Note: Descriptions are shown in the official language in which they were submitted.
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Tissue-Specific Treatment, Diagnostic Methods, and
Compositions Using Replication-Def~lcient Vectors
Background of the Invention
Field of tlle Invention
The present invention relates to gene therapy using recombinant vector
delivery systems and particularly adenovirus vectors. The invention specificallyrelates to replication-deficient vectors which are able to replicate specifically in
certain tissues to provide a the~ ic benefit from the vector per se or from
heterologous gene products encoded by the vector and distributed throughout the
abnormal tissue. Preferably, the tissue is tumor tissue. The invention also relates
to cells for producing recombinant replication-deficient vectors useful for genetherapy. The invention also relates to methods for scleening abnormal tissues,
and especially tumors, for functions or functional deficiencies that permit vector
replication.
Background Art
Targeting Vectors
The introduction of exogenous genes into cells in vitro or in vivo,
systemically or in situ, has been of limited use for mixtures in which it would be
disadvantageous for non-target cells to take up the exogenous gene. One strategyto overcome this problem is to develop ~tlmini~tration procedures or vectors that
target a specific cell-type. Using systemic ~lmini.~tration, attempts have been
made to direct exogenous genes to myocytes and muscle cells by direct injection
of DNA, to direct the exogenous DNA to hepatocytes using DNA-protein
complexes, and to endothelial cells using liposomes.
Using in situ ~tlmini~tration, retroviral replication functions have been
utilized to target cells that are actively replicating. Targeting to replicatively
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active cells in this manner has been augmented by incorporating "suicide" genes
in the vector, which kill actively replicating untargeted, as well as targeted cells
that take up the vector.
Thus far, the ability to target cells has been limited, however, by the lack
of cell-type specificity and low gene transfer efficiencies. The limited ability to
target an exogenous gene to ~ e~ced cells in an org~ni~m, while avoiding
(elimin~ting) uptake ofthe gene by normal, u~ d cells, particularly has been
an obstacle to developing effective gene-transfer-based therapies for fli~e~ces in
~nim~l~ and hllm~n~
One especially difficult challenge is targeting tumor cells, because they
have many of the functions of normal cells. Many seemingly promising
strategies for these cells, moreover, are limited to one or a few cell-types. Thus,
a way to limit vector uptake or viability to targeted cells Itl.lahls to be developed.
The present invention, in one aspect, provides a way to deliver an
exogenous gene in a tumor.
Adenoviruses Generally
Adenoviruses are nonenveloped, regular icosohedrons. The protein coat
(capsid) is composed of 252 capsomeres of which 240 are hexons and 12 are
pentons. Most of the detailed structural studies of the adenovirus polypeptides
have been done for adenovirus types 2 and 5. The viral DNA is 23.85 x 106
daltons for adenovirus 2 and varies slightly in size depending on serotype. The
DNA has inverted terrninal repeats and the length of these varies with the
serotype.
The replicative cycle is divided into early (E) and late (L) phases. The
late phase defines the onset of viral DNA replication. Adenovirus structural
proteins are generally synthesized during the late phase. Following adenovirus
infection, host DNA and protein synthesis is inhibited in eells infected with most
serotypes. The adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very
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efficient and results in approximately 10,000 virions per infected cell along with
the synthesis of excess viral protein and DNA that is not incorporated into the
virion. Early adenovirus transcription is a complicated sequence of interrelatedbiochemical events, but it entails ec~nti~lly the synthesis of viral RNAs prior to
S the onset of viral DNA replication.
The org~ni7~tion of the adenovirus genome is similar in all of the
adenovirus groups and specific functions are generally positioned at identical
locations for each serotype studied. Early cytoplasmic messenger RNAs are
complr..lt;ll~y to four defined, noncontiguous regions on the viral DNA. These
regions are ~ecign~t~ocl (El-E4). The early transcripts have been classified into
an array of il~ early (Ela), delayed early (Elb, E2a, E2b, E3 and E4), and
intermediate (IVa2.IX) regions.
The Ela region is involved in transcriptional transactivation of viral and
cellular genes as well as transcriptional repression of other sequences. The Elagene exerts an important control function on all of the other early adenovirus
messenger RNAs. In normal tissues, in order to transcribe regions Elb, E2a, E2b,E3, or E4 efficiently, active Ela product is required. However, as discussed
below, the Ela function may be bypassed. Cells may be manipulated to provide
Ela-like functions or may naturally contain such functions. The virus may also
be manipulated to bypass the functions as described below.
The Elb region is required for the normal progression of viral events late
in infection. The El b product acts in the host nucleus. Mutants generated within
the Elb sequences exhibit ~limini~hed late viral mRNA accumulation as well as
p~ nt in the inhibition of host cellular transport norrnally observed late in
adenovirus infection (Berkner, K.L., Biotechniques 6:616-629 (1988)). Elb is
required for altering ~nctions of the host cell such that processing and transport
are shifted in favor of viral late gene products. These products then result in viral
packaging and release of virions. Elb produces a 19 kD protein that prevents
apoptosis. Elb also produces a 55 kD protein that binds to p53.
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For a complete review on adenoviruses and their replication, see Horwitz,
M.S., Virology 2d ed., Fields, B.N., eds., Raven Press Limited, New York (1990),Chapter 60, pp. 1679-1721.
Adenovirus as Recombinant DeUvery Vehicle
Adenovirus provides advantages as a vector for adequate gene delivery
for the following reasons. It is a double stranded DNA nonenveloped virus with
tropism for the hurnan respiratory system and ga~ l tract. It causes a
mild flu-like disease. Adenoviral vectors enter cells by receptor me~ ted
endocytosis. The large (36 kilobase) genome allows for the removal of genes
essential for replication and nonessP~ti~l regions so that foreign DNA may be
inserted and ~A~..essed from the viral genome. Adenoviruses infect a wide variety
of cell types in vivo and in vitro. Adenoviruses have been used as vectors for
gene therapy and for ~les~ion of heterologous genes. The ~IJ.ession of viral
or foreign genes from the adenovirus genome does not require a replicating cell.Adenovirus vectors rarely integrate into the host chromosome; the adenovirus
genome remains as an t;"ll~chlolllosomal element in the cellular nucleus. There
is no association of human m~lign~ncy with adenovirus infection; attenuated
strains have been developed and have been used in hllm~n~ for live vaccin~s
For a more detailed discussion of the use of adenovirus vectors for gene
therapy, see Berkner, K.L., Biotechniques 6:616-629 (1988); Trapnell, B.C.,
Advanced DrugDeliveryReviews 12:185-199 (1993).
Adenovirus vectors are generally deleted in the E1 region of the virus.
The E1 region may then be substituted with the DNA sequences of interest. It
was pointed out in-a-recent article on human gene therapy, however, that "the
main disadvantage in the use of adenovirus as a gene transfer vector is that theviral vector generally remains episomal and does not replicate, thus, cell division
leads to the eventual loss of the vector from the r~ ght~r cells" (Morgan, R.A.,e~ al., Annual Review of Biochemistry 62:191-217 (1993)) (emphasis added).
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Non-replication of the vector leads not only to eventual loss of the vector
without e.~ ion in most or all of the target cells but also leads to insufficient
.lession in the cells that do take up the vector, because copies of the gene
whose t;~le3sion is desired are insufficient for maximum effect. The
S insufficiency of gene t;~lession is a general limitation of all non-replicating
delivery vectors. Because of this limitation, for example, the effective amount
of ganciclovir used in conjunction with current thymidine kinase e;~lJression
vectors results in ullw~lled side effects. However, if gene dosage is increased
such that greater amounts of thymidine kinase are produced, less ganciclovir is
required to produce the desired treatment and side effects are minimi7.o~ Thus,
it is desirable to introduce a vector that can provide multiple copies of a gene and
hence greater amounts of the product of that gene.
Published PCT Patent Application No. WO 94/18992 describes methods
and compositions for treating neoplastic conditions based on the use of
replication-deficient viruses in p53-minus or Rb-minus tumor cells. The
publication asserts a virus able to produce a replication phenotype in neoplastic
cells, but unable to produce a replication phenotype in non-replicating non-
neoplastic cells having normal pS3 and/or pRb function. The t;xpelilllental datashow cytopathic effects of the replication-deficient viruses but not the production
of infectious virions. The reference does not disclose producer cell lines for
vector production, and especially for virion production of recombinant viruses
free of wild-type viruses. The reference does not disclose a combination of
diagnosing a tissue type ex vivo for the ability to replicate a replication-defective
virus and subsequent use of such virus to treat the tissue in vivo. The reference
does not disclose methods and compositions wherein cellular functions other thanpS3 and/or Rb are deficient. The reference does not disclose replication of a
replication-defective virus by means of complementing gene functions. To make
a producer cell line or effective therapy involving the spread of the virus
throughout the tissue, requires the production of infectious virus.
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The present invention overcomes all of the disadvantages discussed above
by providing a replicating vector, multiple DNA copies, and increased amounts
of gene product, especi~lly infectious virus.
Pro~ etiD~- of Adenoviral Vectors
Adenoviral vectors for recombinant gene e~les~ion have been produced
in the human embryonic kidney cell line 293 (Graham, F.L. et al., J. Gen. Virol.36:59-72 (1977)). This cell line, initially ~ s~,llled with human adenovirus 5,
now contains the left end of the adenovirus 5 genome and expresses El.
Therefore, these cells are permissive for growth of adenovirus 2 and adenovirus 5
mutants defective in El functions. They have been extensively used for the
isolation and propagation of E1 ~ ; The.erolt;, 293 cells have been used for
helper-independent cloning and ~ ession of adenovirus vectors in m~mm~ n
cells. E1 genes integrated in cellular DNA of 293 cells are expressed at levels
which permit deletion of these genes from the viral vector genome. The deletion
provides a nonessenti~l region into which DNA may be inserted. For a review,
see, Young, C.S.H., et al. in The Adenoviruses, Ginsberg, H.S., ed., Plenum
Press, New York and London (1984), pp. 125-172.
However, 293 cells are subject to severe limitations as producer cells for
adenovirus vectors. Growth rates are low. Titres are limited, especially when the
vector produces a heterologous gene product that proves toxic for the cells, such
as thymidine kinase. Recombination with the viral El sequence in the genome
can lead to the co.-l~"~in~tion of the recombinant defective virus with unsafe
wild-type virus. The quality of certain viral preparations is poor with regard to
the ratio of virus particle to plaque forming unit. Further. the cell line does not
supporf growth of more highly deleted mutants because the expression of E1 in
combination with other viral genes in the cellular genome (required to
complement the further deletion), such as E4, is toxic to the cells. Therefore, the
amount of heterologous DNA that can be inserted into the viral genome is limited
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in these cells. It is desirable, therefore, to produce adenovirus vectors for gene
therapy in a cell that cannot produce wild-type recombinants and can produce
high titres of high-quality virus. The present invention overcomes these
limitations.
Tumor S~ r~s~or Genes
Experiments in somatic cell genetics show that one or more genetic
elements can inhibit tumor cell proliferation. These t;~ hllcnts suggested that
genes contained in the normal cell SU~1C~ the ability of a normal cell to becometurnorigenic.
Since the original ~ h,lents were done, two genes in particular have
been chalnc~ as tumor-su~ressor genes, that is, genes that are able to
suppress the tumorigenic phenotype in tumor cells. The retinoblastoma
susceptibility gene (Rb) and p53 are two of the best studied of the tumor-
~u~,lessor genes. The inheritance of a defective allele of Rb predisposes to
lS retinoblastoma, a childhood tumor of the eye. However, the Rb gene is also
subject to somatic mutation in the development of breast, lung and bladder
carcinomas, osteosarcomas, and soft-tissue sarcomas (Weinberg, R.A., Cancer
Surveys 12:43-57 (1992)). p53 is mutated during the development of various
sporadic human cancers, and germ-line mutations in p53 are associated with Li-
Fraumeni syndrome (Harris, C.C. et al., New Engl. J. Med 329:1318-1327
(1993); Malkin, D., Cancer Genet. Cytogenet. 66:83-92 (1993), Malkin, D. et al.,Science 250:1233-1238 (1990)). Many human tumors show a high frequency of
mutation of both p53 and Rb. Research on these genes has suggested that there
may be cooperative tumorigenic effects of p53 and Rb in transforrnation. For a
detailed review of these genes and their functions, see Levine, A.J., Ann. Rev.
