U.S. patent application number 10/785116 was filed with the patent office on 2004-07-22 for polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells.
Invention is credited to Feinstein, Elena, Pecker, Iris, Vlodavsky, Israel.
Application Number | 20040142427 10/785116 |
Document ID | / |
Family ID | 22982587 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040142427 |
Kind Code |
A1 |
Pecker, Iris ; et
al. |
July 22, 2004 |
Polynucleotide encoding a polypeptide having heparanase activity
and expression of same in genetically modified cells
Abstract
A polynucleotide (hpa) encoding a polypeptide having heparanase
activity, vectors including same, genetically modified cells
expressing heparanase, a recombinant protein having heparanase
activity and antisense oligonucleotides and constructs for
modulating heparanase expression.
Inventors: |
Pecker, Iris; (Rishon
LeZion, IL) ; Vlodavsky, Israel; (Mevaseret Zion,
IL) ; Feinstein, Elena; (Rehovot, IL) |
Correspondence
Address: |
SOL SHEINBEIN
c/o ANTHONY CASTORINA
SUITE 207
2001 JEFFERSON DAVIS HIGHWAY
ARLINGTON
VA
22202
US
|
Family ID: |
22982587 |
Appl. No.: |
10/785116 |
Filed: |
February 25, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10785116 |
Feb 25, 2004 |
|
|
|
09988113 |
Nov 19, 2001 |
|
|
|
09988113 |
Nov 19, 2001 |
|
|
|
09776874 |
Feb 6, 2001 |
|
|
|
09776874 |
Feb 6, 2001 |
|
|
|
09258892 |
Mar 1, 1999 |
|
|
|
09258892 |
Mar 1, 1999 |
|
|
|
PCT/US98/17954 |
Aug 31, 1998 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
435/200; 435/320.1; 435/325; 536/21; 536/23.2 |
Current CPC
Class: |
C12Y 302/01166 20130101;
C12N 9/2402 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/069.1 ;
435/200; 435/320.1; 435/325; 536/023.2; 536/021 |
International
Class: |
C12Q 001/68; C08B
037/10; C07H 021/04; C12N 009/24 |
Claims
What is claimed is:
1. A polynucleotide fragment comprising a polynucleotide sequence
encoding a polypeptide having heparanase catalytic activity,
wherein said polypeptide shares at least 70% homology with SEQ ID
NOs:10, 14 or 44, as determined using default parameters of a DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the University of Wisconsin.
2. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes nucleotides 63-1691 of SEQ ID
NO:9.
3. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes nucleotides 63-721 of SEQ ID
NO:9.
4. The polynucleotide fragment of claim 1, wherein said
polynucleotide is as set forth in SEQ ID NO:9.
5. The polynucleotide fragment of claim 1, wherein said
polynucleotide sequence includes a segment of SEQ ID NO:9, said
segment encodes said polypeptide having said heparanase catalytic
activity.
6. The polynucleotide fragment of claim 1, wherein said polypeptide
includes an amino acid sequence as set forth in SEQ ID NOs:10, 14
or 44.
7. The polynucleotide fragment of claim 1, wherein said polypeptide
includes a segment of SEQ ID NOs:10, 14 or 44 said segment harbors
said heparanase catalytic activity.
8. The polynucleotide fragment of claim 1, wherein said
polynucleotide-sequence is selected from the group consisting of
double stranded DNA, single stranded DNA and RNA.
9. A polynucleotide sequence as set forth in SEQ ID NOs:9, 13, 42
or 43.
10. A polynucleotide sequence at least 70% homologous to SEQ ID
NOs:9, 13, 42 or 43, as determined using default parameters of a
DNA sequence analysis software package developed by the Genetic
Computer Group (GCG) at the University of Wisconsin, wherein said
polynucleotide sequence encodes a polypeptide having heparanase
catalytic activity.
11. A vector comprising a polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity, wherein said
polypeptide shares at least 70% homology with SEQ ID NOs:10, 14 or
44, as determined using default parameters of a DNA sequence
analysis software package developed by the Genetic Computer Group
(GCG) at the University of Wisconsin.
12. The vector of claim 11, wherein said polynucleotide sequence
includes nucleotides 63-1691 of SEQ ID NO:9.
13. The vector of claim 11, wherein said polynucleotide sequence
includes nucleotides 63-721 of SEQ ID NO:9.
