Packaging cell lines for adeno-associated viral vectors

The present invention is directed to novel replication-deficient adenoviral vectors characterized in that they harbor at least two lethal early region gene deletions (E1 and E4) that normally transcribe adenoviral early proteins. These novel recombinant vectors find particular use in human gene therapy treatment whereby the vectors additionally carry a transgene or therapeutic gene that replaces the E1 or E4 regions. The present invention is further directed to novel packaging cell lines that are transformed at a minimum with the adenoviral E1 and E4 gene regions and function to propagate the above novel replication-deficient adenoviral vectors.

FIELD OF THE INVENTION 
The present invention relates to novel replication-deficient adenoviral 
vectors, novel packaging cell lines and recombinant adenoviruses for human 
gene therapy. In particular, the novel packaging cell lines have the 
complementary function for the early gene region E1, E4 and optionally the 
E3 deletions of human adenovirus. 
BACKGROUND OF THE INVENTION 
Replication-defective retroviral vectors as gene transfer vehicles provide 
the foundation for human gene therapy. Retroviral vectors are engineered 
by removing or altering all viral genes so that no viral proteins are made 
in cells infected with the vector and no further virus spread occurs. The 
development of packaging cell lines which are required for the propagation 
of retroviral vectors were the most important step toward the reality of 
human gene therapy. The foremost advantages of retroviral vectors for gene 
therapy are the high efficiency of gene transfer and the precise 
integration of the transferred genes into cellular genomic DNA. However, 
major disadvantages are also associated with retroviral vectors, namely, 
the inability of retroviral vectors to transduce non-dividing cells and 
the potential insertional mutagenesis. 
Human adenoviruses have been developed as live viral vaccines and provide 
another alternative for in vivo gene delivery vehicles for human gene 
therapy Graham & Prevec in New Approaches to Immunological Problems, 
Ellis (ed), Butterworth-Heinemann, Boston, Mass., pp. 363-390 (1992) 
Rosenfeld, et al, Science 252: 431-434 (1991), Rosenfeld, et al, Cell 68: 
143-155 (1992), and Ragot, et al, Nature 361: 647-650 (1993)!. The 
features which make recombinant adenoviruses potentially powerful gene 
delivery vectors have been extensively reviewed Berkner, Biotechniques 6: 
616-629, (1988) and Kozarsky & Wilson, Curr. Opin. Genet. Dev. 3: 499-503, 
(1993)!. Briefly, recombinant adenoviruses can be grown and purified in 
large quantities and efficiently infect a wide spectrum of dividing and 
non-dividing mammalian cells in vivo. 
Moreover, the adenoviral genome may be manipulated with relative ease and 
accommodate very large insertions of DNA. 
The first generation of recombinant adenoviral vectors currently available 
have a deletion in the viral early gene region 1 (herein called E1 which 
comprises the E1a and E1b regions from genetic map units 1.30 to 9.24) 
which for most uses is replaced by a transgene. A transgene is a 
heterologous or foreign (exogenous) gene that is carried by a viral vector 
and transduced into a host cell. Deletion of the viral E1 region renders 
the recombinant adenovirus defective for replication and incapable of 
producing infectious viral particles in the subsequently infected target 
cells Berkner, Biotechniques 6: 616-629 (1988)!. The ability to generate 
E1-deleted adenoviruses is based on the availability of the human 
embryonic kidney packaging cell line called 293. This cell line contains 
the E1 region of the adenovirus which provides the E1 region gene products 
lacking in the E1-deleted virus Graham, et al, J. Gen Virol. 36: 59-72, 
(1977)!. However, the inherent flaws of current first generation 
recombinant adenoviruses have drawn increasing concerns about its eventual 
usage in patients. Several recent studies have shown that E1 deleted 
adenoviruses are not completely replication incompetent Rich, Hum. Gene. 
Ther. 4: 461-476 (1993) and Engelhardt, et al, Nature Genet. 4: 27-34 
(1993)!. Three general limitations are associated with the adenoviral 
vector technology. First, infection both in vivo and in vitro with the 
adenoviral vector at high multiplicity of infection (abbreviated m.o.i.) 
has resulted in cytotoxicity to the target cells, due to the accumulation 
of penton protein, which is itself toxic to mammalian cells (Kay, Cell 
Biochem. 17E: 207 (1993)3. Second, host immune responses against 
adenoviral late gene products, including penton protein, cause the 
inflammatory response and destruction of the infected tissue which 
received the vectors Yang, et al, Proc. Natl, Acad. Sci. USA 91: 
4407-4411 (1994)!. Lastly, host immune responses and cytotoxic effects 
together prevent the long term expression of transgenes and cause 
decreased levels of gene expression following subsequent administration of 
adenoviral vectors Mittal, et al, Virus Res.28: 67-90 (1993)!. 
In view of these obstacles, further alterations in the adenoviral vector 
design are required to cripple the ability of the virus to express late 
viral gene proteins, decreasing host cytotoxic responses and the 
expectation of decreasing host immune response. Engelhardt et al recently 
constructed a temperature sensitive (ts) mutation within the E2A-encoded 
DNA-binding protein (DBP) region of the E1-deleted recombinant adenoviral 
vector Engelhardt, et al, Proc. Natl. Acad. Sci. USA 91: 6196-6200 
(1994)! which fails to express late gene products at non-permissive 
temperatures in vitro. Diminished inflammatory responses and prolonged 
transgene expression were reported in animal livers infected by this 
vector (Engelhart, et al 1994). However, the ts DBP mutation may not give 
rise to a full inactive gene product in vivo, and therefore be incapable 
of completely blocking late gene expression. Further technical advances 
are needed that would introduce a second lethal deletion into the 
adenoviral E1-deleted vectors to completely block late gene expression in 
vivo. Novel packaging cell lines that can accommodate the production of 
second (and third) generation recombinant adenoviruses rendered 
replication-defective by the deletion of the E1 and E4 gene regions hold 
the greatest promise towards the development of safe and efficient vectors 
for human gene therapy. The present invention provides for such packaging 
cell lines and resultant mutant viruses and recombinant viral vectors (for 
example, adenoviral or AAV-derived) carrying the transgene of interest. 
SUMMARY OF THE INVENTION 
Accordingly, the present invention generally aims to provide an improved 
adenoviral vector system to obviate the difficulties found in using the 
first generation adenoviral vectors currently available by providing 
second and third generation viral vectors deleted of at least two early 
region DNA sequences, and that are capable of delivering foreign, 
therapeutic or transgenes to somatic cells. 
In particular, the present invention provides for second and third 
generation recombinant adenoviral vectors (adenoviruses) harboring at 
least two lethal deletions, namely, the E1 and E4 early region genes. 
Optionally, this vector may also be deleted of the E3 early gene region. 
