Note: Descriptions are shown in the official language in which they were submitted.
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MULTIGENE VECTORS
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BACKGROUND OF THE INVENTION
This application claims priority to and specifically incorporates by
reference, the
content of U.S. Provisional Application Serial No. 60/078,205 filed March 16,
1998. The
entire text of each of the above-referenced disclosures is specifically
incorporated by
reference herein without disclaimer.
1. Field of the Invention
The present invention relates generally to the fields of gene transfer and
gene
therapy. More particularly, it concerns the development of viral vectors,
particularly
adenoviral vectors, that deliver specific combinations of genes to a target
cell, in vitro or
in vivo.
2. Description of Related Art
Gene replacement or augmentation is now considered a powerful tool in the
therapeutic 'intervention of a variety of diseases and in particular, in
cancer. The
therapeutic treatment of diseases and disorders by gene therapy involves the
transfer and
stable or transient insertion of new genetic information into cells. The
correction of a
genetic defect by re-introduction of the normal allele of a gene encoding the
desired
function has demonstrated that this concept is clinically feasible (Rosenberg
et al.
(1990)). Indeed, preclinical and clinical studies covering a large range of
genetic
disorders currently are underway to solve basic issues dealing with gene
transfer
efficiency, regulation of gene expression, and potential risks of the use of
viral vectors.
The majority of clinical gene transfer trials that employ viral vectors
perform ex vivo gene
transfer into target cells which are then administered in vivo. Viral vectors
also may be
given in vivo but repeated administration may induce neutralizing antibody.
Of the different viral vectors attempted to mediate gene transfer, the
retrovirus and
adenovirus-based vector systems have been extensively investigated. Recently,
adeno-
associated virus (AAV) has emerged as a potential alternative to the more
commonly
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used retroviral and adenoviral vectors (Muzyczka, 1992; Carter, 1992; Flotte
and Carter,
1995; Chatterjee et al., 1995; Chatterjee and Wong, 1996). While studies with
retroviral
and adenoviral mediated gene transfer raise concerns over potential oncogenic
properties
of the former, and immunogenic problems associated with the latter, AAV has
not been
associated with any such pathological indications (Berns and Bohenzky, 1987;
Berns and
Giraud, 1996). However, the single-stranded nature of the AAV genome
significantly
impacts upon the transduction efficiency since the second-strand viral DNA
synthesis is
the rate-limiting step. Nonetheless, these vectors continue to be used
extensively in gene
therapy application.
It is important, therefore, in gene transfer therapies, to use as little of
the viral
vector as possible whilst effectively killing as many of the cells as quickly
as possible.
This can be achieved using combinations of gene therapy with other traditional
therapies,
as well as, with other gene therapies. Additional gene therapies require the
use of
1 S separate vectors for each therapeutic construct, this presents a variety
of problems
including immunogenicity, oncogenicity and minimal transduction efficiency as
described above. Further, the use of separate delivery vectors does not result
in the
consistent, reproducible expression of both genes in the same target cell.
It would be useful to develop a vector system that allows for the simultaneous
delivery of two or more therapeutic genes to a target cell. Once such a vector
system is
elucidated, it will be possible to increase the efficiency and effectiveness
of a gene based
therapeutic intervention in, for example, hyperproliferative disorders.
SUMMARY OF THE INVENTION
Therefore, the present invention is directed to the use of particular gene
combinations in genetic therapy. Delivery of multiple genes to a target cell
at the same
time augments the action of one or both genes. This is particularly effective
in attacking
diseased cells such as those making up hyperplastic or neoplastic tissues.
Methods and
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compositions for achieving such simultaneous delivery are described in detail
herein
below.
In a preferred embodiment, the present invention provides an expression
construct
comprising at least two different genes selected from the group consisting of
a tumor
suppressor and a cytokine, a tumor suppressor and an enzyme, a tumor
suppressor and an
antisense oncogene, a tumor suppressor and a toxin, a cytokine and an toxin, a
cytokine
and an antisense oncogene, an antisense oncogene, a toxin and an enzyme and a
toxin, a
tumor suppressor and an inducer of apoptosis, a cytokine and an inducer of
apoptosis, an
IO antisense oncogene and an inducer of apoptosis, an enzyme and an inducer of
apoptosis,
and a toxin and an inducer of apoptosis; and a first promoter active in
eukaryotic cells
positioned 5' to the different genes.
In other embodiments, the expression construct may further comprise an
internal
ribosome entry site (IRES), wherein the IRES is positioned 3' to the upstream
gene and
S' to the downstream gene. In alternative embodiments, the expression
construct may
further comprises a second promoter, wherein the second promoter is positioned
3' to the
upstream gene and S' to the downstream gene. In a particularly preferred
embodiment,
the expression construct comprises tumor suppressor and a cytokine. In
alternative
preferred embodiment, the expression construct comprises a tumor suppressor
and an
enzyme. In still further preferred embodiments, the expression construct
comprises a
tumor suppressor and an antisense oncogene. In yet another alternative
embodiment, the
expression construct comprises a tumor suppressor and a toxin. In another
embodiments,
the expression construct comprises a cytokine and an toxin. In yet another
embodiment,
the expression construct comprises a cytokine and an antisense oncogene. In
still another
embodiment, the expression construct comprises an antisense oncogene and a
toxin.
Another alternative provides an expression construct comprising an enzyme and
a toxin.
Also preferred is an expression construct comprising a tumor suppressor and an
inducer
of apoptosis. 1n yet another preferred embodiment, there is provided an
expression
construct comprising a cytokine and an inducer of apoptosis. In yet another
alternative,
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the expression construct comprises an antisense oncogene and an inducer of
apoptosis.
Also contemplated is an expression construct comprising an enzyme and an
inducer of
apoptosis. In yet another embodiment, the expression construct comprises a
toxin and an
inducer of apoptosis.
In those embodiments employing a tumor suppressor as part of the expression
construct, the tumor suppressor may be any tumor suppressor known to those of
skill in
the art. In particularly preferred embodiment, the tumor suppressor may be
selected from
the group consisting of p53, p16, p21, Rb, p15, BRCA1, BRCA2, zacl, p73,
MMAC1,
ATM, HIC-1, DPC-4, FHIT, NF2, APC, DCC, ING1, NOEY1, NOEY2, PML, OVCA1,
MADR2, WT1, PTEN, 53BP2, IRF-1 and C-CAM.
In those embodiments employing a cytokine as part of the expression construct,
the cytokine may be any cytokine known to those of skill in the art. In
especially
1 S preferred embodiments, the cytokine may be selected from the group
consisting of IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,
IL-15, GM-
CSF, G-CSF, TNF, ~i-interferon and y-interferon.
Likewise, those embodiments employing an enzyme as part of the expression
construct may employ an enzyme selected from the group consisting of cytosine
deaminase, adenosine deaminase, hypoxanthine-guanine
phosphoribosyltransferase,
galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,
glucocerbrosidase,
collagenase, sphingomyelinase, a-L-iduronidase, glucose-6-phosphate
dehydrogenase,
HSV thymidine kinase and human thymidine kinase.
Particular embodiments employ oncogenes as part of the expression construct,
any oncogene known to those of skill in the art may be used herein. In
especially
preferred embodiments, the oncogene may be selected from the group consisting
of ras,
myc, neu, raf, erb, src, fms, jun, trk, ret, hst, gsp, bcl-2 and abl. In
specific embodiments,
the expression construct may comprise a toxin, in particularly preferred
embodiments, the
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toxin may be selected from the group consisting of ricin A chain, diphtheria
toxin,
pertussis toxin, Pseudomonas toxin, E coli enterotoxin, and cholera toxin. In
those
embodiment employing an inducer of apoptosis, particularly preferred inducers
of
apoptosis may be selected from the group consisting of Bax, Bak, Bcl-Xs, Bik,
Bid, Bad,
Harakiri, TRAIL, SARP-2, AdElb and an ICE-CED3 protease. Particularly
preferred
examples of gene combinations are listed in Table 1 herein below. Of course
these are
only exemplary combinations and given the teachings of the present invention
one of skill
in the art will be able to produce multigene constructs of any conceivable
combination,
including but not limited to combinations of two, three, four five or more
genes or nucleic
acid constructs. By "nucleic acid constructs" the present invention refers to
a nucleic acid
the can encode a defined portion of or the whole of a particular gene.
In particularly preferred embodiments, the promoters used in the present
invention
may be selected from the group consisting of CMV IE, SV40 IE, RSV, human
ubiquitin
C, ~i-actin, tetracycline regulatable and ecdysone regulatable. These
promoters
independently may be used as the first promoter, or as the second or
substituent promoter.
In particular aspects of the present invention the expression construct may
further
comprise a polyadenylation signal positioned 3' to the downstream gene. In
particularly
preferred embodiments, the expression construct may comprise a first
polyadenylation
signal positioned 3' to the upstream gene and S' to the downstream gene and a
second
polyadenylation signal positioned 3' to the downstream gene. In particularly
preferred
embodiments, the polyadenylation signal may be from BGH, thymidine kinase or
SV40.
In more defined embodiments it is contemplated that the first polyadenylation
signal is
from BGH or SV40, and the second polyadenylation signal is from BGH when the
first
polyadenylation signal is from SV40, and the second polyadenylation signal is
from
SV40 when the first polyadenylation signal is from BGH.
In other aspects of the present invention, the expression construct may be a
viral
vector. In preferred embodiments, the viral vector may be selected from the
group
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consisting of retrovirus, adenovirus, vaccinia virus, herpesvirus and adeno-
associated
virus.
In those embodiments employing an adenoviral vector, it is contemplated that
the
S adenovirus vector is replication deficient. In particularly preferred
aspects, the
adenovirus vector lacks at least a portion of the E 1 region. In other
especially preferred
aspects the adenovirus lacks at least a portion of the E1B region. In
alternative
embodiments, the adenovirus lacks the entire EI region.
Also contemplated by the present invention is an expression construct
comprising
a cytokine gene and an enzyme gene; and a first promoter active in eukaryotic
cells
positioned 5' to the genes, wherein either (i) the cytokine gene is not an IL-
2 gene or (ii)
the enzyme is not a herpesvirus thymidine kinase gene.
I S Further the present invention provides a method for the simultaneous
expression
of two polypeptides in a cell comprising providing an expression construct
comprising at
least two different genes selected from the group consisting of a tumor
suppressor and a
cytokine, a tumor suppressor and an enzyme, a tumor suppressor and an
antisense
oncogene, a tumor suppressor and a toxin, a cytokine and an toxin, a cytokine
and an
antisense oncogene, an antisense oncogene, a toxin and an enzyme and a toxin,
a tumor
suppressor and an inducer of apoptosis, a cytokine and an inducer of
apoptosis, an
antisense oncogene and an inducer of apoptosis, an enzyme and an inducer of
apoptosis,
and a toxin and an inducer of apoptosis; and a first promoter active in
eukaryotic cells
positioned 5' to the different genes; and transferring the expression
construct into the cell,
whereby expression of the gene is effected.
In particularly preferred embodiments, the expression construct is a viral
vector
and the transferring is achieved by viral infection. In other embodiments, the
expression
construct is formulated in a liposome and the transfer is achieved by cellular
uptake of the
liposome. In particular embodiments, the cell is a tumor cell and the cell is
killed by
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expression of the different genes. In more particular embodiments, the tumor
cell is
selected from the group consisting of a prostate cancer cell, a lung cancer
cell, a brain
cancer cell, a skin cancer cell, a liver cancer cell, a breast cancer cell, a
lymphoid cancer
cell, a stomach cancer cell, a testicular cancer cell, an ovarian cancer cell,
a pancreatic
S cancer cell, a bone cancer cell, a bone marrow cancer cell, a head and neck
cancer cell, a
cervical cancer cell, a colon cancer cell, a rectal cancer cell, a blood
cancer cell, an
esophagus cancer cell, an eye cancer cell, a gall bladder cancer cell, a
kidney cancer cells,
an adrenal cancer cell and heart cancer cell.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that
the detailed description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration only, since
various
changes and modifications within the spirit and scope of the invention will
become
apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to
further demonstrate certain aspects of the present invention. The invention
may be better
understood by reference to one or more of these drawings in combination with
the
detailed description of specific embodiments presented herein.
FIG. lA and FIG. 1B - Multigene constructs. FIG. lA: Single cassette, multiple
promoter construct; FIG. 1 B: Single, cassette, single promoter construct;
FIG. 2 - Cloning vector pIN147.
FIG. 3 - Cloning vectors) p~E 1 sp 1 A/B.
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FIG. 4 - Multiple cassette construct pAB26.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. The Present Invention
Gene therapy now is becoming a viable alternative to various conventional
therapies, especially in the area of cancer treatment. Limitations such as
long term
expression of transgenes and immuno-destruction of target cells through the
expression of
vector products, which have been said to limit the implementation of genetic
therapies,
are not concerns in cancer therapies, where destruction of cancer cells is
desired.
It is important in gene transfer therapies, especially those involving
treatment of
cancer, to kill as many of the cells as quickly as possible. Thus, the use of
"combination"
therapies may be favored. Such combinations may include gene therapy and
radiotherapy
or chemotherapy. Roth et al. ( 1996) have demonstrated that a combination of
DNA
damaging agents and p53 gene therapy provides increased killing of tumor cells
in' vivo.
Yet another type of combination therapy involves the use of mufti-gene
therapy.
In this situation, more than one therapeutic gene would be transferred into a
target cell.
The genes could be from the same functional group (e.g., both tumor
suppressors, both
cytokines, etc. ) or from different functional groups {e.g., a tumor
suppressor and a
cytokine). By presenting particular combinations of therapeutic genes to a
target cell, it
may be possible to augment the overall effect of either or both genes on the
physiology of
the target cell.
The present invention seeks, therefore, to provide unique and advantageous
combinations of genes for therapies, particularly where the destruction of a
target cell is
particularly desired. Such conditions include hyperproliferation, such as
hyperplasia and
benign and malignant neoplasias. The primary consideration in this endeavor is
the
combination of genes. The secondary consideration is how to achieve
simultaneous
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expression in a single cell of both therapeutic genes. The present inventors
have chosen
to approach this second issue by utilizing a single viral vector carrying both
genes. Thus,
infection of the cell by the vector ensures uptake and expression of both
genes.
II. Viral Vectors
Although the methods and compositions described herein are directed to
multigene adenoviral constructs, the methods and compositions described may be
applicable to the construction of multigene constructs using other viral
vectors including
but not limited to retroviruses, herpes viruses, adeno-associated viruses,
vaccinia viruses.
The discussion below provides details regarding the characteristics of each of
these
viruses in relation to their application in therapeutic compositions.
A. Adenovirus
One of the preferred methods for in vivo delivery involves the use of an
adenovirus expression vector. "Adenovirus expression vector" is meant to
include those
constructs containing adenovirus sequences sufficient to (a) support packaging
of the
construct and (b) to express an antisense polynucleotide, a protein, a
polynucleotide (e.g.,
a ribozyme, or an mRNA) that has been cloned therein. In this context,
expression does
not require that the gene product be synthesized.
The expression vector comprises a genetically engineered form of adenovirus.
Knowledge of the genetic organization of adenovirus, a 36 kb, linear, double-
stranded
DNA virus, allows substitution of large pieces of adenoviral DNA with foreign
sequences
up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to retroviruses, the
adenoviral
infection of host cells does not result in chromosomal integration because
adenoviral
DNA can replicate in an episomal manner without potential genotoxicity. As
used
herein, the term "genotoxicity" refers to permanent inheritable host cell
genetic alteration.
Also, adenoviruses are structurally stable, and no genome rearrangement has
been
detected after extensive amplification of normal derivatives. Adenovirus can
infect
virtually all epithelial cells regardless of their cell cycle stage.
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Adenovirus is particularly suitable for use as a gene transfer vector because
of its
mid-sized genome, ease of manipulation, high titer, wide target cell range and
high
infectivity. Both ends of the viral genome contain 100-200 base pair inverted
repeats
S (ITRs), which are cis elements necessary for viral DNA replication and
packaging. The
early (E) and late (L) regions of the genome contain different transcription
units that are
divided by the onset of viral DNA replication. The EI region (ElA and ElB)
encodes
proteins responsible for the regulation of transcription of the viral genome
and a few
cellular genes. The expression of the E2 region (E2A and E2B) results in the
synthesis of
the proteins for viral DNA replication. These proteins are involved in DNA
replication,
late gene expression and host cell shut-off (Renan, 1990). The products of the
late genes,
including the majority of the viral capsid proteins, are expressed only after
significant
processing of a single primary transcript issued by the major late promoter
(MLP). The
MLP (located at 16.8 m.u.) is particularly efficient during the late phase of
infection, and
all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL)
sequence
which makes them preferred mRNA's for translation.
The E3 region encodes proteins that appear to be necessary for efficient lysis
of
Ad infected cells as well as preventing TNF-mediated cytolysis and CTL
mediated lysis
of infected cells. In general, the E4 region encodes is believed to encode
seven proteins,
some of which activate the E2 promoter. It has been shown to block host mRNA
transport and enhance transport of viral RNA to cytoplasm. Further the E4
product is in
part responsible for the decrease in early gene expression seen late in
infection. E4 also
inhibits E 1 A and E4 (but not E 1 B) expression during lytic growth. Some E4
proteins are
necessary for efficient DNA replication however the mechanism for this
involvement is
unknown. E4 is also involved in post-transcriptional events in viral late gene
expression;
i.e., alternative splicing of the tripartite leader in lytic growth.
