Note: Descriptions are shown in the official language in which they were submitted.
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Methods for Pseudoadenoviral Vector Production
Introduction
The invention is directed to novel helper adenoviruses which facilitate the
production of pseudoadenoviral vectors (PAV) but which cannot be packaged into
viral
particles. The invention is further directed to novel PAV producer cell lines
expressing
DNA binding and/or repressor proteins that prevent the packaging of the helper
viruses.
The invention is also directed to methods for the production of PAV in such
cell lines
with minimal contamination from helper viruses.
Back~ound of the Invention
Pseudoadenoviral vectors (PAV) are adenoviral vectors derived fibm the genome
of an adenovirus which contain the minimal cis-acting nucleotide sequences
required for
the replication and packaging of the vector genome and which can contain one
or more
transgenes (see, gig,,, allowed U.S. Application Serial No. 08/895,194)
incorporated
herein by reference). Such PAVs are advantageous because the transgene
carrying
capacity of the vector is optimized (up to 36Kb in size), while the potential
for host
immune reaction to viral proteins or for the generation of replication-
competent viruses is
reduced. PAVs contain the adenoviral 5' and 3' inverted terminal repeat (ITR)
nucleotide
sequences containing origins of replication, the 5' cis-acting packaging
signal of the viral
genome, and can accomodate one or more transgenes with operably linked
expression
elements. These minimal viral nucleotide sequences retained in PAVs are
required in cis
for the replication and packaging of the PAV genome into viral particles. In
addition, the
production of PAVs requires the provision of a helper adenovirus to supply the
viral
proteins required for replication of the PAV genome and assembly of the viral
particles.
PAV vectors have been designed to take advantage of the desirable features of
adenovirus which render it a suitable vehicle for gene transfer, as evidenced
by studies
with first and second generation adenoviral vectors. Adenovirus is a non-
enveloped,
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nuclear DNA virus with a genome of about 36 kb, which has been well-
characterized
through studies in classical genetics and molecular biology (Horwitz, M.S.,
"Adenoviruses," in yjroloQV. 3rd edition, Fields et al., eds., Raven Press,
New York,
1996; Hitt, M.M. et al., "Adenovirus Vectors," in The Development of Human
Gene
Theranv, Friedman, T. ed., Cold Spring Harbor Laboratory Press, New York,
1999). The
viral genes are classified into early (known as E1-E4) and late (known as L1-
LS)
transcriptional units, referring to the generation of two temporal classes of
viral proteins.
The demarcation between these events is viral DNA replication. The human
adenoviruses are classified into numerous serotypes (approximately 47,
numbered
accordingly and organized into 6 subgroups: A, B, C, D, E and F), based upon
properties
including hemaglutination of red blood cells, oncogenicity, DNA base and
protein amino
acid compositions and homologies, and antigenic relationships.
Recombinant adenoviruses have several advantages for use as gene transfer
vectors, including tropism for both dividing and non-dividing cells, minimal
pathogenic
potential, ability to replicate to high titer for preparation of vector
stocks, and the
potential to carry large inserts (Hitt, et al. supra); Berkner, K.L., Curr.
Top. Micro.
Immunol. 158:39-66, 1992; Jolly, D., Cancer Gene Therapy 1:51-64, 1994).
The cloning capacity of an adenovirus vector to date has been proportional to
the
size of the adenovirus genome present in the vector. For example, a cloning
capacity of
about 8 kb results from the deletion of regions of the virus genome which are
dispensable
for virus growth, gsg=, E3, and the deletion of a genomic region such as E1
whose func-
tion may be restored in traps from a complementing cell line such as 293 cells
(Graham,
F.L., J. Gen. Virol. 36:59-72, 1977). Such E1-deleted vectors are rendered
replication-
defective, a desirable attribute for a gene transfer vector. The upper limit
of vector DNA
capacity for optimal carrying capacity is about 105%-108% of the length of the
wild-type
genome. Further adenovirus genomic modifications are possible in vector design
using
cell lines which supply other viral gene products in traps, ~,g,.,
complementation of E2a
(Zhou et al., J. Virol. 70:7030-7038, 1996), complementation of E4 (Krougliak
et al.,
Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther. 2:775-783, 1995),
or
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complementation of protein IX (Caravokyri et al., J. Virol. 69:6627-6633,
1995;
Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995).
However, maximal carrying capacity can be achieved with the use of adenoviral
vectors containing deletions of most viral coding sequences, including PAVs
(allowed
U.S. Patent Application Serial No. 08/895,194; Kochanek et al., Proc. Natl.
Acad. Sci.
USA 93:5731-5736, 1996; Parks et al., Proc.Natl.Acad.Sci. USA 93:13565-13570,
1996;
Lieber et al., J.Virol. 70:8944-8960, 1996; Fisher et al., Virology 217:11-22,
1996; U.S.
Patent No. 5,670,488; PCT Publication No. W096/33280, published October 24,
1996;
PCT Publication No. W096/40955, published December 19, 1996; PCT Publication
No.
W097/25446, published July 19, 1997; PCT Publication No. W095/29993, published
November 9, 1995; PCT Publication No. W096/13597, published May 9, 1996; PCT
Publication No. W097/00326, published January 3, 1997; Morral et al., Hum.Gene
Ther.
10:2709-2716, 1998; Burcin et al., Proc. Natl. Acad. Sci. USA 95:355-360,
1999).
A wide variety of transgenes (foreign nucleic acids) have been delivered to
various target cells by first and second-generation adenoviral vectors,
illustrating the
heterogeneity of adenoviral vector transduction. Such transgenes include p53
(Wills et
al., Human Gene Therapy 5:1079-188, 1994); dystrophin (Vincent et al., Nature
Genetics
5:130-134, 1993; erythropoietin (Descamps et al., Human Gene Therapy 5:979-
985,
1994; ornithine transcarbamylase (Stratford-Perricaudet et al., Human Gene
Therapy
1:241-256, 1990; We et al., J. Biol. Chew. 271;3639-3646, 1996;); adenosine
deaminase
(Mitani et al., Human Gene Therapy 5:941-948, 1994); interleukin-2 (Haddada et
al.,
Human Gene Therapy 4:703-711, 1993); and al-antitrypsin (Jaffe et al., Nature
Genetics
1:372-378, 1992); thrombopoietin (Ohwada et al., Blood 88:778-784, 1996); and
cytosine
deaminase (Ohwada et al., Hum. Gene Ther. 7:1567-1576, 1996).
The particular tropism of adenoviruses for cells of the respiratory tract has
relevance to the use of adenoviral vectors for gene transfer in cystic
fibrosis (CF), which
is the most common autosomal recessive disease in Caucasians. Mutations in the
cystic
fibrosis transmembrane conductance regulator (CFTR) gene that disturb the
cAMP-regulated CY channel in airway epithelia result in pulmonary dysfunction
(Zabner
et al., Nature Genetics 6:75-83, 1994). Adenovirus vectors engineered to carry
the CFTR
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gene have been developed (Rich et al., Human Gene Therapy 4:461-476, 1993) and
studies have shown the ability of these vectors to deliver CFTR to nasal
epithelia of CF
patients (Zabner et al., Cell 75:207-216, 1993), the airway epithelia of
cotton rats and
primates (Zabner et al., Nature Genetics 6:75-83, 1994), and the respiratory
epithelium of
CF patients (Crystal et al., Nature Genetics 8:42-51, 1994). Recent studies
have shown
that administering an adenoviral vector containing a DNA sequence encoding
CFTR to
airway epithelial cells of CF patients can restore a functioning chloride ion
channel in the
treated epithelial cells (Zabner et al., J. Clin. Invest. 97:1504-151 I, 1996;
U.S. Patent No.
5,670,488, issued September 23, 1997). Persistent expression of CFTR from
adenoviral
vectors which results in the establishment of functional chloride channels in
the airway
epithelium of immunocompetent animals has recently been achieved (Scaria et
al.,
J.Virol. 72:7302-7309, 1998).
The use of first and second generation adenovirus vectors in gene transfer
studies
to date indicates that persistence of transgene expression in target cells and
tissues is
1 S often transient. This is at least partly due to the generation of a
cellular immune response
to viral proteins which are expressed even from a replication-defective
vector, triggering
a pathological inflammatory response which may destroy or adversely affect the
adenovirus-infected cells (Yang et al., J. Virol. 69:2004-2015, 1995; Yang et
al., Proc.