More particularly, this recombinant viral vector carries a transgene, for 
example, the .beta.-galactosidase gene, introduced into either the E1 or 
E4 regions. In a more particular embodiment, the recombinant adenoviruses 
may contain a therapeutic gene that replaces the E1 or E4 regions (or 
optionally the E3 region), and the therapeutic gene is expressed and/or 
transcribed in a targeted host cell. 
Another object of the present invention is to provide a novel packaging 
cell line which complements functions of the E1, E4 and optionally the E3 
gene regions of a defective adenovirus deleted of the E1, E4 and 
optionally E3 regions, thereby allowing the production of the above 
described second generation recombinant adenoviral vectors deficient of 
the E1, E4 and optionally, the E3 DNA regions. The preferred packaging 
cell line derived from human embryonic kidney cells (293 cell line) 
contains the adenovirus E1 and E4 gene regions integrated into its genome. 
In a particular embodiment, the packaging cell line is identified herein 
as 293-E4 and deposited on Aug. 30, 1994, with the American Type Culture 
Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., under the 
Budapest Treaty, and has there been designated ATCC #CRL 11711. 
Another object of the present invention is to provide a plasmid used to 
introduce the E4 region into the 293 cells. The bacterial plasmid 
comprises the adenovirus E4 region devoid of the E4 promoter and 
substituted with an inhibin promoter. In a particular embodiment, the 
plasmid comprises the adenovirus described above and a mouse alpha 
(.alpha.)-inhibin promoter which is identified as pIK6.1 MIP(.alpha.)-E4 
and deposited at the ATCC on Aug. 30, 1994, under the Budapest Treaty, and 
has there been designated ATCC #75879. 
Yet another object of the present invention is to provide a method of 
infecting a mammalian target cell with the above-identified second or 
third generation recombinant viral vectors that carry transgenes for in 
vivo and ex vivo gene therapy.

DETAILED DESCRIPTION OF THE INVENTION 
One strategy designed to circumvent the problems associated with current 
early region-deleted adenoviral vectors is to introduce a second essential 
gene region deletion into the adenoviral vector. Several adenovirus early 
gene region transformed cell lines which support the growth of E1, E2A or 
E4 mutant virus growth, respectively, have been established Grahan, et 
al, J. Gen Virol. 36: 59-72 (1977), Weinberg, et al, Proc. Natl. Acad. 
Sci. USA 80: 5383-5386 (1983) and Brough, et al, Virology 190: 624-634 
(1992)!. However, no cell line offers the functions of two gene regions 
simultaneously and at permissive temperatures. Establishing such a cell 
line which possesses the capability to complement the E1 and a second 
essential gene region function in trans (eg., E4), and the capacity to 
function as a packaging cell line for the propagation of recombinant viral 
vectors containing such double (or possibly triple or quadruple) 
deletions, may eliminate the drawbacks of the first generation adenoviral 
vectors currently available. 
Studies of the adenovirus early region (ER) gene functions have shown that 
the deletion of the E4 region results in a failure to accumulate viral 
late transcripts; a reduction in viral late protein synthesis; a defective 
viral particle assembly and a failure to inhibit host protein synthesis at 
the late infection stage Sandler, et al, J. Virol.63: 624-630 (1989), 
Bridge & Ketner, Virology 174: 345-353 (1990), Ross & Ziff, J. Virol. 66: 
3110-3117 (1992), Bridge, et al, Virology 193: 794-801 (1993), and Bett, 
et al, J. Virol. 67: 5911-5921 (1993)!. Dual removal of the E1 and E4 gene 
regions from the recombinant adenovirus vectors may therefore dramatically 
minimize or eliminate the pathogenic effects of direct cytotoxicity to the 
targeted cells and inflammatory responses in the human body. The E4 
deletion in a second generation recombinant adenoviral vector would 
provide the additional benefit of increasing the capacity of this vector 
system to accommodate human gene inserts as large as 10 kb. 
In one aspect of the present invention, the successful establishment of a 
novel packaging cell line which supports the growth of both the E1 and E4 
deletions in E1 and E4 deficient adenoviruses has been demonstrated. Since 
one of the E4 gene products 294R protein of open reading frame (ORF) 6! 
in association with the E1b gene product (496R protein) has a function of 
inhibiting cellular mRNA transport resulting in the cessation of cellular 
protein synthesis (Bridge & Ketner, 1990), the overexpression of the E4 
gene region would be expected to ultimately result in cell death. A major 
obstacle to the introduction of the E4 gene region into 293 cells has been 
overcome, i.e., the trans activation of the E1a gene product in the 
parental 293 cells which causes the overexpression of the E4 genes which 
would otherwise result in cell death. In the present invention, the E4 
promoter is replaced with a cellular inducible hormone gene promoter, 
namely, a gene that is regulated by a nuclear factor called CRE binding 
protein (CREB). Particularly, the promoter that replaces the E4 promoter 
is chosen from the CREB regulated gene family such as .alpha.-inhibin, 
beta (.beta.)-inhibin, .alpha.-gonadotropin, cytochrome c, cytochrome c 
oxidase complex (subunit IV), glucagon, etc. listed in Table I on page 
15695 in Kim, et al, J. Biol Chem., 268: 15689-15695 (1993). In a 
preferred embodiment, the CREB regulated gene promoter is a mammalian 
.alpha.-inhibin, most preferably, mouse .alpha.-inhibin. In this instance, 
a 165 base pair sequence of the mouse inhibin promoter region has been 
shown to drive the heterologous gene expression at a low basal level and 
increase the levels of heterologous gene expression in response to the 
induction of cAMP or adenylic cyclase activators Su & Hsueg, Biochem. and 
Biophys. Res. Common. 186, 293-300 (1992)!. An 8 bp palindromic sequence 
called cAMP response element (CRE) is responsible for this inductory 
effect and has been identified within the inhibin promoter region. In 
fact, all adenovirus early gene promoters contain the CRE-like element 
which renders these early genes responsive to the induction of cAMP 
(Jones, et al, Genes Dev. 2: 267-281 (1988)!. It is clear that E1a trans 
activation and the cAMP enhancement act on adenovirus early genes via 
independent mechanisms Leza & Hearing, J. Virol. 63: 3057-3064 (1989) and 
Lee, et al, Mol. Cell. Biol. 9: 4390-4397 (1989)!. The replacement of the 
E4 promoter with the mouse .alpha.-inhibin promoter uncouples the E1a 
trans-activation from the cAMP induction on the E4 gene. In the present 
invention, a full length sequence of the E4 region is introduced into the 
293 cells whereby the cAMP induction is still effective in inducing E4 
gene expression in the transformed cells in a controlled manner. It should 
also be noted that this novel 293-E4 packaging cell line may also rescue 
(supports the growth of) adenoviruses containing the E3 deletion in 
addition to the E1 and E4 deletions because the deletion of the E3 region 
will not affect the viability of the virus. 
