Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
USE OF HELPER-DEPENDENT ADENOVIRAL VECTORS OF
ALTERNATIVE
SEROTYPES PERMITS REPEAT VECTOR ADMINISTRATION
GOVERNMENT SUPPORT:
Portions of the work described in this invention disclosure was supported by a
grant
from the US National Institutes of Health; accordingly, the U.S. Government
may
have certain rights in this invention.
CROSS-REFERENCES TO RELATED INVENTIONS:
This application is a continuation-in-part of co-pending application serial
number
09/251,955, filed February 17, 1999, which was a continuation-in-part of
copending
application serial number 08/473,168, filed June 7, 1995, which was a
continuation-
in-part of copending application serial number 08/250,885, filed May 31, 1994,
which
was a continuation-in-part of application serial number 08/080,727, filed June
24,
1993,abandoned.
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION:
This invention relates to the field of gene therapy and vaccine delivery, and
provides a
significant advance in the art by facilitating repeat administration of a
transgene in a
vector, circumventing anti-vector immune responses, which diminish the
efficacy of
known gene delivery vectors.
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BACKGROUND OF THE INVENTION:
Recently, adenovirus (Ads) vectors have received considerable attention for
transgene
delivery to mammalian cells generally and for gene therapy in particular due
to several
advantages over other vector systems, including high transduction efficiency
of a
variety of cell types comprised of both replicating and non-replicating cells,
ease of
growth, and relative safety (for review see Hitt et al. 1997). However, data
from
preclinical and clinical studies have shown that Ads also have several
disadvantages,
primarily due to the induction of both cellular and humoral immune responses
to
vector-derived antigens (Yang et al. 1994a, 1995a,b, 1996a,b, Dai et al. 1995,
Gilgenkrantz et al. 1995, McCoy et al. 1995, Christ et al. 1997, Morral et al.
1997,
van Ginkel et al. 1997, Kafri et al. 1998). Because of these immune responses,
administration of first-generation Ad vectors (i.e. deleted of early region 1
(E1) or
E1/E3) has generally resulted in only transient transgene expression and poor
expression following repeat vector administration (bong et al. 1996, Kaplan et
al.
1996, St. George et al. 1996, Schulick et al. 1997). Reintroduction of the E3
region,
which encodes functions involved in aiding virus escape from host immune
responses,
can prolong transgene expression in some animal models (Lee et al. 1995,
Poller et al.
1996, Bruder et al. 1997, Ilan et al. 1997, Schowalter et al. 1997), and is
reported to
decrease the formation of anti-Ad neutralizing antibodies (Ilan et al. 1997).
The use
of second-generation Ad vectors, which are further deleted or attenuated in E2
or E4,
can lead to decreased inflammatory responses and a longer duration of
transgene
expression (Engelhardt et al. 1994, Yang et al. 1994b, Goldman et al. 1995,
Gao et al.
1996, Dedieu et al. 1997, Wang et al. 1997, Amalfitano et al. 1998), although
not in
all cases (Fang et al. 1996, Christ et al. 1997, Morral et al. 1997, Lusky et
al. 1998).
However induced antibody titers were similar to those generated against first
generation vectors (Christ et al. 1997), thus compromising the ability to
readminister
the vector.
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The development of systems for the generation of helper-dependent Ad vectors
(hdAd) which are deleted for most if not all viral coding sequences (Mitani et
al.
1995, Fisher et al. 1996, Haeker et al. 1996, Kochanek et al. 1996, Kumar-
Singh and
Chamberlain 1996, Lieber et al. 1996, Parks et al. 1996, Hardy et al. 1997,
reviewed
S by Hitt et al. 1998), has allowed production of hdAd which can provide long
term,
high level transgene expression (Chen et al. 1997, Schiedner et al. 1998,
Morsy et al.
1998), and which result in substantially reduced inflammatory and cellular
immune
responses (Morsy et al. 1998, Schiedner et al. 1998, Morral et al. 1998).
However, as
expected, deletion of all Ad coding sequences does not overcome the humoral
immune response, and neutralizing antibodies are formed (J.L. Bramson, R.J.P.
and
F.L.G., unpublished results), thus reducing the effectiveness of hdAd vector
readministration.
In an attempt to prevent vector-directed immune responses, many groups have
explored the use of transient immune blockage at the time of Ad vector
administration, or the induction of tolerance to Ad (Vilquin et al. 1995,
Jooss et al.
1996, Kass-Eisler et al. 1996, Kolls et al. 1996, Lochmuller et al. 1996,
Sawchuk et al.
1996, Smith et al. 1996, Yang et al. 1996c, Zepeda and Wilson 1996, Kaplan and
Smith 1997, Kuzmin et al. 1997, Lieber et al. 1997, Scaria et al. 1997, Wolff
et al.
1997, Zsengeller et al. 1997). These strategies have been somewhat successful,
and
allow repeat vector administration; however, complications and potential side-
effects
may make immune suppression impractical for clinical use. An alternative
strategy to
allow for repeat vector administration is the sequential use of different Ad
serotypes.
Neutralizing antibodies formed against one serotype should have no effect on
subsequent delivery of a different serotype, and this approach has allowed
repeat
administration of first generation Ad vectors (Kass-Eisler et al. 1996,
Mastrangeli et
al. 1996, Mack et al. 1997, Roy et al. 1998, A.L. Beaudet, unpublished
results). Over
40 different serotypes of human Ads have been isolated, suggesting that, in
theory, Ad
vectors of different serotypes could be administered many times throughout the
life of
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a patient. However, how to achieve this feat does not appear to have been
disclosed
or suggested for helper dependent vectors.
The Cre/loxP system for producing helper-dependent Ad vectors involves the use
of a
helper virus that contains a packaging signal flanked by loxP sites (Parks et
al. 1996).
Upon infection of a 293-derived cell line that stably expresses the
bacteriophage P1
Cre recombinase (Chen et al. 1996), the packaging signal is excised from the
helper
virus DNA, rendering it unpackageable. The helper virus DNA retains the
ability to
replicate and provides all of the functions required in traps for the
replication and
packaging of a hdAd. This system facilitates the generation of high titer hdAd
preparations with substantially reduced quantities of contaminating helper-
virus. A
key feature of the helper-dependent system is that the serotype of the hdAd is
determined only by the helper virus. Therefore, in contrast to first
generation vectors
that require the construction of a new vector to switch serotypes, a series of
genetically identical hdAds of different serotypes could be generated simply
by
changing the serotype of the helper.
