Stocks of recombinant, replication-deficient adenovirus free of replication-competent adenovirus

The present invention provides multiply deficient adenoviral vectors and complementing cell lines. Also provided are recombinants of the multiply deficient adenoviral vectors and a therapeutic method, particularly relating to gene therapy, vaccination, and the like, involving the use of such recombinants.

TECHNICAL FIELD OF THE INVENTION 
The present invention relates to recombinant, multiply deficient adenoviral 
vectors and complementing cell lines and to the therapeutic use of such 
vectors. 
BACKGROUND OF THE INVENTION 
During the winter and spring of 1952-1953, Rowe and his colleagues at the 
National Institutes of Health (NIH) obtained and placed in tissue culture 
adenoids that had been surgically removed from young children in the 
Washington, D.C. area (Rowe et al., Proc. Soc. Exp. Biol: Med., 84, 
570-573 (1953)). After periods of several weeks, many of the cultures 
began to show progressive degeneration characterized by destruction of 
epithelial cells. This cytopathic effect could be serially transmitted by 
filtered culture fluids to established tissue cultures of human cell 
lines. The cytopathic agent was called the "adenoid degenerating" (Ad) 
agent. The name "adenovirus" eventually became common for these agents. 
The discovery of many prototype strains of adenovirus, some of which 
caused respiratory illnesses, followed these initial discoveries (Rowe et 
al., supra; Dingle et al., Am. Rev. Respir. Dis., 97, 1-65 (1968); 
reviewed in Horwitz, "Adenoviridae and their replication," In Virology, 
Fields et al., eds., 2nd ed., Raven Press Ltd., New York, N.Y., pp. 
1679-1721 (1990)). 
Over 40 adenoviral subtypes have been isolated from humans and over 50 
additional subtypes have been isolated from other mammals and birds 
(reviewed in Ishibashi et al., "Adenoviruses of animals," In The 
Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562 
(1984); Strauss, "Adenovirus infections in humans," In The Adenoviruses, 
Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984)). All 
these subtypes belong to the family Adenoviridae, which is currently 
divided into two genera, namely Mastadenovirus and Aviadenovirus. All 
adenoviruses are morphologically and structurally similar. In humans, 
however, adenoviruses show diverging immunological properties and are, 
therefore, divided into serotypes. Two human serotypes of adenovirus, 
namely Ad2 and Ad5, have been studied intensively and have provided the 
majority of information about adenoviruses in general. 
Adenoviruses are nonenveloped, regular icosahedrons, 65-80 nm in diameter, 
consisting of an external capsid and an internal core. The capsid is 
composed of 20 triangular surfaces or facets and 12 vertices (Horne et 
al., J. Mol. Biol., 1, 84-86 (1959)). The facets are comprised of hexons 
and the vertices are comprised of pentons. A fiber projects from each of 
the vertices. In addition to the hexons, pentons, and fibers, there are 
eight minor structural polypeptides, the exact positions of the majority 
of which are unclear. One minor polypeptide component, namely polypeptide 
IX, binds at positions where it can stabilize hexon-hexon contacts at what 
is referred to as the group-of-nine center of each facet (Furcinitti et 
al., EMBO, 8, 3563-3570 (1989)). The minor polypeptides VI and VIII are 
believed to stabilize hexon-hexon contacts between adjacent facets, and 
the minor polypeptide IIIA, which is known to be located in the regions of 
the vertices, is suggested to link the capsid and the core (Stewart et 
al., Cell, 67, 145-154 (1991)). 
The viral core contains a linear, double-stranded DNA molecule with 
inverted terminal repeats (ITRs), which vary in length from 103 bp to 163 
bp (Garon et al., PNAS USA 69, 2391-2394 (1972); Wolfson et al., PNAS USA, 
69, 3054-3057 (1972); Arrand et al., J. Mol. Biol., 128, 577-594 (1973); 
Steenberg et al., Nucleic Acids Res., 4, 4371-4389 (1977); and Tooze, DNA 
Tumor Viruses, 2nd ed., Cold Spring Harbor, N.Y.: Cold Spring Harbor 
Laboratory. pp. 943-1054 (1981)). The ITRs harbor origins of DNA 
replication (Garon et al., supra; Wolfson et al., supra; Arrand et al., 
supra; Steenberg et al., supra). The viral DNA is associated with four 
polypeptides, namely V, VII, .mu., and terminal polypeptide (TP). The 55 
kd TP is covalently linked to the 5' ends of the DNA via a dCMP (Rekosh et 
al., Cell, 11, 283-295 (1977); Robinson et al., Virology, 56, 54-69 
(1973)). The other three polypeptides are noncovalently bound to the DNA 
and fold it in such a way as to fit it into the small volume of the 
capsid. The DNA appears to be packaged into a structure similar to 
cellular nucleosomes as seen from nuclease digestion patterns (Corden et 
al., PNAS USA, 73, 401-404 (1976); Tate et al., Nucleic Acids Res., 6, 
2769-2785 (1979); Mirza et al., Biochim. Biophys. Acta, 696, 76-86 
(1982)). 
The overall organization of the adenoviral genome is conserved among 
serotypes, such that specific functions are similarly positioned. The Ad2 
and Ad5 genomes have been completely sequenced and sequences of selected 
regions of genomes from other serotypes are available. 
Adenovirus begins to infect a cell by attachment of the fiber to a specific 
receptor on the cell membrane (Londberg-Holm et al., J. Virol., 4, 323-338 
(1969); Morgan et al., J. Virol., 4, 777-796 (1969); Pastan et al., 
"Adenovirus entry into cells: some new observations on an old problem," In 
Concepts in Viral Pathogenesis, Notkins et al., eds., Springer-Verlag, New 
York, N.Y., pp. 141-146 (1987)). Then, the penton base binds to an 
adenoviral integrin receptor. The receptor-bound virus then migrates from 
the plasma membrane to clathrin-coated pits that form endocytic vesicles 
or receptosomes, where the pH drops to 5.5 (Pastan et al., Concepts in 
Viral Pathogenesis, Notkins and Oldstone, eds. Springer-Verlag, N.Y. pp. 
141-146 (1987)). The drop in pH is believed to alter the surface 
configuration of the virus, resulting in receptosome rupture and release 
of virus into the cytoplasm of the cell. The viral DNA is partially 
uncoated, i.e., partially freed of associated proteins, in the cytoplasm 
while being transported to the nucleus. 
When the virus reaches the nuclear pores, the viral DNA enters the nucleus, 
leaving most of the remaining protein behind in the cytoplasm (Philipson 
et al., J. Virol., 2, 1064-1075 (1968)). However, the viral DNA is not 
completely protein-free--at least a portion of the viral DNA is associated 
with at least four viral polypeptides, namely V, VII, TP and .mu., and is 
converted into a viral DNA-cell histone complex (Tate et al., Nucleic 
Acids Res., 6, 2769-2785 (1979)). 
The cycle from cell infection to production of viral particles lasts 1-2 
days and results in the production of up to 10,000 infectious particles 
per cell (Green et al., Virology, 13, 169-176 (1961)). The infection 
process of adenovirus is divided into early (E) and late (L) phases, which 
are separated by viral DNA replication, although some events which take 
place during the early phase also take place during the late phase and 
vice versa. Further subdivisions have been made to fully describe the 
temporal expression of viral genes. 