The novel 293-E4 packaging cell lines were stably transformed by the E4 
region and displayed the same morphology and the growth rate as parental 
293 cells. This indicates that the low level of E4 gene expression under 
the control of the mouse .alpha.-inhibin promoter does not cause extensive 
inhibition of host cell protein synthesis. The mutant adenovirus, H5dl1014 
Bridge, et al, Virology 193: 794-801 (1993)!, was used to examine the 
complementing activity of the above described 293-E4 packaging cell line 
because it carries lethal deletions in the E4 region and can only grow in 
W162 cells (Bridge, & Ketner, 1989). The W162 cell line is a Vero monkey 
kidney cell line transformed by adenovirus E4 DNA and complements the 
growth of E4 deletion adenoviruses. The H5dl1014 virus has been shown to 
produce markedly reduced levels of DNA and failed to synthesize late 
protein due to an intact ORP 4 Bridge, et al, (1993)! in its mostly 
deleted E4 region. Cell lines were found that produced the H5dl1014 virus 
at comparable titers to that produced in W162 cells (See Table IV, Groups 
1 and 2 in Example 11, infra). 
In another embodiment, the present invention relates to novel recombinant 
adenoviruses or mutant adenoviruses produced by the novel packaging cell 
lines of the present invention. As described herein, the term "recombinant 
adenovirus" or "recombinant adeno-associated virus" (also known as 
recombinant viral vectors in the art) refers to a virus wherein the genome 
contains deletions, insertions and/or substitutions of one or more 
nucleotides, and the virus further carries a transgene. The term "mutant 
virus" refers herein to a particular virus, for example adenovirus and 
AAV, wherein the genome contains deletions, insertions and/or 
substitutions of one or more nucleotides; however no transgene is carried 
in the mutant virus. In one particular aspect of this embodiment, the 
novel 293-E4 packaging cell lines described above are used to generate a 
second generation of recombinant virus called 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E4. Although the 293-E4 packaging cell 
line contains the adenoviral serotype 5 E1 and E4 gene regions, other 
serotypes of mutant and recombinant adenoviruses, for example, serotype 2, 
7 and 12, may be rescued due to the high degree of structural and 
functional homology among the adenoviral serotypes. Moreover, mutant and 
recombinant adenoviruses from serotypes other than serotype 5 may be 
rescued from the other novel adenoviral packaging cell lines of the 
present invention described infra. 
In vitro studies demonstrate that the infection of the novel recombinant 
adenovirus vectors of the present invention in non-permissive human cells 
show no cytopathic effects and the efficiency of the transgene expression 
is at levels comparable to conventional E1-deleted viruses. It is expected 
that the host immune responses and inflammatory reactions at the sites 
infected with novel second generation recombinant adenoviruses of the 
present invention will be reduced compared to the first generation 
recombinant adenoviruses currently available. The establishment of the 
dual complementing packaging cell line of the present invention marks a 
significant event in the evolution of safer and more effective gene 
transfer adenoviral vectors. The method used in the construction of the 
293-E4 cell lines of the present invention is of general utility in the 
production of other packaging cell lines which contain additional 
adenoviral regions which complement further deletions of the adenoviral 
vectors of the present invention or in the construction of other viral 
vectors. 
Thus, in another embodiment, the present invention relates to novel 
adenoviral packaging cell lines that can rescue deletions in addition to 
E1, E4 and optionally E3 by the methods described above. In this example, 
an adenoviral vector packaging cell line which can rescue the E2A 
mutation, in addition to the E1, E3 and E4 deletions, was constructed 
starting with the novel packaging cell line described above, namely the 
293-E4 packaging cell line. The E2A gene product is a regulatory protein, 
specifically, a DNA binding protein. This gene may be introduced into the 
293-E4 packaging cell line by placing the E2A gene under the control of an 
inducible promoter operably linked to the E2A gene in a similar manner as 
described above. The inducible promoter may be selected from the same 
family of CREB regulated genes described above used to replace the E2 gene 
promoter. 
In yet another embodiment, the present invention relates to an adenoviral 
vector packaging cell line that may rescue the adenovirus recombinant 
virus containing the minimum essential cis-elements (inverted terminal 
repeats (ITRs) and packaging signal sequence) Hering, et al, Virol. 61: 
2555-2558 (1987)! and protein IX sequence Ghosh-Choudury, et al, EMBO J. 
6: 1733-1739 (1987)! only. This cell line may be established by 
introducing the adenovirus DNA sequence from around m.u. 11.2 to 
approximately 99 into the novel 293-E4 cell line described above. This DNA 
sequence represents the sequence from after E1b gene to the 3' end of the 
viral structural gene Sanbrook, et al, Cold Spring Harbor Symp. Quant. 
Biol. 39: 615-632 (1974); Ziff & Evans, Cell 15: 1463-1476 (1978)!. The 
introduced adenovirus sequence contains viral structural genes and almost 
the entire functional gene regions except E1a and E1b. Because the 
constitutive expression or overexpression of viral gene products are very 
toxic to the cells, the introduced adenoviral DNA may be manipulated to 
replace adenoviral native promoters with heterologous promoters. For 
example, the early gene regions which encode viral regulatory proteins may 
be placed under the control of the CREB regulated promoters, which have 
about 2 to 10 fold induction efficiency. In the case of the gene region 
that encodes viral structural proteins, the native major late promoter may 
be replaced by a tightly controlled exogenous promoter such as the 
tetracycline-responsive promoter which has an induction level up to about 
10.sup.5 fold in the presence of tetracycline Manfred & Hermann, PNAS 89: 
5547-5551 (1992)!. 
In another embodiment, the present invention relates to novel 
adenoviral-associated (AAV) packaging cell lines prepared in the following 
manner. The novel complementing cell line contains the E1a, E1b, E2A, and 
E4 gene regions and the DNA sequence encoding virus-associated RNA. This 
cell line may be constructed by introducing the adenovirus DNA sequence 
encoding the virus-associated RNA (around 200 NTs from m.u. 29 Mathews, 
Cell 6: 223-229 (1975) and Petterson & Philipson, Cell 6: 1-4 (1975)! into 
the novel 293-E4 packaging cell line constructed above that rescues the 
E1and E4 deletions, the E2A mutation of adenovirus and optionally E3. The 
wild type AAV produced from this packaging cell line will be free of 
helper adenovirus. The recombinant adeno-associated virus or mutant AAV 
will only contain the minimal essential cis-elements and will be generated 
by co-transfecting a non-packaging complementing AAV plasmid which is 
defective for packaging but supplies the wild type AAV gene products 
Samulski et al, J. Virol. 61: 3096-3101 (1987)!. Moreover, the 
recombinant adeno-associated viral vectors or mutant AAV rescued from this 
cell line will be free of helper viruses, i.e., adenoviruses. 
