U.S. patent application number 11/217386 was filed with the patent office on 2006-01-05 for vectors for tissue-specific replication.
Invention is credited to Yung-Nien Chang, Yawen L. Chiang, Paul L. Hallenbeck.
Application Number | 20060002897 11/217386 |
Document ID | / |
Family ID | 27407819 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002897 |
Kind Code |
A1 |
Hallenbeck; Paul L. ; et
al. |
January 5, 2006 |
Vectors for tissue-specific replication
Abstract
The invention generally relates to targeted gene therapy using
recombinant vectors and particularly adenovirus vectors. The
invention specifically relates to replication-conditional vectors
and methods for using them. Such vectors are able to selectively
replicate in a target tissue to provide a therapeutic benefit from
the presence of the vector per se or from heterologous gene
products expressed from the vector and distributed throughout the
tissue. In such vectors, a gene essential for replication is placed
under the control of a heterologous tissue-specific transcriptional
regulatory sequence. Thus, replication is conditioned on the
presence of a factor(s) that induces transcription or the absence
of a factor(s) that inhibits transcription of the gene by means of
the transcriptional regulatory sequence with this vector;
therefore, a target tissue can be selectively treated.
Inventors: |
Hallenbeck; Paul L.;
(Gaithersburg, MD) ; Chang; Yung-Nien;
(Cockeysville, MD) ; Chiang; Yawen L.; (Potomac,
MD) |
Correspondence
Address: |
Patent Group;DLA PIPER RUDNICK GRAY CARY US LLP
1200 Nineteenth Street, N.W.
Washington
DC
20036-2412
US
|
Family ID: |
27407819 |
Appl. No.: |
11/217386 |
Filed: |
September 2, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10323955 |
Dec 19, 2002 |
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11217386 |
Sep 2, 2005 |
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09210936 |
Dec 15, 1998 |
6551587 |
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10323955 |
Dec 19, 2002 |
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08849117 |
Aug 1, 1997 |
5998205 |
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PCT/US95/15455 |
Nov 28, 1995 |
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09210936 |
Dec 15, 1998 |
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08487992 |
Jun 7, 1995 |
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08849117 |
Aug 1, 1997 |
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08348258 |
Nov 28, 1994 |
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08487992 |
Jun 7, 1995 |
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Current U.S.
Class: |
424/93.2 ;
435/456 |
Current CPC
Class: |
C12N 2820/002 20130101;
C12N 2830/32 20130101; C12N 2830/008 20130101; A61P 35/00 20180101;
A61K 48/0058 20130101; C12N 2710/10343 20130101; C12N 2830/002
20130101; A61P 43/00 20180101; C12N 2840/20 20130101; C12N 2830/85
20130101; A61K 48/00 20130101; C12N 15/86 20130101 |
Class at
Publication: |
424/093.2 ;
435/456 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/861 20060101 C12N015/861 |
Claims
1. A vector capable of tissue-specific replication comprising: a
tissue-specific transcriptional regulatory sequence operably linked
to the coding region of a gene that is essential for replication of
said vector.
2-40. (canceled)
Description
[0001] This application is a continuation of U.S. application Ser.
No. 09/210,936, filed Dec. 15, 1998, which is a continuation of
U.S. application Ser. No. 08/849,117, filed Aug. 1, 1997, now U.S.
Pat. No. 5,998,205, which is the U.S. national phase under 35
U.S.C. .sctn. 371 of PCT application PCT/US95/15455, filed Nov. 28,
1995, which is a continuation-in-part of U.S. application Ser. No.
08/487,992, filed Jun. 07, 1995, now abandoned, which is a
continuation-in-part of U.S. application Ser. No. 08/348,258, filed
Nov. 28, 1994, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention generally relates to targeted gene therapy
using recombinant vectors and particularly adenovirus vectors. The
invention specifically relates to replication-conditional vectors
and methods for using them. Such vectors are able to selectively
replicate in a target tissue to provide a therapeutic benefit from
the presence of the vector per se or from heterologous gene
products expressed from the vector and distributed throughout the
tissue. In such vectors, a gene essential for replication is placed
under the control of a heterologous tissue-specific transcriptional
regulatory sequence. Thus, replication is conditioned on the
presence of a factor(s) that induces transcription or the absence
of a factor(s) that inhibits transcription of the gene by means of
the transcriptional regulatory sequence. With this vector,
therefore, a target tissue can be selectively treated. The
invention also relates to methods of using the vectors to screen a
tissue for the presence or absence of transcriptional regulatory
functions that permit vector replication by means of the
transcriptional regulatory sequence. The invention also relates to
cells for producing recombinant replication-conditional vectors
useful for targeted gene therapy.
[0004] 2. Background Art
Targeting Vectors
[0005] One of the major goals for therapeutic use of exogenous
genes has been cell targeting with high specificity. General
approaches have included systemic introduction of DNA, DNA-protein
complexes, and liposomes. In situ administration of retroviruses
has also been used for cells that are actively replicating.
[0006] However, because of the lack of, or significantly low,
cell-specificity and inefficient gene transfer, the targeting of
desired genes to specific cells in an organism has been a major
obstacle for exogenous gene-based therapy. Thus, the use of such
genes has been limited.
[0007] Tumor cells are among those cell types for which it would be
especially desirable to provide a means for exogenous gene
targeting. In an embodiment of the present invention, compositions
and methods are provided to deliver exogenous genes to tumor cells
safely and efficiently.
Adenoviruses Generally
[0008] 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.times.10.sup.6
daltons for adenovirus 2 and varies slightly in size depending on
serotype. The DNA has inverted terminal repeats and the length of
these varies with the serotype.
[0009] 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 cells infected with most serotypes. The
adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very
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 interrelated biochemical events, but
it entails essentially the synthesis of viral RNAs prior to the
onset of viral DNA replication.
[0010] The organization of the adenovirus genome is similar in all
of the adenovirus groups and specific finctions are generally
positioned at identical locations for each serotype studied. Early
cytoplasmic messenger RNAs are complementary to four defined,
noncontiguous regions on the viral DNA. These regions are
designated (E1-E4). The early transcripts have been classified into
an array of immediate early (E1a), delayed early (E1b, E2a, E2b, E3
and E4), and intermediate (IVa2.IX) regions.
[0011] The E1a region is involved in transcriptional
transactivation of viral and cellular genes as well as
transcriptional repression of other sequences. The E1a gene exerts
an important control finction on all of the other early adenovirus
messenger RNAs. In normal tissues, in order to transcribe regions
E1b, E2a, E2b, E3, or E4 efficiently, active E1a product is
required. However, as discussed below, the E1a function may be
bypassed. Cells may be manipulated to provide E1a-like functions or
may naturally contain such functions. The virus may also be
manipulated to bypass the functions as described below.
[0012] The E1b region is required for the normal progression of
viral events late in infection. The E1b product acts in the host
nucleus. Mutants generated within the E1b sequences exhibit
diminished late viral mRNA accumulation as well as impairment in
the inhibition of host cellular transport normally observed late in
adenovirus infection (Berkner, K. L., Biotechniques 6:616-629
(1988)). E1b is required for altering functions 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. E1b produces a 19 kD protein that prevents
apoptosis. E1b also produces a 55 kD protein that binds to p53.
[0013] 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 Delivery Vehicle
[0014] 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 human respiratory system
and gastrointestinal tract. It causes a mild flu-like disease.
Adenoviral vectors enter cells by receptor mediated endocytosis.
The large (36 kilobase) genome allows for the removal of genes
essential for replication and nonessential regions so that foreign
DNA may be inserted and expressed 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 expression of heterologous genes. The expression 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 extrachromosomal
element in the cellular nucleus. There is no association of human
malignancy with adenovirus infection; attenuated strains have been
developed and have been used in humans for live vaccines.
[0015] 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 Drug Delivery Reviews
12:185-199. (1993).