Biochem. 62:623-651 (1993).
Programmed cell death plays an important part in the regulation of
development of multicellular organi~m~ (see Ellis, R.E. et al., Ann. Rev. Cell
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Biol. 7:663-693 (1991) and Cohen, J.J. et al., Ann. Rev. Immunol. 10:267-293
(1992)). Programrned cell death char~cterictically occurs by apoptosis, a process
of nuclear con-len~tion and cleavage of chromosomes into small DNA
fragments. p53 appears to be an important component in the pathway towards
apoptosis (Yonish-Rouach, C.D. et aL, Nature 352:345-347 (1991)). p53 can
complex with viral and m~mm~ n proteins. p53 can heterodimerize and form
heterodimers with SV40 T-antigen, human papilloma virus E6 protein and
adenovirus E1 b. The binding of viral proteins to wild-type p53 either results in
rapid degradation of p53 or in sequestration in an inactive form. The ability ofviral proteins to inactivate p53 may be a me~h~ni~m by which these viruses
transform cells (see Lee, J.M. et al., Mutation Res. 307:573-581 (1994)).
Accordingly, a block to programmed cell death may occur through the binding
of a viral protein to pS3 or by functional inactivation of p53 in a cell.
Cells that are resting in Go or are in G, have Rb protein which is found in
a complex with a cellular transcription factor called E2F. E2F mediates the
L~ sc~ lion of several viral genes as well as some cellular genes that contribute
enzymatic activities involved in DNA replication. When the Rb/E2F complex is
exposed to adenovirus Ela protein, the E2F protein is released from this complex,
and there is an increased ability of E2F to promote the transcription of an E2F-responsive promotor element. Similarly, the HPV16 E7 protein interacts with Rb
and releases E2F, providing a new activity. In this way, Rb acts during Go or Glin the cell cycle to sequester transcription factor E2F so that it will not transcribe
a set of genes presumably required for entry into the S phase of the cell cycle. Rb
may regulate a number of cellular proteins in this fashion (Levine, A.J., Ann. Rev.
Biochem. 62:623-652 (1993)).
Accordingly, it would be desirable to have a method for ascertaining
whether or not a given cell, and particularly whether a given tumor cell, is
functionally inactivated in pS3 or Rb.
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Summary of fhe Invention
In view of the limitations discussed above, a general object of the
invention is to provide a method for selectively distributing a vector in vivo in a
tissue mass, and preferably in a tumor mass, such that a greater number of cellsare treated than would be treated with a non-replicating vector, and treatment is
avoided or significantly reduced in normal or non-tumor tissue.
Another object of the invention is to provide a method for identifying
turnor cells that contain a factor(s) that allows replication of a replication-
deficient vector or is deficient for a factor(s) that prevents replication of a
replication-deficient vector. A preferable object of the invention is to identify
tumor cells that do not contain functionally active pS3 or pS3 and Rb.
A third object of the invention is to provide producer cell lines for vector
production, and preferably for adenovirus vector production. Preferably, the cell
lines have one or more of the~llowing characteristics: high titer virus
production, resistance to toxic effects due to heterologous gene products
expressed in the vector, lack of production of wild-type virus cont~min~ting thevirus plcp~lion and resulting from recombination between cellular viral
sequences and vector sequences, growth to high density and in suspension,
~lnlimite(l passage potential, high growth rate, and by permitting the growth ofhighly deleted viruses that are impaired for viral functions and able to
accommodate large pieces of heterologous DNA.
Accordingly, the invention is generally directed to tissue-specific gene
therapy using a vector that selectively replicates only in a target tissue. The
invention is specifically directed to a method for distributing a polynucleotide in
a tissue in vivo, comprising introducing a replication deficient vector Cont~ining
the polynucleotide into the tissue, allowing the vector to replicate in cells of the
tissue, and in which cells the replication deficiency is complimented by one or
more endogenous, naturally-occurring factors that allow the replication of the
replication deficient vector in this cell or in which an endogenous, naturally-
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occurnng inhibitor of vector replication in functionally inactive in the cells,
thereby allowing vector replication to occur. Thus, the invention is also directed
to a method for distributing a vector in a target tissue and a method for
distributing a gene product produced from a gene on the vector in a target tissue.
In a plcf~ d embodiment, the tissue is abnormally proliferating, and
especi~lly is tumor tissue. However, the invention is also directed to other
abnormal tissue as described herein, as well as normal tissue.
In a ~CÇt;ll~ d embodiment, the replication-deficient vector is a DNA
tumor viral vector. In plcr~ d embo.lim~nt~, the DNA tumor viral vector is a
vector selected from the group con~i~ting of herpesvirus, papovavirus,
papillomavirus, and hepatitus virus vectors.
In a most l,lefelled embodiment, the vector is an adenovirus vector.
In a further preferred embodiment, the E1 region, and preferably the Ela
region, is deleted from the adenovirus.
In a further plcr~llcd en~iment of the invention, the cells are
functionally inactive for tumor ~u~lcs~ion gene products, and preferably, for at least p53, and more preferably, also for Rb.
In a further preferred embodiment, the cells contain functionally active
p53 and Rb, but also contain an endogenous cellular factor(s) that inactivate atleast p53 and preferably, also Rb. Such a function can arise from an endogenous
cellular element(s) resulting from natural virus infection, such as an integrated
provirus(es) that expresses proviral functions from the cellular genome that
inactivate the functions that interfere with or prevent viral replication.
In a further embodiment of the invention, the vector encodes a
heterologous gene product and expresses this heterologous gene product in the
cells of the target tissue.
In a further embodiment of the invention, the heterologous gene product
is toxic for cells in the target tissue.
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In a further embodiment of the invention, the heterologous gene product
acts on a non-toxic prodrug, converting the non-toxic prodrug into a form that is
toxic for cells in the target tissue. Preferably, the toxin has anti-tumor activity.
In a further embodiment of the invention, the vector is introduced into the
tissue by virus infection. In a i~lcr~llcd embodiment, the virus is an adenovirus.
Replication can be vector replication alone or can also include virus
replication (i.e., virion production). Thus, either DNA or virions or both may be
distributed in the target tissue.
In another embodiment of the invention, a method is provided for
assaying vector utility for tumor tre~tm~nt comprising the steps of removing a
tumor biopsy from a patient, explanting the biopsy into tissue culture, introducing
a replication-deficiont vector into the cells of the biopsy, and assaying for vector
replication in the cells. In a p~f~ d embodiment of the invention, the vector isan adenovirus vector.
In a further embodiment of the invention, a method is providing for
identifying a tumor, the cells of which contain a factor that allows replication of
a replication-deficient vector, or the deficiency of a factor that prevents
replication of a replication-deficient vector, by explanting a tumor biopsy,
introducing a replication deficient vector into the cells of the biopsy, and
qual~ lhlg vector DNA replication in the cells. Accordingly, a method is
provided for screening tumors for the presence of factors that allow replicationof a replication vector, or for a deficiency of a factor that prevents replication of
a replication-deficient vector. Such a screen is useful, among other things, foridentifying tumors prior to treatment which will be amenable to treatment with
a particular vector to be replicated in the tumor cell. In specific embodiments, the
tissue to be tested or screened is assayed for tumor-suppressor function, the
presence of which prevents vector replication, and the absence of which permits
vector replication. In specific disclosed embodiments, the tumor-suppressor
functions are the p53 and Rb functions.
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Therefore, in a further embodiment of the invention, a method is provided
for identifying a tumor with functionally inactivated pS3, Rb, or both p53 and Rb,
comprising explanting a tumor biopsy, introducing a vector that is unable to
replicate in the presence of p53, Rb, or both p53 and Rb, into cells of the biopsy,
S and qu~Li~ vector DNA replication in the cells. Testing or screening of
tissues or tumors, however, is not limited to an assay for viral DNA replicationbut may also include an assay for virus replication. Potentially, any tissue maybe screened for the functions described above by an assay for DNA or virus
replication.
In a plert;lled embodiment, the replication-deficient vector is a DNA
tumor viral vector. In ple~lled embo-lim~nt~, the DNA tumor viral vector is a
vector selected from, but not limited to, the group consisting of herpesvirus,
papovavirus, papillomavirus, and hepatitus virus vectors. In a most preferred
embodiment, the vector is an adenovirus vector. In a futther p,ere,-ed
embodiment, the E1 region, and preferably the Ela region, is deleted from the
adenovlrus.
In another embodiment of the invention, a cell line is provided col ll;~ i l .g
a virion produced in the cell by replication in the cell of a replication-defective
vector, and wherein the replication deficiency is complem~nted by an
endogenous, naturally-occ.nTing cellular function or by the deficiency of an
endogenous, naturally-occurring cellular function that inhibits vector replication.
In a preferred embodiment, the endogenous, naturally-occurring inhibitor of
vector replication that is functionally activated in the producer cell line is the
product of a tumor-suppressor gene. In a preferred embodiment, the endogenous,
naturally-occurring inhibitor of vector replication that is functionally inactivated
in the producer cell line is either pS3 or Rb, or both p53 and Rb, or functionalequivalents of p53 and/or Rb. The endogenous, naturally-occurring inhibitor of
vector replication can preexist in the producer cell. Alternatively, the function
can be inactivated by standard recombinant or other methods prior to introduction
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or replication of the vector in the cell. In further embodiments, the cell contains
multiple copies of the vector or virions.
In a preferred embodiment, the vector is a DNA tumor viral vector and
preferably is selected from the group, but is not limited to, herpesvirus,
S papovavirus, papillomavirus, and hepatitus virus. In a l,le~lled embodiment, the
virion is an adenoviral virion and the vector is an adenovirus vector.
In a further preferred embotlimP.nt, the E1 region, and preferably the Ela
region, is deleted from the adinovirus. In a further pl~rt;.led embodiment, the cell
is functionally inactivated for at least p53 and preferably also for Rb.
In further ~lefclled embodiments, the cell line is a small cell carcinoma
cell line. In further p~ ed embodiments of the invention, the cell line is a
hepatoma cell line. Most preferably, the hepatoma cell line is the Hep3B cell
line.
In a further preferred embodiment, the vector encodes a heterologous gene
product. The heterologous gene product may be a directly toxic drug, or a
product that metabolizes a prodrug into a directly toxic drug. Alternatively, the
gene product could be a beneficial gene product to be produced in large amounts
for further therapeutic application. Further, the producer cell lines are used to
produce vectors per se, or virions, for further use in gene therapy.
In a further embodiment of the invention, a method is provided for
producing a replication-deficient vector or virion, comprising the steps of
culturing the cell line described above and recovering the vector or virion fromthe cell. In preferred embodiments, the cell is a tumor cell. In a still furtherembodiment, a method is provided for producing replication-deficient DNA
tumor virion free of wild-type virions, or DNA tumor viral vector free of wild-
type tumor viral vectors, comprising the steps of culturing the producer cell line
~ described above and recovering replication-deficient virions or vectors from the
cell.
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Brief Description of fhe Figures
Figure 1: Expression of thymidine kinase activity in AV.TK1 (for construction,
see Example 1) infected AVl replication permissive HuH7 cells.
Figure 2: Replese~ e Southern blot demonstrating cell line specific DNA
replicationofAV1 vector.
Figure 3: Southem blot demonslld~ g that cell lines which support replication
of AV 1 vectors do not contain endogenous E1 genes.
Figure 4: Replesel~ e plaque titer demonstrating cell line specific virus
replicationofAVl vector.
Figure 5: Complete table sllmm~n7ing viral cytotoxicity data, DNA replication,
and virus production in a variety of tumor cell lines and normal cells.