SUMMARY OF THE INVENTION
We have developed a new helper adenovirus (Ad) based on serotype 2,
Ad2LC8cCARP,
for use in the Cre/loxP system (Parks et al. 1996, Proc. Natl. Acad. Sci. USA
93:13565-
13570) to generate Ad vectors deleted of all protein coding sequences (helper-
dependent
Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our helper virus developed
previously (based on serotype 5, Ad5LC8cluc) showed that the two helper
viruses
amplified hdAd with a similar efficiency, and resulted in a similar yield and
purity after
large scale preparation of vector. In vitro, the resulting hdAd2 had a similar
transduction
efficiency and expressed levels of transgene ((3-gal) identical to those
produced by
hdAdS. An important feature of the helper-dependent system is that all virion
components, except the virion DNA, derive from the helper virus. Consequently
vectors
produced with help from Ad2LC8cCARP were not neutralized by antibodies against
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AdS, and vectors produced with Ad5 helper were resistant to neutralizing
antibodies
against Ad2. Analysis of transgene expression in vivo after transduction of
mouse liver
by intravenous inj ection of the Ad2-based hdAd showed that the vector could
efficiently
transduce hepatocytes, and produce high levels of a foreign transgene (human
secreted
5 alkaline phosphatase), similar to those expressed by the hdAd generated with
the Ad5
helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies,
which
did not cross-react with hdAdS. To determine if successful repeat Ad vector
administration could be achieved by sequential use of alternative Ad
serotypes, we
injected mice with hdAd2 (hSEAP) followed three months later by a lacZ-
expressing
hdAd of either the same or different serotype. Administration of a vector of
the same
serotype resulted in a 30- to 100-fold reduction in transgene expression
compared to
naive animals. In contrast, no decrease in transgene expression was observed
when the
second vector was of a different serotype. These results suggest that
effective vector
readministration can be achieved by the sequential use of hdAds based on
alternative
serotypes.
Accordingly, this invention responds to a long felt need, this invention
provides, in
one embodiment, a helper virus based on the Ad2 serotype for use in the
Cre/loxP
system for the generation of Ad vectors deleted of all Ad protein coding
sequences.
Using this and helper virus based on AdS, genetically identical hdAd that
differ only
in the virion protein components, which are derived from the helper virus,
were
produced. The vectors have identical expression characteristics in vitro,
regardless of
the serotype, and the sequential use of hdAd of different serotypes allows for
successful repeat vector administration in vivo.
Accordingly, it is one object of this invention to provide a helper-dependent
adenovirus vector (hdAd) administration system whereby repeat administration
of a
gene of interest is facilitated by using hdAd wherein all protein present in
said hdAd
is derived from a helper virus, the serotype of which is switched in the
production of a
vector to be used in a repeat hdAd administration.
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Another object of this invention is to provide a generally applicable
strategy, not
restricted to adenoviruses, whereby repeat administration of a gene is
facilitated, such
that high level gene expression occurs on each administration.
Another object of this invention is to provide a system whereby helper
adenoviruses
of different serotypes are used to generate a series of hdAd vectors against
which
humoral and cellular immune responses are minimized, while providing for
repeat
administration of genes of interest.
Other objects and advantages of this invention will become apparent from a
review of
the complete disclosure and the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. pRP1045 (shown in linear form) is deleted of all Ad protein coding
sequences but contains an Ad5 head-to-tail inverted terminal repeat (ITR)
junction
and packaging signal, and encodes the E. coli ~3-galactosidase gene under the
regulation of the murine cytomegalovirus immediate-early promoter and Simian
virus
40 polyadenylation sequence. pRP 1045 also contains a ~22 kb fragment of the
human
hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene as stuffer, in
order to
maintain the size of the resulting vector within the limits for efficient Ad
DNA
packaging (Parks and Graham 1997). pRP1050 is similar to pRP1045, but is
deleted
of a 1.2 kb StuI fragment from the HPRT sequence, and the resulting vectors
(AdRP1045 and AdRP1050) have essentially identical expression characteristics.
pRP 1046 is similar in structure to pRP 1050, but encodes a cDNA for human
secreted
alkaline phosphatase gene (hSEAP, Tropix) in place of the lacZ gene. The HPRT
genomic sequence was obtained from Dr. Andrew J. Bett (Merck Research
Laboratories, West Point, PA). All hdAd vectors were amplified using the
appropriate helper virus in 293Cre4 cells, as previously described (Parks et
al. 1996,
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Parks and Graham 1997), and the DNA structures of all hdAd vectors were
confirmed
by restriction digestion analysis of DNA isolated from virions. The titer of
each
vector was determined on 293 cells as the number of transducing particles, or
blue
forming units (BFU), per ml. For AdRP1046, the total particle count, as
determined
spectrophotometrically (lAZbo - 1.1x10'2 particles per ml), was used to
estimate the
number of transducing particles, assuming a particlearansducing particle ratio
of
100:1. For clarity, hdAd are designated with the appropriate serotype. For
example,
AdSRP1050 and Ad2RP1050 are generated using Ad5LC8cluc and Ad2LC8cCARP,
respectively.
Figure 2. Construction of Ad2LC8cCARP. Panel A: Strategy for the generation of
an Ad2 virus with a loxP-flanked packaging signal. Panel B: Strategy for the
rescue
of a stuffer segment into the E3 region of Ad2LC8c. Ad2LC8cCARP was
constructed
by a combination of molecular cloning and in vivo recombination techniques, as
detailed in the Materials and Methods. The final viral construct, Ad2LC8cCARP,
is
deleted of Ad sequences between 339-3533 by (E1), and contains a packaging
signal
flanked by loxP sites. Ad2LC8cCARP also contains a 5.6 kb fragment of lambda
DNA inserted within the E3 region. Regions of the Ad genome are delineated by
whether they are of Ad2 or Ad5 origin, and the appropriate nucleotide number
according to the conventional Ad2 or Ad5 map. Restriction enzyme sites used in
virus construction are also shown. Ad5 packaging signal ('I'), loxP sites
(black
triangles), Ad5 ITR (black arrow).
Figure 3. Amplification of Ad2RP1050 using Ad2LC8cCARP. After each serial
passage, an aliquot of the resulting crude vector lysates was titered for the
presence of
lacZ-transducing particles (blue forming units - bfu), as previously described
(Parks et
al. 1996). Amplifications were performed in duplicate, and the average bfu/ml
is
reported.
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Figure 4. Effect of Ad5 neutralizing antibodies on hdAd2 or hdAdS. Serial
dilutions
of Ad5 neutralizing antibodies were incubated for 1 hr with 106 bfu of
Ad2RP1050 or
Ad5RP1045, or 106 pfu of Ad5CA35, a first generation Ad vector containing an
identical expression cassette in place of the El region (Addison et al. 1997).
The
resulting vector was used to infect 22-mm dishes of A549 cells, and the
quantity of (3-
gal assayed at 24 hr post-transduction. Using this assay, the quantity of (3-
gal
expressed is proportional to the transduction efficiency (ie titer) of the
vector.
Figure 5. hZ vitro expression from an Ad2- versus Ad5-based hdAd. Ad2RP1050
and Ad5RP1045 are similar in structure, and contain an identical MCMV-lacZ
expression cassette, but were generated using Ad2LC8cCARP and AdLCBcluc,
respectively. Monolayers of A549 cells in 60-mm dishes were transduced in
duplicate
with 106 bfu of vector, crude protein lysates prepared at various times post-
transduction, and assayed for (3-gal activity. The average of the duplicate
samples is
reported.
Figure 6. In vivo expression from an hdAd2. Adult FVB/n mice were injected
through the tail vein with 5x10'° particles (approximately 5x108
transducing particles)
of Ad2RP1046 (n=6). At various times post-injection, blood samples were
removed
by orbital bleed, and the serum isolated. Aliquots of serum were assayed for
hSEAP
activity using a chemiluminescent assay, and compared to a standard curve of
purified
hSEAP to determine the quantity of hSEAP in each sample. The average hSEAP for
all mice is reported.