[0016] 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 the viral vector
generally remains episomal and does not replicate, thus, cell
division leads to the eventual loss of the vector from the daughter
cells" (Morgan, R. A., et al., Annual Review of Biochemistry
62:191-217 (1993)) (emphasis added).
[0017] Non-replication of the vector leads not only to eventual
loss of the vector without expression in most or all of the target
cells but also leads to insufficient expression in the cells that
do take up the vector, because copies of the gene whose expression
is desired are insufficient for maximum effect. The insufficiency
of gene expression is a general limitation of all non-replicating
delivery vectors. 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. The present invention overcomes the
disadvantages discussed above by providing a tissue-specific, and
especially a tumor-specific replicating vector, multiple DNA
copies, and thus increased amounts of gene product.
Production of Adenoviral Vectors
[0018] Adenoviral vectors for recombinant gene expression 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
transformed with human adenovirus 5, now contains the left end of
the adenovirus 5 genome and expresses E1. Therefore, these cells
are permissive for growth of adenovirus 2 and adenovirus 5 mutants
defective in E1 functions. They have been extensively used for the
isolation and propagation of E1 mutants. Therefore, 293 cells have
been used for helper-independent cloning and expression of
adenovirus vectors in mammalian 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 nonessential 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.
[0019] 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. Recombination with
the viral E1 sequence in the genome can lead to the contamination
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 support 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 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.
SUMMARY OF THE INVENTION
[0020] In view of the limitations discussed above, a general object
of the invention is to provide novel vectors for tissue-specific
vector replication and gene expression from the replicating vector.
Accordingly, the invention is directed to a vector that contains a
gene which is essential for replication, and which gene is operably
linked to a heterologous transcriptional regulatory sequence, such
that a vector is created whose replication is conditioned upon the
presence of a trans-acting transcriptional regulatory factor(s)
that permits transcription from the transcriptional regulatory
sequence, or the absence of a transcriptional regulatory factor(s)
that normally prevents transcription from that transcriptional
regulatory sequence. Thus, these regulatory sequences are
specifically activated or derepressed in the target tissue so that
replication of the vector proceeds in that tissue.
[0021] Another object of the invention is to provide
tissue-specific treatment of an abnormal tissue. Thus, a further
object of the invention is to provide a method to selectively
distribute a vector in vivo in a target tissue, such that a greater
number of cells are treated than would be treated with a
non-replicating vector, and treatment is avoided or significantly
reduced in non-target tissue. Accordingly, a method is provided for
selectively distributing a vector in a target tissue by introducing
the replication-conditional vector of the present invention into a
target tissue that contains a transcriptional regulatory factor(s)
that allows replication of the vector or is deficient in a
transcription-inhibiting factor(s) that prevents replication of the
vector.
[0022] For providing tissue-specific treatment, another object of
the invention is to selectively distribute a polynucleotide in a
target tissue in vivo. Accordingly, the invention is directed to a
method for selectively distributing a polynucleotide in a target
tissue in vivo by introducing the replication-conditional vector of
the present invention, containing the polynucleotide, into the
target tissue that contains a transcriptional regulatory factor(s)
that allows replication of the vector or is deficient in a
transcription-inhibiting factor(s) that prevents replication of the
vector.
[0023] For providing tissue-specific treatment, a further object of
the invention is to selectively distribute a heterologous gene
product in a target tissue. Accordingly, the
replication-conditional vectors of the present invention are
constructed so that they contain a heterologous DNA sequence
encoding a gene product that is expressed in the vector. When the
vector replicates in the target tissue, effective quantities of the
desired gene product are also produced in the target tissue.
[0024] Another object of the invention is to provide a method to
identify abnormal tissue that can be treated by the vectors of the
present invention. Therefore, a further object of the invention is
to identify a tissue in which the replication-conditional vectors
of the present invention can be replicated by means of the
transcriptional regulatory sequence contained on the vector.
Accordingly, the invention is further directed to a method wherein
the replication-conditional vectors of the present invention are
exposed to a given abnormal tissue. If that tissue contains a
transcriptional regulatory factor(s) that allows replication of the
vector or is deficient in a transcription-inhibiting factor(s) that
prevents replication of the vector, then replication of the vector
will occur and can be detected. Following identification of such a
tissue, targeted treatment of that tissue can be effected by
tissue-specific transcription and the consequent vector replication
in that tissue in vivo.
[0025] Thus, a method is provided for assaying vector utility for
tissue treatment comprising the steps of removing a tissue biopsy
from a patient, explanting the biopsy into tissue culture,
introducing a replication-conditional vector into the cells of the
biopsy, and assaying for vector replication in the cells.
[0026] Another object of the invention is to provide producer cell
lines for vector production. Preferably, the cell lines have one or
more of the following 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
contaminating the virus preparation and resulting from
recombination between integrated viral sequences and vector
sequences, growth to high density and in suspension, unlimited
passage potential, high growth rate, and by permitting the growth
of highly deleted viruses that are impaired for viral functions and
able to accommodate large pieces of heterologous DNA.
[0027] Accordingly, in a further embodiment of the invention, a
cell line is provided containing the replication-conditional vector
of the present invention, the cells of which cell line contain a
transcriptional regulatory factor(s) that allows replication of the
vector or is deficient in a transcription-inhibiting factor(s) that
prevents replication of the vector.
[0028] In further embodiments of the invention, the cell line
contains nucleic acid copies of the replicated vector. In other
embodiments, the cell line contains virions produced in the cell by
replication in the cell of the replication-conditional vector.
[0029] In further embodiments, a method is provided for producing a
replication-conditional vector or virion comprising the steps of
culturing the producer cell line described above and recovering the
vector or virion from the cells. In still further embodiments, a
method is provided for producing replication-conditional virions
free of wild-type virions or viral vectors free of wild-type
vectors, comprising the steps of culturing the producer cell line
described above and recovering the replication-deficient virions or
vectors from the cells.
[0030] In a preferred methods of treatment and diagnosis, the
tissue is abnormally proliferating, and especially is tumor tissue.
However, the methods are also directed to other abnormal tissue as
described herein.
[0031] In preferred embodiments of the invention, the
replication-conditional vector is a DNA tumor viral vector. In a
further preferred embodiment, the DNA tumor viral vector is a
vector selected from the group consisting of herpesvirus,
papovavirus, papillomavirus, parvovirus, and hepatitis virus
vectors. In a most preferred embodiment, the vector is an
adenovirus vector. However, it is to be understood that potentially
any vector source is useful if it contains a gene essential for
replication that can be operably linked to a tissue-specific
transcriptional regulatory sequence.
[0032] In further methods of treatment and diagnosis, the vector is
introduced into the tissue by infection.
[0033] Replication can be vector nucleic acid 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.
[0034] In a further preferred embodiment of the invention, a gene
in the adenovirus E1 region is operably linked to the
tissue-specific transcriptional regulatory sequence. Preferably,
the E1a or E1b gene is operably linked to the tissue-specific
transcriptional regulatory sequence.
[0035] In a further embodiment of the invention, the vector encodes
a heterologous gene product. This heterologous gene product is
expressed from the vector replicating in the target tissue.
[0036] In a further embodiment of the methods of treatment, the
heterologous gene product is toxic for the target tissue.
[0037] In a further embodiment of the methods, the heterologous
gene product acts on a non-toxic prodrug, converting the non-toxic
prodrug into a form that is toxic for the target tissue.
Preferably, the toxin has anti-tumor activity or eliminates cell
proliferation.
[0038] In preferred embodiments of the invention, the
transcriptional regulatory sequence is a promoter. Preferred
promoters include, but are not limited to, carcinoembryonic antigen
(CEA), DE3, .alpha.-fetoprotein (AFP), Erb-B2, surfactant, and
especially lung surfactant, and the tyrosinase promoter. However,
any genetic control region that controls transcription of the
essential gene can be used to activate (or derepress) the gene.