In another embodiment, the present invention relates to yet another novel 
AAV packaging cell line constructed by starting with the AAV packaging 
cell line described above. This packaging cell line contains the E1a, E1b, 
E2A and E4 gene regions, the DNA encoding virus-associated RNA and 
additionally, the AAV virus replication (rep) gene regions. The rep gene 
region encodes at least four replication (Rep) proteins that are essential 
for AAV DNA replication and trans-regulation of AAV gene expression (for 
review, see Bervis & Bolienzsky, Adv. Virus Res. 32: 243-306 (1987)!. It 
is constructed by introducing the AAV rep gene region into the AAV 
packaging line described above that already contains the E1, E2A, E4 gene 
regions and DNA sequences encoding the virus-associated RNA in the manner 
that replaces the P5 promoter (Yang, et al, J. Virol. 68: 4847-4856 
(1994)! with an inducible promoter chosen from the CREB regulated gene 
family described previously. The novel AAV virus and its recombinant virus 
rescued from the cell line will be free of helper viruses (adenoviruses) 
and is Rep-Muzyczka, Curr. Top. Microbiol. Immunol. 158: 97-129 (1992)!. 
In another embodiment, the present invention relates to another novel AAV 
packaging cell line constructed by starting with the AAV packaging cell 
line described in the previous paragraph. This packaging cell line 
contains the E1a, E1b, E2A, E4 gene regions, the DNA encoding the 
virus-associated RNA, the AAV virus replication (rep) gene region, and 
additionally the AAV cap gene region. The cap gene region encodes a family 
of capsid proteins, i.e., VP1, VP2 and VP3 Janik, et al, J. Virol. 52: 
591-597 (1984)!. The synthesis of all three mRNAs are started from a 
single promoter called P40 Janik, et al, (1984)!. This gene region will 
be introduced into the AAV packaging cell line described above by 
replacing the P40 promoter with an inducible promoter selected from either 
the CREB regulated promoters or the tetracycline responsive promoter. The 
novel AAV virus and its recombinant virus rescued from the cell line will 
be free of helper viruses (adenoviruses) and only contain the minimal 
essential cis-elements Muzyczka, Curr. Top. Microbiol. Immunol. 158: 
97-129 (1992)!. 
The present invention further provides the production of novel mutant 
viruses (particularly, adenoviruses and AAV), and novel recombinant 
adenoviruses and AAV (also referred to herein as recombinant 
adenoviral-derived and AAV-derived vectors) containing a transgene which 
will be expressed in the target cells. The recombinant adenoviral-derived 
and AAV-viral vectors are prepared using the packaging cell lines 
described above which comprise one or more distinct nucleotide sequences 
capable of complementing the part of the adenovirus or AAV genome that is 
essential for the virus' replication and which is not present in the novel 
recombinant adenoviral-derived and AAV-derived vectors. Recombinant 
adenoviral-derived and AAV-derived vectors will no longer contain genes 
required for the virus replication in infected target cells. More 
particularly, the recombinant adenoviral vectors will only contain the 
minimum essential cis-elements (i.e., ITRs and packaging signal sequence) 
and protein IX sequence, and be free of the E1 (specifically, E1a and E1b) 
and E4 regions, and may additionally be free of E3 and E2A regions and the 
viral structural genes. In the case of the recombinant AAV vectors, these 
vectors will contain deletions of the AAV virus Rep protein coding region 
or will only contain the minimal essential cis-elements. The latter will 
be generated from the AAV packaging cell line which contains the E1a, E1b, 
E2A and E4 gene regions, and the DNA encoding virus-associated RNA by 
co-transfecting a non-packaging complementing AAV plasmid which is 
defective for packaging but supplies the wild type AAV gene products 
Samulski, et al, (1987)!. 
The recombinant adenovirus-derived or AAV-derived vector is also 
characterized in that it is capable of directing the expression and the 
production of the selected transgene product(s) in the targeted cells. 
Thus, the recombinant vectors comprise at least all of the sequences of 
the adenoviral or AAV DNA sequence essential for encapsidation and the 
physical structures for infection of the targeted cells and a selected 
transgene which will be expressed in the targeted cells. 
The transgene may be a therapeutic gene that will ameliorate hereditary or 
acquired diseases when expressed in a targeted cell by using gene transfer 
technology methods well known in the art. In one particular aspect, the 
therapeutic gene is the normal DNA sequence corresponding to the defective 
gene provided in Table I below, for example, the normal DNA sequence 
corresponding to LDL receptors and .alpha. 1-antitrypsin. In another 
aspect, the transgene may encode a cytokine gene, suicide gene, tumor 
suppressor gene or protective gene, or a combination thereof chosen from 
the list provided in Table II. If a cytokine gene is selected, the 
expression of the gene in a targeted cell may provide a treatment to 
malignancies by stimulating cellular immune responses which result in 
suppression of tumor growth and/or killing of tumor cells. If a suicide 
gene is chosen, the gene when expressed in the tumor cell will enable the 
tumor cell to be destroyed in the presence of specific drugs. For example, 
the thymidine kinase gene when expressed in tumor cells will enable the 
tumor to be destroyed in the presence of gancyclovir. 
In yet another embodiment, the transgene may encode a viral immunogenic 
protein that is utilized as a vaccine for prevention of infectious 
diseases (See Table III). Procedures for preparing and administering such 
vaccines are known in the art (see e.g., Estin, et al, Proc. Nat. Acad. 
Sci. 85:1052 (1988)). 
The present invention further relates to therapeutic methods for the 
treatment of hereditary and acquired diseases, cancer gene therapies, and 
vaccines for prevention of infectious diseases. The transgene may be 
expressed under the control of a tissue specific promoter. For example, a 
suicide gene under the control of the tyrosinase promoter or tyrosinase 
related protein-1 promoter will only be expressed in melanocytes in the 
case of cancer therapy for melanoma Vile & Hart, Cancer Res. 53: 962-967 
(1993) and Lowings, et al, Mol. Cell. Biol. 12: 3653-3663 (1992)!. Various 
methods that introduce an adenoviral or AAV vector carrying a transgene 
into target cells ex vivo and in vivo have been previously described and 
are well known in the art. See for example, Brody & Crystal, Annals of 
N.Y. Acad. Sci. 716: 90-103, 1993!. The present invention provides for 
therapeutic methods, vaccines, and cancer therapies by infecting targeted 
cells with the recombinant adenoviral or AAV vectors containing a 
transgene of interest, and expressing the selected transgene in the 
targeted cell. 
