Note: Descriptions are shown in the official language in which they were submitted.
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ASSAY TO DETECT REPLICATION COMPETENT VIRUSES
RELATED APPLICATIONS
Benefit of priority is claimed under 35 U.S.C. ~119(e) to U.S. provisional
application Serial No. 60/459,375, filed March 27, 2003, entitled "ASSAY TO
DETECT REPLICATION COMPETENT ADENOVIRUSES," to Mark Bowe,
Russette M. Lyons, and Tracey Walker, and to U.S. provisional application
Serial No.
60/387,251, filed June 7, 2002 entitled "ASSAY TO DETECT REPLICATION
COMPETENT ADENOVIRUSES," to Mark Bowe, Russette M. Lyons, and Tracey
Walker. Both of these applications are incorporated herein by reference in
their
entireties.
FIELD OF THE INVENTION
The present invention generally relates to methods and assays useful for
detecting replication competent viruses in preparations of selectively
replicating
viruses.
BACI~.GROUND OF THE INVENTION
Viruses that replicate selectively in tumor cells are being developed as
anticancer agents ("oncolytic viruses"). Such oncolytic viruses amplify the
input virus
dose due to viral replication in the tumor, leading to spread of the virus
throughout the
tumor mass. In situ replication of viruses leads to cell lysis. This in situ
replication
may allow relatively Iow, non-toxic doses to be highly effective in the
selective
elimination of tumor cells.
These oncolytic viral viruses can be based on any virus that can be designed
to
preferentially replicate in tumors cells. Non-limiting examples of viruses
designed to
preferentially replicate in tumors cells include those derived from
adenoviruses (US
patent 5,998,205; WO 02/067861), herpesvirus (PCT Publication number WO
96139841), reovirus (Yin, H.S.,. J Virol Methods, 1997. 67:93- 101; Strong,
J.E. and
P.W. Lee,. J Virol, 1996. 70:612-616; Strong, J.E., et al, Virology, 1993.
197:405-
41I; Minuk, G.Y., et al., J Hepatol, 1987. 5:8-13; Rozee, K.R., et al., Appl
Environ
Microbiol, 1978. 35:297-300; Coffey, M.C., et al., Science, 1998. 282:1332-
1334;
Strong, 3.E., et al., Embo J, 1998 17:3351-1362; Mundschau, L.J. and D.V.
Faller, J
Biol Chem, 1992. 267:23092-23098), Paramyxovirus (e.g., Newcastle Disease,
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measles and mumps virus; PCT Publication number WO 99/18799), Togavirus (e.g.,
Sindbis PCT Publication number WO 99/18799), Parvoviruses, Poxvirus (e.g.,
Vaccinia), Picornavirus (e.g., Poliovirus), Orthonyxoviruses (e.g.,
influenza), and
rhabdovirus (PCT Publication number WO 01/19380).
One approach to achieving selectivity is to introduce loss-of function
mutations in viral genes that are essential for growth in non-target cells but
not in
tumor cells. This strategy is exemplified by the use of Add11520, which has a
deletion
in the Elb-55KD gene. (WO 94/189992; Ganly, et al., "A Phase I Study of Onyx-
015,
an ElB Attenuated Adenovirus, Administered Intratumorally to Patients with
Recurrent Head and Neck Cancer," Clinical Cancer Research, 6:798-806 (March
2000). In normal cells, the adenoviral Elb-55KD protein is needed to bind to
p53 to
prevent apoptosis. In p53-deficient tumor cells, Elb-55K binding to p53 is
unnecessary. Thus, deletion of Elb-55KD should theoretically restrict virus
replication to p53-deficient tumor cells.
Another approach is to use tissue-selective promoters to control the
expression
of one or more early viral genes required for replication (WO 96/17053; WO
99/25860; WO 02/067861; and WO 02/068627). Thus, in this approach the
adenoviruses will selectively replicate and Iyse tumor cells if a gene that is
essential
for replication is under the control of a promoter or other transcriptional
regulatory
element that is tissue-selective.
One concern when using an adenoviral virus preparation is the possibility of
contamination with a replication competent adenovirus (RCA) or even a non
adenoviral replication competent virus. When the adenovirus is a standard
replication
defective virus (e.g., ~El, DE2, and/or 4E4), detection of RCA can usually be
accomplished using either PCR methods or biological cell-based assays (Dion,
et al.,
"Supernatant rescue assay vs. polymerase chain reaction for detection of wild
type
adenovirus-contaminating recombinant adenovirus stocks," J. Vii°ol
Methods,
56(1):99-107 (January 1996); Murakami, et al., "A single short stretch of
homology
between adenoviral vector and packaging cell line can give rise to cytopathic
effect-
inducing, helper-dependent el-positive particles," Hum Gene Tlaer, 13(8):909-
920
(May 20, 2002)). The PCR methods utilize primers based on predicted
recombination
events that could lead to the production of RCA; for example, recombination
events
with the genome of the production cell line, wherein the complementing gene
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provided by the cell line is recombined into the adenovirus, thus creating an
RCA.
These PCR-based assays are limited to only detecting predicted recombination
events.
The cell-based assays, when used with a replication defective virus, comprise
infecting non-complementing cell lines with the virus preparations and
serially
passaging the virus on the non-complementing cell line (Dion, et al.,
sz~pj°a). The
desired replication defective virus should not replicate on the non-
complementing cell
line, whereas an RCA would amplify to a detectable level through serial
passaging.
Detection of the RCA can usually be determined through the appearance of
cytopathic
effect of the cells during the amplification steps. This biological cell-based
assay
should theoretically detect the presence of any recombinant or contaminant
RCAs,
whereas the PCR-based assays only detect predicted RCAs. The present invention
is
meant to include any selectively replicating virus and not just "oncolytic
viruses."
This would include any virus that preferentially replicates in a specific
tissue or cell
type.
It is therefore an object of the present invention to provide a method for
detecting replication competent viruses in preparations of selectively
replicating
viruses.
SUMMARY OF THE INVENTION
The present invention addresses the need for a biological assay to detect
replication competent virus (RCV) in replication selective virus (a.k.a.
selectively
replicating; e.g., oncolytic virus) preparations.
Selectively replicating viruses present a unique challenge with regard to
testing for the presence of RCV. Unlike replication defective viruses,
selectively
replicating adenoviruses are replication competent. The degree of replication
of
selectively replicating viruses will depend on both the design of the virus
and the cells
infected. It is unlikely that cell lines can be identified that will support
effcient
replication of a fully replication competent virus while being absolutely non-
permissive for replication of a selectively replicating virus such that
testing similar to
that currently used for replication defective viruses can be applied to these
novel
products. Rather, a biological testing strategy based on the relative
difference in
replication between a fully replication competent virus and the selectively
replicating
virus is presented herein.
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Accordingly, the present invention provides a method for detecting a non-
selective replication competent virus (RCV) in a preparation of selectively-
replicating
virus, comprising:
(a) passaging the virus preparation at least once on cells that differentially
amplify the selectively-replicating virus versus a replication competent
virus; and
(b) analyzing the virus preparation amplified in (a), thereby detecting the
presence of a replication competent virus, wherein analyzing the virus
preparation
comprises one or more of the following: detecting increased cytopathic effect;
detecting increased virus production; detecting increased potency to kill
normal cells;
detecting an altered restriction digest pattern; detecting an altered viral
genome
sequence; detecting anticipated recombinants with PCR amplification; and
detecting
acute hepatotoxicity in an animal administered the virus preparation.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic of PCR detection promoter recombinations.
Figure 2 shows the potency of Ar6pAE2fE3F as compared to wild-type Ad5
on HS-68 cells. The difference in potency (LD50 values) is approximately 30
fold.
Figure 3 shows the potency of Ar6pAE2fE3F (PO), Ar6pAE2fE3F (P4) and
Ad5 wild-type on MRC-5 cells. Potency of virus is measured by cell killing
with an
MTS readout. Potency increased more than 2 logs after amplification on MRC-5
cells. Potency is greater than wild-type Ad5 on MRC-5 cells.
Figure 4 shows the potency on MRC-5 cells of Ad5 wild-type after passaging
on MRC-5 cells. No significant change in Ad5 potency after amplification was
observed.
Figure 5 shows the structure/sequence some of the RCAs generated in the
assay from Ar6pAE2fE3F. Right end of rearranged vector contains the packaging
signal, suggesting recombination mechanisms of either intermolecular
recombination
or polymerase jumping. These RCAs had a deletion of some or all of E2F
promoter,
deletion of the p(A), duplication of part or all of E4 promoter and/or
duplication of the
packaging signal.
Figure 6 shows a comparison of cell killing (potency) by MTS.
Figure 7 shows a comparison of replication measured by hexon FACS assay in
human cells.
Figure 8 shows the potency on MRC-5 cells for Ar5pAE2fF and Ar6pAE2fF.
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Figure 9 shows potency on MRC-5 and human aortic endothelial cells for
Ar6pAE2fE3F and wild-type Ad5 virus.
Figure 10 shows schematic representations of selectively replicating,
oncolytic
adenoviral vectors Ar6pAE2fE3F, Ar6pAE2fF and Ar5pAE2fF. The Ar6pAE2fE3F
vector is based on the human adenovirus serotype 5 (Ad5) backbone, and is
approximately 34kb in size (Jakubczak et al. 2002). The native adenoviral ElA
promoter has been replaced with the human E2F-1 promoter (E2f P). The
packaging
signal (yr) has been moved to the right end of the genome, and an SV40
polyadenylation sequence (pA) has been inserted after the left inverted
terminal repeat
(ITR). The 14.7K gene of the E3 region has also been deleted. The Ar6pAE2fF
vector
is similar to Ar6pAE2fE3F, except that the entire adenoviral E3 region (E3)
has been
removed. The Ar5pAE2fF vector is similar to Ar6pAE2fF, except that the
packaging
signal remains at the left end of the genome. Schematics are not drawn to
scale. ElA,
adenoviral ElA coding sequence; E4, adenoviral E4 coding sequence.
Figure 11 shows a potency analysis by cytotoxicity assay. Wild-type Ad5 and
Ar6pAE2fE3F, either before (P0) or after (P4) four passages on MRC-5 cells,
are
diluted to the concentrations shown (ppc; particles per cell) and applied to
MRC-5
cells in 96-well plates. The cells are incubated for 10 or 7 days,
respectively, and then
analyzed for viability using an MTS assay. Measurements of viability at each
dilution
are expressed as the mean ~ SEM (N = 4). EC50's are calculated by regression
analysis of a four parameter logistic equation, using the pooled data for each
vector.
Figure 12 shows a potency analysis by cytotoxicity assay. Wild-type Ad5 and
Ar6pAE2fE3F, either before (PO) or after (P4) four passages on MRC-5 cells,
are
diluted to the concentrations shown (ppc; particles per cell) and applied to
primary
human aortic endothelial cells in 96-well plates. The cells are incubated for
10 or 7
days, respectively, and then analyzed for viability using an MTS assay.
Measurements
of viability at each dilution are expressed as the mean ~ SEM (N = 4). EC50's
are
calculated by regression analysis of a four parameter logistic equation, using
the
pooled data for each vector.
Figure 13 shows the structure of Ar6pAE2fE3F recombinants. Twenty-six
clonal isolates are prepared from the Ar6pAE2fE3F that has been passaged 4
times on
MRC-5. Each clone is sequenced. Seven individual recombinants were found (A-
G).
The labeled schematic indicates the potential components of the recombined
left end,
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while the bars below indicate the sequence of each recombinant analyzed. The
columns to the right indicate whether each recombinant has been isolated using
PER.C6 or MRC-5 cells or both. The forward (-~) and reverse (F-) arrows
indicate
positions of the forward and reverse primers, respectively, for a quantitative
PCR
assay that recognizes recombinants A and E (REC133 Q-PCR), while the
asterisked
bar (*-) indicates the position of the probe. The schematics are not drawn to
scale.
ITR, inverted terminal repeat; pA, poly A site; E2f P, human E2F-I promoter; E
1 A,
adenoviral ElA coding sequence; E4, adenoviral E4 coding sequence; E4 P,
adenoviral E4 promoter; 'f packaging signal; Rec P, recombinant promoter.
Figure 14 shows a potency analysis by cytotoxicity assay. The vector indicated
(Ar6pAE2fF or Ar5pAE2fF) is initially cloned and produced on the cell line
indicated
in parentheses (defined as P0), prior to being passaged sequentially four
times on
MRC-5 cells (P4). The vector samples are diluted to the concentrations shown
(ppc;
particles per cell) and applied to MRC-5 cells in 96-well plates. The cells
are
incubated for 10 days, and then analyzed for viability using an MTS assay.
Measurements of viability at each dilution are expressed as the mean ~ SE of
four
replicates. EC50's are calculated by regression analysis of a four parameter
logistic
equation, using the pooled data for each vector.
DETAILED DESCRIPTION OF THE INVENTION
Just as for any viral vector or other therapeutic, safety testing is a
component
of both early development of replication-selective viruses and to release of
individual
lots for clinical use. Viruses have a propensity to recombine, and so a key
concern
with viral vectors is the potential presence of recombinant replication
competent virus
{Lochmuller, Jani, et al. 1994} {Hehir, Armentano, et al. 1996} {Smith & Eck
1999} {Murakami, Pungor, et al.}. Because a typical replication defective
virus is
deleted for one or more crucial genes, PCR may be used to test for the
presence of
recombinants containing the deleted gene {Zhang, Koch, et al. 1995 } {Dion,
Fang, et
al. 1996} {Melcher, Murphy, et al. 1999}. A more general approach, and an
advisable
one due to its ability to detect the unexpected recombinant, is to use a
biological assay
to detect the replication competent recombinant {Dion, Fang, et al. 1996}
{Hehir,
Armentano, et al. 1996} {Zhu, Grace, et al. 1999} {Melcher, Murphy, et al.
1999} {Roitsch, Achstetter, et al.} {Murakami, Pungor, et al.}. A biological
assay for
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replication competent virus in a replication defective virus preparation
typically
involves inoculating a sufficient quantity of the virus into one or more cell
lines which
are non-permissive to the replication defective virus but permissive to the
parental
wild-type virus or replication competent recombinant. After passaging to
amplify any
replication competent virus that might be present, the cultures are examined
for
evidence of viral infection. Cytopathic effect (CPE) is a common read-out for
viral
vectors in this type of assay {Lelunberg, McCaman, et al. 2002}. These assays
are
endpoint assays with established limits of detection that have been determined
by
spike-in experiments using known quantities of replication competent positive
control
virus.
Selectively replicating viruses present a unique challenge with regard to
testing for the presence of replication competent virus. Unlike replication
defective
viruses, replication-selective viruses are to some degree replication
competent. For
example, the the tumor selective adenovirus Ar6pAE2fE3F, which is described in
detail in PCT/US02105280 (WO 02/068627) and PCT/US02/05300 (WO 02/067861),
is a genetically modified human adenovirus designed to preferentially
replicate in
tumor cells, resulting in tumor cell death due to oncolysis. Ar6pAE2fE3F
utilizes the
E2F-1 promoter to control the expression of Ela. Although the E2F-1 promoter
is
permanently de-repressed in Rb-defective tumor cells, it is transiently de-
repressed in
normal, proliferating cells. Therefore, a low level of replication of
replication-
selective viruses controlled by the E2F-1 promoter may be expected in normal
cells in
vitro.
The E2F-1 promoter takes advantage of multiple defects in the Rb pathway, a
common feature of many tumor cells, that results in hyper-phosphorylated Rb,
release
of Rb from E2F/DP-1 complexes and concomitant activation of promoters
containing
E2F binding domains, such as the E2F-1 promoter itself. Although the E2F-1
promoter is activated in Rb-defective tumor cells due to mutation or loss of
RB
pathway checkpoint proteins, RB is also transiently active during the cell
cycle of
normal cells. Therefore, a low level of replication of Ar6pAE2fE3F may be
expected
in normal cells in vitro.