Nevertheless, E4 functions
are not absolutely required for DNA replication but their lack will delay
replication.
Other functions include negative regulation of viral DNA synthesis, induction
of sub-
nuclear reorganization normally seen durinb adenovirus infection, and other
functions
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that are necessary for viral replication, late viral mRNA accumulation, and
host cell
transcriptional shut off.
In a current system, recombinant adenovirus is generated from homologous
recombination between shuttle vector and provirus vector. Possible
recombination
between the proviral vector and Ad sequences in 293 cells, or in the case of
pJMl7
plasmid spontaneous deletion of the inserted pBR322 sequences, may generate
full length
wild-type Ad5 adenovirus. Therefore, it is critical to isolate a single clone
of virus from
an individual plaque and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which are
replication deficient, depend on a unique helper cell line, designated 293,
which was
transformed from human embryonic kidney cells by Ad5 DNA fragments and
constitutively expresses E1 proteins (Graham et al., 1977). Since the E3
region is
dispensable from the adenovirus genome (Jones and Shenk, 1978), the current
adenovirus
vectors, with the help of 293 cells, carry foreign DNA in either the El, the
E3 or both
regions (Graham and Prevec, 1991 ). In nature, adenovirus can package
approximately
105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing
capacity for
about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is
replaceable in the E l and E3 regions, the maximum capacity of the current
adenovirus
vector is under 7.5 kb, or about 15% of the total length of the vector. More
than 80% of
the adenovirus viral genome remains in the vector backbone and is the source
of vector-
borne cytotoxicity. Also, the replication deficiency of the El-deleted virus
is incomplete.
For example, leakage of viral gene expression has been observed with the
currently
available vectors at high multiplicities of infection (MOI) (Mulligan, 1993;
Shenk, 1978).
Helper cell Lines may be derived from human cells such as human embryonic
kidney cells, muscle cells, hematopoietic cells or other human embryonic
mesenchymal
or epithelial cells. Alternatively, the helper cells may be derived from the
cells of other
mammalian species that are permissive for human adenovirus. Such cells
include, e.g.,
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Vero cells or other monkey embryonic mesenchymal or epithelial cells. As
stated above,
the preferred helper cell line is 293.
Racher et al. (1995) disclosed improved methods for culturing 293 cells and
S propagating adenovirus. In one format, natural cell aggregates are grown by
inoculating
individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge,
UK)
containing 100-200 ml of medium. Following stirring at 40 rpm, the cell
viability is
estimated with trypan blue. In another format, Fibra-Cel microcarners (Bibby
Sterlin,
Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5
ml of
medium, is added to the carrier (SO ml) in a 250 ml Erlenmeyer flask and left
stationary,
with occasional agitation, for 1 to 4 h. The medium is then replaced with 50
ml of fresh
medium and shaking is initiated. For virus production, cells are allowed to
grow to about
80% confluence, after which time the medium is replaced (to 25% of the final
volume)
and adenovirus added at an MOI (pfu/mL) of 0.05. Cultures are left stationary
overnight,
following which the volume is increased to 100% and shaking commenced for
another 72
h.
Other than the requirement that the adenovirus vector be replication
defective, or
at least conditionally defective, the nature of the adenovirus vector is not
believed to be
crucial to the successful practice of the invention. The adenovirus may be of
any of the
42 different known serotypes or subgroups A-F. Adenovirus type S of subgroup C
is the
preferred starting material in order to obtain the conditional replication-
defective
adenovirus vector for use in the present invention. This is because Adenovirus
type 5 is a
human adenovirus about which a great deal of biochemical, medical and genetic
information is known, and it has historically been used for most constructions
employing
adenovirus as a vector.
As stated above, the typical vector according to the present invention is
replication defective and will not have an adenovirus E 1 region. Thus, it
will be most
convenient to introduce the polynucleotide encoding the gene of interest at
the position
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from which the E1-coding sequences have been removed. However, the position of
insertion of the construct within the adenovirus sequences is not critical to
the invention.
The polynucleotide encoding the gene of interest may also be inserted in lieu
of the
deleted E3 region in E3 replacement vectors as described by Karlsson et al. (
1986), or in
the E4 region where a helper cell line or helper virus complements the E4
defect.
Adenovirus is easy to grow and manipulate and exhibits broad host range in
vitro
and in vivo. This group of viruses can be obtained in high titers, e.g., 109-
10' ~ plaque-
forming units per ml, and they are highly infective. The life cycle of
adenovirus does not
require integration into the host cell genome. The foreign genes delivered by
adenovirus
vectors are episomal and, therefore, have low genotoxicity to host cells. No
side effects
have been reported in studies of vaccination with wild-type adenovirus (Couch
et al.,
1963; Top et al., 1971 ), demonstrating their safety and therapeutic potential
as in vivo
gene transfer vectors.
IS
Adenovirus vectors have been used in eukaryotic gene expression investigations
(Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development
{Grunhaus and
Horwitz, 1992; Graham and Prevec, 1992). Recently, animal studies suggested
that
recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet
and
Perncaudet, 1991; Stratford-Perricaudet et al., 1990; Rich et al., 1993).
Studies in
administering recombinant adenovirus to different tissues include trachea
instillation
(Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et
al., 1993),
peripheral intravenous injections (Herz and Gerard, 1993), intranasal
inoculation
(Ginsberg et al., 1991 ), aerosol administration to lung (Bellon, 1996) infra-
peritoneal
administration (Song et al., 1997), infra-pleural injection (Elshami et al.,
1996)
administration to the bladder using infra-vesicular administration (Werthman,
et al.,
1996), subcutaneous injection (Ogawa, 1989), ventricular injection into
myocardium
(heart, French et al., 1994), liver perfusion (hepatic artery or portal vein,
Shiraishi et al.,
1997) and stereotactic inoculation into the brain (Le Gal La Salle et al.,
1993).
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B. Retrovirus
The retroviruses are a group of single-stranded RNA viruses characterized by
an
ability to convert their RNA to double-stranded DNA in infected cells by a
process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates
into
cellular chromosomes as a provinls and directs synthesis of viral proteins.
The
integration results in the retention of the viral gene sequences in the
recipient cell and its
descendants. The retrovira( genome contains three genes, gag, pol, and env
that code for
capsid proteins, polymerase enzyme, and envelope components, respectively. A
sequence found upstream from the gag gene contains a signal for packaging of
the
genome into virions. Two long terminal repeat (LTR) sequences are present at
the 5' and
3' ends of the viral genome. These contain strong promoter and enhancer
sequences and
are also required for integration in the host cell genome (Coffin, 1990).
In order to construct a retroviral vector, a nucleic acid encoding a gene of
interest
is inserted into the viral genome in the place of certain viral sequences to
produce a virus
that is replication-defective. In order to produce virions, a packaging cell
line containing
the gag, pol, and env genes but without the LTR and packaging components is
constructed (Mann et al., 1983). When a recombinant olasmid cnntainino a ~1~NA
together with the retroviral LTR and packaging sequences is introduced into
this cell line
(by calcium phosphate precipitation for example), the packaging sequence
allows the
RNA transcript of the recombinant plasmid to be packaged into viral particles,
which are
then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin,
1986; Mann
et al., 1983). The media containing the recombinant retroviruses is then
collected,
optionally concentrated, and used for gene transfer. Retroviral vectors are
able to infect a
broad variety of cell types. However, integration and stable expression
require the
division of host cells (Paskind et al., 1975).
A novel approach designed to allow specific targeting of retrovirus vectors
was
recently developed based on the chemical modification of a retrovirus by the
chemical
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addition of lactose residues to the viral envelope. This modification could
permit the
specific infection of hepatocytes via sialoglycoprotein receptors.
A different approach to targeting of recombinant retroviruses was designed in
S which biotinylated antibodies against a retroviral envelope protein and
against a specific
cell receptor were used. The antibodies were coupled via the biotin components
by using
streptavidin (Roux et al., 1989). Using antibodies against major
histocompatibility
complex class I and class II antigens, they demonstrated the infection of a
variety of
human cells that bore those surface antigens with an ecotropic virus in vitro
(Roux et al.,
1989).
There are certain limitations to the use of retrovirus vectors in all aspects
of the
present invention. For example, retrovirus vectors usually integrate into
random sites in
the cell genome. This can lead to insertional mutagenesis through the
interruption of host
genes or through the insertion of viral regulatory sequences that can
interfere with the
function of flanking genes (Varmus et al., 1981). Another concern with the use
of
defective retrovirus vectors is the potential appearance of wild-type
replication-competent
virus in the packaging cells. This can result from recombination events in
which the
intact- sequence from the recombinant virus inserts upstream from the gag,
pol, env
sequence integrated in the host cell genome. However, new packaging cell lines
are now
available that should greatly decrease the likelihood of recombination
(Markowitz et al.,
1988; Hersdorffer et al., 1990).
C. Herpesvirus
Because herpes simplex virus {HSV) is neurotropic, it has generated
considerable
interest in treating nervous system disorders. Moreover, the ability of HSV to
establish
latent infections in non-dividing neuronal cells without integrating in to the
host cell
chromosome or otherwise altering the host cell's metabolism, along with the
existence of
a promoter that is active during latency makes HSV an attractive vector. And
though
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much attention has focused on the neurotropic applications of HSV, this vector
also can
be exploited for other tissues given its wide host range.
Another factor that makes HSV an attractive vector is the size and
organization of
the genome. Because HSV is large, incorporation of multiple genes or
expression
cassettes is less problematic than in other smaller viral systems. In
addition, the
availability of different viral control sequences with varying performance
(temporal,
strength, etc.) makes it possible to control expression to a greater extent
than in other
systems. It also is an advantage that the virus has relatively few spliced
messages, further
easing genetic manipulations.
HSV also is relatively easy to manipulate and can be grown to high titers.
Thus,
delivery is less of a problem, both in terms of volumes needed to attain
sufficient MOI
and in a lessened need for repeat dosings. For a review of HSV as a gene
therapy vector,
I 5 see Glorioso et al. ( I 995).
HSV, designated with subtypes 1 and 2, are enveloped viruses that are among
the
most common infectious agents encountered by humans, infecting millions of
human
subjects worldwide. The large, complex, double-stranded DNA genome encodes for
dozens of different gene products, some of which derive from spliced
transcripts. In
addition to virion and envelope structural components, the virus encodes
numerous other
proteins including a protease, a ribonucleotides reductase, a DNA polymerase,
a ssDNA
binding protein, a helicase/primase, a DNA dependent ATPase, a dUTPase and
others.
HSV genes form several groups whose expression is coordinately regulated and
sequentially ordered in a cascade fashion (Honess and Roizman, 1974; Honess
and
Roizman 1975; Roizman and Sears, 1995). The expression of a genes, the first
set of
genes to be expressed after infection, is enhanced by the virion protein
number 16, or
a-transinducing factor (Post et al. , I 981; Batterson and Roizman, 1983;
Campbell, et al. ,
1983). The expression of ~3 genes requires functional a gene products, most
notably
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ICP4, which is encoded by the a4 gene (DeLuca et al., 1985). y genes, a
heterogeneous
group of genes encoding largely virion structural proteins, require the onset
of viral DNA
synthesis for optimal expression (Holland et al., 1980).
In line with the complexity of the genome, the life cycle of HSV is quite
involved.
In addition to the lytic cycle, which results in synthesis of virus particles
and, eventually,
cell death, the virus has the capability to enter a latent state in which the
genome is
maintained in neural ganglia until some as of yet undefined signal triggers a
recurrence of
the lytic cycle. Avirulent variants of HSV have been developed and are readily
available
for use in gene therapy contexts (U.S. Patent No. 5,672,344).
D. Adeno-Associated Yirus
Recently, adeno-associated virus (AAV) has emerged as a potential alternative
to
the more commonly used retroviral and adenoviral vectors. While studies with
retroviral
and adenoviral mediated gene transfer raise concerns over potential oncogenic
properties
of the former, and immunogenic problems associated with the latter, AAV has
not been
associated with any such pathological indications.
In addition, AAV possesses several unique features that make it more desirable
than the other vectors. Unlike retroviruses, AAV can infect non-dividing
cells; wild-type
AAV has been characterized by integration, in a site-specific manner, into
chromosome
19 of human cells (Kotin and Berns, 1989; Kotin et al., 1990; Kotin el al.,
1991;
Samulski et al., 1991); and AAV also possesses anti-oncogenic properties
(Ostrove et al.,
1981; Berns and Giraud, 1996). Recombinant AAV genomes are constructed by
molecularly cloning DNA sequences of interest between the AAV ITRs,
eliminating the
entire coding sequences of the wild-type AAV genome. The AAV vectors thus
produced
lack any of the coding sequences of wild-type AAV, yet retain the property of
stable
chromosomal integration and expression of the recombinant genes upon
transduction
both in vitro and in vivo (Bems, 1990; Berns and Bohensky, 1987; Bertran et
al., 1996;
Kearns et al., 1996; Ponnazhagan et al., 1997a). Until recently, AAV was
believed to
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infect almost all cell types, and even cross species barriers. However, it now
has been
determined that AAV infection is receptor-mediated (Ponnazhagan et al., 1996;
Mizukami et al., 1996).
AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted
terminal repeats flank the genome. Two genes are present within the genome,
giving rise
to a number of distinct gene products. The first, the cap gene, produces three
different
virion proteins (VP), designated VP-l, VP-2 and VP-3. The second, the rep
gene,
encodes four non-structural proteins (NS). One or more of these rep gene
products is
responsible for transactivating AAV transcription. The sequence of AAV is
provided by
Srivastava et al. (1983), and in U.S. Patent 5,252,479 (entire text of which
is specifically
incorporated herein by reference).
The three promoters in AAV are designated by their location, in map units, in
the
genome. These are, from left to right, p5, p 19 and p40. Transcription gives
rise to six
transcripts, two initiated at each of three promoters, with one of each pair
being spliced.
The splice site, derived from map units 42-46, is the same for each
transcript. The four
non-structural proteins apparently are derived from the longer of the
transcripts, and three
virion proteins all arise from the smallest transcript.
AAV is not associated with any pathologic state in humans. Interestingly, for
efficient replication, AAV requires "helping" functions from viruses such as
herpes
simplex virus I and II, cytomegalovirus, pseudorabies virus and, of course,
adenovirus.
The best characterized of the helpers is adenovirus, and many "early"
functions for this
virus have been shown to assist with AAV replication. Low level expression of
AAV rep
proteins is believed to hold AAV structural expression in check, and helper
virus
infection is thought to remove this block.
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E. vaccinia virus
Vaccinia virus vectors have been used extensively because of the ease of their
construction, relatively high levels of expression obtained, wide host range
and large
capacity for carrying DNA. Vaccinia contains a linear, double-stranded DNA
genome of
about 186 kb that exhibits a marked "A-T" preference. Inverted terminal
repeats of about
10.5 kb flank the genome. The majority of essential genes appear to map within
the
central region, which is most highly conserved among poxviruses. Estimated
open
reading frames in vaccinia virus number from 1 SO to 200. Although both
strands are
coding, extensive overlap of reading frames is not common.
IO
At least 25 kb can be inserted into the vaccinia virus genome (Smith and Moss,
1983). Prototypical vaccinia vectors contain transgenes inserted into the
viral thymidine
kinase gene via homologous recombination. Vectors are selected on the basis of
a
tk-phenotype. Inclusion of the untranslated leader sequence of
encephalomyocarditis
virus, the level of expression is higher than that of conventional vectors,
with the
transgenes accumulating at 10% or more of the infected cell's protein in 24 h
(Elroy-Stein
et al., 1989).
III. Non-viral transfer
Although the present invention describes the use of adenoviral multigene
constructs, the present invention may also employ non-viral gene transfer.
This section
provides a discussion of methods and compositions of non-viral gene transfer.
DNA constructs of the present invention are generally delivered to a cell, and
in
certain situations, the nucleic acid to be transferred may be transferred
using non-viral
methods.
Several non-viral methods for the transfer of expression constructs into
cultured
mammalian cells are contemplated by the present invention. These include
calcium
phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987;
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Rippe et al., 1990) DEAE-dextran (copal, 1985), electroporation (Tur-Kaspa et
al., 1986;
Potter et al., 1984), direct microinjection (Harland and Weintraub, 1985), DNA-
loaded
liposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication
(Fechheimer et
al., 1987), gene bombardment using high velocity microprojectiles (Yang et
al., 1990),
and receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988).
Once the construct has been delivered into the cell the nucleic acid encoding
the
therapeutic gene may be positioned and expressed at different sites. In
certain
embodiments, the nucleic acid encoding the therapeutic gene may be stably
integrated
into the genome of the cell. This integration may be in the cognate location
and
orientation via homologous recombination (gene replacement) or it may be
integrated in a
random, non-specific location (gene augmentation). In yet further embadiments,
the
nucleic acid may be stably maintained in the cell as a separate, episomal
segment of
DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to
permit
maintenance and replication independent of or in synchronization with the host
cell cycle.
How the expression construct is delivered to a cell and where in the cell the
nucleic acid
remains is dependent on the type of expression construct employed.
In a particular embodiment of the invention, the expression construct may be
entrapped in a liposome. Liposomes are vesicular structures characterized by a
phospholipid bilayer membrane and an inner aqueous medium. Multilamellar
Iiposomes
have multiple lipid layers separated by aqueous medium. They form
spontaneously when
phospholipids are suspended in an excess of aqueous solution. The lipid
components
undergo self rearrangement before the formation of closed structures and
entrap water
and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991 ).