Figure 6: Table showing the Rb/pS3 status of selected cell lines.
Figure 7: Multiplicity of infection vs. cell killing of HuH7.
Figure 8: Ganciclovir mediated bystander effect in HuH7 cells.
Figure 9: Carcinoma cell line specific killing.
Figure 10: Cure rate of nude mice bearing Hep 3B subcutaneous hepatoma
treated with AVl.TK1.
Figure 11: pS3 dependent ablation of AVI DNA replication in HuH7 cells.
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Figure 12: Survival curve for Hep3B tumor-bearing animals treated with various
adenovirus vectors.
Det~ile~ Description of the Preferred Embodimenfs
Def ni~ions
S The term "distributing" is int~n~led to mean the spreading of a vector and
its ~ttentl~nt heterologous gene (product) (when present on the vector) throughout
a target tissue, but especially throughout a tissue mass such as a mass of
~hnnrm~lly proliferating tissue, a non-m~lign~nt or m~lign~nt tissue. The objectof the distribution is to deliver the gene product or the effects of the gene product
(as by a bystander effect, for example) to suba~ 11y all or a significant numberof cells of the target tissue, so as to treat the entire target tissue.
The term "gene product" is int.on-led to mean DNA, RNA, protein,
peptides, or viral particles. Thus, the distribution, for the purposes of the
invention, is of any of these components.
The term "heterologous gene product" is inten~ed to mean a gene product
encoded by a gene not found in the native viral genome.
The terms "replication deficient", "replication minus", or "replication
defective" are used interchangeably. These terms are intended to mean a vector
or virus having a genetic deficiency resulting in the inability of the virus to
replicate in the norrnal host cell.
The term "abnormally proliferating" is intended to mean a cell having a
higher mitotic index than its normally-functioning counterpart, such that there is
an abnormal accumulation of such cells.
- The term "anti-tumor activity" is intended to mean any activity which
inhibits, prevents, or destroys the growth of a tumor.
The term "functional inactivation" is intended to mean a genetic lesion
that prevents the normal activity of a gene product. Thus, functional inactivation
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could result from a mutation in the gene encoding the gene product. Such a
lesion includes insertions, deletions, and base changes. Alternatively, functional
inactivation may occur by the abnormal interaction of the normal gene product
with one or more other cellular gene products which bind to or otherwise preventthe functional activity of said gene product. Thus, the gene product may be a
protein produced from a normal gene but which cannot perform its ordinary and
normal function because of an interaction with a second factor.
The term "complemented" is int~n~le(l to mean the provision of a gene
product which provides the function of a gene product inactivated on the viral
genome.
The terms "endogenous, naturally-occuIring factor" or "endogenous,
naturally-occurring function" are intPnde~l to mean a gene product encoded by
native sequences in the cellular genome. Such factors are to be distinguished
from those engineçred by recombinant or laboratory methods (as, for example,
by the introduction and integration of viral sequences into a cell in the
laboratory). I.e., such sequences are a result of natural events and not as a result
of human engineering of the cell.
The term "functional equivalent" is intP.n-lecl to mean a gene product
arising from a dirre~ll~ gene, but having the same biological function; e.g., with
respect to p53, such a functional equivalent would interact with viral gene
products to achieve the same effect on cells and on viral replication as interaction
with E1 a does.
The disclosures of all patents, publications (including published patent
applications), and cl~t~b~e entries referred to in this specification are specifically
2S incorporated herein by reference in their entirety to the same extent as if each
such individual patent, publication, and database entry were specifically and
individually indicated to be incorporated by reference in its entirety.
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Treatmen~
The present invention first provides methods for selectively distributing
a polynucleotide in a given tissue in vivo, and particularly in tumor tissue,
significantly redllcing or avoiding distribution in non-target tissue. The
S polynucleotide is provided in a vector which is selectively distributed in a given
tissue, and particularly in a tumor tissue, re~ cing or avoiding distribution in non-
tumor tissue.
The present invention also provides m~tho~1~ for selectively t:~plessing a
gene product in a given tissue, and particularly in tumor tissue, avoiding or
significantly re(l~-cing t;~lei,~ion in non-target or non-tumor tissue. The
invention provides methods for distribution of the above-mentioned to a greater
number of target cells than would be reached using a non-replicating vector.
Cells in addition to those first exposed to the polynucleotide, vector, or gene
product, are thus potentially reached by the methods.
The methods are specifically directed to the introduction into a target
tissue of a normally replication-defective vector, which replicates in the cells of
the target tissue. In the target tissue, replication occurs because either the
replication deficiency is complem~nted by a cellular function that allows viral
replication, or there is a deficiency in a cellular function that normally prevents
or inhibits vector replication. The presence or absence of such functions provides
the selectivity that allows the treatment of a specific tissue with minimum effect
on the surrounding tissue(s).
In the target tissue, DNA replication alone may occur. Late viral
functions that result in packaging of vector DNA into virions may also occur.
The vector may be introduced into the target tissue as naked DNA or by
- means of encapsidation (as an infectious virus particle or virion). In the latter
case, the distribution is accomplished by successive infections of cells in the
tissue by the virus such that substantially all or a significant number of the
daughter cells are infected.
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In a fi~rther embodiment of the invention, the vector encodes a
heterologous gene product which is expressed from the vector in the tissue cells.
The heterologous gene product can be toxic for the cells in the targeted tissue or
confer another desired property.
A gene product produced by the vector can be distributed throughout the
tissue, because the vector itself is distributed throughout the tissue. Alternatively,
although the ~l,res~ion of the gene product may be localized, its effect may be
more far-reaching because of a bystander effect or the production of molecules
which have long-range effects such as chemokines. The gene product can be
RNA, such as antisense RNA or ribozyme, or protein. Exarnples of toxic
products include, but are not limited to, thymidine kinase in conjunction with
ganciclovir.
A wide range of toxic effects are possible. Toxic effects can be direct or
indirect. Indirect effects may result from the conversion of a prodrug into a
1.5 directly toxic drug. For example, Herpes simplex virus thymidine kinase
phosphorylates ganciclovir to produce the nucleotide toxin ganciclovir phosphate.
This compound functions as a chain terrnin~tor and DNA polymerase inhibitor,
and thus is cytotoxic. Another exarnple is the use of cytosine ~e~min~e to
convert 5 '-fluorocytosine to the anti-cancer drug 5 '-fluorouracil. For a discussion
of such "suicide" genes, see Blaese, R.M. et al., Eur. J. Cancer 30A: 1190-1193
(1994).
Direct toxins include, but are not limited to, diphtheria toxin (Brietman
et al., Mol. Cell Biol. 10:474-479 (1990)), pseudomonas toxin, cytokines
(Blankenstein, T., et al., J. Exp. Med. 1 73: 1047-1052 (1991), Colombo, M.P.,
et al., J. Exp. Med. 173:889-897 (1991), Leone, A., et al., Cell 65:25-35 (1991)),
~nti~çn~e RNAs and ribozymes (Zaia, J.A. et al., Ann. N. ~ Acad. Sci. 660:95- 106
(1992)), tumor vaccination genes, and DNA encoding for ribozymes.
In accordance with the present invention, the agent which is capable of
providing for the inhibition, prevention, or destruction of the growth of the target
tissue or tumor cells upon expression of such agent can be a negative selective
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marker; i.e., a material which in combination with a chemotherapeutic or
interaction agent inhibits, prevents or destroys the growth of the target cells.Thus, upon introduction to the cells of the negative selective marker, an
interaction agent is ~ ed to the host. The interaction agent interacts with
the negative selective marker to prevent, inhibit, or destroy the growth of the
target cells.
Negative selective m~rker~ which may be used include, but are not limited
to, thymidine kinase and cytosine de~min~ce. In one embodiment, the negative
selective marker is a viral thymidine kinase selected from the group con~i~tin~ of
0 Herpes simplex virus thymidine kinase, cytomegalovirus thymidine kinase, and
varicella-zoster virus thymidine kinase. When viral thymidine kinases are
employed, the interaction or chemotherapeutic agent preferably is a nucleoside
analogue, for example, one selected from the group con~i~ting of ganciclovir,
acyclovir, and 1-2-deoxy-2-fluoro-~-D-arabinofuranosil-5-iodouracil (FIAU).
Such intt-r~ction agents are utilized~iciently by the viral thymidine kinases assubstrates, and such interaction agents thus are incorporated lethally into the
DNA of the tumor cells expressing the viral thymidine kin~es, thereby resulting
in the death of the target cells.
When cytosine ~le~min~ce is the negative selective marker, a preferred
interaction agent is 5-fluorocytosine. Cytosine de~min~e converts
5-fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the target
cells which express the cytosine ~le~min~e gene convert the 5-fluorocytosine to
5-fluorouracil and are killed.
The interaction agent is ~riministered in an amount effective to inhibit,
prevent, or destroy the growth of the target cells. For example, the interactionagent is ~tlmini~tered in an amount based on body weight and on overall toxicity- to a patient. The interaction agent preferably is :~tlmini~tered systemically, such
as, for example, by intravenous ~mini.~tration, by l,~elllel~l ~tlmini~tration, by
hl~r~cl;loneal ~Amini~tration, or by intramuscular ~(lmini~tration.
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When the vectors of the present invention induce a negative selective
marker and are ~fimini~t~red to a tissue or tumor in vivo, a "bystander effect" may
result, i.e., cells which were not originally tr~n~duced with the nucleic acid
sequence encoding the negative selective marker may be killed upon
S iq-lnnini~tration of the interaction agent. Although the scope of the present
invention is not int~n(led to be limited by any theoretical reasoning, the
tr~n~d~lced cells may be producing a diffusible forrn of the negative selective
marker that either acts extracellularly upon the interaction agent, or is taken up
by ~ cent~ non-target cells, which then become susceptible to the action of the
interaction agent. It also is possible that one or both of the negative selective
marker and the interaction agent are communicated between target cells.
In one embodiment, the agent which provides for the inhibition,
prevention, or destruction of the growth of the tumor cells is a cytokine. In one
embodiment, the cytokine is an interleukin. Other cytokines which may be
employed include hllelr~lulls and e~r}y-stimulating factors, such as NM-CSF.
Interleukins include, but are not limited to, interleukin-l, interleukin-l,~,
interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6,
interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, andinterleukin-12. In one embodiment, the interleukin is interleukin-2.
In a preferred embodiment ofthe invention, the target tissue is abnorrnally
proliferating, and preferably tumor tissue. The vector or virus is distributed
throughout the tissue or tumor mass.
All turnors are potentially amenable to treatment with the methods of the
invention. Tumor types include, but are not limited to hematopoietic, pancreatic,
neurologic, hepatic, gastrointestin~l tract, endocrine, biliary tract. sino-
pulmonary, head and neck, soft tissue sarcoma and carcinoma, dermatologic,
reproductive tract, and the like. Preferred tumors for treatrnent are those with a
high mitotic index relative to normal tissue. Preferred tumors are solid tumors,and especially, tumors of the brain, most preferably glioma.
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The methods can also be used to target other abnormal cells, for example,
cytomegalovirus, Epstein-Barr virus, and human papilloma virus-infected cells.
Cytomegalovirus infects a variety of cells, causing many dirrel~lll types of
disease (Virology, second edition, Fields, B.N. et al., eds., Raven Press Ltd.
(1990)). Cytom~ogalovirus inactivates the p53 gene (Ohno, T., Science 265:781-
784 (1994); Speir et al., Science 265:391-394 (1994)), leading to enhanced
proliferation of cells and in some cases detrimental affects. Restenosis is one
example of an ablloll,lal cell pathology treatable by the methods of the invention.