During the early phase, viral mRNA, which constitutes a minor proportion of 
the total RNA present in the cell, is synthesized from both strands of the 
adenoviral DNA present in the cell nucleus. At least five regions, 
designated E1-4 and MLP-L1, are transcribed (Lewis et al., Cell, 7, 
141-151 (1976); Sharp et al., Virology, 75, 442-456 (1976); Sharp, 
"Adenovirus transcription," In The Adenoviruses, Ginsberg, ed., Plenum 
Press, New York, N.Y., pp. 173-204 (1984)). Each region has a distinct 
promoter(s) and is processed to generate multiple mRNA species, and, 
therefore, each region may be thought of as a gene family. 
The products of the early (E) regions serve regulatory roles for the 
expression of other viral components, are involved in the general shut-off 
of cellular DNA replication and protein synthesis, and are required for 
viral DNA replication. The intricate series of events regulating early 
mRNA transcription begins with expression of immediate early regions E1A, 
L1 and the 13.5 kd gene (reviewed in Sharp (1984), supra; Horwitz (1990), 
supra). Expression of the delayed early regions E1B, E2A, E2B, E3 and E4 
is dependent on the E1A gene products. Three promoters, the E2 promoter at 
72 map units (mu), the protein IX promoter, and the IVa promoter are 
enhanced by the onset of DNA replication but are not dependent on it 
(Wilson et al., Virology, 94, 175-184 (1979)). Their expression 
characterizes an intermediate phase of viral gene expression. The result 
of the cascade of early gene expression is the start of viral DNA 
replication. 
Adenoviral DNA replication displaces one parental single-strand by 
continuous synthesis in the 5' to 3' direction from replication origins at 
either end of the genome (reviewed in Kelly et al., "Initiation of viral 
DNA replication," In Advances in Virus Research, Maramorosch et al., eds., 
Academic Press, Inc., San Diego, Calif., 34: 1-42 (1988); Horwitz (1990), 
supra; van der Vliet, "Adenovirus DNA replication in vitro," In The 
Eukaryotic Nucleus, Strauss et al., eds., Telford Press, Caldwell, N.J. 1: 
1-29 (1990)). Three viral proteins encoded from E2 are essential for 
adenoviral DNA synthesis: the single-stranded DNA binding protein (DBP), 
the adenoviral DNA polymerase (Ad pol), and the pre-terminal protein 
(pTP). In addition to these, in vitro experiments have identified many 
host cell factors necessary for DNA synthesis. 
DNA synthesis is initiated by the covalent attachment of a dCMP molecule to 
a serine residue of pTP. The pTP-dCMP complex then functions as the primer 
for Ad pol to elongate. The displaced parental single-strand can form a 
panhandle structure by base-pairing of the inverted terminal repeats. This 
terminal duplex structure is identical to the ends of the parental genome 
and can serve as an origin for the initiation of complementary strand 
synthesis. 
Initiation of viral DNA replication appears to be essential for entry into 
the late phase. The late phase of viral infection is characterized by the 
production of large amounts of the viral structural polypeptides and the 
nonstructural proteins involved in capsid assembly. The major late 
promoter (MLP) becomes fully active and produces transcripts that 
originate at 16.5 mu and terminate near the end of the genome. 
Post-transcriptional processing of this long transcript gives rise to five 
families of late mRNA, designated L1-5 (Shaw et al., Cell, 22, 905-916 
(1980)). The mechanisms which control the shift from the early to late 
phase and result in such a dramatic shift in transcriptional utilization 
are unclear. The requirement for DNA replication may be a cis-property of 
the DNA template, since late transcription does not occur from a 
superinfecting virus at a time when late transcription of the primary 
infecting virus is active (Thomas et al., Cell, 22, 523-533 (1980)). 
Assembly of the virion is an intricate process from the first step of 
assembling major structural units from individual polypeptide chains 
(reviewed in Philipson, "Adenovirus Assembly," In The Adenoviruses, 
Ginsberg, ed., Plenum Press, New York, N.Y. (1984), pp. 309-337; Horwitz 
(1990), supra). Hexon, penton base, and fiber assemble into trimeric 
homopolymer forms after synthesis in the cytoplasm. The 100 kd protein 
appears to function as a scaffolding protein for hexon trimerization and 
the resulting hexon trimer is called a hexon capsomere. The hexon 
capsomeres can self-assemble to form the shell of an empty capsid, and the 
penton base and fiber trimers can combine to form the penton when the 
components are inside the nucleus. The facet of the icosahedron is made up 
of three hexon capsomeres, which can be seen by dissociation of the 
capsid, but the intermediate step of formation of a group-of-nine hexons 
has not been observed. Several assembly intermediates have been shown from 
experiments with temperature-sensitive mutants. The progression of capsid 
assembly appears dependent on scaffolding proteins, 50 kd and 30 kd, and 
the naked DNA most probably enters the near-completed capsid through an 
opening at one of the vertices. The last step of the process involves the 
proteolytic trimming of the precursor polypeptides pVI, pVII, pVIII and 
pTP, which stabilizes the capsid structure, renders the DNA insensitive to 
nuclease treatment, and yields a mature virion. 
Recombinant adenoviral vectors have been used in gene therapy. The use of a 
recombinant adenoviral vector to transfer one or more recombinant genes 
enables targeted delivery of the gene or genes to an organ, tissue, or 
cells in need of treatment, thereby overcoming the delivery problem 
encountered in most forms of somatic gene therapy. Furthermore, 
recombinant adenoviral vectors do not require host cell proliferation for 
expression of adenoviral proteins (Horwitz et al., In Virology, Raven 
Press, New York, 2, 1679-1721 (1990); and Berkner, BioTechniques, 6, 616 
(1988)) and, if the diseased organ in need of treatment is the lung, has 
the added advantage of being normally trophic for the respiratory 
epithelium (Straus, In Adenoviruses, Plenum Press, New York, pp. 451-496 
(1984)). 
Other advantages of adenoviruses as potential vectors for human gene 
therapy are as follows: (i) recombination is rare; (ii) there are no known 
associations of human malignancies with adenoviral infections despite 
common human infection with adenoviruses; (iii) the adenoviral genome 
(which is linear, double-stranded DNA) currently can be manipulated to 
accommodate foreign genes ranging in size from small peptides up to 
7.0-7.5 kb in length; (iv) an adenoviral vector does not insert its DNA 
into the chromosome of a cell, so its effect is impermanent and unlikely 
to interfere with the cell's normal function; (v) the adenovirus can 
infect non-dividing or terminally differentiated cells, such as cells in 
the brain and lungs; and (vi) live adenovirus, having as an essential 
characteristic the ability to replicate, has been safely used as a human 
vaccine (Horwitz, M. S. et al.; Berkner et al.; Straus et al.; Chanock et 
al., JAMA, 195, 151 (1966); Haj-Ahmad et al., J. Virol., 57, 267 (1986); 
and Ballay et al., EMBO, 4, 3861 (1985)). 
Until now, adenoviral vectors used to express a foreign gene have been 
deficient in only a single early region (E1) that is essential for viral 
growth, i.e., singly functionally deficient. Only the essential region E1 
or, alternatively, the nonessential region E3 has been removed for 
insertion of a foreign gene into the adenoviral genome. If the region 
removed from the adenovirus is essential for the virus to grow, a 
complementing system, such as a complementing cell line is necessary to 
compensate for the missing viral function. In other words, the 
complementing cell line will express the missing viral function so that 
the singly deficient adenovirus can grow inside the complementing cell. 