14. The vector of claim 11, wherein said polynucleotide sequence is
as set forth in SEQ ID NO:9.
15. The vector of claim 11, wherein said polynucleotide sequence
includes a segment of SEQ ID NO:9, said segment encodes said
polypeptide having said heparanase catalytic activity.
16. The vector of claim 11, wherein said polypeptide includes an
amino acid sequence as set forth in SEQ ID NOs:10, 14 or 44.
17. The vector of claim 11, wherein said polypeptide includes a
segment of SEQ ID NOs:10, 14 or 44, said segment harbors said
heparanase catalytic activity.
18. The vector of claim 11, wherein said polynucleotide sequence is
selected from the group consisting of double stranded DNA, single
stranded DNA and RNA.
19. The vector of claim 11, wherein said vector is a baculovirus
vector.
20. A host cell comprising an exogenous polynucleotide fragment
including a polynucleotide sequence encoding a polypeptide having
heparanase catalytic activity, wherein said polypeptide shares at
least 70% homology with SEQ ID NOs:10, 14 or 44, as determined
using default parameters of a DNA sequence analysis software
package developed by the Genetic Computer Group (GCG) at the
University of Wisconsin.
21. The host cell of claim 20, wherein said polynucleotide sequence
includes nucleotides 63-1691 of SEQ ID NO:9.
22. The host cell of claim 20, wherein said polynucleotide sequence
includes nucleotides 63-721 of SEQ ID NO:9.
23. The host cell of claim 20, wherein said polynucleotide sequence
is as set forth in SEQ ID NO:9.
24. The host cell of claim 20, wherein said polynucleotide sequence
includes a segment of SEQ ID NO:9, said segment encodes said
polypeptide having said heparanase catalytic activity.
25. The host cell of claim 20, wherein said polypeptide includes an
amino acid sequence as set forth in SEQ ID NOs:10, 14 or 44.
26. The host cell of claim 20, wherein said polypeptide includes a
segment of SEQ ID NOs:10, 14 or 44 said segment harbors said
heparanase catalytic activity.
27. The host cell of claim 20, wherein said polynucleotide sequence
is selected from the group consisting of double stranded DNA,
single stranded DNA and RNA.
28. A host cell expressing a recombinant heparanase, wherein said
recombinant heparanase shares at least 70% homology with SEQ ID
NOs:10, 14 or 44, as determined using default parameter of a DNA
sequence analysis software package developed by the Genetic
Computer (Group (GCG) at the University of Wisconsin.
29. A heparanase overexpression system comprising a cell
overexpressing heparanase catalytic activity, wherein said
heparanase catalytic activity is effected by a heparanase sharing
at least 70% homology with SEQ ID NOs:10, 14 or 44, as determined
using default parameters of a DNA sequence analysis software
package developed by the Genetic Computer Group (GCG) at the
University of Wisconsin.
30. The host cell of claim 20, wherein said cell is an insect cell.
Description
[0001] This is a continuation of U.S. patent application Ser. No.
09/776,874, filed Feb. 6, 2001, which is a continuation of U.S.
patent application Ser. No. 09/258,892, filed Mar. 1, 1999, which
is a continuation-in-part of PCT/US98/17954, filed Aug. 31, 1998,
which claims priority from U.S. patent application Ser. No.
09/109,386, filed Jul. 2, 1998, now abandoned, which is a
continuation-in-part of U.S. patent application Ser. No.
08/922,170, filed Sep. 2, 1997, now, U.S. Pat. No. 5,968,822.
FIELD AND BACKGROUND OF THE INVENTION
[0002] The present invention relates to a polynucleotide, referred
to hereinbelow as hpa, encoding a polypeptide having heparanase
activity, vectors (nucleic acid constructs) including same and
genetically modified cells expressing heparanase. The invention
further relates to a recombinant protein having heparanase activity
and to antisense oligonucleotides, constructs and ribozymes for
down regulating heparanase activity. In addition, the invention
relates to heparanase promoter sequences and their uses.