Natl. Acad. Sci. USA 91:4407-4411, 1994; Zsengeller et al., Hum Gene Ther.
6:457-467,
1995; Worgall et al., Hum. Gene Ther. 8:37-44, 1997; Kaplan et al., Hum. Gene
Ther.
8:45-56, 1997). Because adenovirus does not integrate into the cell genome, an
adverse
immune response poses a serious obstacle for high dose administration of an
adenovirus
vector or for repeated administration (Crystal, R., Science 270:404-410,
1995).
In order to circumvent the host immune response which limits the persistence
of
transgene expression, various strategies have been employed, generally
involving either
the modulation of the immune response itself or the engineering of a vector
that decreases
the immune response. The administration of immunosuppressive agents together
with
vector administration has been shown to prolong transgene persistence (Fang et
al., Hum.
Gene Ther. 6:1039-1044, 1995; Kay et al., Nature Genetics 11:191-19?, 1995;
Zsellenger
et al., Hum. Gene Ther. 6:457-467, 1995; Scaria et al., Gene Therapy 4:611-
617, 1997).
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Modifications to the adenovirus genome in the recombinant vector can decrease
the host
immune response (Yang et al., Nature Genetics 7:362-369, 1994; Lieber et al.,
J. Virol.
70:8944-8960, 1996; Gorziglia et al., J. Virol. 70:4173-4178; Kochanek et al.,
Proc. Natl.
Acad. Sci. USA 93:5731-5736, 1996; Fisher et al., Virology 217:11-22, 1996).
For
example, the adenovirus E3 gpl9K protein can complex with MHC Class I antigens
and
retain them in the endoplasmic reticulum, which prevents cell surface
presentation and
killing of infected cells by cytotoxic T-lymphocytes (CTLs) (Wold et al.,
Trends
Microbiol. 437-443, 1994), suggesting that the presence of its encoding gene
in a
recombinant adenoviral vector may be beneficial.
The lack of persistent expression of adenoviral vector-delivered transgenes
may
also be due to limitations imposed by the choice of promoter and/or transgene
contained
in the transcription unit (Guo et al., Gene Therapy 3:802-801, 1996; Tripathy
et al.,
Nature Med. 2:545-550, 1996. Further optimization of minimal adenoviral
vectors for
persistent transgene expression in target cells involves the synergistic
choice of
expression control elements and vector genome design such that expression is
maximized
and host immune response is limited (W098/46780 Scaria et al., J.Virol.
72:7302-7309,
1998).
It is desirable to provide PAV with mimimal viral coding sequences that cannot
elicit a strong host immune response, but which can take advantage of the
ability of
adenoviral vectors to deliver transgenes to a wide variety of target cells.
Production of
PAV requires the presence of adenovirus proteins in traps which facilitate the
replication
and packaging of a PAV genome into viral vector particles. Most commonly, such
proteins are provided by infecting a producer cell with a helper adenovirus
containing the
genes encoding such proteins. However, such helper viruses are potential
sources of
contamination of a PAV stock during purification if they are able to replicate
and be
packaged into viral particles. It is advantageous, therefore, to increase the
purity of a
PAV stock by reducing or eliminating any production of helper viruses which
can
contaminate the preparation. Several strategies to reduce the production of
helper viruses
in the preparation of a PAV stock are disclosed in allowed U.S. Patent
Application Serial
No. 08/895,194 and U.S. Patent No. 5,670,488, issued September 23, 1997,
incorporated
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herein by reference. For example, the helper virus can contain mutations in
the
packaging sequence of its genome which prevent packaging, or may contain an
oversized
adenoviral genome which cannot be packaged due to size constraints of the
virion.
Other strategies include the design of a helper virus with a packaging signal
flanked by the excision target site of a recombinase, such as the cre-lox
system (Parks
et al., Proc. Natl. Acad. Sci.USA 93:13565-13570, 1996; Hardy et al., J.Virol.
71:1842-
1849, 1997).
Detailed analysis of the structure of the adenovirus packaging signal has
revealed
that it is organized into a minimum of seven functional elements, identified
as A repeats
(Schmid et al., J. Virol. 71:3375-3384, 1997). Using this information, a PAV
production
system which comprises helper adenoviruses and producer cell lines optimized
for PAV
production is provided such that the packaging signal region is available for
the
production of a helper virus stock but is disabled during the production of a
PAV vector
stock. The present invention provides for an increased preferential packaging
of PAVs in
the production of purified vector stocks to further the development and
widespread use
of these vectors for gene transfer.
Summar;r of the Invention
The invention is directed to novel helper adenoviruses which facilitate the
production and packaging of pseudoadenoviral vectors (PAV) by providing for
the
production of essential viral proteins in trans production and packaging
required for PAV
the helper viruses of the inventors are packaging defective due to the
inclusion in the
packaging signal region of their genomes of binding sequences for DNA binding
and/or
repressor proteins that prevent access by packaging proteins to this signal.
The invention
is also directed to to PAV producer cell lines expressing such nucleic acids
encoding
DNA binding and/or repressor proteins. The invention is further directed to
methods for
the production of PAVs with minimal contamination from helper viruses, using
the helper
viruses and producer cell lines of the invention.
Brief Descrintiori of the Drawines
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Figure 1 shows a schematic diagram of strategies for the production of
packaging-
defective helper adenoviruses.
Figure 2 shows a schematic diagram of the FLP recombinase/FRT helper system
for
excision of packaging sequences from helper adenoviruses.
Figure 3a shows a schematic diagram of a helper adenovirus containing a
packaging
signal flanked by lambda operator binding sequences; Figure 3b shows the
structure of
the junction of the packaging signal and operator sequences.
Figure 4a shows a schematic diagram of a helper adenovirus containing a
packaging
signal flanked by lambda operator binding sequences; Figure 4b shows the
structure of
the junction of the packaging signal and operator sequences.
Figure 5 shows a schematic diagram of a helper adenovirus containing a
packaging signal
flanked by lambda operator binding sequences.
Figure 6 shows a schematic diagram of a helper adenovirus containing a
packaging signal
flanked by lambda operator binding sequences.
Figure 7 shows a schematic diagram of a series of helper adenoviruses which
contain a
packaging signal flanked by FRT binding sequences.
Figure 8 shows a schematic diagram of Ad2HeIpFRT containing a packaging signal
flanked by FRT binding sequences.
Figure 9 shows a schematic diagram of Ad2HeIpFRTIuc containing a packaging
signal
and luciferase marker gene flanked by FRT binding sequences.
Figure 10 shows a schematic diagram of helper adenovirus TBTP.
Figure 11 is a schematic diagram of a helper adenovirus with internal ITR
sequences
facilitating virus replication.
Figure 12 shows a schematic diagram of plasmid pTRE/FLPe6.
Figure 13a shows a schematic diagram of a tetracycline induction system for
FLPe6
expression; Figure 13b shows tetracycline induction system for FLPe6
expression in 293-
tet-OFF cells.
Figure 14 shows a schematic diagram of plasmid pTRE/FLPe6.
Figure 15 shows a schematic diagram of plasmid pCEP/FLPe6.
Figure 16 shows a schematic diagram of plasmid pOG-FLPe6.
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petailed Descri tin on o f the Invention
The invention is directed to novel helper adenoviruses which facilitate the
production and packaging of pseudoadenoviral vectors (PAV) by providing for
the
production of essential viral proteins in traps required for PAV production
and packaging.
The helper viruses of the invention are packaging defective due to the
inclusion in the
packaging signal region of their genome of binding sequences for DNA binding
and/or
repressor proteins that either prevent access by packaging proteins to this
signal region or
which facilitate enzymatic excision of the packaging signal. The invention is
also
directed to to PAV producer cell lines expressing nucleic acids encoding such
DNA
binding and/or repressor proteins. The invention is further directed to
methods for the
production of PAV with minimal contamination from helper viruses, using the
helper
viruses and producer cell lines of the invention.