Currently, there are only a few cell lines that exist that will complement 
for essential functions missing from a singly deficient adenovirus. 
Examples of such cell lines include HEK-293 (Graham et al., Cold Spring 
Harbor Svmp. Ouant. Biol., 39, 637-650 (1975)), W162 (Weinberg et al., 
PNAS USA, 80, 5383-5386 (1983)), and gMDBP (Klessig et al., Mol. Cell. 
Biol., 4, 1354-1362 (1984); Brough et al., Virology, 190, 624-634 (1992)). 
Foreign genes have been inserted into two major regions of the adenoviral 
genome for use as expression vectors. Insertion into the E1 region results 
in defective progeny that require either growth in complementary cells or 
the presence of an intact helper virus (Berkner et al., J. Virol., 61, 
1213-1220 (1987); Davidson et al., J. Virol., 61, 122-1239 (1987); and 
Mansour et al., Mol. Cell Biol., 6, 2684-2694 (1986)). This region of the 
genome has been used most frequently for expression of foreign genes. Such 
E1-defective expression vector viruses usually have been grown in the 
HEK-293 cell line, which contains and expresses the complementing 
adenoviral E1 region. The inserted genes have been placed under the 
control of various promoters and most produce large amounts of the foreign 
gene product, dependent on the expression cassette. These adenoviral 
vectors, however, are defective in noncomplementing cell lines. In 
contrast, the E3 region is nonessential for virus growth in tissue 
culture, and replacement of this region with a foreign gene expression 
cassette leads to a virus that can productively grow in a noncomplementing 
cell line. The insertion and expression of the hepatitis B surface antigen 
in the E3 region with subsequent inoculation and formation of antibodies 
in the hamster has been reported (Morin et al., PNAS USA, 84, 4626-4630 
(1987)). 
The problem with singly deficient adenoviral vectors is that they limit the 
amount of usable space within the adenoviral genome for insertion and 
expression of a foreign gene. Due to similarities, or overlap, in the 
viral sequences contained within the singly deficient adenoviral vectors 
and the complementing cell lines that currently exist, recombination 
events can take place and create replication competent viruses within a 
vector stock. This event can render a stock of vector unusable for gene 
therapy purposes as a practical matter. 
Accordingly, it is an object of the present invention to provide multiply 
deficient adenoviral vectors that can accommodate insertion and expression 
of larger pieces of foreign DNA. It is another object of the present 
invention to provide cell lines that complement the present inventive 
multiply deficient adenoviral vectors. It is also an object of the present 
invention to provide recombinants of multiply deficient adenoviral vectors 
and therapeutic methods, particularly relating to gene therapy, 
vaccination, and the like, involving the use of such recombinants. These 
and other objects and advantages of the present invention, as well as 
additional inventive features, will be apparent from the following 
detailed description. 
BRIEF SUMMARY OF THE INVENTION 
The present invention provides multiply deficient adenoviral vectors and 
complementing cell lines. The multiply deficient adenoviral vectors can 
accommodate insertion and expression of larger fragments of foreign DNA 
than is possible with currently available singly deficient adenoviral 
vectors. The multiply deficient adenoviral vectors are also replication 
deficient, which is particularly desirable for gene therapy and other 
therapeutic purposes. Accordingly, the present invention also provides 
recombinant multiply deficient adenoviral vectors and therapeutic methods, 
for example, relating to gene therapy, vaccination, and the like, 
involving the use of such recombinants.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides, among other things, multiply deficient 
adenoviral vectors for gene cloning and expression. The multiply deficient 
adenoviral vectors of the present invention differ from currently 
available singly deficient adenoviral vectors in being deficient in at 
least two essential gene functions and in being able to accept and express 
larger pieces of foreign DNA. 
Any subtype, mixture of subtypes, or chimeric adenovirus may be used as the 
source of DNA for generation of the multiply deficient adenoviral vectors. 
However, given that the Ad5 genome has been completely sequenced, the 
present invention will be described with respect to the Ad5 serotype. 
Preferably, the adenoviral vector of the present invention is at least 
deficient in a function provided by early region 1 (E1) and/or one or more 
functions encoded by early region 2 (E2), such as early region 2A (E2A) 
and early region 2B (E2B), and/or early region 3 (E3), and/or early region 
4 (E4) of the adenoviral genome. Any one of the deleted functional regions 
then may be replaced with a promoter-variable expression cassette to 
produce a novel gene product. The insertion of a novel gene into the E2A 
region, for example, may be facilitated by the introduction of a unique 
restriction site, such that the novel gene product may be expressed from 
the E2A promoter. 
The present invention, however, is not limited to adenoviral vectors that 
are deficient in gene functions only in the early region of the genome. 
Also included are adenoviral vectors that are deficient in the late region 
of the genome, adenoviral vectors that are deficient in the early and late 
regions of the genome, as well as vectors in which essentially the entire 
genome has been removed, in which case it is preferred that at least 
either the viral inverted terminal repeats and some of the promoters or 
the viral inverted terminal repeats and a packaging signal are left 
intact. One of ordinary skill in the art will appreciate that the larger 
the region of the adenoviral genome that is removed, the larger the piece 
of exogenous DNA that can be inserted into the genome. For example, given 
that the adenoviral genome is 36 kb, by leaving the viral inverted 
terminal repeats and some of the promoters intact, the theoretical 
capacity of the adenovirus is approximately 35 kb. Alternatively, one 
could generate a multiply deficient adenoviral vector that contains only 
the ITR and a packaging signal. This could then effectively allow for 
expression of 37-38 kb of foreign DNA from this vector. 
In general, virus vector construction relies on the high level of 
recombination between fragments of adenoviral DNA in the cell. Two or 
three fragments of adenoviral DNA, containing regions of similarity (or 
overlap) between fragments and constituting the entire length of the 
genome, are transfected into a cell. The host cell's recombination 
machinery constructs a full-length viral vector genome. Similar procedures 
for constructing viruses containing alterations in various single regions 
have been previously described (Berkner et al., Nucleic Acids Res., 12, 
925-941 (1984); Berkner et al., Nucleic Acids Res., 11, 6003-6020 (1983); 
Brough et al., Virol., 190, 624-634 (1992)) and can be used to construct 
multiply deficient viruses as can in vitro recombination and ligation, for 
example. 
The first step in virus vector construction is to construct a deletion or 
modification of a particular region of the adenoviral genome in a plasmid 
cassette using standard molecular biological techniques. After extensive 
analysis, this altered DNA (containing the deletion or modification) is 
then moved into a much larger plasmid that contains up to one half of the 
adenovirus genome. The next step is to transfect the plasmid DNA 
(containing the deletion or modification) and a large piece of the 
adenovirus genome into a recipient cell. Together these two pieces of DNA 
encompass all of the adenovirus genome plus a region of similarity. Within 
this region of similarity a recombination event will take place to 
generate a complete intact viral genome with the deletion or modification. 
In the case of multiply deficient vectors, the recipient cell will provide 
not only the recombination functions but also all missing viral functions 
not contained within the transfected viral genome. The multiply deficient 
vector can be further modified by alteration of the ITR and/or packaging 
signal, for example, such that the multiply deficient vector only 
functions in a complementing cell line. 