For example, in vivo delivery of recombinant adenoviral or AAV vectors 
containing a transgene of the present invention may be targeted to a wide 
variety of organ types including brain, liver, blood vessels, muscle, 
heart, lung and skin. The delivery route for introducing the recombinant 
vectors of the present invention include intravenous, intramuscular, 
intravascular and intradermal injection to name a few routes. (See also 
Table I in the Brody & Crystal article and the references cited.) 
In the case of ex vivo gene transfer, the target cells are removed from the 
host and genetically modified in the laboratory using AAV-vectors of the 
present invention and methods well known in the art Walsh, et al, PNAS 
89: 7257-7261, (1992) and Walsh et al, Proc. Soc. Exp. Bio. Med. 204: 
289-300 (1993)!. 
Thus, the recombinant adenoviral or AAV vectors of the invention can be 
administered using conventional modes of administration including, but not 
limited to, the modes described above. The recombinant adenoviral or AAV 
vectors of the invention may be in a variety of dosages which include, but 
are not limited to, liquid solutions and suspensions, microvesicles, 
liposomes and injectable or infusible solutions. The preferred form 
depends upon the mode of administration and the therapeutic application. 
TABLE I 
__________________________________________________________________________ 
Gene Therapy for Hereditary Disease 
DISEASES DEFECTIVE GENES 
GENE PRODUCTS 
__________________________________________________________________________ 
Familial hypercholesterolemia 
LDL receptor LDL receptor 
(type II hyperlipidemias) 
Familial lipoprotein lipase 
Lipoprotein lipase 
Lipoprotein lipase 
deficiency (type I 
hyperlipidmias) 
Phenylketonuria 
Phenylalanine hydroxylase 
Phenylalanine hydroxylase 
Urea cycle deficiency 
Ornithine transcarbamylase 
Ornithine transcarbamylase 
Von Gierke's disease (glycogen 
G6Pase Glucose-6-phosphotase 
storage disease, type I) 
Alpha I-antitrypsin deficiency 
Alpha 1-antitrypsin 
Alpha 1-antitrypsin 
Cystic fibrosis 
Cystic tibrosis transmembrane 
Membrane chlorine channel 
conductant regulator 
Von Willebrand's disease and 
Factor VIII Clotting factor VIII 
Hemophilia A 
Hemophilia B Factor IX Clotting factor Ix 
Sickle cell anemia 
Beta globin Beta globin 
Beta thalassemias 
Beta globin Beta globin 
Alpha thalassemias 
Alpha globin Alpha globin 
Hereditary sperocytosis 
Spectrin Spectrin 
Severe combined immune 
Adenosine deaminase 
Adenosine deaminase 
deficiency 
Duchenne muscular dystrophy 
Dystrophin minigene 
Dystrophin 
Lesch-Nyhan syndrome 
Hypoxanthine guanine 
HGPRT 
phosphoribosyl 
transferase (HGPRT) 
Gaucber's disease 
Beta-glucocerebrosidase 
Beta-glucocerebrosidase 
Nieman-Pick disease 
Sphingomyelinase 
Sphingomyelinase 
Tay-Sachs disease 
Lysosomal hexosaminidase 
Lysosomal hexosaminidase 
Maple syrup urine disease 
Branched-chain keto acid 
Branced-chain keto acid 
dehydrogenase 
dehydrogenase 
__________________________________________________________________________ 
TABLE II 
______________________________________ 
Cancer Gene Therapy 
TUMOR 
CYTOKINE SUICIDE SUPPRESSOR PROTECTIVE 
GENES GENES GENES GENES 
______________________________________ 
IFN-gamma, IL-2, 
thymidine kinase, 
p53, Rb, multiple drug 
IL4, and granu- 
cytosine deamin- 
and Wt-1 resistant 
locyte-macrophage 
ase, diphtheria 
colony stimulation 
toxin, and TNF 
factor 
______________________________________ 
TABLE III 
______________________________________ 
Vaccine for Infectious Disease 
DISEASES VACCINE 
______________________________________ 
Hepatitis HBV surface antigen 
HIV infection and AIDS 
HIV envelope proteins 
Rabies Rabies glycoproteins 
______________________________________ 
The following examples are presented to illustrate the present invention 
and are not intended in any way to otherwise limit the scope of this 
invention. 
EXAMPLES 
Example 1 
Construction of Plasmids 
This example describes the construction of the plasmids used to introduce 
the E4 gene region into the 293 cells. The constructed plasmids are 
diagrammatically represented in FIG. 1. The parental plasmid pIK6.1 
MMSV-E4 (.DELTA.E4 pro.) derived from the pIK6.1 MMSV enpoNhe(Hpa) Finer, 
et al, Blood 83: 43-50, (1994)! contains the promoterless E4 region from 
15 bp upstream of the transcription start site to 810 bp downstream of the 
E4 polyadenylation site. The E4 gene is linked to the Moloney murine 
sarcoma virus U3 fragment. The pIK6.1. MIP(.alpha.)-E4 was constructed by 
ligation of a 238 bp fragment of the Hind III-XbaI PCR product of mouse 
alpha inhibin promoter MIP(.alpha.)! (Su, & Hsueh, Biochem. and Biophys. 
Res. Common. 186: 293-300, 1992) with the 2.9 kb XbaI-StuI fragment and 
the 3.9 kb Stu I-Hind III fragment of the PIK6.1 MMSV-E4 (E4 pro.). The 
primers used for PCR of the MIP (.alpha.) were 
5'-gcgcaagcttcGGGAGTGGGAGATAAGGCTC-3' (SEQ ID NO:1) and 
5'-ggcctctagaAGTTCACTTGCCCTGATGACA-3' (SEQ ID NO:2). The sequences 
containing either the Hind III site or Xba I site in lower case are 
present to facilitate cloning. The cloned .alpha.-inhibin promoter was 
sequenced to verify the accuracy of the sequence. 
The plasmid ADV-.beta.-gal used to generate recombinant adenoviruses was 
constructed as shown in FIG. 2. The starting plasmid ADV-1 contains the 
left end of adenovirus 5 Xho I C fragment (m.u. 0-15.8) with a deletion 
from nucleotides 469-3326 (m.u. 1.3-9.24) on the backbone of PCR II (In 
Vitrogen, San Diego, Calif.). A polylinker cassette was inserted into the 
deletion site. Several restriction sites at the left end of the adenovirus 
sequence can be conveniently used to linearize the plasmid. The resulting 
ADV-.beta.-gal plasmid was constructed by insertion of a Bst BI-Xba I 
fragment of the E. coli .beta.-galactosidase gene driven by the mouse pgk 
promoter into the ADV-1 compatible sites Spe I and Cla I in the E1 region 
and was later used to generate the recombinant virus. 