[0003] Heparan sulfate proteoglycans: Heparan sulfate proteoglycans
(HSPG) are ubiquitous macromolecules associated with the cell
surface and extra cellular matrix (ECM) of a wide range of cells of
vertebrate and invertebrate tissues (1-4). The basic HSPG structure
includes a protein core to which several linear heparan sulfate
chains are covalently attached. These polysaccharide chains are
typically composed of repeating hexuronic and D-glucosamine
disaccharide units that are substituted to a varying extent with N-
and O-linked sulfate moieties and N-linked acetyl groups (1-4).
Studies on the involvement of ECM molecules in cell attachment,
growth and differentiation revealed a central role of HSPG in
embryonic morphogenesis, angiogenesis, neurite outgrowth and tissue
repair (1-5). HSPG are prominent components of blood vessels (3).
In large blood vessels they are concentrated mostly in the intima
and inner media, whereas in capillaries they are found mainly in
the subendothelial basement membrane where they support
proliferating and migrating endothelial cells and stabilize the
structure of the capillary wall. The ability of HSPG to interact
with ECM macromolecules such as collagen, laminin and fibronectin,
and with different attachment sites on plasma membranes suggests a
key role for this proteoglycan in the self-assembly and
insolubility of ECM components, as well as in cell adhesion and
locomotion. Cleavage of the heparan sulfate (HS) chains may
therefore result in degradation of the subendothelial ECM and hence
may play a decisive role in extravasation of blood-borne cells. HS
catabolism is observed in inflammation, wound repair, diabetes, and
cancer metastasis, suggesting that enzymes which degrade HS play
important roles in pathologic processes. Heparanase activity has
been described in activated immune system cells and highly
metastatic cancer cells (6-8), but research has been handicapped by
the lack of biologic tools to explore potential causative roles of
heparanase in disease conditions.
[0004] Involvement of Heparanase in Tumor Cell Invasion and
Metastasis: Circulating tumor cells arrested in the capillary beds
of different organs must invade the endothelial cell lining and
degrade its underlying basement membrane (BM) in order to invade
into the extravascular tissue(s) where they establish metastasis
(9, 10). Metastatic tumor cells often attach at or near the
intercellular junctions between adjacent endothelial cells. Such
attachment of the metastatic cells is followed by rupture of the
junctions, retraction of the endothelial cell borders and migration
through the breach in the endothelium toward the exposed underlying
BM (9). Once located between endothelial cells and the BM, the
invading cells must degrade the subendothelial glycoproteins and
proteoglycans of the BM in order to migrate out of the vascular
compartment. Several cellular enzymes (e.g., collagenase IV,
plasminogen activator, cathepsin B, elastase, etc.) are thought to
be involved in degradation of BM (10). Among these enzymes is an
endo-.beta.-D-glucuroni- dase (heparanase) that cleaves HS at
specific intrachain sites (6, 8, 11). Expression of a HS degrading
heparanase was found to correlate with the metastatic potential of
mouse lymphoma (11), fibrosarcoma and melanoma (8) cells. Moreover,
elevated levels of heparanase were detected in sera from metastatic
tumor bearing animals and melanoma patients (8) and in tumor
biopsies of cancer patients (12).
[0005] The control of cell proliferation and tumor progression by
the local microenvironment, focusing on the interaction of cells
with the extracellular matrix (ECM) produced by cultured corneal
and vascular endothelial cells, was investigated previously by the
present inventors. This cultured ECM closely resembles the
subendothelium in vivo in its morphological appearance and
molecular composition. It contains collagens (mostly type III and
IV, with smaller amounts of types I and V), proteoglycans (mostly
heparan sulfate- and dermatan sulfate-proteoglycans, with smaller
amounts of chondroitin sulfate proteoglycans), laminin,
fibronectin, entactin and elastin (13, 14). The ability of cells to
degrade HS in the cultured ECM was studied by allowing cells to
interact with a metabolically sulfate labeled ECM, followed by gel
filtration (Sepharose 6B) analysis of degradation products released
into the culture medium (11). While intact HSPG are eluted next to
the void volume of the column (Kav<0.2, Mr
.about.0.5.times.10.sup.6), labeled degradation fragments of HS
side chains are eluted more toward the V.sub.t of the column
(0.5<kav<0.8, Mr=5-7.times.10.sup.3) (11).