Following angioplasty, the smooth muscle cells are prone to cytomegalovirus
infection or reactivation (Ohno, T., Science 265:781-784 (1994); Speir et al.,
Science 265:391-394 (1994)). Thus, the cells proliferate and cause serious
blockage of the artery in which angioplasty was performed. These cells could be
selectively killed by the methods of the invention, since pS3 is inactivated in
those cells. In addition, other intraocular lesions develop as a result of
cytomegalovirus infection or reactivation. These cells could be selectively
elimin~t~d by the methods described herein. Finally, Epstein-Barr virus causes
a variety of fii~e~ces, including Burkitt's lymphoma. Cells cont~ining this virus,
or cells in which this virus has inactivated the pS3 gene, can be targeted. Cells
cont~ining human papilloma virus (85% of all cervical carcinomas, and several
other tumors of the head and neck as well as many papillomas) are also known
to elimin~te pS3, and could be treated with the methods described herein.
In a further embodiment of the invention, the vectors of the present
invention can be used to replicate in specific subsets of normal cells. For
exarnple, in certain stages of embryogenesis, factors complimenting replication
deficiency are known to occur. Vector replication can be exploited in such cell
types for the purpose of directed gene t;xples~ion in these cell types. Therapeutic
- interventions are achievable in normal tissues if similar conditions favoring DNA
replication are produced by the pharmacologic manipulations.
Further, treatment can be ex vivo. Ex vivo transduction of tumor cells
would overcome many of the problems with current viral delivery systems.
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Tissue is harvested under sterile conditions, dissociated mechanically and/or
enzym~ti~lly and cultured under sterile conditions in applopliate media. Vector
pl~ ions demonstrated to be free of endotoxins and bacterial cont~min~tion
are used to tr~n.cdllce cells under sterile conditions in vitro using standard
protocols. The accessibility of virus to cells in culture is currently superior to in
vivo injection and permits introduction of vector viral sequences into essentially
all cells. Following removal of virus-co..~ i"g media cells are immediately
returned to the patient or are m~int~in~d for several days in culture while testing
for function or sterility is performed.
For example, patients with hypercholesterolemia have been treated
successfully by removing portions of the liver, explanting the hepatocytes in
culture, genetically modifying them by exposure to retrovirus, and re-infusing the
corrected cells into the liver (Grossman et al., 1994).
Viral transduction also has potential applications in the area of
~ nt:~l medicine. Transient expression of biological modifiers of immune
system function such as IL-2, Ifn-g~mm~, GM-CSF or the B7 co-stimulatory
protein has been proposed as a potential means of inducing anti-tumor responses
in cancer patients.
In a further preferred embodiment, the cellular function that prevents virus
replication, and in the absence of which function, virus replication occurs, is a
turnor-suppressor gene function.
In a further preferred embodiment of the invention, the tumor cells in
which the vector or gene product is distributed lack at least functionally active
pS3 and preferably also lack Rb. The invention also encompasses, however, cells
lacking other tumor suppressor functions, such as plO7, pl30, and p300. For
known tumor suppressor genes, see Levine, A.J., Ann. ~el~. Biochem. 62:623-651
(1993).
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Producer Cells
In a further embodiment of the invention, a cell is provided which
contains a virion produced in the cell by replication in the cell of a replication-
defective vector, wherein the replication deficiency is comp~ e~l by an
S endogenous, naturally-occurring cellular function or replication occurs because
of a deficiency in a function that inhibits vector replication. Thus, the invention
provides "producer cells" for the efficient and safe production of recombinant
replication-defective vectors for further use for gene therapy in vivo.
One of the major problems with the l;ullelllly available producer cells is
that such cells contain, in the genome, viral sequences that provide
complementing functions for the replicating vector. Because the cell contains
such sequences, homologous recombination can occur between the viral sequence
in the genome and the viral vector sequences. Such recombination can regenerate
recombinant wild-type viruses which co~ le the vector or virus ~ ~dlion
produced in the producer cell. Such co.. l;.. i.~tion is undesirable, as the wild-
type viruses or vectors can then replicate in non-target tissue and thereby impair
or kill non-target cells. Therefore, one of the primary advantages of the producer
cells of the present invention is that they do not contain endogenous viral
sequences homologous to sequences found in the vector to be replicated in the
cells. The absence of such sequences avoids homologous recombination and the
production of wild-type viral recombinants that can affect non-target tissue.
Accordingly, the invention embodies methods for constructing and
producing replication-deficient virions in a cell comprising introducing a
replication-deficient vector into the cell wherein the genome of the cell is devoid
of vector sequences, replicating the vector in the cell wherein the replication-- deficiency is compensated by a naturally-occ~lrring endogenous cellular function
or deficiency in a function, forming the virion, and purifying the virion from ~e
cell. In preferred embodiments of the invention, the endogenous cellular function
is provided by tumor-suppressor genes. In more preferable embodiments, the
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tumor-~ul,l,~ssor functions are the p53 and Rb tumor-suppressor functions.
Preferred vectors are DNA viral vectors, including but not limited to herpesvirus,
papillomavirus, hepatitus virus, and papovavirus vectors. In prefelled
embo-limçrlS~ of the invention, the virion is an adenoviral virion and the vector
S is an adenoviral vector. In further embo-liment~ of the invention, the cell is a
tumor cell which is functionally inactive at least for p53 and preferably also for
Rb. In a specific disclosed embor~im~nt, the tumor cell is Hep3b.
In a further preferred embo-limlont, the vector encodes a heterologous gene
product such that the virion also encodes the gene product, and when the vector
or virion are used for gene therapy, the therapy is f~ilit~ted by eA~i~ ssion of the
heterologous gene product. Alternatively, the producer cell can be used for the
production of a heterologous gene productper se encoded by the vector. When
the vector replicates in the producer cell, the gene product is expressed from the
multiple copies ofthe gene encoding the gene product. Following ~A~ ssion, the
gene product can be purified from the producer cells by conventional lysis
procedures, or secreted from the producer cell by a~plo~l;ate secretion signals
linked to the heterologous gene by known methods. The tr~n~ rtion of cells by
adenoviral vectors has been described. Transfection of plasmid DNA into cells
by calcium phosphate (~n~h~n, D., J. Mol. Biol. 166:577 (1983)), lipofection
(Feigner et al., PNAS 84:7413 (1987)), or electroporation (Seed, B., Nature
329:840 ( )) has been described. DNA, RNA, and virus purification procedures
are described (Graham et al., J. Gen. Virol. 36:59-72 (1977).
Preferred producer cells are at least p53 and preferably also Rb-minus.
Primary tumors from which cell lines can be derived, or existing cell lines, canbe tested for this phenotype. Alternatively, any of the cell lines previously shown
to contain Rb and p53 mutations can be used. Examples of primary tumors that
could be explanted and developed into producer cells for the vectors of the
present invention include small cell lung carcinoma, cervical carcinoma, marly
tumors of the brain, prostate tumors, colon tumors, lung tumors, sarcomas, and
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pancreatic tumors. Cell lines bearing these mutations are also known in the
literature and provide candidates for producer cells.
Alternatively, any cell can be chosen whether or not the phenotype is p53
and/or Rb-minus. The cell that is selected on the basis of other desirable
char~ct~rictics can be manipulated such that the endogenous p53 and/or Rb
function is reduced or elimin~tPfl For example, a mutation in either or both of
these genes can be introduced by homologous recombination with exogenous
mutated DNA. Alternatively, ~nti~Pn~e RNA may be used to block p53/Rb
expression. Further, functional protein antagonists (that bind and thus inactivate
either or both of the proteins) may be introduced into such producer cells as byadditional exogenous c~lession vectors. Such recombinant manipulations for
inactivating gene function are well-known to those of oldin~y skill in the art.
The llltim~te goal for a producer cell line, and particularly an adenoviral
producer line, is to produce the highest yield of vector with the least possibility
of co~ tion by wild-type vector. Yield depends upon the number of cells
infected. Thus, the more cells that it is possible to grow and infect, the more
virus it is possible to generate. Accordingly, candidate cells would have a highgrowth rate and will grow to a high density. The cell should also have a high
arnount of viral receptor so that the virus can easily infect the cell. Another
characteristic is the quality of the vector produced (i.e., the ~lc~udlion should not
include a high amount of non-infectious viral particles). Accordingly, candidateproducer cells would have a low particle-to-plaque-forrning-unit ratio. For
example, small cell lung carcinoma in general is known to grow well in
suspension culture and is one of the fastest growing cancers known (Gazden, A.F.et al., Cancer Res. 40:3502 (1980)). In addition, lung cells in general have a high
propensit~v for adenoviral infection (Rosenfeld, M.A. ef al., Cell 68:143 (1992),
and the majority studied to date (Horowitz et al., PNAS 87:2775 (1990)) have Rb
and pS3 mutations. Thus, these cells are a preferred cell type for deriving a
producer cell line. Primary explants or the known cell lines can be used.
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Further, the p53 and/or Rb in any cell may be mutated or functionally
inactivated by homologous recombination with mutated genes (Williarns, B.D.
et al., Nature: Genetics 7:440 (1994)), or by transduction of that cell line with
genes that will inactivate these gene products (Yei et al., Nature 357:82-85
(1992), Crook et al., Cell 67:547 (1991), and DeCaprio et al., Cell 54:275
(1988)). Thus, such obtainable cells can serve as producer cells for recombinantreplication-deficient vectors, viruses, and gene products.
Dia~,~ osti~
The ability to have a method for detPrmining the loss of function of
tumor-~up~,lessor genes is hllpol~ll for ~le~ . ",i~ the stage, prognosis, and
potential treatment of patients with tumors. In particular, a method is most
desirable for detPrmining the loss of function of the tumor-~u~ssor genes p53
and Rb. The p53 and Rb status is known to play a major role in tumor
progression. Further, it is important to know whether current vectors will
replicate in a given patient's tumor. If replication is found to be beneficial in
therapy, then a screen is provided for those patients who may best respond to this
therapy. If it is found to be harmful, then there is a screen for prevention of the
treatment of patients who would have an adverse response to the tre~tment
Currently, the only non-biological assays that are commonly used are expression
screening, PCR, and seqllPn~ing These often result in false negatives, are time-con~llrning expensive, and yield only information in the best of cases about thestatus of the genes and not their biological function.
In a further embodiment of the invention, therefore, a method is provided
for assaying vector utility in tumor treatment by removing a tumor biopsy from
a patient, explanting the biopsy into tissue culture, introducing a replication-deficient vector into the biopsy, and assaying vector replication in the cells of the
biopsy. In pler~led embodiments of the invention, the vector is an adenovirus
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vector. Other vectors, however, may be used in the invention, as discussed
herein.
Thus, the invention provides a method for screening a primary tumor for
complementation functions that allow vector replication in the presence of turnor-
~u~rcssor genes and especially p53 and Rb, or for the absence of functions and
especially tumor-~u~ressor funetions which prevent the replieation of a virus
veetor. The d~ ion, therefore, is done by removing a tumor biopsy from
the tumor, explanting the biopsy, introdueing a replication-deficient vector into
cells of the biopsy, and assaying veetor replieation in the eells. In partieular, a
method is provided for sclce~ lg a tumor for functionally inaetivated pS3 and Rbby means of assaying replieation in explanted tumor biopsies. It should be
appreeiated, however, that sueh diagnostie methods are not useful only for
screel~illg turnors, but for sereening any abnormal or normal tissue in whieh sueh
complementation funetions may be present, or in whieh ~U~ lCSso~ funetions may
be absent.
Intro~r~ion of Vecfors into Cells
A variety of ways have been developed to introduce vectors into cells in
culture, and into eells and tissues of an animal or a human patient. Methods forintroducing vectors into m~mm~ n and other animal cells include calcium
phosphate transfection, the DEAE-dextran technique, microinjection, liposome
mediated techniques, cationic lipid-based techniques, transfection using
polybrene, protoplast fusion techniques, electroporation and others. These
techniques are well known to those of skill, are described in many readily
available publications and have been extensively reviewed. Some of the
techniques are reviewed in Transcription and Translation, ~ Practical Approach,
Hames, B.D. and Higgins, S.J., eds., IRL Press, Oxford (1984), herein
incorporated by reference in its entirety, and Molecular Cloning, Second Edition,
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Maniatis et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New
York (1989), herein incorporated by reference in its entirety.