A helper virus of the invention is defined as an adenovirus which is able to
supply
the viral proteins required in traps for the production of PAV or other
minimal adenoviral
vectors. In accordance with the invention, the helper virus genome is disabled
for
packaging, thereby allowing for preferential packaging of the PAV genomes into
viral
particles. The helper virus genome comprises at least those genes and/or
regions of the
adenovirus genome that are required to produce the viral proteins required in
traps for the
production of PAV. The adenovirus proteins supplied by the helper virus
include inter
alia the regulatory proteins from the adenovirus early (E) genomic regions,
the capsid
proteins encoded by the viral late (L) genomic regions, and other structural
and non-
structural proteins. The production of the proteins encoded by a helper virus
genome
facilitates the replication of the PAV genome during the production of vector
stock. The
adenovirus genes required in traps are not limited by virus serotype, and the
helper
viruses of the invention can contain adenovirus genes from more than one
serotype.
Structural proteins, which are supplied by the helper virus, can therefore be
chosen so that
the capsid proteins are derived from a desired serotype or serotypes and
optimized for a
particular use.
The helper virus genome is desirably modified according to the present
invention
such that packaging of helper virus particles is impaired or eliminated. Such
disability
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reduces or eliminates the production of helper virus in the preparation of a
PAV stock,
while allowing the helper virus itself to be propagated during the separate
production of a
helper virus stock. A helper virus of the invention is rendered packaging-
defective by
incorporation into its genome of a reduced length packaging signal or the
insertion
therein of heterologous nucleotide sequences (defined as binding sequences)
into in or
near the packaging signal region of the helper virus genome. Such binding
sequences are
capable of binding to specific DNA binding and/or repressor proteins, thereby
either
blocking the utilization of the cis-acting packaging signal (packaging-signal
masked) or
causing excision of the signal from the helper virus genome (packaging-signal
deleted)
(Figure 1 ).
A binding protein is defined herein as any protein or peptide ( 1 ) which is
capable
of binding to the binding sequences inserted into or near the packaging signal
region of a
helper virus genome so as to prevent utilization of the signal and repress
packaging or (2)
which is capable of binding to and induce cleavage at specific target sites.
Binding sequences are defined as nucleotide sequences inserted in proximity
to,
adjacent to or into the packaging signal region of the helper virus genome
which are
capable of binding to specific DNA binding and/or repressor proteins. A
binding
sequence of the invention, alone or in combination or tandem with other
binding
sequences, is capable of avidly binding one or more DNA binding and/or
repressor
proteins such that the packaging signal of a helper virus genome cannot be
accessed by
the viral packaging proteins or becomes excised from the genome. Preferably,
the
binding sequences interact with and bind DNA binding and/or repressor
proteins. The
binding sequences which are incorporated into or near or near the packaging
signal region
of the helper virus genome can be of various lengths, ~, from about 8-30
nucleotides,
but can also be tandem arrays of such sequences.
Preferred specific binding sequences of the invention include, but are not
limited
to, those derived from the bacteriophage lambda operator (Ptashne, M. A
Genetic Switch,
Cell Press and Blackwell Scientific Publications, 1986) and the papillomavirus
E2
binding sequence (McBride et al., J.Biol. Chem. 266:18411-18414, 1991;
Androphy et
al., Nature 235:70-73, 1987). Most preferably, the packaging signal region in
a helper
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virus is flanked by the recognition site for a FLP recombinase, an enzyme
which
recognizes the FLP recombination target (FRT) and can catalyze site-specific
excision of
flanked nucleotide sequences (Senecoff et al., Proc. Natl. Acad.Sci. 82:7270-
7274, 1985;
O'Gorman et al., Science 251:1351-1355, 1991). Upon recognition of the FRT
nucleotide sequences by a FLP recombinase, the flanked packaging signal is
excised from
the helper virus genome, thereby preventing the packaging of the helper virus
genome
and the production of helper virus particles (Figure 2). In a preferred
embodiment of the
invention, the FLP recombinase is used in a producer cell of the invention for
enzymatic
cleavage of the packaging signal in a helper virus because it exhibits
increased
thermostability at 37°C (Buchholz et al., NAR 24:4256-4262, 1996;
Buchholz et al.,
Nature Biotech. 16:657-662, 1998). The invention contemplates the use of an
FLP
recombinase which is optimized for particular uses as needed in the form of
monomers,
dimers, tetramers or other multimeric forms.
Specific binding sequences which can be inserted in to an adenovirus genome at
a
site located in or near the adenovirus packaging signal region to accomplish
the goals of
the invention include the following:
Lambda operator binding sequences (Ptashne, M. A Genetic Switch, Cell Press
and Blackwell Scientific Publications, 1986}:
OL1: TATCACCGCCAGTGGTA (SEQ ID NO:1)
ATAGTGGCGGTCACCAT
OR1: TATCACCGCCAGAGGTA (SEQ ID N0:2)
ATAGTGGCGGTCTCCAT
0,,2: TATCTCTGGCGGTGTTG (SEQ >D N0:3)
ATAGAGACCGCCACAAC
0~3: TATCACCGCAGATGGTT (SEQ >D N0:4)
ATAGTGGCGTCTACCAA
OR2: TAACACCGTGCGTGTTG (SEQ ID NO:S)
ATTGTGGCACGCACAAC
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OR3: TATCACCGCAAGGGATA (SEQ ID N0:6)
ATAGTGGCGTTCCCTAT
In a preferred embodiment, the binding sequence is OL1:
TATCACCGCCAGTGGTA (SEQ ID NO:1)
ATAGTGGCGGTCACCAT
Specific binding sequences for use by the papilloma virus E2 proteins include:
ACCGAAATCGGT (SEQ ID N0:7) Romanczuk et al., J.Virol. 64:2489-
2859, 1990
ACCGAAACCGGT (SEQ ID N0:8) Romanczuk et al., J.Virol. 64:2489-
2859, 1990
ACCN(6)GGT (SEQ ID N0:9) Androphy et al., Nature 325:70-73, 1985
Other papilloma virus E2 protein consensus sequences which can be used as
binding sequences in accordance with the present invention are those described
in
McBride et al., J.Biol. Chem. 266:18411-18414, 1991.
The binding sequences responsive to the FLP recombinase inciude its
recognition
site, FLP recombinase target (FRT) repeated motifs underlined) (Senecoff et
al.,
Proc.Natl.Acad.Sci. USA 82:7270-7274, 1985):
FRT:GAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTC
(SEQ ID NO:10)
Another binding site for the FLP recombinase is:
CGAAGTTCCTATTCTCTAGAAAGtATAGGAACTTC (SEQ m NO:11 )
The FRT binding sequences can be derived from plasmid pOG45 (Stratagene, La
Jolla,CA) or can be synthesized using standard techniques for oligonucleotide
synthesis.
Truncated FRT sequences which also facilitate cleavage of an adjacent
packaging signal
in a helper virus are also within the scope of the invention. The use of any
binding
sequences which facilitate FLP-mediated excision of a packaging signal from a
helper
a,denovirus is within the scope of the invention in the construction of
packaging-defective
helper adenoviruses.
Locations for the insertion of the binding sequences within or near a
packaging
signal in an adenovirus genome region can be identified by reference to the
nomenclature
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of the packaging signal region which defines a minimum of seven AT-rich
elements,
denoted AI-AVII. These seven AT-rich elements are located in the adenovirus
genome
from nucleotides 194-380 (referencing adenovirus serotype 5, Schmid et al., J.
Virol.
71:3375-3384, 1997). For example, one or more binding sequences can be
inserted into
one or more sites flanking the A elements, or can be inserted into or between
the A
repeats. The redesign of the adenovirus packaging signal region according to
the present
invention also contemplates the deletion or multiplication of one or more A
repeat
elements. It will be evident to the skilled artisan that strategies for
adenovirus genome
design, which include various combinations of insertions of binding sequences
or in close
proximity or adjacent to the A repeats as well as deletions or repetitions
thereof can
accomplish the goal of the invention, i.e., disabling the packaging signal of
the helper
virus genome. Where the binding sequences facilitate the excision of a
packaging signal,
they may be inserted into the adenovirus genome in order to flank a desired
packaging
signal region at the 5' and 3' ends thereof, such that the entire signal
region is excised by a
site-specific recombinase.