In addition, the present invention also provides complementing cell lines 
for propagation of the present inventive multiply deficient adenoviral 
vectors. The preferred cell lines of the present invention are 
characterized in complementing for at least one gene function of the gene 
functions comprising the E1, E2, E3 and E4 regions of the adenoviral 
genome. Other cell lines include those that complement adenoviral vectors 
that are deficient in at least one gene function from the gene functions 
comprising the late regions, those that complement for a combination of 
early and late gene functions, and those that complement for all 
adenoviral functions. One of ordinary skill in the art will appreciate 
that the cell line of choice would be one that specifically complements 
for those functions that are missing from the recombinant multiply 
deficient adenoviral vector of interest and that can be generated using 
standard molecular biological techniques. The cell lines are further 
characterized in containing the complementing genes in a nonoverlapping 
fashion, which eliminates the possibility of the vector genome recombining 
with the cellular DNA. Accordingly, replication-competent adenoviruses are 
eliminated from the vector stocks, which are, therefore, suitable for 
certain therapeutic purposes, especially gene therapy purposes. This also 
eliminates the replication of the adenoviruses in noncomplementing cells. 
The complementing cell line must be one that is capable of expressing the 
products of the two or more deficient adenoviral gene functions at the 
appropriate level for those products in order to generate a high titer 
stock of recombinant adenoviral vector. For example, it is necessary to 
express the E2A product, DBP, at stoichiometric levels, i.e., relatively 
high levels, for adenoviral DNA replication, but the E2B product, Ad pol, 
is necessary at only catalytic levels, i.e., relatively low levels, for 
adenoviral DNA replication. Not only must the level of the product be 
appropriate, the temporal expression of the product must be consistent 
with that seen in normal viral infection of a cell to assure a high titer 
stock of recombinant adenoviral vector. For example, the components 
necessary for viral DNA replication must be expressed before those 
necessary for virion assembly. In order to avoid cellular toxicity, which 
often accompanies high levels of expression of the viral products, and to 
regulate the temporal expression of the products, inducible promoter 
systems are used. For example, the sheep metallothionine inducible 
promoter system can be used to express the complete E4 region, the open 
reading frame 6 of the E4 region, and the E2A region. Other examples of 
suitable inducible promoter systems include, but are not limited to, the 
bacterial lac operon, the tetracycline operon, the T7 polymerase system, 
and combinations and chimeric constructs of eukaryotic and prokaryotic 
transcription factors, repressors and other components. Where the viral 
product to be expressed is highly toxic, it is desirable to use a 
bipartite inducible system, wherein the inducer is carried in a viral 
vector and the inducible product is carried within the chromatin of the 
complementing cell line. Repressible/inducible expression systems, such as 
the tetracycline expression system and lac expression system also may be 
used. 
DNA that enters a small proportion of transfected cells can become stably 
maintained in an even smaller fraction. Isolation of a cell line that 
expresses one or more transfected genes is achieved by introduction into 
the same cell of a second gene (marker gene) that, for example, confers 
resistance to an antibiotic, drug or other compound. This selection is 
based on the fact that, in the presence of the antibiotic, drug, or other 
compound, the cell without the transferred gene will die, while the cell 
containing the transferred gene will survive. The surviving cells are then 
clonally isolated and expanded as individual cell lines. Within these cell 
lines are those that will express both the marker gene and the genes of 
interest. Propagation of the cells is dependent on the parental cell line 
and the method of selection. Transfection of the cell is also dependent on 
cell type. The most common techniques used for transfection are calcium 
phosphate precipitation, liposome, or DEAE dextran mediated DNA transfer. 
Many modifications and variations of the present illustrative DNA sequences 
and plasmids are possible. For example, the degeneracy of the genetic code 
allows for the substitution of nucleotides throughout polypeptide coding 
regions, as well as in the translational stop signal, without alteration 
of the encoded polypeptide coding sequence. Such substitutable sequences 
can be deduced from the known amino acid or DNA sequence of a given gene 
and can be constructed by conventional synthetic or site-specific 
mutagenesis procedures. Synthetic DNA methods can be carried out in 
substantial accordance with the procedures of Itakura et al., Science, 
198, 1056 (1977) and Crea et al., PNAS USA, 75, 5765 (1978). Site-specific 
mutagenesis procedures are described in Maniatis et al., Molecular 
Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed. 1989). 
Therefore, the present invention is in no way limited to the DNA sequences 
and plasmids specifically exemplified. Exemplified vectors are for gene 
therapy of cystic fibrosis and, therefore, contain and express the CFTR 
gene but the vectors described are easily convertible to treat other 
potential diseases including, but not limited to, other chronic lung 
diseases, such as emphysema, asthma, adult respiratory distress syndrome, 
and chronic bronchitis, as well as cancer, coronary heart disease, etc. 
Accordingly, any gene or DNA sequence can be inserted into a multiply 
deficient adenoviral vector. The choice of gene or DNA sequence should be 
one that will achieve a therapeutic effect, for example, in the context of 
gene therapy, vaccination, and the like. 
One skilled in the art will appreciate that suitable methods of 
administering a multiply deficient adenoviral vector of the present 
invention to an animal for therapeutic purposes, e.g., gene therapy, 
vaccination, and the like (see, for example, Rosenfeld et al., Science, 
252, 431-434 (1991), Jaffe et al., Clin. Res., 39(2), 302A (1991), 
Rosenfeld et al., Clin. Res., 39(2), 311A (1991), Berkner, BioTechniques, 
6, 616-629 (1988)), are available, and, although more than one route can 
be used to administer the vector, a particular route can provide a more 
immediate and more effective reaction than another route. Pharmaceutically 
acceptable excipients are also well-known to those who are skilled in the 
art, and are readily available. The choice of excipient will be determined 
in part by the particular method used to administer the composition. 
Accordingly, there is a wide variety of suitable formulations of the 
pharmaceutical composition of the present invention. The following 
formulations and methods are merely exemplary and are in no way limiting. 
However, oral, injectable and aerosol formulations are preferred. 
Formulations suitable for oral administration can consist of (a) liquid 
solutions, such as an effective amount of the compound dissolved in 
diluents, such as water, saline, or orange juice; (b) capsules, sachets or 
tablets, each containing a predetermined amount of the active ingredient, 
as solids or granules; (c) suspensions in an appropriate liquid; and (d) 
suitable emulsions. Tablet forms can include one or more of lactose, 
mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, 
gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium 
stearate, stearic acid, and other excipients, colorants, diluents, 
buffering agents, moistening agents, preservatives, flavoring agents, and 
pharmacologically compatible excipients. Lozenge forms can comprise the 
active ingredient in a flavor, usually sucrose and acacia or tragacanth, 
as well as pastilles comprising the active iningredient in an inert base, 
such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and 
the like containing, in addition to the active ingredient, such excipients 
as are known in the art. 
The vectors of the present invention, alone or in combination with other 
suitable components, can be made into aerosol formulations to be 
administered via inhalation. These aerosol formulations can be placed into 
pressurized acceptable propellants, such as dichlorodifluoromethane, 
propane, nitrogen, and the like. They also may be formulated as 
pharmaceuticals for non-pressured preparations such as in a nebulizer or 
an atomizer. 
