferably, the
gene OV20.0L is not transcribed. Such attenuated or modified
parapoxvirus orf virus would not be able to replicate in normal
cells that do not have an activated ras pathway, however, it would
be able to infect and replicate in cells having an activated ras
pathway.
[0103] The term "attenuated vaccinia virus" or "modified vaccinia
virus" means a vaccinia virus in which the gene product or products
which prevents the activation of PKR is lacking, inhibited or
mutated such that PKR activation is not blocked. Preferably, the
E3L gene and/or the K3L gene is not transcribed. Such attenuated or
modified vaccinia virus would not be able to replicate in normal
cells that do not have an activated ras pathway, however, it would
be able to infect and replicate in cells having an activated ras
pathway.
[0104] The term "attenuated influenza virus" or "modified influenza
virus" means an influenza virus in which the gene product or
products which prevents the activation of PKR is lacking, inhibited
or mutated such that PKR activation is not blocked. Preferably, the
NS1 gene is not transcribed. Such attenuated or modified influenza
virus would not be able to replicate in normal cells that do not
have an activated ras pathway, however, it would be able to infect
and replicate in cells having an activated ras pathway.
[0105] Methods
[0106] The present invention provides a method of detecting
adventitious agents in a composition comprising microorganisms. An
embodiment of the method is demonstrated in Example 1. Thus, to
determine if an adventitious agent is present in a reovirus
preparation, a plasmid that encodes a ribozyme, Rz-553 (Shahi et
al., 2001), is constructed. Rz-553 is a "hammerhead" ribozyme
consisting of a catalytic region flanked by two eight-nucleotide
sequences that hybridize to the S1 segment of the reovirus genome.
S1 codes for the protein o1, which binds to the reovirus receptor
on the cell. Therefore, cleavage of S1 RNA leads to reduced
infectivity of any resultant virus. Also constructed is a plasmid
encoding a mutant of Rz-553 in which a single nucleotide in the
catalytic region is mutated (G to U). The mutant is known to be
completely inactive.
[0107] The plasmids are introduced into COS-1 cells, and the
transfected cells are exposed to an aliquot of the reovirus
preparation being tested. Mock-infected cells are used as a
control. As expected, cells expressing the mutant ribozyme had
extensive CPE. However, cells expressing Rz-553 also show moderate
CPE when compared to the mock-infected cells. Therefore, the
reovirus preparation contains a non-reovirus agent that caused the
CPE on COS-1 cells.
[0108] Any ribozyme capable of cleaving the target RNA sequence of
a microorganism to inhibit replication or infection of the
microorganism is useful in the present invention. The target
sequence may be, for example, RNAs encoding structural proteins
(particularly outer coat proteins), proteins of the replication
machinery, or proteins important for cellular entry, such as the
receptor protein. Methods for constructing sequence-specific
ribozymes are well known in the art. For example, U.S. Pat. No.
5,254,678 describes the hammer-head ribozymes, which have a central
catalytic region flanked by two hybridization regions. Upon
hybridizing to the pre-selected target sequence through the
hybridization regions, the catalytic region forms a secondary
structure that facilitates cleavage, and cleaves the target
sequence. Although this patent describes ribozymes in which at
least one hybridization region has a minimum of nine hybridizing
nucleotides, such minimal length is not required in the present
invention. In the present invention, each hybridization region may
contain six, seven, eight or more hybridizing nucleotides.
[0109] U.S. Pat. No. 6,307,041 describes derivatives of hammerhead
ribozymes, including the circular, hairpin, circular/hairpin,
lariat and hairpin-lariat forms of hammerhead ribozymes. These
ribozymes have increased specific activity and different co-factor
requirement, and may also be used in the present invention. Other
examples include the hairpin ribozymes as described in, for
example, U.S. Pat. Nos. 5,631,359 and 5,631,115.
[0110] All these ribozymes contain at least one hybridization
region and a catalytic region. The hybridization region is designed
according to the target sequence. The catalytic region can be
derived from, for example, a hammerhead ribozyme (U.S. Pat. No.