In one preferred embodiment of a helper virus packaging signal, the helper
virus
genome is modified by the deletion of packaging elements AI-AIV, retaining
only the
packaging elements AV, AVI and AVII. A binding sequence comprising a 17 by
bacteriophage lambda operator sequence (preferably, OL1) is inserted into the
helper
genome upstream and downstream fibm elements AV and AVII (sites #1 and #2),
adjacent to nucleotides 334 and 385 of the adenovirus genome (Figure 3:Ad(AV-
VI-
AVII). Alternatively, the A repeat elements AV, AVI and AVII can be repeated
as a
motif, e.g., (AV-AVI-AVII)Z and flanked by inserted lambda operator sequences
as
shown in Figure 4:Ad(AV-AVI-AVII)2. Helper vectors Ad(AV-AVI-AVII) and Ad(AV-
AVI-AVII)2 can be used directly as helpers [packaging impaired, in the case of
Ad(AV-
AVI-AVID] or in conjunction with lambda repressor, in a cell line expressing a
nucleic
acid encoding that protein (packaging-masked helper vector).
In another preferred embodiment, the packaging signal region of a helper virus
genome is modified by the deletion of packaging elements AI-AII-AIII-AN,
retaining
only the packaging elements AV, AVI and AVII. A binding sequence comprising a
17
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by operator sequence (preferably, OL1) from bacteriophage lambda is inserted
into the
helper virus genome between elements AV and AVI (site #3), as well as upstream
of AV
and downstream from AVII (sites #1 and #2) (Figure 5).
In a further preferred embodiment, the packaging signal region of a helper
virus
genome is modified by the deletion of packaging elements AVI and AVII. A
binding
sequence comprising a 17 by operator sequence from bacteriophage lambda
(preferably,
OL1) is inserted into the helper virus genome between All and AIII (site #5),
as well as
upstream from AI and downstream from AV (sites #4 and #6) (Figure 6).
Preferred sites within the packaging signal region of the helper virus genome
for
insertion of any binding sequences (with reference to the A repeats numbered
from I-VII)
include, but are not limited to:
Table 1
Location relative to A repeat
1 5' to AV
2 3' to AVII
3 Between AV and AVI
4 5' to AI
5 Between All and AIII
6 3' to AV
In another embodiment of a helper virus, binding sequences that bind,
papilloma
virus E2 protein are inserted into the helper virus genome to flank alI or
part of an
packaging signal region in order to bind the E2 protein. Insertion sites set
forth in Table
1 are preferred.
Where the binding sequences comprise a lambda operator sequence used with the
lambda repressor to prevent the packaging of the helper virus during the
production of
PAV, it is preferable to flank the packaging signal region with the operator
sequences,
since cooperative binding of tetramers to pairs of operator sites is required
for full
repression (Ptashne, M. A Genetic Switch, Cell Press and Blackwell Scientific
Publications, 1986). Prior studies have demonstrated the feasibility of
blocking cis-acting
signals using the lambda operator/repressor system, g4g~, the TATA
transcriptional
element (Wedler et al., Mol. Gen. Genet. 248:499-505, 1995).
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Where the binding sequences comprise a FRT binding sequence, specific designs
of the packaging signal region of a helper virus genome (with reference to the
A repeats
I-VII) include, but are not limited to (Figure 7):
FRT(V-VI-VII)FRT
FRT{V-VI-VII)zFRT
FRT(V-VI-VII)3FRT
FRT(VI)IZFRT
FRT(VI),zlucFRT
A preferred embodiment of a helper adenovirus which comprises the packaging
signal regions FRT(VI},zFRT or FRT(V-VI-VII)zFRT includes Ad2HeIpFRT (Figure
8).
It will be recognized by those skilled in the art that other combinations of
deletions and insertions into and in proximity to the packaging signal region
of a helper
virus genome are within the scope of the invention and will create a helper
virus which is
disabled for packaging PAV in a cell line of the invention. Where a binding
sequence is
denoted (X) and an A repeat is denoted by a Roman numeral from I through VII
and a
subscript number denotes the number of times such a motif is repeated, other
recombinant
packaging signal regions include the following:
XVXVIXVII
XIXIIXIIIXIVXVXVIXVIIX
X(I-II-III-IV-V-VI-VII}X
X(I-II-III-N-V-VI-VII)zX
X(I-II-III-N-V-VI-VI)3X
X(I-II-III-N-V)X
X(I-II-III-IV-V)zX
X(I-II-III-IV-V)3X
X(V-VI-VII)X
X(V-VI-VII)zX
X(V-VI-VII)3X
X(VI),zX
X[~I)iz~zX
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X~~I)~z~3X
Another aspect of the invention is directed to helper viruses which are
designed to
reduce or prevent recombination between nucleotide sequences in a helper virus
packaging signal region and the PAV or between a packaging signal region or
other
regions in a helper virus genome and the adenovirus genome sequences in a
packaging
cell line, g~g,,, E1 sequences in 293 cells (nucleotides 1-4344 of adenoviras
serotype 5), so
as to prevent the generation of viable helper adenoviruses upon recombination.
Recombination between replication-defective adenoviral vectors and 293 cells
leading to the generation of replication-competent adenoviruses has been
demonstrated
(Hehir et al., J.Virol. 70:8459-8467, 1996). Where a helper virus genome
comprises
binding sequences that flank the packaging signal region (~, lox sites which
can be
cleaved by the cre enzyme provided by a packaging cell line, Parks et al.,
Proc.Natl.Acad.Sci.USA 93:13565-13570, 1996, or contain the FRT target sites
for the
FLP recombinase), recombination between homologous nucleotide sequences in the
helper virus genome and a producer cell could lead to the deletion of the
flanking target
sequences, e.g, the lox sites or the FRT sites. Such a deletion would
therefore prevent the
FLP or cre-mediated excision of the packaging signal in the helper virus which
reduces
packaging of the helper virus genomes during PAV production and packaging.
Accordingly, therefore, the invention also provides helper adenoviruses
contain
packaging signal regions that are not homologous with a PAV genome. In a
particular
embodiment of this aspect of the invention, a helper virus genome is provided
which
contains one or more AVI packaging signal sequences (reference for signal
elements,
Schmid et al., J. Virol. 71:3375-3384, 1997) which only minimally overlap with
any
nucleotide sequences in a PAV vector (~, nucleotides 1-356, containing only
packaging
signal elements AI-AV; disclosed in allowed U.S. Patent Application Serial No.
08/895,194 and U.S. Patent No. 5,670,488). The possibility of recombination
between the
PAV and the helper virus genome, therefore, is significantly reduced.
In a particularly preferred embodiment of a helper virus genome, the signal
region
comprises a (12 x AVI repeats) nucleotide sequence, or contains tandem repeats
of this
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signal. Such helper virus genome contains no overlapping nucleotide sequences
with a
PAV genome, which utilizes a packaging signal containing nucleotides sequences
from
the A repeats I-V. Such a helper packaging signal region can be flanked by
binding
sequences that facilitate excision of the packaging signal by a recombinase
(~,,g" lox,
FRT) or by binding sequences that facilitate binding to the packaging signal
by a
repressor protein (g~g~,, lambda repressor) in accordance with the invention.
A marker gene also may be inserted into a helper virus packaging signal region
in
order to act as a marker for the excision of the packaging signal by loss of
the
characteristic signal encoded by the marker gene, e.g., luciferase (assay kits
available
from Promega, Madison, WI). A preferred genome of a marker helper adenovirus
is
Ad2HeIpFRTIuc (Figure 9) which is constructed such that, upon excision of the
FRT-
flanked packaging signal by an FLP recombinase, the signal from a luciferase
protein can
no longer be detected. However, recombination with homologous nucleotide
sequences
in a producer cell line would also result in loss of the marker signal,
although the
packaging signal will not be deleted. Therefore, a marker helper adenovirus is
therefore
most optimally used in a producer cell line which does not contain nucleotide
sequences
which may generate recombination with the 5' region in a helper virus and
confound
interpretation of a marker assay for excision of a packaging signal by an FLP
recombinase. For example, a marker helper adenovirus may be used in a cell
line, such as
PER.C6 cells (containing adenovirus serotype 5 nucleotides 459-3510), whose
genome
comprises adenovirus sequences which only minimally overlap with any 5'
nucleotide
sequences in a helper virus, thereby reducing the likelihood of recombination
(Fallaux et
al., Hum.Gene Ther. 1:1909-1917, 1998). PER.C6 cells are preferably used in
the
production methods of the invention in order to minimize recombination between
producer cells and a helper adenovirus.