Thus, other genetic control elements, such as enhancers,
repressible sequences, and silencers, can be used to regulate
replication of the vector in the target cell. The only requirement
is that the genetic element be activated, derepressed, enhanced, or
otherwise genetically regulated by factors in the host cell and,
with respect to methods of treatment, not in the non-target.
cell.
[0039] Preferred enhancers include the DF3 breast cancer-specific
enhancer and enhancers from viruses and the steroid receptor
family. Other preferred transcriptional regulatory sequences
include NF1, SP1, AP1, and FOS/JUN.
[0040] In further embodiments, promoters are not necessarily
activated by factors in the target tissue, but are derepressed by
factors present in the target tissue. Thus, in the target tissue,
repression is lifted.
[0041] Transcriptional regulatory factors include, but are not
limited to, transactivating factors produced by endogenous viral
sequences such as from cytomegalovirus (CMV), HIV, Epstein-Barr
virus (EBV), Herpes simplex virus (HSV), SV40, and other such
viruses that are pathogenic in mammals and, particularly, in
humans.
[0042] Methods for making such vectors are well known to the person
of ordinary skill in the art. The art adequately teaches the
construction of recombinant vectors with deletions or modifications
in specific coding sequences and operable linkage to a heterologous
transcription control sequence such that expression of a desired
coding region is under control of the heterologous transcriptional
regulatory sequence. Many viral sequences have been adequately
mapped such that it is routine to identify a gene of choice and use
appropriate well known techniques (such as homologous recombination
of the virus with deleted or otherwise. modified plasmids) to
construct the vectors for tissue-specific replication and
expression.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1A-B. Cloning of pAVE1a02i: pAVSAFP.TK1 was digested
with NheI/MunI. A 10667 bp fragment was isolated. pSE280-E1 was
digested with SpeI/MunI. A 3397 bp fragment was isolated. The
isolated fragments were ligated to form pAVE1a02i.
[0044] FIG. 2A-C. PCR identification of recombinant adenovirus with
E1a expressed from the hepatoma-specific AFP promoter. FIG. 2A
shows that viral plaques are produced by viral genomes containing
the AFP promoter operably linked to E1a. FIG. 2B shows that there
was no contamination with wild-type virus. FIG. 2C shows that there
was no contamination with AV11acZ DNA.
[0045] FIG. 3A-F. Tissue specific adenovirus with E1a expressed
from the AFP promoter. The experiment shows cytopathic effects and
spreading of cell death following infection with the virus
AVAFPE1a. FIGS. 3A-3C show uninfected controls in A549.30, A549,
and HuH 7 cells, respectively. FIGS. 3D-3F show the results of
infection with the virus in A549.30, A549, and HuH 7 cells,
respectively.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Definitions
[0046] 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.
[0047] The term "anti-tumor activity" is intended to mean any
activity which inhibits, prevents, or destroys the growth of a
tumor.
[0048] The term "distributing" is intended to mean the spreading of
a vector and its attendant heterologous gene (product) (when
present on the vector) throughout a target tissue, and especially
throughout abnormally proliferating tissue (non-malignant or
malignant). The object of the distribution is to deliver the
vector, gene product or the effects of the gene product (as by a
bystander effect, for example). to substantially all or a
significant number of cells of the target tissue, so as to treat
substantially the entire target tissue.
[0049] The term "enhancer" is used according to its art-recognized
meaning. It is intended to mean a sequence found in eukaryotes and
certain eukaryotic viruses which can increase transcription from a
gene when located (in either orientation) up to several kilobases
from the gene being studied. These sequences usually act as
enhancers when on the 5' side (upstream) of the gene in question.
However, some enhancers are active when placed on the 3' side
(downstream) of the gene. In some cases, enhancer elements can
activate transcription from a gene with no (known) promoter.
[0050] The term "functional inactivation" is intended to mean a
genetic lesion that prevents the normal activity of a gene product.
Thus, functional inactivation 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 prevent the 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.
[0051] The term "gene essential for replication" refers to a
genetic sequence whose transcription is required for the vector to
replicate in the target cell.
[0052] The term "gene product" is intended to mean DNA, RNA,
protein, peptides, or viral particles. Thus, the distribution, for
the purposes of the invention, is of any of these components.
[0053] The term "heterologous" means a DNA sequence not found in
the native vector genome. With respect to a "heterologous
transcriptional regulatory sequence", "heterologous" indicates that
the transcriptional regulatory sequence is not naturally ligated to
the DNA sequence for the gene essential for replication of the
vector.
[0054] The term "promoter" is used according to its art-recognized
meaning. It is intended to mean the DNA region, usually upstream to
the coding sequence of a gene or operon, which binds RNA polymerase
and directs the enzyme to the correct transcriptional start
site.
[0055] The term "replication" means duplication of a vector. This
duplication, in the case of viruses, can occur at the level of
nucleic acid, or at the level of infectious viral particle. In the
case of DNA viruses, replication at the nucleic acid level is DNA
replication. In the case of RNA viruses, nucleic acid replication
is replication into plus or minus strand (or both). In the case if
retroviruses, replication at the. nucleic acid level includes the
production of cDNA as well as the further production of RNA viral
genomes. The essential feature is nucleic acid copies of the
original viral vector. However, replication also includes the
formation of infectious DNA or RNA viral particles. Such particles
may successively infect cells in a given target tissue thus
distributing the vector through all or a significant portion of the
target tissue.
[0056] The term "replication-conditional vector" refers to a vector
which when introduced into a tissue will not replicate unless a
transcriptional regulatory sequence in that vector is activated or
derepressed in that tissue. That is, replication depends upon
transcription by means of that transcriptional regulatory sequence.
Such a vector is replication-conditional as described because it
has been modified in the following manner. A gene that is essential
for replication has been modified by replacing the transcriptional
regulatory sequence on which transcription of that gene normally
depends with a heterologous transcriptional regulatory sequence.
This transcriptional regulatory sequence depends upon the presence
of transcriptional regulatory factors or the absence of
transcriptional regulatory inhibitors. The presence of these
factors in a given tissue or the absence of such inhibitors in a
given tissue provides the replication-conditionality. Accordingly,
the native transcriptional regulatory sequence may be replaced with
the heterologous transcriptional regulatory sequence.
Alternatively, the native transcriptional regulatory sequence may
be disabled or rendered dysfunctional by partial removal (deletion)
or other mutation (one or more base changes, insertions,
inversions, etc.).
[0057] The gene sequence may be a coding sequence. It may contain
one or more open reading frames, as well as intron sequences.
However, such a sequence is not limited to a coding sequence, but
includes sequences that are transcribed into RNA, which RNA is
itself essential for vector replication. The essential feature is
that the transcription of the gene sequences does not depend on the
native transcriptional regulatory sequences.
[0058] The term "silencer," used in its art-recognized sense, means
a sequence found in eucaryotic viruses and eucaryotes which can
decrease or silence transcription of a gene when located within
several kilobases of that gene.
[0059] The term "tissue-specific" is intended to mean that the
transcriptional regulatory sequence to which the gene essential for
replication is operably linked functions specifically in that
tissue so that replication proceeds in that tissue. This can occur
by the presence in that tissue, and not in non-target tissues, of
positive transcription factors that activate the transcriptional
regulatory sequence. It can also occur by the absence of
transcription inhibiting factors that normally occur in non-target
tissues and prevent transcription as a result of the transcription
regulatory sequence. Thus, when transcription occurs, it proceeds
into the gene essential for replication such that in that target
tissue, replication of the vector and its attendant functions
occur.