In normal cells the RB pathway is tightly regulated. Without being bound by
theory, the inventors believe that the mechanism of action is as follows. In
GO and
Gl, the ternary complex of E2F-DP-1-pRB represses transcription from promoters
containing E2F binding sites, including the E2f 1 promoter in Ar6pAE2fE3F. pRB
is
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temporally regulated by phosphorylation during the cell cycle. Phosphorylation
reversibly inactivates pRB, resulting in transcriptional activation by E2F-DP-
1 dimers
and entry into S phase of the cell cycle. Dephosphorylation of pRB after
mitosis
causes re-entry into Gl phase. In tumor cells, any one or several of the cell
cycle
checkpoint proteins may be modified and lead to cell cycle deregulation and
unrestricted cell cycling. Loss of the pRB-E2F-DP-1 interaction, or abundance
of
"free E2F," results in derepression/activation of promoters having E2F sites.
Derepression of the E2f 1 promoter in Ar6pAE2fE3F leads to transcription of
Ela,
viral replication, and oncolysis.
As shown in the examples below, the RCVS that can be generated can be
unpredictable. The characteristics of each RCV and how they were generated
will
vary based on the specific design of the particular selctively-replicating
virus. It is the
entire viral genome that must be considered, as each element, whether
engineered into
the virus or an endogenous viral sequence, may influence the nature of any
recombinant that is formed. These data in the provided examples suggest that a
combination of factors contribute to the generation of RCV in a background of
replication-selective virus. The relative importance of each of these factors
may vary
with each virus construct. An interpretation concerning virus structure to be
made
from the data in the examples is that recombination events can be surprising,
thus
calling for the use of sequence-independent biological assays, as described
herein, for
more comprehensive detection of undesirable recombinants that have formed.
The degree of replication of replication-selective viruses will depend on both
the design of the virus and the cells that are infected. It is unlikely that
cell lines can
be identified that will support efficient replication of a non-selective
replication
competent virus while being absolutely non-permissive for replication of a
selectively
replicating viral vector. Therefore, biological assays that rely solely on the
presence
of, for example, CPE would be unlikely to be able to differentiate easily and
objectively between a replication-selective vector and a replication competent
virus.
Accordingly, in one aspect, the present invention provides a method for
detecting a non-selective replication competent virus in a viral preparation.
The
method includes passaging the viral preparation at least once on cells. The
cells can
be any cells that are selected prior to performing the assay and they are
selected for
their ability to differentially amplify the selectively-replicating virus
versus an RCV.
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RCV may be defined as a recombinant or contaminant virus which lacks the
selectivity of the replication-selective virus.
In one embodiment, the viral preparation is comprised of a virus from the
group consisting of adenovirus, herpesvirus, reovirus, paramyxovirus, sindbis,
parvoviruses, poxvirus, picornavirus, orthonyxoviruses, and rhabdovirus.
In a one embodiment, the viral preparation is an adenoviral preparation. When
an adenoviral preparation is employed, detected RCV would most likely be a
replication competent adenovirus (RCA), although the assay will detect a non-
adenovirus RCV.
In another embodiment, the virus is a tissue-specific replication-conditional
virus.
In another embodiment of the invention, the replication-conditional virus
comprises a heterologous tissue-specific transcriptional regulatory sequence
operatively linked to the coding region of a gene that is essential for
replication of the
virus, wherein the transcriptional regulatory sequence functions in the cell
so that
replication of the virus occurs in the cell.
In another embodiment of the invention the selectively-replicating virus
comprises a heterologous tissue-specific transcriptional regulatory sequence
operatively linked to the coding region of a gene that is essential for
replication of the
virus.
In another embodiment of the invention, the transcriptional regulatory
sequence is selected from the group consisting of an E2F-responsive promoter,
a
human telomerase reverse transcriptase (hTERT) promoter, an osteocalcin
promoter, a
carcinoembryonic antigen (CEA) promoter, a DF3 promoter, an oc-fetoprotein
promoter, an ErbB2 promoter, a surfactant promoter, a tyrosinase promoter, a
MUCl/DF3 promoter, a TK promoter, a p21 promoter, a cyclin promoter, an HKLK2
promoter, a uPA promoter, a HER-2neu promoter, a prostate specific antigen
(PSA)
promoter, a probasin promoter, a glandular kallikrein transcriptional
regulatory
element (U.S. Patent Application Publication No. US 2002/0136707 A1), a
uroplakin
II-derived transcriptional regulatory element (U.S. Patent Application
Publication No.
US 200210120117 Al), and a melanoma cell specific transcriptional regulatory
element (TRE), e.g. a tyrosinase, tyrosinase-related protein 2 (TRP2), MIA,
microphthalmia associated transcription factor (MITF); melanocyte-specific
gene l;
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melanocyte-specific tyrosinase-related protein-l, or MART-1 derived THE (all
disclosed in U.S. Patent Application Publication No. US 2003/0039633 Al).
In another embodiment of the invention, the transcriptional regulatory
sequence is an E2F~ 1 promoter.
In another embodiment of the invention, the virus further comprises a second
heterologous tissue-specific transcriptional regulatory sequence operatively
linked to
the coding region of a second gene that is essential for replication of the
virus,
wherein the second transcriptional regulatory sequence functions in the cell
so that
replication of the virus occurs in the cell. In various embodiments, the first
and second
heterologous tissue-specific transcriptional regulatory sequences are the same
or
different.
In another embodiment of the invention, the selectively-replicating virus
comprises first and second genes co-transcribed as a single mRNA, wherein the
first
and the second genes are under transcriptional control of a heterologous,
target cell-
specific TRE, wherein the second gene has a mutation in or deletion of its
endogenous
promoter and is under translational control of an internal ribosome entry site
(IRES)
(WO 01/73093).
In another embodiment of the invention, the virus further comprises a
heterologous coding sequence. In one embodiment, the product of the coding
sequence provides anti-tumor activity in the cells of a target tissue. In one
embodiment, the heterologous coding sequence encodes a GM-CSF.
In another embodiment of the invention, the virus is replication-competent in
a
neoplastic cell and overexpresses an adenovirus death protein.
In another embodiment of the invention, the tissue-specific replication-
conditional virus is an adenovirus.
In another embodiment of the invention, the tissue-specific replication-
conditional adenovirus is tumor-specific.
In a preferred embodiment the tissue-specific replication-conditional virus or
the tumor-specific replication-conditional virus is an adenovirus.
In another embodiment of the invention, the tumor-specific replication-
conditional adenovirus comprises a mutation or deletion in the Elb gene,
wherein the
encoded Elb protein lacks the capacity to bind p53.
In another embodiment of the invention, the tumor-specfic replication-
conditional adenovirus comprises a mutation or deletion in the Elb gene.
Preferably,
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the encoded Elb protein lacks the capacity to bind p53. For examples, See U.S.
Pat.
No. 5,677,178. This modification of the Elb region may be combined with
viruses
where all or a part of the E3 region is present.
In another embodiment of the invention, the tumor-specific replication-
conditional adenovirus comprises a mutation or deletion in the Ela gene. For
examples, see U.S. Pat. No. 5,677,178. Preferably the encoded Ela protein
lacks the
capacity to bind RB. This modification of the Ela region may be combined with
viruses where all or a part of the E3 region is present.
In another embodiment of the invention, the selectively-replicating virus
comprises (a) an E3 sequence; and (b) an ElA gene under transcriptional
control of a
probasin transcriptional regulatory element and an E1B gene under
transcriptional
control of a prostate specific antigen transcriptional regulatory element. (WO
00/39319). For example, the adenovirus vector comprises the entire E3 region,
the
probasin transcriptional regulatory element is the rat probasin promoter, and
the
prostate specific antigen transcriptional regulatory element is the human
prostate-
specific enhancer/promoter. (Yu et al., Cancer Research 59:4200-4203, 1999).
In another embodiment, the replication-conditional adenovirus comprises a
mutation or deletion in the E3 region. However, in an alternative embodiment,
all or a
part of the E3 region may be preserved or re-inserted. See, e.g., U.S. Pat.
No.
6,495,130. Presence of all or a part of the E3 region may decrease the
immunogenicity of the virus. It also may increase cytopathic effect in tumor
cells and
decrease toxicity to normal cells. Preferably, the virus expresses more than
half of the
E3 proteins.
In one embodiment of the invention, the transcriptional regulatory sequence is
a promoter or an enhancer.
In another embodiment of the invention, the adenovirus coding region that is
operatively linked to the transcriptional regulatory sequence is an El, E2, or
E4
coding region. In various embodiments, the El coding region is an Ela or Elb
coding
region. In other various embodiments, the E2 coding region is an E2a or E2b
coding
region.
In another embodiment of the invention, the adenovirus comprises an E2F-1
promoter operatively linked to the Ela coding region.
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In another embodiment of the invention, the adenovirus comprises an E2F-1
promoter operatively linked to the Ela coding region and an hTERT promoter
operatively linked to the E4 coding region.
In another embodiment of the invention, the adenovirus comprises an
adenovirus early gene essential for propagation under the transcriptional
control of a
prostate cell specific response element for transcription of prostate specific
antigen
comprising an enhancer and promoter specifc for a prostate cell.
In another embodiment of the invention, the adenovirus comprises at least one
of the genes E 1 A, E 1 B, or E4 under the transcriptional control of a
prostate cell
specific response element
In another embodiment of the invention, the adenovirus comprises a transgene
under the transcriptional control of a prostate cell specific response element
and
lacking at least one of ElA, E1B, or E4 as a functional gene.
In another embodiment of the invention, the adenovirus is replication
competent only in mammalian cells expressing prostate specific antigen.
In another embodiment of the invention, the adenovirus comprises an
adenovirus gene essential for propagation under transcriptional control of a
prostate
specific response element, said prostate cell specific response element
comprising an
enhancer specific for prostate specific antigen and a promoter.
In another embodiment of the invention, the adenovirus comprises E 1 A and
E1B, wherein ElA and E1B arc both under transcriptional control of separate a
fetoprotein transcription regulatory elements .
In another embodiment of the invention, the adenovirus comprises a first
adenovirus gene under transcriptional control of a first heterologous
transcriptional
regulatory element (TRE) and at least a second gene under transcriptional
control of a
second heterologous TRE, wherein the first heterologous TREs is cell-specific,
the
first heterologous THE is different from the second heterologous TRE, and the
heterologous TREs are functional in the same cell.
In another embodiment of the invention, the adenovirus comprises an
adenovirus gene under transcriptional control of an alpha-fetoprotein
transcription
regulatory element .
In another embodiment of the invention, the adenovirus comprises an
adenovirus gene under transcriptional control of a transcriptional regulatory
element
comprising a cell status- specific TRE.
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In another embodiment of the invention, the adenovirus comprises (a) an
adenovirus gene under transcriptional control of a target cell- specific
transcriptional
regulatory element; and (b) an E3 sequence.
In another embodiment of the invention, the adenovirus comprises g (a} an E3
sequence; and (b) a first adenovirus gene under transcriptional control of a
first target
cell-specific transcriptional response element and a second gene under
transcriptional
control of a second target cell-specific TRE.
In another embodiment of the invention, the adenovirus comprises a
therapeutic gene and a disease specific gene regulatory region operationally
linked to
at least one replication gene wherein the cancer cells activate the tumor
specific gene
regulatory region causing the adenoviral vector to replicate.
In another embodiment of the invention, the adenovirus possesses enhanced
infectivity towards a specific cell type due to a modification or replacement
of the
fiber of a wildtype adenovirus, said modifcation or replacement resulting in
enhanced
infectivity relative to said wildtype adenovirus, and wherein said infectivity-
enhanced
conditionally-replicative adenovirus has at least one conditionally regulated
early
gene, said early gene conditionally regulated such that replication of said
infectivity-
enhanced conditionally-replicative adenovirus is limited to said specific cell
type.
In another embodiment of the invention, the adenovirus is derived from a
human adenovirus 5 genome or a human adenovirus 35 genome.
In describing the present invention, the following terms are employed and are
intended to be def ned as indicated below.
As used herein, the terms "virus" and "viral particle" are used to include any
and all viruses that infect a human or anima cells.
As used herein, the terms "virus preparation" and "viral preparation" are used
interchangeably and refer to any batch of a virus. This includes batches
prepared for
research, preclinical or clinical use. A viral preparation may be purified,
concentrated, non-purified or partially purified.
As used herein, the terms "adenovirus" and "adenoviral particle" are used to
include any and all viruses that may be categorized as an adenovirus,
including any
adenovirus that infects a human or an animal, including all groups, subgroups,
and
serotypes. Adenovirus serotypes 1 through 47 are currently available from ATCC
and
the invention contemplates the production of any other serotype of adenovirus
available from any source. The adenoviruses that can be produced according to
the
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invention may be of human or non-human origin. For instance, an adenovirus can
be
of subgroup A (e.g., serotypes 12, 18, 31), subgroup B (e.g., serotypes 3, 7,
1 l, 14, 16,
21, 34, 35), subgroup C (e.g., serotypes 1, 2, S, 6}, subgroup D (e.g.,
serotypes 8, 9,
10, 13, 1 S, 17, 19, 20, 22-30, 32, 33, 36-39, 42-47), subgroup E (serotype
4),
subgroup F (serotype 40, 41), or any other adenoviral serotype. Preferred
serotypes
are adenovirus serotypes 2(Ad2), S (AdS) and 3S (Ad3S).
Thus, as used herein, "adenovirus" and "adenovirus particle" refer to the
virus
itself or derivatives thereof and cover all serotypes and subtypes and both
naturally
occurring and recombinant forms, except where indicated otherwise. Preferably,
such adenoviruses are ones that infect human cells. Such adenoviruses may be
wildtype or may be modified in various ways known in the art or as disclosed
herein.
Such modifications include modifications to the adenovirus genome that is
packaged
in the particle in order to make an infectious virus. Such modifications
include
deletions known in the art, such as deletions in one or more of the Ela, Elb,
E2a,
E2b, E3, or E4 coding regions. Such modifications also include deletions of
all of the
coding regions of the adenoviral genome. Such adenoviruses are known as
"gutless"
adenoviruses. The terms also include replication-conditional adenoviruses;
that is,
viruses that preferentially replicate in certain types of cells or tissues but
to a lesser
degree or not at all in other types. In a preferred embodiment of the
invention, the
adenoviral particles replicate in abnormally proliferating tissue, such as
solid tumors
and other neoplasms. These include the viruses disclosed in EPOS14603 U.S.
Patent
Nos. 5,677,178, 5,698,443, S,87I,726, 5,801,029, 5,998,205, 6,432,700,
U62S4862
and PCT publications WO 97/01358, WO 98139464, WO 98/39465, WO 00/15820,
WO 00/39319, WO 96/34969, WO 97/04805, WO 01/36650, , WO 00/67576, WO
01/23004, and WO 01/04282. Such viruses are sometimes referred to as
"cytolytic"
or "cytopathic" viruses (or vectors), and, if they have such an effect on
neoplastic
cells, are referred to as "oncolytic" viruses (or vectors).
The terms "adenovirus vector" and "adenoviral vector" are used
interchangeably and are well understood in the art to mean a polynucleotide
comprising all or a portion of an adenovirus genome. An adenoviral vector of
this
invention may be in any of several forms, including, but not limited to, naked
DNA,
DNA encapsulated in an adenovirus capsid, DNA packaged in another viral or
viral-
like form (such as herpes simplex, and AAV), DNA encapsulated in liposomes,
DNA
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complexed with polylysine, complexed with synthetic polycationic molecules,
conjugated with transferrin, complexed with compounds such as PEG to
immunologically "mask" the molecule and/or increase half life, or conjugated
to a
non-viral protein.