[0006] The heparanase inhibitory effect of various
non-anticoagulant species of heparin that might be of potential use
in preventing extravasation of blood-borne cells was also
investigated by the present inventors. Inhibition of heparanase was
best achieved by heparin species containing 16 sugar units or more
and having sulfate groups at both the N and O positions. While
O-desulfation abolished the heparanase inhibiting effect of
heparin, O-sulfated, N-acetylated heparin retained a high
inhibitory activity, provided that the N-substituted molecules had
a molecular size of about 4,000 daltons or more (7). Treatment of
experimental animals with heparanase inhibitors (e.g.,
non-anticoagulant species of heparin) markedly reduced (>90%)
the incidence of lung metastases induced by B16 melanoma, Lewis
lung carcinoma and mammary adenocarcinoma cells (7, 8, 16). Heparin
fractions with high and low affinity to anti-thrombin III exhibited
a comparable high anti-metastatic activity, indicating that the
heparanase inhibiting activity of heparin, rather than its
anticoagulant activity, plays a role in the anti-metastatic
properties of the polysaccharide (7).
[0007] Heparanase activity in the Urine of cancer patients: In an
attempt to further elucidate the involvement of heparanase in tumor
progression and its relevance to human cancer, urine samples for
heparanase activity were screened (16a). Heparanase activity was
detected in the urine of some, but not all, cancer patients. High
levels of heparanase activity were determined in the urine of
patients with an aggressive metastatic disease and there was no
detectable activity in the urine of healthy donors.
[0008] Heparanase activity was also found in the urine of 20% of
normal and microalbuminuric insulin dependent diabetes mellitus
(IDDM) patients, most likely due to diabetic nephropathy, the most
important single disorder leading to renal failure in adults.
[0009] Possible involvement of heparanase in tumor angiogenesis:
Fibroblast growth factors are a family of structurally related
polypeptides characterized by high affinity to heparin (17). They
are highly mitogenic for vascular endothelial cells and are among
the most potent inducers of neovascularization (17, 18). Basic
fibroblast growth factor (bFGF) has been extracted from the
subendothelial ECM produced in vitro (19) and from basement
membranes of the cornea (20), suggesting that ECM may serve as a
reservoir for bFGF. Immunohistochemical staining revealed the
localization of bFGF in basement membranes of diverse tissues and
blood vessels (21). Despite the ubiquitous presence of bFGF in
normal tissues, endothelial cell proliferation in these tissues is
usually very low, suggesting that bFGF is somehow sequestered from
its site of action. Studies on the interaction of bFGF with ECM
revealed that bFGF binds to HSPG in the ECM and can be released in
an active form by HS degrading enzymes (15, 20, 22). It was
demonstrated that heparanase activity expressed by platelets, mast
cells, neutrophils, and lymphoma cells is involved in release of
active bFGF from ECM and basement membranes (23), suggesting that
heparanase activity may not only function in cell migration and
invasion, but may also elicit an indirect neovascular response.
These results suggest that the ECM HSPG provides a natural storage
depot for bFGF and possibly other heparin-binding growth promoting
factors (24, 25). Displacement of bFGF from its storage within
basement membranes and ECM may therefore provide a novel mechanism
for induction of neovascularization in normal and pathological
situations.
[0010] Recent studies indicate that heparin and HS are involved in
binding of bFGF to high affinity cell surface receptors and in bFGF
cell signaling (26, 27). Moreover, the size of HS required for
optimal effect was similar to that of HS fragments released by
heparanase (28). Similar results were obtained with vascular
endothelial cells growth factor (VEGF) (29), suggesting the
operation of a dual receptor mechanism involving HS in cell
interaction with heparin-binding growth factors. It is therefore
proposed that restriction of endothelial cell growth factors in ECM
prevents their systemic action on the vascular endothelium, thus
maintaining a very low rate of endothelial cells turnover and
vessel growth. On the other hand, release of bFGF from storage in
ECM as a complex with HS fragment, may elicit localized endothelial
cell proliferation and neovascularization in processes such as
wound healing, inflammation and tumor development (24, 25).
[0011] Expression of heparanase by cells of the immune system:
Heparanase activity correlates with the ability of activated cells
of the immune system to leave the circulation and elicit both
inflammatory and autoimmune responses. Interaction of platelets,
granulocytes, T and B lymphocytes, macrophages and mast cells with
the subendothelial ECM is associated with degradation of HS by a
specific heparanase activity (6). The enzyme is released from
intracellular compartments (e.g., lysosomes, specific granules,
etc.) in response to various activation signals (e.g., thrombin,
calcium ionophore, immune complexes, antigens, mitogens, etc.),
suggesting its regulated involvement in inflammation and cellular
immunity.