Formulations suitable for parenteral administration include aqueous and 
non-aqueous, isotonic sterile injection solutions, which can contain 
anti-oxidants, buffers, bacteriostats, and solutes that render the 
formulation isotonic with the blood of the intended recipient, and aqueous 
and non-aqueous sterile suspensions that can include suspending agents, 
solubilizers, thickening agents, stabilizers, and preservatives. The 
formulations can be presented in unit-dose or multi-dose sealed 
containers, such as ampules and vials, and can be stored in a freeze-dried 
(lyophilized) condition requiring only the addition of the sterile liquid 
excipient, for example, water, for injections, immediately prior to use. 
Extemporaneous injection solutions and suspensions can be prepared from 
sterile powders, granules, and tablets of the kind previously described. 
Additionally, the vectors employed in the present invention may be made 
into suppositories by mixing with a variety of bases such as emulsifying 
bases or water-soluble bases. 
Formulations suitable for vaginal administration may be presented as 
pessaries, tampons, creams, gels, pastes, foams, or spray formulas 
containing, in addition to the active ingredient, such carriers as are 
known in the art to be appropriate. 
The dose administered to an animal, particularly a human, in the context of 
the present invention will vary with the gene or other sequence of 
interest, the composition employed, the method of administration, and the 
particular site and organism being treated. The dose should be sufficient 
to effect a desirable response, e.g., therapeutic or immune response, 
within a desirable time frame. 
The multiply deficient adenoviral vectors and complementing cell lines of 
the present invention also have utility in vitro. For example, they can be 
used to study adenoviral gene function and assembly. 
The following examples further illustrate the present invention and, of 
course, should not be construed as in any way limiting its scope. Enzymes 
referred to in the examples are available, unless otherwise indicated, 
from Bethesda Research Laboratories (BRL), Gaithersburg, Md. 20877, New 
England Biolabs Inc. (NEB), Beverly, Mass. 01915, or Boehringer Mannheim 
Biochemicals (BMB), 7941 Castleway Drive, Indianapolis, Ind. 46250, and 
are used in substantial accordance with the manufacturer's 
recommendations. Many of the techniques employed herein are well known to 
those in the art. Molecular biology techniques are described in detail in 
laboratory manuals, such as Maniatis et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor, N.Y. (2d ed. 1989) and Current 
Protocols in Molecular Bioloqy (Ausubel et al., eds. (1987)). One of 
ordinary skill in the art will recognize that alternate procedures can be 
substituted for various procedures presented below. Although the examples 
and figures relate to Ad.sub.GV.10, Ad.sub.GV.11, Ad.sub.GV.12, and 
Ad.sub.GV.13 which contain the cystic fibrosis transmembrane regulator 
gene (CFTR), namely Ad.sub.GV CFTR.10, Ad.sub.GV CFTR.11, Ad.sub.GV 
CFTR.12, and Ad.sub.GV CFTR.13, these vectors are not limited to 
expression of the CFTR gene and can be used to express other genes and DNA 
sequences. For example, therefore, the present invention encompasses such 
vectors comprising any foreign gene (e.g., for use in gene therapy), any 
DNA sequence capable of expressing in a mammal a polypeptide capable of 
eliciting an immune response to the polypeptide (e.g., for use in 
vaccination), and any DNA sequence capable of expressing in a mammal any 
other therapeutic agent (e.g., an antisense molecule, particularly an 
antisense molecule selected from the group consisting of MRNA and a 
synthetic oligonucleotide). 
EXAMPLE 1 
This example describes the generation of one embodiment involving 
AD.sub.GV.10, namely Ad.sub.GV CFTR.10. 
Ad.sub.GV CFTR.10 expresses the CFTR gene from the cytomegalovirus (CMV) 
early promoter. Two generations of this vector have been constructed and 
are designated Ad.sub.GV CFTR.10L and Ad.sub.GV CFTR.10 R, dependent on 
the direction in which the CFTR expression cassette is placed in the E1 
region in relation to the vector genome as shown in FIG. 1, which is a set 
of schematic diagrams of Ad.sub.GV CFTR.10L and Ad.sub.GV CFTR.10R. 
The CFTR expression cassette was constructed as follows. pRK5 (Genentech 
Inc., South San Francisco, Calif.) was digested with Kpn I (New England 
Biolabs (NEB), Beverly, Mass.), blunt-ended with Mung Bean nuclease (NEB), 
and an Xho I linker (NEB) was ligated in place of the Kpn I site. The 
resulting vector was named pRK5-Xho I. pRK5-Xho I was then digested with 
Sma I (NEB) and Hin dIII (NEB) and blunt-ended with Mung bean nuclease. A 
plasmid containing the CFTR gene, pBQ4.7 (Dr. Lap-Chee Tsui, Hospital for 
Sick Children, Toronto, Canada), was digested with Ava I (NEB) and Sac I 
(NEB) and blunt-ended with Mung bean nuclease. These two fragments were 
isolated and ligated together to produce pRK5-CFTRl, the CFTR expression 
cassette. 
pRK5-CFTR1 was digested with Spe I (NEB) and Xho I and blunt-ended with 
Klenow (NEB). pAd60.454 (Dr. L. E. Babiss, The Rockefeller University, New 
York, N.Y.), which contains Ad5 sequences from 1-454/3325-5788, was 
digested with Bal II (NEB) and blunt-ended with Klenow. These two 
fragments were purified from vector sequences by low-melt agarose 
technique (Maniatis et al., Molecular Cloning: a laboratory manual, Cold 
Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2nd ed. (1989)) and 
ligated together to produce the left arm plasmids pGVCFTR.10L and 
pGVCFTR.10R. 
The left arm plasmid from pGVCFTR.10L or pGVCFTR.10R was digested with Nhe 
I (NEB). The right arm of the virus was produced by digesting Ad5d1324 
(Dr. Thomas E. Shenk, Princeton University, Princeton, N.J.) with Cla I 
(NEB). The two fragments, a small 918 bp fragment and a large 
approximately 32,800 bp fragment, were separated by sucrose gradient 
centrifugation (Maniatis et al., supra). The large fragment was mixed with 
the left arm plasmid fragments and transfected into 293 cells by standard 
calcium phosphate protocol (Graham et al., Virology, 52, 456 (1973)). The 
resulting recombinant viruses were plaque-purified on 293 cells, and viral 
stocks were established using standard virology techniques (e.g., Berkner 
et al., (1983) and (1984), supra). 
EXAMPLE 2 
This example describes the generation of Ad.sub.GV CFTR.11. 
Ad.sub.GV CFTR.11 was constructed by means of a single in vivo 
recombination between 1-27082, i.e., the left arm, of Ad.sub.GV CFTR.10 
and a plasmid (pGV11A, pGV11B, pGV11C, or pGV11D; described in detail 
below) containing 21562-35935, i.e., the right arm, of Ad5 linearized with 
Bam HI (NEB) and Sal I (NEB) and into which the various E3 and E4 
alterations as described below were introduced. 
The left arm from Ad.sub.GV CFTR.10 was isolated on a concave 10-40% 
sucrose gradient, wherein 1/th of the total solution was 40%, after intact 
Ad.sub.GV CFTR.10 was digested with Spe I (NEB) and Srf I (Stratagene, La. 
Jolla, Calif.) to yield the 1-27082 bp fragment. 