The
addition of DNA to cationic liposomes causes a topological transition from
liposomes to
optically birefringent liquid-crystalline condensed globules (Radler et al.,
1997). These
DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
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Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro
has been very successful. Using the (3-lactamase gene, Wong et al. ( 1980)
demonstrated
the feasibility of liposome-mediated delivery and expression of foreign DNA in
cultured
chick embryo, HeLa, and hepatoma cells. Nicolau et al. (1987) accomplished
successful
S liposome-mediated gene transfer in rats after intravenous injection. Also
included are
various commercial approaches involving "lipofection" technology.
In certain embodiments of the invention, the liposome may be complexed with a
hemagglutinating virus (HVJ). This has been shown to facilitate fusion with
the cell
membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al.,
1989).
In other embodiments, the liposome may be complexed or employed in conjunction
with
nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet
further
embodiments, the liposome may be complexed or employed in conjunction with
both
HVJ and HMG-1. In that such expression constructs have been successfully
employed in
transfer and expression of nucleic acid in vitro and in vivo, then they are
applicable for
the present invention.
Other vector delivery systems which can be employed to deliver a nucleic acid
encoding a therapeutic gene into cells are receptor-mediated delivery
vehicles. These
take advantage of the selective uptake of macromolecules by receptor-mediated
endocytosis in almost all eukaryotic cells. Because of the cell type-specific
distribution
of various receptors, the delivery can be highly specific (Wu and Wu, 1993).
Receptor-mediated gene targeting vehicles generally consist of two components:
a cell receptor-specific ligand and a DNA-binding agent. Several ligands have
been used
for receptor-mediated gene transfer. The most extensively characterized
ligands are
asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al.,
1990).
Recently, a synthetic neoglycoprotein, which recognizes the same receptor as
ASOR, has
been used as a gene delivery vehicle (Ferkol et al., 1993; Perales et al.,
1994) and
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epidermal growth factor (EGF) has also been used to deliver genes to squamous
carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a
liposome. For example, Nicolau et al. (1987) employed lactosy'1-ceramide, a
galactose-
terminal asialganglioside, incorporated into liposomes and observed an
increase in the
uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic
acid encoding
a therapeutic gene also may be specifically delivered into a cell type such as
prostate,
epithelial or tumor cells, by any number of receptor-ligand systems with or
without
liposomes. For example, the human prostate-specific antigen (Watt et al.,
1986) may be
used as the receptor for mediated delivery of a nucleic acid in prostate
tissue.
In another embodiment of the invention, the expression construct may simply
consist of naked recombinant DNA or plasmids. Transfer of the construct may be
I S performed by any of the methods mentioned above which physically or
chemically
permeabilize the cell membrane. This is applicable particularly for transfer
in vitro,
however, it may be applied for in vivo use as well. Dubensky et al. (1984)
successfully
injected polyomavirus DNA in the form of CaP04 precipitates into liver and
spleen of
adult and newborn mice demonstrating active viral replication and acute
infection.
Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal
injection of
CaP04 precipitated plasmids results in expression of the transfected genes. It
is
envisioned that DNA encoding a CAM may also be transferred in a similar manner
in
vivo and express CAM.
Another embodiment of the invention for transferring a naked DNA expression
construct into cells may involve particle bombardment. This method depends on
the
ability to accelerate DNA coated microprojectiles to a high velocity allowing
them to
pierce cell membranes and enter cells without killing them (Klein et al.,
1987). Several
devices for accelerating small particles have been developed. One such device
relies on a
high voltage discharge to generate an electrical current, which in turn
provides the motive
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force (Yang et al., 1990). The microprojectiles used have consisted of
biologically inert
substances such as tungsten or gold beads.
IV. Gene Combinations
A. Tumor Suppressors
p53 (designated.l in Table 1) currently is recognized as a tumor suppressor
gene.
High levels of mutant p53 have been found in many cells transformed by
chemical
carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a
frequent
target of mutational inactivation in a wide variety of human tumors and is
already
documented to be the most frequently-mutated gene in common human cancers. It
is
mutated in over 50% of human NSCLC (Hollstein et al., 1991 ) and in a wide
spectrum of
other tumors.
The p53 gene encodes a 393-amino acid phosphoprotein that can form complexes
with host proteins such as SV40 large-T antigen and adenoviral EIB. The
protein is
found in normal tissues and cells, but at concentrations which are minute by
comparison
with transformed cells or tumor tissue. Interestingly, wild-type p53 appears
to be
important in regulating cell growth and division. Overexpression of wild-type
p53 has
been shown in some cases to be anti-proliferative in human tumor cell lines.
Thus, p53
can act as a negative regulator of cell growth (Weinberg, 1991) and may
directly suppress
uncontrolled cell growth or indirectly activate genes that suppress this
growth. Thus,
absence or inactivation of wild-type p53 may contribute to transformation.
However,
some studies indicate that the presence of mutant p53 may be necessary for
full
expression of the transforming potential of the gene.
Wild-type p53 is recognized as an important growth regulator in many cell
types.
Missense mutations are common for the p53 gene and are essential for the
transforming
ability of the oncogene. A single genetic change prompted by point mutations
can create
carcinogenic p53, in as much as mutations in p53 are known to abrogate the
tumor
suppressor capability of wild-type p53. Unlike other oncogenes, however, p53
point
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mutations are known to occur in at least 30 distinct codons, often creating
dominant
alleles that produce shifts in cell phenotype without a reduction to
homozygosity.
Additionally, many of these dominant negative alleles appear to be tolerated
in the
organism and passed on in the germ line. Various mutant alleles appear to
range from
S minimally dysfunctional to strongly penetrant, dominant negative alleles
(Weinberg,
1991 ).
Casey and colleagues have reported that transfection of DNA encoding wild-type
p53 into two human breast cancer cell lines restores growth suppression
control in such
cells (Casey et al., 1991). A similar effect also has been demonstrated on
transfection of
wild-type, but not mutant, p53 into human lung cancer cell lines (Takahasi et
al., 1992).
p53 appears dominant over the mutant gene and will select against
proliferation when
transfected into cells with the mutant gene. Normal expression of the
transfected p53
does not affect the growth of normal or non-malignant cells with endogenous
p53. Thus,
such constructs might be taken up by normal cells without adverse effects. It
is thus
proposed that the treatment of p53-associated cancers with wild-type p53 will
reduce the
number of malignant cells or their growth rate.
The major transitions of the eukaryotic cell cycle are triggered by cyclin-
dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4),
regulates
progression through the G,. The activity of this enzyme may be to
phosphorylate Rb at
late G~. The activity of CDK4 is controlled by an activating subunit, D-type
cyclin, and
by an inhibitory subunit pl6~K°. The p16~K4 has been biochemically
characterized as a
protein that specifically binds to and inhibits CDK4, and thus may regulate Rb
2S phosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since the
p16~'K4 protein is
a CDK4 inhibitor (Serrano, 1993), deletion of this gene may increase the
activity of
CDK4, resulting in hyperphosphorylation of the Rb protein. p16 also is known
to
regulate the function of CDK6.
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pl6~N~4 belongs to a newly described class of CDK-inhibitory proteins that
also
includes I S ~N~4a 21 WAF1 KIPI INK4
p , p , and p27 . The p I 6 gene maps to 9p21, a chromosome
region frequently deleted in many tumor types. Homozygous deletions and
mutations of
the pl6~N~4 gene are frequent in human tumor cell lines. This evidence
suggests that the
p I 6~N~'4 gene is a tumor suppressor gene. This interpretation has been
challenged,
however, by the observation that the frequency of the p 16~K4 gene alterations
is much
lower in primary uncultured tumors than in cultured cell lines (Caldas et al.,
1994; Cheng
et al., 1994; Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994;
Mori et al.,
1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al., 1994; Arap et
al., 1995).
However, it was later shown that while the pl6 gene was intact in many primary
tumors,
there were other mechanisms that prevented pl6 protein expression in a large
percentage
of some tumor types. pl6 promoter hypermethylation is one of these mechanisms
(Merio
et al., 1995; Herman, 1995; Gonzalez-Zulueta, 1995). Restoration of wild-type
pl6iN~4
function by transfection with a plasmid expression vector reduced colony
formation by
some human cancer cell lines (Okamoto, 1994; Arap, 1995). Delivery of pl6 with
adenovirus vectors inhibits proliferation of some human cancer lines and
reduces the
growth of human tumor xenografts.
C-CAM (designated 2 in Table 1 ) is expressed in virtually all epithelial
cells
(Odin and Obrink, 1987). C-CAM, with an apparent molecular weight of 105 kD,
was
originally isolated from the plasma membrane of the rat hepatocyte by its
reaction with
specific antibodies that neutralize cell aggregation (Obrink, 1991 ). Recent
studies
indicate that, structurally, C-CAM belongs to the immunoglobulin (Ig)
superfamily and
its sequence is highly homologous to carcinoembryonic antigen (CEA; designated
3 in
Table 1 ) (Lin and Guidotti, 1989). Using a baculovirus expression system,
Cheung et al.
(1993) demonstrated that the first Ig domain of C-CAM is critical for cell
adhesive
activity.
Cell adhesion molecules, or CAM's are known to be involved in a complex
network of molecular interactions that regulate organ development and cell
differentiation
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(Edelman, i 985). Recent data indicate that aberrant expression of CAM's maybe
involved in the tumorigenesis of several neoplasms; for example, decreased
expression of
E-cadherin, which is predominantly expressed in epithelial cells, is
associated with the
progression of several kinds of neoplasms (Edelman and Crossin, 1991; Frixen
et al.,
S 1991; Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al., 1992).
Also,
Giancotti and Ruoslahti (1990) demonstrated that increasing expression of
a5~3~ integrin
by gene transfer can reduce tumorigenicity of Chinese hamster ovary cells in
vivo. C-
CAM now has been shown to suppress tumor growth in vitro and in vivo.
Other tumor suppressors that may be employed according to the present
invention
include p21 (designated 4 in Table 1 ), p 1 S (designated 5 in Table I ), BRCA
1 (designated
6 in Table 1 ), BRCA2 (designated 7 in Table I ), IRF-I (designated 8 in Table
1 ), PTEN
(MMAC 1; designated 9 in Table i ), RB (designated 11 in Table 1 ), APC
(designated 12
in Table 1 ), DCC (designated 13 in Table 1 ), NF-1 (designated 14 in Table 1
), NF-2
(designated 15 in Table 1 ), WT-1 (designated 16 in Table I ), MEN-I
(designated I7 in
Table 1 ), MEN-II (designated 18 in Table I ), zac 1 (designated 19 in Table 1
), p73
(designated 20 in Table 1), VHL (designated 21 in Table I), FCC (designated 23
in Table
1 ), MCC (designated 24 in Table 1 ), DBCCR 1 (designated 133 in Table 1 ),
DCP4
(designated 137 in Table 1) and p57 (designated 138 in Table 1).
B. Inducers of Apoptosis
Inducers of apoptosis, such as Bax (designated 25 in Table I ), Bak
(designated 26
in Table 1 ), Bcl-XS (designated 27 in Table 1 ), Bad (designated 28 in Table
1 ), Bim
(designated 29 in Table 1 ), Bik (designated 30 in Table 1 ), Bid (designated
31 in Table
1 ), Harakiri (designated 32 in Table I ), Ad E1 B (designated 33 in Table 1
), Bad
(designated 34 in Table 1 ), ICE-CED3 proteases (designated 35 in Table I ),
TRAIL
(designated 125 in Table 1 ), SARP-2 (designated 126 in Table 1 ) and apoptin
(designated
132 in Table 1), similarly could find use according to the present invention.
In addition,
the delivery and regulated expression of cytotoxic genes have been described
in the U.S.
Patent Application entitled, "Induction of Apoptic or Cytotoxic Gene
Expression by
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Adenoviral Mediated Gene Codelivery," filed March I t, 1999 (specifically
incorporated
herein by reference).
C. Enrymes
S Various enzyme genes are of interest according to the present invention,
Such
enzymes include cytosine deaminase (designated 36 in Table 1 ), adenosine
deaminase
(designated 37 in Table I ), hypoxanthine-guanine phosphoribosyltransferase
(designated
38 in Table 1), galactose-I-phosphate uridyltransferase (designated 39 in
Table I),
phenylalanine hydroxylase (designated 40 in Table 1 ), glucocerbrosidase
(designated 41
in Table 1 ), sphingomyelinase (designated 42 in Table I ), a-L-iduronidase
(designated 43
in Table i), glucose-6-phosphate dehydrogenase (designated 44 in Table 1), HSV
thymidine kinase (designated 45 in Table I ) and human thymidine kinase
(designated 46
in Table I) and extracellular proteins such as collagenase (designated 118 in
Table I),
matrix metalloprotease (designated 119 in Table 1), RSKB (designated 128 in
Table 1},
1 S RSK1 (designated 129 in Table 1 ), RSK2 (designated 130 in Table 1 ), RSK3
(designated
131 in Table 1 ), thrombospondin (designated 134 in Table 1 ), fibronectin
(designated 135
in Table 1) and plasminogen (designated 136 in Table 1). In other embodiments
of the
present invention, the use of anti-angiogenic factors are contemplated.
D. Cytokines
Another class of genes that is contemplated to be inserted into the adenoviral
vectors of the present invention include interleukins and cytokines.
Interleukin I (IL-I;
designated 47 in Table I ), IL-2 (designated 48 in Table 1 ), IL-3 (designated
49 in Table
I), IL-4 (designated 50 in Table 1), IL-5 (designated S1 in Table 1), IL-6
(designated 52
in Table 1 ), IL-7 (designated 53 in Table I ), IL-8 (designated 54 in Table I
), IL-9
(designated 55 in Table 1), IL-10 (designated 56 in Table 1), IL-11
(designated 57 in
Table I), IL-12 (designated 58 in Table 1), IL-13 (designated 59 in Table I),
IL-14
(designated 60 in Table I), IL-IS (designated 61 in Table I), ~3-interferon
(designated 62
in Table 1 ), a-interferon (designated 63 in Table 1 ), y-interferon
(designated 122 in Table
1 ), angiostatin (designated 64 in Table I ), thrombospondin (designated 65 in
Table 1 ),
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endostatin (designated 66 in Table 1), METH-1 (designated 67 in Table 1), METH-
2
(designated 68 in Table 1 ), GM-CSF (designated 69 in Table 1 ), G-CSF
(designated 70 in
Table 1 ), M-CSF (designated 123 in Table 1 ) and tumor necrosis factor
(designated I24
in Table 1 ).
E Toxins
Various toxins are also contemplated to be useful as part of the expression
vectors
of the present invention, these toxins include bacterial toxins such as ricin
A-chain
(Burbage, 1997; designated 71 in Table 1 ), diphtheria toxin A (Massuda et
al., 1997;
Lidor, 1997; designated 72 in Table 1 ), pertussis toxin A subunit (designated
73 in Table
1 ), E. coli enterotoxin toxin A subunit (designated 74 in Table 1 ), cholera
toxin A subunit
(designated 75 in Table 1 ) and pseudomonas toxin c-terminal (designated 76 in
Table 1 ).
Recently, it was demonstrated that transfection of a plasmid containing the
fusion protein
regulatable diphtheria toxin A chain gene was cytotoxic for cancer cells.
Thus, gene
transfer of regulated toxin genes might also be applied to the treatment of
cancers
(Massuda et al., 1997).
F. Antisense Constructs
Antisense methodology takes advantage of the fact that nucleic acids tend to
pair
with "complementary" sequences. By complementary, it is meant that
polynucleotides
are those which are capable of base-pairing according to the standard Watson-
Crick
complementarity rules. That is, the larger purines will base pair with the
smaller
pyrimidines to form combinations of guanine paired with cytosine (G:C) and
adenine
paired with either thymine (A:T) in the case of DNA, or adenine paired with
uracil (A:U)
in the case of RNA. Inclusion of less common bases such as inosine, 5-
methylcytosine,
6-methyladenine, hypoxanthine and others in hybridizing sequences does not
interfere
with pairing.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix
formation; targeting RNA will lead to double-helix formation. Antisense
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polynucleotides, when introduced into a target cell, specifically bind to
their target
polynucleotide and interfere with transcription, RNA processing, transport,
translation
and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA
may be employed to inhibit gene transcription or translation or both within a
host cell,
either in vitro or in vivo, such as within a host animal, including a human
subject.
Antisense constructs may be designed to bind to the promoter and other control
regions, exons, introns or even exon-intron boundaries of a gene. It is
contemplated that
the most effective antisense constructs will include regions complementary to
intron/exon
splice junctions. Thus, it is proposed that a preferred embodiment includes an
antisense
construct with complementarity to regions within 50-200 bases of an intron-
exon splice
junction. It has been observed that some exon sequences can be included in the
construct
without seriously affecting the target selectivity thereof. The amount of
exonic material
included will vary depending on the particular exon and intron sequences used.
One can
readily test whether too much exon DNA is included simply by testing the
constructs in
vitro to determine whether normal cellular function is affected or whether the
expression
of related genes having complementary sequences is affected.