Several of these techniques have been used to introduce vectors into
tissues and cells in ~nim~1~ and human patients. Chief among these have been
S systemic ~lmini~tration and direct injection into sites in situ. Depending on the
route of ~ ion and the vector, the techniques have been used to introduce
naked DNA, DNA complexed with cationic lipid, viral vectors and vector
producer cell lines into normal and abnorrn~l cells and tissues, generally by direct
injection into a targeted site.
The aforementioned techniques for introducing polynucleotide, viral and
other vectors into cells in culture, in ~nim~l~ and in patients can be used to
develop, test and produce, as well as use vectors in acco,d~ce with the invention.
For in~t~ncç7 cells co..~ i..g a vector introduced by these methods can be used
for producing the vector. In addition, cells co.~l~i"in~ a vector can be used asproducer-cells and introduced into cells or tissues of an animal to produce the
vector in sit2l.
Assay of DNA and Viral Repli~ ntio~.
Replication of a polynucleotide, viral or other vector can be assayed by
well-known techniques. Assays for replication of a vector in a cell generally
involve detecting a polynucleotide, virions or infective virus. A variety of well-
known methods that can be used for this purpose involve de~errnining the amount
of a labelled substrate incorporated into a polynucleotide during a given periodin a cell.
When replication involves a DNA polynucleotide, 3H-thymidine often is
used as the labelled substrate. In this case, the amount of replication is
detçrrnined by separating DNA of the vector from the bulk of cellular DNA and
measuring the amount of tritium incorporate specifically into vector DNA.
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Replication of a polynucleotide vector also may be detecte-l by Iysing or
p~nn~ting cells to release the polynucleotide, then isolating the polynucleotideand quan~ g directly the DNA or RNA that is recovered. Polynucleotide
replication also may be detected by quan~i~live PCR using primers that are
specific for the assay polynucleotide.
Virions may be assayed by EM counting techniques well known to the art,
by isolating the virions and clt;l~ l;..g protein and nucleic acid content, and by
labelling viral genomic polynucleotides or virion proteins and ~let~rmining the
amount of virion from the amount of polynucleotide or protein.
It is well known that virions may not all be viable and where infectivity
is hlllJoll~l~, infectious titer may be ~etennine~ by cytopathic effect or plaque
assay.
Any of these well-known techniques, among others, can be employed to
assay replication of a vector in a cell or tissue in accordance with the invention.
It will be appreciated that different techniques will be better suited to some
vectors than others and to some cells or tissues than others.
Vectors
The plefelled vectors of the present invention are adenoviral vectors.
Adenovirus is preferred, among other things, because it rarely integrates into the
host genome, and, therefore, if used to express a beneficial heterologous gene, it
will not cause host genetic mutation. In a ~lel~lled embodiment of the invention,
an adenovirus vector is deleted in the El region.
The adenovirus Elb 55 kDa protein functions both in the disruption of
pS3-mediated apoptosis (for review, Moran, E., FASEB J. 7:880-885 (1933)) and
- 25 in cooperative interactions with Ad E4 34 kDa protein to facilitate accumulation
of late viral mRNAs (Ornelles & Shenk, J: Virol. 65:425-439 (1991). El -deleted
adenovirus DNA replication which occurs in pS3 negative (dysfunctional (e.g.,
inactivated by other cellular functions) or deleted p53) cells is only rarely
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accompanied by production of infectious virus (Figure 5). This phenomenon may
be related to the absence of Elb 55 kDa-E4 34 kDa interaction.
Addl 312 (Jones & Shenk, Cell 1 7:683-689 (1979)) is deleted for E1 a but
retains a functional Elb transcription unit as well as the wild-type E4 region.
Mutagenesis ofthe Elb region in Addl312, followed by selection on cells known
to be deleted or dysfunctional for the genes encoding p53 (such as C-33, a
cervical carcinoma cell line) should allow selection of viral Elb 55 kDa mutantsretaining ~ossenti~l interactions with E4 34 kDa but lacking p53 interaction
capabilities.
Therefore, in a further preferred embodiment, the methods use a vector
that retains its ability to replicate DNA in p53 defective cells and furthermoredisplays increased virion production by virtue of the more efficient proces~ing
and transport of late viral RNAs, the step at which Ela-Elb deleted vector virion
production in p53 negative cells appears blocked. Vectors of this class are
capable of spreading among p53 negative (deleted and/or dysfunctional) cells as
replicating virions, though replication of either vector viral DNA or virions would
remain impossible or greatly reduced in cells with normal p53 function.
In one embodiment, both Ela and Elb are deleted. In an alternative
embodiment, only the E I a region is deleted from the El region; the Elb region
remains intact. In the latter case, a cellular compl~ment~tion function may allow
virus replication to proceed by means of a factor that compensates for Ela
deficiency and the viral Elb region allows cellular processing and transport
functions to be utilized for late virus product and virus p~cl~ging and release.This Elb function is particularly desirable in cells that are p53-minus (i.e., that
do not contain a functionally active p53). Further, either one or both of the E 1 b
functions may be included or deleted from the Ela vector.
In a specific disclosed embodiment, a vector for use in the methods of the
invention is the AVl.TKl vector. For a detailed description of vector
construction, see Example 1 herein. The generation of AVl .TKl is identical to
the generation of AV l .LacZ4, except that the thymidine kinase gene is inserted
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in place of the LacZ gene. This (as well as AVl .LacZ4) vector is constructed byoperably linking the Herpes simplex virus type 1 thymidine kinase gene (or the
~-gal gene co. .~ -g a nuclear loc~li7ing signal (GenBank J01636)) to the Rous
Sarcoma Virus promotor on pAVS6, an adenoviral shuttle plasmid co..l 1irling theleft ITR of Ad5 and another region homologous to AdS (Trapnell, B. et al., Adv.
Drug Deliv. Rev 12:185-189 (1994)). After linP~ri7~tion of the plasmid, it is
co~ rected into 293 cells ~,vith a replication-inco~ elll subgenomic fragment
of AdS that has the E3 region deleted (Addl327) (Yei, S. et al., Human Gene
Therapy 5:731-744 (1994)). Upon homologous recombination, a recombinant
virus is generated. Virus is then plaque-purified on 293 cells, and purified as
previously described (Graham, F.L. et al., J. Gen. Virol. 36:59-72 (1977)).
Addl312 is an Ela deletion mutant generated as previously described (Shenk
et al., Cold Spring Harbor Symp. Quant. Biol. 44:367-375 ( )).
In alternative embodiments, adenovirus vectors are provided with
deletions in any of the other genes ese~nti~l for replication, such as E2-E4.
In further alternative embodiments, the vector is derived from another
DNA tumor virus. Such viruses generally include, but are not limited to the
herpesviruses (such as Epstein-Barr virus, cytomegalovirus, Herpes zoster, and
Herpes simplex), papillomaviruses, papovaviruses (such as polyoma and SV40),
and hepatitis viruses. The alternative viruses preferably are selected from the
group of viruses that inactivate Rb or p53. All serotypes are included. The
comrnon IJlU~ y is a viral gene that inactivates a cellular function(s) that
prevents DNA replication or promotes apoptosis. Most preferably, the viral
gene(s) perforrn a subst~nti~lly identical function to the Ela and Elb genes found
in adenovirus and its serotypes. Examples of genes include, but are not limited
to, the E6 and E7 regions of human papilloma virus, 16 and 18 T antigen of
SV40, and CMV immediate early genes. Any of the viruses which infect cells,
and particularly human cells, could have either specific portions or the entire E1
region counterpart deleted and thus be used in all the methods described herein,in which methods an adenovirus deletion mutant could be used.
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The invention further embodies the use of plasmids and vectors having
only the essential regions of adenovirus needed for replication with either Ela,Elb 19kDa gene, or Elb 55kDa gene, or some combination thereof, deleted.
Such a plasmid, lacking any structural genes, would be able to undergo DNA
replication as the E1-deleted adenovirus vectors do.
The vectors described herein can be constructed using standard molecular
biological techniques. Standard techniques for the construction of such vectors
are well-known to those of ordinary skill in the art, and can be found in references
such as Sambrook et al., in Molecular Cloning: ~ Laboratory Manual, Cold
Spring Harbor, New York (1989), or any of the myriad of laboratory m~nu~l~ on
recombinant DNA technology that are widely available. A variety of strategies
are available for ligating fragm~ntc of DNA, the choice of which depends on the
nature of the termini of the DNA i~gm~nt~ and can be readily ~ d by the
skilled artisan.
The adenoviral vector may be a modified adenoviral vector in which at
least a portion of the adenoviral genome has been deleted or mutated so that a
functional viral product is not produced (Shenk et al., Curr. Top. Microbiol.
Immunol. 111(3): 1-39 (1984)).
In one embodiment, the vector comprises an adenoviral 5 ITR; an
adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence
containing a heterologous gene. The vector is free of at least the majority of
adenoviral El and E3 DNA sequences, but is not necessarily free of all of the E2and E4 DNA sequences, and DNA sequences encoding adenoviral proteins
promoted by the adenoviral major late promoter.
In still another embodiment, the gene in the E2a region that encodes the
72 kilodalton binding protein is mutated to produce a temperature sensitive
protein that is active at 32~C, the temperature at which viral particles are
produced but is inactive at 37~C, the temperature of the animal or human host.
This telllp~ re sensitive mutant is described in Ensinger et al., J. Virology
10:328-339 (1972); Van der Vliet et al., J. Virology 15:348-354 (1975); and
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Friefeld et aL, T~irology 124:380-389 (1983); Englehardt et al., Proc. Nat. Acad.
Sci 91:6196-6200 (1994); Yang et al., Nature: Genetics 7:362-369 (1994).
Such a vector, in a plefelled embodiment, is constructed first by
;ling, according to standard techniques, a shuttle plasmid which contains,
begirming at the 5' end, the "critical left end elements," which include an
adenoviral 5' ITR, an adenoviral enr~psid~tion signal, and an Ela enh~n~er
sequence; a promoter (which may be an adenoviral promoter or a foreign
promoter); a tripartite leader sequence, a multiple cloning site (which may be as
herein described); a poly A signal; and a DNA segment which corresponds to a
segment of the adenoviral genome. Such DNA segm~nt serves as a substrate for
homologous recombination with a modified or mllt~tçd adenovirus. The plasmid
may also include a selectable marker and an origin of replication. The origin ofreplication may be a bacterial origin of replication. Representative examples ofsuch shuttle plasmids include pAVS6, as discussed herein and see Trapnell, B.
e~ al., Adv. Drug Deliv. Rev 12:185-189 (1994). A desired DNA sequence
Co~ g a heterologous gene may then be inserted into the multiple cloning site
to produce a plasmid vector.
This construct then is used to produce an adenoviral vector. Homologous
recombination then is effected with a modified or mutated adenovirus in which
at least the majority of the E1 and E3 adenoviral DNA sequences have been
deleted. Such homologous recombination may be effected through co-
transfection of the plasmid vector and the modified adenovirus into a helper cell
line by CaPO4 precipitatlon.
Through such homologous recombination, a vector is formed which
includes an adenoviral 5' ITR, an adenoviral encapsidation signal; an Ela
enhancer sequence; a promoter; a tripartite leader sequence; a DNA sequence
containing the heterologous gene; a poly A signal; adenoviral DNA free of at
least the majority of the E1 and E3 adenoviral DNA sequences; and an adenoviral
3' ITR. This vector may then be transfected into a helper cell line for viral
replication and to generate infectious viral particles. Transfections may take
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place by electroporation, calcium phosphate precipitation, microinjection, or
through proteoliposomes.
The vector may include a multiple cloning site to facilitate the insertion
of DNA sequence(s) co..l~;..ing the heterologous gene into the cloning vector.
In general, the multiple cloning site includes "rare" restriction enzyme sites; i.e.,
sites which are found in eukaryotic genes at a frequency of from about one in
every 10,000 to about one in every 100,000 base pairs. An a~rol.l;ate vector is
thus formed by cutting the cloning vector by standard techniques at applopl;ate
restriction sites in the multiple cloning site, and then ligating the DNA sequence
co.. ~ g the heterologous gene into the cloning vector.