The right arm was obtained by Bam HI-Sal I digestion of a modified pGEM 
vector (pGBS). pGBS was generated as follows. pGemI (Promega, Madison, 
Wis.) was digested with Eco RI and blunt-ended with Klenow, and a Sal I 
linker was ligated into the vector. The resulting DNA was then digested 
with Sal I and religated, thereby replacing the Eco RI site with a Sal I 
site and deleting the sequence between the two Sal I sites, to generate 
pGEMH/P/S, which was digested with Hin dIII and blunt-ended with Klenow, 
and a Bam HI linker was ligated into the vector to generate pGEMS/B. 
pGEMS/B was digested with Bam HI and Sal I and ligated with an .about.14 
kb Bam HI-Sal I fragment (21562-35935 from Ad5) from a pBR plasmid called 
p50-1000 (Dr. Paul Freimuth, Columbia University, N.Y.) to generate pGBS. 
Three different versions of the right arm plasmid have been constructed in 
order to introduce into the adenoviral vector two Ad E3 gene products 
having anti-immunity and anti-inflammatory properties. The large E3 
deletion in pGBS.DELTA.E30RF6, designated pGV11(0) (Example 7), was 
essentially replaced with three different versions of an expression 
cassette containing the Rous sarcoma viruslong terminal repeat (RSV-LTR) 
promoter driving expression of a bicistronic mRNA containing at the 5' end 
the Ad2 E3 19 kDa anti-immunity gene product and at the 3' end the Ad5 E3 
14.7 kDa anti-inflammatory gene product. One additional virus was 
constructed by deleting the 19 kDa cDNA fragment by Bst BI (NEB) fragment 
deletion. This virus, designated Ad.sub.GV CFTR.11(D), contains the 
RSV-LTR promoter driving expression of a monocistronic mRNA containing 
only the E3 14.7 kDa anti-inflammatory gene product. 
The Spe I (27082)--Nde I (31089) fragment from pGBS.DELTA.E3 (Example 5) 
was subcloned into pUC 19 by first cloning the Eco RI (27331)--Nde I 
(31089) fragment into identical sites in the pUC 19 polylinker. A Hin dIII 
(26328)--Eco RI (27331) fragment generated from pGBS was then cloned into 
the Eco RI site of this clone to generate pHN.DELTA.E3. Using appropriate 
primers, a PCR fragment with flanking Xba I sites was generated containing 
the RSV-LTR promoter, the Ad2 E3 19 kDa gene product, and the Ad5 E3 14.7 
kDa gene product. The amplified fragment was digested with Xba I and 
subcloned into pUC 19 to generate pXA. After analysis of the Xba I 
fragment, the fragment was ligated into pHN.DELTA.E3 to generate pHNRA. 
Using appropriate primers, two PCR fragments with flanking Bst BI sites 
were generated that encode internal ribosomal entry sites (IRES), which 
are known to enhance the translation of mRNAs that contain them (Jobling 
et al., Nature, 325, 622-625 (1987); Jang et al., Genes and Development, 
4, 1560-1572 (1990)). One fragment (version B) contains a 34 bp IRES from 
the untranslated leader of the coat protein mRNA of alfalfa mosaic virus 
(AMV RNA 4 leader) (Jobling et al., supra). The other fragment (version C) 
contains a 570 bp IRES from the 5' nontranslated region of 
encephalomyocarditis virus (EMCV) mRNA (Jang et al., supra). Each Bst BI 
fragment from version B or C was cloned in place of the Bst BI fragment in 
pXA. The resulting plasmids, named pXB and pXC, respectively, were moved 
into pHN.DELTA.E3 to generate pHNRB and PHNRC, respectively, after 
sequence analysis of the Xba I fragments. 
The Spe I (27082)--Nde I (31089) fragment from pGBS.DELTA.E30RF6 was 
replaced with the Spe I--Nde I fragments from PHNRA, PHNRB, pHNRC and 
PHNRD to generate pGV11A, pGV11B, pGV11C and pGV11D, respectively. 
The pGBV plasmid DNA was linearized with Bam HI and Sal I and mixed with 
the purified left arm DNA fragment in varying concentrations to give about 
20 .mu.g total DNA, using salmon sperm or calf thymus DNA (Life 
Technologies, Gaithersburg, Mass.) to bring the amount of DNA to about 20 
.mu.g as needed. The mixed fragments were then transfected into 293 cells 
using standard calcium phosphate techniques (Graham et al., supra). 
Five days after transfection, the cell monolayer was harvested by 
freeze-thawing three times. The resulting hybrid virus was titered onto 
293 cells and isolated plaques were picked. The process of plaque 
isolation was repeated twice more to ensure a single recombinant virus 
existed in the initial plaque stock. The plaque isolate stock was then 
amplified to a large viral stock according to standard virology techniques 
as described in Burlseson et al., Virology: a Laboratory Manua, Academic 
Press Inc. (1992). 
FIG. 2 is a set of schematic diagrams of the various AD.sub.GV CFTR.11 
viral vectors. The diagrams are aligned with that of AD.sub.GV CFTR.10L 
for comparison. 
EXAMPLE 3 
This example describes the generation of Ad.sub.GV CFTR.12. 
Ad.sub.GV.12 is characterized by complete elimination of the E4 region. 
This large deletion allows for insertion of up to about 10 kb of exogenous 
DNA. More importantly, another region of the genome has become accessible 
for introduction of foreign gene expression cassettes. This deletion now 
enables the incorporation of larger expression cassettes for other 
products. For example, soluble receptors, i.e., TNF or IL-6 without a 
transmembrane domain so that they are now not attached to the membrane, 
and antisense molecules, e.g., those directed against cell cycle 
regulating products, such as cdc2, cdk kinases, cyclins, i.e., cyclin E or 
cyclin D, and transcription factors, i.e., E2F or c-myc, to eliminate 
inflammation and immune responses. pGV11 (O) is altered to produce a right 
arm plasmid in which the entire E4 region is deleted. The resulting 
plasmid in which the entire E3 and E4 regions are deleted is named 
pGV12(O). This is done by introducing a Pac I restriction site at the Afl 
III site at 32811 and the Bsg I site at 35640. Deletion of the Pac I 
fragment between these two sites effectively eliminates all of the E4 
sequences including the E4 TATA element within the E4 promoter and the E4 
poly A site. 
Virus construction is performed as previously described except that the 
293/E4 cell line or the 293/ORF6 cell line is used. The left arm from 
Ad.sub.GV CFTR.10L, the right arm pGV12(O) plasmid, and all other general 
techniques are as described in Example 2. Since E4 contains essential gene 
products necessary for viral growth, the resulting E4 deletion mutant 
virus cannot grow in the absence of exogenously expressed E4. Therefore, 
all manipulations for viral construction are carried out in the new 293/E4 
cell line or 293/ORF6 cell line (described in Examples 8 and 9, 
respectively). The resulting virus is Ad.sub.GV CFTR.12. 
EXAMPLE 4 
This example describes the generation of Ad.sub.GV CFTR.13. 
Ad.sub.GV.13 is characterized by not only complete elimination of E1, and 
E4 (as Ad.sub.GV.12) but also complete elimination of E2A. The complete 
coding region of E2A is deleted by fusing together the DNA from two E2A 
mutant viruses, namely H5in800 and H5in804, containing insertions of Cla I 
restriction sites at both ends of the open reading frame (Vos et al., 
Virology, 172, 634-642 (1989); Brough et al., Virology, 190, 624-634 
(1992)). The Cla I site of H5in800 is between codons 2 and 3 of the gene, 
and the Cla I site of H5in804 is within the stop codon of the E2A gene. 