[0060] As described herein, tissue specificity is particularly
relevant in the treatment of the abnormal counterpart of a normal
tissue. Such counterparts include, but are not limited to, liver
tissue and liver cancer, breast tissue and breast cancer, melanoma
and normal skin tissue. Tissue specificity also includes the
presence of an abnormal tissue type interspersed with normal tissue
of a different tissue type, as for example in the case of
metastases of colon cancer, breast cancer, and the like, into
tissue such as liver. In this case, the target tissue is the
abnormal tissue, and tissue specificity reflects the restriction of
vector replication to the abnormal tissue interspersed in the
normal tissue. It is also to be understood that tissue specificity,
in the context of treatment, is particularly relevant in vivo..
However, as described herein, ex vivo treatment and tissue
replacement also falls within the concept of tissue specificity
according to the present invention.
[0061] The term "transcriptional regulatory function" or
"transcriptional regulatory factor" is intended to mean any
cellular function whose presence activates the heterologous
transcriptional regulatory sequence described herein or whose
absence permits transcription as a result of the transcriptional
regulatory sequences described herein.. It is understood that in
the given target tissue, a tissue that "lacks the transcriptional
regulatory factor" or is "deficient" in the transcriptional
regulatory factor could refer to either the absence of the factor
or the functional inactivation of the factor in the target
tissue.
[0062] The term "transcriptional regulatory sequence" is used
according to its art-recognized meaning. It is intended to mean any
DNA sequence which can, by virtue of its sequence, cause the linked
gene to be either up- or down-regulated in a particular cell. In
one embodiment of the present invention, the native transcriptional
regulatory sequence is completely deleted from the vector and
replaced with a heterologous transcriptional regulatory sequence.
The transcriptional regulatory sequence may be adjacent to the
coding region for the gene that is essential for replication, or
may be removed from it. Accordingly, in the case of a promoter, the
promoter will generally be adjacent to the coding region. In the
case of an enhancer, however, an enhancer can be found at some
distance from the coding region such that there is an intervening
DNA sequence between the enhancer and the coding region. In some
cases, the native transcriptional regulatory sequence remains on
the vector but is non-functional with respect to transcription of
the gene essential for replication.
[0063] Various combinations of transcriptional regulatory sequences
can be included in a vector. One or more may be heterologous.
Further, one or more may have the tissue-specificity. For example,
a single transcriptional regulatory sequence could be used to drive
replication by more than one gene essential for replication. This
is the case, for example, when the gene product of one of the genes
drives transcription of the further gene(s). An example is a
heterologous promoter linked to a cassette containing an E1a coding
sequence (E1a promoter deleted) and the entire E1b gene. In such a
cascade, only one heterologous transcriptional regulatory sequence
may be necessary. When genes are individually (separately)
controlled, however, more than one transcriptional regulatory
sequence can be used if more than one such gene is desired to
control replication.
[0064] The vectors of the present invention, therefore, also
include transcriptional regulatory sequence combinations wherein
there is more than one heterologous transcriptional regulatory
sequence, but wherein one or more of these is not tissue-specific.
For example, one transcriptional regulatory sequence can be a basal
level constitutive transcriptional regulatory sequence. For
example, a tissue-specific enhancer can be combined with a basal
level constitutive promoter.
Vectors
[0065] The preferred vectors of the present invention are
adenoviral vectors. In a preferred embodiment of the invention, an
adenovirus vector contains a tissue-specific transcriptional
regulatory sequence linked to a gene in the E1 region.
[0066] In one embodiment, both E1a and E1b are operably linked to
heterologous tissue-specific transcriptional regulatory sequences.
In an alternative embodiment, only E1a is linked; E1b remains
intact. In still another embodiment, E1b is linked, and E1a remains
intact or is deleted. In any case, one or more tissue-specific and
promoter-specific cellular transcriptional regulatory factors
allows virus replication to proceed by transcribing the E1a and/or
E1b gene functionally linked to the promoter. Further, either one
or both of the E1b functions may be linked to the transcriptional
regulatory sequence.
[0067] In alternative embodiments, adenovirus vectors are provided
with any of the other genes essential for replication, such as E2,
E4, under control of a heterologous transcriptional regulatory
sequence.
[0068] The invention further embodies the use of plasmids and
vectors having only the essential regions of adenovirus needed for
replication with either E1a, E1b 19 kDa gene, or E1b 55 kDa gene,
or some combination thereof, modified. Such a plasmid, lacking any
structural genes, would be able to undergo DNA replication.
Accordingly, the vectors of the invention may consist essentially
of the transcriptional regulatory sequence and one or more genes
essential for replication of the vector. In the case of viral
vectors, the vectors may consist essentially of the transcriptional
regulatory sequence and the gene or genes essential for replication
or life-cycle functions of the virus. It is also understood that
these vectors may also further consist essentially of a DNA
sequence encoding one or more toxic heterologous gene products when
such vectors are intended as expression vectors for treatment.
[0069] In broader embodiments, the vector is derived from another
DNA tumor virus. Such viruses generally include, but are not
limited to, Herpesviruses (such as Epstein-Barr virus,
cytomegalovirus, Herpes zoster, and Herpes simplex),
papillomaviruses, papovavirises (such as polyoma and SV40), and
hepatitis viruses.
[0070] The alternative viruses preferably are selected from any
group of viruses in which the essential genes for replication of
the virus can be placed under the control of a tissue-specific
transcriptional regulatory sequence. All serotypes are included.
The only common property of such viruses, therefore, is that they
are transducible into target tissue, are genetically manipulatable,
and are non-toxic when not replicating.
[0071] The relevant viral gene(s) are those that are essential for
replication of the viral vector or of the virus. 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, polymerases from retroviruses and the like.
Essentially, these include any gene that is necessary for the life
cycle of the virus.
[0072] In further embodiments, the vector is derived from an RNA
virus. In still further embodiments, the vector is derived from a
retrovirus. It is understood, however, that potentially any
replicating vector can be made and used according to the essential
design disclosed herein.
[0073] 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: A Laboratory Manual, Cold
Spring Harbor, New York (1989), or any of the myriad of laboratory
manuals on recombinant DNA technology that are widely available. A
variety of strategies are available for ligating fragments of DNA,
the choice of which depends on the nature of the termini of the DNA
fragments and can be readily determined by the skilled artisan.
[0074] An adenovirus vector, in a preferred embodiment, is
constructed first by constructing, according to standard
techniques, a shuttle plasmid which contains, beginning at the 5'
end, the "critical left end elements," which include an adenoviral
5' ITR, an adenoviral encapsidation signal, and an E1a enhancer
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 segment serves as a substrate for homologous recombination
with a modified or mutated adenovirus. The plasmid may also include
a selectable marker and an origin of replication. The origin of
replication may be a bacterial origin of replication.
Representative examples of such shuttle plasmids include pAVS6, as
discussed herein and see Trapnell, B.. et al., Adv. Drug Deliv. Rev
12:185-189 (1994). A desired DNA sequence containing a heterologous
gene may then be inserted into the multiple cloning site to produce
a plasmid vector.
[0075] This construct then is used to produce an adenoviral vector.
Homologous recombination then is effected with a modified or
mutated adenovirus in which one or more of the native
transcriptional regulatory sequences have been deleted and replaced
with the desired transcriptional regulatory sequence. Such
homologous recombination may be effected through co-transfection of
the plasmid vector and the modified adenovirus into a helper cell
line by CaPO.sub.4 precipitation.
[0076] Through such homologous recombination, a vector is formed
which includes adenoviral DNA free of one or more of the native
transcriptional regulatory sequences. This vector may then be
transfected into a helper cell line for viral replication and to
generate infectious viral particles. Transfections may take place
by electroporation, calcium phosphate precipitation,
microinjection, or through proteoliposomes.
[0077] The vector may include a multiple cloning site to facilitate
the insertion of DNA sequence(s) containing 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 appropriate
vector is thus formed by cutting the cloning vector by standard
techniques at appropriate restriction sites in the multiple cloning
site, and then ligating the DNA sequence containing the
heterologous gene into the cloning vector.