As stated above, "complementary" or "antisense" means polynucleotide sequences
that are substantially complementary over their entire length and have very
few base
mismatches. For example, sequences of fifteen bases in length may be termed
complementary when they have complementary nucleotides at thirteen or fourteen
positions. Naturally, sequences which are completely complementary will be
sequences
which are entirely complementary throughout their entire length and have no
base
mismatches. Other sequences with lower degrees of homology also are
contemplated.
For example, an antisense construct which has limited regions of high
homology, but also
contains a non-homologous region (e.g., ribozyme; see below) could be
designed. These
molecules, though having less than 50% homology, would bind to target
sequences under
appropriate conditions.
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It may be advantageous to combine portions of genomic DNA with cDNA or
synthetic sequences to generate specific constructs. For example, where an
intron is
desired in the ultimate construct, a genomic clone will need to be used. The
cDNA or a
synthesized polynucleotide may provide more convenient restriction sites for
the
remaining portion of the construct and, therefore, would be used for the rest
of the
sequence.
Particular oncogenes that are targets for antisense constructs are ras
(designated
77 in Table 1 ), myc (designated 78 in Table 1 ), neu (designated 79 in Table
1 ), raf
(designated 80 in Table 1 ), erb (designated 81 in Table 1 ), src (designated
82 in Table 1 },
fms (designated 83 in Table 1 ), jun (designated 84 in Table 1 ), trk
(designated 85 in Table
1 ), ret (designated 86 in Table 1 ), hst (designated 87 in Table 1 ), gsp
(designated 88 in
Table 1 ), bcl-2 (designated 89 in Table 1 ) and abl (designated 90 in Table 1
). Also
contemplated to be useful will be anti-apoptotic genes and angiogenesis
promoters.
G. Riborymes
Although proteins traditionally have been used for catalysis of nucleic acids,
another class of macromolecules has emerged as useful in this endeavor.
Ribozymes are
RNA-protein complexes that cleave nucleic acids in a site-specific fashion.
Ribozymes
have specific catalytic domains that possess endonuclease activity (Kim and
Cook, 1987;
Gerlach et al., 1987; Forster and Symons, 1987). For exampie, a large number
of
ribozymes accelerate phosphoester transfer reactions with a high degree of
specificity,
often cleaving only one of several phosphoesters in an oligonucleotide
substrate (Cook et
al., 1981; Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This
specificity
has been attributed to the requirement that the substrate bind via specific
base-pairing
interactions to the internal guide sequence ("IGS") of the ribozyme prior to
chemical
reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific
cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al.,
1980. For
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example, U.S. Patent No. 5,354,855 reports that certain ribozymes can act as
endonucleases with a sequence specificity greater than that of known
ribonucleases and
approaching that of the DNA restriction enzymes. Thus, sequence-specific
ribozyme-
mediated inhibition of gene expression may be particularly suited to
therapeutic
applications (Scanlon et al., 1991; Sarver et al., 1990). Recently, it was
reported that
ribozymes elicited genetic changes in some cells lines to which they were
applied; the
altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of
this work
involved the modification of a target mRNA, based on a specific mutant codon
that is
cleaved by a specific ribozyme. Targets for this embodiment will include
angiogenic
genes such as VEGFs and angiopoeiteins as well as the oncogenes (e.g., ras,
myc, neu,
raj, erb, src, fms, jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and ably.
H. Single Chain Antibodies
In yet another embodiment, one gene may comprise a single-chain antibody.
Methods for the production of single-chain antibodies are well known to those
of skill in
the art. The skilled artisan is referred to U.S. Patent No. 5,359,046,
(incorporated herein
by reference) for such methods. A single chain antibody is created by fusing
together the
variable domains of the heavy and light chains using a short peptide linker,
thereby
reconstituting an antigen binding site on a single molecule.
Single-chain antibody variable fragments (scFvs) in which the C-terminus of
one
variable domain is tethered to the N-terminus of the other via a I S to 25
amino acid
peptide or linker, have been developed without significantly disrupting
antigen binding or
specificity of the binding (Bedzyk et al., 1990; Chaudhary et al., 1990).
These Fvs lack
the constant regions (Fc) present in the heavy and light chains of the native
antibody.
Antibodies to a wide variety of molecules are contemplated, such as oncogenes,
growth factors, hormones, enzymes, transcription factors or receptors. Also
contemplated
are secreted antibodies, targeted to serum, against angiogenic factors
(VEGF/VSP
designated 91 in Table l; ~iFGF designated 92 in Table l; ocFGF designated 93
in Table
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l; others) and endothelial antigens necessary for angiogenesis (i.e., V3
integrin,
designated 94 in Table 1 ). Specifically contemplated are growth factors such
as
transforming growth factor (designated I20 in Table 1 ) and platelet derived
growth factor
(designated 121 in Table 1 ).
S
I. Transcription Factors and Regulators
Another class of genes that can be applied in an advantageous combination are
transcription factors. Examples include C/EBPa (designated 95 in Table 1 ),
IKB
(designated 96 in Table 1), NficB (designated 97 in Table 1), Par-4
(designated 98 in
Table 1 ) and C/EBPa (designated 127 in Table 1 )
J. Cell Cycle Regulators
Cell cycle regulators provide possible advantages, when combined with other
genes. Such cell cycle regulators include p27 (designated 99 in Table 1), p16
(designated
100 in Table 1 ), p21 (designated 4 in Table 1 ), p57 (designated 101 in Table
1 ), p 18
(designated 102 in Table 1 ), p73 (designated 103 in Table 1 ), p 19
(designated 104 in
Table 1 ), p 15 (designated 5 in Table 1 ), E2F-1 (designated 105 in Table 1
), E2F-2
(designated 106 in Table 1 ), E2F-3 (designated 107 in Table 1 ), p 107
(designated 109 in
Table 1), p130 (designated 110 in Table 1) and E2F-4 (designated 111 in Table
1). Other
cell cycle regulators include anti-angiogenic proteins, such as soluble Fltl
(dominant
negative soluble VEGF receptor; designated 112 in Table 1), soluble Wnt
receptors
(designated 113 in Table 1 ), soluble Tie2/Tek receptor (designated 114 in
Table 1 ),
soluble hemopexin domain of matrix metalloprotease 2 (designated 115 in Table
1) and
soluble receptors of other angiogenic cytokines (e.g. VEGFR1/KDR (designated
116 in
Table 1), VEGFR3/Flt4 (designated 117 in Table 1), both VEGF receptors).
h'. Chemokines
Genes that code for chemokines also may be used in the present invention.
Chemokines generally act as chemoattractants to recruit immune effector cells
to the site
of chemokine expression. It may be advantageous to express a particular
chemokine gene
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in combination with, for example, a cytokine gene, to enhance the recruitment
of other
immune system components to the site of treatment. Such chemokines include
RANTES
(designated 10 in Table 1 ), MCAF, MIP 1-alpha (designated 108 in Table 1 ),
MIP 1-Beta
(designated 22 in Table 1), and IP-10. The skilled artisan will recognize that
certain
cytokines are also known to have chemoattractant effects and could also be
classified
under the term chemokines.
L. Gene Combinations
As described herein, it is contemplated that any one particular gene may be
combined with any other particular gene. Particularly preferred examples of
gene
combinations are listed in Table 1 herein below. Thus, any gene in the first
column of
Table 1 may be advantageously combined with any other gene depicted in the
first row of
Table 1. Of course these are only exemplary combinations and given the
teachings of the
present invention one of skill in the art will be able to produce multigene
constructs of
any conceivable combination, including but not limited to combinations two,
three, four,
five or more genes or nucleic acid constructs. The following graphic may be
helpful to
one of ordinary skill in the art viewing Table 1. This Table is split into 36
leaves labeled
consecutively leaf a, leaf b, leaf c, leaf d, leaf e, leaf f, leaf g, leaf h,
leaf i, leaf j, leaf k,
leaf l, leaf m, leaf n, leaf o, leaf p, leaf q, leaf r, leaf s, leaf t, leaf
u, leaf v, leaf w, leaf x,
leaf y, leaf z, leaf aa, leaf bb, leaf cc, leaf dd, leaf ee, leaf ff, leaf gg,
leaf hh, leaf ii and
leaf jj; in order to view the Table as a whole the leaves are arranged in the
following
spatial order:
a b c d a f g h hh
i k m o q s a w ii
j 1 n p r t v x jj
y z as bb cc dd ee ff gg
Thus, Table 1 has been split into separate pages in order to conform to the
specification however it is under stood that Row 1 of table 1 contains the
individual
numbers from I through to 140 (inclusive) and Column 1 of Table 1 contains the
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individual numbers from 1 through to 140 (inclusive). These numerals refer to
individual
genes as designated throughout the specification for example numeral 1 refers
to p53,
numeral 2 refers to C-CAM, numeral 3 refers to CEA, numeral 4 refers to p2 I ,
numeral S
refers to p 15, numeral 6 refers to BRCA 1. Each box marked with an "X"
denotes a
combination comprising the gene located in that horizontal row and with the
gene located
in the vertical column. Given the format of this Table it should be easy for
one of skill in
the art to add further genes to this table to work out possible combinations
contemplated.
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Table 1 (leaf a) Specific Gene Combinations.
1 2 3 4 5 6 7 C8 9 10 11 12 13 14 15
I X X X X X X _X X X X X X X X
2 X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X
X X _X X X X X X X X X X X X
6 X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X
8 X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
I1 X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X
17 X X X X X X X X X X X _ X X X
X
is x x x x x x x x x x x x x x x
I9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X
140 X X X X X X X~ X X X X X X X X
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Table 1 (Continued; leaf b)
16 i7 18 19 20 ,~2122 23 24 25 26 27 28 29 30
1 X X X X X X X X X X X X X X X
2 X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X
4 X X X X X X_ X X X X X X X X X
X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X
8 X _X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
11 X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X _
~ X
38 X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
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Table 1 (Continued; leaf c)
31 32 33 34 35 36 ~I 38 39 40 41 42 43 44 45
37 I
1 X X X X X X X X X X X X X X X
2 X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X
8 X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
11 X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X
is x x x x x x x x x x x x x x x
16 X X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X
X X X X X X X X X X X X X X
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Table 1 (Continued; leaf d)
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
1 X X X X X X X X X X X X X X X
2 X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X
~
7 X X X X X X X X _ X X X X X X
X
8 X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
11 X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X
l4oix~ x~ x~ x~ x~ x~ x~ x~ x~ xp x x x x x
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Table 1 (Continued; leaf e)
61 62 63 64 65~6b 67 68 69 70 71 72 73 74 75
~ ~ .
I X X X X X X X X X X X X X X X
2 X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X
8 X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
I1 X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
ZI X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X
I
X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
~~ ~ ~ ( ~ ~ ~ ~ ~ ~ J
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Table 1 (Continued; leaf f)
76 77 78 79 80 81 82 83 84 85 86 87 ~8889 ~90
1 X X X X X X X X X X X X X _ X
X
2 X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X
8 X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
il X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X
2s x x x x x x x x x x x x x x x
29 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X
( X X X X X X X X X X X X X X X
~ ~ ~ ~ ~ ~ ~ ~
~~
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Table 1 (Continued; leaf g)
91 92 93 94 95 96 97 98 99 100101 102 103I04 105 106107
1 X X X X X X X X X X X X X X _ X X
X
2 X X X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X X X
S X X X X X X X X X X X X X X X X X
6 X X X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X X X
8 X X X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
11 X X X X X X X X X _X X X X X X X X
12 X X X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X X X
~
X X X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X X X
i7 X X X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X X X
I9 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X X X
i X X X X X X X X X X X X X X X X X
I X X X X X X X X X X X X X X X X X
36
37 X X X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X X X
~ X X X X X X X X X X X . X X X X X
~ ~ ~ ~ ~ ~ ~ ~ ~ ~ I X 1
~f ~
-42-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf h)
108 109110 I11 112113 114115 116 117118 119120 121122 123124
1 x x x x x x x x x x x x x x x x x
2 X X X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X x X X X
X X X _ X X X X X X X X X X X _ X
X X
6 X X X X X X X X X X X X X X X X X
7 X X X X X X X X X X X X X X X X X
8 x x x x x x x x x x x x x x x x x
9 x x x x x x x x x x X -x x x x x x
1o x x x x x x x x x x x x x x x x x
I1 x x x x x x x x x x x x x x x x x
12 X X X X X X X X X X X X X X X _ X
X
13 X X X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
u6 x x x x x x x x x x x x x x x x x
I7 X X X X X X X X X X X X X X X X X
18 X X X X X X X X X X X X X X X X X
19 X X X X X X X X X X X X X X X X X
2o x x x x x x x x x x x x x x x x x
21 x x x x x x x x x x x x x x x x x
22 X X X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X X
-4s-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf i)
1 2 _3 4 5 6 ~ S r 10 11 12 t 14 15
1 9~ 3
41 X X X X X X X X X X X X X X X
~
42 X X X X X X X X X X X X X X X
43 X X X X X _X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X
46 X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X
69 X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X
80 X X X X X X X X X X X X X X X
-44-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf j)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
81 X X X X X X X X X X X X X X X
82 X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X
~
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X
115 X X X X X X X X X X X X X X X
llb X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X
-45-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf k)
16 17 18 19 20 21 22 23 24 25 26 27 ~2829 30
n n
N i
41 X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X X X X X X X X X _ X X X X
X
46 X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X
48 X X X X X X_ X X X X X X X X X
49 X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X
61 X X X X X X X X -X X X X X X X
62 X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X
~
69 X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X
76 X X X X X X X _ X X X X X X X
X
77 X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X
80 X X X X X X X X X X X X X X X
~~ ~ ~ ~ ~ ~ ~ ~ ~
-46-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf 1)
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
s1 x x x x x x x x x x x x x x x
82 X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
l08 x x x x x x x x x x x x x x x
~
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X
115 X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X
l22 X X X X X X X X X X X X X X X
i23 X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X
-47-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf m)
31 32 33 34 35 3b 37 38 39 40 41 42 43 44 45
41 X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X
46 X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X
64' X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X _ X
X
69 X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X
L X X x x x x x X x x - x x x x
8~ I ~ ~ ~ ~ ~ ~ ~ ~ I -x
II
-48-
CA 02323112 2000-09-14
WO 99/47690 PGT/US99/05781
Table 1 (Continued; leaf n)
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
81 X X X X X X X X X X X. X X X X
82 X X X X X X X X X X X _ X X X
X
83 X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X
90 X X X X X X X X X X X _ X X X
X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X
96 X X X X X X X X X X X _ X X X
X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
111 _X X X X _X X X X X X X X X X X
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
II4 X X X X X X X X X X X X X X X
115 X X X X _X X X X X X X X X X X
116 X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X
~ X X X X X X X X X X X X X X X
- ~ ~ ~ ~ ~ ~
124
~~
-49-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf o)
46 47 48 49 50 51 52 53 54 55 56 57 ~ 59 60
58
41 X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X * X X X X X X X X X X X X
46 X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X
~
52 X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X
b0 X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X
69 X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X
18~IIX x x x x x x x x x x x x x x
I I ~ ~ ~
-so-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/OS781
Table 1 (Continued; leaf p)
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
~
81 X X X X X X X X X X X X X X X
82 X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
9s X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
l04 x x x x x x x x x x x x x x x
los x x x x x x x x x x x x x x x
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X
115 X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X
( X X X X X X X X X X X X X X X
124 ~ ~ ~ ~
~~
-51-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf q)
61 62 63 64 ~ ~ 67 68 69 70 71 72 73 74 75
65 66
41 X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X
46 X _X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X ~X X
54 X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X
62 X X X X X X X ## X X X X X X
63 X X X X X X X # X X X X X X
64 X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X
69 X ## # X X X X X X _ X X X X
X
70 X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X
~ X X X X X X X X X X X X X X X
80 ~ ~ ~ ~
~~
-52-
CA 02323112 2000-09-14
WO 99/47690 PCTNS99/05781
Table 1 (Continued; leaf r)
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
81 X X X X X X X X X X X X X X X
82 X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X
s7 x x x x x x x x x x x x x x x
ss x x x x x x x x x x x x x x x
89 X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
~
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X
115 X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X 'Y X X X X X X
121 X X X X X X X X ~ X X X X X X
122 X X X X X X X X cp X X X X X X
123 X X X X X X X X 8 X X X X X X
124 X X X X X X X X p X X X X X X
-53-
CA 02323112 2000-09-14
WO 99/47690 PCTNS99/05781
Table 1 (Continued; leaf s)
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
41 X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X
46 X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X
'
60 X X X X X X X X X X X X X X X
6I X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X
69 X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X
a X X X X ~ X X X X X X X X X X
80 ~ ~ ~ ~
~~
-54-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Tabic 1 (Continued; leaf t)
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
81 X X X X X X X X X X X X X X
82 x x x x x x x x x x x x x x
s3 x x x x x x x x x x x x x x