In a further embodiment of the invention, a helper virus is provided that
reduces
the possibility of recombination between the helper virus genome and the
overlapping
adenovirus nucleotide sequences in a complementing cell line (g~gt, E1
sequences in 293
cells) or in a PAV that can result in the production of viable helper viruses
which
contaminate a PAV preparation. The genome of helper virus comprises a
packaging
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signal which is prevent in a reverse orientation such that, even though
recombination may
occur between the helper virus and either the packaging cell or the PAV
genome, the
resulting recombination products do not constitute viable adenovirus. The
packaging
signal of such helper virus comprises any combination of the A repeat elements
(Schmid
S et al., J. Virol. 71:3375-3384, 1997) sufficient to confer packaging
capability, inserted
into the genome in a reverse orientation. In a particularly preferred
embodiment, the
packaging signal region includes a (12 x AVI repeats} sequence in a reverse
orientation.
In a further embodiment of the invention, a helper virus is provided which is
inactivated upon the acquisition of homologous sequences from a recombination
event
with a complementing cell line, preferably an El-complementing cell line, such
as 293
cells. For example, this helper virus is engineered such that its size, upon a
recombination
event, renders the virus genome too large for packaging. A preferred
embodiment is the
helper virus TBTP whose genome contains a FRT-flanked packaging signal (Figure
10),
and in which the E3 region of the adenovirus genome is deleted for 2.9 kb, but
into which
the 1.8 kb EGFP gene operably linked to a CMV promoter and a 5.0 kb fragment
of the
human alpha-antitrypsin gene are inserted. The size of the helper virus is
36.9 kb (102%
of wild-type), but upon recombination with adenovirus E 1 sequences in 293
cells, the
helper virus genome size becomes approximately 39.6 kb (110%), which is too
large to be
packaged.
To create the particular helper viruses of the invention, the adenovirus
genome,
whether wild-type or recombinant, is modified by the insertion thereto of
binding
sequences in proximity to or into the A repeats of the packaging signal region
so as to
prevent access to the cis-acting packaging signal upon the binding of a DNA
binding
and/or repressor protein to the specific binding sequences. Standard techniqes
of
molecular biology such as restriction enzyme digestion and ligation,
polymerase chain
reaction and site-directed mutagenesis can be used to create a recombinant
packaging
signal region within a helper adenovirus genome. Such a packaging signal
region can
then be inserted into the appropriate site in the 5' region of an adenovirus
genome utilizing
a plasmid comprising the signal. Such a plasmid can be co-transfected into a
cell line
with DNA encoding the remainder of the adenovirus helper genome to be
contained in the
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helper virus, such that homologous recombination occurs, thereby generating a
helper
adenovirus with the desired recombinant packaging signal. The helper viral
genome can
also be constructed in bacteria, thus simplifying the procedure (Chartier et
al., J.Virol.
70:4805-4810, 1998). The entire viral genome can be provided by transfection
of a
plasmid from which any non-viral (i.e., bacterial) sequences have been
removed. Other
methods for the production of recombinant adenoviruses are known to those
skilled in the
art and can be used to produce the helper viruses of the invention.
The helper viruses of the invention can be derived from any wild-type,
truncated
or mutant adenovirus whose genome encodes the viral proteins required in trans
to
produce PAVs, and are not limited by serotype. Preferably, the helper viruses
of the
invention are also replication-defective, as an additional safety feature for
the use in
generating PAVs for use in gene transfer. Replication-defective viruses can be
created by,
for example, deletion of the E1 region of the adenovirus genome. Such helper
viruses can
be propagated in E1-complementing cell lines such as the 293 cell line (Graham
et al.,
J.Gen.Viro1.36:59-72, 1977).
Although the invention provides packaging-defective helper adenoviruses, the
genomes of such viruses are desirably optimized for replication and gene
expression in
order to ensure adequate levels of adenoviral helper proteins. Therefore, in
one particular
embodiment of the invention, a helper virus genome contains an internal ITR
sequence
located downstream from the blocked packaging signal region which allows for
adequate
replication and expression of the helper virus genome in the PAV producer cell
line,
thereby ensuring adequate provision of the viral proteins required in fans.
Although a
repressor protein can occupy the binding sequences inserted into or near a
helper virus
packaging signal, this particular embodiment of the invention provides an
exposed ITR
which is available to the adenovirus DNA polymerise and terminal protein for
replication
of the helper virus genome (Figure 11).
In a further aspect of the invention, a method for the production of PAV
vectors is
provided which uses a PAV genome and a helper adenovirus that contain
packaging
signal regions or ITR sequences from different adenovirus serotypes such that
sequence
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overlap between PAV genome and helper adenovirus is minimized and the
possibility of
recombination is reduced.
The invention is further directed to PAV producer cell lines that produce DNA
binding andJor repressor proteins which can bind to the binding sequences
inserted into or
near the packaging signal region of the helper virus genome. As a result of
this
interaction, the helper virus is disabled for packaging or deleted during the
production of
PAV stocks. In a preferred embodiment of the invention, 293 cells comprising
and
express a nucleic acid encoding a DNA binding and/or repressor protein,
thereby creating
a PAV producer cell line of the invention. Other cell lines can be used,
including, but not
limited to, VK2-20, as well as any cell lines designed to complement deletions
of
adenoviral genomic regions E2a (Zhou et al., J. Virol. 70:7030-7038, 1996), E4
(Krougliak et al., Hum. Gene Ther. 6:1575-1586, 1995; Wang et al., Gene Ther.
2:775-
783, 1995), or protein IX (Caravokyri et al., J. Virol. 69:6627-6633, 1995;
Krougliak et
al., Hum. Gene Ther. 6:1575-1586, 1995).
Preferably, 293 cells comprise and express a nucleic acid encoding a DNA
binding
protein, preferably the FLP or FLPe recombinase, under the control of the
tetracycline
gene control system (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89:5547-
5551,
1992; and Gossen et al., Science 268:1766-1769, 1995, both incorporated here
by
reference). In this system, gene expression from a minimal promoter is under
strict
control of the tetracycline repressor (TetR), expressed as a fusion protein
with the herpes
virus VP 16 transcriptional activation domain (TetR/VP 16). Two versions of
the
TetR/VP16 protein exist: the wild type TetR is active only in the absence of
tetracycline
or doxycycline, while a mutated form of TetR (reverse TetR or rTetR) is active
only in the
presence of doxycycline. When linked to VP16, the TetR form activates
transcription
only in the absence of tetracycline and the rTetR form activates transcription
only in the
presence of doxycycline. Each of these transcriptional control factors,
TetR/VP16 and
rTetR/vpl6 controls the expression of genes linked to a minimal promoter
cloned adjacent
to tetracycline transcriptional regulatory elements (TRE), such as a gene for
FLP
recombinase stably integrated into 293 cells. A 293 cell line incorporating a
nucleic acid
encoding the Tet-off fusion protein (TetR-VP16) can be constructed by the
manufacturer
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(Clontech, Palo Alto, CA} or can be produced by transfection of a Tet-
offplasmid
available from Clontech. It will be noted that a 293-Tet-off cell line which
further
expresses a FLP recombinase can also be used to inducibly and preferentially
excise a
helper adenovirus packaging signal region which is flanked by FTR binding
sequences or
could also be used to excise any desired segment of an adenoviral genome which
is
flanked by the requisite FRT binding sequences.
In this embodiment of the invention, therefore, a nucleic acid encoding the
FLP
recombinase is cloned into a pTRE plasmid (Clontech, Palo Alto, CA) (Figure
12), such
that the FLP recombinase is under the control of a minimal CMV promoter
operably
linked thereto and seven tet operator sites which are responsive to the TetR-
VP 16 fusion
protein (Figure 13a). When tetracycline is provided to the 293 Tet-off
producer cells,
transcription from the FLP recombinase gene does not occur (Figure 13b).