[0078] 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 promoter, the
Rous sarcoma virus promoter; inducible promoters, such as the mouse
mammary tumor virus (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 foreign genes or
promoters.
[0079] In one embodiment, the adenovirus may be constructed by
using a yeast artificial chromosome containing an adenoviral genome
according to the method described in Ketner, et al., Proc. Nat.
Acad. Sci. 91:6186-6190 (1994), in conjunction with the teachings
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 containing 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.
[0080] The infectious viral particles may then be administered in
vivo to a host. The host may be an animal host, including
mammalian, non-human primate, and human hosts.
[0081] The viral particles may be administered in combination with
a pharmaceutically acceptable carrier suitable for administration
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.
Treatment
[0082] In preferred embodiments, the methods are specifically
directed to the introduction into a target tissue of a
replication-conditional adenoviral vector. This vector selectively
replicates in the cells of the target tissue. The replication is
conditioned upon the function of a transcriptional regulatory
sequence to which a viral gene is operably linked, which gene is
necessary for vector replication. Thus, in the target tissue,
replication can occur because either a cellular function in the
target tissue allows transcription. Alternatively, there is a
deficiency in a cellular function in the target tissue that
normally prevents or inhibits transcription. 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).
[0083] The present invention thus provides methods for selectively
distributing a polynucleotide in a given tissue in vivo,
significantly reducing or avoiding distribution in non-target
tissue. The polynucleotide is provided in the
replication-conditional vector which is selectively distributed in
the given tissue.
[0084] The present invention also provides methods for selectively
expressing a gene product in a given tissue, avoiding or
significantly reducing expression 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. Successive infection
provides a "domino effect" so that all or substantially all of the
cells in the target tissue is reached. Cells in addition to those
first exposed to the polynucleotide, vector, or gene product, are
thus potentially reached by the methods.
[0085] Such treatment is particularly necessary in cases in which
surgical intervention is not feasible. For example, in patients
with abnormal tissue intimately associated with neural tissue,
surgery may be precluded or highly dangerous. Further, in the case
of multiple metastases or microscopic metastases, surgery is not
feasible.
[0086] In the target tissue, DNA replication alone may occur. Late
viral functions that result in packaging of vector DNA into virions
may also occur.
[0087] 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.
[0088] Tissue specificity is particularly relevant with respect to
targeting an abnormal counterpart of a particular tissue type while
avoiding the normal counterpart of the tissue, or avoiding
surrounding tissue of a different type than the abnormal tissue,
while treating the abnormal tissue. For example, the vectors of the
present invention are useful for treating metastases to the liver.
One specific example is colon cancer, which often metastasizes into
the liver. It has been found that even when colon cancer
metastasizes into the liver, the CEA promoter is active in the
cells of the metastases but not in normal liver cells. Accordingly,
normal human adult liver should not support replication of a virus
that has viral genes essential for replication linked to the colon
cancer CEA-specific promoter. Replication should occur in the
primary cancer cells. Another example is breast cancer, which also
metastasizes to the liver. In this case, the DF3 mucin enhancer is
linked to a gene essential for replication such as both E1a and
E2a. Replication should occur in breast cancer but not in normal
liver. A further example is the .alpha.-fetoprotein promoter, which
is active in hepatocellular carcinoma. This promoter is linked to a
gene essential for replication. It has been found that the promoter
is active only in the hepatocellular carcinoma. Accordingly, a
virus is used that has a gene essential for replication linked to
this promoter. Replication should be limited to hepatocellular
carcinoma. A further example is the tyrosinase promoter. This
promoter is linked to a gene essential for replication. Replication
should occur in melanoma and not in normal skin. In each case,
replication is expected in the abnormal but not the normal
cells.
[0089] In a further 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.
[0090] 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 expression 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. Examples of toxic products include, but are not limited
to, thymidine kinase in conjunction with ganciclovir.
[0091] A wide range of toxic effects is possible. Toxic effects can
be direct or indirect. Indirect effects may result from the
conversion of a prodrug into a 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 terminator and DNA polymerase inhibitor,
prevents DNA synthesis, and thus is cytotoxic. Another example is
the use of cytosine deaminase 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).
[0092] 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.
173:1047-1052 (1991), Colombo, M. P., et al., J. Exp. Med.
173:889-897 (1991), Leone, A., et al., Cell 65:25-35 (1991)),
antisense RNAs and ribozymes (Zaia, J. A. et al., Ann. NY. Acad.
Sci. 660:95-106 (1992)), tumor vaccination genes, and DNA encoding
for ribozymes.
[0093] 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 marker; i.e., a material
which in combination with a chemotherapeutic or interaction agent
inhibits, prevents or destroys the growth of the target cells.
[0094] Thus, upon introduction to the cells of the negative
selective marker, an interaction agent is administered to the host.
The interaction agent interacts with the negative selective marker
to prevent, inhibit, or destroy the growth of the target cells.
[0095] Negative selective markers which may be used include, but
are not limited to, thymidine kinase and cytosine deaminase. In one
embodiment, the negative selective marker is a viral thymidine
kinase selected from the group consisting of 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 consisting of ganciclovir, acyclovir, and
1-2-deoxy-2-fluoro-.beta.-D-arabinofuranosil-5-iodouracil (FIAU).
Such interaction agents are utilized efficiently by the viral
thymidine kinases as substrates, and such interaction agents thus
are incorporated lethally into the DNA of the tumor cells
expressing the viral thymidine kinases, thereby resulting in the
death of the target cells.
[0096] When cytosine deaminase is the negative selective marker, a
preferred interaction agent is 5-fluorocytosine. Cytosine deaminase
converts 5-fluorocytosine to 5-fluorouracil, which is highly
cytotoxic. Thus, the target cells which express the cytosine
deaminase gene convert the 5-fluorocytosine to 5-fluorouracil and
are killed.
[0097] The interaction agent is administered in an amount effective
to inhibit, prevent, or destroy the growth of the target cells. For
example, the interaction agent is administered in an amount based
on body weight and on overall toxicity to a patient. The
interaction agent preferably is administered systemically, such as,
for example, by intravenous administration, by parenteral
administration, by intraperitoneal administration, or by
intramuscular administration.
[0098] When the vectors of the present invention induce a negative
selective marker and are administered to a tissue or tumor in vivo,
a "bystander effect" may result, i.e., cells which were not
originally transduced with the nucleic acid sequence encoding the
negative selective marker may be killed upon administration of the
interaction agent. Although the scope of the present invention is
not intended to be limited by any theoretical reasoning, the
transduced cells may be producing a diffusible form of the negative
selective marker that either acts extracellularly upon the
interaction agent, or is taken up by adjacent, 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.
[0099] 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
interferons and colony-stimulating factors, such as GM-CSF.
Interleukins include, but are not limited to, interleukin-1,
interleukin-1.beta., and interleukins-2-15. In one embodiment, the
interleukin is interleukin-2.
[0100] In a preferred embodiment of the invention, the target
tissue is abnormally proliferating, and preferably tumor tissue.
The vector or virus is distributed throughout the tissue or tumor
mass.
[0101] All tumors are potentially amenable to treatment with the
methods of the invention. Tumor types include, but are not limited
to hematopoietic, pancreatic, neurologic, hepatic, gastrointestinal
tract, endocrine, biliary tract, sino-pulmonary, head and neck,
soft tissue sarcoma and carcinoma, dermatologic, reproductive
tract, and the like. Preferred tumors for treatment 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.
[0102] The methods can also be used to target other abnormal cells,
for example, any cells in which are harmful or otherwise unwanted
in vivo. Broad examples include cells causing autoimmune disease,
restenosis, and scar tissue formation.
[0103] Further, treatment can be ex vivo. Ex vivo transduction of
tumor cells would overcome many of the problems with current viral
delivery systems. Tissue is harvested under sterile conditions,
dissociated mechanically and/or enzymatically and cultured under
sterile conditions in appropriate media. Vector preparations
demonstrated to be free of endotoxins and bacterial contamination
are used to transduce 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-containing media cells are immediately returned to
the patient or are maintained for several days in culture while
testing for function or sterility is performed.