84 X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X
96 X X X X X X X X X ~X X X X X X
97 X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X
II1 X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X
I15 X X X _X X X X X X X _ X X X X
X
116 X X X X' X X X X X X X X X X X
117 X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X
l22 X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X
~~ ~ ~ ~ I ~
-55-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf u)
91 92 93 94 95 96 97 98 99 100101 102103104105 106107 108
41 X X X X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X X X X
46 X X X X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X X X X
69 X X X X X X X X _ X X X X X X X X X
X
70 X X X X X X X X X X X X X X X X X X
71 x x x x x x x x x x x x x x x x x x
72 X X X X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X X X X
80 X X X X X X X X X X X X X X X X X X
CA 02323112 2000-09-14
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Table 1 (Continued; leaf v)
91 92 93 94 95 96 97 98 99 100 101102103104 105106107
sl x x x x x x x x x x x x x x x x x
82 x x x x x x x x x x x x x x x x x
83 X X X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X X
106 X X X X 3C X X X X X X X X X X X
107 X X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X X X
llo x x x x x x x x x x x x x x x x x
111 X X X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X _ X
X
113 X X X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X X X X X X
115 X X X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X X X
-5 7-
CA 02323112 2000-09-14
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Table 1 (Continued; leaf W)
109 110111 112 113114 115116 117118 119120 121 i22123 124
41 X X X X X X X X X X X X X X X X
42 X X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X X X X X
46 X X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X X
67 X X X X X X X X X X X X X X X X
68 X X X X X X X X X X X X X X X X
69 X X X X X X X X X X X 'Y ~ cp 8 p
70 X X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X X
yg~llx x x x x x x x x x x x x x x x
I I ~ ~ p
-ss-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf x)
108 109110 ill112 113114 115 116117 118119 120 121122 123124
81 X X X X X X X X X X X X X X X X X
82 X X X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X X~ X
85 X X X X X X X X X X X X X X X X X
86 X X X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X X X
88 X X X X X X X X X X X X X X X X X
89 X X X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X _ X
X
92 X X X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X _ X
X
10I X X X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X X X
~
106 X X X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X X
114 X X X X X X X X X X X X. X X X X
115 X X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X X
-59-
CA 02323112 2000-09-14
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Table 1 (Continued; leaf y)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
125 X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X _ X X
X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
-60-
CA 02323112 2000-09-14
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Table 1 (Continued; leaf z)
16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
125 X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
!34 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 _X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
-61-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Table 1 (Continued; leaf aa)
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
~~
125 X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
-62-
CA 02323112 2000-09-14
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Table 1 (Continued; leaf bb)
46 47 48 49 50 51 52 53 54 55 56 57 ~58~~5960
125 X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
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Table 1 (Continued; leaf cc)
61 62 63 64 65 66 67 68 69 70 71 72 73 74 75
125 X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
~
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
~~ ~ ~ ~ ~ ~ ~
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Table 1 (Continued; leaf dd)
76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
125 X X X X X
X X X X X X X X X X
126 X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X X 3C X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
_(~j_
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Table 1 (Continued; leaf ee)
91 92 93 94 95 9G 97 98 99 100101 102 103104 105106 107
~
125 X X X X X X X X X X X X X X ~ x X
X
126 X X X X X X X X X x X X X X X X X
127 X X X X X X X X X x X X X X X X X
128 X X X X X X X X X X X X X X X X X
129 X X X X X X X X x X X X -'XX X X X
130 X X X X X X X X X X X X X X X X X
131 x X X X X X X X X X X x X X x X X
132 X X X X X X X X X X X X X X X X X
133 x X X X X X X X X X X X X X X X X
134 x X X X X X X X X X X X X X X x X
135 X X x X X X X X X X X X X x X X X
136 X X x X X X X X X X X X X X X X X
137 X X X x X X X X X x X X x X x X X
138 X X X X X X X X X X X X X X X X X
139 x X X X X X X X X X X X x X X X X
~ x X x x X x x x x x X x x x x x x
14o ~ ~ ~ ~ ~ ~ ~
p
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Table 1 (Continued; leaf ff)
108109 110 111112 113114 115116 117 118119 ,120121122 123124
125 X X X X X X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X X X
129 X X X X X X X X X X X X -'X X X X X
130 X X X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X X X
I X X X X X X X X X X X X X X X X X
139
140 X X X X X X X X X X X X X X X X X
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Table 1 (Continued; leaf gg)
125 126 127128 129130 131132 133 134135 136137 138139 140
125 X X X
X X X X X X X X X X X X
126 X X X X X X X X X X X X X X X X
127 X X X X X X X X X X X X X X
128 X X X X X X X X X X X X X X X
129 X X X X X X X X X X X --X X X X
130 X X X X X X X X X X X X X X X
131 X X X X X X X X X X X X X X X
132 X X X X X X X X X X X X X X X
133 X X X X X X X X X X X X X X X
134 X X X X X X X X X X X X X X X
135 X X X X X X X X X X X X X X X
136 X X X X X X X X X X X X X X X
137 X X X X X X X X X X X X X X X
138 X X X X X X X X X X X X X X X
139 X X X X X X X X X X X X X X X
140 X X X X X X X X X X X X X X X
_~g_
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Table 1 (Continued; leaf hh)
125126 127128 129 130131 ' 133 134135 136 137138 139140
132 a n
1
1 X X X X X X X X X X X X X X X X
2 X X X X X X X X X X X X X X X X
3 X X X X X X X X X X X X X X X X
4 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X -X X X X
6 X X X X X X X X X X X X X X X X
7 X X X X X X X X _ X X X X X X X
X
8 X X X X X X X X X X X X X X X X
9 X X X X X X X X X X X X X X _ X
X
X X X X X X X X X X X X X X X X
11 X X X X X X X X X X X X X X X X
12 X X X X X X X X X X X X X X X X
13 X X X X X X X X X X X X X X X X
14 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
16 X X X X X X X X X X X X X X X X
17 X X X X X X X X X X X X X X X X
Is x x x x x x x x x x x x x x x x
19 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
21 X X X X X X X X X X X X X X X X
22 X X X X X X X X X X X X X X X X
23 X X X X X X X X X X X X X X X X
24 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
26 X X X X X X X X X X X X X X X X
27 X X X X X X X X X X X X X X X X
28 X X X X X X X X X X X X X X X X
29 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
31 X X X X X X X X X X X X X X X X
32 X X X X X X X X X X X X X X X X
33 X X X X X X X X X X X X X X X X
34 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
36 X X X X X X X X X X X X X X X X
37 X X X X X X X X X X X X X X X X
38 X X X X X X X X X X X X X X X X
39 X X X X X X X X X X X X X X X X
X X X X X X X X X X X X X X X X
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Table 1 (Continued; leaf ii)
125126 127128 129 130i31 132133 134135 136 137138 139140
41 _X X X X X X X X X X X X X X X X
42 ~X X X X X X X X X X X X X X X X
43 X X X X X X X X X X X X X X X X
44 X X X X X X X X X X X X X X X X
45 X X X X X X X X X X X X 'X X X X
46 X X X X X X X X X X X X X X X X
47 X X X X X X X X X X X X X X X X
48 X X X X X X X X X X X X X X X X
49 X X X X X X X X X X X X X X X X
50 X X X X X X X X X X X X X X X X
51 X X X X X X X X X X X X X X X X
52 X X X X X X X X X X X X X X X X
53 X X X X X X X X X X X X X X X X
54 X X X X X X X X X X X X X X X X
55 X X X X X X X X X X X X X X X X
56 X X X X X X X X X X X X X X X X
57 X X X X X X X X X X X X X X X X
58 X X X X X X X X X X X X X X X X
59 X X X X X X X X X X X X X X X X
60 X X X X X X X X X X X X X X X X
61 X X X X X X X X X X X X X X X X
62 X X X X X X X X X X X X X X X X
63 X X X X X X X X X X X X X X X X
64 X X X X X X X X X X X X X X X X
65 X X X X X X X X X X X X X X X X
66 X X X X X X X X X X X X X X X X
67 X X X X X ~ X X X X X X X X X X
68 X X X X X X X X X X X X X X X X
69 X X X X X X X X X X X X X X X X
70 X X X X X X X X X X X X X X X X
71 X X X X X X X X X X X X X X X X
72 X X X X X X X X X X X X X X X X
73 X X X X X X X X X X X X X X X X
74 X X X X X X X X X X X X X X X X
75 X X X X X X X X X X X X X X X X
76 X X X X X X X X X X X X X X X X
77 X X X X X X X X X X X X X X X X
78 X X X X X X X X X X X X X X X X
79 X X X X X X X X X X X X X X X X
80 X X X X X X X X X X X X X X X X
X
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Table 1 (Continued; leaf jj)
125126 127128 129I30 131132 133 134135 136137 138139 140
81 X X X X X X X X X X X X X X X X
82 X _X X X X X X X X X X X X X X X
83 X X X X X X X X X X X X X X X X
84 X X X X X X X X X X X X X X X X
85 X X X X X X X X X X X X --X X X X
86 X X X X X X X X X X X X X X X X
87 X X X X X X X X X X X X X X X X
ss x x x x x x x x x x x x x x x x
89 X X X X X X X X X X X X X X X X
90 X X X X X X X X X X X X X X X X
91 X X X X X X X X X X X X X X X X
92 X X X X X X X X X X X X X X X X
93 X X X X X X X X X X X X X X X X
94 X X X X X X X X X X X X X X X X
95 X X X X X X X X X X X X X X X X
96 X X X X X X X X X X X X X X X X
97 X X X X X X X X X X X X X X X X
98 X X X X X X X X X X X X X X X X
99 X X X X X X X X X X X X X X X X
100 X X X X X X X X X X X X X X X X
101 X X X X X X X X X X X X X X X X
102 X X X X X X X X X X X X X X X X
103 X X X X X X X X X X X X X X X X
104 X X X X X X X X X X X X X X X X
105 X X X X X X X X X X X X X X X X
106 X X X X X X X X X X X X X X X X
107 X X X X X X X X X X X X X X X X
108 X X X X X X X X X X X X X X X X
109 X X X X X X X X X X X X X X X X
110 X X X X X X X X X X X X X X X X
111 X X X X X X X X X X X X X X X X
112 X X X X X X X X X X X X X X X X
113 X X X X X X X X X X X X X X X X
114 X_ X X X _X X X X X X X X X X X X
IIS X X X X X X X X X X X X X X X X
116 X X X X X X X X X X X X X X X X
117 X X X X X X X X X X X X X X X X
118 X X X X X X X X X X X X X X X X
119 X X X X X X X X X X X X X X X X
120 X X X X X X X X X X X X X X X X
121 X X X X X X X X X X X X X X X X
122 X X X X X X X X X X X X X X X X
123 X X X X X X X X X X X X X X X X
124 X X X X X X X X X X X X X X X X
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Footnotes pertaining to Table 1: * combination of IL-2 with HSV-tk in an
adenovirus
vector was described by O'Malley et al., (1997 and 1996 both incorporated
herein by
reference). # denotes the combination of GM-CSF and IFNa described in WO
97/32481
(incorporated herein by reference). ## denotes the combination of GM-CSF and
IFN~3
described in WO 97/32481 (incorporated herein by reference). 'I' denotes the
combination of GM-CSF and TGF as described in WO 97/32481 (incorporated herein
by
reference). ~ denotes the combination of PDGF and GM-CSF as described in WO
97/32481 (incorporated herein by reference). cp denotes the combination of
IFNy and
GM-CSF as described in WO 97/32481 (incorporated herein by reference). 0
denotes
the combination of M-CSF and GM-CSF as described in WO 97/32481 (incorporated
herein by reference). p denotes the combination of tumor necrosis factor and
GM-CSF as
described in WO 97/32481 (incorporated herein by reference).
V. Regulatory Elements
A. Promoters
Throughout this application, the term "expression construct" is meant to
include
any type of genetic construct containing a nucleic acid coding for gene
products in which
part or all of the nucleic acid encoding sequence is capable of being
transcribed. The
transcript may be translated into a protein, but it need not be. In certain
embodiments,
expression includes both transcription of a gene and translation of mRNA into
a gene
product. In other embodiments, expression only includes transcription of the
nucleic acid
encoding genes of interest.
The nucleic acid encoding a gene product is under transcriptional control of a
promoter. A "promoter" refers to a DNA sequence recognized by the synthetic
machinery of the cell, or introduced synthetic machinery, required to initiate
the specific
transcription of a gene. The phrase "under transcriptional control" means that
the
promoter is in the correct location and orientation in relation to the nucleic
acid to control
RNA polymerase initiation and expression of the gene.
The term promoter will be used here to refer to a group of transcriptional
control
modules that are clustered around the initiation site for RNA polymerase II.
Much of the
thinking about how promoters are organized derives from analyses of several
viral
promoters, including those for the HSV thymidine kinase (tk) and SV40 early
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transcription units. These studies, augmented by more recent work, have shown
that
promoters are composed of discrete functional modules, each consisting of
approximately
7-20 by of DNA, and containing one or more recognition sites for
transcriptional
activator or repressor proteins.
S
At least one module in each promoter functions to position the start site for
RNA
synthesis. The best known example of this is the TATA box, but in some
promoters
lacking a TATA box, such as the promoter for the mammalian terminal
deoxynucleotidyl
transferase gene and the promoter for the SV40 late genes, a discrete element
overlying
the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptionai
initiation.
Typically, these are located in the region 30-110 by upstream of the start
site, although a
number of promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter elements
frequently
is flexible, so that promoter function is preserved when elements are inverted
or moved
relative to one another. In the tk promoter, the spacing between promoter
elements can
be increased to 50 by apart before activity begins to decline. Depending on
the promoter,
it appears that individual elements can function either co-operatively or
independently to
activate transcription.
The particular promoter employed to control the expression of a nucleic acid
sequence of interest is not believed to be important, so long as it is capable
of directing
the expression of the nucleic acid in the targeted cell. Thus, where a human
cell is
targeted, it is preferable to position the nucleic acid coding region adjacent
to and under
the control of a promoter that is capable of being expressed in a human cell.
Generally
speaking, such a promoter might include either a human or viral promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early
gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal
repeat, (3-
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actin, rat insulin promoter, human ubiquitin C promoter and glyceraldehyde-3-
phosphate
dehydrogenase can be used to obtain high-level expression of the coding
sequence of
interest. The use of other viral or mammalian cellular or bacterial phage
promoters which
are well-known in the art to achieve expression of a coding sequence of
interest is
contemplated as well, provided that the levels of expression are suffcient for
a given
purpose. By employing a promoter with well-known properties, the level and
pattern of
expression of the protein of interest following transfection or transformation
can be
optimized.
Selection of a promoter that is regulated in response to specific physiologic
or
synthetic signals can permit inducible expression of the gene product. For
example in the
case where expression of a transgene, or transgenes when a multicistronic
vector is
utilized, is toxic to the cells in which the vector is produced in, it may be
desirable to
prohibit or reduce expression of one or more of the transgenes. Examples of
transgenes
that may be toxic to the producer cell line are pro-apoptotic and cytokine
genes. Several
inducible promoter systems are available for production of viral vectors where
the
transgene product may be toxic.
The ecdysone system (Invitrogen, Carlsbad, CA) is one such system. This system
is designed to allow regulated expression of a gene of interest in mammalian
cells. It
consists of a tightly regulated expression mechanism that allows virtually no
basal level
expression of the transgene, but over 200-fold inducibility. The system is
based on the
heterodimeric ecdysone receptor of Drosophila, and when ecdysone or an analog
such as
muristerone A binds to the receptor, the receptor activates a promoter to turn
on
expression of the downstream transgene high levels of mRNA transcripts are
attained. In
this system, both monomers of the heterodimeric receptor are constitutively
expressed
from one vector, whereas the ecdysone-responsive promoter which drives
expression of
the gene of interest is on another plasmid. Engineering of this type of system
into the
gene transfer vector of interest would therefore be useful. Cotransfection of
plasmids
containing the gene of interest and the receptor monomers in the producer cell
line would
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then allow for the production of the gene transfer vector without expression
of a
potentially toxic transgene. At the appropriate time, expression of the
transgene could be
activated with ecdysone or muristeron A.
Another inducible system that would be useful is the Tet-~ffrM or Tet-OnTM
system (Ciontech, Palo Alto, CA) originally developed by Gossen and Bujard
(Gossen
and Bujard, 1992; Gossen et al., 1995). This system also allows high levels of
gene
expression to be regulated in response to tetracycline or tetracycline
derivatives such as
doxycycline. In the Tet-OnTM system, gene expression is turned on in the
presence of
doxycycline, whereas in the Tet-OffrM system, gene expression is turned on in
the
absence of doxycycline. These systems are based on two regulatory elements
derived
from the tetracycline resistance operon of E coli. The tetracycline operator
sequence to
which the tetracycline repressor binds, and the tetracycline repressor
protein. The gene of
interest is cloned into a plasmid behind a promoter that has tetracycline-
responsive
elements present in it. A second plasmid contains a regulatory element called
the
tetracycline-controlled transactivator, which is composed, in the Tet-OffrM
system, of the
VP16 domain from the herpes simplex virus and the wild-type tetracycline
repressor.
Thus in the absence of doxycycline, transcription is constituitively on. In
the Tet-OnTM
system, the tetracycline repressor is not wild type and in the presence of
doxycycline
activates transcription. For gene therapy vector production, the Tet-OffrM
system would
be preferable so that the producer cells could be grown in the presence of
tetracycline or
doxycycline and prevent expression of a potentially toxic transgene, but when
the vector
is introduced to the patient, the gene expression would be constituitively on.
In some circumstances, it may be desirable to regulate expression of a
transgene
in a gene therapy vector. For example, different viral promoters with varying
strengths of
activity may be utilized depending on the level of expression desired. In
mammalian
cells, the CMV immediate early promoter if often used to provide strong
transcriptional
activation. Modified versions of the CMV promoter that are less potent have
also been
used when reduced levels of expression of the transgene are desired. When
expression of
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a transgene in hematopoetic cells is desired, retroviral promoters such as the
LTRs from
MLV or MMTV are often used. Other viral promoters that may be used depending
on
the desired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus
promoters such as from the EIA, E2A, or MLP region, AAV LTR, cauliflower
mosaic
virus, HSV-TK, and avian sarcoma virus.