5,254,678), a hairpin ribozyme (European Application No. 89 117
424), a hepatitis delta ribozyme (WO 91/04319 and WO 91/04324), an
RNase P ribozyme (U.S. Pat. No. 5,168,053), a group I intron (U.S.
Pat. No. 4,987,071), or a group II intron (Pyle, 1993).
[0111] It is also contemplated that more than one ribozyme can be
combined in the present invention. For example, multiple ribozymes
recognizing different regions of the same RNA can be combined.
Alternatively and preferably, ribozymes specific for different RNAs
of the same microorganism can be used together to increase the
efficiency of inactivation of the microorganism. When multiple
ribozymes are used, they can be encoded in the same expression
vector or separately encoded.
[0112] The present invention may be used to detect the presence of
adventitious agents in any microbial preparation. It should be
noted that while ribozymes cleave RNA only, the application of the
present invention is not limited to microorganisms with an RNA
genome. Microorganisms with a DNA genome necessarily need to
replicate through an RNA transcript, and/or synthesize proteins
through an RNA transcript, as part of the infection process. Even
prions need an RNA transcript to replicate and infect. Therefore,
replication and infection of any microorganism can be inhibited by
a ribozyme that specifically cleaves the microbial RNA
transcript/genome.
[0113] The microorganism of the present invention is preferably a
virus and more preferably an oncolytic virus. An oncolytic virus
can selectively infect and kill neoplastic cells, thus is useful in
virus therapy. In addition to reovirus, these viruses include, but
are not limited to, modified adenovirus, modified HSV, modified
vaccinia virus, modified parapoxvirus orf virus, modified influenza
virus, p53-expressing viruses, the ONYX-015 virus, the Delta24
virus, vesicular stomatitis virus, the herpes simplex virus 1
mutant which is defective in hrR3, Newcastle disease virus,
encephalitis virus, herpes zoster virus, hepatitis virus, influenza
virus, varicella virus, and measles virus.
[0114] Adenovirus, HSV, vaccinia virus, and parapoxvirus orf virus
are viruses which have developed a mechanism to overcome the double
stranded RNA kinase (PKR). Normally, when virus enters a cell, PKR
is activated and blocks protein synthesis, and the virus can not
replicate in this cell. However, adenovirus makes a large amount of
a small RNA, VA1 RNA. VA1 RNA has extensive secondary structures
and binds to PKR in competition with the double stranded RNA
(dsRNA) which normally activates PKR. Since it requires a minimum
length of dsRNA to activate PKR, VA1 RNA does not activate PKR.
Instead, it sequesters PKR by virtue of its large amount.
Consequently, protein synthesis is not blocked and adenovirus can
replicate in the cell. It should be noted, however, that although
the protein synthesis machinery is not blocked, host cell protein
synthesis is inhibited by the virus to facilitate viral protein
synthesis.
[0115] Vaccinia virus encodes two gene products, K3L and E3L, which
down-regulate PKR with different mechanisms. The K3L gene product
has limited homology with the N-terminal region of eIF-2.alpha.,
the natural substrate of PKR, and may act as a pseudosubstrate for
PKR. The E3L gene product is a dsRNA-binding protein and apparently
functions by sequestering activator dsRNAs.
[0116] Similarly, herpes simplex virus (HSV) gene .sub..gamma.134.5
encodes the gene product infected-cell protein 34.5 (ICP34.5) that
can prevent the antiviral effects exerted by PKR. The parapoxvirus
orf virus encodes the gene OV20.0L that is involved in blocking PKR
activity. Thus, these viruses can successfully infect cells without
being inhibited by PKR.