Figure 7. Formation of Ad2-specific neutralizing antibodies in animals
immunized
with an hdAd2. Adult FVB/n mice (n=3) were injected through the tail vein with
5x10'° particles of Ad2RP1046. At Day 28 post-injection, serum samples
were
collected and assayed for Ad neutralizing antibodies, as described in the
Materials and
Methods. Serial dilutions of antibody were incubated with Ad2RP1050 (Panel A)
or
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AdSRP1050 (Panel B) for 1 hr, and assayed for transduction efficiency on A549
cells.
The data from all 3 mice are presented.
Figure 8. Transgene expression from pre-immunized mice (hdAd2) using either
the
same (hdAd2) or alternative (hdAdS) serotype. FVB/n mice were immunized with
10'° particles of Ad2RP1046 and, 90 days later, injected i.v. with 108
bfu of
Ad2RP1050 or Ad5RP1050. Three or six days after the second vector was
administered, the mice were sacrificed and the livers assayed for (3-gal
activity. Each
bar represents the average of two mice, and the error bars represent the
maximum
value.
DETAILED DISCLOSURE OF THE PREFERRED EMBODIMENTS AND BEST
MODE
The present invention provides a significant advance in the art of gene
therapy and
vaccine delivery, in that it provides a method whereby repeat administration
of a gene
vector according to this method can circumvent anti-vector immune responses.
This is
accomplished by modifying the protein coat of a viral gene vector on each
repeat
administration. While this may be accomplished using any helper-dependent
viral
system, the invention is exemplified with reference to helper-dependent
adenoviruses.
According to this embodiment of the invention, a helper-dependent adenovirus
is
produced encoding a gene, the expression of which is desired, either to induce
a specific
desired immune response against the encoded gene product, for vaccine
applications, or
because a particular genetic function is desired. For example, complementation
of a
genetic defect such as in cystic fibrosis, (e.g. provision of the CFTR gene
product, see US
Patent No. 5,882,877 and 5,670,488, hereby incorporated by reference), or
provision of
anti-sense RNA, or provision of an enzyme or enzyme inhibitor, or structural
gene
product or required hormone or cytokine or immunomodulator~protein, all may be
accomplished according to the present methodology[,] without inhibition by
recipient's
humoral or cellular immune responses elicited by a previous exposure to the
gene vector.
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In one exemplary application of the present invention, a desired gene of
interest is cloned
into a helper-dependent adenoviral vector (hdAd), comprising the left
adenoviral ITR,
the right adenoviral ITR, an adenoviral packaging signal, and sufficient
additional
sequences to ensure efficient packaging of the hdAd vector thus produced. The
desired
S gene of interest is cloned into the hdAd vector with required
transcriptional initiation
(promoter) and termination signals, as are known in the art, in order to
ensure efficient
transcription and translation of the gene of interest, upon introduction into
an appropriate
host cell, by means of the adenoviral vector. The hdAd vector preferably has a
genomic
size between about 75% and 100% of the natural genome size of an adenovirus,
to ensure
10 efficeint packaging of the adenoviral veector genome. The hdAd is co-
transfected into
an appropriate cell in vitro with the helper virus, to generate a stock of
hdAd vector.
Preferably, the cell provides functions necessary for replication or packaging
of the hdAd
vector, as in 293 cells which complement adenoviral vector deletions in the E1
coding
sequences, or such complementation may be provided by the helper virus. In
addition,
it is preferred that an efficient method is provided for elimination of helper
virus from the
stock of hdAd vector that is produced. As disclosed in co-pending applications
related
to the present application, and in PCT publications W096/40955, W095/00655,
and
W098/13510, all of which are hereby incorporated by reference for this
purpose, a
system may be employed whereby a helper virus having a packaging signal is
flanked by
lox sites. Co-transfection of such a virus into a cell in which the Cre
recombinase is
expressed results in excision of the helper virus packaging signal, making the
genome of
the helper virus non-packageable. Since the hdAd vector genome encoding the
gene of
interest has a packaging signal, the hdAd vector is efficiently packaged,
essentially free
of helper viral genome contamination. Additional methodologies may be employed
to
eliminate helper viral genome contamination, and those technologies are
applicable to
this invention, whether previously reported on, or when such technologies
become
publicly available. Thus, regardless of the methodology by which helper viral
genome
contamination is limited, an essentially pure preparation of hdAd vector is
produced
which contains a vector genome encoding a desired gene of interest. The capsid
of the
packaged hdAd vector is completely defined by structural proteins of the
helper virus.
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Accordingly, by preparing a helper virus of a first adenoviral serotype, e.g.
serotype S,
for packaging of a first hdAd vector preparation, and a subsequent helper
virus from a
second adenoviral serotype, e.g. human Ad serotype 2, serotype 1, serotype 6,
or other
human adenoviruses or adenoviruses derived from non-human serotypes, for
packaging
of a second hdAd vector preparation, and a subsequent helper virus from a
third
adenoviral serotype for packaging of a third hdAd vector preparation, and so
forth,
essentially unlimited variations in the capsid serotype maybe produced,
without the need
to make any modifications to the hdAd vector genome. Accordingly, in one
aspect of this
invention, there is provided a kit comprising a series of helper adenoviruses
of different
serotypes, such that upon production of any given hdAd vector, a complete
regimen of
an essentially unlimited number ofbooster hdAd vector administrations maybe
initiated,
without inhibition by anti-vector immune responses previously elicited in a
recipient
thereof. In addition, upon decay of immune responses directed against a first
serotype,
that same serotype may once again be used, thereby expanding the number of
consecutive administrations of the vector that may be employed.
In reviewing the detailed disclosure which follows, it should be borne in mind
that any
publications referenced herein are hereby incorporated by reference in this
application in
order to more fully describe the state of the art to which the present
invention pertains.
It is important to an understanding of the present invention to note that all
technical and
scientific terms used herein, unless otherwise defined, are intended to have
the same
meaning as commonly understood by one of ordinary skill in the art. The
techniques
employed herein are also those that are known to one of ordinary skill in the
art, unless
stated otherwise.
Reference to particular buffers, media, reagents, cells, culture conditions
and the like, or
to some subclass of same, is not intended to be limiting, but should be read
to include all
such related materials that one of ordinary skill in the art would recognize
as being of
interest or value in the particular context in which that discussion is
presented. For
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example, it is often possible to substitute one buffer system or culture
medium for
another, such that a different but known way is used to achieve the same goals
as those
to which the use of a suggested method, material or composition is directed.
The terms used herein are not intended to be limiting of the invention. For
example, the
term "gene" includes cDNAs, RNA, or other polynucleotides that encode gene
products.
"Foreign gene" denotes a gene that has been obtained from an organism or cell
type other
than the organism or cell type in which it is expressed; it also refers to a
gene from the
same organism that has been translocated from its normal situs in the genome.
In using
the terms "nucleic acid", "RNA", "DNA", etc., we do not mean to limit the
chemical
structures that can be used in particular steps. For example, it is well known
to those
skilled in the art that RNA can generally be substituted for DNA, and as such,
the use of
the term "DNA" should be read to include this substitution. In addition, it is
known that
a variety of nucleic acid analogues and derivatives is also within the scope
of the present
invention. "Expression" of a gene or nucleic acid encompasses not only
cellular gene
expression, but also the transcription and translation of nucleic acids) in
cloning systems
and in any other context. The term "recombinase" encompasses enzymes that
induce,
mediate or facilitate recombination, and other nucleic acid modifying enzymes
that cause,
mediate or facilitate the rearrangement of a nucleic acid sequence, or the
excision or
insertion of a first nucleic acid sequence from or into a second nucleic acid
sequence.