[0012] Some of the observations regarding the heparanase enzyme
were reviewed in reference No. 6 and are listed hereinbelow:
[0013] First, a proteolytic activity (plasminogen activator) and
heparanase participate synergistically in sequential degradation of
the ECM HSPG by inflammatory leukocytes and malignant cells.
[0014] Second, a large proportion of the platelet heparanase exists
in a latent form, probably as a complex with chondroitin sulfate.
The latent enzyme is activated by tumor cell-derived factor(s) and
may then facilitate cell invasion through the vascular endothelium
in the process of tumor metastasis.
[0015] Third, release of the platelet heparanase from o-granules is
induced by a strong stimulant (i.e., thrombin), but not in response
to platelet activation on ECM.
[0016] Fourth, the neutrophil heparanase is preferentially and
readily released in response to a threshold activation and upon
incubation of the cells on ECM.
[0017] Fifth, contact of neutrophils with ECM inhibited release of
noxious enzymes (proteases, lysozyme) and oxygen radicals, but not
of enzymes (heparanase, gelatinase) which may enable diapedesis.
This protective role of the subendothelial ECM was observed when
the cells were stimulated with soluble factors but not with
phagocytosable stimulants.
[0018] Sixth, intracellular heparanase is secreted within minutes
after exposure of T cell lines to specific antigens.
[0019] Seventh, mitogens (Con A, LPS) induce synthesis and
secretion of heparanase by normal T and B lymphocytes maintained in
vitro. T lymphocyte heparanase is also induced by immunization with
antigen in vivo.
[0020] Eighth, heparanase activity is expressed by pre-B lymphomas
and B-lymphomas, but not by plasmacytomas and resting normal B
lymphocytes.
[0021] Ninth, heparanase activity is expressed by activated
macrophages during incubation with ECM, but there was little or no
release of the enzyme into the incubation medium. Similar results
were obtained with human myeloid leukemia cells induced to
differentiate to mature macrophages.
[0022] Tenth, T-cell mediated delayed type hypersensitivity and
experimental autoimmunity are suppressed by low doses of heparanase
inhibiting non-anticoagulant species of heparin (30).
[0023] Eleventh, heparanase activity expressed by platelets,
neutrophils and metastatic tumor cells releases active bFGF from
ECM and basement membranes. Release of bFGF from storage in ECM may
elicit a localized neovascular response in processes such as wound
healing, inflammation and tumor development.
[0024] Twelfth, among the breakdown products of the ECM generated
by heparanase is a tri-sulfated disaccharide that can inhibit
T-cell mediated inflammation in vivo (31). This inhibition was
associated with an inhibitory effect of the disaccharide on the
production of biologically active TNFo by activated T cells in
vitro (31).
[0025] Other potential therapeutic applications: Apart from its
involvement in tumor cell metastasis, inflammation and
autoimmunity, mammalian heparanase may be applied to modulate:
bioavailability of heparin-binding growth factors (15); cellular
responses to heparin-binding growth factors (e.g. bFGF, VEGF) and
cytokines (IL-8) (31a, 29); cell interaction with plasma
lipoproteins (32); cellular susceptibility to certain viral and
some bacterial and protozoa infections (33, 33a, 33b); and
disintegration of amyloid plaques (34). Heparanase may thus prove
useful for conditions such as wound healing, angiogenesis,
restenosis, atherosclerosis, inflammation, neurodegenerative
diseases and viral infections. Mammalian heparanase can be used to
neutralize plasma heparin, as a potential replacement of protamine.
Anti-heparanase antibodies may be applied for immunodetection and
diagnosis of micrometastases, autoimmune lesions and renal failure
in biopsy specimens, plasma samples, and body fluids. Common use in
basic research is expected.
[0026] The identification of the hpa gene encoding for heparanase
enzyme will enable the production of a recombinant enzyme in
heterologous expression systems. Availability of the recombinant
protein will pave the way for solving the protein structure
function relationship and will provide a tool for developing new
inhibitors.