Modulation of
expression of the FLP recombinase, and therefore of FLP-catalyzed excision of
a helper
adenovirus packaging signal in a dose-dependent manner can occur relative to
the level of
tetracycline provided to the producer cell.
Preferably, a producer cell line of the invention is a 293 cell line
comprising a
nucleic acid encoding Tet-off fusion protein and the FLP recombinase under the
control of
the THE operator sequences and the minimal CMV promoter such that the cell
line
provides modulated production of the FLP recombinase when provided with a PAV
genome and a helper virus of the invention containing a packaging signal
flanked by FRT
binding sequences, such that the helper virus is preferentially packaging-
disabled during
PAV vector production.
Producer cell lines which stably express a nucleic acid encoding the FLP
recombinase can also be constructed using plasmids which contain the gene
encoding FLP
recombinase under the control of any suitable promoter, such as CMV or SV40.
Examples of such plasmids which contain a FLP recombinase gene are pSVK/FLPe6
(containing FLP recombinase gene under the control of the SV40 promoter)
(Figure 14)
and pCEP/FLP36, containing FLP recombinase gene operably Iinked to the CMV
promoter (Figure 15). Where it is desirable to provide the FLP recombinase
gene to a cell
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for immediate expression therein and assay, a plasmid which is maintained
extrachromosomally, e.g., pCEP/FLPe6 (Figure 15) can be used.
Nucleic acids encoding the DNA binding and/or repressor proteins are
engineered
into PAV producer cell lines by standard techniques of molecular biology, and
can be
stably or inducibly expressed. Preferred DNA binding and/or repressor proteins
to be
used in the invention include, but are not limited to, the bacteriophage
lambda repressor
(wild-type and/or N-terminal fragment) Cro protein (Ptashne, M. A Genetic
Switch, Cell
Press and Blackwell Scientific Publications, 1986); the bovine papillomvirus
E2 protein
(McBride et al., J.Biol. Chem. 266:18411-18414, 1991), tetracycline repressor
(Gossen et
al., Proc.Natl.Acad.Sci. USA 89:5547-5551, 1992),tryptophan repressor, and
others
known to those skilled in the art. Specif c recombinases of the invention
which can be
used to excise a packaging signal flanked by the appropriate binding sequences
include,
but are not limited to, FLP recombinase (Broach et al., CeII 21:501, 1980) or
FLPe
recombinase (Buchholz et al., NAR 24:4256-4262, 1996; Buchholz et al., Nature
Biotech. 16:657-662, 1998). Other DNA binding and/or repressor proteins which
are
capable of binding to the relevant binding sequences in a helper virus genome
can be used
in the cell lines of the invention. Fusion proteins of such repressor proteins
can also be
constructed, for example, by creating a nucleic acid encoding E. coli ~3-
galactosidase
linked to the lambda repressor protein, thereby providing a substantial
physical obstacle to
packaging of the helper virus genome when such a fusion protein binds to the
binding
sequence inserted into or near a packaging signal in a helper virus genome.
Fusion
proteins can also be generated by creating a hybrid nucleic acid encoding a
DNA binding
and/or repressor protein fused to an activator protein (see tet repressor/HSV
VP16,
Gossen et al., Proc.Natl.Acad.Sci. USA 89:5547-5551, 1992).
The lambda repressor protein monomer is 236 amind acids in length (26KD); a
repressor protein dimer binds to one 17 by lambda operator sequence. The 6
lambda
operator sites are recognized by the lambda repressor protein dimer in order
of their
intrinsic affinities for a lambda repressor dimer, each with a central base
pair, the axis of
symmetry. The N-terminal protein domain of the lambda repressor recognizes the
operator and can be used for binding; in one embodiment of the invention, the
C-terminal
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domain of the repressor can be replaced with, for example, a dimeric leucine
zipper
protein.
To create the PAV producer cell lines of the invention, a nucleic acid
encoding a
DNA binding and/or repressor protein and the nucleotide sequences for any
operably
linked regulatory elements are introduced into a cell line by any method of
nucleic acid
transfer, including, but not limited to, transfection, electmporation, or
viral-mediated
transfer. A plasmid comprising a nucleic acid encoding a DNA binding and/or
repressor
protein and nucleotide sequences corresponding to any regulatory elements can
be
transfected into a cell of interest. If the plasmid further contains a nucleic
acid encoding a
selectable marker, integration of the exogenous plasmid DNA can be detected
using such
marker. For example, a nucleic acid encoding neomycin resistance can be
introduced in
parallel with the nucleic acid encoding a repressor protein, and the cells
which are stably
transfected thereby can be selected by cultivation in the presence of 6418.
Alternatively,
a nucleic acid encoding such the repressor protein can be provided to a cell
on an
extrachromosomal plasmid which is maintained episomally (gsg~,, EBNA-based
system),
such as pCEP-4 (Invitrogen, San Diego, CA).
It is within the scope of the invention to use any binding sequence-repressor
protein pair in the design of the helper viruses and cell lines of the
invention which is able
to effectuate a binding interaction that prevents utilization of the packaging
signal in a
helper virus genome or which facilitates the excision of the signal, thereby
preventing
packaging of the helper virus.
The helper adenoviruses and producer cell lines are useful in high-level
production
of PAV, allowing for preferential packaging of PAV genomes into gene transfer
vectors
relative to the helper viruses, thereby providing a means to produce helper-
dependent
PAVs with minimal contamination by helper viruses.
The invention is also directed to methods for the production of PAVs in high
yield, using the helper viruses and producer cell Lines of the invention. In
such methods
PAV is preferentially produced, generating an enriched preparation. To produce
a PAV
stock, the PAV genome which comprises the adenovirus 5' ITR and packaging
signal and
3' ITR, and further comprises one or more transgenes up to 36 kb in size,
operably linked
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to expression control sequences, can be engineered into a plasmid using
standard
techniques of molecular biology (see e.g., Allowed U.S. Patent Application
Serial No.
08/895,194 and U.S. Patent No. 5,670,488, incorporated herein by reference).
The DNA
fragment comprising the PAV genome is then enzymatically excised from the
plasmid
and co-transfected with a helper virus of the invention into a producer cell
line of the
invention. In accordance with the invention, PAVs are preferentially packaged
because
the PAV genome contains a wild-type packaging signal, in contrast to the
helper virus in
which the packaging signal has been disabled or deleted.
When the PAV genome is delivered to a producer cell on a plasmid, such plasmid
can be introduced into a cell line of the invention by any method of nucleic
acid transfer,
including, but not limited to, transfection, lipofection and electroporation.
The cell line
can he infected with an adenovirus helper which is available to provide the
adenovirus
proteins needed in trans to produce and package the PAV genome. In a preferred
embodiment of the invention, 2-20 pg of DNA which encodes a PAV genome is
delivered
to a cell by lipofection using a kit such as Profectin (Promega, Madison, WI},
and the
cells are infected with a helper virus of the invention using a multiplicity
of infection
(MOI) from 0.5 to 10.
In another aspect of the invention, a PAV producer cell line comprises an
integrated PAV genome, as well as a nucleic acid encoding DNA binding and/or
repressor
protein, such that the PAV genome can be conditionally excised and the nucleic
acid
encoding the binding and/or repressor protein can be conditionally expressed.
In a
preferred embodiment, a PAV vector genome flanked by lox nucleotide sequences
is
stably integrated into a PAV producer cell line which is engineered to express
a nucleic
acid encoding the Cre recombinase {Sternberg et al., J.MoI. Biol. 150:467-486,
1981;
Sauer et al., Meth. Enzymol. 225:890-910, 1993) and a repressor protein. Both
Cre
recombinase and the repressor protein can be inducibly produced using one or
more
inducible promoters susceptible to induction by such agents as tetracycline or
ecdysone,
among others. Upon infection by a helper virus of the invention, and induction
of the
recombinase and repressor, the PAV genome is excised and available for
replication and
packaging by the helper virus, while the helper virus is rendered packaging-
disabled by
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the repressor protein. This strategy for the production of PAV requires only
infection of
the producer cell by a helper virus and induction of the recombinase and
repressor
proteins to generate a preferentially packaged PAV stock. The use of other
site-specific
recombinases is also within the scope of this embodiment of the invention,
g,g~, the FLP
recombinase and its target sequence, FRT.