The resultant virus contains an open reading frame consisting of 23 amino 
acids that have no similarity to the E2A reading frame. More importantly, 
this cassette offers yet another region of the virus genome into which a 
unique gene can be introduced. This can be done by inserting the gene of 
interest into the proper reading frame of the existing mini-ORF or by 
introducing yet another expression cassette containing its own promoter 
sequences, polyadenylation signals, and stop sequences in addition to the 
gene of interest. 
Adenovirus DNA is prepared from H5in800 and H5in804. After digestion with 
the restriction enzyme Hin dIII (NEB), the Hin dIII A fragments from both 
H5in800 and H5in804 are cloned into pKS+(Stratagene). The resulting 
plasmids are named pKS+H5in800Hin dIIIA and pKS+H5in804Hin dIIIA, 
respectively. The Cla I (NEB) fragment from pKS+H5in800Hin dIIIA is then 
isolated and cloned in place of the identical Cla I fragment from 
PKS+H5in804Hin dIIIA. This chimeric plasmid, pHin dIIIA.DELTA.E2A 
effectively removes all of the E2A reading frame as described above. At 
this point, the E2A deletion is moved at Bam HI (NEB) and Spe I (NEB) 
restriction sites to replace the wild-type sequences in pGV12(O) to 
construct pGV13(O). 
Ad.sub.GV CFTR.13 virus is constructed as previously described by using 
Ad.sub.GV CFTR.10 left arm DNA and pGV13(O) right arm plasmid DNA. 
However, the recipient cell line for this virus construction is the triple 
complementing cell line 293/E4/E2A . 
EXAMPLE 5 
This example describes the generation of pGBS.DELTA.E3. 
This plasmid was generated to remove the majority of the E3 region within 
pGBS, including the E3 promoter and existing E3 genes, to make room for 
other constructs and to facilitate introduction of E3 expression 
cassettes. This plasmid contains a deletion from 28331 to 30469. 
A PCR fragment was generated with Ad5s(27324) and A5a(28330)X as primers 
and PGBS as template. The resulting fragment was digested with Eco RI 
(27331) and Xba I (28330) and gel-purified. This fragment was then 
introduced into pGBS at the Eco RI (27331) and Xba I (30470) sites. 
EXAMPLE 6 
This example describes the generation of pGBS.DELTA.E3.DELTA.E4. 
A large deletion of the Ad5E4 region was introduced into pGBS.DELTA.E3 to 
facilitate moving additional exogenous sequences into the adenoviral 
genome. The 32830-35566 E4 coding sequence was deleted. 
A Pac I site was generated in place of the Mun I site at 32830 by treating 
PGBS Mun I-digested DNA with Klenow to blunt-end the fragment and by 
ligating a Pac I linker to this. The modified DNA was then digested with 
Nde I and the resulting 1736 bp fragment (Nde I 31089-Pac I 32830) was 
gel-purified. A PCR fragment was prepared using A5 (35564)P (IDT, 
Coralville, Ind.) and T7 primers (IDT, Coralville, Ind.) and PGBS as 
template. The resulting fragment was digested with Pac I and Sal I to 
generate Pac I 35566-Sal I 35935. A Sma I site within the polylinker 
region of pUC 19 was modified to a Pac I site by ligating in a Pac I 
linker. The Pac I 35566 Sal I 35935 fragment was moved into the modified 
pUC 19 vector at Pac I and Sal I sites, respectively, in the polylinker 
region. The modified Nde I 31089-Pac I 32830 fragment was moved into the 
pUC 19 plasmid, into which the Pac I 35566-Sal I 35935 fragment already 
had been inserted, at Nde I and Pac I sites, respectively. The Nde I 
31089-Sal I 35935 fragment from the pUC 19 plasmid was purified by gel 
purification and cloned in place of the respective Nde I and Sal I sites 
in pGBS.DELTA.E3 to yield pGBS.DELTA.E3.DELTA.E4. 
EXAMPLE 7 
This example describes the generation of pGBS.DELTA.E3ORF6. 
The Ad5 894 bp E4 ORF-6 gene was placed 3' of the E4 promoter in 
pGBS.DELTA.E3 .DELTA.E4. ORF-6 is the only absolutely essential E4 product 
necessary for virus growth in a nonE4 complementing cell line. Therefore, 
this product was re-introduced into the right arm plasmid (Example 2) 
under its own promoter control so that Ad.sub.GV CFTR.11 virus can be 
propagated in 293 cells. 
A PCR fragment was generated using A5s(33190)P (32bp; 
5'CACTTAATTAAACGCCTACATGGGGGTAGAGT3') (SEQ ID NO:1) and A5a(34084)P (34 
bp; 5'CACTTAATTAAGGAAATATGACTACGTCCGGCGT3') (SEQ ID NO:2) as primers (IDT, 
Coralville, Ind.) and pGBS as template. This fragment was digested with 
Pac I and gel-purified. The product was introduced into the single Pac I 
site in pGBS.DELTA.E3.DELTA.E4 to generate pGV11(O), which was the plasmid 
that was E3-modified for expression of the 19 kDa and 14.7 kDa Ad E3 
products. 
EXAMPLE 8 
This example describes the generation of the 293/E4 cell line. 
The vector pSMT/E4 was generated as follows. A 2752 bp Mun I (site 32825 of 
Ad2)--Sph I (polylinker) fragment was isolated from pE4 (89-99), which is 
a pUC19 plasmid into which was subcloned region 32264-35577 from Ad2, 
blunt-ended with Klenow, and treated with phosphatase (NEB). The 2752 bp 
Mun I-Sph I fragment was then ligated into pMTO10/A.sup.+ (McNeall et 
al., Gene, 76, 81-89 (1989)), which had been linearized with Bam HI, 
blunt-ended with Klenow and treated with phosphatase, to generate the 
expression cassette plasmid, pSMT/E4. 
The cell line 293 (ATCC CRL 1573; American Type Culture Collection, 
Rockville, Md.) was cultured in 10% fetal bovine serum Dulbecco's modified 
Eagle's medium (Life Technologies, Gaithersburg, Mass.). The 293 cells 
were then transfected with pSMT/E4 linearized with Eco RI by the calcium 
phosphate method (Sambrook et al., Molecular Cloning: a Laboratory Manual, 
Cold Spring Harbor Laboratory Press (1989)). Approximately 24-48 hours 
post-transfection, medium (as above) containing 100 .mu.M methotrexate and 
amethopterin (Sigma Chemical Co., St. Louis, Mo.) was added. The presence 
of methotrexate in the medium selects for expression of the dihydrofolate 
reductase (DHFR) gene, which is the selectable marker on the pSMT/E4 
plasmid. 
The normal cell DHFR gene is inhibited by a given concentration of 
methotrexate (cell type-specific), causing cell death. The expression of 
the additional DHFR gene in transfected cells containing pSMT/E4 provides 
resistance to methotrexate. Therefore, transfected cells containing the 
new genes are the only ones that grow under these conditions (for review, 
see Sambrook et al., supra). 