As used herein, the terms "vector," "polynucleotide vector," "polynucleotide
vector construct," "nucleic acid vector construct," and "vector construct" are
used
interchangeably herein to mean any nucleic acid constrict for gene transfer,
as
understood by those skilled in the art.
As used herein, the term "viral vector" is used according to its art-
recognized
meaning. It refers to a nucleic acid vector construct that includes at least
one element
of viral origin and may be packaged into a viral vector particle. The viral
vector
particles may be utilized for the purpose of transferring DNA, RNA or other
nucleic
acids into cells either i~a vitro or ih vivo. Viral vectors include, but are
not limited to,
retroviral vectors, vaccinia vectors, lentiviral vectors, herpes virus vectors
(e.g.,
HSV), baculoviral vectors, cytomegalovirus (CMV) vectors, papillomavirus
vectors,
simian virus (SV40) vectors, Sindbis virus vectors, semliki forest virus
vectors, phage
vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors. For
purposes
of the present invention, the viral vector is preferably an adenoviral vector.
The terms "virus," "viral particle," "vector particle," "viral vector
particle,"
and "virion" are used interchangeably and are to be understood broadly as
meaning
infectious viral particles that are formed when, e.g., a viral vector of the
invention is
transduced into an appropriate cell or cell line for the generation of
infectious
particles. Viral particles according to the invention may be utilized for the
purpose of
transferring nucleic acids into cells either in vitf°o or ih vivo. For
purposes of the
present invention, these terms preferably refer to adenoviruses, including
recombinant
adenoviruses formed when an adenoviral vector of the invention is encapsulated
in an
adenovirus capsid.
As used herein, the term "Replication Competent Virus" or "RCV" is
understood broadly as meaning any virus in a viral preparation that does not
retain the
same selective replication pattern as the original (parental) selectively-
replicating
virus. This includes a virus that does not retain the same selective
replication pattern
as the originally constructed selectively-replicating virus. For example, the
originally
constructed virus has mutated, so that viruses in the preparation are
different from the
parental virus and the different virus has a replication selectivity profile
different from
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the parental virus. An RCV may be of the same type as the parental virus (e.g.
both
the parental virus and the RCV are based on adenoviruses) or may be different
types
(e.g. the parental virus is based on a herpes virus and the RCV is an
adenovirus).
The term "Replication Competent Adenovirus" or "RCA" is an RCV that is
based on any adenovirus.
The terms "selectively-replicating virus", "replication-conditional"
"conditionally replicating", "replication-selective" and "replication
restricted viruses"
are use interchangeably and understood to mean any virus that has been
designed to
selectively replicate in a type or types of cells. It is not meant to
encompass standard
replication defective viruses that are not used as replicating viruses, except
for the
purpose of amplifying more virus. Replication defective viruses usually have a
certain function mutated or deleted, which is meant to eliminate or
substantially
reduce replication of the virus in the cells that are to be infected.
Although, deletion
of a viral function that may reduce viral replication in a cell, but is
intended to allow
replication in another cell type would be by definition and is still
considered herein as
selectively replicating. For example, the adenovirus Add11520 has the Elb
region
deleted, but is being used as a selectively-replicating virus for killing
tumor (cancer)
cells (WO 94/189992; Ganly, et al., "A Phase I Study of Onyx-015, an EIB
Attenuated Adenovirus, Administered Intratumorally to Patients with Recurrent
Head
and Neck Cancer," Clinical Cancer Research, 6:798-806 (March 2000)).
"Tumor-specific", "tumor-selective", "tumor-selective replication-conditional"
or "Tumor-specific replication-conditional" viruses are understood to be
selectively-
replicating viruses that preferentially replicate in at least one tumor cell
type as
compared to another cell type. One type of tumor specific virus uses tumor-
selective
regulatory elements to control the expression of early viral genes essential
for
replication. (See, e.g., WO 96/17053, WO 99/25860, WO 02/067861, WO 02/068627,
W09701358, WO9839464, W09839465, W00015820, W00039319, W09634969,
W00136650, W00067576, W00123004 and U.S. Patent Nos. 5,698,443, 5,871,726,
5,998,205, 6,432,700, and US6254862), Such oncolytic viruses will selectively
replicate and lyse tumor cells if the gene that is essential for replication
is under the
control of a promoter or other transcriptional regulatory element that is
tumor-
selective.
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Other tumor-specific replication-conditional adenoviruses have one or more
mutations or deletions in the El a and/or Elb genes such that the El a protein
lacks the
capacity to bind RB and such that the Elb protein lacks the capacity to bind
p53. (See,
e.g., U.S. Pat. No. 5,677,178.)
The term "gene essential for replication" refers to a nucleic acid sequence
whose transcription is required for a viral vector to replicate in a target
cell. For
example, in an adenoviral vector of the invention, a gene essential for
replication may
be one or more of the Ela, Elb, E2a, E2b, or E4 genes, such as the Ela gene or
the
E 1 a and E4 genes.
"Regulatory elements" are sequences involved in controlling the expression of
a nucleotide sequence. Regulatory elements include promoters, enhancers, and
termination signals. They also typically encompass sequences required for
proper
translation of the nucleotide sequence.
The term "promoter" refers to an untranslated DNA sequence usually located
upstream of the coding region that contains the binding site for RNA
polymerase II
and initiates transcription of the DNA. The promoter region may also include
other
elements that act as regulators of gene expression.
Promoters and other transcriptional regulatory elements that are tumor-
selective include, but are not limited to, an E2F responsive promoter such as
the E2F-
1 promoter, a human telomerase reverse transcriptase (hTERT) promoter, an
osteocalcin promoter, a carcinoembryonic antigen (CEA) promoter, a DF3
promoter,
an a.-fetoprotein promoter, an ErbB2 promoter, a surfactant promoter, a
tyrosinase
promoter, a MUCl/DF3 promoter, a TIC promoter, a p21 promoter, a cyclin
promoter,
an HKLK2 promoter, a uPA promoter, a HER-2neu promoter, a prostate specific
antigen (PSA) promoter, a probasin promoter, a glandular kallikrein
transcriptional
regulatory element (U.S. Patent Application Publication No. US 2002/0136707
A1), a
uroplakin II-derived transcriptional regulatory element (U.S. Patent
Application
Publication No. US 2002/0120117 Al), and a melanoma cell specific
transcriptional
regulatory element (TRE), e.g. a tyrosinase, tyrosinase-related protein 2
(TRP2),
MIA, microphthalmia associated transcription factor (MITF); melanocyte-
specific
gene l; melanocyte-specific tyrosinase-related protein-l, or MART-1 derived
THE
(all disclosed in U.S. Patent Application Publication No. US 2003/0039633 A1).
Also, see, e.g., WO 96117053, WO 98/13508, WO 98/14593, WO 99/25860, WO
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WO 03/104476 PCT/US03/18243
00/46355, WO 02/067861, WO 02/068627, and U.S. Pat. Nos. 5,648,478 and
6,495,130.
As used herein, the terms "cancer," "cancer cells," "neoplastic cells,"
"neoplasia," "tumor," and "tumor cells" (used interchangeably) refer to cells
that
exhibit relatively autonomous growth, so that they exhibit an aberrant growth
phenotype characterized by a significant loss of control of cell
proliferation.
Neoplastic cells can be malignant or benign.
The terms "coding sequence" and "coding region" refer to a nucleic acid
sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense
RNA or antisense RNA. Preferably the RNA is then translated in a cell to
produce a
protein.
The term "expression" refers to the transcription and/or translation of an
endogenous gene or a transgene in a cell. In the case of an antisense
construct,
expression may refer to the transcription of the antisense DNA only.
The term "gene" refers to a defined region that is located within a genome and
that, in addition to the aforementioned coding sequence, comprises other,
primarily
regulatory, nucleic acid sequences responsible for the control of expression,
i.e.,
transcription and translation of the coding portion. A gene may also comprise
other 5'
and 3' untranslated sequences and termination sequences. Depending on the
source of
the gene, further elements that may be present are, for example, introns.
The terms "heterologous" and "exogenous" as used herein with reference to
nucleic acid molecules such as promoters and gene coding sequences, refer to
sequences that originate from a source foreign to a particular virus or host
cell or, if
from the same source, are modified from their original form. Thus, a
heterologous
gene in a virus or cell includes a gene that is endogenous to the particular
virus or cell
but has been modified through, for example, codon optimization. The terms also
includes non-naturally occurring multiple copies of a naturally occurring
nucleic acid
sequence. Thus, the terms refer to a nucleic acid segment that is foreign or
heterologous to the virus or cell, or homologous to the virus or cell but in a
position
within the host viral or cellular genome in which it is not ordinarily found.
The term "homologous" as used herein with reference to a nucleic acid
molecule refers to a nucleic acid sequence naturally associated with a host
virus or
cell.
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The term "native" refers to a gene that is present in the genome of wildtype
virus or cell.
The term "naturally occurring" or "wildtype" is used to describe an object
that
can be found in nature as distinct from being artificially produced by man.
For
example, a protein or nucleotide sequence present in an organism (including a
virus),
which can be isolated from a source in nature and which has not been
intentionally
modified by man in the laboratory, is naturally occurring.
The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and
polymers thereof ("polynucleotides") in either single- or double-stranded
form.
Unless specifically limited, the term encompasses nucleic acids containing
known
analogues of natural nucleotides that have similar binding properties as the
reference
nucleic acid and are metabolized in a manner similar to naturally occurnng
nucleotides. Unless otherwise indicated, a particular nucleic acid
molecule/polynucleotide also implicitly encompasses conservatively modified
variants thereof (e.g. degenerate codon substitutions) and complementary
sequences
and as well as the sequence explicitly indicated. Specifically, degenerate
codon
substitutions may be achieved by generating sequences in which the third
position of
one or more selected (or all) codons is substituted with mixed-base and/or
deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991);
Ohtsuka et
al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes
8: 91-98
(1994)). In the context of the present invention, the nucleic acid
molecule/polynucleotide is preferably a segment of DNA. Nucleotides are
indicated
by their bases by the following standard abbreviations: adenine (A), cytosine
(C),
thymine (T), and guanine (G).
A nucleic acid sequence is "operatively linked" when it is placed into a
functional relationship with another nucleic acid sequence. For example, a
promoter
or regulatory DNA sequence is said to be "operatively linked" to a DNA
sequence
that codes for an RNA or a protein if the two sequences are operatively
linked, or
situated such that the promoter or regulatory DNA sequence affects the
expression
level of the coding or structural DNA sequence. Operatively linked DNA
sequences
are typically, but not necessarily, contiguous.
The teen "ORF" means Open Reading Frame.
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The term "Ar5" refers to the virus Ar5pAE2fF which is shown in Figure 10.
Ar5pAE2fF is the same as Ar6pAE2fF except that the packaging sequence is
located
on the "left" end of the viral genome.
The virus Ar6pAE2fF is described in PCT/US02/05300 (W002/067861)
"OAV" and "Ar6pAE2fE3F" are used interchangeably and are described in
detail in PCT/US02/05280 (WO 02/068627) and PCT/US02/05300 (WO 02/067861),
and is a genetically modified human adenovirus designed to preferentially
replicate in
tumor cells, resulting in tumor cell death due to oncolysis. Ar6pAE2fE3F
utilizes the
E2F-1 promoter to control the expression of Ela. Selective activation of the
E2F-1
promoter in tumor cells is based on the repression of E2F-1 promoter
activation in
normal cells and both the derepression and activation of the E2F-1 promoter in
tumor
cells. Several reports indicate that defects in the Rb-pathway in tumor cells
lead to
elimination of cellular factors necessary to repress transcription from the
E2F-1
promoter. These defects also lead to an accumulation of "free" E2F-1 that in
turn
activates the E2F-1 promoter. The majority of cancers are expected to have
defects in
the Rb-pathway either through mutation in the Rb gene itself, or in factors up
stream
or down stream of Rb. Thus, Ar6pAE2fE3F is expected to preferentially
replicate and
kill tumor cells that have defects in the Rb-pathway as compared to cells with
an
intact Rb-pathway, i. e. normal cells.
Arl7pAE2fFTrtex, which is described in detail in PCT/US02/05300 (WO
02/067861), is a tumor-selective oncolytic adenovirus designed to be delivered
systemically for the treatment of a broad range of cancer indications. The
replication
of Arl7pAE2fFTrtex is engineered to be dependent on the presence of the two
most
common alterations in human cancer, namely defects in the Rb-pathway (~85% of
all
cancers) and overexpression of telomerase (~85% of all cancers). As with
Ar6pAE2fE3F, Arl7pAE2fFTrtex utilizes the E2F-1 promoter to control expression
of the adenoviral Ela gene. To increase tumor selectivity appropriate for
systemic
delivery, the adenoviral E4 gene in Arl7pAE2fFTrtex is controlled by the hTERT
(human telomerase reverse transcriptase) promoter. Arl7pAE2fFTrtex is expected
to
replicate in the majority of cancer cells, leading to tumor selective-
expression of toxic
viral proteins, cytolysis, and enhancement of sensitivity to chemotherapy,
cytokines
and cytotoxic T lymphocytes.
In another embodiment, an adenovirus produced according to the invention
further comprises at least one heterologous coding sequence, such as a
therapeutic
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gene coding sequence. The therapeutic gene coding sequence, for example in the
form
of cDNA, can be inserted in any position that does not adversely affect the
infectivity
or replication of the virus. Preferably, it is inserted in the E3 region in
place of at
least one of the polynucleotide sequences coding for the E3 proteins. For
example, the
therapeutic gene coding sequence may be inserted in place of the l9kD or 14.7
kD E3
gene.
A therapeutic gene coding sequence can be one that exerts its effect at the
level of RNA or protein. Therapeutic gene coding sequences that may be
introduced
into the adenovirus include a factor capable of initiating apoptosis,
antisense or
ribozymes, which among other capabilities may be directed to mRNAs encoding
proteins essential for proliferation, such as structural proteins,
transcription factors,
polymerases, etc., sequences encoding cytotoxic proteins, sequences that
encode an
engineered cytoplasmic variant of a nuclease (e.g. RNase A) or protease (e.g.
trypsin,
papain, proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and the
like.
Other therapeutic genes of interest include, but are not limited to,
immunostimulatory, anti-angiogenic, and suicide genes. Immunostimulatory genes
include, but are not limited to, genes that encode cytokines (GM-CSF, ILl,
IL2, IL4,
ILS, IFNa, IFNy, TNFa, IL12, IL18, and flt3), proteins that stimulate
interactions
with immune cells (B7, CD28, MHC class I, MHC class II, TAPs), tumor-
associated
antigens (immunogenic sequences from MART-l, gp100(pmel-17), tyrosinase,
tyrosinase-related protein 1, tyrosinase-related protein 2, melanocyte-
stimulating
hormone receptor, MAGEl, MAGE2, MAGE3, MAGE12, BAGE, GAGE, NY-ESO-
l, (3-catenin, MUM-1, CDK-4, caspase 8, KIA 0205, HLA-A2R1701, a-fetoprotein,
telomerase catalytic protein, G-250, MUC-1, carcinoembryonic protein, p53,
Her2/neu, triosephosphate isomerase, CDC-27, LDLR-FUT, telomerase reverse
transcriptase, and PSMA), cDNAs of antibodies that block inhibitory signals
(CTLA4
blockade), chemokines (MIPIa, MIP3a, CCR7 ligand, and calreticulin), and other
proteins. Anti-angiogenic genes include, but are not limited to, genes that
encode
METH-1, METH -2, TrpRS fragments, proliferin-related protein, prolactin
fragment,
PEDF, vasostatin, various fragments of extracellular matrix proteins and
growth
factor/cytokine inhibitors. Various fragments of extracellular matrix proteins
include,
but are not limited to, angiostatin, endostatin, kininostatin, fibrinogen-E
fragment,
thrombospondin, tumstatin, canstatin, and restin. Growth factor/cytokine
inhibitors
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include, but are not limited to, VEGF/VEGFR antagonist, sFlt-l, sFlk, sNRPl,
angiopoietin/tie antagonist, sTie-2, chemokines (IP-10, PF-4, Gro-beta, IFN-
gamma
(Mig), IFNa., FGF/FGFR antagonist (sFGFR), Ephrin/Eph antagonist (sEphB4 and
sephrinB2), PDGF, TGF(3 and IGF-1.