Purification of PAVs from a cell line of the invention can be performed by
standard techniques of virus purification known to those skilled in the art.
For example,
viruses in cell lysates from producer cells can be purified on a standard CsCI
gradient.
The PAV particles are of lower density relative to the helper viruses and will
band at a
higher position in the gradient, allowing for direct isolation and recovery.
Alternatively,
PAV purification can be performed using chromatographic techniques, g~gr., as
set forth in
published PCT Application W097/08298, incorporated herein by reference.
PAV yield is calculated by measuring the DNA and protein composition of the
purified preparation. Maizel et al. (Virology 36:115-125,1968) determined that
an
adenovirus virion comprises 13% DNA, with the remainder being protein.
Transgene activity of a PAV preparation is monitored by immunoflourescent
techniques by infecting 293 cells with the PAV helper, then using an antibody
against a
PAV-encoded transgene expression product (protein) to determine infectious
particles.
Alternatively, enzyme activity encoded by a transgene can be measured (g,g,.,
~-
galactosidase, a-galactosidase, a-antitrypsin), by, for example, an ELISA
assay. The
ability of PAV to enter cells is determined by measuring the amount of viral
capsids that
bind to the cells with anti-adenovirus antibodies. Demonstration of the entry
of the PAV
genome into a cell can be performed by fluorescent in situ hybridization
(FISH).
Minimal contamination of the PAV stock is expected using the novel helper
viruses of the invention. Helper virus production, if any, can be scored, for
example, by
standard plaque assays on 293 cells. Preferably, the ratio of PAV to helper
virus will be
greater than 10,000 to 1.
The practice of the invention employs, unless otherwise indicated,
conventional
techniques of protein chemistry, molecular virology, microbiology, recombinant
DNA
technology, and pharmacology, which are within the skill of the art. Such
techniques are
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explained fully in the literature. See, ~,,g,,, Current Protocols. in Molec~
Biology,
Ausubel et al., eds., John Wiley & Sons, Inc., New York, 1995, and Remington's
Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, PA, 1985.
The invention is further illustrated by the following specific examples which
are
not intended in any way to limit the scope of the invention.
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EXAMPLE 1: Construction of FRT-containing helper adenoviruses
Construction of pAD/FRT(,AV-AVI-AVII)2 RF T and pAD/FRT AV-AVI-AVIIl3
~. Plasmid pAd/ITR (1-194)Mlu2 was digested with SpeI and Mlu I.
Oligonucleotides containing a 5'-~3' FRT site upstream from two copies of the
AV-AVI-
AVII packaging sequence were annealed together and ligated into the SpeI/Mlu I
site of
pAD/ITR(1-194)Mlu 2. The resulting vector was designated pAD/FRT(AV-AVI-
AVII)2.
Oligonucleotides 4758 {5' - CGC GTG AAG TTC CTA TTC CGA AGT TCC TAT TCT
CTA GAA AGT ATA GGA ACT TCA) (SEQ ID N0:12) and 4759 (5' - CGC GTG AAG
TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC GGA ATA GGA ACT TCA)
(SEQ ID N0:13), containing a second FRT site oriented in the same direction as
the first,
were annealed together and ligated into the Mlu I site of pAD/FRT(AV-AVI-
AVII)2 and
designated pAD/FRT(AV-AVI-AVII)ZFRT {Figure 7). Alternatively,
oligonucleotides
4760 (5' - CGC GTC GCG TAA TAT TTG TCT AGG GCC GCG GGG ACT TTG ACC
GTT TAG AAG TTC CTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA
ACT TCA) (SEQ ID N0:14) and 4761 (5' - CGC GTG AAG TTC CTA TAC TTT CTA
GAG AAT AGG AAC TTC GGA ATA GGA ACT TCT AAA CGG TCA AAG TCC
CCG CGG CCC TAG ACA AAT ATT ACG CGA) (SEQ ID NO:15), containing a third
copy of AV-AVI-AVII upstream from a second FRT site, were annealed together
and
ligated into the Mlu I site of pAD/FRT (AV-AVI-AVII)2 and designated
pAD/FRT(AV-
AVI-AVII)3FRT {Figure 7).
Construction of pAD/FRT(,~,~/luc/FRT usine olisonucleotides contai~n_g
twelve cosies of AVI. Oligonucleotides containing a 5'-~3' FRT site upstream
from
twelve head-to-tail copies of the AVI packaging sequence were annealed
together and
ligated into the Spe I / Mlu I site of pAD/ITR(1-194)Mlu2, generating
pAD/FRT(AVI)12
pGL3 control vector (Promega, Madison, WI) was digested with Mlu I and Bam HI
and
the 2427 by fragment containing the luciferase cDNA under the control of the
SV40
promoter/enhancer elements was isolated. Oligonucleotides containing a second
FRT site
were annealed together and coligated with a 2427 by luciferase fragment into
the Mlu I
site of pAD/FRT(AVI),2_ The resulting vector containing an FRT flanked
(AVI)12/luc
cassette was designated pAD/FRT(AVI),2/luclFRT {Figure 9).
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Construction of pAD/FR'~~1~FRT using an oligonucleotide containing one
,cony of AVI. Head-to-tail copies of the AVI packaging sequence were
constructed by
concatomerizing oligonucleotides 4755 (5' - TCG ACC GCG GGG ACT TTG ACC)
(SEQ ID N0:16) and 4754 (5' - TCG AGG TCA AAG TCC CCG CGG) (SEQ ID
N0:17) in the presence of T4 DNA ligase. Following ligation, the reaction was
digested
with Xho I to eliminate head-to-head and tail-to-tail ligation products, and
cloned into the
Xho I site of pSL1180 (Amersham Pharmacia Biotech, Piscataway, NJ) generating
pSL/(AVI),2 FRT sites flanking the AVI repeats were inserted in two
consecutive cloning
steps. pSL/(AVI)1z was digested with Spe I and partially digested with Xho I.
Oligonucleotides HR100 (5' - CTA GTG AAG TTC CTA TTC CGA AGT TCC TAT
TCT CTA GAA AGT ATA GGA ACT TCC) (SEQ ID N0:18) AND HR101 (5' - TCG
AGG AAG TTC CTA TAC TTT CTA GAG AAT AGG AAC TTC GGA ATA GGA
ACT TCA) (SEQ ID N0:19) containing an FRT site was ligated in to the Spe I /
Xho I
sites upstream from (AVI},2. The resulting vector, pSL/FRT(AVI)~2 was digested
with
Mlu I and partially digested with Xho I. Oligonucleotides HR102 (5' - TCG AGG
AAG
TTC CTA TTC CGA AGT TCC TAT TCT CTA GAA AGT ATA GGA ACT TCA)
(SEQ ID N0:20) and HR103 (5' - CGC GTG AAG TTC CTA TAC TTT CTA GAG
AAT AGG AAC TTC GGA ATA GGA ACT TCC) (SEQ ID N0:21) containing a second
FRT site was ligated into the Spe I /Xho I site downstream from (AVI),2. The
resulting
vector was designated pSL/FRT(AVI)12FRT.
Helper adenoviruses containing the above-described designs of the 5'
adenovirus
packaging signal region are constructed in vitro using standard ligation
techniques or by
homologous recombination in vivo with any desired adenovirus.
EXAMPLE 2: Construction of helper adenoviruses containing lambda operator
sites
Helper vector Ad(AV-AVI-AVID. Adenovirus nucleotides I through 194 were
isolated by PCR and cloned into pAdvantage (Genzyme Gene Therapy, Framingham
MA}, in place of Ad nucleotides 1 through 357. pAdvantage encodes the Ad2
genome
(~E1, E302.9) in pBR322. Sequences encoding a deleted packaging site (AV-AVI-
AVII)
(nucleotides 334-385) were cloned into an SpeI / MIuI site within the vector
and flanked
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by binding sites for the lambda repressor (Figure 3a; junction sequences are
shown in
Figure 3b). This vector is 40-fold impaired in packaging, compared to a wild-
type vector.