[0104] 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).
[0105] Viral transduction also has potential applications in the
area of experimental medicine. Transient expression of biological
modifiers of immune system function such as IL-2, IFN-.gamma.,
GM-CSF or the B7 co-stimulatory protein has been proposed as a
potential means of inducing anti-tumor responses in cancer
patients.
[0106] In broader embodiments, the vector is derived from another
DNA tumor virus. Such viruses generally include, but are not
limited to, Herpesviruses (such as Epstein-Barr virus,
cytomegalovirus, Herpes zoster, and Herpes simplex),
papillomaviruses, papovaviruses (such as polyoma and SV40), and
hepatitis viruses.
[0107] The relevant viral gene(s) are those that are essential for
replication of the viral vector or of the virus. 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, polymerases from retroviruses and the like.
Essentially, these include any gene that is necessary for the life
cycle of the virus.
[0108] In further embodiments, the vector is derived from an RNA
virus. In still further embodiments, the vector is derived from a
retrovirus. It is understood, however, that potentially any
replicating vector can be made and used according to the essential
design disclosed herein.
Diagnostic
[0109] It is important to know whether the vectors of the invention
will replicate in a specific tissue from a patient. If vector
replication is found to be beneficial for therapy, then a screen is
provided for those patients who best respond to the therapy
disclosed herein. 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 treatment. Currently, the only
non-biological assays that are commonly used are expression
screening, PCR, and sequencing. These often result in false
negatives, are time-consuming, expensive, and yield only
information in the best of cases about the status of the genes and
not their biological function.
[0110] Accordingly, a method is provided for identifying an
abnormal tissue, the cells of which contain a transcription factor
that allows replication of a replication-conditional vector, or are
deficient for an inhibitory factor for transcription.
[0111] In this method, a tissue biopsy is explanted, a
replication-conditional vector is introduced into the cells of the
biopsy, and vector DNA replication in the cells is quantitated.
Accordingly, a method is provided for screening tissue for the
presence of factors that allow vector replication, or for a
deficiency of a factor that inhibits transcription. Such a screen
is useful, among other things, for identifying tissue, prior to
treatment, which will be amenable to treatment with a particular
vector to be replicated in the tissue.
[0112] Therefore, a method is provided for assaying vector utility
for treatment by removing a tissue biopsy from a patient,
explanting the biopsy into tissue culture, introducing the
replication-conditional vector into the biopsy, and assaying vector
replication in the cells of the biopsy.
[0113] Testing or screening of tissues includes an assay for vector
nucleic acid replication or for virus replication, when the vector
is capable of forming infectious virions.
[0114] Thus, the invention provides a method for screening a tumor
for transcription regulatory functions that allow vector
replication or for the absence of these functions which would
normally prevent the replication of a virus vector.
[0115] However, any abnormal tissue can be screened for the
functions described above by an assay for nucleic acid or virus
replication.
Producer Cells
[0116] 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 the replication-conditional vectors of the present
invention. Thus, the invention provides "producer cells" for the
efficient and safe production of recombinant
replication-conditional vectors for further use for targeted gene
therapy in vivo.
[0117] One of the major problems with the currently 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 contaminate the vector or virus
preparation produced in the producer cell. Such contamination 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.
[0118] Accordingly, the invention embodies methods for constructing
and producing replication-conditional virions in a cell comprising
introducing the replication-conditional vector of the present
invention into the cell wherein the genome of the cell is devoid of
vector sequences, replicating the vector in the cell, forming the
virion, and purifying the virion from the cell. Preferred vectors
are DNA viral vectors, including but not limited to herpesvirus,
papillomavirus, hepatitis virus, and papovavirus vectors. In
preferred embodiments of the invention, the virion is an adenoviral
virion and the vector is an adenoviral vector. In further
embodiments of the invention, the cell is a tumor cell.
[0119] In a further preferred embodiment, 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 facilitated by expression of the
heterologous gene product. Alternatively, the producer cell can be
used for the production of a heterologous gene product per se
encoded by the vector. When the vector replicates in the producer
cell, the gene product is expressed from the multiple copies of the
gene encoding the gene product. Following expression, the gene
product can be purified from the producer cells by conventional
lysis procedures, or secreted from the producer cell by appropriate
secretion signals linked to the heterologous gene by known methods.
The transduction of cells by adenoviral vectors has been described.
Transfection of plasmid DNA into cells by calcium phosphate
(Hanahan, 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).
[0120] Preferred hosts for producer cell lines include but are not
limited to HuH7, SW480, BIGF10, HepG2, MCF-7, and SK-MEL2. Primary
tumors from which cell lines can be derived, or existing cell
lines, can be tested for the ability to allow replication by means
of the tissue-specific transcriptional regulatory sequence. Any
primary tumor could be explanted and developed into producer cells
for the vectors of the present invention. As long as the cell does
not contain endogenous vector or viral sequences that could
recombine with the vector or virus to produce wild-type vector or
virus, the cell is potentially useful as a host. It is understood
that any cell is potentially useful, not only tumor cells.
[0121] The ultimate 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 contamination 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 high growth rate and will grow to a high density. The cell should
also have a high amount of viral receptor so that the virus can
easily infect the cell. Another characteristic is the quality of
the vector produced (i.e., the preparation should not include a
high amount of non-infectious viral particles). Accordingly,
candidate producer cells would have a low
particle-to-plaque-forming-unit ratio. Thus, these cells are a
preferred cell type for deriving a producer cell line. Primary
explants or the known cell lines can be used.
[0122] Thus, such obtainable cells can serve as producer cells for
recombinant replication-conditional vectors, viruses, and gene
products.
Introduction of Vectors into Cells
[0123] A variety of ways have been developed to introduce vectors
into cells in culture, and into cells and tissues of an animal or a
human patient. Methods for introducing vectors into mammalian 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, A 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, Maniatis et al., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York (1989), herein incorporated by
reference in its entirety.
[0124] Several of these techniques have been used to introduce
vectors into tissues and cells in animals and human patients. Chief
among these have been systemic administration and direct injection
into sites in situ. Depending on the route of administration 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 abnormal cells and tissues,
generally by direct injection into a targeted site.
[0125] The aforementioned techniques for introducing
polynucleotide, viral and other vectors into cells in culture, in
animals and in patients can be used to develop, test and produce,
as well as use vectors in accordance with the invention. For
instance, cells containing a vector introduced by these methods can
be used for producing the vector. In addition, cells containing a
vector can be used as producer-cells and introduced into cells or
tissues of an animal to produce the vector in situ.
Assay of DNA and Viral Replication
[0126] 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 determining the amount of a
labelled substrate incorporated into a polynucleotide during a
given period in a cell.
[0127] When replication involves a DNA polynucleotide,
.sup.3H-thymidine often is used as the labelled substrate. In this
case, the amount of replication is determined by separating DNA of
the vector from the bulk of cellular DNA and measuring the amount
of tritium incorporate specifically into vector DNA.
[0128] Replication of a polynucleotide vector also may be detected
by lysing or permeating cells to release the polynucleotide, then
isolating the polynucleotide and quantitating directly the DNA or
RNA that is recovered. Polynucleotide replication also may be
detected by quantitative PCR using primers that are specific for
the assay polynucleotide.
[0129] Virions may be assayed by EM counting techniques well known
to the art, by isolating the virions and determining protein and
nucleic acid content, and by labelling viral genomic
polynucleotides or virion proteins and determining the amount of
virion from the amount of polynucleotide or protein.
[0130] It is well known that virions may not all be viable and
where infectivity is important, infectious titer may be determined
by cytopathic effect or plaque assay.
[0131] 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.
[0132] Having thus described herein the invention in general terms,
the following examples are presented to illustrate the invention.