When small colonies of cells formed from the initial single cell having the 
selectable marker, they were clonally isolated and propagated (for review, 
see Sambrook et al., supra). These clones were expanded to produce cell 
lines that were screened for expression of the product--in this case, 
E4--and screened for functionality in complementing defective viruses--in 
this case, both E1 and E4 defective viruses. 
The result of this process produced the first 293/E4 cell lines capable of 
complementing adenoviral vectors defective in both E1 and E4 functions, 
such as Ad.sub.GV CFTR. 12. 
EXAMPLE 9 
This example describes the generation of the 293/ORF-6 cell line. 
The primers A5s(33190)P and A5a(34084)P were used in a polymerase chain 
reaction (PCR) (PCR Protocols. A guide to Methods and Applications, Innis 
et al., eds., Academic Press, Inc. (1990)) to amplify the ORF-6 gene of 
Ad5 E4 and generate Pac I sites at the ends for cloning. The amplified 
fragment was blunt-ended with Klenow and cloned into pCR-Script SK(+) 
(Stratagene, La Jolla, Calif.). The resulting plasmid, pCR/ORF-6, was 
sequenced and then the ORF-06 insert was transferred into the pSMT/puro 
expression vector, which was generated by ligation of a blunt-ended Eco 
RI--Hin dIII fragment containing the SMT promoter into the blunt-ended Mlu 
I-Hin dIII site in pRCpuro, to generate pSMT/ORF-6. 
The 293 cell line was cultured and transfected with pSMT/ORF-6 as described 
in Example 8, except that the transfected cells were placed under 
selection for the puromycin resistance gene, which allows cells that 
express it to grow in the presence of puromycin. Colonies of transformed 
cells were subcloned and propagated and were screened as described in 
Example 8. 
This cell line is suitable for complementing vectors that are deficient in 
the E1 and E4 regions, such as the Ad.sub.GV CFTR.12 series of vectors. 
EXAMPLE 10 
This example describes the generation of the 293/E4/E2A cell line. The 
293/E4/E2A cell line allows E1, E4 and E2A defective viral vectors to 
grow. 
The E2A expression cassette for introduction into 293/E4 cells is produced 
as follows. The first step is to alter surrounding bases of the ATG of E2A 
to make a perfect Kozak consensus (Kozak, J. Molec. Biol., 196, 947-950 
(1987)) to optimize expression of E2A . Two primers are designed to alter 
the 5' region of the E2A gene. Ad5s(23884), an 18 bp oligonucleotide 
(5'GCCGCCTCATCCGCTTTT3') (SEQ ID NO:3), is designed to prime the internal 
region flanking the Sma I site of the E2A gene. DBP(ATG)R1, a 32-bp 
gonucleotide (5'CCGGAATTCCACCATGGCGAGtcGGAAGG3') (SEQ ID NO:4), is 
designed to introduce the translational consensus sequence around the ATG 
of the E2A gene modifying it into a perfect Kozak extended consensus 
sequence and to introduce an Eco RI site just 5' to facilitate cloning. 
The resulting PCR product using the above primers is digested with Eco RI 
and Sma I (NEB) and cloned into the idetical polylinker sites of 
pBluescript IIKS+(Stratagene, La Jolla, Calif.). The resulting plasmid is 
named pKS/ESDBP. 
A Sma I-Xba I fragment is isolated from pHRKauffman (Morin et al., Mol. 
Cell. Biol., 9, 4372-4380 (1989)) and cloned into the corresponding Sma I 
and Xba I sites of pKS/ESDBP to complete the E2A reading frame. The 
resulting plasmid is named pKSDBP. In order to eliminate all homologous 
sequences from vector contained within the expression cassette, the Kpn I 
to Dra I fragment from pKSDBP is moved into corresponding Kpn I and Pme I 
sites in PNEB193 (NEB) in which the Eco RI sites in the polylinker have 
been destroyed (GenVec). The resulting clone, pE2A , contains all of the 
E2A reading frame without any extra sequences homologous to the E2A 
deleted vector in Example 4. 
A 5' splice cassette is then moved into pE2A to allow proper nuclear 
processing of the mRNA and to further enhance expression of E2A . To do 
this, pRK5, described in Example 1, is digested with Sac II (NEB), 
blunt-ended with Mung Bean nuclease (NEB), and digested with Eco RI (NEB). 
The resulting approx. 240 bp fragment of interest containing the splicing 
signals is cloned into the Cla I (blunt-ended with Klenow) to Eco RI sites 
of pE2A to generate p5'E2A . The blunt-ended (Klenow) Sal I to Hin dIII 
fragment from p5'E2A containing the E2A sequences is moved into the 
blunt-ended (Klenow) Xba I site of pSMT/puro and pSMT/neo. The resulting 
E2A is named pKSE2A. 
The Xba I fragment from pKSE2A that contained all the E2A gene is moved 
into the Xba I site of pSMT/puro and pSMT/neo. The resulting E2A 
expression plasmids, pSMT/E2A /puro and pSMT/E2A /neo, are transfected 
into 293/E4 and 203/ORF-6 cells, respectively. Cells transfected with 
pSMT/E2A /puro are selected for growth in standard media plus puromycin 
and cells transfected with pSMT/E2A /neo are selected for growth in 
standard media plus G418. Clonal expansion of isolated colonies is as 
described in Example 8. The resulting cell lines are screened for their 
ability to complement E1, E4 and E2A defective viral vectors. 
These cell lines are suitable for complementing vectors that are deficient 
in the E1, E4 and E2A regions of the virus, such as those described in the 
Ad.sub.GV CFTR.13 series of viral vectors. 
EXAMPLE 11 
This example describes the generation of complementing cell lines using the 
cell line A549 (ATCC) as the parental line. 
Ad2 virus DNA is prepared by techniques previously described. The genomic 
DNA is digested with Ssp I and Xho I and the 5438 bp fragment is purified 
and cloned into Eco RV/Xho I sites of pKS+(Stratagene) to produce 
pKS341-5778. After diagnostic determination of the clone, an Xho I 
(blunt-ended with Klenow) to Eco RI fragment is moved into Nru I (blunt) 
to Eco RI sites in pRC/CMVneo to produce pE1neo. Transformation of A549 
cells with this clone yields a complementing cell line (similar to 293), 
wherein additional expression cassettes can be introduced, in a manner 
similar to that described for the 293 cell, to produce multicomplementing 
cell lines with excellent plaqueing potential. 
All references, including publications and patents, cited herein are hereby 
incorporated by reference to the same extent as if each reference were 
individually and specifically indicated to be incorporated by reference 
and were set forth in its entirety herein. 
While this invention has been described with emphasis upon preferred 
embodiments, it will be obvious to those of ordinary skill in the art that 
the preferred embodiments may be varied. It is intended that the invention 
may be practiced otherwise than as specifically described herein. 
Accordingly, this invention includes all modifications encompassed within 
the spirit and scope of the appended claims. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 4 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (synthetic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - CACTTAATTA AACGCCTACA TGGGGGTAGA GT - # - # 
32 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (synthetic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - CACTTAATTA AGGAAATATG ACTACGTCCG GCGT - # - 
# 34 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (synthetic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - GCCGCCTCAT CCGCTTTT - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 32 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: DNA (synthetic) 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - CCGGAATTCC ACCATGGCGA GTCGGGAAGA GG - # - # 
32 
__________________________________________________________________________