The "target site" of a recombinase is the nucleic acid sequence or region that
is
recognized (e.g., specifically binds to) and/or acted upon (excised, cut or
induced to
recombine) by the recombinase. The term "gene product" refers primarily to
proteins and
polypeptides encoded by other nucleic acids (e.g., non-coding and regulatory
RNAs such
as tRNA, sRNPs). The term "regulation of expression" refers to events or
molecules that
increase or decrease the synthesis, degradation, availability or activity of a
given gene
product.
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The present invention is also not limited to the use of the cell types and
cell lines used
herein. Cells from different tissues (breast epithelium, colon, lymphocytes,
etc.) or
different species (human, mouse, etc.) are also useful in the present
invention.
It is important in this invention to detect the generation and expression of
recombinant
nucleic acids and their encoded gene products. The detection methods used
herein
include, for example, cloning and sequencing, ligation of oligonucleotides,
use of the
polymerise chain reaction and variations thereof (e.g., a PCR that uses 7-
deaza GTP), use
of single nucleotide primer-guided extension assays, hybridization techniques
using
target-specific oligonucleotides that can be shown to preferentially bind to
complementary sequences under given stringency conditions, and sandwich
hybridization
methods.
Sequencing may be carned out with commercially available automated sequencers
utilizing labeled primers or terminators, or using sequencing gel-based
methods.
Sequence analysis is also carried out by methods based on ligation of
oligonucleotide
sequences which anneal immediately adjacent to each other on a target DNA or
RNA
molecule (Wu and Wallace, Genomics 4: 560-569 (1989); Landren et al., Proc.
Natl.
Acid. Sci. 87: 8923-8927 (1990); Barany, F., Proc. Natl. Acid. Sci. 88: 189-
193 (1991)).
Ligase-mediated covalent attachment occurs only when the oligonucleotides are
correctly
base-paired. The Ligase Chain Reaction (LCR), which utilizes the thermostable
Taq
ligase for target amplification, is particularly useful for interrogating late
onset diabetes
mutation loci. The elevated reaction temperatures permits the ligation
reaction to be
conducted with high stringency (Barany, F., PCR Methods and Applications 1: 5-
16
(1991)).
The hybridization reactions may be carried out in a filter-based format, in
which the
target nucleic acids are immobilized on nitrocellulose or nylon membranes and
probed
with oligonucleotide probes. Any of the known hybridization formats may be
used,
including Southern blots, slot blots, "reverse" dot blots, solution
hybridization, solid
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support based sandwich hybridization, bead-based, silicon chip-based and
microtiter
well-based hybridization formats.
The detection oligonucleotide probes range in size between 10-1,000 bases. In
order to
obtain the required target discrimination using the detection oligonucleotide
probes, the
hybridization reactions are generally run between 20°-60°C, and
most preferably
between 30°-50 ° C. As known to those skilled in the art,
optimal discrimination between
perfect and mismatched duplexes is obtained by manipulating the temperature
and/or salt
concentrations or inclusion of formamide in the stringency washes.
The cloning and expression vectors described herein are introduced into cells
or tissues
by any one of a variety of known methods within the art. Such methods are
described for
example in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold
Spring
Harbor Laboratory, New York (1992), which is hereby incorporated by reference.
See,
also, Ausubel et al., Current Protocols in Molecular Biolo~y, John Wiley and
Sons,
Baltimore, MD (1989); Hitt et al, "Construction and propagation of human
adenovirus
vectors," in Cell Biology: A Laboratory Handbook, Ed. J.E. Celis., Academic
Press. 2°a
Edition, Volume 1, pp: 500-512, 1998; Hitt et al, "Techniques for human
adenovirus
vector construction and characterization," in Methods in Molecular Genetics,
Ed. K.W.
Adolph, Academic Press, Orlando, Florida, Volume 7B, pp:l2-30, 1995; Hitt, et
al.,
"Construction and propagation of human adenovirus vectors," in Cell Biology
Laboratory Handbook," Ed. J. E. Celis. Academic Press. pp:479-490, 1994, also
hereby
incorporated by reference. The methods include, for example, stable or
transient
transfection, lipofection, electroporation and infection with recombinant
viral vectors.
The protein products of recombined and unrecombined coding sequences may be
analyzed using immune techniques. For example, a protein, or a fragment
thereof is
injected into a host animal along with an adjuvant so as to generate an immune
response.
Immunoglobulins which bind the recombinant fragment are harvested as an
antiserum,
and are optionally further purified by affinity chromatography or other means.
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Additionally, spleen cells may be harvested from an immunized mouse host and
fused
to myeloma cells to produce a bank of antibody-secreting hybridoma cells. The
bank of
hybridomas is screened for clones that secrete immunoglobulins which bind to
the variant
polypeptides but poorly or not at all to wild-type polypeptides are selected,
either by pre-
y absorption with wild-type proteins or by screening of hybridoma cell lines
for specific
idiotypes that bind the variant, but not wild-type, polypeptides.
Nucleic acid sequences capable of ultimately expressing the desired variant
polypeptides
are formed from a variety of different polynucleotides (genomic or cDNA, RNA,
10 synthetic olignucleotides, etc.) as well as by a variety of different
techniques.
The DNA sequences are expressed in hosts after the sequences have been
operably linked
to (i.e., positioned to ensure the functioning of) an expression control
sequence. These
expression vectors are typically replicable in the host organisms either as
episomes or as
15 an integral part of the host chromosomal DNA. Commonly, expression vectors
contain
selection markers (e.g., markers based on tetracycline resistance or
hygromycin
resistance) to permit detection and/or selection ofthose cells transformed
with the desired
DNA sequences. Further details can be found in U.S. Patent No. 4,704,362.
Polynucleotides encoding a variant polypeptide include sequences that
facilitate
transcription (expression sequences) and translation of the coding sequences
such that the
encoded polypeptide product is produced. Construction of such polynucleotides
is well
known in the art. For example, such polynucleotides include a promoter, a
transcription
termination site (polyadenylation site in eukaryotic expression hosts), a
ribosome binding
site, and, optionally, an enhancer for use in eukaryotic expression hosts, and
optionally,
sequences necessary for replication of a vector.
E. Coli is one prokaryotic host useful particularly for cloning DNA sequences
of the
present invention. Other microbial hosts suitable for use include bacilli,
such as Bacillus
subtilus, and other enterobacteriaceae, such as Salmonella, Serratia, and
various
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16
Pseudomonas species. Expression vectors are made in these prokaryotic hosts
which will
typically contain expression control sequences compatible with the host cell
(e.g., an
origin of replication). In addition, any number of a variety of well-known
promoters are
used, such as the lactose promoter system, a tryptophan (Trp) promoter system,
a beta-
s lactamase promoter system, or a promoter system from phage lambda. The
promoters
typically control expression, optionally with an operator sequence, and have
ribosome
binding site sequences, for example, for initiating and completing
transcription and
translation.
Other microbes, such as yeast, are used for expression. Saccharomyces is a
suitable host,
with suitable vectors having expression control sequences, such a promoters,
including
3-phosphoglycerate kinase or other glycolytic enzymes, and an origin of
replication,
termination sequences, etc. as desired.