A "suicide gene" encodes a protein that itself can lead to cell death, as with
expression of diphtheria toxin A, or the expression of the protein can render
cells
selectively sensitive to certain drugs, e.g., expression of the Herpes simplex
thymidine
kinase gene (HSV-TK) renders cells sensitive to antiviral compounds, such as
acyclovir, gancyclovir and FIAU (1-(2-deoxy-2-fluoro-(3-D-arabinofuranosil)-5-
iodouracil). Other suicide genes include, but are not limited to, genes that
encode
carboxypeptidase G2 (CPG2), carboxylesterase (CA), cytosine deaminase (CD),
cytochrome P450 (cyt-450), deoxycytidine kinase (dCK), nitroreductase (NR),
purine
nucleoside phosphorylase (PNP), thymidine phosphorylase (TP), varicella zoster
virus
thymidine kinase (VZV-TK), and xanthine-guanine phosphoribosyl transferase
(XGPRT). Alternatively, the therapeutic gene can exert its effect at the level
of RNA,
for instance, by encoding an antisense message or ribozyme, a protein that
affects
splicing or 3' processing (e.g., polyadenylation), or a protein that affects
the level of
expression of another gene within the cell, e.g. by mediating an altered rate
of mRNA
accumulation, an alteration of mRNA transport, and/or a change in post-
transcriptional regulation. The addition of a therapeutic gene to the virus
results in a
virus with an additional antitumor mechanism of action. Thus, a single entity
(i.e., the
virus carrying a therapeutic transgene) is capable of inducing multiple
antitumor
mechanisms.
Alternately, the therapeutic gene coding sequence encodes thymidine kinase,
Nos, Fast, sFasR (soluble Fas receptor), or granulocyte macrophage colony
stimulating factor (GM-CSF; U.S. Patent No. 5,908,763).
The therapeutic gene coding sequence is under the control of a suitable
promoter. Suitable promoters that may be employed include, but are not limited
to,
adenoviral promoters, such as the adenoviral major late promoter and/or the E3
promoter; or heterologous promoters, such as the cytomegalovirus (CMV)
promoter;
the Rous Sarcoma Virus (RSV) promoter; inducible promoters, such as the MMT
promoter, the metallothionein promoter; heat shock promoters; the albumin
promoter;
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the ApoAI promoter; and a tissue-selective promoter such as those disclosed in
PCT/EP98/07380 (WO 99/25860).
The invention further contemplates combinations of two or more transgenes
with synergistic, complementary and/or nonoverlapping toxicities and methods
of
action. The resulting oncolytic adenovirus would retain the viral oncolytic
functions
and would, for example, additionally have the ability to induce immune and
anti-
angiogenic responses, etc.
In another embodiment, adenoviruses produced according to the invention
further comprise a targeting ligand included in a capsid protein of the
particle. In one
embodiment, the capsid protein is a fiber protein and the ligand is in the HI
loop of
the fiber protein. The adenoviral vector particle may also include other
mutations to
the fiber protein. Examples of these mutations include, but are not limited to
those
described in US application no. 101351,890, WO 98/07877, WO 01/92299, and US
Patent Nos. 5,962,311, 6,153,435, and 6,455,314. These include, but are not
limited
to mutations that decrease binding of the viral vector particle to a
particular cell type
or more than one cell type, enhance the binding of the viral vector particle
to a
particular cell type or more than one cell type and/or reduce the immune
response to
the adenoviral vector particle in an animal. In addition, the adenoviral
vector particles
produced according to the invention may also contain mutations to other viral
capsid
proteins. Examples of these mutations include, but are not limited to those
described
in US Patent Nos. 5,731,190, 6,127,525, and 5,922,315. Other adenoviruses that
can
be produeed according to the invention are described in U.S. Patent,Nos.
6,057,155,
5,543,328 and 5,756,086.
Viruses are made by transferring vectors into packaging cells by techniques
known to those skilled in the art. Packaging cells may complement functions
deleted
from the wild-type virus genome. The production of such particles requires
that the
vector be replicated and that those proteins necessary for assembling an
infectious
virus be produced. The packaging cells are cultured under conditions that
permit the
production of the desired viral particle. The particles are recovered by
standard
techniques.
Selectively replicating viral vectors are promising candidates for our arsenal
to
treat cancer including metastatic disease. A biological assay is described
herein for
unintended recombinant adenoviruses which are no longer replication-selective,
but
rather replicate non-selectively much like parental wild-type viruses. This
biological
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assay or a variation thereof is broadly applicable to selectively replicating
viral
products. A biological assay has a distinct advantage in testing for
replication
competent virus (as is well illustrated in the examples below by the discovery
of the
REC133 RCV and related RCVs in the Ar6pAE2fE3F vector particle preparation).
The biological assays of the present invention are capable of detecting
recombinants
due not only to predicted but also due to unpredicted recombination events. As
described in detail in the examples, the assay detected RCV's that are not
predicted
(e.g. REC133) and would not have been detected by PCR assays that would have
been
developed in the anticipation of predicted recombinants (e.g. between the
vector and
packaging sequences).
In one embodiment the biological assay for RCV can be considered to have
two phases. The first phase is the relative amplification of the RCV compared
to the
intended selectively-replication virus by passaging the viral sample on the
cell line
(e.g. MRC-5 in the case of Ar6pAE2fE3F as described in the examples). The
second
phase includes all appropriate analyses of the amplified material in order to
detect and
confirm the presence of an RCV Increased CPE, increased virus production,
increased potency to kill normal cells, altered restriction digest patterns or
other
means of characterization of the virus may be used in any combination as
detection
methods to indicate the presence or absence of an RCV that is relatively
amplified in
the cells selected for the assay. In the case of the assay developed for
Ar6pAE2fE3F,
the MRC-5 cells do allow some replication of the Ar6pAE2fE3F virus and some
CPE
is observed at the time of optimum harvest. The CPE observed can be variable,
and
'scoring' it is subjective. This does not exclude that assays developed for
other
selectively-replicating viruses may be able to use CPE as observable
indication of
RCV. Parameters may be optimized whereas selectively-replicating viruses only
develop CPE (or an observable difference in CPE) when an RCV is present at
what is
considered unacceptable amounts.
The RCV detection assays of the invention are not meant to detect every virus
present in a preparation that has even a slightly altered pattern of
selectivity or is
present in acceptable amounts, but to detect RCVs that may pose problems for
the
desired purpose of the viral preparation. Acceptable amounts is meant to be an
amount that does not significantly effect the intended use of the virus.
For example, in the case of in vitro killing of cancerous cells in a
population of
non-cancerous cells the purpose may be obtaining a culture free of cancerous
cells,
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although the death of some noncancerous cells is considered acceptable.
Therefore, in
this case a contaminating virus present in the preparation that has only a
slight change
in selectivity that would cause the killing of a limited amount of
noncancerous cells
would be acceptable. Also, in the example a low level contamination of a
potent RCV
maybe acceptable. For instance, if the initial input virus is only 1x10s virus
particles
of the selectively-replicating per experiment and the level contamination is
found to
be at a level of less than 1 RCV in 1x108 total virus particles, then the
level of
contamination would probably be considered acceptable, since theoretically
less than
0.1 % of the experiments would ever be compromised by the contaminating RCV.
The sensitivity to detect RCV will likely depend on both the nature of the
replication-selective virus and that of the RCV. A greater difference in their
relative
selectivity should allow a greater sensitivity of detection by these methods.
Another read out for the assays of the invention is total viral particle
production. In some cases, as compared across passages total viral particle
production may be a more quantitative and robust indicator. The cytotoxicity
potency
assay is another very useful indicator of amplified RCV, and in some cases may
be
more reliable than even viral particle production. Restriction digests provide
the most
concrete visual indication of altered structure, but are dependent upon: (1)
selecting a
restriction endonuclease that yields a detectably different restriction
pattern for the
recombinant, and (2) amplification of the recombinant to a frequency high
enough
that it is detected by this relatively insensitive method. Banding patterns
may be
misleading if an aberrant fragment is not adequately resolved from expected
fragments. As described in the examples below, the REC133 recombinants in
Ar6pAE2fE3F amplified to more than 10% of the total genomes. However, they
were
resolved with only one of the five endonucleases chosen. Another read out in
vivo
toxicity. For example, both the original virus preparation (PO) and a
preparation from
assay amplification (e.g., P4) can be injected in an animal in vivo and
various data
collected and compared. For example, inject the virus intravenously into mice
and
measure parameters of toxicity. For example, serum may be collected at various
time
points after injection and liver enzyme measured, which may be indicators of
hepatotoxicity. Increased hepatotoxicity in the amplified preparation (e.g.,
P4) as
compared to the original (PO) preparation may indicate the presence of an RCV.
The
use of multiple detection methods for an RCA Bioamplification Assay of the
present
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invention is contemplated and in fact will probably increase the sensitivity
of the
assay.
Analysis of the amplified material can be extended to include some
characterization of the recombinants detected. One concern with a biological
amplification assay such as this is that a previously absent recombinant might
be
generated during the amplification on MRC-5 cells. Thus, it may be desirable
to clone
out the RCV(s) and develop a method for detecting the RCV(s). For example, the
method of detection may be based on PCR. In the examples below, a developed
PCR
assay for the identified recombinant sequence REC133 is able to confirm that
the
recombinant RCA was indeed present in the initial (PO) Ar6pAE2fE3F material at
a
frequency exceeding 1 copy in 1 x 107 vp. In contrast, Ar5pAE2fF that is
cloned and
produced on HeLa-S3 cells shows no change in any of the read-out methods,
indicating that no RCV is present in the starting material.
The disclosure presented herein describes a biological assay for detection of
RCV in selectively-replicating virus products, and that these products may
contain
RCV's with genomes that cannot be predicted. A biological assay such as this
that
permits differential amplification of a RCV and the selectively-replicating
virus is of
general utility for testing selectively-replicating viral products, regardless
of the
arrangement of the virus genome.
As shown below in the examples, a Bioamplification Assay for Replication-
Selective Vectors has been successfully developed to detect RCV in a
preparation of
selectively-replicating virus. The examples describe the assay for detecting
RCV in
an adenoviral preparation, but the teachings described herein are not limited
to an
assay to detect RCAs or to performing the assay on only adenoviral
preparations.
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EXAMPLES
The present invention is described by reference to the following Examples,
which are offered by way of illustration and are not intended to limit the
invention in
any manner. Standard techniques well known in the art or the techniques
specifically
described below are utilized.
EXAMPLE 1: Cell culture and virus production.
The tumor cell line derivative HeLa-S3 and the normal diploid human
fibroblast cell lines MRC-5, Hs68 and WI-38 are obtained from American Type
Culture Collection (Manassas, VA). MRC-5 and WI-38 cells are cultured in EMEM
containing 10% fetal bovine serum (10% EMEM). HeLa-S3 and Hs68 cells are
cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Small airway
epithelial cells (SAEC), bronchial epithelial cells (NHBE), renal epithelial
cells
(HRE), mammary epithelial cells (HMEC), aortic endothelial cells (HAEC),
microvascular endothelial cells (HMVEC), lung fibroblasts (NHLF), and prostate
epithelial cells (PrEC) are all primary human cells obtained from
CloneticsBiowhittaker (Walkersville, MD), and are cultured in the
manufacturer's
cell type-specific media. AEl-2A is an adenoviral vector complementing cell
line
derived from A549 {Gorziglia, Kadan, et al. 1996}, and is cultured in
Richter's
medium containing 10% FBS. PER.C6 is also an adenoviral vector complementing
cell line (Crucell, Leiden, The Netherlands; {Fallaux, Bout, et al. 1998}) and
is
cultured in DMEM supplemented with 10% FBS and 10 mM MgCl2. All cells are
cultured at 37°C and 5% C02, except Hs68, HeLa-S3 and PER.C6 cells,
which are
cultured at 37°C and 10% C02.
Adenoviral vector particles are produced in the indicated cell line and
purified
on CsCI gradients using methods described previously {Jakubczak, Rollence, et
al.} {Jakubczak, Ryan, et al. 2002}.
EXAMPLE 2: Limiting dilution cloning of virus.
Thirty-one 96-well tissue culture plates are seeded with 5 x 103 PER.C6,
MRC-5 or HeLa-S3 cells/well as indicated, in 100 ~.l/well. For a given virus
to be
cloned in a given cell line, three dilutions of the viral vector particles are
prepared and
added to 10 plates/dilution, in a volume of 100 ~,l/well. The three dilutions
of viral
vector particles are chosen such that the middle dilution is calculated to
yield ~3
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positive wells per 10 plates (e.g. an infecting concentration of 0.03 pfu/ml),
with the
other two dilutions differing by 10-fold higher and lower. The 31 st plate for
each
vector is infected with higher concentrations of the viral vector particles to
serve as
positive controls for observable CPE. The virus-infected cells are incubated
for 10-14
days and then scored for CPE. Whenever cloning on HeLa-S3 and sometimes when
cloning on PER.C6, scoring of positive wells is achieved with a secondary
infection
of S8 cells. In these cases, S8 cells are seeded on 96-well tissue culture
plates at 5 x
104 cells/well in Richter's medium with 10% FBS, 2 mM glutamine, and 0.5 ~.M
dexamethasone added (100 ~1/well). Secondary infection is performed after 1
freeze
and thaw of the primary infection plates, by transferring 50 ~1 of the
contents of each
well of the primary infection plates to the corresponding well of the S8
plates. The
primary infection plates are refrozen and stored at -80°C. After three
days, the
secondary infection plates are observed for CPE. Primary infection plates
corresponding to secondary infection plates containing CPE-positive wells are
thawed
and the clonal isolates in the primary infection plates are then amplified,
preferably in
the same cell line in which they had been cloned, first in 6-well plates and
then in T-
175 flasks. Crude viral lysates (CVL's) are prepared from the T-175 flasks and
the
clonal isolates are analyzed by restriction digest and agarose gel
electrophoresis.
Based on the restriction digest analysis of genome structure, selected clones
are
chosen for production of purified vector particle lots and further analysis.
This example is one of many methods to clone a virus by limiting dilution
(e.g. plaque purification) that are known to one skilled in the art.
EXAMPLE 3: Secondary hexon titer assay.
The secondary hexon titer assay is an indirect measure of adenoviral titer.
Samples are applied to a cell line, AEl-2A, that is permissive to viral vector
replication. After 3 days, the cells are analyzed by an ELISA for total
adenoviral
hexon produced. The hexon signal is proportional to the input virus titer. The
titer of
unknown samples is determined by generating a standard curve using dilutions
of a
reference standard vector.