Example 2 
Transfection and Selection of 293-E4 Cell Lines 
This example describes the transfection and selection process employed to 
establish 293-E4 cell lines. The 293 cells, obtained from the American 
Type Culture Collection, ATCC #CRL 1573, were grown in Dulbecco's modified 
Eagle's medium (DMEM), 1 g/L glucose (JRH Biosciences), 10% donor calf 
serum (Tissue Culture Biologics) . Cells were seeded at 5.times.10.sup.5 
per 10-cm plate 48 hours prior to the transfection experiment. Ten .mu.g 
of pIK.MIP(.alpha.)-E4 and 1 .mu.g of pGEM-pgkNeo.pgkpolyA containing the 
Neo.sup.r gene were co-transfected into 293 cells by calcium phosphate 
co-precipitation Wigler, et al, Cell 57: 777-785 (1979)!. The transfected 
cells were split 1:20 in normal medium at 24 hours post-transfection. 
After the cells were attached to the plate, the medium was changed to 
selective medium containing 1 mg/ml G418 (Sigma, St Louis, Mo.). The cells 
were refed with fresh selective medium every 3 days for about 2-3 weeks. 
Isolated clones were picked, expanded and maintained in the selective 
medium for 5-6 passages. The established 293-E4 cell lines were routinely 
maintained in the normal medium. 
Example 3 
Southern Transfers and Hybridization 
Genomic DNA from 293-E4 cell lines were digested with desired restriction 
enzymes and purified with phenol/chloroform. 10 .mu.g of digested DNA were 
run on 0.8%-1% agarose gel and transferred to a nylon membrane (Zetabind, 
America Bioanalytical, Natick, Mass.). DNA from the 293-E4 cell lines were 
digested with restriction enzymes and analyzed. DNA from wild type 
adenovirus 5, pIK6.1 MIP(.alpha.)-E4 plasmid and parental 293 cells were 
also digested with the same enzymes and used as controls. Restriction 
fragments of the E4 region, .alpha.-inhibin promoter sequence, and the E1 
region were detected by hybridization to the appropriate .sup.32 P-labeled 
probes and subsequent autoradiography. 
Example 4 
Preparation of Viral Stocks 
W162 cells were grown in DMEM, 4.5 g/L glucose and 10% CS. The W162 cell 
line is a Vero monkey kidney cell line transformed by adenovirus E4 DNA 
and supports the growth of E4 deleted adenovirus mutants Weinberg, & 
Ketner, Proc. Natl. Acad. Sci. USA 80: 5383-5386 (1983)!. The H5dl1014 
virus has been previously described in Bridge & Ketner, J. Virol. 63: 
631-638, (1989). This adenovirus 5 virus strain has two deletions within 
the E4 region and can only grow in W162 cells (Bridge, & Ketner 1989). 
Propagation and titration of H5dl1014 virus were done on W162 cells. For 
evaluation of the production of H5dl1014 virus from 293-E4 cell lines of 
the present invention, the W162, 293 and 293-E4 cell lines were counted 
and plated in the 6-well plate at 1.times.10.sup.5 /well and infected with 
H5dl1014 at a multiplicity of infection (m.o.i.) of 50 plaque-forming 
units (p.f.u.) per cell. The viral stocks were prepared by harvesting the 
cells at 48 hr post-infection. The cells were precipitated and resuspended 
in 200 .mu.l of serum free medium. The cell suspensions underwent 3 cycles 
of freeze and were thawed to release the viral particles from the cells. 
The cell debris was discarded by centrifugation. The titers of the virus 
produced from the infected cells were determined by plaque formation on 
monolayers of W162 cells. 
Example 5 
Construction of Recombinant Viruses 
The 293 cell line and 293-E4 cell line were plated in 10-cm plate at 
2.5.times.10.sup.6 /plate 48 hours before the experiment. One hour prior 
to the co-transfection, cells were fed with 10 ml fresh medium. 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus was made by co-transfection of 10 
.mu.g of ADV-.beta.-gal linearized by Bst BI with 4 .mu.g of H5dl327 
(Thimmappaya, et al, Cell 31: 543-551 1982) digested with Cla I. 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus was generated by co-transfection 
of 10 .mu.g of Bst BI linearized ADV-.beta.-gal and 4 .mu.g of Cla I 
digested H5dl1014 on 293-E4 cell lines by calcium phosphate precipitation 
technique. Twenty-four hours after co-transfection, the medium was removed 
and the monolayers of the culture were overlaid with 10 ml DMEM medium 
containing 20 mM MgCl.sub.2, 5% of CS and 0.5% of noble agar (DIFCO Lab. 
Detroit, Mich.). The plaques were picked and resuspended in 100 .mu.l of 
PBS. Diluted plaque samples were immediately subjected to 2 to 3 rounds of 
blue plaque purification. The blue plaque purification was carried out as 
a regular plaque assay except that the cultures were overlaid with a 
second layer of soft agar containing 1 mg/ml X-gal when plaques appeared. 
After incubation for 2 hours, plaques which contained the recombinant 
virus carrying the .beta.-galactosidase gene were stained blue. The purity 
of the recombinant virus was determined by no contamination of white 
plaques. The purified plaques were expanded and the DNA of the lysate was 
analyzed (FIG. 6) as previously described Graham & Prevec (1992)!. 
Adenoviral DNA was digested with Sma I and fractionated on 0.8% agarose 
gel. DNA samples of H5dI1014 and the Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 
viruses were extracted from CsCl gradient purified viral stocks. DNA of 
the Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 was extracted from the virus 
infected cells. 
Example 6 
Histochemical Staining 
Forty-eight hours following recombinant viral infection with 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus (E1 and E3 deletion viruses) and 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus (E1 and E4 deletion viruses) at 
20 m.o.i. the monolayers of cells are washed once in PBS and fixed for 10 
min. at room temperature with 0.5% glutaraldehyde (Sigma, St. Louis, Mo.) 
in PBS. The cells were washed three times with PBS containing 1 mM 
MgCl.sub.2 and then stained with 5-bromo-4-chloro-3-indolyl-.beta., 
D-galactosidase (X-gal, Sigma) as previously described (Thimmappaya et al, 
1982). The X-gal solution at 40 mg/ml in dimethylformamide was diluted to 
1 mg/ml in KC solution (PBS containing 5 mM K.sub.3 Fe (CN).sub.6, 5 mM 
K.sub.4 Fe (CN).sub.6.3H.sub.2 O). After staining, for 2-4 hours the cells 
were washed with H.sub.2 O and inspected under a light microscope. 
Example 7 
.beta.-galactosidase Activity Assay 
Cells were infected with either Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus 
and Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus at 20 m.o.i. assayed for 
enzyme activity as described in MacGregor, et al, Somatic Cell Mol. 