[0117] In the modified adenovirus, modified HSV, modified vaccinia
virus, or modified parapoxvirus orf virus, the viral anti-PKR
mechanism has been mutated or otherwise inactivated. Therefore,
these modified viruses are not capable of replicating in normal
cells which have normal PKR function. Ras-activated neoplastic
cells, however, are not subject to protein synthesis inhibition by
PKR, because ras inactivates PKR. These cells are therefore
susceptible to infection by the modified adenovirus, modified HSV,
modified vaccinia virus, or modified parapoxvirus orf virus.
[0118] The viruses can be modified or mutated according to the
known structure-function relationship of the viral PKR inhibitors.
For example, since the amino terminal region of E3 protein of the
vaccinia virus interacts with the carboxy-terminal region domain of
PKR, deletion or point mutation of this domain prevents anti-PKR
function (Chang et al., 1992, 1993, 1995; Sharp et al., 1998;
Romano et al., 1998). The K3L gene of vaccinia virus encodes pK3, a
pseudosubstrate of PKR. There is a loss-of-function mutation within
K3L. By either truncating or by placing point mutations within the
C-terminal portion of K3L protein, homologous to residues 79 to 83
in eIF-2.alpha. abolish PKR inhibitory activity
(Kawagishi-Kobayashi et al., 1997).
[0119] The modified HSV include, but are limited to, R3616 (both
copies of the .sub..gamma.134.5 gene have been deleted), R4009 (two
stop codons have been inserted in the .sub..gamma.134.5 gene), and
G207 (mutated in the ribonucleotide reductase and the
.sub..gamma.134.5 genes) (Andreansky et al., 1996). These modified
viruses have been used in brain tumor therapy, and it has been
recently shown that R3616 preferentially infects ras-activated
cells (Farassati et al., 2001).
[0120] Similarly, the delNS1 virus (Bergmann et al., 2001) is a
genetically engineered influenza A virus that can selectively
replicate in ras-activated neoplastic cells. The NS1 protein of
influenza virus is a virulence factor that overcomes the
PKR-mediated antiviral response by the host. NS1 is knocked out in
the delNS1 virus, which fails to infect normal cells, presumably
due to PKR-mediated inhibition, but replicates successfully in
ras-activated neoplastic cells. Therefore, a modified influenza
virus in which NS1 is modified or mutated, such as the delNS1
virus, is also useful in the present invention.
[0121] Other oncolytic viruses include the viruses which
selectively kill neoplastic cells by carrying a tumor suppressor
gene. For example, p53 is a cellular tumor suppressor which
inhibits uncontrolled proliferation of normal cells. However,
approximate half of all tumors have a functionally impaired p53 and
proliferate in an uncontrolled manner. Therefore, a virus which
expresses the wild type p53 gene can selectively kill the
neoplastic cells which become neoplastic due to inactivation of the
p53 gene product. Such a virus has been constructed and shown to
induce apoptosis in cancer cells that express mutant p53
(Blagosklonny et al., 1996).
[0122] Adenoviruses carrying the E2 gene have also been developed
(WO 02/095042). The E2 gene encodes the E2 protein, which inhibits
oncogene expression and induces cellular senescence. Therefore,
adenoviruses expressing the E2 gene can be used in gene therapy,
particularly for cancer patients in the terminal stage.
[0123] A similar approach involves viral inhibitors of tumor
suppressors. For example, certain adenovirus, SV40 and human
papilloma virus include proteins that inactivate p53, thereby
allowing their own replication (Nemunaitis 1999). For adenovirus
serotype 5, this protein is a 55 Kd protein encoded by the E1B
region. If the E1B region encoding this 55 kd protein is deleted,
as in the ONYX-015 virus (Bischoff et al, 1996; Heise et al., 2000;
WO 94/18992), the 55 kd p53 inhibitor is no longer present. As a
result, when ONYX-015 enters a normal cell, p53 functions to
suppress cell proliferation as well as viral replication, which
relies on the cellular proliferative machinery. Therefore, ONYX-015
does not replicate in normal cells. On the other hand, in
neoplastic cells with disrupted p53 function, ONYX-015 can
replicate and eventually cause the cell to die. Accordingly, this
virus can be used to selectively infect and kill p53-deficient
neoplastic cells. A person of ordinary skill in the art can also
mutate and disrupt the p53 inhibitor gene in adenovirus 5 or other
viruses according to established techniques.