1 S In addition to microorganisms, mammalian tissue cell culture is used to
express and
produce the polypeptides of the present invention. Eukaryotic cells are
preferred, because
a number of suitable host cell lines capable of secreting intact human
proteins have been
developed in the art, and include the CHO cell lines, various COS cell lines,
HeLa cells,
myeloma cell lines, Jurkat cells, and so forth. Expression vectors for these
cells include
expression control sequences, such as an origin of replication, a promoter, an
enhancer,
and necessary information processing sites, such as ribosome binding sites,
RNA splice
sites, polyadenylation sites, and transcriptional terminator sequences.
Preferred
expression control sequences are promoters derived from immunoglobin genes,
SV40,
Adenovirus, Bovine Papilloma Virus, Herpes Virus, and so forth. The vectors
containing
the DNA segments of interest (e.g., polypeptides encoding a variant
polypeptide) are
transferred into the host cell by well-known methods, which vary depending on
the type
of cellular host. For example, calcium chloride transfection is commonly
utilized for
prokaryotic cells, whereas calcium phosphate treatment or electroporation is
useful for
other cellular hosts.
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17
The method lends itself readily to the formulation of test kits for use in
diagnosis or kits
for production of vectors for gene therapy or vaccination. Such a kit
comprises a carrier
compartmentalized to receive in close confinement one or more containers
wherein a first
container contains reagents useful in the localization of the labeled probes,
such as
enzyme substrates. Still other containers contain restriction enzymes, buffers
etc.,
together with instructions for use.
Those skilled in the art will appreciate that for viral DNA replication and
packaging of
viral DNA into virion particles, only three regions of the viral DNA are known
to be
required in cis. These are the left inverted terminal repeat , or ITR, (bp 1
to approximately
103) the packaging signals (approximately 194 to 358 bp) (Hearing and Shenk,
1983,
Cell 33: 695-703; Grable and Hearing 1992, J. Virol. 64: 2047-2056) and the
right ITR.
Among the regions of the viral genome that encode proteins that function in
traps, two
have been most important in the design and development of adenovirus vectors.
These
are early region 3 (E3) located between approximately 76 and 86 mu (mu = %
distance
from the left end of the conventionally oriented genome) and early region 1 (E
1 ) located
between approximately 1 and 11 mu. E3 sequences have long been known to be
nonessential for virus replication in cultured cells and many viral vectors
have deletions
of E3 sequences so that the capacity of the resulting vector backbone for
insertion of
foreign DNA is thereby increased significantly over that allowable by the wild-
type virus
(Bett, A. J., Prevec, L., and Graham, F. L. Packaging capacity and stability
of human
adenovirus type 5 vectors. J. Virol. 67: 5911- 5921, 1993.). E1 encodes
essential
functions. However, E1 can also be deleted, providing that the resulting virus
is
propagated in host cells, such as the 293 cell line, PER-C6 cells, 911 cells,
and the like,
which contain and express E1 genes and can complement the deficiency of El (-)
viruses.
Viruses with foreign DNA inserted in place of E 1 sequences, and optionally
also carrying
deletions of E3 sequences are conventionally known as "first generation"
adenovirus
vectors. First generation vectors are of proven utility for many applications.
They can be
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18
used as research tools for high-efficiency transfer and expression of foreign
genes in
mammalian cells derived from many tissues and from many species. First
generation
vectors can be used in development of recombinant viral vaccines when the
vectors
contain and express antigens derived from pathogenic organisms. The vectors
can be
used for gene therapy, because of their ability to efficiently transfer and
express foreign
genes in vivo, and due to their ability to transduce both replicating and
nonreplicating
cells in many different tissues. Adenovirus vectors are widely used in these
applications.
There are many known ways to construct adenovirus vectors. As discussed above,
one
of the most commonly employed methods is the so called "two plasmid"
technique. In
that procedure, two noninfectious bacterial plasmids are constructed with the
following
properties: each plasmid alone is incapable of generating infectious virus.
However, in
combination, the plasmids potentially can generate infectious virus, provided
the viral
sequences contained therein are homologously recombined to constitute a
complete
infectious virus DNA. According to that method, typically one plasmid is large
(approximately 30,000-35,000 nt) and contains most of the viral genome, save
for some
DNA segment (such as that comprising the packaging signal, or encoding an
essential
gene) whose deletion renders the plasmid incapable of producing infectious
virus. The
second plasmid is typically smaller (eg 5000-10,000 nt), as small size aids in
the
manipulation of the plasmid DNA by recombinant DNA techniques. Said second
plasmid contains viral DNA sequences that partially overlap with sequences
present in
the larger plasmid. Together with the viral sequences of the larger plasmid,
the
sequences of the second plasmid can potentially constitute an infectious viral
DNA.
Cotransfection of a host cell with the two plasmids produces an infectious
virus as a
result of homologous recombination between the overlapping viral DNA sequences
common to the two plasmids. One particular system in general use by those
skilled in the
art is based on a series of large plasmids known as pBHGlO, pBHGl l and pBHGE3
described by Bett, A. J., Haddara, W., Prevec, L. and Graham, F.L: "An
efficient and
flexible system for construction of adenovirus vectors with insertions or
deletions in early
regions 1 and 3," Proc. Natl. Acad. Sci. US 91: 8802-8806, 1994 and in US
patent
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19
application S/N 08/250,885, and published as W095/00655 (hereby incorporated
by
reference). Those plasmids contain most of the viral genome and are capable of
producing infectious virus but for the deletion of the packaging signal
located at the left
end of the wild-type viral genome. The second component of that system
comprises a
series of "shuttle" plasmids that contain the left approximately 340 nt of the
Ad genome
including the packaging signal, optionally a polycloning site, or optionally
an expression
cassette, followed by viral sequences from near the right end of E1 to
approximately 15
mu or optionally to a point further rightward in the genome. The viral
sequences
rightward of E1 overlap with sequences in the pBHG plasmids and, via
homologous
recombination in cotransfected host cells, produce infectious virus. The
resulting viruses
contain the packaging signal derived from the shuttle plasmid, as well as any
sequences,
such as a foreign DNA inserted into the polycloning site or expression
cassette located
in the shuttle plasmid between the packaging signal and the overlap sequences.
Because
neither plasmid alone has the capability to produce replicating virus,
infectious viral
vector progeny can only arise as a result of recombination within the
cotransfected host
cell. Site-specific methods for achieving recombination may also be employed
when
practising the present invention.
It has been shown that use of hdAds can lead to prolonged transgene expression
and
reduced immune and inflammatory responses compared to first generation Ad
vectors
(Morral et al. 1988, Morsy et al. 1998, Scheidner et al. 1998). HdAds retain
the other
beneficial properties of Ad vectors, mainly virion stability during vector
propagation and
purification, and high transduction efficiency of replicating and quiescent
cells, while
eliminating some of the obstacles and concerns that have been raised with
respect to first
and second-generation Ads.
The instant disclosure demonstrates that, should transgene expression levels
decrease
over time, the use of hdAds of alternative serotypes permits readministration
of a vector
with the identical genotype. It is important to note that, in our experiments,
repeat
administration was performed with a different reporter gene than was carried
by the
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vector used in the initial animal immunization. Since vector persistence (and
hence
transgene expression) is influenced by immune responses to both vector and
transgene
(Dai et al. 1995, Dong et al. 1996, Tripathy et al. 1996, Christ et al. 1997,
Michou et al.
1997, Morral et al. 1997), the effectiveness of vector readministration using
hdAd's may
5 ultimately depend primarily on the immunogenicity ofthe therapeutic gene.