The DNA sequence encoding the heterologous gene product is under the
control of a suitable promoter. Suitable promoters which may be employed
include, but are not limited to, adenoviral promoters, such as the adenoviral major
late promoter; or heterologous promoters, such as the cytomegalovirus (CMV)
promoter; the Rous Sarcoma Virus promoter; inducible promoters, such as the
MMTV promoter, the metallothionein promoter; heat shock promoters; the
albumin promoter; the ApoE promoter; and the ApoAI promoter. It is to be
understood, however, the scope of the present invention is not limited to specific
fore1gn genes or promoters.
In one embodiment, the adenovirus may be constructed by using a yeast
artificial chromosome (or YAC) cont~ining an adenoviral genome according to
the method described in Ketner, et al., Proc. Nat. ,4cad. Sci 91:6186-6190
(1994), in conjunction with the ~e~ching~ contained herein. In this embodiment,
the adenovirus yeast artificial chromosome is produced by homologous
recombination in vivo between adenoviral DNA and yeast artificial chromosome
plasmid vectors carrying segments of the adenoviral left and right genomic
termini. -A DNA sequence cont~ining the heterologous gene then may be cloned
into the adenoviral DNA. The modified adenoviral genome then is excised from
the adenovirus yeast artificial chromosome in order to be used to generate
infectious adenoviral particles.
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The infectious viral particles may then be ~1mini~tered in vivo to a host.
The host may be an anirnal host, including m~mm~ n, non-human primate, and
human hosts.
The viral particles may be a~lminictçred in combination with a
ph~ elltically acceptable carrier suitable for ~lminictration to a patient. The
carrier may be a liquid carrier (for example, a saline solution), or a solid carrier,
such as, for example, microcarrier beads.
Having ~us described herein the invention in general terms, the following
exarnples are presented to illustrate the invention.
0 Example I
Construction of pA VS6
The adenoviral construction shuttle plasmid pAVS6 was constructed in
several steps using standard cloning techniques including polymerase chain
reaction based cloning techniques. First, the 2913 bp BglII, HindIII fragment was
removed from Addl327 and inserted as a blunt fragment into the XhoI site of
pBluescript II KS (Stratagene, La Jolla, CA). Addl 327 (Thimm~ppaya et al.,
Cell 31:543-551 (1983), incorporated herein by reference) is identical to
Adenovirus 5 except that an XbaI fragment including bases 28591 to 30474 (or
map units 78.5 to 84.7) of the Adenovirus 5 genome, and which is located in the
E3 region, has been deleted. The complete Adenovirus 5 genome is registered
as Genbank accession #M73260, incorporated herein by reference, and the virus
is available from the American Type Culture Collection, Rockville, Maryland,
U.S.A. under accession number VR-5.
Addl327 was constructed by routine methods from Adenovirus S (AdS).
The method is outlined briefly as follows an previously described by Jones and
Shenk, Cell 13:181-188, (1978). Ad5 DNA is isolated by proteolytic digestion
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of the virion and partially cleaved with XbaI restriction endonuclease. The XbaIfragments are then re~c~emhled by ligation as a mixture of fr~gment~. This
results in some ligated genomes with a sequence similar to Ad5, except excludingsequences 28591 bp to 30474 bp. This DNA is then transfected into suitable cells(e.g. KB cells, HeLa cells, 293 cells) and overlaid with soft agar to allow plaque
formation. Individual plaques are then isolated, amplified, and screened for theabsence of the 1878 bp E3 region XbaI fr~gm.ont
The orientation of this fragment was such that the BglII site was nearest
the T7 RNA polymerase site of pKSII and the HindIII site was nearest the T3
RNA polymerase site of Pbluescript II KS. This plasmid was ~lecign~tetl pHR.
Second, the ITR, en~p~ tion signal, Rous Sarcoma Virus promoter, the
adenoviral tripartite leader (TPL) sequence and linking sequences were assembledas a block using PCR amplification. The ITR and encapsidation signal
(sequences 1-392 of Addl327 [identical to sequences from AdS, Genbank
accession #M73260], incorporated herein by reference) were amplified
(amplification 1) together from Addl327 using primers cont~inin~ NotI or AscI
restriction sites. The Rous Sarcoma Virus LTR promoter was arnplified
(amplification 2) from the plasmid pRC/Rous sarcoma virus (sequences 209 to
605; Invitrogen, San Diego, CA) using primers co~ ;"ing an AscI site and an
SfiI site. DNA products from amplifications 1 and 2 were joined using the
"overlap" PCR method (amplification 3) (Horton et aL, Biotechniques 8:528-535
(1990)) with only the NotI primer and the SfiI primer. Complementarily between
the AscI cont~ining end of each initial DNA amplification product from reactions1 and 2 allowed joining of these two pieces during amplification. Next the TPL
was amplified (arnplification 4) (sequences 6049 to 9730 of Addl327 [identical
to similar sequences from Ad5, Genbank accession #M73260]) from cDNA made
from mRNA isolated from 293 cells (ATCC accession No. CRL 1573) infected
for 16 hrs. with Addl327 using primers cont~inin~ SfiI and XbaI sites
respectively. DNA fragments from amplification reactions 3 and 4 were then
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joined using PCR (amplification 5) with the NotI and XbaI primers, thus creatingthe complete gene block.
Third, the ITR-encapsidation signal-TPL fragment was then purified,
cleaved with NotI and XbaI and inserted into the NotI, XbaI cleaved PHR
plasmid. This plasmid was ~l~si~n~ted pAvS6A and the orientation was such that
the NotI site of the fragment was next to the T7 RNA polymerase site.
Four~, the SV40 early polyA signal was removed from SV40 DNA as an
HpaI-BamHI fragmPnt, treated with T4 DNA polymerase and inserted into the
SalI site of the plasmid pAVS6a to create pAVS6.
0 Construction of AvlLacZ4
The recombinant, replication-deficient adenoviral vector AvlLacZ4,
which ~AI,lesses a nuclear-targetable B-galactosidase enzyme, was constructed
in two steps. First, a transcripti~-~nit con~i~ting of DNA encoding amino
acids 1 through 4 of the SV40 T-antigen followed by DNA encoding amino acids
127 through 147 of the SV40 T-antigen (co~ ,g the nuclear targeting peptide
Pro-Lys-Lys-Lys-Arg-Lys-Val), followed by DNA encoding amino acids 6
through 1021 of E. coli B-galactosidase, was constructed using routine cloning
and PCR techniques and placed into the EcoRV site of pAVS6 to yield pAVS6-
nlacZ.
The infectious, replication-deficient, AvlLacZ4 was assembled in 293
cells by homologous recombination. To accomplish this, plasmid pAVS6-Nlacz
was lineari~d by cleavage with KpnI. Genomic adenoviral DNA was isolated
from purified Addl327 viruses by Hirt extraction, cleaved with ClaI, and the
large (approximately 3S kb) fragment was isolated by agarose gel electrophoresisand purified. The ClaI fragment was used as the backbone for all first generation
adenoviral vectors, and the vectors derived from it are known as AV l .
Five micrograrns of linearized plasmid DNA (pAVS6n-LacZ) and 2.5 ~lg
ofthe large ClaI fragment of Addl327 then were mixed and co-transfected into
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a dish of 293 cells by the calcium phosphate precipitation method. After 16
hours, the cells were overlaid with a 1: 1 mixture of 2% Sea Plaque agar and 2x
medium and incubated in a humidified, 37~C, 5% CO2/air environment until
plaques al~pe~cd (approximately one to two weeks). Plaques were selected and
intracellular vector was released into the medium by three cycles of freezing and
thawing. The Iysate was cleared of cellular debris by centrifugation. The plaque(in 300 Ill) was used for a first round of infection of 293 cells, vector release, and
clarification as follows:
One 35 mrn dish of 293 cell was infected with 100 ~ll of plaque lysate plus
400 ~ll of IMEM-2 (IMEM plus 2% FBS, 2mM glutamine (Bio Whittaker
046764)) plus 1.5 ml of IMEM-10 (Improved minim~l ec.c~nti~l medium (Eagle's)
with 2x glutamine plus 10% vol./vol. fetal bovine serum plus 2mM gl~ e
plus (Bio Whittaker 08063A) and in~.ub~te(l at 37~C for approximately three daysuntil the cytopathic effect, a rounded appearance and "grapelike" clusters, was
observed. Cells and supe."~ t were collected and clesi~n~ted as CVL-A.
AvlLacZ4 vector (a sch~m~tic of the construction of which is shown in Figure
10) was released by three cycles of freezing and thawing of the CVL-A. Then,
a 60 mm dish of 293 cells was infected with 0.5 ml of the CVL-A plus 3 ml of
IMEM-10 and incubated for approximately three days as above. Cells and
sup~ from this infection then were processed by three freeze/thaw cycles
in the same manner. AvlLacZ4 also is described in Yei et al., Human Gene
Therapy 5:731 -744 (1994); Trapnell, ~dvanced Drug Delivery Rev. 12:185-199
(1993), and Smith et al., Nature: Genetics 5:397-402 (1993), which are
incorporated herein by reference.
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Example 2
Cytopafhic effect of El-deleted adenovirus in El- cell lines
A survey of tumor cell lines was undertaken to identify potential
candidates for in vi~ro and in vivo models of tumor killing using adenovirus-
thymidine kinase constructs in conjunction with ganciclovir. A number of cell
lines were tested, including human glioma cell lines U87, U373, and U118, a
hepatocellular carcinoma cell line Hep 3B, and Y79, a human retinoblastoma cell
line. The most prominent effect occurred with Hep 3B. The cell line proved to
be exquisitely sensitive to adenovirus exposure, showing significant cytopathic
effects, even at low levels of input virus.
To test the hypothesis that viral replication was causing the cytopathic
effects, cells were exposed to varying amounts of virus and observed for the
development of cytopathic effects.
Hep3B cells were plated at 1 million cells per well of 6-well dishes in
RPMI 1640 media co~ g 10% fetal calf serum. Transductions of Hep3B
cells were performed by adding AV.lacZ4 virus (6 x 10'~ plaque forrning units
(pfu)/ml) at ratios of 5000, 1600, 530, 175, 55, 18, 6, 2, 0.2, and 0.06 pfu/celi
Uninfected cells served as a control. Cells were observed for cytopathic effectsfor a period of 15 days. Cells not showing cytopathic effects at day 8 were
tryp~ini7P.d and re-plated at 5 x 105 cells per well and followed for an additional
7 days. Cytopathic effects were vacuole formation in the cytoplasm progressing
to displacement of the nucleus, nuclear densities, crenulations of the cell
membrane, and cell rounding with loss of adherence and/or cell-cell contacts.
The results are shown in Table 1.
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-40-
,.,
o
oo X
t' _1
X
~&
X X _~ +
X X ~ ~ + +
_l I + + +
D
+ + + + ~ ~ C
O l ' ' ' ' ' ' ' ' ~ _ O
''O _. -- O
C ~ ~
o o o C ~ C .,
D ~ O O O ~ ~ o5
O .'~ t~
~" O
U~
~ ~ o 11 11 11 11
CC C ~ I + _l X
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41-
The results show morphological changes consistent with virus replication.
Such changes previously had only been known to occur in cells infected with
wild type adenovirus or in cells constructed to specifically trans-complement
mutant adenoviruses (293).
The absence of the E1 region genes in the mutant AV.lacZ should have
rendered this virus incompetent for replication. Nonetheless, evidence of
replication was present. The results indicate that the turnor cells either contain
viral DNA sequences which complement the El deficiency or that the cells have
acc~ tçd sufficient mutations to create an environrnent which would perrnit
virus replication in the absence of E1 genes or E1 homologs (for exarnple, HPV
16 or 18 E6/E7, CMV irnrnediate early genes, SV40 T-antigen).