Examples 1-4 show the replacement of the constitutive E1A promoter
on an adenoviral vector with a tumor-specific promoter. Constructs
made this way have the E1a protein expressed only in tumor cells
and therefore, will replicate only in tumor cells.
EXAMPLE 1
The Hepatoma-specific Promoter, .alpha.-fetoprotein Promoter,
Linked to E1a
[0133] The .alpha.-fetoprotein promoter has been previously shown
to be highly active in hepatoma cells and silent in adult
hepatocytes and other adult tissues. A 4.9 kb .alpha.-fetoprotein
promoter containing construct was used to derive the promoter.
Alternatively, the promoter could also be made based on available
references.
[0134] The adenovirus shuttle plasmid pAVS21.TK1 (FIG. 1), which
has the TK gene under the control of the 4.9 kb .alpha.-fetoprotein
promoter, was made exactly as described in FIGS. 11 and 12 of U.S.
patent application Ser. No. 08/444,284 of Chiang et al. for "Gene
Therapy of Hepatocellular Carcinoma Through Cancer-Specific Gene
Expression", filed on May 18, 1995, which is incorporated herein by
reference for its relevant teaching. pAVE1a02i (FIG. 1) which
places the E1a/E1b genes under the control of the
.alpha.-fetoprotein promoter in an adenovirus shuttle plasmid was
cloned by purifying a restriction fragment which contained the E1a
coding region only and all of E1b gene by cleaving the plasmid
pSE280-E1 (FIG. 1) with SpeI and MunI and ligating this to
pAVS21.TK1 cleaved with MunI and NheI. Plasmid SE280-E1, which
contains the E1A ORF and all of E1b, was constructed as described
in U.S. patent application Ser. No.08/458,403 to Kadan et al. for
"Improved Adenoviral Vectors and Producer Cells," filed Jun. 2,
1995, which is incorporated herein by reference for its relevant
teaching. pAVE1a02i is cotransfected with the large ClaI fragment
of Add327 by standard methods into 293 cells to generate
recombinant virus.
Construction of a Virus with the Hepatoma-specific AFP Promoter
Operably Linked to the E1a Gene
[0135] The adenovirus AVE1a04i was constructed by homologous
recombination of the shuttle plasmid, pAVE1a04i (See FIG. 2), with
the large (Cla1) fragment of AV1lacZ4 DNA in 293 cells. The
construction of the plasmid pAVE1a02i is described above. The
construction of pAVE1a04i is almost identical to that of pAVE1a02i.
pAVE1a02i contains the entire AFP promoter. pAVE1a04i utilizes a
derivative of this promoter, which has six silencer elements and a
duplicated enhancer region.
[0136] The plasmid pAF(AB).sub.2(Sd).sub.6-CAT was constructed by
placing six copies of the distal silencer immediately upstream of
the basal 200 base pair AFP promoter. Two copies of the enhancer AB
region, in opposite orientation, are placed immediately upstream of
the silencer elements. This promoter, extending from the enhancer
element through the basal AFP promoter, was used to make the AV/AFP
short E1a virus with the shuttle plasmid described herein. The
distal silencer element, the basal promoter, and the enhancer
elements are as described in Nakabayashi et al. (Molec. & Cell.
Biol. 11:5885-5893 (1991)).
[0137] The plasmid pAVE1a04i was grown in STBL2 cells and was
purified by standard cesium banding methods prior to use in
transfection. Genomic AV1lacZ4 DNA was isolated from cesium
gradient-purified virus (herein described). The AV1lacZ4 purified
virus was digested with proteinase K and the DNA isolated by
phenouchloroform extraction. The purified DNA was digested with
Cla1 and the large fragment was isolated by gel electrophoresis and
quantified. 5 .mu.g of the plasmid pAVE1a04i and 2.5 .mu.g of the
large Clal fragment of AV1lacZ4 were co-transfected into 293 cells
using a calcium phosphate-mediated transfection procedure (Promega,
E1200 kit). The transfection plate was overlayered with a 1%
agarose overlay and incubated until plaques formed. Once plaques
had formed, they were picked and the virus was released into 500
.mu.l of IMEM media by alternate cycles of freezing and thawing
(5.times.). The eluted viral plaques were reamplified on A30 cells
for 48 hours and then the cells were lysed for use in screening by
PCR.
[0138] Primers specific for the short AFP (sAFP) promoter in
plasmid pAVE1a04i were used to identify the putative plaques. FIG.
2A shows that viral plaques contain a sAFP-specific band of the
predicted molecular weight and specific for the sAFP primers. To
confirm that this recombinant virus was not contaminated with
Ad5d1327 (wild type), E1a primers were used. FIG. 2B demonstrates
that no wild type virus was present and that pAVE1a04i plasmid
sequences were present in the recombinant virus. FIG. 2C
demonstrates that little or no AV1lacZ4 was present. The data
indicate the construction of a virus with E1a under control of a
tissue-specific promoter and that the virus is capable of
replication in A30 cells.
[0139] Individual plaques were grown in A30 cells and analyzed by
PCR for the presence of the AFP promoter (FIG. 2). The arrow
indicates the AFP-specific band generated from PCR. The figure
shows that the band is present in each of the viruses in the
selected plaques (L6, L10, L11, M1 and M2). The control in the
experiment was an A30 cell lysate, expected not to contain the
band. The experiment also included the PCR reaction with the
plasmid pAVE1a04i (the shuttle plasmid from which the virus was
made and which therefore should produce the AFP-specific fragment).
Thus, FIG. 2A confirms the presence of a recombinant virus
containing the AFP promoter. FIGS. 2B and 2C confirm that these
results were not the result of contamination in the individual
plaques. FIG. 2B uses E1a-specific primers to detect the presence
of any contaminating wild-type virus. The arrow shows the band
produced with E1a-specific primers. The figure shows that none of
the recombinant viruses produced the relevant band. FIG. 2C
confirms that there is no AV1.lacZ contamination in the viral
plaques (since the viruses were made using AV1.lacZ DNA). The
figure indicates that only the lane containing AV1.lacZ DNA
produced the band.
Tissue-specific Viral Replication
[0140] Cytopathic viral lysate of this virus ("AVAFPE1a") was
serially diluted in logs of 10 on A549.30 cells, A549 cells, and
HuH 7 cells. A549.30 cells express the E1a from the glucocorticoid
receptor element (GRE) promoter in the presence of dexamethasone
since this construct is integrated into the genome of this cell
line. Thus, any E1a-deleted virus or any virus not expressing E1a
should be able to replicate in this cell line. This has previously
been shown for E1-deleted vectors (unpublished communication). As
can be seen from FIGS. 3A and 3D, the AVAFPEI a vector replicates
in the infected cells as indicated by characteristic cytopathic
effects and spreading of cell death. The A549 cells do not express
AFP and should not be capable of transactivating the AFP promoter.
In addition, A549 cells do not express E1a. Thus, AVAFPE1a should
not be able to replicate in this cell line. As can be seen from
FIGS. 3B and 3E, both uninfected and infected wells appear
identical with no characteristic cytopathic effects or spreading
observed at all dilutions tested. HuH 7 cells do express AFP,
should transactivate the AFP promoter, and should make E1a with
subsequent replication. As shown in FIGS. 3C and 3F, AVAFPE1a
clearly replicates, as indicated by the cytopathic effects. In
addition, on several wells of infected HuH 7 cells, the replication
began with a single plaque which spread throughout the rest of the
well within one week. All HuH 7 wells showing cytopathic effects
were tested by PCR and demonstrated to be free of wild-type virus
and AV1 LacZ4 virus, and to contain an intact AFP promoter. These
data clearly indicate that a virus has been constructed that is
capable of replicating specifically in tumor cells expressing
AFP.