AEl-2A cells are seeded into 96-well tissue culture plates on the day of
infection at 5 x 10~ cells/well (in 100 p.l), in Richter's medium supplemented
with
10% FBS, 2 mM glutamine, and 1 pM dexamethasone. Only the inner 60 wells of
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each plate are seeded with cells, while the outer wells of each plate are
filled with 200
~l DPBS. Serial dilutions of standards and unknowns are prepared (1:2 for
known
concentrations of Ad5 and Ar6pAE2fE3F to serve as the standard curves and 1:4
for
each unknown production sample) in the same medium. Secondary infection of the
AE1-2A cells takes place by adding 100 ~.l of each dilution of production
sample or
standard curve to triplicate wells of AEl-2A cells and incubating the plates
for three
days at 37°C with 5% C02, with no further media changes. After three
days, the
secondary infection plates are harvested and three freeze-thaws are performed.
An ELISA is performed on these samples by first coating the inner 60 wells of
the appropriate number of Immulon 96-well flat-bottom plates. Plates are
coated
overnight at 4°C with 100 ~l anti-hexon purified antibody (2.5 ~.g/ml;
Chemicon
#MAB8052, diluted in 20 mM Tris + 150 mM NaCI). The next day, the plates are
washed three times (20 mM Tris, 150 mM NaCI, 0.05% Tween 20), blocked with
Pierce SuperBlock (Pierce Cat #37545), and washed again. CVLs from the
secondary
infection plates are diluted 1:500 with dilution buffer (20 mM Tris, 150 mM
NaCI,
1% bovine albumin fraction V), and 100 ~l of each diluted lysate is added to
the pre-
coated wells. The plates are incubated at room temperature (R.T.) for 1 hour,
followed
by washing as above. Anti-hexon biotinylated antibody (2.5 ~.g/ml, diluted in
dilution
buffer) is then added to each well (100 ~,l/well) and the plates are incubated
at room
temperature (R.T.) for 1 hour. After incubation, plates are washed and 100 ~.1
Streptavidin alkaline phosphatase (1:2000 dilution of 1 mg/ml stock solution;
Pierce
Cat #21324) is added to each well. After a 30 min incubation at R.T., the
plates are
washed and pNPP substrate (p-Nitrophenyl Phosphate, Disodium; Sigma #N-2770)
is
added to each well (100 pl/well). The plates are incubated at R.T. for 1 hour.
Absorbance is read on a microplate reader at a test wavelength of 405 nm while
subtracting the absorbance at the reference wavelength of 490 nm. Standard
curves
and sample data are analyzed using GraphPad Prism.
EXAMPLE 4: Generation of an Ar6pAE2fE3F preparation
A research viral seed lot of Ar6pAE2fE3F is generated on AEl-2A cells, a
derivative of A549 cells (Gorziglia MI, I~adan MJ, Yei S, Lim J, Lee GM,
Luthra R,
Trapnell BC, "Elimination of both E1 and E2 from adenovirus vectors ftirther
improves prospects for in vivo human gene therapy." J Virol 1996
Jun;70(6):4173-8).
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Following limiting dilution cloning on PER.C6 cells, a single clone of
Ar6pAE2fE3F
is expanded on PER.C6 cells to generate an Accessionary Virus Bank (AcVB). The
AcVB is purified by centrifugation on a cesium chloride gradient. Molecular
and
biologic characterization indicates that the AcVB is acceptable for generation
of
further viral banks. It should be noted that limiting dilution cloning
followed by
production of an AcVB may require a significant number of viral passages.
EXAMPLE 5: Procedure for screening cell lines for differential amplification
of
"wild-type" versus selectively replicating adenoviruses
To develop an assay capable of detecting an RCA in a background of
replication-selective adenoviuses, normal cell lines are evaluated for
increased
production of an RCA compared to the selectively-replicating adenovirus, i.e.
"relative amplification". Cell lines that may be evaluated include, but are
not limited
to, the fibroblast cell lines MRC-5, Hs68 and WI-38, as well as primary human
Small
Airway Epithelial Cells, SAEC (Clonetics). For example, for detecting RCA in a
preparation of Ar6pAE2fE3F, a wild-type Ad5 is used to model potential RCA,
and
the oncolytic virus Ar6pAE2fE3F (Figure 10) is used as the test article.
Production
tests are performed in 75 cm2 tissue culture flasks, with cells cultured to 50-
70%
confluence. Multiple multiplicities of infection (0.4-50 particles per cell;
ppc) and
times of harvest (3-1? days post-infection) are assessed. At harvest, cells
are
collected, a cell pellet is formed by centrifugation, and a crude viral lysate
(CVL) is
prepared by: (1) resuspending the cell pellet in a, small volume of Dulbecco's
phosphate buffered saline (DPBS), (2) three cycles of freeze-thawing, and (3)
clarification by low speed centrifugation. Productivity of virus is determined
by
titering CVL's using a secondary hexon titer assay. For example, for detecting
RCA
in a preparation of Ar6pAE2fE3F, these initial production tests are performed
with
wild-type Ad5 and Ar6pAE2fE3F in separate flasks. The goal of these initial
experiments is to select conditions for further development using mixtures of
Ad5 and
and the test article (e.g. Ar6pAE2fE3F).
Criteria used to select a cell line and culture conditions for further
development
include the relative production of Ad5 wild-type compared to the test article
(e.g.
Ar6pAE2fE3F), the absolute virus productivity, the general growth
characteristics of
the cells, and the overall feasibility of assay development. The required
virus yield is
set at approximately 1 x 1012 purified viral particles (vp) to enable
molecular analyses
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such as quantitative PCR, restriction digest mapping and sequencing, if
warranted.
More or less virus may suffice depending on which diagnostic assays are
needed.
EXAMPLE 6: Screening cell lines and initial development of an RCA
bioamplification assay for Ar6pAE2fE3F preparations
The following are results from when cell lines are screened with a wild-type
Ad5 as a model for potential RCA, and the oncolytic virus Ar6pAE2fE3F
preparation
is used as the test article. The procedure in example 5 is used. Cell lines
evaluated
include the fibroblast cell lines MRC-5, Hs68 and WI-38, as well as primary
human
Small Airway Epithelial Cells, SAEC (Clonetics). WI-38 and SAEC were both
rejected early due to poor characteristics for growth to large scales required
for this
assay.
The principle criteria used to select a cell line and culture conditions for
further
development is the relative production of Ad5 wild-type compared to
Ar6pAE2fE3F,
the absolute virus productivity, the general growth characteristics of the
cells, and the
overall feasibility of assay development. The required virus yield is set at
approximately 1 x 1012 purified viral particles (vp) to enable molecular
analyses such
as quantitative PCR, restriction digest mapping and sequencing, if warranted.
In
general, longer times before harvest or greater MOI increase total
productivity, but
also decrease the difference in the amount of Ad5 produced compared to
Ar6pAE2fE3F. Based on these comparisons, MRC-5 cells are selected for
assessment
of biological amplification of Ad5 spiked into Ar6pAE2fE3F to determine
whether
this method can be used to detect low levels of RCA in an oncolytic adenoviral
virus
preparation. The harvest time is set to 6 days post-infection, after infecting
with an
MOI of 10-50 ppc.
EXAMPLE 7: Detection of an RCA in a preparation of Ar6pAE2fE3F
Based on these data from Example 6, MRCS cells are selected for assessment of
biological amplification of Ad5 spiked in Ar6pAE2fE3F. The assay is designed
to
include 4 passages of virus on MRCS cells with PCR detection of only 2
predicted
classes of potential RCA. These predicted RCAs are based on the potential for
recombination between viral vector and packaging sequences in the cell line
(Figure
1 ).
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The RCA Bioamplification Assay is initially tested at full scale using
Ar6pAE2fE3F, either alone or spiked with wild-type Ad5 at ratios ranging from
1
plaque forming unit (pfu) Ad5 (wild-type) per 3 x 101° vp Ar6pAE2fE3F
to 1 x 104
pfu Ad5 per 3 x 101° vp Ar6pAE2fE3F. One viral production passage is
defined as
including infection of the MRC-5 cells with a known amount of virus, culturing
for 6
days with one change of medium, and harvesting the virus produced. Each virus
sample is passaged sequentially four times in MRC-5 cells. In the starting
sample (PO)
and at each subsequent passage (Pl-P4) the total number of adenoviral
particles
present is determined using an OD260/SDS method, and the number of copies of
Ad5
present is determined using a quantitative PCR assay specific for the wild-
type E 1 a
promoter (absent in Ar6pAE2fE3F). The ratio of Ad5 to total adenoviral
particles is
compared between each passage to determine whether relative amplification of
Ad5
(the model RCA) compared to the test virus is achieved.
In preliminary experiments, relative amplification of Ad5 was consistently
observed for samples spiked at 100 pfu Ad5 or greater per 3 x 101° vp
Ar6pAE2fE3F.
Preliminary results with 1 pfu Ad5 per 3 x 101° vp Ar6pAE2fE3F were
inconclusive
due to the presence of an unexpected RCA present in the lot of Ar6pAE2fE3F
used,
and which is described below.
During the amplification of Ar6pAE2fE3F described above, cytopathic effect
(CPE) was observed to be minimal at the time of harvest (6 days) for Pl, P2
and P3 in
all spiked and unspiked samples. It should be noted that if the cultures were
incubated
for additional days, CPE was observed to increase. However, at P4 the amount
of CPE
was noticeably increased at the time of harvest compared to earlier passages.
At P1,
P2 and P3 it was necessary at harvest to incubate the cells with EDTA in order
to
release the cells from the plastic substrate. At P4 the cells easily entered
suspension
with mild shaking of the roller bottles. Moreover, the yield of virus was
greater at P4
then at earlier passages. These effects were observed in the Ar6pAE2fE3F
samples
that were spiked with AdS, and unexpectedly in unspiked samples. The increase
in
CPE and increase in virus yield are consistent with a change in the virus
composition
during passaging on MRC-5 cells, so that a virus or virus mixture less
selective for
tumor cells and more productive on normal cells was present at P4 than at P0.
These
observations lead us to suspect the presence of a non-selective RCV in the
Ar6pAE2fE3F virus preparation and was later confirmed that an RCA was present
in
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relatively low levels in the initial adenovirus preparation and the percentage
of RCA
was amplified at each passage.
The assay was designed to include 4 passages of virus on MRCS cells with PCR
detection of only 2 classes of potential RCA. These predicted RCAs are based
on the
potential for recombination between virus and packaging plasmid sequences
(Figure
1). However, during the development of the amplification portion of the assay,
a
biological change in the MRCS cells was observed after the third passage of
Ar6pAE2fE3F even though no model RCA had been spiked into the initial sample.
The yield of virus increased at passage 3 and by passage 4 an obvious
cytopathic
effect was observed.
In the description above, MRCS cells are used for the amplification steps of
the
assay. The present invention is not meant to be limited to a particular cell
line. For
example selectively-replicating (e.g. oncolytic) viruses may for the purposes
of the
assay be passaged ("amplified") on any "normal" or non-target cell type.
Normally
several of these non-target cell types are screened based on the differential
or selective
amplification of the RCA as compared to a selectively-replicating virus.
Also, the number of passages on the non-target cells may be increased or
decreased to optimize for the differential amplification of the selective
replicating
virus as compared to an RCA. See example 5.
The "read-out" of the assay may include any of the many factors or
combinations thereof including: observation of CPE, measurement of total virus
or
yield, analysis for molecular structure (e.g. restriction digestion analysis,
nucleotide
sequencing), measurement of viral potency (e.g. cell killing assays). These
factors
may be measured/observed at the end of the amplification process or at each
passage
during this process. Then measurements/observations may be compared to those
made from the first passage or starting viral preparation.
EXAMPLE ~: General procedure for characterizing an RCA in a preparation of
adenovirus
If an RCA is detected the RCA may be molecularly characterized. One way to
characterize the RCA is to perform a restriction digest on viral DNA isolated
from
one of the passages, preferably the last passage, of the assay. If an
uncharacteric
restriction pattern is displayed, the unexpected fragment is cloned and
sequenced.
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To determine whether a rearranged virus is present in the AcVB, is generated
as
a consequence of forced virus production on a poorly permissive cell line or
generated
by another means, a PCR assay is developed to specifically detect the
rearranged viral
vector. Preferably, this PCR assay has a sensitivity ~of at least 10-100
rearranged
vector in 10$ viral vectors. Using this assay, viral preparations are tested.
EXAMPLE 9: Characterization of an RCA in a preparation of Ar6pAE2fE3F
The following describes a procedure that may be used to characterize the viral
DNA from passage 7 of example 2. Diagnostics of the passage 4 virus particles
revealed a restriction pattern that is not characteristic of Ar6pAE2fE3F.
The unexpected fragment is cloned and sequenced. The sequence analysis
suggests that viral vector rearrangement has occurred resulting in a loss of
most of the
E2F-1 promoter and replacement with most of the viral E4 promoter (Figure 5)
such
that the predicted tumor selectivity of Ela expression and viral replication
are lost.
Surprisingly, the assay detected an RCA that was not predicted by the
inventors and
therefore would not have been detected by the PCR designed to detect the two
classes
of predicted RCA (Figure 1).
To determine whether the rearranged virus is present in the AcVB, a PCR assay
is developed to specifically detect the rearranged vector. Preferably, this
PCR assay
has a sensitivity of at least 10-100 rearranged viral vectors in 108 viral
vectors. Using
this assay, the AcVB, Master Virus Bank (MVB) and Pre-Clinical Material are
tested.
When performed on the above mentioned lots of Ar6pAE2fE3F all of these virus
banks and product lots are positive at levels between 30-200 RCA vectors per
108
vector particles (Table 1). Further, these results show that passaging of a
replication-
selective virus in the less permissive cells such as MRC-5 does not lead to
the
generation of RCV, which would be a false positive. The most prevalent RCA has
greater cytotoxicity on some normal cells than wild type Ad5 virus (Figures 3
and 6).
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Table 1: Quantitation of Recombinant in Ar6pAE2fE3F by Q-PCR
a
REC133 Copies °
Cell Line Viral Passage 8 /o REC133
per 1 X 10 vp
Production cells pOb 2.0 X 10~ 0.000020%
MRC-5 P1 2.2 X 103 0.0022%
P2 1.9 X 105 0.19%
P3 4.5 X 106 4.5%
P4 4.0 X 107 40%
Production cells P2 3.4 X 10~ 0.000034%
p4 1.6 X 102 0.00016%
aPCR results are the average of triplicate determinations.
bThe initial test material grown in standard production cells is defined as
viral passage
0 ("PO"). Subsequent viral passages on either cell line are numbered relative
to the
initial test material.
Without being bound by theory, the inventors believe that the mechanism of
action that leads to the production of these particular RCAs in Ar6pAE2fE3F
preparations is as follows. The recombinants probably either are created
through
intermolecular recombination or by a mechanism of "polymerase jumping." Either
one of these mechanisms leads to the viral sequences from the right end of the
vector
being duplicated, as is seen in the recombinant. This leads to part or all of
the tumor
selective promoter (E2F-1) being replaced by part or all of the viral promoter
for E4.
These recombinants could then be packaged and are found to replicate non-
selectively
or least with a different selectivity as compared to Ar6pAE2fE3F.
In summary, RCAs were detected in preparations of Ar6pAE2fE3F using a cell-
based biological assay that involved serially passaging the virus. The RCAs
were
fortuitously detected while developing the assay. The recombinants that were
detected through this assay were unexpected recombinants that were not
predicted by
the inventors. A PCR-based assay based on predicted recombinants would not
have
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detected these recombinants. This demonstrates a clear advantage of the cell-
based
biologic amplification assay over the PCR detection methods for detecting
replication
competent viruses.