Accordingly,
this disclosure demonstrates that, in the absence of transgene effects, the
sequential use
of hdAd of alternative serotype is an effective strategy for vector
readministration.
Accordingly, therapeutic genes encoding products of low immunogenicity may be
repeatedly administered according to the instant disclosure. In addition, in
vaccine
10 applications, in which repeat administration of a gene encoding a
particular gene product
against which an immune response is desired, or when administration of a
second, third,
fourth etc. gene is desired, ability to overcome unwanted immune responses
induced by
a previous exposure to a vector is highly desirable.
1 S Having generally described the present invention, the following specific
examples
provide additional written and illustrative description of the invention and
the
methods of practicing the invention, including the best mode thereof. However,
those
skilled in the art will appreciate that modifications and variations on the
specifics of
the invention as disclosed in these examples may be made, without departing
from the
20 essential features of this invention, which are defined by the appended
claims, and the
equivalents thereof.
EXAMPLE 1
Cell and virus culture:
All cell culture media and reagents were obtained from Gibco Laboratories
(Grand
Island, NY). 293 (Graham et al. 1977) and A549 (human lung carcinoma, ATCC CCL
185) cells were grown in monolayer in F-11 minimum essential medium
supplemented
with 100 U of penicillin per ml, 100 mg of streptomycin per ml, 2.5 mg
fungizone per
ml, and 10% fetal bovine serum for cell maintenance or 5% horse serum after
virus
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infection. Recombinant Ad helper viruses were grown and titered on 293 cells,
as
previously described (Hitt et al. 1995). The 293-derived cell line that stably
expresses the
Cre recombinase, 293Cre4 (Chen et al. 1996), was propagated in complete F-11
medium
supplemented with 0.4 mg/ml 6418.
S
EXAMPLE 2
Helper-Dependent Adenovirus Vector Constructs
With reference to Figure 1, pRP1045 (shown in linear form) is deleted of all
Ad
protein coding sequences but contains an Ad5 head-to-tail inverted terminal
repeat
(ITR) junction and packaging signal, and encodes the E. coli (3-galactosidase
gene
under the regulation of the murine cytomegalovirus immediate-early promoter
and
Simian virus 40 polyadenylation sequence. pRP1045 also contains a ~22 kb
fragment
of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene as
stuffer, in order to maintain the size of the resulting vector within the
limits for
efficient Ad DNA packaging (Parks and Graham 1997). pRP 1050 is similar to
pRP 1045, but is deleted of a 1.2 kb StuI fragment from the HPRT sequence, and
the
resulting vectors (AdRP1045 and AdRP1050) have essentially identical
expression
characteristics. pRP1046 is similar in structure to pRP1050, but encodes a
cDNA for
human secreted alkaline phosphatase gene (hSEAP, Tropix) in place of the lacZ
gene.
The HPRT genomic sequence was obtained from Dr. Andrew J. Bett (Merck Research
Laboratories, West Point, PA). All hdAd vectors were amplified using the
appropriate helper virus in 293Cre4 cells, as previously described (Parks et
al. 1996,
Parks and Graham 1997), and the DNA structures of all hdAd vectors were
confirmed
by restriction digestion analysis of DNA isolated from virions. The titer of
each
vector was determined on 293 cells as the number of transducing particles, or
blue
forming units (BFU), per ml. For AdRP 1046, the total particle count, as
determined
spectrophotometrically (IAZ~o - 1.1x10'2 particles per ml), was used to
estimate the
number of transducing particles, assuming a particlearansducing particle ratio
of
100:1. For clarity, hdAd are designated with the appropriate serotype. For
example,
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Ad5RP1050 and Ad2RP1050 are generated using Ad5LC8cluc and Ad2LC8cCARP,
respectively.
EXAMPLE 3
Production of Helper-Dependent Adenovirus Vectors'
Helper-dependent Ad vectors were propagated and titered as previously
described (Parks
et al. 1996). pRP 1045 is a hdAd deleted of all Ad protein coding sequences,
but
containing an Ad5 head-to-tail inverted terminal repeat (ITR) junction and
packaging
signal, as well as the E. coli b-galactosidase gene under the regulation of
the murine
cytomegalovirus immediate-early promoter (MCMV) and Simian virus 40
polyadenylation (pA) sequence (Figure 1 [2]). In order to maintain the size of
the vector
above the limit for efficient DNA packaging (~28 kb, Parks and Graham 1997),
pRP 1045
also contains a ~22 kb fragment of eukaryotic DNA derived from the human
hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene, as described
elsewhere
(Parks et al. submitted). pRP 1050 is essentially identical to pRP 1045, but
is deleted of
a 1.2 kb StuI fragment from the HPRT sequence, and has expression
characteristics
identical to pRP1045 (R.J.P. and F.L.G., unpublished results). pRP1046 is
similar in
structure to pRP1050, but contains the human secreted alkaline phosphatase
cDNA
(hSEAP, Tropix) replacing the lacZ gene. For clarity, hdAd will be designated
with the
appropriate serotype. For example, Ad5RP1050 and Ad2RP1050 are generated using
Ad5LC8cluc and Ad2LC8cCARP, respectively. For Ad2RP1046, the titer of the
vector
was determined spectrophometrically, assuming lA2~o = 1.1x10'2 particles.
EXAMPLE 4
Construction of a Cre/loxP helper virus based on Ad2:
The Ad2-based helper virus, Ad2LC8cCARP was constructed using both molecular
cloning and in vivo genetic recombination techniques (Fig. 2A). pLCBc was used
to
provide the "left end" of the helper virus (i.e. left ITR and floxed packaging
signal),
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and has been previously described (Parks et al. 1996). Initially, an AatII
fragment was
removed from pLCBc in order to reduce the Ad5 sequences contained within the
plasmid. The resulting plasmid, pCElO, was cotransfected into 293 cells with
Ad2
genomic DNA digested with PshAI. The resulting viruses were screened by
restriction analysis for those which contained the majority of protein coding
sequences
derived from the Ad2 virus. One virus, designated Ad2LC8c, had resulted from a
recombination event between an MfeI site located at 9622 by of the
conventional Ad2
map and a BspHI site located at 7882 by of the Ad5 map. Thus, in Ad2LC8c all
coding sequences for virion capsid proteins, with the exception of pIX, are
derived
from Ad2.
Because of the requirement for multiple serial passages in order to increase
the titer of
hdAd, the formation of replication competent adenovirus (RCA) is of concern.
Once
generated, RCA can rapidly outgrow the vector, leading to highly contaminated
vector
stocks. We have shown that the inclusion of a "stuffer" segment within the E3
region,
such that recombination between the helper virus and the Ad5 sequences
contained in the
293 or 293Cre cells results in a virus which is above the upper packaging
limit for Ad
virions (approximately 105% of the wildtype genome, Bett et al. 1993), can
eliminate the
potential for RCA (Parks et al. 1996). We therefore designed an Ad2-based
stuffer
plasmid which contained a fragment of lambda DNA located within the E3 region
as
follows (Fig. 2B). pFG28, which contains the right 40 map units of Ad2
(F.L.G.,
unpublished), was digested with PacI and ligated with PacI digested pCARPABSI
(Addison 1997), which contains a 5.6 kb fragment of lambda DNA (22346-27972 by
of
the conventional lambda map) cloned into the unique BamHI site of pABS 1
(Neo', Bett
1995). The resulting plasmid, pFG28CARP, was partially digested with HpaI,
resulting
in the loss of the neomycin resistance gene, part of the lambda DNA, and other
bacterial
sequences derived from pABSl, and recircularized, generating pFG28CARPc. To
transfer the stuffer segment to Ad2LC8c, DNA isolated from Ad2LC8c virions was
digested with Srfl and cotransfected with pFG28CARPc into 293 cells Fi . 2B .