Genetic. 13: 253-264, (1987) with the following modifications. Cells in 
6-well plate were washed with PBS twice and lysed in the well by addition 
of 200 .mu.l of 2.times. Z buffer (1.times. Z buffer: 60 mM Na.sub.2 
PO4.7H.sub.2 O, 40 mM NaH.sub.2 PO.sub.4.H.sub.2 O, 10 mM KCl, 1 mM 
MgSO.sub.4.7H.sub.2 O) and 200 .mu.l of 0.2% Triton X-100. After 
incubation at room temperature for 5-10 min, 100 .mu.l of each sample was 
transferred to the 96-well microtiter plate. After addition of 50 .mu.l of 
2-nitrophenyl-.beta.-D-galactopyranoside (2mg/ml), the reaction was 
allowed to proceed for 5 min at room temperature and stopped by adding 50 
.mu.l of stop solution (1M Na.sub.2 CO.sub.3) . Fluorescence was measured 
at 420 nm on a microtiter plate reader (Molecular Devices Co. Menlo Park, 
Calif.). 
Example 8 
Construction of 293-E4 Cell Lines 
The purpose of introducing the Ad5 E4 gene region into 293 cells is that 
the derived cell line is able to package the recombinant adenoviruses 
containing two lethal deletions (E1 and E4). The plasmid, 
PIk.MIP(.alpha.)-E4 carries the full length region of the Ad5 E4 region 
from 15 bp upstream of transcription start site to 810 bp downstream of 
the polyadenylation site (FIG. 1). The E4 gene region (m.u. 88.9-98.8) was 
directly linked to 238 bps of the mouse .alpha.-inhibin promoter 
containing the first 159 bps of the promoter region and 5' untranslated 
region. This promoter sequence is required for basal expression (Su & 
Hseuh (1992)). Within this promoter region, there is a cyclic adenosine 
3', 5'-monophosphate (cAMP) response element (CRE) which allows an 
increased level of gene expression induced by either cAMP or adenylic 
cyclase activator Paei, et al, Mol. Endocrinol. 5: 521-534 (1991)!. The 
pIK.MIP(.alpha.)-E4 was introduced into 293 cells together with the 
PGEM-pgkNeo.pghpolyA which bears a neomycin resistant gene by calcium 
phosphate precipitation at a molar ratio equivalent to 10:1. A total of 66 
G418 resistant clones were picked for further analysis. 
Example 9 
Identification of E4 Transfectants 
To examine the integration of the introduced adenovirus E4 region, genomic 
DNA from each clone was digested with either Hind III and Sfi I, or Nco I 
restriction enzymes and analyzed by Southern transfer. FIG. 3A shows a 
restriction map of the introduced .alpha.-inhibin-E4 region and 
corresponding regions of the E4 probe (Sma I H fragment of Ad5) and the 
inhibin promoter probe. 17 clones out of a total of 66 presented the 
correct DNA patterns as predicted for a full length E4 region DNA 
integration in the screen blots of both digestions. Other clones showed 
either no integration or integration with variable sizes of E4 region. 
FIGS. 3B-3E represent the Southern blots of genomic DNA extracted from the 
17 clones with full length integration and two clones which contains 
variable sizes of E4 region integration on the initial screening blots. 
The DNA was extracted after maintaining these 19 cell lines in the 
non-selective medium for more than 30 passages. As shown in FIGS. 3B and 
3C, 15 cell lines represent the characteristic 0.9 kb and 3.2 kb fragments 
in HindIII/Sfi I digestion and 1.6 kb and 2.1 kb fragments in Nco I 
digestion. There were no detectable E4 region sequences in two cell lines 
(lines 13 and 29) which had the same integration patterns as the other 15 
lines in the screening blots, indicating an unstable integration event in 
these two lines. Lines 16 and 19 are examples of cell lines which retained 
the E4 gene region with variable restriction patterns. The 0.9 kb band of 
all 15 lines hybridized to the mouse inhibin promoter sequence in the Hind 
III/Sfi digestion (FIG. 3D). The 3.1 kb fragment along with the 2.1 kb 
fragment was hybridized to the inhibin promoter probe in the Nco I 
digestion blot. These results indicate that a full length gene region of 
E4 was stably integrated into these 15 cell lines. To rule out the 
possibility that these cell lines can survive and maintain a full length 
of the E4 region due to a loss of the E1 gene region, the blots were 
reprobed with the Ad5 Hind III E fragment (m.u. 7.7-17.1). All 19 lines 
have a same sized fragment detected by the E1 probe as that in the 
parental 293 cell line (FIG. 3E). Therefore, the E1 gene was not altered 
in the 293-E4 cell lines. 
Example 10 
Screen of Biological Activity of 293-E4 Cell Lines 
To determine whether these cell lines were capable of supporting the E4 
deletion virus growth, each of the cell lines was infected with an 
adenovirus E4 deletion mutant virus H5dl1014 Bridge & Ketner, (1989)!. 
The E4 defective strain H5dl1014 contains two deletions from m.u. 92 to 
93.8 and m.u. 96.4 to 98.4. The deletions destroy all the open reading 
frames of the E4, region except ORF 4. This virus produces substantially 
less viral DNA and late viral proteins in Hela cells similar to that seen 
in cells infected with H2dl808 and H5dl366 Halbert, et al, J. Virol. 56: 
250-256 (1985)!. The only permissive cell line for the growth of H5dl1014 
is W162 Weinberg & Ketner, (1983)!. When the parental 293 cells, W162 
cells and all 15 lines were infected with H5dl1014 at m.o.i. 25 with or 
without addition of the 1 mM cAMP, 6 cell lines showed comparable 
cytopathic effect (CPE) as observed on W162 cells at 3-4 days of 
post-infection (FIG. 4). The CPE appeared much faster in the presence of 
cAMP both in W162 cells and in some of the 293-E4 cell lines. The parental 
293 cells showed CPE at much milder level (FIG. 4). This result shows that 
293-E4 cell lines (containing both E1 and E4 gene regions) support the 
growth of E4 deleted viruses (eg., H5dl1014 virus) as efficiently as cell 
lines containing the E4 gene region only (eg., W162 cell line). 
Example 11 
Induction of H5dl1014 Production On 293-E4 Cell Lines 
To quantitatively examine the ability of 293-E4 cell lines to produce 
H5dl1014 mutant virus and to determine whether there is a specific 
induction of E4 gene expression in the 293-E4 cell lines, the titer of the 
H5dl1014 produced from the 293-E4 cell lines was measured in the presence 
or absence of cAMP. Viral stocks were prepared from each cell line by 
infecting the same number of cells with H51014 at m.o.i. 50. At 48 hr 
post-infection, the supernatant of each cell line was removed and the 
cells were resuspended in 1/10 of the original volume of serum free 
medium. Titration of the viral stocks were performed on W162 cells by 
plaque assay. As presented in Table 1, the phenomenon of virus production 
from these 15 lines can be generally classified into three groups. Group 1 
which includes lines 8, 50 and 51 showed increased viral titers by 4 to 6 
orders of magnitude compared to the titer produced from 293 cells. Line 8 
and 51 had a 10 fold increase of the viral titers in the presence of cAMP. 