Similarly tissue specific promoters may be used to effect transcription in
specific
tissues or cells so as to reduce potential toxicity or undesirable effects to
non-targeted
tissues. For example, promoters such as the PSA, probasin, prostatic acid
phosphatase or
prostate-specific glandular kallikrein (hK2) may be used to target gene
expression in the
prostate. Similarly, the following promoters may be used to target gene
expression in
other tissues (Table 2).
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Table 2. Tissue specific promoters
Tissue Promoter
Pancreas insulin
elastin
amylase
pdr-1 pdx-1
glucokinase
Liver albumin PEPCK
HB V enhancer
alpha fetoprotein
apolipoprotein C
alpha-1 antitrypsin
. vitellogenin, NF-AB
Transthyretin
Skeletal muscle myosin H chain
muscle creatine kinase
dystrophin
calpain p94
skeletal alpha-actin
fast troponin 1
Skin keratin K6
keratin K1
Lung CFTR
human cytokeratin 18 (K18)
pulmonary surfactant proteins A, B
and C
CC-10
P1
Smooth muscle sm22 alpha
SM-alpha-actin
Endothelium endothelin-1
E-selectin
von Willebrand factor
TIE (Korhonen et al., 1995)
KDR/flk-1
Melanocytes tyrosinase
Adipose tissue lipoprotein lipase (Zechner et al.,
1988)
adipsin (Spiegelman et al., 1989)
acetyl-CoA carboxylase (Page and Kim,
1989)
glycerophosphate dehydrogenase (Dani
et al., 1989)
adipocyte P2 (Hunt et al., 1986)
Blood ~3-globin
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In certain indications, it may be desirable to activate transcription at
specific times
after administration of the gene therapy vector. This may be done with such
promoters as
those that are hormone or cytokine regulatable. For example in gene therapy
applications
where the indication is a gonadal tissue where specific steroids are produced
or routed to,
use of androgen or estrogen regulated promoters may be advantageous. Such
promoters
that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco. Other
hormone regulated promoters such as those responsive to thyroid, pituitary and
adrenal
hormones are expected to be useful in the present invention. Cytokine and
inflammatory
protein responsive promoters that could be used include K and T Kininogen
(Kageyama
et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al., 1988),
haptoglobin
(Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poll and
Cortese,
1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-I acid glycoprotein
(Prowse
and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al.,
1988),
l5 angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible by phorbol
esters, TNF-
alpha, UV radiation, retinoic acid, and hydrogen peroxide), collagenase
(induced by
phorbol esters and retinoic acid), metallothionein (heavy metal and
glucocorticoid
inducible), Stromelysin (inducible by phorbol ester, interleukin-1 and EGF),
alpha-2
macroglobulin and alpha-I antichymotrypsin.
It is envisioned that cell cycle regulatable promoters may be useful in the
present
invention. For example, in a bi-cistronic gene therapy vector, use of a strong
CMV
promoter to drive expression of a first gene such as p16 that arrests cells in
the G1 phase
could be followed by expression of a second gene such as p53 under the control
of a
promoter that is active in the G1 phase of the cell cycle, thus providing a
"second hit" that
would push the cell into apoptosis. Other promoters such as those of various
cyclins,
PCNA, galectin-3, E2F 1, p53 and BRCA 1 could be used.
Tumor specific promoters such as osteocalcin, hypoxia-responsive element
{HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be
used
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to regulate gene expression in tumor cells. Other promoters that could be used
according
to the present invention include Lac-regulatable, chemotherapy inducible (e.g.
MDR),
and heat (hyperthermia) inducible promoters, radiation-inducible (e.g., EGR
(Joki et al.,
1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acid promoters, Ul
snRNA (Bartlett et al., 1996), MC-1, PGK, (3-actin and a-globin. Many other
promoters
that may be useful are listed in Walther and Stein (1996).
It is envisioned that any of the above promoters alone or in combination with
another may be useful according to the present invention depending on the
action desired.
In addition, this list of promoters is should not be construed to be
exhaustive or limiting,
those of skill in the art will know of other promoters that may be used in
conjunction with
the promoters and methods disclosed herein.
B. Enhancers
Enhancers are genetic elements that increase transcription from a promoter
located at a distant position on the same molecule of DNA. Enhancers are
organized
much like promoters. That is, they are composed of many individual elements,
each of
which binds to one or more transcriptional proteins. The basic distinction
between
enhancers and promoters is operational. An enhancer region as a whole must be
able to
stimulate transcription at a distance; this need not be true of a promoter
region or its
component elements. On the other hand, a promoter must have one or more
elements that
direct initiation of RNA synthesis at a particular site and in a particular
orientation,
whereas enhancers lack these specificities. Promoters and enhancers are often
overlapping and contiguous, often seeming to have a very similar modular
organization.
Below is a list of promoters additional to the tissue specific promoters
listed
above, cellular promoters/enhancers and inducible promoters/enhancers that
could be
used in combination with the nucleic acid encoding a gene of interest in an
expression
construct (Table 3 and Table 4). Additionally, any promoter/enhancer
combination (as
per the Eukaryotic Promoter Data Base EPDB) could also be used to drive
expression of
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the gene. Eukaryotic cells can support cytoplasmic transcription from certain
bacterial
promoters if the appropriate bacterial polymerase is provided, either as part
of the
delivery complex or as an additional genetic expression construct.
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TABLE 3
ENHANCER
ImmunoglobulinHeavy Chain
ImmunoglobulinLight Chain
T-Cell Receptor
HLA DQ a and DQ ~i
(3-Interferon
Interleukin-2
Interleukin-2 Receptor
MHC Class II 5
MHC Class II HLA-DRa
(3-Actin
Muscle Creatine Kinase
Prealbumin (Transthyretin)
Elastase I
Metallothionein
Collagenase
Albumin Gene
a-Fetoprotein
T-Globin
(3-Globin
e-fos
c-HA-ras
Insulin
Neural Cell Adhesion Molecule (NCAM)
a 1-Antitrypsin
H2B (TH2B) Histone
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ENHANCER
Mouse or Type I Collagen
Glucose-RegulatedProteins (GRP94 and GRP78)
Rat Growth Hormone
Human Serum Amyloid A (SAA)
Troponin I (TN I)
Platelet-Derived Growth Factor
Duchenne Muscular Dystrophy
S V40
Polyoma
Retroviruses
Papilloma Virus
Hepatitis B Virus
Human ImmunodeficiencyVirus
Cytomegalovirus
Gibbon Ape Leukemia Virus
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TABLE 4
Element Inducer
MT II Phorbol Ester (TPA)
Heavy metals
MMTV (mouse mammary tumorGlucocorticoids
virus)
13-Interferon poly(rI)X
poly(rc)
Adenovirus 5 E2 Ela
c J~ Phorbol Ester (TPA), H20z
Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL-1
SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle Disease Virus
GRP78 Gene A23187
a-2-Macroglobulin IL-6
V imentin Serum
MHC Class I Gene H-2kB Interferon
HSP70 Ela, SV40 Large T Antigen
Proliferin Phorbol Ester-TPA
Tumor Necrosis Factor FMA
Thyroid Stimulating HormoneThyroid Hormone
a
Gene
Insulin E Box Glucose
C. Polyadenylation Signals
Where a cDNA insert is employed, one will typically desire to include a
polyadenylation signal to effect proper polyadenylation of the gene
transcript. The nature
of the polyadenylation signal is not believed to be crucial to the successful
practice of the
invention, and any such sequence may be employed such as human or bovine
growth
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hormone and SV40 polyadenylation signals. Also contemplated as an element of
the
expression cassette is a terminator. These elements can serve to enhance
message levels
and to minimize read through from the cassette into other sequences.
D. IRES
In certain embodiments of the invention, the use of internal ribosome entry
site
(IRES) elements is contemplated to create multigene, or polycistronic,
messages. IRES
elements are able to bypass the ribosome scanning model of 5' methylated Cap
dependent
translation and begin translation at internal sites (Pelletier and Sonenberg,
1988). IRES
elements from two members of the picornavirus family (poliovirus and
encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as
well an
IRES from a mammalian message (Macejak and Sarnow, 1991 ). IRES elements can
be
linked to heteroiogous open reading frames. Multiple open reading frames can
be
transcribed together, each separated by an IRES, creating polycistronic
messages. By
virtue of the IR.ES element, each open reading frame is accessible to
ribosomes for
efficient translation. Multiple genes can be efficiently expressed using a
single
promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This
includes genes for secreted proteins, multi-subunit proteins, encoded by
independent
genes, intracellular or membrane-bound proteins and selectable markers. In
this way,
expression of several proteins can be simultaneously engineered into a cell
with a single
construct and a single selectable marker.
VI. Methods for Producing Viral Particles
The traditional method for the generation of adenoviral particles is co-
transfection
followed by subsequent in vivo recombination of a shuttle piasmid (usually
containing a
small subset of the adenoviral genome and the gene of interest in an
expression cassette)
and an adenoviral helper plasmid (containing most of the entire adenoviral
genome) into
either 293 or 91 1 cells (obtained from Introgene, 1'he Netherlands). After
transfection,
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the adenoviral plaques are isolated from the agarose overlaid cells and the
viral particles
are expanded for analysis. For detailed protocols the skilled artisan is
referred to Graham
and Prevac ( I 991 ).
Alternative technologies for the generation of novel adenouiral vectors (i.e.,
vectors including the adenoviral genes necessary for 293 cell dependent viral
replication
and expression cassettes) involving genes) of interest, include utilization of
the bacterial
artificial chromosome (BAC) system, in vivo bacterial recombination in a recA+
bacterial
strain utilizing two plasmids containing complementary adenoviral sequences
and
expression cassettes, and the yeast artificial chromosome (YAC) system).
Incorporated
herein by reference are PCT publications 95/27071 and 96/33280 which provide
details
of adenoviral production methodologies. Methods for improved production and
purification of adenoviral vectors have been described in U.S. Patent
Application
08/975,519, filed November 20, 1997 (specifically incorporated herein by
reference).
The following protocol provides an example of virus production and expansion
used in the present invention. Of course this is only an exemplary protocol
and one of
ordinary skill in the art will be able to modify steps therein according to
individual
requirements.
A. Cotransfection of Adenoviral Plasmids to Make Adenovirus
Day 1:
In the morning, seed ATCC 293 cells at I x 106 celis per 60 mm dish
Day 2:
Using the protocol for calcium phosphate transfections, transfect 293 cells
with
5pg of DNA per plate (2.5~g of each plasmid, i.e. pJMl7 and shuttle vector).
-Transfect 2-3 plates each for the control (no DNA) and test plasmid:
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-Transfect 1-2 plates with 13-gal to check effciency.
Day 3:
Change media on cells in morning
In afternoon, trypsinize transfected plates, count cells, and seedat 1.5 -
1.75 x 106
cells per well of a 6-well dish.
Day 4:
X-gal stain ~3-gal plate to check efficiency.
Day 5:
Overlay transfected cells using procedures from SOP #TM001-04
Wait to see plaques (probably 4 - 7 days, but could be 3 - 4 wk when using YAC
DNA)
IS
B. Expansion of Adenovirus Following Plaque Purification
Day 1:
Seed a 24-well plate at 2 - 2.5 x 105 ATCC 293 cells/well two days prior to
infection
Day 3:
Seed 60 mm dishes at 2 x 106 ATCC 293 cells/dish for infection on Day 5
Pick a plaque, using a sterile capillary pipette, into 1 SO ul of serum-free
media
Vortex briefly to break up agarose
Aspirate media off 24-well plate
Infect with 100 pl of viral-containing media/well for 1 hr, rocking after 15
and
4~ min
Mock infect control well with 100 pl of serum-free media
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Add 1 ml of DMEM + 10% FBS
Wait to see CPE activity (about 48 h).
Day 5:
S Seed 2 x 150 mm dishes (for each plaque isolated) at 1.2 x 10' ATCC 293
cells/dish for infection on Day 7 (or ~6 x 106 cells/dish for infection on Day
8).
After CPE appears in 24-well dish, harvest cells and supernatant into a 1 S ml
Corning tube.
Freeze/thaw 3X in liquid nitrogen and 37C H20 bath.
Spin at 2000 rpm for 5 min in clinical centrifuge.
Filter supernatant through a 0.22 pm filter.
Aspirate media off 60 mm dish.
Infect with 550 pl of viral-containing media/60 mm dish for 1 hr, rocking
after 15
and 45 min.
1 S Mock infect control dish with 550 tcl of serum-free media.
Add 5 ml DMEM + 10% FBS.
Wait to see CPE.
Day 7:
After CPE appears in 60 mm dish, harvest cells and supernatant into a 50 ml
Corning tube.
Freeze/thaw 3X in liquid nitrogen and 37C H20 bath.
Spin at 2000 rpm for 5 min in clinical centrifuge.
Filter supernatant through a 0.22 pm filter (reserve .5 - 1 ml for DNA
isolation
and PCRTM).
Aspirate media off 150 mm dishes.
Infect with 2 ml of viral-containing media + 2 ml of serum-free medial150 mm
dish for 1 hr, rocking after 15 and 45 min.
Mock infect control dish with 4 ml of serum-free media.
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Add 20 ml DMEM + 10% FBS.
Wait to see CPE.
Day 9: ,
After CPE appears in 1 SO mm dishes, harvest cells and supernatant into 3 x 50
ml
Corning tubes.
Freeze/thaw 3X in liquid nitrogen and 37C H20 bath.
Spin at 2000 rpm for S min in clinical centrifuge.
Filter supernatant through a 0.22 pm filter into a sterile SO ml Corning tube.
Viral lysate is ready to be titered.
VII. Disease States
The present invention deals with the treatment of disease states that involve
hyperproliferative disorders including benign and malignant neoplasias. Such
disorders
I S include restinosis, cancer, multi-drug resistant cancer, primary,
psoriasis, inflamatory
bowel disease, rheumatoid arthritis, osteoarthritis and metastatic tumors.
In particular, the present invention is directed at the treatment of human
cancers
including cancers of the prostate, lung, brain, skin, liver, breast, lymphoid
system,
stomach, testicular, ovarian, pancreatic, bone, bone marrow, head and neck,
cervical,
esophagus, eye, gall bladder, kidney, adrenal glands, heart, colon, rectum and
blood.
VIII. Screening For Anti-Tumor Activity In Multigene Adenoviral Constructs
Using Animal Models
Animal models may be used as a screen for tumor suppressive effects of genes
or
gene combinations. Preferably, orthotopic animal models will be used so as to
closely
mimic the particular disease type being studied and to provide the most
celevant results.
One type of orthotopic model is that for head and neck cancer, which involves
the
development of an animal model for the analysis of microscopic residual
carcinomas and
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microscopic seeding of body cavities. "Carcinoma," as used herein, may refer
to a single
cell or a multicellular tumor mass. In microscopic disease, the "tumor" will
consist or
one or a few carcinoma cells which cannot be observed with the naked eye. The
animal
model described herein is particularly advantageous mimicking (i) the post
surgical
environment of head and neck cancer patients, particularly in advanced stages
of disease
and (ii) the body cavity of an affected subject wherein microscopic carcinoma
has been
established. The model, similar to~ other animal models for cancer, derives
from
inoculation of tumor cells into an animal. A distinction, however, lies in the
creation,
subcutaneously, of a pouch that is a physiologic equivalent of a natural body
cavity or a
post-surgical cavity created by the excision of a tumor mass.
The instant invention preferably uses nude mice as the model organism.
Virtually
any animal may be employed, however, for use according to the present
invention.
Particularly preferred animals will be small mammals that are routinely used
in laboratory
protocols. Even more preferred animals will be those of the rodent group, such
as mice,
rats, guinea pigs and hamsters. Rabbits also are a preferred species. The
criteria for
choosing an animal will be largely dependent upon the particular preference of
an
investigator.
The first step is to create a tissue flap in the experimental animal. The term
"tissue flap" means any incision in the flesh of the animal that exposes the
target tissue.
It is generally preferred that an incision be made in the dorsal flank of an
animal, as this
represents a readily accessible site. However, it will be understood that an
incision could
well be made at other points on the animal, and the choice of tissue sites may
be
dependent upon various factors such as the particular type of therapeutics
that are being
investigated.
Once a target tissue site is exposed, carcinoma cells, either individually or
in
microscopic tumors, are contacted with the tissue site. The most convenient
manner for
seeding the cancer cells into the tissue site is to apply a suspension of
tissue culture media
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containing the cells to the exposed tissue. Cancer cell application may be
achieved
simply using a sterile pipette or any other convenient applicator. Naturally,
this
procedure will be conducted under sterile conditions.
In a particular example, 2.5 x 106 cells are inoculated into the exposed
tissue flap
of a nude mouse. Those of skill in the art will be able to readily determine,
for a given
purpose, what the appropriate number of cells will be. The number of cells
will be
dependent upon various factors, such as the size of the animal, the site of
incision, the
replicative capacity of the tumor cells themselves, the time intended for
tumor growth,
the potential anti-tumor therapeutic to be tested, and the like. Although
establishing an
optimal model system for any particular type of tumor may require a certain
adjustment
in the number of cells administered, this in no way represents an undue amount
of
experimentation. Those skilled in the area of animal testing will appreciate
that such
optimization is required.