[0124] Another example is the Delta24 virus which is a mutant
adenovirus carrying a 24 base pair deletion in the E1A region
(Fueyo et al., 2000). This region is responsible for binding to the
cellular tumor suppressor Rb and inhibiting Rb function, thereby
allowing the cellular proliferative machinery, and hence virus
replication, to proceed in an uncontrolled fashion. Delta24 has a
deletion in the Rb binding region and does not bind to Rb.
Therefore, replication of the mutant virus is inhibited by Rb in a
normal cell. However, if Rb is inactivated and the cell becomes
neoplastic, Delta24 is no longer inhibited. Instead, the mutant
virus replicates efficiently and lyses the Rb-deficient cell.
[0125] Yet other oncolytic viruses include the interferon sensitive
viruses. Vesicular stomatitis virus (VSV) selectively kills
neoplastic cells in the presence of interferon. Interferons are
circulating factors which bind to cell surface receptors which
ultimately lead to both an antiviral response and an induction of
growth inhibitory and/or apoptotic signals in the target cells.
Although interferons can theoretically be used to inhibit
proliferation of tumor cells, this attempt has not been very
successful because of tumor-specific mutations of members of the
interferon pathway.
[0126] However, by disrupting the interferon pathway to avoid
growth inhibition exerted by interferon, tumor cells may
simultaneously compromise their anti-viral response. Indeed, it has
been shown that VSV, an enveloped, negative-sense RNA virus rapidly
replicated in and killed a variety of human tumor cell lines in the
presence of interferon, while normal human primary cell cultures
were apparently protected by interferon. An intratumoral injection
of VSV also reduced tumor burden of nude mice bearing subcutaneous
human melanoma xenografts (Stojdl et al., 2000).
[0127] Other interferon-sensitive viruses (WO 99/18799), namely
viruses which do not replicate in a normal cell in the presence of
interferons, can be identified by growing a culture of normal
cells, contacting the culture with the virus of interest in the
presence of varying concentrations of interferons, then determining
the percentage of cell killing after a period of incubation.
Preferably, less than 20% normal cells is killed and more
preferably, less than 10% is killed.
[0128] It is also possible to take advantage of the fact that some
neoplastic cells express high levels of an enzyme and construct a
virus which is dependent on this enzyme. For example,
ribonucleotide reductase is abundant in liver metastases but scarce
in normal liver. Therefore, a herpes simplex virus 1 (HSV-1) mutant
which is defective in ribonucleotide reductase expression, hrR3,
was shown to replicate in colon carcinoma cells but not normal
liver cells (Yoon et al., 2000).
[0129] In addition to the viruses discussed above, a variety of
other viruses have been associated with tumor killing, although the
underlying mechanism is not always clear. Newcastle disease virus
(NDV) replicates preferentially in malignant cells, and the most
commonly used strain is 73-T (Reichard et al., 1992; Zorn et al,
1994; Bar-Eli et al, 1996). Clinical antitumor activities wherein
NDV reduced tumor burden after intratumor inoculation were also
observed in a variety of tumors, including cervical, colorectal,
pancreas, gastric, melanoma and renal cancer (WO 94/25627;
Nemunaitis, 1999).
[0130] Moreover, encephalitis virus was shown to have an oncolytic
effect in a mouse sarcoma tumor, but attenuation may be required to
reduce its infectivity in normal cells. Tumor regression have been
described in tumor patients infected with herpes zoster, hepatitis
virus, influenza, varicella, or measles virus (for a review, see
Nemunaitis, 1999). These viruses are thus also candidate oncolytic
viruses.