The
resulting viruses were screened by restriction analysis for those containing
the lambda
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24
stuffer segment within the E3 region, and one positive isolate, designated
Ad2LC8cCARP, was used for subsequent experiments.
Generation of Ad2-based helper virus:
We have previously shown that hdAd can efficiently transduce cells in vitro
and in vivo,
and can lead to long term transgene expression with dramatically reduced
cellular and
inflammatory responses, compared to first generation Ad vectors (Schiedner et
al. 1998,
Morsy et al. 1998, Morral et al. 1998). Although the hdAd DNA does persist
within non-
dividing or slowly cycling cells for long times, the episomal nature of Ads
(and hdAds)
may mean that the vector DNA will eventually be lost from the cell. Thus,
although
hdAds allow for longer-term transgene expression than observed with first
generation
Ads, there may be a requirement for repeat vector administration in order to
"boost"
transgene expression levels. The formation of neutralizing antibodies in
immunized
animals, which would occur due to the processing and presentation of virion
proteins,
would reduce the effectiveness of repeat administrations of hdAds, as found
with first
generation Ad vectors. Use of alternative Ad serotypes, either of the same
(Mack et al.
1997, Roy et al. 1998, A.L. Beaudet, unpublished results) or different
(Mastrangeli et al.
1996, Kass-Eisler et al. 1996) subgroup, allows repeat first generation vector
administration. We therefore undertook to (1) construct and characterize a
helper virus
based on a different serotype than our Ad5-based helper virus, and (2)
determine if
sequential use of vectors derived from different serotypes could permit
efficient hdAd
vector readministration.
Ad2LC8cCARP contains an Ad5 left-end identical to that of our previous helper
virus
Ad5LC8cluc including the "floxed" packaging signal. We would therefore expect
that
the efficiency of Cre-mediated excision of the packaging signal ('1') would be
similar for
the two viruses. Experiments to test for excision in the 293Cre4 cell line
showed that 'l'
was indeed excised from Ad2LC8cCARP with an efficiency similar to that
observed for
Ad5LC8cluc. Thus, Ad2LC8cCARP should act as an equally effective helper virus
in
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our Cre/loxP system, resulting in only very low levels of helper virus
contamination in
the resulting vector stocks.
Ad2LC8cCARP encodes all structural proteins derived from Ad2, with the
exception of
5 pIX. The pIX gene is located immediately adjacent to E1, and encodes a minor
virion
structural component that has not been shown to be a major target for
neutralizing
antibody activity (Wohlfart 1988, Gahery-Segard et al. 1998), and is highly
conserved
between the two serotypes (138 of 139 amino acids are identical between Ad2
and Ad5
pIX). Thus, the presence of an Ad5-pIX in our "Ad2" hdAd vectors should not
interfere
10 with their ability to transduce cells in the presence of anti-Ad5
antibodies. We also
included a fragment of lambda DNA within the E3 region of Ad2LC8cCARP as a
stuffer
to prevent RCA formation. Were Ad2LC8cCARP to recombine with the Ad5 sequences
contained in the 293 or 293Cre4 cells, it would generate a virus of
approximately 39 kb,
which greatly exceeds the upper limit for Ad DNA packaging (Bett et al. 1993).
15 Consequently, as with our earlier helper Ad5LC8cluc, we have not observed
RCA in our
helper virus preparations or in stocks of vector produced using Ad2LC8cCARP.
Amplification of a hdAd using Ad2LC8cCARP:
20 To determine if Ad2LC8cCARP could amplify hdAd with an efficiency equal to
that of
AdSLCBcIuc, we transfected duplicate 60-mm dishes of 293Cre cells with 5 ~g of
pRP1050 and, next day, infected the monolayers at an moi of 5 with
Ad2LC8cCARP.
After complete CPE (approximately 72 hr), the monolayers were harvested into
the
medium, freeze/thawed, and an aliquot of the lysates analyzed for lacZ-
transducing
25 particles. Ad2RP 1050 was rescued at a frequency of approximately 400 bfu
per pmol of
transfected DNA, which is similar to the efficiency previously observed for
plasmids of
comparable size using Ad5LC8cluc (Parks and Graham 1997), suggesting that
Ad2LC8cCARP could indeed act as an effective helper virus in the 293Cre4
cells. We
then subjected an aliquot of the crude lysates (500 ~l) to serial passage on
Ad2LC8cCARP-infected 293Cre cells, in order to determine the kinetics of
vector
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26
amplification. As shown in Fig. 3, Ad2RP1050 was amplified using Ad2LC8cCARP
at
a rate similar to that previously observed for the Ad5LC8cluc helper virus
(R.J.P. and
F.L.G., unpublished results) and, after 4 serial passages on the helper virus-
infected
293Cre cells, reached a titer of 3.4x 10' bfu/ml. A large scale preparation of
Ad2RP 1050
was performed, yielding 3x10" bfu from 20 150-mm dishes, with a helper virus
contamination of 3.2x10' pfu/ml 00.02 % of the Ad2RP1050 titer). We conclude
that
Ad2LC8cCARP can act as a helper virus in 293Cre4 cells with an efficiency
similar to
that of Ad5LC8cluc.
Effect of Ad5 neutralizing antibodies on hdAd2:
In theory, an hdAd based on an Ad2 serotype should not be affected by Ad5
neutralizing
antibodies, allowing for vector readministration in animals previously treated
with an
Ad5-based hdAd. To determine if Ad2RP1050 was sensitive to antibodies
generated
against AdS, 106 bfu of Ad2RP1050, Ad5RP1045 or Ad5CA35 were incubated with
serial dilutions of Ad5-neutralizing serum, and then used to infect A549
cells. Twenty-
four hours later, crude cell extracts were prepared from the infected cells
and assayed for
(3-gal activity. In this assay, infectivity directly correlates with (3-gal
activity and thus
neutralizaton of the vector by antibody leads to a corresponding reduction in
(3-gal
activity. Both Ad5CA35 and Ad5RP1045 were neutralized by incubation with the
Ad5
antibodies, with an almost 100-fold decrease in (3-gal activity at the highest
antibody
concentration (Fig. 4). In contrast, no decrease in (3-gal activity (or virus
infectivity) was
noted for Ad2RP 1050. These results indicate that the virion protein
components derived
from Ad2LC8cCARP and present in Ad2RP1050 are not sensitive to Ad5
neutralizing
antibodies.
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EXAMPLE S
Transg;ene expression studies:
Methods for preparation of cell samples and assays for (3-gal are described
elsewhere
(Parks et al. submitted) and are known in the art. Assays for hSEAP activity
were
performed using a chemiluminescence kit, as described by the manufacturer
(Tropix),
with the exception that the serum samples were not heat treated prior to
assay,
resulting in a slightly higher background level of AP activity. For in vivo
expression
studies, adult female FVB/n mice (Harlan) were injected through the tail vein
with
5x10'° particles of vector in a volume of 200 ~1. At various times post-
injection,
blood samples were removed by orbital bleed, incubated overnight at
4°C, and the
serum cleared by two rounds of centrifugation at 16,OOOxg for 5 min in a
microcentrifuge. The serum samples were stored at -70°C until the end
of the
experiment. The method for preparation and analysis of (3-gal levels in the
liver of
mice is well known.