Group 2, which includes lines 12, 27 and 61, produced similar titers of 
virus as that produced from W162 cells. The titers increased 1,000-10,000 
fold with the exception of line 12 in which the level of virus production 
increased by 7 orders of magnitude in the presence of cAMP. These results 
indicate an induced E4 gene expression in these three cell lines. Group 3 
includes the remaining cell lines which produced the virus titers 
essentially at levels similar to that produced from parental 293 cells in 
the presence or absence of cAMP. The induced E4 gene expression is also 
indicated in several cell lines in this group. 
The 10 fold induction was also observed in the W162 cells and parental 293 
cells when the cells were treated with cAMP. It is possible that this 10 
fold increase in the virus yield is due to the enhancement effect of cAMP 
on other adenovirus early gene expression Leza & Hearing, J. Virol. 63: 
3057-3064 (1989)! which also contains CRE elements causing an increase in 
viral DNA synthesis. 
TABLE IV 
______________________________________ 
Titers of H5d11014 produced from 
cell lines W162, 293, and 293-E4 
TITER pfu/ml!.sup..dagger. 
GROUP CELL LINE No cAMP ImM cAMP 
______________________________________ 
control W162 2.2 .times. 10.sup.13 
1.2 .times. 10.sup.14 
293 1.6 .times. 10.sup.4 
2.7 .times. 10.sup.5 
1 293-E4-8 8.9 .times. 10.sup.12 
3.3 .times. 10.sup.13 
293-E4-50 6.7 .times. 10.sup.10 
4.5 .times. 10.sup.10 
293-E4-51 8.9 .times. 10.sup.8 
2.2 .times. 10.sup.9 
2 293-E4-12 4.5 .times. 10.sup.5 
8.9 .times. 10.sup.12 
293-E4-27 6.7 .times. 10.sup.9 
2.2 .times. 10.sup.13 
293-E4-61 1.3 .times. 10.sup.10 
8.0 .times. 10.sup.13 
3 293-E4-6 1.1 .times. 10.sup.4 
8.9 .times. 10.sup.4 
293-E4-15 1.3 .times. 10.sup.5 
6.7 .times. 10.sup.6 
293-E4-33 6.7 .times. 10.sup.4 
1.6 .times. 10.sup.6 
293-E4-34 6.7 .times. 10.sup.6 
1.3 .times. 10.sup.7 
293-E4-35 1.3 .times. 10.sup.5 
1.1 .times. 10.sup.6 
293-E4-48 6.7 .times. 10.sup.4 
6.7 .times. 10.sup.6 
293-E4-52 1.8 .times. 10.sup.4 
1.3 .times. 10.sup.7 
293-E4-59 3.3 .times. 10.sup.3 
6.7 .times. 10.sup.6 
293-E4-62 1.6 .times. 10.sup.5 
6.7 .times. 10.sup.6 
______________________________________ 
.sup..dagger. The titer was determined by plaque assay on W162 monolayer 
culture. 
Values in the table are the averages of titers measured on duplicate 
samples. 
Example 12 
Generation of Ad5/.DELTA.E1 (.beta.-gal).DELTA.E4 Virus 
To rescue recombinant virus which harbors lethal deletions in both the E1 
region and the E4 region the two most efficient cell lines, line 8 and 
line 61, were utilized. The ADV-.beta.-gal plasmid was linearized by BstBl 
and co-transfected with Cla I digested H5dl1014 into the monolayers of 
293-E4 cell lines (FIG. 5). The recombinant virus was generated by in vivo 
recombination between the overlapping adenoviral sequence of 
ADV-.beta.-gal and the H5dl1014 large Cla I fragment (m.u. 2.55-100). 
Plaques appearing at 7-10 days post-transfection were isolated and 
purified by blue plaque assay. The final purified blue plaque and the 
viral DNA were analyzed (FIG. 6). For the following comparative studies of 
the double deletion recombinant virus, the 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus was generated. This virus was 
generated by co-transfection of Bst BI linearized ADV-.beta.-gal plasmid 
with Cla I digested H5dl327 Thimmappaya, et al, (1982)! into 293 cells 
(FIG. 5). 
Example 13 
In Vitro Evaluation of the Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 Virus 
To evaluate the infectivity of this second generation of recombinant virus, 
infectivity was compared with the .beta.-gal gene expression of the double 
lethal deletion virus and single lethal deletion virus in Hela, 293, W162 
and line 61 cells. The cells were infected with these two strains of 
recombinant viruses at 20 m.o.i. for 48 hrs. Expression was observed in 
both infections as detected both by histochemical staining and the 
.beta.-galactosidase activity assay described supra. The abolished 
cytopathic effect of the Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus was also 
tested by the plaque assay. The 293-E4 was the only permissive cell line 
for all three strains of virus (Ad5/.DELTA.E1(.beta.-gal).DELTA.E4, 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 and H5dl1014). The 293 cells were 
permissive for the Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus, 
semi-permissive (low level of virus production) for the H5dl1014 virus but 
non-permissive to Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus. The W162 cell 
line was permissive for H5dl1014 virus, but non-permissive for 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E3 virus and 
Ad5/.DELTA.E1(.beta.-gal).DELTA.E4 virus. Hela cells are non-permissive 
for all three strains of viruses. These results demonstrate that the 
double deletion virus does not cause any cytopathic effect to the human 
cell lines tested. Absence of cytopathic effects following infection of 
the double deletion viruses at m.o.i. 20 suggests that in vivo these 
viruses will not express late gene products. This should eliminate the 
immune response against cells infected with recombinant virus, thereby 
prolonging transgene expression. 
All publications cited in this specification are herein incorporated by 
reference in their entirety as if each individual publication was 
specifically and individually indicated to be incorporated by reference. 
As will be apparent to those skilled in the art to which the invention 
pertains, the present invention may be embodied in forms other than those 
specifically disclosed above without departing from the spirit or 
essential characteristics of the invention. The particular embodiments of 
the invention described above, are, therefore, to be considered as 
illustrative and not restrictive. The scope of the invention is as set 
forth in the appended claims rather than being limited to the examples 
contained in the foregoing description. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
GCGCAAGCTTCGGGAGTGGGAGATAAGGCTC31 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
GGCCTCTAGAAGTTCACTTGCCCTGATGACA31 
__________________________________________________________________________