[0131] It is contemplated that for the modified oncolytic viruses,
in which a nucleic acid is modified to result in replication in
tumor cells, the ribozyme can be designed to cleave the RNA that
corresponds to the modified nucleic acid. The ribozyme can be
introduced into the susceptible tumor cells to prepare the
indicator cells. Since the ribozyme cleaves the modified RNA, which
is responsible for infectivity in the tumor cells, no infection
will occur unless there is an adventitious agent in the virus
preparation.
[0132] Indicator cells
[0133] The indicator cell should be prepared from a cell that is
susceptible to infection of the microorganism to be validated. A
construct encoding the appropriate ribozyme can be introduced into
the susceptible cell by any method known in the art, such as
calcium phosphate precipitation, liposome fusion, lipofectin.RTM.,
electroporation, and viral infection.
[0134] Both transient and stable introduction of the construct may
be useful in the present invention. However, the indicator cells
preferably express the ribozyme in a permanent manner. To this end,
an expression vector encoding the ribozyme may be integrated into
the genome of the cell or introduced as an episomal vector (such as
a bovine papilloma virus vector). Typically, the expression vector
contains a selectable marker, cells harboring the expression
vectors are selected using the selectable marker and maintained
under selection. Methods for constructing such vectors,
transfection and selection are well known in the art (see, for
example, Sambrook, 2001).
[0135] The following examples are offered to illustrate this
invention and are not to be construed in any way as limiting the
scope of the present invention.
EXAMPLES
[0136] In the examples below, the following abbreviations have the
following meanings. Abbreviations not defined have their generally
accepted meanings.
1 .degree. C. = degree Celsius hr = hour min = minute .mu.M =
micromolar mM = millimolar M = molar ml = milliliter .mu.l =
microliter mg = milligram .mu.g = microgram FBS = fetal bovine
serum PBS = phosphate buffered saline DMEM = Dulbecco's modified
Eagle's medium .alpha.-MEM = .alpha.-modified Eagle's medium
.beta.-ME = .beta.mercaptoethanol MOI = multiplicity of infection
PFU = plaque forming units PKR = double-stranded RNA activated
protein kinase EGF = epidermal growth factor PDGF = platelet
derived growth factor CPE = cytopathic effect
EXAMPLE 1
Testing a Reovirus Preparation
[0137] Reovirus is prepared and purified according to U.S. patent
application Ser. No. 09/920,012. Briefly, human embryonic kidney
293 SF (293/SF) cells are cultured in 15 L spinner flasks and
infected with reovirus at a multiplicity of infection of 0.5 when
cell density reach 10.sup.6/ml. The culture is incubated until cell
lysis begins, as evidenced by the culture media color change from
red to orange due to the presence of phenol red in the media, or by
a viable cell count under the microscope. At this point, the cells
are harvested by centrifugation, resuspended and disrupted by
freeze/thaw. The virus is then purified by a cesium chloride
gradient.
[0138] COS-1 cells are used as an indicator cell line. Thus, COS-1
cells are grown to 60% confluency in 6-well plates and transfected
with Rz-553 encoding DNA or control DNA (mutant S1-Rz-553) by using
Lipofectin (GIBCO/BRL) as described in Shahi et al. (2001). pSVLacZ
(Promega) is co-transfected in all the experiments to ensure
uniform transfection efficiency. 12 hours later, the cells are
washed once with fresh medium and infected with an aliquot of the
reovirus preparation described above at an m.o.i. of 1 PFU/cell.
Mock-infected cells are treated in the same manner without the
reovirus preparation. 8 hours after infection, the cells are
observed for cytopathic effects (CPE).
[0139] The mutant S1-Rz-553 transfected cells show extensive CPE as
expected, since the mutant ribozyme does not inactivate reovirus
and reovirus infection causes CPE on the infected cells. However,
the Rz-553 transfected cells also display detectable CPE as
compared to mock-infected cells. Since Rz-553 is known to
inactivate reovirus, these results indicate that this reovirus
preparation contains a non-reovirus agent that causes cytopathic
effects on COS-1 cells.
* * * * *