This can be accomplished, for example, by conducting preliminary studies in
which differing numbers of cells are delivered to the animal and the cell
growth is
monitored following resealing of the tissue flap. Naturally, administering
larger numbers
of cells will result in a larger population of microscopic residual tumor
cells.
In the present study the flaps were effectively sealed using mattress sutures.
However, it is envisioned that persons skilled in the art may use any of a
variety of
methods routinely used to seal the incision such as the use of adhesives,
clamps, stitches,
sutures, etc., depending on the particular use contemplated.
Other orthotopic animal models are well known in the art. The orthotopic lung
cancer model, for example has been described in the literature. This protocol
involves
injection of tumor cells into the bronchus of a mouse wherein tumors will form
in the
bronchus and bronchioles, mimicking tumors commonly found in non-small cell
lung
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cancer patients. The skilled artisan will readily be able to adapt or modify
each particular
model for his intended purpose without undue experimentation.
IX. Treatment Protocols
Clinical protocols may be developed to facilitate the treatment of disease
using
the multigene constructs discussed herein and above. Patients may, but need
not have
received previous chemo-, radio- or gene therapies. Optimally, patients will
have
adequate bone marrow function (defined as peripheral absolute granulocyte
count of >
2,000/mm3 and platelet count of 100,000/mm3), adequate liver function
(bilirubin < 1.5
mg/dl) and adequate renal function (creatinine < 1.5 mg/dl).
The protocol calls for single dose administration, via intratumoral injection,
of a
pharniaceutical composition containing between 106 and 109 infectious
particles of the
expression construct. For tumors of > 4 cm, the volume administered will be 4-
10 ml
(preferably 10 ml), while for tumors < 4 cm, a volume of 1-3 ml will be used
(preferably
3 ml). Multiple injections will be delivered for a single dose, in 0.1-0.5 ml
volumes, with
spacing of approximately 1 cm or more.
The treatment course consists of about six doses, delivered over two weeks.
Upon
election by the clinician, the regimen may be continued, six doses each two
weeks, or on
a less frequent (monthly, bimonthly, quarterly, etc.) basis.
Where patients are eligible for surgical resection, the tumor will be treated
as
described above for at least two consecutive two-week treatment courses.
Within one wk
of completion of the second (or more, e.g., third, fourth, fifth, sixth,
seventh, eighth, etc.)
course, the patient will receive surgical resection. Prior to close of the
incision, 10 ml of
a pharmaceutical composition containing the expression construct (106-109
infectious
particles) will be delivered to the surgical site (operative bed) and allowed
to remain in
contact for at least 60 min. The wound is closed and a drain or catheter
placed therein.
On the third post-operative day, additional 10 ml of the pharmaceutical
composition is
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administered via the drain and allowed to remain in contact with the operative
bed for at
least two h. Removal by suction is then performed, and the drain removed at a
clinically
appropriate time.
A. Treatment ofArtificial and Natural Body Cavities
One of the prime sources of recurrent tumor growth is the residual,
microscopic
disease that remains at the primary tumor site, as well as locally and
regionally, following
tumor excision. In addition, there are analogous situations where natural body
cavities
are seeded by microscopic tumor cells. The effective treatment of such
microscopic
disease would present a significant advance in therapeutic regimens.
Thus, in certain embodiments, a cancer may be removed by surgical excision,
creating a "cavity." Both at the time of surgery, and thereafter (periodically
or
continuously), the therapeutic composition of the present invention is
administered to the
body cavity. This is, in essence, a "topical" treatment of the surface of the
cavity. The
volume of the composition should be sufficient to ensure that the entire
surface of the
cavity is contacted by the expression construct.
In one embodiment, administration simply will entail injection of the
therapeutic
composition into the cavity formed by the tumor excision. In another
embodiment,
mechanical application via a sponge, swab or other device may be desired.
Either of
these approaches can be used subsequent to the tumor removal as well as during
the
initial surgery. In still another embodiment, a catheter is inserted into the
cavity prior to
closure of the surgical entry site. The cavity may then be continuously
perfused for a
desired period of time.
In another form of this treatment, the "topical" application of the
therapeutic
composition is targeted at a natural body cavity such as the mouth, pharynx,
esophagus,
larynx, trachea, pleural cavity, peritoneal cavity, or hollow organ cavities
including the
bladder, colon or other visceral organs. In this situation, there may or may
not be a
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significant, primary tumor in the cavity. The treatment targets microscopic
disease in the
cavity, but incidentally may also affect a primary tumor mass if it has not
been previously
removed or a pre-neoplastic lesion which may be present within this cavity.
Again, a
variety of methods may be employed to affect the "topical" application into
these visceral
S organs or cavity surfaces. For example, the oral cavity in the pharynx may
be affected by
simply oral swishing and gargling with solutions. However, topical treatment
within the
larynx and trachea may require endoscopic visualization and topical delivery
of the
therapeutic composition. Visceral organs such as the bladder or colonic mucosa
may
require indwelling catheters with infusion or again direct visualization with
a cystoscope
or other endoscopic instrument. Cavities such as the pleural and peritoneal
cavities may
be accessed by indwelling catheters or surgical approaches which provide
access to those
areas.
B. Monitoring Gene Expression FollowingAdministration
Another aspect of the present invention involves the monitoring of gene
expression following administration of the therapeutic composition. Because
destruction
of microscopic tumor cells cannot be observed, it is important to determine
whether the
target site has been effectively contacted with the expression construct. This
may be
accomplished by identifying cells in which the expression construct is
actively producing
the gene product. It is important, however, to be able to distinguish between
the
exogenous gene product and that present in tumor and non-tumor cells in the
treatment
area. Tagging of the exogenous protein with a tracer element would provide
definitive
evidence for expression of that molecule and not an endogenous version
thereof.
One such tracer is provided by the FLAG biosystem (Hopp et al., 1988). The
FLAG polypeptide is an octapeptide (AspTyrLysAspAspAspAspLys} and its small
size
does not disrupt the expression of the delivered gene therapy protein. The
coexpression
of FLAG and the protein of interest is traced through the use of antibodies
raised against
FLAG protein.
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Other immunologic marker systems, such as the 6XHis system (Qiagen) also may
be employed. For that matter, any linear epitope could be used to generate a
fusion
protein so long as (i) the immunologic integrity of the epitope is not
compromised by the
fusion and (ii) the functional integrity is not compromised by the fusion.
S
X. Therapeutic Formulations and Routes of Administration
Where clinical applications are contemplated, it will be necessary to prepare
the
viral expression vectors of the present invention as pharmaceutical
compositions, i.e., in a
form appropriate for in vivo applications. Generally, this will entail
preparing
compositions that are essentially free of pyrogens, as well as other
impurities that could
be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render
delivery vectors stable and allow for uptake by target cells. Aqueous
compositions of the
I S present invention comprise an effective amount of the vector, dissolved or
dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such compositions also
are
referred to as inocula. The phrase "pharmaceutically or pharmacologically
acceptable"
refer to molecular entities and compositions that do not produce adverse,
allergic, or other
untoward reactions when administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents and
the like. The use of such media and agents for pharmaceutically active
substances is well
know in the art. Except insofar as any conventional media or agent is
incompatible with
the vectors of the present invention, its use in therapeutic compositions is
contemplated.
Supplementary active ingredients also can be incorporated into the
compositions.
The active compositions of the present invention include classic
pharmaceutical
preparations. Administration of these compositions according to the present
invention
will be via any common route so long as the target tissue is available via
that route. This
includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively,
administration may
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be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or
intravenous
injection. Such compositions would normally be administered as
pharmaceutically
acceptable compositions, described supra.
The active compounds may be administered via any suitable route, including
parenterally or by injection. Solutions of the active compounds as free base
or
pharmacologically acceptable salts can be prepared in water suitably mixed
with a
surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared
in glycerol,
liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary
conditions
of storage and use, these preparations contain a preservative to prevent the
growth of
microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile
injectable solutions or dispersions. In all cases the form must be sterile and
must be fluid
to the extent that easy syringability exists. It must be stable under the
conditions of
manufacture and storage and must be preserved against the contaminating action
of
microorganisms, such as bacteria and fungi. The carrier can be a solvent or
dispersion
medium containing, for example, water, ethanol, polyoi (for example, glycerol,
propylene
glycol, and liquid polyethylene glycol, and the like), suitable mixtures
thereof, and
vegetable oils. The proper fluidity can be maintained, for example, by the use
of a
coating, such as lecithin, by the maintenance of the required particle size in
the case of
dispersion and by the use of surfactants. The prevention of the action of
microorganisms
can be brought about by various antibacterial an antifungal agents, for
example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases,
it will be
preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged
absorption of the injectable compositions can be brought about by the use in
the
compositions of agents delaying absorption, for example, aluminum monostearate
and
gelatin.
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Sterile injectable solutions are prepared by incorporating the active
compounds in
the required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions
are prepared by incorporating the various sterilized active ingredients into a
sterile
vehicle which contains the basic dispersion medium and the required other
ingredients
from those enumerated above. In the case of sterile powders for the
preparation of sterile
injectable solutions, the preferred methods of preparation are vacuum-drying
and freeze-
drying techniques which yield a powder of the active ingredient plus any
additional
desired ingredient from a previously sterile-filtered solution thereof.
For oral administration the polypeptides of the present invention may be
incorporated with excipients and used in the form of non-ingestible
mouthwashes and
dentifrices. A mouthwash may be prepared incorporating the active ingredient
in the
required amount in an appropriate solvent, such as a sodium borate solution
(Dobell's
1 S Solution). Alternatively, the active ingredient may be incorporated into
an antiseptic
wash containing sodium borate, glycerin and potassium bicarbonate. The active
ingredient may also be dispersed in dentifrices, including: gels, pastes,
powders and
slurries. The active ingredient may be added in a therapeutically effective
amount to a
paste dentifrice that may include water, binders, abrasives, flavoring agents,
foaming
agents, and humectants.
The compositions of the present invention may be formulated in a neutral or
salt
form. Pharmaceutically-acceptable salts include the acid addition salts
(formed with the
free amino groups of the protein) and which are formed with inorganic acids
such as, for
example, hydrochloric or phosphoric acids, or such organic acids as acetic,
oxalic,
tartaric, mandelic, and the like. Salts formed with the free carboxyl groups
also can be
derived from inorganic bases such as, for example, sodium, potassium.
ammonium,
calcium, or ferric hydroxides, and such organic bases as isopropylamine,
trimethylamine,
histidine, procaine and the like.
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Upon formulation, solutions will be administered in a manner compatible with
the
dosage formulation and in such amount as is therapeutically effective. The
formulations
are easily administered in a variety of dosage forms such as injectable
solutions, drug
release capsules and the like. For parenteral administration in an aqueous
solution, for
example, the solution should be suitably buffered if necessary and the~liquid
diluent first
rendered isotonic with sufficient saline or glucose. These particular aqueous
solutions are
especially suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal
administration.
"Unit dose" is defined as a discrete amount of a therapeutic composition
dispersed
in a suitable carrier. For example, in accordance with the present methods,
viral doses
include a particular number of virus particles or plaque forming units (pfu).
For
embodiments involving adenovirus, particular unit doses include 103, 104, 105,
106, 10',
10g, 109, 10'°, 10~ ~, 1012, 1013 or 10'4 pfu. Particle doses may be
somewhat higher (10 to
100-fold) due to the presence of infection defective particles.
In this connection, sterile aqueous media which can be employed will be known
to those of skill in the art in light of the present disclosure. For example,
a unit dose
could be dissolved in 1 ml of isotonic NaCI solution and either added to 1000
ml of
hypodermoclysis fluid or injected at the proposed site of infusion, (see for
example,
"Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-
1580).
Some variation in dosage will necessarily occur depending on the condition of
the subject
being treated. The person responsible for administration will, in any event,
determine the
appropriate dose for the individual subject. Moreover, for human
administration,
preparations should meet sterility, pyrogenicity, general safety and purity
standards as
required by FDA Office of Biologics standards.
In a preferred embodiment, the present invention is directed at the treatment
of
human malignancies. A variety of different routes of administration are
contemplated.
For example, a classic and typical therapy will involve direct, intratumoral
injection of a
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discrete tumor mass. The injections may be single or multiple; where multiple,
injections
are made at about i cm spacings across the accessible surface of the tumor.
Alternatively, targeting the tumor vasculature by direct, local or regional
infra-arterial
injection are contemplated. The lymphatic systems, including regional lymph
nodes,
present another likely target given the potential for metastasis along his
route. Further,
systemic injection may be preferred when specificaIiy targeting secondary
(i.e.,
metastatic) tumors.
In another embodiment, the viral gene therapy may precede or following
resection
of the tumor. Where prior, the gene therapy may, in fact, permit tumor
resection where
not possible before. Alternatively, a particularly advantageous embodiment
involves the
prior resection of a tumor (with or without prior viral gene therapy),
followed by
treatment of the resected tumor bed. This subsequent treatment is effective at
eliminating
microscopic residual disease which, if left untreated, could result in
regrowth of the
tumor. This may be accomplished, quite simply, by bathing the tumor bed with a
viral
preparation containing a unit dose of viral vector. Another preferred method
for
achieving the subsequent treatment is via catheterization of the resected
tumor bed,
thereby permitting continuous perfusion of the bed with virus over extended
post-
operative periods.
XI. Examples
The following examples are included to demonstrate preferred embodiments of
the invention. It should be appreciated by those of skill in the art that the
techniques
disclosed in the examples which follow represent techniques discovered by the
inventor
to function well in the practice of the invention, and thus can be considered
to constitute
preferred modes for its practice. However, those of skill in the art should,
in light of the
present disclosure, appreciate that many changes can be made in the specific
embodiments which are disclosed and still obtain a like or similar result
without
departing from the spirit and scope of the invention.
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EXAMPLE l: CONSTRUCTION OF MULTIGENE ADENOVIRAL
CONSTRUCTS
Several different gene vectors will be constructed according to the present
invention. A first expression cassette will be constructed in a plasmid vector
before
subcloning in to pINl47 (FIG. 2). The plasmid pIN147 contains bases 1 to 456
and 3333
to 5788 of Ad5 and the CMV IE promoter. The first cassette contains a first
gene,
followed by the BGH polyA signal, which in turn is followed by an SV40
promoter, a
second gene and optionally the SV40 polyadenylation signal, in that order.
FIG. lA
shows the resulting cassette with CMV IE promoter from the vector.
A second expression cassette will utilize only one promoter, the CMV IE
promoter, derived from pIN147. In this cassette, an IRES will be included
downstream
of a first gene and a synthetic intron (IVS) and upstream of the second gene
and a polyA.
FIG. 1B shows the cassette including the upstream pIN147 promoter. The IRES
allows
for efficient translation of the second gene, and the IVS improves the
stability of the
mRNA. The IRES and IVS are obtained from plasmids available from Clontech,
Palo
Alto, CA.
After insertion of the cassettes into the multipurpose cloning site of pIN
147, the
recombinant vector is contransfected with either pBGHlO or pBGHI l, available
from
Microbix Biosystems, Inc. (Toronto, Ontario, Canada). These plasmids contain
bases 1
to 187 and 1340 to 35935 of AdS, less deletions in the E3 region (28133 to
30818 and
27865 to 30995 and respectively). Recombination will result in the generation
of an
adenoviral vector containing the multigene cassettes in the E1 region and
having an E3-
deletion.
Additional constructs will utilize different approaches. Using cassettes
including
all of the elements shown in FIG. 1 A and FIG. 1 B, constructs will be
generated by
inserting the cassettes into pOElsplA and p~ElsplB (Microbix Biosystems, Inc.)
(FIG.
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3), which contain bases 22 to 342 and 3523 to 5790 of AdS, flanking a
multipurpose
cloning site. Cotransfection with pBGH 10 or pBGH 11 permits recombination and
generation of adenoviral vectors having E 1-inserted cassettes and E3
deletions.
In another construction scheme to generate an adenoviral vector containing two
expression cassettes, an expression cassette will be cloned into the
adenoviral E3 region
of the plasmid pAB26 (Microbix Biosystems, Inc.) (FIG. 4) and Srf! digested
adenoviral
DNA containing an expression cassette cloned into the El region (termed Ad5-
expression
cassette # 1 ) will be co-transfected into 293 cells and viral particles will
be generated as
the result of in vivo recombination.
To begin this construction scheme, the Ad5-expression cassette #1 vector will
be
constructed by the co-transfection of pIN 147, containing the expression
cassette # I
cloned into the E1 region, and pJMl7, which contains the entire adenoviral
genome
including an insertion in the E1 region, into 293 cells. After plaque
purification, viral
expansion, and subsequent adenoviral DNA purification, the Ad5-expression
cassette # 1
DNA will be digested with the restriction enzyme Srfl, the restriction
fragments will be
separated by agarose gel electrophoresis, and the band corresponding to
approximately 28
kb (or roughly 78% of the adenoviral genome) will be agarose gel purified. The
plasmid
pAB26 is comprised of Ad5 sequences corresponding to bases 1 to 353, 3825 to
5787,
and 24797 to 35935.
An expression cassette will be cloned into the adenoviral E3 multicloning site
present within pAB26, and thus will be termed pAB26 expression cassette #2.