EXAMPLE 10: Investigation of detected RCA in Ar6pAE2fE3F
A key characteristic of selectively replicating viruses is decreased potency
to
kill normal or non-target cells. To help determine whether a non-selective RCA
is
amplified in the Ar6pAE2fE3F sample, the potencies of the PO and P4 virus
particles
to kill normal cells are compared, using a cytotoxicity assay with MTS
readout. On
MRC-5 cells and on primary Human Aortic Endothelial Cells (HAEC's, Clonetics)
the Ar6pAE2fE3F MRC-5 P4 viral particles are more potent than the PO viral
particles by greater than 2 logs (Figures 11 and 12). Surprisingly, the P4
virus is also
more potent than wild-type AdS. A panel of normal primary human cell types are
evaluated with varying results ranging from no difference in potency between
the PO
and P4 viral particles to a 3+ log increase in potency of the P4 viral
particles
compared to PO (Table 2). On no cell type tested is the potency of the PO
viral
particles greater than that of the P4 viral particles.
Table 2: Relative potency of Ar6pAE2fE3F, P4 compared to P0.
Cell lo~(dose ratio)S.E.M.
Line
HMEC 0.23 O.OS2
SAEC *0.30 0.079
NHBE *0.35 0.11
PrEC *0.57 0.11
HRE *0.90 0.17
NHLF *2.1 0.25
HMVEC *2.6 0.23
HAEC *3.6 0.16
MRC-5 *2.6 0.29
Log(dose ratio) is calculated as:
log [ECSO of P4] - log [ECso of PO]
*Potency of P4 significantly different from potency of P0, p < 0.05, Student's
t-test
To further explore whether an RCA is amplified, restriction digest analyses of
the PO and P4 viral vectors are performed. A panel of restriction
endonucleases are
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selected to provide banding patterns that together might indicate genetic
changes in
many of the critical regions of the vector. All five digests result in the
expected
pattern with the exception of SspBl digestion of the P4 virus, which yields a
fragment
of unexpected size. The altered potency and restriction digest pattern of the
P4 vectors
compared to the starting vector strongly suggests that the P4 material
contains a
significant portion of vector particles with altered molecular structure and
decreased
replication selectivity. The RCA Bioamplification Assay of Ar6pAE2fE3F virus
at
P0, including analysis by potency assay and restriction digest, is then
repeated in its
entirety, which yields equivalent results, confirming the initial finding.
The unexpected restriction fragment of the P4 virus is extracted from the gel,
cloned into a plasmid, and sequenced. Sequence analysis of the fragment, and
subsequently of cloned recombinant viruses described below, indicates that a
rearrangement of the viral vector genome has occurred resulting in a
duplication of
the right end of the vector in place of the left end. This results in the loss
of most or all
of the E2F-1 promoter previously driving ElA expression, and replacement with
part
or all of the adenoviral E4 promoter.
To determine whether the observed recombinant virus is an assay artifact
generated during the RCA Bioamplification Assay, or is present in the original
test
material (Ar6pAE2fE3F at PO), a quantitative PCR assay is developed capable of
detecting one of the recombinant sequences, termed "REC 133". This PCR assay
has a
sensitivity of 10 rearranged vector copies in 10$ vector particles. PCR
analysis for
REC133 is performed on Ar6pAE2fE3F virus before and after passaging on MRC-5.
REC 133 is present in Ar6pAE2fE3F at PO at a frequency of 20 copies per 1 x
108 vp
Ar6pAE2fE3F (Table 3). Thus, the RCA Bioamplification Assay detects a
recombinant that was present in the original Ar6pAE2fE3F test sample. The
frequency of REC 133 increased dramatically with each passage on MRC-5 cells,
indicating that "relative amplification" of this non-selective recombinant
virus
compared to the Ar6pAE2fE3F was achieved as hypothesized in the design of the
assay. It was further postulated that in a cell line in which this selective
advantage was
reduced or eliminated, such as PER.C6, the relative amplification of REC133
would
be reduced or absent. To investigate this hypothesis, an aliquot of the
original
Ar6pAE2fE3F test sample (PO) is passaged sequentially on PER.C6 cells, and the
resulting viral lots tested for REC133. The frequency of REC133 increases with
each
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passage when Ar6pAE2fE3F is passaged in PER.C6 cells, although at a much
slower
rate than when passaged on MRC-5 cells (Table 3).
Table 3: Quantitation of REC133 and PGK Recombinants by PCR
Vector Passages on REC 133 % REC 133 PGK Copiesb % PGK
(Cell line MRC-5 Copiesb per 5 X 10'° vp
for PO) Per 1 X 108 vp
Ar6pAE2fF PO 3.4 X 102 0.00034% ~.4 X 105 0.00048%
(PER.C6)
P4 1.1 X 104 0.011 % 5.6 X 1 O6 0.011
Ar6pAE2fF PO 4.5 X 10' 0.000045% b~dv 0%
(HeLa-S3)
P4 3.1 X 106 3.1% b'd' 0%
Ar5pAE2fF PO b.d.~ 0% 4.0 X 106 0.0080%
(PER.C6)
P4 b.d. 0% 2.8 X 109 5.6%
Ar5pAE2fF PO b.d. 0% b.d. 0%
(HeLa-S3)
P4 b.d. 0% b.d. 0%
aThe initial test material was cloned and produced on the cell line indicated
in
parentheses below the vector name, and is defined as P0. This lot was then
passaged
sequentially on MRC-5 cells for four passages.
bPCR results are the average of triplicate determinations.
°b.d. - below detection. Limit of detection for REC133 is 10 copies per
1 X 108 vp.
Limit of detection for PGK promoter sequence is 50 copies per 1 X
10'° vp.
Ar6pAE2fE3F was originally cloned and produced on PER.C6 cells, which
provide ElA to support viral replication. During the RCA Bioamplification
Assay
REC 133 would likely co-infect cells with parental Ar6pAE2fE3F. Therefore it
is
possible that the recombinant observed is not truly replication competent but
is
dependent upon ElA provided by either the cells when grown in PER.C6, or
helper
virus, in this case Ar6pAE2fE3F, when passaged in MRC-5. To more fully
characterize the RCA, clonal isolates were prepared from the P4 virus. Because
there
is no definitive evidence that the recombinant in the P4 virus is replication
competent,
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limiting dilution cloning is done on both PER.C6 cells and MRC-5 cells to
maximize
the potential to obtain the recombinant virus. Clones are successfully
isolated on both
PER.C6 cells and MRC-5 cells. Nineteen clones from PER.C6 and seven from MRC-
S are analyzed by restriction enzyme digest and also by sequencing. The clones
isolated include the parental (non-recombined) Ar6pAE2fE3F virus (3 of 26
analyzed). Surprisingly, the restriction digest analyses indicate the presence
of
multiple recombinants isolated from both PER.C6 and MRC-5 cells, which is
confirmed by sequence analysis (Figure 13). Sequencing demonstrates that all
of the
sequenced RCAs shared the characteristic of a right end duplication to the
left end of
the vector, with an altered promoter containing all or part of the viral E4
promoter
presumably driving ElA expression. In some recombinants a chimeric E4lE2F-1
promoter is present, while in others a complete E4 promoter is present at that
position.
The clones are also analyzed using the REC133 PCR assay. Only recombinants A
and E are detected by this PCR assay, as is predicted from the location of the
primers
and probe relative to the sequence of each recombinant (Figure 13),
reaffirming the
limitations of PCR methodology for comprehensive assessment andlor detection
of
recombinant vectors.
The clonal isolation of some of the recombinants on MRC-5 cells (Figure 13)
indicates that at least recombinants A, B, E and F are truly replication
competent since
MRC-5 cells do not provide any helper function. One additional clone
(recombinant
C) that is not originally isolated from MRC-5 cells is subsequently shown to
replicate
on these and other normal cells. Moreover, in those experiments recombinants
A, B
and C replicate more efficiently than Ad5 in the normal cell lines evaluated
(MRC-5,
primary human renal epithelial cells, primary human lung diploid fibroblast
cells), in
agreement with the greater potency of the P4 virus compared to Ad5 in the
cytotoxicity assay (Figures 1 l and 12). Recombinants D-G are not similarly
evaluated
for productivity. Because a relatively small number of clones from MRC-5 are
analyzed, the lack of cloning of recombinants D and G using MRC-5 cells should
not
be taken to suggest that they are necessarily replication incompetent or not
capable of
independent replication.
EXAMPLE 11: Further Characterization of the RCA Bioamplification Assay
The preceding results and examples demonstrate that by using an RCA
Bioamplification Assay it is possible to detect an RCA in the presence of a
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replication-selective adenoviral vector particle preparation. In order to
further
characterize the assay, the assay is performed using two additional viruses,
Ar6pAE2fF and Ar5pAE2fF (Figure 10). Each virus is cloned and produced on two
different cell lines, PER.C6 and HeLa-S3. PER.C6 is a derived cell line that
expresses
the adenoviral E1 a gene under the control of the human PGK promoter [
{Fallaux,
Bout, et al. 1995}]. PER.C6 cells were originally developed to support
production of
E1-deleted replication defective adenoviral viruses. In the context of
selectively-
replicating viruses, however, the presence of the Ela sequence preceded by the
PGK
promoter could allow recombination with the vector to generate a virus with El
a
under the control of the PGK promoter, which is constitutive and non-selective
[{Murakami, Pungor, et al.}]. The other cell line used for production, HeLa-
S3, is a
clonal derivative of the tumor cell line HeLa. This transformed cell line
supports
replication of E2F-1 promoted oncolytic adenoviruses, and so also serves as an
excellent production platform for these viruses.
One sample of each virus preparation produced on PER.C6 cells and two
samples of each virus preparation produced on HeLa-S3 cells (4 x 10'°
vp/sample) are
passaged sequentially four times on MRC-5 cells. The degree of CPE increases
slightly in the Ar6pAE2fF samples at P3 at the time of harvest while at P4
there is a
severe increase in the degree of CPE, regardless of whether the virus is
produced on
PER.C6 or HeLa-S3 cells. A smaller increase in CPE is observed in the
Ar5pAE2fF
virus that has been produced on PER.C6 cells. In contrast, an increase in CPE
is not
observed with the Ar5pAE2fF virus that has been produced on HeLa-S3 cells.
The virus particle concentrations of the purified virus samples are determined
by an OD260 method, and the total particles produced at each passage are
calculated.
The patterns in productivity are similar to the observable patterns of CPE.
Productivity increases at P4 with all of the Ar6pAE2fF samples regardless of
producer cell platform, and to a smaller degree with the Ar5pAE2fF virus
sample that
has been cloned and produced in PER.C6 cells. However, no change in
productivity
during sequential passaging on MRC-5 cells is observed in the Ar5pAE2fF sample
that has been cloned and produced in HeLa-S3 cells. Restriction digest
analyses are
also performed using the same enzymes as are used previously. Aberrant
restriction
patterns are observed after 4 passages on MRC-5 with Ar6pAE2fF that has been
cloned and produced on either PER.C6 or HeLa-S3, and with Ar5pAE2tF that has
been cloned and produced on PER.C6. The Ar5pAE2fF sample that is initially
cloned
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and produced on HeLa-S3 shows the expected restriction digest patterns with
all
enzymes.
Finally, analysis of potency of the PO and P4 samples to kill normal cells are
determined using MRC-5 cells. The Ar5pAE2fF (HeLa-S3) virus shows no change in
potency after four passages on MRC-5 cells (Table 4 and Figure 14). However,
the
MRC-5 P4 samples of Ar5pAE2fF (PER.C6) and Ar6pAE2fF (PER.C6 or HeLa-S3)
all are more potent than the corresponding viral particles at P0. Thus, the
observation
of CPE and quantitation of production during passaging on MRC-5, potency to
kill
normal cells before and after passaging on MRC-5 cells, and restriction digest
patterns
all suggest that Ar5pAE2fF virus that is cloned and produced on HeLa-S3 cells
contains no RCA, but that the other 3 virus samples do.
Table 4: Virus Production During RCA BioAmplification on MRC-5 Cells
Virus (Cell Line for PO)
Ar6pAE2fF Ar6pAE2fF Ar6pAE2fF ArSpAE2fF Ar5pAE2fF Ar5pAE2fF
(PER.C6) (HeLa-S3) (HeLa-S3) (PER.C6) (HeLa-S3) (HeLa-S3)
P1 5.2E+0123.9E+012 3.1E+012 3.8E+0123.8E+012 2.4E+012
P2 3.9E+012S.SE+012 S.SE+012 6.6E+0123.SE+012 6.6E+012
P3 5.9E+012S.lE+012 7.1E+012 1.SE+0122.9E+012 8.6E+012
P4 3.3E+0132.1E+013 2.8E+013 1.4E+0134.3E+012 4.3E+012
Each value represents total viral particles purified at viral passage 1-4 (Pl,
P2...P4)
from one experiment.
Murakami and colleagues [ {Murakami, Pungor, et al. 2002 f ] have described
adenoviral recombinants that have incorporated the PGK-promoted Ela of the
PER.C6 cell line when using that cell line for production of viruses
containing overlap
with the Ela sequence of the cell line. To partially characterize the
recombinants
detected by our RCA Bioamplification Assay in the Ar6pAE2fF and Ar5pAE2fF
virus preparations, the PO and P4 viral particles are analyzed by quantitative
PCR for
both the PGK promoter and REC133. Ar6pAE2fF and Ar5pAE2fF that are originally
cloned and produced on PER.C6 cells do contain PGK sequence at moderate
frequencies prior to passaging on MRC-5 cells. In both viral preparations, the
frequency of PGK sequence in the viral genomes increases after passaging on
MRC-5
cells. Neither virus cloned and produced on HeLa-S3 cells contains detectable
PGK
sequences. Ar6pAE2fF virus that is produced on either PER.C6 or HeLa-S3 cells
contain sequences detected by the REC133 PCR. Ar5pAE2fF produced on either
cell
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line contained no sequences detected by the REC133 PCR. Further
characterization
was not performed on the PGK promoter-containing recombinants) detected in our
assay.
Without being bound by theory, molecular structures of the REC133-related
recombinants, could have been the result of intermolecular recombination, or
intramolecular polyrnerase jumping. The formation of the REC133-related
recombinants may have happened due to at least three structural factors within
the
Ar6pAE2fE3F and Ar6pAE2fF viruses: (1) the location of the packaging signal on
the
right end of the molecule; (2) the replacement of the endogenous viral E1
promoter
with the human E2F-1 promoter; and (3) the endogenous viral E4 promoter
(adjacent
to the packaging signal). The lack of detectable RCA in Ar5pAE2fF, in which
the
packaging signal is located at the left end of the vector genome, suggests
that leaving
the packaging signal on the left end was sufficient to eliminate similar
recombinations
in this family of vectors. However, this data should not be interpreted to
indicate that
placing the packaging signal on the right end in and of itself will lead to
the formation
of undesirable recombined viruses, nor that leaving the packaging signal on
the left
end will prevent their formation. Rather, it is the entire vector genome that
must be
considered, as each element, whether engineered into the vector or an
endogenous
viral sequence, may influence the nature of any recombinant that is forned.
These
data suggest that a combination of factors contribute to the generation of RCA
in a
background of replication-selective virus. The relative importance of each of
these
factors is likely to vary with each virus construct. An interpretation
concerning virus
structure to be made from the data shown here is that recombination events can
be
surprising, thus calling for the use of sequence-independent biological assays
such as
this one for more comprehensive detection of undesirable recombinants that
have
formed.
EXAMPLE 12: A general description of an RCA bioamplification assay for
selectively replicating adenovirus preparations on MRC-5 cells.