As the function ofthe adenovirus E1 genes include specific inactivation
of cellular p53 and Rb gene products, the results suggest that a deficiency in the
function of these cellular genes allows replication.
Example 3
Expression of thymidine kinase activity in A V. TKl infected A VI r~pli~ ntion
.",.~ssi.c HuH7 cells
In order to determine the quantity of activity of a gene product expressed
from an AV1 vector in cells which support replication of AV1 vectors the human
hepatoma cell line HuH7 was infected with AVl.TKl. This vector expresses
thymidine kinase activity from the Rous sarcoma virus promoter in the vector.
This vector was chosen since previous work (Culver et al., Science 256:1550-
1557) has demonstrated that thymidine kinase actively positively correlates withcell-killing by ganciclovir and the bystander effect (the ability of cells expressing
thymidine kinase in the presence of ganciclovir to kill neighboring cells not
~.essillg thyrnidine kinase). HuH 7 cells were chosen because as demonstrated
herein, these cells support replication of AV 1 vectors.
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For each thymidine kinase activity measurement, 1 x 107 cells were
infected with either AVl.LacZ4 at an MOI of 100 as a negative control for
thymidine kinase activity or with AVl.TK1 at MOIs of 10 and 100. Infections
were performed as previously described (Graham, F.L. et al., J. Gen. Virol.
36:56-72 (1977)). NIH 3T3 thymidine kinase-cells were also harvested and used
as a negative control. PA317/GlTKlSvNa#7 is a cell line that produces high
amounts of thymidine kinase. The line was previously optimized to express
thymidine kinase from multiple endogenous copies of a retrovirus thymidine
kinase gene. This line was used as a positive control for thymidine kinase
activity. Equivalent amounts of protein were assayed from the samples for
thymidine kinase activity ~ previously described. Thymidine kinase activity was
measured two days following infection.
The results are shown in Figure 1. No thymidine kinase activity was
found in NIH 3T3 thymidine kinase-cells alone or in HuH 7 cells infected with
AVl.LacZ4 at an MOI of 100. High levels of thymidine kinase activity were
present in PA3 1 7/GlTK1 SVNa#7 cells. HuH 7 cells infected with an AVl .TKI
at an MOI of 10 showed a significant amount of thymidine kinase activity.
However, HuH 7 cells infected with AVl.TKl at an MOI of 100 showed
approximately 3 fold higher thymidine kinase activity than the optimized
producer cell line PA3 1 7/GlTKI SVNa#7 and 30 fold higher activity than HuH
7 cells infected with AVl.TKl. This result suggests that the high thymidine
kinase activity in AVl.TKl infected cells is due to higher gene dosage from
replication of AVl . This conclusion is b~ed on the observation that thymidine
kinase activity in HuH 7 cells appears to be exponential with increasing MOI:
greater than 30 fold higher activity was found in cells infected with an MOI of
100 vs an MOI of 10. If thymidine kinase activity linearly increased with
increasing MOI, a 10 fold increase would be expected, as has been demonstrated
for other cell lines which may not support replication of AV I vectors (Cancer
Gene Therapy 1:107-112 (1994)).
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The levels of thymidine kinase activity were also shown to correlate with
the amount of protein by a quantitative Western analysis using a monoclonal
antibody specific to thymidine kinase. That DNA replication of adenovirus leads
to a drarnatically ~nh~n-~ecl gene dosage has been demonstrated previously
~ltili7.ing a luciferase reporter gene (Mittal, S.K. et al., Virus Res. 28(1):67-90
(1994))
R~ t..~ri.c Sou~hern blot demo..~ ,g cell line specif c DNA replication
of A VI vector
The possibility was investig~tecl that El deleted adenoviral vectors could
replicate in a variety of tumor cell lines. Cell lines were infected with the
AVl.LacZ4 vector. Adenoviral DNA accumulation over time indicates DNA
replication.
1 x 105 cells of each of the various cell lines were plated into 6 well
dishes. Semi-confluent monolayers were formed within 24h. 24h later, cells
were infected with either AVl.LacZ4 E1 deleted vector, or as a positive control
for replication, wild-type virus and Addl327, both at an MOI of 10. Infections
were performed as described for Fig. 1. Samples were harvested at 4h, t~,vo days,
and six days, after infection by standard trypsinization of adherent cells
immediately following a 2 ml wash of the monolayer by PBS. Cells were
collected by centrifugation at 3000 x g to recover the residual cell pellet. The cell
pellet was resuspended once with 1 ml PBS and respun. Pellets were frozen on
dry ice and stored at -70 until isolation of DNA. DNA was isolated from frozen
cell pellets as previously described (Molecular Cloning: A Laboratory Manual,
second edition. Sambrook, J. et al., eds., Cold Spring Harbor Laboratory Press
(1989)). One third of the total DNA isolated from each well was digested to
completion by Hind III and electrophoresed on a 1% agarose gel. Gels were
transferred onto Nytran, prehybridized, and hybridized, as previously described
(Molecular Cloning: A Laboratory Manual, second edition, Sambrook, J. et al.,
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44-
eds., Cold Spring Harbor Laboratory Press (1989)). Probes were a 400 bp PCR
generated and purified probe specific for the E2b region of adenovirus (which
should hybridize to a specific Hind III restriction fragment of adenovirus), anda 200 bp PCR generated and purified probe specific for the Ela region (which
should hybridize to a specific Hind III fragment of adenovirus). The E2b probe
should hybridi~ to both AV1 DNA and Addl327 DNA, whereas the Ela probe
should only hybridize to the AV1 DNA since this region is deleted in AV1
vectors.
The results are shown in Figure 2. The results demonstrate that the wild-
type viral DNA was subst~nti~lly increased in every cell line tested. The
AVl.LacZ4 vector replicated in 293 cells. This result is not unexpected since
293 cells harbor and express the E1 region deleted in most ~ ;ntly used
adenoviral vectors (including the AVl vectors herein). However, DNA
replication of the AVI vector was observed in Hep3B cells, at a level
approaching that found in 293 cells. DNA replication was also observed in HuH
7 cells and, on a much longer exposure, A549 cells. Although there was no
a~p~,~ replication at an M01 of 10 of the AV l .LacZ4 vector in SW480 cells,
preliminary e~-~cl;lllental evidence has been obtained that replication of AVl
DNA does occur at the slightly higher MOI of 30 in SW480 cells.
To further conclude that what was observed with the E2b probe was
replication of AVl DNA, and not replication of a small amount of Cont~min~ting
wild-type virus, the blot was stripped by boiling twice in 0.1 M EDTA
(Molecular Cloning: A Laboratory Manual, second edition, Sambrook, J. et al.,
eds., Cold Spring Harbor Laboratory Press (1989)) and reprobed with the Ela
probe. The Ela probe hybridized only to the expected Hind III restriction
fragment and only in cells infected with wild-type virus. This confirms that theAVl.DNA is replicated in the turnor cell lines. These results indicate that
replication of AV 1 occurs in cell lines that are not known to harbor the E1 genes.
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D~ - -n~trafion that ceU lines whiclt support replicafion of A Vl vectors do notco~ oi~1 endogenous El genes
Although it was excee.lin~ly unlikely that human cell lines would have
an integrated adenoviral sequence (Virology, second edition, Fields, B.N. et al.,
S eds., Raven Press Ltd. (1990)), to ~lçt~nnin~ unambiguously that there were no
integrated E1 sequences in Hep3B and SW480, DNA from these lines was
analyzed by Southern blot.
1 X 107 cells from actively growing cultures from each cell line were
harvested by centrifugation at 3000 xg. DNA was harvested by an automated
system per the m~mlf~tllrer's instructions (Genepure Extractor Model 341,
Perkin Elmer/Applied Biosystems Division, Foster City, CA). 10 ~lg of DNA,
digested with Hind III, was electrophoresed on a 1% gel, and transferred to
nitrocellulose. Hyhritli7~tion conditions and the ~ lion of the E1 probe were
as described for Figure 2. Another probe for the entire adenovirus of Addl327
(which is identical to Ad 5 except that the E3 region is deleted) was made by
random priming of purified viral DNA. The results are shown in Figure 3.
Although both probes hybridized to 293 DNA as expected, neither probe
hybridi~d to DNA isolated from either Hep 3B or SW480 cells. This indicates
that the factor compl~menting the E1 defect in the AV1 vector is not related to
genes present on the adenovirus backbone.
Repr~ser~tntive plaque titer demo..sl,ulirlg cell line specif c virus replication of
AVl vector
Since high levels of AV1 vector DNA replication were observed in a
variety of turnor cell lines, the inventors determined if infectious virus also could
be produced from tumor cell lines supporting DNA replication. Each cell line
was plated at a density of 1 x 105 cells in a six well plate and allowed to adhere
for 24h before infection. Cells from 293 cells (positive control), Hep3B, SW480,
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~6-
and A549 were infected at an MOI of 10 with AVl.LacZ4. Plates were washed
at 4h with PBS and the infection was allowed to proceed for 6d before pre~ ,ng
a CVL as described (Graham, F.L. et al., J: Gen. Virol. 36:56-72 (1977)). Plaquetiters on 293 cells (Graham, F.L. et al., J. Gen. Virol. 36:56-72 (1977)) were
performed on each CVL to determine the amount of infectious virus produced
from the cells over the 6d period.
The results are shown in Figure 4. No detectable virus was found in any
of the 4h time points on any of the cell lines. This result was anticipated because
the virus cannot replicate in this short time period. 293 cells produced
~plo~ ly 4500 pfus/starting cell as expected. Although A549 and SW480
produced no tletect~hle virus (although they supported a low level of DNA
replication), a large amount of virus was produced in Hep3B cells. These cells
produced nearly as much infectious virus as 293 cells without optimi7ing the
conditions (1500 pfus/starting cell). The results demonstrate that Hep3B cells,
and potentially other tumor cells, are fully capable of complementing the E1
deletion in AV 1 vectors.
Since the AV1 vector does not contain the gene which expresses Elb 55
kDa polypeptide (which has the function of transporting viral MRNA into the
cytoplasm for translation) (Ornelles, D.A. and Shenk, T., J. Virology 65:424-439(1991)), the question was asked whether the presence of this protein might
enhance virus production because of higher translation of viral encoded proteins.
Addl312 (Shenk, T. et al., Cold Spring Harbor Symp. Quant. Biol. 44:367-375),
which is deleted in Ela but contains Elb, was utilized. When Hep3B cells were
infected with Addl312 at an MOI of 10, approximately six-fold more virus was
produced compared to virus produced with AV l .LacZ4.
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Complete table ~a.,,. ,.,,i~.g viral cyto~o.~ieity data, DNA replienfion, and virus
production in a variety of tumor cell lines and normal cells -
Other cell lines were examined for each of the three properties of AV 1
infected cells (cytotoxicity, DNA replication, and virus production) to ~et~ nnine
S the extent of turnor cell lines capable of pc~ g replication of an AV1 vector.
Normal cells were also ex~min~-l
Each assay was ~c.r~llned as described above. The results are shown in
Figure 5. + refers to ~bi~ grading system for simplicity; for DNA replication,
I I I I = m~iml-m observed effect (col~lpaled to effect observed with Hep 3B
cells); I I I = 50-75%; t~ = 25-50%; + = 10-25%;--= none det~ct~hle. For
cytotoxicity (Cyto) only I I I I, I I I, and ~ were used. Less than ~ is a - in this
assay. NT = not tested. IP = In progress. pub = published.
The data demonstrate that AV1 infection leads to slowed growth
(cytotoxicity) in many tumor cell lines, but not in normal fibroblasts, hepatocytes,
or lung cells. When DNA replication was measured, it was found that DNA
replication occurred in all cells demon~ ing any significant CPE (as well as in
several others). However, no DNA replication was observed at this MOI in
normal fibroblasts (Figure S), a normal lung line (Yei, S. et al., Hum. Gene Ther.