The co-
transfection of the 28 kb Srfl digested Ad5-expression cassette # 1 DNA and
the pAB26
expression cassette #2 plasmid into 293 cells and the subsequent in vivo
recombination
will generate an adenoviral vector containing two expression cassettes, one in
the E1 and
the other in the E3 region of the adenoviral genome.
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EXAMPLE 2: IN VITRO MONITORING OF GROWTH INHIBITION AND
APOPTOSIS
Apoptosis assays
DNA Fragmentation Analysis. Following incubation of the cells with the gene
therapy construct, cells are harvested, resuspended in 300 pl of PBS with the
addition of 3
ml of extraction buffer ( 10 mM Tris, pH 8.0, 0.1 M EDTA, 20 p.g/ml RNAse,
0.5% SDS)
and incubated at 37°C for 1-2 h. At the end of incubation, proteinase K
is added to a final
concentration of 100 p.glml and the solution placed in a 50°C water
bath for at least 3 h.
DNA is extracted once with equal volumes of 0.5 M Tris (pH 8.0) saturated
phenol and then
the extraction is repeated with phenol/chloroform. Precipitated DNA is
analyzed in a 1%
agarose gel.
Ceil Fixation. For TUNEL method, the cells are fixed in 1 % formaldehyde in
PBS
(pH 7.4) for 30 min on ice. Cells are then washed with 3 ml of PBS,
resuspended in 70%
1 S ice-cold ethanol and stored at -20°C until used. For cell-cycle
analysis, cells are fixed in
70% ice-cold ethanol only.
Terminal Deoxynucleotidyl Transferase Assay. The assay is performed
according to the Gorczyca et al., procedure (Gorczyca et al., 1993). Briefly,
after fixation
and washing, cells are resuspended in 50 ~l of TdT buffer containing 0.2 M
sodium
cacodylate (pH 7.0), 2.5 mM Tris-HCI, 2.5 mM C°Cl2 (Sigma Chemical
Company, St.
Louis, MO), 0.1 mM DTT (Sigma Chemical Company), 0.25 mg/ml BSA (Sigma
Chemical Company), 5 units of terminal transferase (Boehringer Mannheim
Biochemicals,
Indianapolis,1N), and 0.5 nmoles biotin-16-dUTP along with dATP, dGTP and dCTP
at a
concentration of 20 pM. Controls are prepared by incubating a separate aliquot
of each test
sample without d-UTP. The cells are incubated in the solution at 37°C
for 30 min, rinsed in
PBS, and resuspended in 100 pl of, FITC, the staining solution containing 4X
SSC, 0.1%
Triton X-100 and 2.5 p.g/ml fluoresceined avidin (Vector Labs. Inc.,
Burlingame, CA).
Tubes are incubated for 30 min in the dark at room temperature. Cells are
rinsed in PBS
with 0.1 % Triton X-100 and resuspended in 0.5 ml PBS containing propidium
iodide (5
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flg/ml) and 70 pl (1 mg/ml) RNAse. Tubes are incubated in the dark on ice for
30 min prior
to flow cytometric analysis.
Flow Cytometry Analysis. Samples are analyzed using an EPICS Profile II flow
cytometer (Coulter Corp., Hialeah, FL) with the standard optical conf
guration. At least
5,000 events are collected for each sample. Positively for TdT end-labeling is
determined
by subtracting the control histogram from the test histogram using the immuno-
4 program
of the Elite workstation software (Coulter Corp., Hialeah, FL).
Cell Growth Assay
Cell growth can be measured by cell counting or tritiated thymidine
incorporation
assays.
Growth Assay by Cell Counting
For cell growth measurements, cells are generally inoculated at densities of
1 x 104 cells in 12 well plates. Cells were trypsinized and counted using a
hemocytometer.
Tritiated Tltymidine Incorporation Assay
Growth of cells can be measured by analysis of DNA synthesis. Briefly, a stock
solution of 100 p.Ci/ml of 3H-thymidine (Amersham) is prepared by dilution
into high
glucose DMEM. 3H-thymidine to a final concentration of 1 pCi/ml is added to
each well
in 20 pl. The reaction is stopped 6 or 15 h later by removal of supernatant
from recipient
cells. The cells are harvested by the addition of 100X trypsin/EDTA to each
well for five
min at room temperature. Cells are collected using a Packard Filtermate cell
harvester
following manufacturer's protocol and washed in distilled deionized water and
methanol.
Alternatively, the reaction also can be stopped by removing the supernatant
from
recipient cells and the cells washed once with PBS + O.SmM MgCl2/1mM CaCl2 and
30
~l of lysis buffer (0.05% SDS/1mM MgCl2/1mM CaCl2) added. The cells are
scraped,
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adsorbed onto Whatman filters and non-specific radioactivity removed by
washing with
TCA. Filters are placed into S ml scintillant and counted in a gamma counter.
The rate
of incorporation of activity into DNA is indicative of the rate of cell
growth.
$ ***********************
All of the compositions and/or methods disclosed and claimed herein can be
made
and executed without undue experimentation in light of the present disclosure.
While the
compositions and methods of this invention have been described in terms of
preferred
embodiments, it will be apparent to those of skill in the art that variations
may be applied
to the compositions and in the steps or in the sequence of steps of the method
described
herein without departing from the concept, spirit and scope of the invention.
More
specifically, it will be apparent that certain agents which are both
chemically and
physiologically related may be substituted for the agents described herein
while the same
I S or similar results would be achieved. All such similar substitutes and
modifications
apparent to those skilled in the art are deemed to be within the spirit, scope
and concept
of the invention as defined by the appended claims.
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REFERENCES
The following references, to the extent that they provide exemplary procedural
or
other details supplementary to those set forth herein, are specifically
incorporated herein
by reference:
Arap et al., Cancer Res., 55:1351-1354, 1995.
Arcone et al., Nucleic Acids Research, 16(8):3195-3207, 1988.
Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum
Press, pp. I 17-148,1986.
Bartlett et al., Proc. Natl. Acad. Sci. USA, 93:8852-8857, 1996.
Battersonand Roizman,J. Virol., 46:371-377,1983.
Bedzyk et al., J. Biol. Chem., 265:18615,1990
Bellon et al., de Ses Filiales,190(1):109-142, 1996.
Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83:9551-9555, 1986.
Bems and Bohenzky, Adv. Virus Res., 32:243-307, 1987.
Berns and Giraud, Curr. Top. Microbiol. Immunol., 218:1-23, 1996.
Burbage et al., Leuk Res, 21(7):681-690, 1997.
Bussemakers et al., Cancer Res., 52:2916-2922, 1992.
Caldas et al., Nat. Genet., 8:27-32, 1994.
Campbell et al., J. Mol. Biol., 180: I-19,1984.
Carter et al., BiolTechnology, 10:163-167, 1992.
Casey et al., Oncogene, 6:1791-1797, 1991.
Chatterjee and Wong, Jr., Curr. Top. Microbiol. Immunol., 218:61-73, 1996.
Chaudhary et al. Proc. Natl. Acad Sci., 87:9491,1990
Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987.
Cheng et al., Nature, 379:554, 1996.
Cheng et al., Cancer Res., 54:5547-5551, 1994.
Coffin, In: Virology, ed., New York: Raven Press, pp. 1437-1500,1990.
Cook et al., Cell, 62:671-680, 1990.
Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.
- I 04-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Dani, et al., J. Biol. Chem., 264:10119-10125, 1989.
DeLuca et al., J. Virol., 56:558-570,1985.
Dubensky et al., Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984.
Edelman and Crossin, Annu. Rev. Biochem., 60:1 SS-190, 1991.
Edelman, Annu. Rev. Biochem., 54:135-169, 1985.
Elroy-Stein et al., Proc. Nat'1 Acad. Sci. USA , 1989.
Elshami et al., Gene Therapy, 7(2):141-148, 1996.
Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987.
Ferkol et al., FASEBJ., 7:1081-1091,1993.
Forster and Symons, Cell, 49:211-220, 1987.
Fraley et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979.
French, et al., Circulation, 90(5}:2414-2424, 1994.
Frixen et al., J. Cell Biol., 113:173-185, 1991.
Gerlach et al., Nature (London), 328:802-805, 1987
Ghosh-Choudhury et al., EMBO J., 6:1733-1739, 1987.
Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using
specific receptors and ligands, (Wu G, Wu C ed.), New York: Marvel
Dekker, pp. 87-104,1991.
Giancotti and Ruoslahti, Cell, 60:849-859, 1990.
Ginsberg et al., Proceedings of the National Academy of Sciences of the United
States ofAmerica, 88(5)1651-1655, 1991.
Glorioso et al., Ann. Rev. Microbiol. 49:675-710,1995.
Gomez-Foix et al., J. Biol. Chem., 267:25129-25134, 1992.
Gonzalez-Zulueta et al., Cancer Research, 55(20):4531-4535, 1995.
Gopal, Mol. Cell Biol., 5:1188-1190, 1985.
Gorczyca et al., Int'I J. Oncol., 1:639-648, 1992.
Gossen and Bujard, Proc. Natl. Acad. Sci. USA, 89:5547-SS51, 1992.
Gossen et al., Science, 268:1766-1769, 1995.
Graham and Prevec, Biotechnology, 20:363-390, 1992.
-1 OS-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Graham and Prevec, In: Methods in Molecular Biology: Gene Transfer and
Expression Protocols 7, E.J. Murray (ed.}, Clifton, N.J., Humana Press,
pp. 205-225. 1991.
Graham and Van Der Eb, Virology, 52:456-467, 1973.
Grunhaus and Horwitz, Seminar in Virology, 3:237-252, 1992.
Harland and Weintraub,J. Cell Biol., 101:1094-1099,1985.
Herman et al., Cancer Research, 55(20):4525-4530, 1995.
Hersdorffer et al., DNA Cell Biol., 9:713-723, 1990.
Herz and Gerard, Proc. Nat'1 Acad. Sci. USA, 90:2812-2816, 1993.
Holland et al., Virology 101:10-18,1980.
Hollstein et al., Science, 253:49-53, 1991.
Honess and Roizman,J. Virol., 14:8-19,1974.
Honess and Roizman, J. Virol., 16:1308-1326,1975.
Hunt et al., Proc. Natl. Acad. Sci. USA, 83:3786-3790, 1986.
Hussussian et al., Nature Genetics, 15-21, 1994.
Joki, et al., Human Gene Ther., 6:1507-1513, 1995.
Jones and Shenk, Cell, 13:181-188, 1978.
Joyce, Nature, 338:217-244, 1989.
Kageyama, et al., J. Biol. Chem., 262(5):2345-2351, 1987.
Kamb et al., Nature Genetics, 8:22-26, 1994.
Kamb et al., Science, 2674:436-440, 1994.
Kaneda et al., Science, 243:375-378,1989.
Karlsson et al., EMBO J., 5:2377-2385, 1986.
Kato et al, J. Biol. Chem., 266:3361-3364, 1991.
Kearns, et al., Gene Ther., 3:748-755, 1996.
Kim and Cook, Proc. Natl. Acad. Sci. USA, 84:8788-8792, 1987.
Klein et al , Nature, 327:70-73,1987.
Korhonen et al., Blood, 86(5):1828-1835, 1995.
Kotin and Berns, Virol., 170:460-467, 1989.
Kotin et al., Genomics, 10:831-834, 1991.
-106-
CA 02323112 2000-09-14
WO 99/47690 PCTNS99/05781
Kotin et al., Proc. Natl. Acad Sci. USA, 87:2211-2215, 1990.
Le Gal La Salle et al. , Science, 259:988-990, 1993.
Levrero et al., Gene, 101:195-202, 1991.
Lidor et al., Am J Obstet Gynecol;177(3):579-585, 1997.
Lin and Guidotti, J. Biol. Chem., 264:14408-14414, 1989.
Macejak and Sarnow, Nature, 353:90-94, 1991.
Mann et al., Cell, 33:153-159, 1983.
Markowitz et al., J. Virol., 62:1120-1124, 1988.
Massuda et al., Proc Natl Acad Sci USA , 94(26):14701-14706, 1997.
Matsura et al., Brit. J. Cancer, 66:1122-1130, 1992.
Michel and Westhof, J. Mol. Biol., 216:585-610, 1990.
Mizukami et al., Virology, 217:124-130, 1996.
Mori et al., Cancer Res., 54:3396-3397,1994.
Mulligan et al., Proc. Nat'I Acad Sci. USA, 78: 2072, 1981.
1 S Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129, 1992.
Myers, EPO 0273085.
Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and
their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp.
493-513, 1988.
Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.
Nicolau et al., Methods Enzymol., 149:157-176, 1987.
Nobri et al., Nature, 368:753-756, 1995.
O'Malley, et al., Cancer. Res., 56(8)1737-1741, 1996.
O'Malley, et al., Mol. Endocrinol, 11 (6):667-673, 1997.
Obrink, BioEssays, 13:227-233, 1991.
Odin and Obrink, Exp. Cell Res., 171:1-15, 1987.
Ogawa, W., Neuropathologica, 77(3):244-253, 1989.
Okamoto et al., Proc. Nat'I Acad. Sci. USA, 91:11045-11049, 1994.
Olivierio et al., EMBO J., 6(7):1905-1912, 1987.
Orlow et al., Cancer Res., 54:2848-2851, 1994.
-I07-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Ostrove et al., Virology, 113:532-533, 1981.
Pape and Kim, Mol. Cell. Biol., 974-982, 1989.
Paskind et al., Virology, 67:242-248,1975.
PCT 95/27071
S PCT 96/33280
Perales et al., Proc. Natl. Acad Sci. 91:4086-4090,1994.
Poli and Cortese, Proc. Natl. Acad. Sci. USA, 86:8202-8206, 1989.
Ponnazhagan, et al., Hum. Gene Ther., 8:275-284, 1997a.
Ponnazhagan, et al., J. Gen. Virol., 77:1111-1122, 1996.
Post et al., Cell 24:555-565,1981.
Potter et al., Proc. Nat'I Acad. Sci. USA, 81:7161-7165,1984.
Racher et al., Biotechnology Techniques, 9:169-174, 1995.
Radler et al., Science, 275:810-814, 1997.
Ragot et al., Nature, 361:647-650, 1993.
Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.
Remington's Pharmaceutical Sciences, 15th ed., pp. 1035-1038 and 1570-1580.
Renan, Radiother. Oncol., 19:197-218, 1990.
Rich et al., Hum. Gene Ther., 4:461-476, 1993.
Ridgeway, In: Vectors: A survey of molecular cloning vectors and their uses,
Rodriguez RL, Denhardt DT, ed., Stoneham:Butterworth, pp. 467-492,
I 988.
Rippe et al., Mol. Cell Biol., 10:689-695, 1990.
Roizman and Sears, In Fields' Virology, 3rd Edition, eds. Fields, et al.
(Raven
Press, New York, N.Y.), pp. 2231-2295,1995.
Ron et al., Mol. Cell. Biol., 2887-2895, 1991.
Rosenberg, P., Autotransfusion (editorial)"Duodecim., 106 (14) p1027-9, 1990.
Rosenfeld et al., Cell, 68:143-155, 1992.
Rosenfeld et al., Science, 252:431-434, 1991.
Roux et al., Proc. Natl Acad. Sci. USA, 86:9079-9083,1989.
Samulski, et al, EMBOJ., 10:3941-3950, 1991.
-108-
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Sarver et al., Science, 247:1222-1225, 1990.
Scanlon et al., Proc Natl Acad Sci USA, 88:10591-10595, 1991.
Serrano et al., Nature, 366:704-707, 1993.
Serrano et al., Science, 267:249-252, 1995.
S Shiraishi, et al., Transplant International, 1-0(3):202-206, 1997.
Smith and Moss, Gene 25:21-28, 1983.
Song et al., Hum. Gene. Ther., 8(10):1207-1217, 1997
Speigelman, et al., J. Biol. Chem., 264(3):181 I-1815, 1989.
Srivastava et al., J. Virol., 45:555-564, 1983.
Stratford-Perricaudetet al., Hum. Gene. Ther. 1:241-256,1991.
Stratford-Perricaudet et al., Hum. Gene Ther., 1:241-256, 1990.
Takahashi et al., Cancer Res., 52:734-736, 1992.
Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-
188, 1986.
IS Top et al., J. Infect. Dis., 124:155-160, 1971.
Tur-Kaspa et al., Mol. Cell Biol., 6:716-718,1986.
U. S. Patent 5,252,479
U. S. Patent 5,354,855
U. S. Patent 5,359,046
U. S. Patent 5,672,344
U. S. Patent 4,554,1 O1
Umbas et al., Cancer Res., 52:5104-S 109, 1992.
Varmus et al., Cell, 25:23-36, 1981.
Wagner et al., Proc. Natl. Acad Sci. 87(9):3410-3414, 1990.
Walther and Stein, J. Mol. Med, 74:379-392, 1996.
Watt et al., Proc. Natl Acad Sci., 83(2): 3166-3170, 1986.
Weinberg, Science, 254:1138-1145, 1991.
Werthman et al., Journal of Urology, 155(2):753-756, 1996.
Wilson, et al., Mol. Cell. Biol., 6181-6191, 1990.
Wong et al., Gene, 10:87-94, 1980.
- I 09-
.. ...r",.=."""",~rw"-""a"""~... . .............~.".~............ ...
CA 02323112 2000-09-14
WO 99/47690 PCT/US99/05781
Wu and Wu, Biochem., 27:887-892, 1988.
Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.
Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990.
Zechner, et al., Mol. Cell. Biol., 2394-2401, 1988.
-110-