The biological assay comprises of four sequential passages of each virus in 20
roller bottles of MRC-5 cells per virus, with CsCl purification of virus at
each
passage. For each passage, roller bottles (RB) are seeded at 2 x 107 cells/RB
and
incubated at 37°C with 5% C02. Two days after seeding, the cells are
infected at 50
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vp/cell with an estimated cell concentration of 4 x 107 cells/RB (for a total
of 4 x l Olo
vp tested per sample). Each roller bottle receives 60 ml infection medium
(virus
diluted in 10% EMEM). Roller bottles are incubated at 37°C with 5% CO2
for 4
hours and then further filled with 240 ml/RB 10% EMEM supplemented with
antibiotics (100 ug/ml streptomycin, 100 U/ml penicillin, 2.5 ug/ml Fungizone;
BioWhittaker, Walkersville, MD). Two days after infection, all roller bottles
receive a
medium change with 300 ml/RB fresh 10% EMEM supplemented with antibiotics.
On Day 6 after infection, cells are harvested. Harvesting the infected cells
may
be performed by various methods. For example, one method is to aspirate the
medium and add 50 ml Versene (0.2 mg/ml EDTA in phosphate buffered saline) to
each roller bottle. After 15 to 20 min incubations at 37°C, cells are
sharply banged off
of the roller bottles and the bottles are rinsed with 10% EMEM. This method is
preferred when the cells show lower levels of CPE when infected and incubated
in the
manner described, and are not easily removed from the surface of the roller
bottles.
Another harvest method is to simply shake the roller bottles with the medium
in them
to remove the cells. This method is preferred when the cells show more CPE
and/or
are already starting to detach from the roller bottles. After harvesting,
cells are
pelleted by low-speed centrifugation and resuspended in DPBS twice. A crude
viral
lysate (CVL) is formed by subjecting the cell suspension to 5 freeze-thaw
cycles and
removing cellular debris by low speed centrifugation. CVL from each virus
passage is
purified through two rounds of CsCI gradients and dialyzed in THPG dialysis
buffer
.(200 mM Tris, 50 mM HEPES, phosphoric acid to pH 8.0, 10% glycerol) to remove
the CsCI.
EXAMPLE 13: A detailed description of an RCA bioamplification assay for
selectively replicating adenovirus preparations on MRC-5 cells.
1.0 SEEDING CELLS
1.1 Trypsinize 2 10-stack cell factories of MRC-5 (ATCC #CCL-171) cells
that are ready to be split. Combine the cell suspensions, and count the
cells per mL of this suspension.
1.2 Determine the volume of cell suspension required for each roller bottle
to be seeded at 2x107 cells/RB.
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1.3 Label the roller bottles with the cell line information (name, passage #,
population doubling level, concentration of cells/RB, and date) and add
approximately 200mL culture medium (EMEM + 10% FBS) to each
bottle.
NOTE: MRC-5 cells have a finite life span and should not be used at
the point where the growth starts to decline. ATCC estimates that they
are capable of attaining 42 to 46 population doublings.
1.4 Add the amount of cell suspension required to each bottle and place all
bottles on a roller rack at 37° C in a humidified 5% COZ incubator.
1.5 Repeat this procedure with 2 more cell factories each time, until the
appropriate number of roller bottles have been seeded.
2.0 INFECTION OF CELLS (2 DAYS POST-SEEDING)
2.1 Microscopically examine the roller bottles to ensure that the cells are
healthy and about 50% confluent.
2.2 Prepare the infection medium need for each group of 20 roller bottles
(Falcon, expanded surface 1450 cm2 surface area) to be infected.
2.2.1 The infection medium for each group of 20 roller bottles
contains 1260mL of EMEM + 10% FBS. Each roller bottle is
infected with 60mL infection medium and this allows an
excess of 1 roller bottle.
2.2.2 Calculate amount of virus needed for infection of each group at
an MOI of 50 particles/cell.
2.2.2.1 The average number of cells per roller bottle has been
determined to be 4x107 cells on the day of infection.
2.2.2.2 To obtain an MOI of 50, each roller bottle will be
infected with 2x109 virus particles.
2.2.2.3 For each group of 20 roller bottles, 4.2x10° virus
particles are required for each 1260mL of infection
medium.
2.2.3 Thaw virus quickly to room temperature and add the
appropriate amount virus to the infection medium just before
use.
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2.3 Remove the roller bottles from the incubator one group at a time, label
each bottle (with the group identification and number of times the virus
has been passaged on MRC-5 cells).
2.4 Aspirate the medium from each roller bottle and add 60mL of infection
medium (with virus added) to each roller bottle.
2.5 Place roller bottles back on a roller rack at 37°C in a humidified
5%
C02 incubator.
2.6 Clean hood and start infection of the next group of roller bottles (steps
2.2 through 2.5).
2.7 After the roller bottles have been incubated for at least 4 hours on a
roller rack, add EMEM Recovery Medium (EMEM + 10% FBS + 1
Pen-Strep + 1 % Fungizone) to each group in the same order as
infection, cleaning hood between each group.
2.7.1 If there is a partial bottle of medium remaining after all roller
bottles from the group are completed, discard the medium and
do not use it for the next group.
2.8 Incubate infected cells on a roller rack at 37°C in a humidified 5%
C02
incubator.
3.0 REFEEDING INFECTED ROLLER BOTTLES
3.1 Refeed all groups of roller bottles in the same order as infected.
3.1.1 Refeeding is performed two days after infection, if infection
occurs on Wednesday. This means that roller bottles were
seeded on Monday, infected on Wednesday, and refed on
Friday.
3.1.2 Refeeding is performed three days after infection, if infection
occurs on Friday. This means that roller bottles were seeded
on Wednesday, infected on Friday, and refed the following
Monday.
3.2 Aspirate medium from one group of roller bottles and add 300mL
EMEM Recovery Medium to each bottle. Do not use the same bottle
of medium for more than one group of roller bottles.
3.3 Repeat the process until all groups have been refed, cleaning the hood
between groups.
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3.4 Incubate infected cells on a roller rack at 37°C in a humidified 5%
COZ
incubator.
4.0 CELL HARVEST (6 DAYS POST-INFECTION)
4.1 Observe the infected roller bottles under the microscope on the day of
harvest to see how much CPE is present.
NOTE: As passage on MRC-5 cells increases, the CPE might also
increase, if a more potent recombinant is present. It is possible that by
the fourth passage on MRC-5 cells, the cells will be showing so much
CPE that they will come off of the roller bottles as in a normal prep.
These roller bottles are harvested without removal of the medium and
addition of Versene. They are harvested by shaking the roller bottles
to remove cells and centrifuging the whole suspension. Day of harvest
may be decreased to 5 days post-infection for those samples in which
CPE is present earlier.
4.2 Harvest roller bottles, one group at a time, in the order of infection.
4.2.1 Repeat the entire harvesting process below (steps 4.3 through
4.14) for each group, cleaning the hoods) between groups.
4.2.2 Two groups can be harvested at the same time, in an
overlapping fashion, by using a separate hood for each group.
4.3 Aspirate medium from each roller bottle in the group and add SOml
Versene to each roller bottle. Do not use the same bottle of Versene
for more than one group of roller bottles.
4.4 Place roller bottles back on the roller rack and incubate for
approximately 20 min. at 37°C in a humidified 5% COZ incubator.
NOTE: The time required for incubation with Versene will depend on
the level of CPE.
4.5 Make sure caps are closed tightly and bang the roller bottles very
sharply. Try to get all cells off of the surface.
4.6 Pipette the Versene from each roller bottle into SOOml centrifuge tubes.
Add contents of 5 roller bottles to each SOOmI tube. This means you
will use 4 tubes for each group of 20 roller bottles.
4.7 Slowly add SOml EMEM Recovery Medium to each roller bottle,
rotating the bottle as you drip the medium down the sides to rinse.
Add this medium to the same tube containing the Versene suspension
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from these roller bottles. Do not use the same bottle of medium for
more than one group.
4.8 Centrifuge tubes at 2000 rpm for 5 minutes at room temperature with
no brake.
4.9 Pour off or aspirate supernatant and combine cell pellets into one
SOOmL tube with 400 to 450mL DPBS. Reserve about 50 to 100mL
DPBS for the final resuspension of the pellet. Do not use the same
bottle of DPBS for more than one group.
4.10 Centrifuge the SOOmL tube again at 2000 rpm for 5 minutes at room
temperature with no brake.
4.11 Aspirate supernatant and estimate the volume of the pellet. Resuspend
the pellet in SmL of DPBS. Use a sterile lOmL pipette to measure the
suspension.
4.12 Pipette the cell suspension up and down to make a slurry. Transfer the
slurry into SOmL conical tubes as l OmL aliquots. The tubes are labeled
with group identification and tube # of total #.
4.13 Determine the volume of DPBS needed to bring the final volume of
cell suspension to 2.5 times the initial volume of cell pellet, rinse the
SOOmL tube with at least this volume of DPBS, and add this volume
evenly to the tubes of cell suspension.
4.14 Store tubes at -65° C to -85°C until purification is
initiated.
5.0 PURIFICATION PROCEDURE
5.1 Freeze/Thaws
5.1.1 Using dry ice and a 37°C water-bath, perform a series of 3
freeze/thaw cycles on the cell suspension. The thaw time will
vary due to the differing volume of CVL in each tube. Check
the tubes in the thaw cycle periodically since excess time at
37°C may result in lower virus viability.
5.1.2 Vortex tubes for at least 10 seconds after each thaw. The
vortexing is required to help release the virus into the DPBS.
NOTE: The CVL may be stored at -65 to -85°C during any of
the freeze cycles.
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S.l .3 One final thaw is performed immediately before beginning the
discontinuous gradient.
5.2 Discontinuous Gradient
5.2.1 After the final thaw, centrifuge the tubes at 3000 rpm with the
brake off for 5 minutes at room temperature, to clear the final
freeze/thaw suspension.
5.2.2 Remove the cleared CVL with a pipette from the centrifuge
tube. Place cleared CVL in sterile SOmL tube(s).
NOTE: If there is a question as to whether or not you have
produced enough virus to purify, the cleared CVL can be
tested by HPLC to determine particle titer. The CVL should
be kept frozen at -80°C while waiting for results. Once
particle titer is available, the decision can be made to purify
(need to start with approximately 1.5 to 2x1012 virus particles)
or to infect the next passage using CVL.
5.2.3 Divide the total volume of the CVL by seven to get the number
of gradients. For each gradient to be run, remove 3.SmL
aliquots each of CsCI (1.25 g/mL) and CsCl (1.40 g/mL) to
sterile SOmL tubes in the clean hood. Do not open stock
bottles in the virus hood. Remove aliquots for preparing a
balance tube if one will be required.
5.2.4 Set up one Beckman SW28.1 ultraclear tube for each gradient
in the virus hood, and place a sterile Pasteur pipet into the
neck of each tube.
5.2.5 Add 3mL of CsCI (1.25 g/mL) through the Pasteur pipet for
each tube. Slowly underlay 3mL of CsCI (1.40 g/mL) through
the Pasteur pipet. Carefully remove the pipette and place in
the sharp container.
NOTE: Check to see that the gradient interface has formed.
This appears as a silver or gray line in the middle of the
liquid. If the line hasn't formed, dispose of the cesium and
set up a new tube.
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5.2.6 Attach an 18 gauge, 1.5-inch needle to a 1 Occ syringe with the
plunger removed. Insert the needle into the neck of the tube in
place of the Pasteur pipet.
5.2.7 Add 5-7 mL of CVL into the syringe. Do not load more than
7mL of CVL on a single gradient. To avoid disrupting the
gradient, allow the CVL to drip from the needle. Dispose of
the needle in the sharp container.
5.2.8 With a clean syringe/needle add DPBS into each tube,
completely filling each tube excluding the neck.
5.2.9 In the virus hood, weigh the tubes and balance pairs within
O.OSg, using DPBS to balance the tubes.
5.2.10 Using the Beckman heat sealer, heat-seal the tubes. Place the
metal caps on top of each tube. Pressing the red button, touch
the sealer to the metal cap. Allow the sealer to melt the tube
until the metal cap reaches the rounded part of the tube. After
the cap reaches the top of the tube, hold the metal cap in place
with the black rod and allow the plastic to cool. Once the
plastic has set pull the metal cap off. Rinse metal caps with
anti-viral disinfectant and alcohol.
5.2.11 Place the tubes into the SW 28.1 buckets. Then add the SW
28.1 black spacers to the top of the tube.
5.2.12 Match the lid of the bucket to the number on the bucket. Screw
the lid on until it is tight and the numbers on the lid and bucket
match up. The balanced pairs must be run as a pair. Bucket 1
and 4, 2 and 5, and 3 and 6, are the pairs.
5.2.13 Load buckets onto SW 28 swinging bucket rotor. This also
requires the numbers on the rotor and the bucket to match up.
Give the bucket a slight tug to ensure that the bucket is
securely attached to the rotor. All six buckets must be run.
Run buckets empty if fewer than 6 tubes are set up.
5.2.14 Turn on the Beckman Ultracentrifuge. Place the rotor in the
centrifuge and close the door. Go to program library. Find
the program number that corresponds to 1 hour at 28,000 rpm
at 20°C. Press the number and enter. Then press the rotor
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button and pick the correct serial number for the rotor being
run and press enter. Press enter one more time (total of twice)
and press the start button.
5.2.15 After centrifugation, press the vacuum button on the centrifuge.
Remove the rotor. Open the lid and pull out the spacers and
then the tube using hemostats. Return to the virus hood. Two
bands should be visible. The bottom band is the purified virus.
The upper band is cellular debris. A dark or black background
behind the tube may make it easier to clearly see the bands.
NOTE: If tube is stuck in the bucket, grasp tube with
hemostats and twist the tube while lifting. Be careful not to
jiggle the tube too much. If the tube is leaking because of
collapsing, return the rotor/bucket to the virus hood and
remove the tube.
5.2.16 Wipe or spray the tube with alcohol and allow to dry. Puncture
the top of the tube with a 1-inch needle to break the vacuum.
5.2.17 Using an 18 gauge, 1.5-inch needle attached to a syringe,
carefully puncture the tube about 1 cm below the bottom band.
Slowly pull back on the plunger of the syringe to collect the
band, being sure to harvest only the bottom band. Remove the
needle and place the virus in a sterile SOmL tube. The tube
from which you collect the virus with cesium in it is discarded
in the sharp container. Discard all needles as sharp waste.
5.3 Continuous Gradient (Overnight)
5.3.1 The total volume of virus that has been collected from the
discontinuous gradients in a sterile SOmL tube should be
divided by seven for the number of gradients that will be set
up for the overnight gradient.
5.3.2 Set up one NVT polyallomer tube for each gradient in the virus
hood, and place a sterile Pasteur pipet into the neck of each
tube. Add the virus with a pipette through the Pasteur pipet.
Do not load more than 7mL of virus per tube.
5.3.3 In the clean hood, remove enough CsCI (1.33 g/mL) to fill each
tube completely with CsCI. The total capacity of the tube is
CA 02488778 2004-12-07
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approximately l3mL. Remember to remove aliquots for
preparing a balance tube if one is needed.
5.3.4 After filling the NVT tube with virus, fill each tube to be
centrifuged completely with CsCI (1.33 g/mL), using a
syringe/needle. If running a blank, fill the tube completely
with CsCl (1.33 g/mL).
5.3.5 Balance pairs of tubes within O.OSg using CsCI ( 1.33 g/mL) to
balance and heat seal as before.
5.3.6 Place tubes in the NVT rotor and place the blue inserts on the
tops of the tubes. Place the rotor lids on. Using the torque
wrench, tighten the rotor lids to 120 inch pounds. Empty
chambers should be run without caps or inserts.
5.3.7 Place the rotor in the centrifuge and close the door. Go to
program library. Find the program number that corresponds to
"hold" at 60,000 rpm at 20°C. Press the number and enter.
Then press the rotor button and pick the correct serial number
for the rotor being run and press enter. Press enter one more
time (total of twice) and press the start button. Record
information needed in the Beckman Log Book.