In vitro trans~ene expression:
Although Ad2RP1050 and AdSRP1050 are genetically identical, and would be
predicted to have virtually identical expression characteristics, the
efficiency of cell
transduction could be affected by the presence of different virion capsid
proteins. It is
also possible that subtle differences in the protein coat or core proteins
contained
within the virion might influence the efficiency of transport of the hdAd DNA
to the
nucleus or affect promoter activity. We therefore examined transgene
expression of
Ad2RP1050 and Ad5RP1050 in transduced A549 cells. We chose A549 since E1-
deleted first generation vectors do not undergo productive infection in these
cells, and
our analysis of transgene expression over time would not be complicated by the
presence of small quantities of helper virus. Monolayers of A549 cells in 60-
mm
dishes were transduced in duplicate with 106 bfu of Ad2RP1050 or Ad5RP1050
and,
at various times post-transduction, crude protein extracts were prepared and
analyzed
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for (3-gal activity. The hdAd2- and hdAdS-lacZ vectors had virtually identical
expression characteristics in vitro over the duration of the experiment (Fig.
5). We
conclude that hdAd generated using an Ad2- or Ad5-based helper virus have
identical
transduction efficiencies and transgene expression characteristics in vitro.
Trans expression in vivo:
We next examined the ability of the hdAd2 to transduce mouse hepatic cells in
vivo.
We injected FVB/n mice through the tail vein with 5x10'° particles of
Ad2RP1046,
and determined the level of hSEAP in the serum of animals at various times
post-
transduction. Injection of Ad vectors through the tail vein results in the
majority of
the vector being delivered to, and retained in, the liver resulting in high
efficiency
transduction of hepatocytes (Guo et al. 1996), and, for proteins efficiently
secreted
from the transduced cell, can lead to high levels of transgene products in the
serum of
treated animals (Morral et al. 1998, Morsy et al. 1998, Scheidner et al.
1998). As
shown in Fig. 6, high levels of hSEAP were detected in the serum of transduced
animals. Mice injected with a control Ad vector showed only a small increase
in
serum AP levels (approximately 3-fold above background, data not shown),
presumably due to minor liver toxicity. Maximum levels of expression
(approximately
14 ng per ml of serum) were obtained within one week of vector injection,
remained
constant for approximately 2 weeks, and declined to background levels within 3
weeks. The maximum level of protein expression is similar to that observed for
a first
generation Ad vector with an identical expression cassette (~10 ng per ml of
serum,
G. Maelandsmo, R.J.P. and F.L.G., unpublished results). The duration of hSEAP
expression (~14 days) is consistent with that observed in other studies with a
hdAd
encoding a potentially immunogenic transgene (Parks et al. submitted). We
conclude
that hdAds generated using the Ad2 helper virus are able to efficiently
transduce cells
in vivo, and can lead to high levels of transgene expression, similar to Ad5-
based
hdAd.
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29
EXAMPLE 6
Virus neutralization assays:
The Ad5 neutralizing antibodies used in these experiments were generated in
rabbits by
injection of a first-generation Ad5 vector (M. Anton and F. L. Graham,
unpublished) and
Ad2 antibodies were made in mice by inj ecting a serotype 2 hdAd. One skilled
in the art
would appreciate that neutralizing antibodies could also be generated in mice
or rabbits
or other animals b~j ection with wild type Ad2 or Ad5 viruses. Aliquots
[Aliqouts] ( 1 O6
bfu in 100 ~l) of a first generation Ad vector (Ad5CA35, Addison et al. 1997),
Ad5RP1045, AdRP1050, or Ad2RP1050, all containing an identical (3-gal
expression
cassette, were incubated with serial dilutions (100 ~1) of antibody-containing
serum.
After a 1 hr at 37°C, the treated vectors were used to infect 22 mm
dishes of A549 cells
for 1 h, the monolayers washed twice with PBS, maintenance medium replaced,
and the
quantity of (3-gal present within the cells assayed 24 hr later. In this
assay, the quantity
of (3-gal produced in the cells correlates directly with the efficiency of
cell transduction.
Generation of neutralizing antibodies in hdAd2-immunized animals:
We next wished to determine whether animals immunized with a hdAd2 would
generate neutralizing antibodies to Ad2, and whether these antibodies would
have any
effect on a hdAdS. We therefore analysed serum collected at 28 days post-
injection
from mice injected i.v. with 5x10'° particles of Ad2RP1046 for
neutralizing
antibodies to Ad2 or AdS. Serum samples were serially diluted and incubated
with
Ad2RP1050 or Ad5RP1050, and the resulting vector assayed for the ability to
transduce A549 cells and express lacZ, as described above. All of the animals
immunized with Ad2RP1046 produced neutralizing antibodies to Ad2, which
resulted
in a 30- to 100-fold decrease in Ad2RP1050 transduction at the highest
antibody
concentrations examined (Fig. 7). In contrast, there was no effect on AdSRP
1050,
indicating that the hdAd2-immunized animals produced antibodies that were
specific
CA 02367390 2001-10-03
WO 00/60106 PCT/US00/09149
to Ad2. Therefore, it appears that helper-dependent Ad vectors based on
alternative
serotypes have the same general virion characteristics, with respect to
presentation of
surface antigens, as first generation Ad vectors. Based on these observations,
we
would predict that use of hdAd based on alternative Ad serotypes should permit
5 vector readministration.
Use of hdAd of alternative serotype allows for repeat vector administration
Since mice immunized with the hdAd2 generated antibodies to Ad2, and those
10 antibodies did not cross-react with AdS, we next determined whether
subsequent
delivery of hdAdS to mice preimmunized with hdAd2 could overcome the effects
of
neutralizing antibodies against Ad2 and result in higher levels of transgene
expression compared to readministration of hdAd2. Mice were immunized with
10'°
particles of Ad2RP1046 and, 90 days later, injected with 108 bfu of either
15 Ad2RP1050 or Ad5RP1050. As a control, naive animals were injected in
parallel
with the same set of lacZ-expressing vectors. At three and six days after
administration of the lacZ vector, the animals were euthanized and the livers
removed
and assayed for (3-gal activity. Administration of a serotype 2 vector into
animals
immunized against Ad2 resulted in an over 30-fold reduction in transgene
expression
20 (4.0x105 versus 2.1x10' rlu per tissue) at day 3, and a 100-fold reduction
(2.5x105
versus 2.6x 10' rlu per tissue) by day 6 post-inj ection compared to naive
animals (Fig.
8A). In contrast, surprisingly, no decrease in transgene expression was
observed
relative to that in naive animals when the re-administered vector was of a
different
serotype (Fig. 8B). Interestingly, we did not observe a decrease in expression
of lacZ
25 between days 3 and 6 in the hdAd2-hdAdS treatment, as has been observed for
similar
treatments using first generation Ad vectors (Mack et al. 1997). The decrease
in
expression observed by Mack et al. (1997) was attributed to cellular immune
processes, and suggests that either hdAd do not elicit such destructive
processes or,
alternatively, are poor targets.
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WO 00/60106 PCT/US00/09149
31
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