5:731-744 (1994)), or in primary hepatocytes (Kozarsky, K. et al., Somal. Cell
Mol. Genet. 19(5):449-58 (1993)). Ofthe cell lines tested, only Hep3B cells werecapable of producing infectious virus at the sensitivity examined.
Table sllowing tlle pS3/~b genotypes of cell lines
The function of the Ela and Elb region in adenovirus is to inactivate the
cellular Rb and p53 gene products, respectively. Since the E1 deleted adenoviralvector was replicating in a variety of tumor cells, it was possible that the tumor
cell lines in which DNA replication was observed harbored functionally
inactivated Rb and p53, and could thus complement the El defect.
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All tumor cell lines which supported DNA replication (shown in bold in
Figure 6) were found in the literature to contain either mutated or null p53 genes
or a functionally inactivated p53 protein (SIHA/HeLa). All lines which did not
support DNA replication had a wild type p53 gene. Thus, the ability of AV1
S vectors to replicate in tumor cell lines correlates with the p53 status of the line.
Although an Rb mutation alone did not allow DNA replication ofthe AV1 vector
(DU-145), many of the cell lines harboring a functionally inactivated or mutatedp53 gene were also shown to contain a functionally inactivated or mutated Rb
gene.
A~OI vs cell killing of HuH7
An t;A~ ont was p~ o,llled to eY~ ninto if replication could enhance the
ability of a vector carrying a suicide gene. The gene is the Herpes simplex type1 thymidine kinase gene. The g~ ploduct is known to render tumor cells
sensitive to the nucleotide analogue of ganciclovir. The human hepatoma cell
line HuH7 was infected. The results are shown in Figure 7. The cell line, which
supports DNA replication of the vector, showed greater than 90% kill rate by a
relatively MOI of 5 in the presence of 50 llm ganciclovir. In addition, when a
MOI of 50 was used, greater than 80% of the cells were killed in a two-day time
period in the absence of ganciclovir, and 100% of the cells were killed in the
presence of ganciclovir. In similar studies lltili~in~ other cells which may notsupport DNA replication, much higher MOIs were necessary to achieve a similar
degree of tumor cell killing by ganciclovir, and in the absence of ganciclovir, no
tumor cell killing was observed (Smythe, W.R. et al., Cancer Res. 54~8):2055-
2059 (1994))
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~nci(~lovir .~ int~d bystn~ r effect in HuH7 cells
Cells bearing herpesvirus thymidine kinase-~ re~ g vectors are known
to cause neighboring cells not t;"l"e~ g the thymidine kinase gene to become
sensitive to ganciclovir by a "bystander effect." The inventors have obtained data
suggesting that at least in vitro (and ple~LI.llably in vivo), the higher the thymidine
kinase ~lc;~sion in tumor cells, the more neighboring tumor cells can be killed.Therefore, the question of whether a cell line which supports replication could
cause a high bystander effect was t;.~ .e~l
HuH7 cells infected with a MOI of 50 were mixed with increasing
quantities of uninfected cells (holding the total number of cells constant) and
were treated with 50 ~lM ganciclovir. The results are shown in Figure 8. If no
bystander effect occurred, a linear les~ se between 100% infected cells (all
killed) and 0% infected cells (all living) would have been expected. However, itwas observed that when only 1% of the cells were infected, nearly 80% of the
cells were killed. This dramatic bystander effect may be due to the enh~n~ed
gene dosage expected from DNA replication of the vector. The bystander effect
should significantly contribute to tumor cell killing, since it is difficult to achieve
high tr~n~d~lction rates of in situ tumors with any vector (Blaese, R.M. et al., Eur.
J. Cancer 30A: 1191-1193 (1994)). These results, combined with the results
described above, suggest that replication significantly enhances the ability of E1-
deleted vectors to specifically kill tumor cells.
Carcinorna ceU line specif c killing
In order to det~rrnine if infection of AV 1 vectors caused a cytotoxic effect
in certain infected tumor cells, a crystal violet staining assay was utilized
(Yamamoto et al., in Practical Methods in Chemical Immunology, Volume 9:
Investigation of Cell Mediated Immunity, Yoshida, T., ed., Churchill Livingstone,
Edinburgh, New York (1985), pp. 127-134), in which the level of crystal violet
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uptake is proportional to the amount of adherent live cells. 1 x 104 cells of each
turnor cell line was plated into each of three wells in a 96 well plate. 24h after
plating, cells were infected with either diluent, Addl327, or AVl.LacZ4, both atan MOI of 10. Samples were assayed for crystal violet uptake in triplicate, 2d,
6d, and 9d following infection.
The results are shown in Figure 9. As expected, cells treated with diluent
in each of the four cell lines tested showed in increase in crystal violet staining
over time, in-lir~tin~ cell growth. 293 cells were rapidly killed by either the wild-
type or AV1 vector, as expected, since this cell line ~u~ol~ replication of the
virus. Addl327 killed either A549 Hep3B or HuH7 cells within the six-day
period. However, the AV1 vector killed greater than 90% of either the Hep3B
cells or HuH7 cells within the six-day period. This result was unexpected, sinceE1-deleted adenoviral vectors have not caused cytotoxic effects at this MOI in awide variety of normal tissues (Kozarsky, K. et al., Somat. Cell Mol. Genet.
19(5):449-458 (1993); Yei, S. et al., Hum. Gene Ther. 5:731-744 (1994)) and
other cell lines (Smythe, W.R. et aL, Cancer Res. 54(8):2055-2059 (1994)). The
AV1 vector had a reproducible effect on cell growth of the A549 cells as well.
The results suggest that: (1) replication of the AV 1 vector occurs in a cell-
specific manner, since cytotoxicity and cytopathic effects are indicative of
replication, and (2) replication was tumor-cell-line-specific at these MOIs. An
absence of cytotoxic effects of this vector in primary human hepatocytes even upto a MOI of 100 has been demonstrated by the inventors and also by others.
Furthermore, a number of tumor cell lines and normal cells have been screened,
with the result that cytotoxicity was found only in cells which have either mutated
or inactivated p53 (see Figures 5 and 6).
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Cure rate oSnude mice bearing Hep 3B sub~r t~1eous hepatoma treated with
A Vl. TKl
Since Hep 3B cells were shown to support both virion and DNA
replication, the ability of AV1 .TKl vector was tested to either kill or slow growth
of a preestablished Hep 3B tumor in nude mice. Hep 3B tumors were established
by injecting 1 x 107 cells s~lbc.lt~n~usly on the flank of a nude mouse. Tumors
were allowed to grow until they reached an average size of 50 rnrn3. Tumors
were then injected with 2 x 109 pfus of either AVl.null or AVl.TK1 or diluent
alone. Virus was injected in a total volume of 100 ul with two injections through
the outer skin. One half of each group was treated with gancyclovir (ganciclovir),
at 300 mg/kg/day.
The results are shown in Figure 10. All ~nim~lc treated with AVl.TK1
and ganciclovir showed complete regression of tumors with 50% rt~n-~inin~
tumor free after 30 days. No adverse effect was observed in mice treated this way.
These results show that a vector which speçifi~ ~lly replicates in turnor cells can
be used for cancer therapy.
p53-dependent ~7b1~ . of A Vl DNA repli~ti~n in HuH7 cells
To directly demonstrate that reintroduction of wild-type p53 in cells
which were devoid of wild-type p53 and which ~u~ Led replication of the AVI
vector, pS3 was introduced into HuH7 cells and cells were assayed for
replication. An AVl.p53 vector was constructed and produced in 293 cells as
described herein. Since this vector expresses wild-type p53, cells infected withthis virus should not be capable of replicating the vector as they do AV 1 .LacZ4.
The results are shown in Figure 11. Cells infected with Addl327, or
AVl.LacZ4 showed an increase in viral DNA over time, indicating replication.
However, cells infected with AVl.p53 showed no increase in DNA over time.
These results demonstrate that reintroduction of p53 prevents replication.
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Survival curve for Hep3B fumor-bearing onim~ trea~ed witl~ various
adenoviral vectors
Athymic nude mice with Hep3B tumors averaging 95 mm3 were treated
by direct illL~ lloral injection of AVl.lacZ4 (Ela/Elb-deleted virus t;~lessillgS an irrelevant transgene), AV1 .TKl (Ela/Elb-deleted virus t;~ies~ g a transgene
which converts gancyclovir (GCV) to a toxic metabolite), or dl327 (Ela/Elb-
co,-l~;";"g adenovirus which replicates in a wild-type fashion in human cells invitro). Survival curves are based upon the sacrifice of ~nim~l~ when tumor
volume ~xceeclecl 2,000 rnm3 or ~nim~l~ were found dead.
The ability of Ela/Elb-deleted adenovirus to ablate tumors in vivo is
demonstrated in Figure 12. In the context of Hep3B cells, which support the
replication of ElaJElb-deleted adenovirus at a level similar to wild-type
replication, tumor ablation can occur as a direct consequence of infection with
dl327 (wild type). Impo~ ly, AVl.lacZ4 ablates the tumor as well as dl327,
and does so as ~rr~clively as an El-deleted virus co~ ini"g a suicide gene
(AVl.TKl plus gancyclovir).
Compl~m~-f~fiQn of viral repli~lti~n in fumor cells
Results obtained herein using Hep3B cells and an AVI vector show
evidence for a complçm.ont~tion function for defective replication functions in the
vector. In the t;~ hllents described in the exemplary material herein, it is shown
that of all the tumor cell lines screened, many of which were pS3- and/or Rb-,
only Hep3B cells were capable of making infectious viral particles. Most of the
Rb- and/or p53- tumor cells replicated the DNA of the adenoviral genome, but
only Hep3B cells produced infectious virus. See, for example, Figure 5.
Thus, the data support involvement of complementing factors for full
infectious virus production. The data also show that CPE and cytotoxicity can
occur in the complete absence of any infectious virus production.
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Cells which complement the replication deficiencies of Ela/Elb-deleted
adenoviruses must permit several biochPn-ic~l events to proceed. These events
include blockage of apoptosis and the accumulation of the transcription factor
E2F (E2F exerts much of the positive llallsc~ ional control over the activity ofthe E4 and E2 promoters and subsequently over the progression of adenoviral
replication from the DNA synthetic phase to the phase of capsid production and
assembly). Naturally-occurring cellular factors which completely overcome the
block to infectious virus production by El a-deleted viral vectors include nflL-6
(Spergel etal., Proc. Natl. Acad. Sci. (ZJSA) 88(15):6472-6476 (1991)). The needfor mutations in, or inactivation of, the pRb farnily (and possibly pS3 as well) of
gene products by Ela is merely a means of increasing the concentration of activeE2F. Further, the active form of this factor is found in all mitotically-active cells
(including tumor cells) as part of cell cycle progression. E1b-me~ ted blockage
of apoptosis may also be dispensable, as other naturally-occurring cellular
peptides can block apoptosis as well (e.g., proteins from the bc1-2 locus).
However, cells permissive for virion production by Ela/Elb deleted
vectors must also possess additional factors. These factors are necessary to
transcomplement functions formally produced by interactions between Ela/Elb
gene products and other viral regulatory proteins (e.g., the interaction of Elb with
E4 gene products, n~cçss~ry for the preferential processing and transport of
mRNAs encoding the viral late gene products (Ornelles et al., J. T~irol. 65(1):425-
439 (1991)). Most cells lacking either p53 or pRb function do not efficiently
produce infectious virions (see Figs. 5 and 6). In the absence of
transcomplementing activities which supplant the need for protein interactions
between El a!E1 b-encoded proteins and products of the other adenovirus genes,
cells may support viral DNA replication but can not efficiently support infectious
virion production (Fig. S). Hep3B cells, which support vigorous viral replication,
must possess one or more factors that will transcomplement the defect arising
from the absent Ela/Elb interactions with other viral gene products. It has beenshown (Martuza et al., Science 252:854-856 (1991)) that a thymidine kinase
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defect in a Herpesvirus can be overcome in tumor cells, which produce
significantly more TK than normal cells do. This complementation allows the
virus to replicate in the tumor cells.