EXAMPLE 2
The Breast Cancer-specific DF3-Mucin Enhancer
[0141] The DF3 breast carcinoma associated antigen (MUC1) is highly
overexpressed in human breast carcinomas. The expression of the
gene is regulated at the transcriptional level. The DNA sequence
between -485-588 is necessary and sufficient for conferring a
greater than 10-fold increase in transcription of the reporter gene
CAT when placed immediately upstream of a basal promoter derived
from the Herpesvirus TK promoter in transient transfection assays
performed in the human breast cancer cell line MCF-7. A specific
transcription factor which binds to this region of DNA has also
been found within cells derived from the breast cancer cell line
MCF-7 but not a non-breast cancer cell line HL-60. The same region
of DNA has been found to promote breast cancer-specific expression
of the TK gene in the context of a retroviral construct or an
adenoviral construct.
[0142] The DF3 enhancer from -598 to 485 (obtained from GenBank)
was synthesized by constructing four oligonucleotides synthesized
in such a way as they would overlap and anneal. The
oligonucleotides are shown in Table 1. Additional restriction sites
were added on both ends for future ease of cloning. One end was
kept blunt to enable cloning into the Smal site of the vector
pTK-Luc. This vector contains the basal promoter of the Herpesvirus
TK gene which gives low level basal activity in a variety of cells.
It was used as a source of this basal promoter. The other end had
an overlapping BglII site for ease in cloning into the BglII site
of pTK-Luc. 1,000 ng of each oligonucleotide were annealed in 0.017
M Tris, pH 8.0, 0.16 M NaCl in a total volume of 26.5 .mu.l by
heating at 95.degree. C. for two minutes and allowing to cool to
room temperature after several hours. Finally, 1 .mu.l of this
mixture was ligated to 100 ng of previously SmaI/BglII--and glass
milk (BIO 101)--purified vector by standard conditions. Following
transformation into DH5.alpha. cells (GIBCO), colonies were
screened for the presence of the insert by standard restriction
digests. DNA derived from this vector is then cleaved with HindIII
and blunted by Klenow. It is then cleaved by AscI. This fragment,
which contains the DF3 enhancer lined to the basal TK promoter, is
then purified by agarose gel electrophoresis and glass milk and
ligated to the plasmid pAVE1a02i, cleaved with Spe I and
blunt-ended with AscI and purified as above. The resultant plasmid
has the E1A gene product under the control of the DF3 enhancer and
basal TK promoter and is in an adenoviral shuttle plasmid. 5 .mu.g
of this plasmid, pAVE1a03i, is cotransfected with 5 .mu.g of the
right ClaI fragment arm, derived from Add1327, into 293 cells.
Plaques are screened for the expected recombinant virus by standard
methods.
[0143] A crude virus lysate is used to infect MCF-7 at an MOI of
10. Virus stocks are confirmed to replicate specifically in breast
cancer cells by standard methods. Virus is scaled up in MCF-7 cells
and/or 293 cells as described for scaleup and purification on 293
cells. Virus stocks are tested for replication in vivo by using a
mode mouse model of MCF-7 and, as a negative control, a cervical
cancer (Hela) derived tumor is used. Virus is tested for a
recombinational event in 293 cells which would generate a wild-type
virus by PCR assay of the original E1A promoter which would only be
in a wild-type virus. A variety of other human and rat breast
cancer cell lines and non-related cell lines are also tested. The
TK gene can be inserted into the E3 region and have TK driven
either by the E1A-dependent promoter present there or under the
control of the RSV or CMV promoter.
EXAMPLE 3
The Melanoma-specific Tyrosinase Promoter
[0144] PCR primers and PCR were used to clone a fragment of DNA 800
bp upstream of the tyrosinase gene from mouse genomic DNA using PFU
and the described primers as described by Stratogene. The resultant
PCR fragment was cloned into pCRSCRIPT and then recloned into
pAVE1a02i by digesting the new plasmid with AscI/SpeI and pAVE1a01i
with AscI/SpeI and ligating the two together. The final shuttle
plasmid, pAVE1a04i, which has E1a/E1b under the control of the
tyrosinase promoter, is utilized to make a recombinant virus
identically as described above.
EXAMPLE 4
The Colon Cancer-specific CEA Promoter
[0145] The CEA promoter was cloned from human genomic DNA as
described above and cloned in a similar way into the pAVE1a01i
plasmid using the primers shown in Table 1. The final shuttle
plasmid, pAVE1a05i, is used to generate recombinant virus as
described above.
EXAMPLE 5
[0146] A. Replacing the Promoter of E2a on an adenoviral vector
with a tumor specific promoter Constructs made as above will have
the E2a protein (essential for viral replication expressed only in
tumor cells. Therefore, replication of the vector occurs only in
tumor cells. All four of these very specific promoters (in the
examples above) are used to place the E2a coding region obtained
from pSE280-E2a (see U.S. patent application Ser. No. 08/458,403 of
Kadan et al., "Improved adenoviral vectors and producer cells"
filed Jun. 2, 1995). under the control of that tumor-specific
promoter. The resultant plasmid is recombined with Add1327, using
standard methods of homologous recombination. The final virus is
grown in the cell lines described in the aforementioned patent
application or in the tumor specific cell lines. The E2a protein,
because it is needed in stoichiometric amounts, has the ability to
regulate the degree of replication over a broad range. This is
desirable for therapy. The methods used are the same as those
described for E1a. The difference is that a shuttle plasmid is used
that places E2a under the control of the tumor specific promoter
and returns it to a virus backbone (by homologous recombination)
that has the E2a and E3 genes deleted.
[0147] B. Replacement of Other Therapeutic Toxic Genes into the
Tumor-specific Replication Competent Vectors
[0148] Genes such as TK, cytokines, or any therapeutic genes can be
placed into the E3 region of the vector backbone by standard
plasmid construction and homologous recombination. Those genes can
be placed under the control of an E1a-dependent promoter, or a
constitutive promoter such as RSV or CMV.
[0149] The disclosures of all patents, publications (including
published patent applications), and database accession numbers
referred to in this specification are specifically incorporated
herein by reference in their entirety to the same extent as if each
such individual patent, publication, and database accession numbers
were specifically and individually indicated to be incorporated by
reference in its entirety. TABLE-US-00001 TABLE 1 Oligonucleotide
Primers for Constructing Tissue- Specific Promoters 1. DF3 (Breast
Cancer) 5' GGG CGC GCC CTG GAA AGT CCG GCT (SEQ ID NO: 1) GGG GCG
GGG ACT GTG GGT TTC AGG GTA GAA CTG CGT GTG GAA 3' 5' CGG GAC AGG
GAG CGG TTA GAA GGG (SEQ ID NO: 2) TGG GGC TAT TCC GGG AAG TGG TGG
GGG GAG GGA ACT AGT A 3' 5' GAT CTA CTA GTT CCC TCC CCC CAC (SEQ ID
NO: 3) CAC TTC CCG GAA TAG CCC CAC CCT TCT AAC CGC TCC CTG 3' 5'
TCC CGT TCC ACA CGC AGT TCT ACC (SEQ ID NO: 4) CTG AAA CCC ACA GTC
CCC GCC CCA GCC GGA CTT TCC AGG GCG CGC CC 3' 2. Tyrosinase
(Melanoma) 5' GAC CCG GGC GCG CCG GAG CAG TGC (SEQ ID NO: 5) TAT
TCA AAC CAT CCA G 3' 5' CGA GAT CTA CTA GTT CTG CAC CAA (SEQ ID NO:
6) TAG GTT AAT GAG TGT C 3' 3. CEA Promoter (Hepatocellular
Carcinoma) 5' GAC CCG GGC GCG CCT CTG TCA CCT (SEQ ID NO: 7) TCC
TGT TGG 3' 5' CGA GAT CTA CTA GTT CTC TGC TGT (SEQ ID NO: 8) CTG
CTC TGT C 3'
[0150]
Sequence CWU 1
1
* * * * *