5.3.8 The overnight run must be a minimum of 15 hours. Press the
stop button. After the spin is complete, press the vacuum
button. Take the rotor from the centrifuge and pull the bottom
bands as before.
5.4 Dialysis
5.4.1 Add glycerin to the virus to make a final concentration that is
approximately 10% glycerin.
5.4.2 Thoroughly mix the virus and the glycerin by pipetting up and
down in the tube.
5.4.3 Remove up to 3.SmL of virus with a syringe and an I 8 gauge,
1-inch needle. Inject the virus into the Slide-A-Lyzer dialysis
cassette (10,000 MW cut-off, Pierce Cat #66425) through port
#1.
5.4.4 Before removing the needle from the cassette, pull back on the
plunger of the syringe to remove the air and utilize all of the
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surface area of the cassette. It may be necessary to go to
another port to remove all of the air. Use each port only once
when injecting virus because ports may leak if used more than
once.
5.4.5 Each cassette is dialyzed in one liter of cold THPG dialysis
buffer (200mM Tris, 50 mM Hepes, 0.3% o-Phosphoric acid,
10% Glycerin).
5.4.5.1 Place the cassettes in an autoclaved foil-covered
beaker with a stir bar, one group per beaker.
5.4.5.2 Beakers are placed at 2-~°C on a stir plate set on
medium-low speed.
5.4.5.3 There will be a total of three changes of dialysis
buffer. There is at least one hour between changes.
For the changes, aspirate the buffer from the beaker,
then add the same amount of buffer as before. One
change continues overnight, for a minimum of 10
hours.
6.0 PARTICLE TITER DETERMINATION AND FILL
6.1.1 Turn on the UV lamp on the spectrophotometer prior to use.
Turn on the visible light lamp immediately before use.
6.1.2 Take syringe with needle and pull the plunger back to capture 1
cc of air. Insert the needle into the cassette port. Slowly push
down on the plunger to inject air into the dialysis cassette.
6.1.3 Flip the cassette upside down and pull back on the plunger to
remove the virus from the cassette. Remove the needle from
the syringe using hemostats, place the virus in a sterile SOmL
tube.
6.1.4 Deep the virus in the refrigerator until aliquoted.
6.1.5 Determine the particle titer by optical density (OD26o).
6.1.5.1 Mix 3.75mL of TE buffer and 40p.1 of 10% SDS in a
lSmL tube, to serve as Lysis buffer.
6.1.5.2 Label 2 non-sterile Eppendorf tubes per virus
sample and one for a blank.
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NOTE: It may be necessary to use a lower dilution (such as
1:5) if the virus is suspected to have a lower particle titer.
Volume of Lysis Volume of virus
buffer
Sample-1:10 dilution360pL 40p.L
(lOx)
Sample-1:20 dilution380pL 20p.L
(20x)
Blank 400 L None
6.1.5.3 Using a 1000pL pipetter add the correct amount of
Lysis buffer to the corresponding tubes from the
chart above. Using a 20pL or 200p.L pipetter add
the amount of virus indicated in the chart to the 20x
and l Ox tubes. Be sure to use a new tip each time to
ensure that Lysis buffer does not get into the virus
and to keep the virus sterile.
6.1.5.4 Vortex the tubes for approximately 10 seconds at a
setting of 5.
6.1.5.5 Allow the solutions to sit at RT for 15-30 minutes
before taking the readings.
6.1.5.6 Rinse the cuvette thoroughly with rinse water before
adding the blank. Use all 400~L of the blank when
reading. Read the blank as a blank at a wavelength
of 260nm and then as a sample on the
spectrophotometer.
6.1.5.7 Rinse the cuvette thoroughly with rinse water before
adding the samples. Read the 20x dilution before
the 1 Ox dilution. Rinse the cuvette with rinse water
in between each reading.
6.1.5.8 After the results appear on the screen print out the
data using the "print" command. Use the
Save/Clear command to allow the machine to exit.
Write on the printout indicating which line is the
blank, l Ox dilution, and 20x dilution of each sample.
Initial and date the printout.
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6.1.5.9 Determine the concentration of the virus by
multiplying the OD26o by the dilution ( 10 or 20) and
conversion factor (1.1x10'2). Final results should be
rounded to three significant figures.
6.2 Aliquot the virus.
6.2.1 Label the cryovials with the following:
Sample Number
Name of virus and Passage # on MRC-5
# of particles/mL
Date aliquoted Volume of aliquot
Store at -80C
6.2.2 Virus aliquots are stored in a -80°C freezer.
7.0 FURTHER PASSAGES OF VIRUS ON MRC-5 CELLS
7.1 Repeat the full procedure three more times using the purified virus or
CVL produced from the passage before.
7.2 Once four passages have been completed, perform endpoint analyses
and data interpretation as described.
8.0 ENDPOINT ANALYSES
8.1 Determine virus production at the end of each passage
8.2 Assess cytopathic effect (CPE) at the end of each passage
8.3 If there is evidence of increased virus yield and CPE, the following
additional analyses may be performed on virus harvested from any
passage:
8.3.1 Perform restriction digests on harvested virus.
8.3.2 Determine potency of harvested virus on MRC-5 cells using a
cytotoxicity assay with MTS readout.
8.3.3 Assess replication competence by hexon FAGS analyses
following infection of indicator cells
9.0 DATA INTERPRETATION
9.1 CPE and increased production at the end of four passages is interpreted
as the presence of a replication competent adenovirus (RCA)
9.2 Aberrant banding patterns on restriction digest gels after passage on
MRCS cells also is interpreted as the presence of recombinant
adenovirus.
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9.3 Increased potency after passage on MRCS cells is interpreted as a
recombinant that has lost selectivity.
9.4
EXAMPLE 14: Cytotoxicity potency assay with MTS readout.
MRC-5 cells are seeded in 96-well tissue culture dishes at 10,000 cells per
well
in 100 ~l 10% EMEM. The next day, adenoviruses are serially diluted 1:3 in 10%
EMEM. The growth medium from each plate is removed and 100 ~,l of each viral
dilution are added to the appropriate wells on each of four plates. The
dilution ranges
tested are 3.3 x 10$ to 7.0 x 101 vp/mL (or 3.3 x 103 to 7.0 x 10-4 particles
per cell,
ppc). Cells are exposed to virus for ten days after which the Promega
CellTiter 96~
AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) is performed
according to the manufacturer's instructions (Promega Tech. Bulletin #TB 169).
Absorbance units are converted to percent uninfected control values (% Max
Control)
and replicates are averaged (~ SD) between the four plates. A sigmoidal dose-
response curve of average % Max Control plotted versus vector viral particle
dose
(ppc) in logarithmic scale is fit to the data and an EC50 value is determined
for each
virus tested, using GraphPad Prism software.
EXAMPLE 15: Determination of viral vector particle concentration by
OD260/SDS procedure.
Samples are diluted at 1:10 and 1:20 into lysis buffer (0.106% SDS in TE
buffer; BioWhittaker #16-013B) and incubated for 15-30 min at room temperature
prior to determining absorbance readings. Samples are analyzed using a Beckman
DU
640 spectrophotometer. The absorbance reading using lysis buffer only is
adjusted to
zero at 260 nm, and then absorbance readings of each dilution are obtained.
Viral
particle concentrations are determined using the following formula (Maizel, et
al
1968): Concentration (particles/ml) _ (A260 nm) x (1.1 x 102 particles/ml) x
( 1 /dilution factor)
A similar procedure is described by Mittereder et al. ( J Virol.l996 Nov
;70(11):
7498-509)
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EXAMPLE 16: Quantitative PCR.
All PCR reactions are performed in an ABI PRISM 7700 Sequence Detection
System (Applied Biosystems). PCR data is analyzed using the Sequence Detection
System software version 1.6.3 (Applied Biosystems).
To quantify the amount of wild type Ad5 in purified oncolytic virus samples, a
quantitative real-time PCR is designed to detect the wild-type ElA promoter. A
standard curve is generated using 1.2 ~.g (3 x 10'° viral genomes) of
Ar6pAE2fE3F
viral genomic DNA as background mixed with known amounts of Ad5 viral genomic
DNA, ranging from 10 to 1 x 106 viral genomes. A negative control of 1.3 ~g (3
x
101° plasmid copies) of plasmid containing the Ar6pAE2fE3F sequence is
included in
each assay. Each 100 pl reaction of standard or unknown contains: 1.2 ~.g DNA
(3 x
101° viral genomes), lx TaqMan Universal PCR Master Mix (Applied
Biosystems),
0.3 ~M of each primer (forward primer 5'-GACCGTTTACGTGGAGACTCG-3'
(SEQ ID NO: l ) and reverse primer 5'-TCGGAGCGGCTCGGA-3' (SEQ ID N0:2))
and 0.1 ~M of the probe (5'-FAM-TTTTCCGCGTTCCGGGTCAAAGT-TAMRA-3'
((SEQ ID N0:3)). The PCR reactions are performed under the following
conditions:
50°C for 2 min, 95°C for 10 min, followed by 60 cycles of
95°C for 15 sec and 60°C
for 1.5 min.
Adenoviruses can be grown (amplified) on PerC.6 cells. PerC.6 cells contain an
expression cassette with Ad5 E 1 a operably linked to a PGK promoter. If
adenoviruses are grown on a PerC.6 cell line there is a potential for the PGK
promoter
to be incorporated in to the adenovirus. The PGK promoter has been reported to
incorporate into the adenovirus gemone between the ITR and the El region in
such a
way that it is operably linked to the adenovirus E1 coding regions. Thus
replacing or
interfering with any promoter that may have been operably linked to the E 1
coding
region to selectively control replication of the virus.
To determine the amount of PGK promoter-containing recombinants in
adenoviral preparations, (if any) the PCR assay is designed to detect
adenoviral
sequences containing the PGK promoter/ElA junction as found in PER.C6 cells. A
standard curve is generated using a background of 5 x 101° PGK promoter-
negative
adenoviral genomes (2 ug of adenoviral genomic DNA) mixed with 50 to 1 x 106
copies of a plasmid containing the PGK/Ela junction sequence. Each 100 ~.l
reaction
of either standard or unknown sample contains: 2 ug adenoviral genomic DNA, lx
TaqMan Universal PCR Master Mix, 0.3 ~,M of each primer (forward primer 5'-
56
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CCGCACGTCTCACTAGTACCC-3' (SEQ ID N0:4) and reverse primer 5'-
ACACGATCGAATTCGGAACG-3' (SEQ ID NO:S)) and 0.1 pM of the probe (probe
5'-6FAM-CGGAGCGGGATCGAGCCCTCT-TAMRA-3' (SEQ ID N0:6)). The PCR
reactions are carried out under the following conditions: 50°C for 2
min, 95°C for 10
min, followed by 60 cycles of 95°C for 15 sec and 60°C for 1.5
min. In one of these
reactions the limit of detection of the assay was 50 copies of PGK/Ela per 5 x
10'°
adenoviral genomes.
To quantitate REC133, a standard curve is generated using 10 to 1 x 106 copies
of a plasmid containing a cloned REC133 restriction fragment in a background
of 1 x
l Og adenoviral genomes (4 ng adenovioral genomic DNA). Each 100 ~1 PCR
reaction
contains: 4 ng adenoviral genomic DNA, 1 x TaqMan Universal PCR Master Mix, 1
~M of each primer (forward primer 5'-TTTCTGGGCGTAGGTTCGC-3' (SEQ ID
N0:7) and reverse primer 5'-GGTAATAACACCTCCGTGGCA-3' (SEQ ID N0:8))
and 0.2 p.M of the probe (probe 5'-6FAM-CGGAGCGGGATCGAGCCCTCT-
TAMRA-3' (SEQ ID N0:9)). The PCR reactions are carried out under the following
conditions: 50°C for 2 min, 95°C for 10 min, followed by 45
cycles of 95°C for 15 sec
and 64°C for 1 min. In one of these reactions the limit of detection of
the assay was
copies of REC133 per 1 x 108 adenoviral genomes.
REFERENCES
Adams, P.D. and Kaelin, W.G.J. (1995) Semin. Cancer Biol. 6, 99-108.
Alemany, R., et al. (2000) Nat. Biotechnol. 1~, 723-727.
Curiel, D.T. (2000) Clin. Cancer Res. 6, 3395-3399.
Dion, L.D., Fang, J., and Carver, R.LJ. (1996) J. Virol. Methods 56, 99-107.
Fallaux, F.J., et al. (1998) Hum. Gene Ther. 9, 1909-1917.
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Hawkins, L.K., et al. (2002) Lancet Oncol. 3, 17-26.
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pathway defective tumors: dependence on ElA, the E2F-1 promoter and viral
replication for selectivity and efficacy. Cancer Research in pf-ess..
Lehmberg, E., et al. (2002). Analytical assays to characterise adenoviral
vectors and their applications. In Manufacturing of Gene Therapeutics;
Methods,
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Processing, Regulation, and Validation, G.Subraminian, ed. (New York: Kluwer
Academic / Plenum Publishers), pp. 201-225.
Lochmuller, H., et al. (1994) Hum. Gene Ther. 5, 1485-1491.
Lu, K., et al. Cancer Chemother. Pharmacol. (2000) 46:293-304.
Maizel, J.V.J., et al. (1968) Virology 36, 115-125.
Melcher, A., et al. (1999) Hum. Gene Ther.10, 1431-1442.
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Parr, M.J., et al. (1997) Nat. Med. 3, 1145-1149.
Ring, C.J. (2002) J. Gen. Virol. 83, 491-502.
Roitsch, C., et al., B. Biomed. Sci. Appl. 752, 263-280.
Sladek, T.L. (1997) Cell Prolif. 30, 97-105.
Smith, J.G. and Eck, S.L. (1999) Cancer Gene Ther. 6, 475-481.
Zhang, W.W., et al. (1995) Biotechniques 1~, 444-447.
Zhu, J., et al. (1999) Hum. Gene Ther. 10, 113-121.
Zwicker, J. and Muller, R. (1995) Prog. Cell Cycle Res. l: 91-99.
The disclosures of all patents, patent applications, publications (including
published patent applications), and database accession numbers referred to in
this
specification are specifically incorporated herein by reference in their
entirety to the
same extent as if each such individual patent, patent application,
publication, and
database number were specifically and individually indicated to be
incorporated by
reference in its entirety.
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SEQUENCE LISTING
<110> Bowe, Mark
Lyons, Russette
Walker, Tracey
<120> ASSAY TO DETECT REPLICATION COMPETENT VIRUSES
<130> 4-32523A
<150> US 60/387,251
<151> 2002-06-07
<150> US 60/459,375
<151> 2003-03-31
<160> 9
<170> Patentln version 3.1
<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> PCR Primer
<400> 1
gaccgtttac gtggagactc g 21
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<211> 15
<212> DNA
<213> Artificial Sequence
Page 1
CA 02488778 2004-12-07
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<220>
<223> PCR Primer
<400> 2
tcggagcggc tcgga 15
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<Z20>
<223> Probe
<400> 3
ttttccgcgt tccgggtcaa agt 23
<210> 4
<211> 21
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<213> Artificial Sequence
<220>
<223> PCR primer
<400> 4
ccgcacgtct cactagtacc c 21
<210>5
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<213>Artificial sequence
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acacgatcga attcggaacg 20
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Page 2
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<212> DNA
<213> Artificial Sequence
<Z20>
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<400> 6
cggagcggga tcgagccctc t 21
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tttctgggcg taggttcgc 19
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<223> PCR primer
<400> 8
ggtaataaca cctccgtggc a 21
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<212> DNA
<213> Artificial sequence
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<400> 9
cggagcggga tcgagccctc t 21
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