Helper vector Ad(AV-AVI-AVII)2 was built similarly to Ad(AV-AVI-AVID
(Figure 4a; junction sequences are shown in Figure 4b). This helper vector
incorporates 2
copies of the (AV-AVI-AVID packaging repeat sequences, giving it a packaging
efficiency equivalent to wild-type
EXAMPLE 3: Construction of TBTP helper adenovirus
The E3 gene of Ad2 (nts 27970 - 30937) is replaced with a l.8kb EGFP
expression cassette (CMV / EGFP / SvpA), at a genetically engineered RsrII
site in the
E3-deleted genome. A 5.0 kb Hinp l I/AccI fragment from the human genomic AAT
gene
(ghAAT) is cloned in an AccI site just upstream of the EGFP expression
cassette. The
size of the completed vector is 36.9 kb (102% wild-type) (Figure 10). After
recombination with adenovirus sequences from 293 cells, the vector size will
be
approximately 39.6 kb (110%).
EXAMPLE 4: Construction of producer cells expressing FLP recombinase
pFLPe6. pOG-Flpe6 was received from Francis Stewart (EMBL, Heidelberg,
Germany) (Figure 16). This gene contains mutations of the FIp gene that make
the
encoded protein more stable at 37°C. The gene, with upstream and
downstream
regulatory elements, is cloned into pOG44 ~Stratagene, La Jolla, CA) behind
the CMV
promoter (Figure 16}.
pSVK/FLPe6. The 2.0 kb XbaI / SaII fragment of pOG-Flpe6 was cloned directly
into the XbaI / SaII site of the 3.9 kb pSVK3 (Pharmacia, Piscataway, NJ). The
plasmid
pSVK/FLPe6 has the Flpe6 gene under the control of both eukaryotic (SV40e) and
Prokaryotic (T7) promoters (Figure 14).
pCEP/FLPe6. The 2.0 kb XbaI / SaII fragment of pOG-Flpe6 was cloned into the
Acc65I / XhoI site of the 10.4 kb pCEP-4 (Invitrogen, San Diego, CA ) using
adapter
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linkers. The plasmid pCEP/FLPe6 has the Flpe6 gene under the control of the
CMV
promoter. The plasmid retains the EBNA-1 gene and EBV origin for
extrachromosomal
plasmid maintenance and the hygromycin gene for plasmid selection (Figure 1
S).
pTRE/FLPe6. The 2.0 kb XbaI / SaII fragment of pOG-Flpe6 was cloned into
Acc65I / XhoI site of the 3.1 kb pTRE plasmid (Clontech, Palo Alto, CA) using
adapter
linkers. The Flpe6 gene in pTRE/FLPe6 is under the control of a minimal CMV
promoter
(hCMV*) and 7 operator sites (tet O, 1 through 7).
pCEP/Flpe6 cells. 293 and PER.C6 cells are transfected with 10-20~cg of
pCEP/Flpe6 DNA. Following hygromycin selection, cells expressing high levels
of Flpe6
are selected based on a functional assay for FLP activity, such as FRT-
mediated excision
of a target sequence. Cells with a stable extrachromosomal plasmid copy number
are
selected for PAV amplification.
pSVK/Flpe6 cells. Cells are transfected similarly as described above, with the
1 S addition of a plasmid bearing a neo resistance gene marker. Following neo
selection, cells
expressing high levels of Flpe6 are selected based on a functional assay for
FLP activity,
such as FRT-mediated excision of a target sequence. Cells with stable,
integrated
pSVKlFlpe6 DNA are selected for PAV amplification.
pTRE/Flpe6 cells. Cells are transfected as described above, with the addition
of
the pTK/hygro plasmid (Clontech, Palo Alto, CA) in the transfection. Following
hygromycin selection, cells expressing low background and high tetracyline-
induced
levels levels of Flpe6 are selected based on a functional assay for FLP
activity, such as
FRT-mediated excision of a target sequence. Cells with stable, integrated,
inducible
pTRE/Flpe6 are selected for PAV amplification.
EXAMPLE 5: Propagation of PAV using helper adenoviruses
PAV DNA excised from backbone DNA (4-20,ug) is used to transfect semi-
confluent PER.C6 or 293 cells. To select for PAV packaging at the expense of
helper
vector, the cells express a repressor protein, which, upon binding to cognate
sequences in
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the helper vector, prevents its packaging. After overnight incubation, cells
are infected at
an MOI of 1 to 10 with an E1-deleted helper adenovirus harboring the
appropriate
packaging impaired sequences. Following the observation of complete cytopathic
effect,
cells and lysates are collected and the lysate is used for serial propagayion
and expansion
of PAV. Following several amplifications, the cell lysate is CsCI banded for
the isolation
of PAV.
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SEQUENCE LISTING
<110> Samuel C. Wadsworth
Helen Romanczuk
Richard J. Gregory
Donna Armentano
<120> METHODS FOR PSEUDOADENOVIRAL VECTOR
PRODUCTION
<130> 31428-A-A-pct
<150> 60/074,761
<151> 1998-02-17
<150> 60/086,528
<151> 1998-05-22
<160> 21
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 17
<212> DNA
<213> LAMBDA
<400> 1
PCT/C1S99/03483
tatcaccgcc agtggta 17
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<210> 2
<211> 17
<212> DNA
<213> LAMBDA
<400> 2
tatcaccgcc agaggta 17
<210> 3
<211> 17
<212> DNA
<213> LAMBDA
<400> 3
tatctctggc ggtgttg 17
<210> 4
<211> 17
<212> DNA
<213> LAMBDA
<400> 4
tatcaccgca gatggtt 17
<210> 5
<211> 17
<212> DNA
<213> LAMBDA
<400> 5
taacaccgtg cgtgttg 17
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<210> 6
<211> 17
<212> DNA
< 213 > LAMBDA
<400> 6
tatcaccgca agggata 17
<210> 7
<211> 12
<212> DNA
<213> PAPILLOMA VIRUS
<400> 7
accgaaatcg gt 12
<210> 8
<211> 12
<212> DNA
<213> PAPILLOMA VIRUS
<400> 8
accgaaaccg gt 12
PCT/US99/03483
<210> 9
<211> 12
<212> DNA
<213> papilloma virus
<400> 9
accnnnnnng gt 12
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<210> 10
<211> 48
<212> DNA
<213> yeast
PCT/US99/03483
<400> 10
gaagttccta ttccgaagtt cctattctct agaaagtata ggaacttc 48
<210> 11
<211> 35
<212> DNA
<2I3> yeast
<400> 11
cgaagttcct attctctaga aagtatagga acttc 35
<210> 12
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 12
cgcgtgaagt tcctattccg aagttcctat tctctagaaa 40
gtataggaac ttca
54
<210> 13
<221> 54
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S
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 13
cgcgtgaagt tcctatactt tctagagaat aggaacttcg 40
gaataggaac ttca 54
<210> 14
<211> 96
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 14
cgcgtcgcgt aatatttgtc tagggccgcg gggactttga 40
ccgtttagaa gttcctattc cgaagttcct attctctaga 80
aagtatagga acttca 96
<210> 15
<211> 96
<212> DNA
<213> Artificial Sequence
<220>
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<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 15
cgcgtgaagt tcctatactt tctagagaat aggaacttcg 40
gaataggaac ttctaaacgg tcaaagtccc cgcggcccta 80
gacaaatatt acgcga 96
<210> 16
<211> 21
<212> DNA
<213> adenovirus
<400> 16
tcgaccgcgg ggactttgac c 21
<210> 17
<211> 21
<212> DNA
<213> adenovirus
<400> 17
tcgaggtcaa agtccccgcg g 21
<210> 18
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
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adenovirus sequences
PCT/ITS99/03483
<400> 18
ctagtgaagt tcctattccg aagttcctat tctctagaaa 40
gtataggaac ttcc 54
<210> 19
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 19
tcgaggaagt tcctatactt tctagagaat aggaacttcg 40
gaataggaac ttca 54
<210> 20
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 20
tcgaggaagt tcctattccg aagttcctat tctctagaaa 40
gtataggaac ttca 54
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<210> 21
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> synthesized as a hybrid of yeast and
adenovirus sequences
<400> 21
cgcgtgaagt tcctatactt tctagagaat aggaacttcg 40
gaataggaac ttcc 54