[0027] Viral Infection: The presence of heparan sulfate on cell
surfaces have been shown to be the principal requirement for the
binding of Herpes Simplex (33) and Dengue (33a) viruses to cells
and for subsequent infection of the cells. Removal of the cell
surface heparan sulfate by heparanase may therefore abolish virus
infection. In fact, treatment of cells with bacterial heparitinase
(degrading heparan sulfate) or heparinase (degrading heparan)
reduced the binding of two related animal herpes viruses to cells
and rendered the cells at least partially resistant to virus
infection (33). There are some indications that the cell surface
heparan sulfate is also involved in HIV infection (33b).
[0028] Neurodegenerative diseases: Heparan sulfate proteoglycans
were identified in the prion protein amyloid plaques of
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease and Scrape
(34). Heparanase may disintegrate these amyloid plaques which are
also thought to play a role in the pathogenesis of Alzheimer's
disease.
[0029] Restenosis and Atherosclerosis: Proliferation of arterial
smooth muscle cells (SMCs) in response to endothelial injury and
accumulation of cholesterol rich lipoproteins are basic events in
the pathogenesis of atherosclerosis and restenosis (35). Apart from
its involvement in SMC proliferation (i.e., low affinity receptors
for heparin-binding growth factors), HS is also involved in
lipoprotein binding, retention and uptake (36). It was demonstrated
that HSPG and lipoprotein lipase participate in a novel catabolic
pathway that may allow substantial cellular and interstitial
accumulation of cholesterol rich lipoproteins (32). The latter
pathway is expected to be highly atherogenic by promoting
accumulation of apoB and apoE rich lipoproteins (i.e. LDL, VLDL,
chylomicrons), independent of feed back inhibition by the cellular
sterol content. Removal of SMC HS by heparanase is therefore
expected to inhibit both SMC proliferation and lipid accumulation
and thus may halt the progression of restenosis and
atherosclerosis.
[0030] Gene Therapy:
[0031] The ultimate goal in the management of inherited as well as
acquired diseases is a rational therapy with the aim to eliminate
the underlying biochemical defects associated with the disease
rather then symptomatic treatment. Gene therapy is a promising
candidate to meet these objectives. Initially it was developed for
treatment of genetic disorders, however, the consensus view today
is that it offers the prospect of providing therapy for a variety
of acquired diseases, including cancer, viral infections, vascular
diseases and neurodegenerative disorders.
[0032] The gene-based therapeutic can act either intracellularly,
affecting only the cells to which it is delivered, or
extracellularly, using the recipient cells as local endogenous
factories for the therapeutic product(s). The application of gene
therapy may follow any of the following strategies: (i)
prophylactic gene therapy, such as using gene transfer to protect
cells against viral infection; (ii) cytotoxic gene therapy, such as
cancer therapy, where genes encode cytotoxic products to render the
target cells vulnerable to attack by the normal immune response;
(iii) biochemical correction, primarily for the treatment of single
gene defects, where a normal copy of the gene is added to the
affected or other cells.
[0033] To allow efficient transfer of the therapeutic genes, a
variety of gene delivery techniques have been developed based on
viral and non-viral vector systems. The most widely used and most
efficient systems for delivering genetic material into target cells
are viral vectors. So far, 329 clinical studies (phase I, I/II and
II) with over 2,500 patients have been initiated Worldwide since
1989 (50).
[0034] The approach of gene addition pose serious barriers. The
expression of many genes is tightly regulated and context
dependent, so achieving the correct balance and function of
expression is challenging. The gene itself is often quite large,
containing many exons and introns. The delivery vector is usually a
virus, which can infect with a high efficiency but may, on the
other hand, induce immunological response and consequently
decreases effectiveness, especially upon secondary administration.
Most of the current expression vector-based gene therapy protocols
fail to achieve clinically significant transgene expression
required for treating genetic diseases. Apparently, it is difficult
to deliver enough virus to the right cell type to elicit an
effective and therapeutic effect (51)
[0035] Homologous recombination, which was initially considered to
be of limited use for gene therapy because of its low frequency in
mammalian cells, has recently emerged as a potential strategy for
developing gene therapy. Different approaches have been used to
study homologous recombination in mammalian cells; some involve DNA
repair mechanisms. These studies aimed at either gene disruption or
gene correction and include RNA/DNA chimeric oligonucleotides,
small or large homologous DNA fragments, or adeno-associated viral
vectors. Most of these studies show a reasonable frequency of
homologous recombination, which warrants further in vivo testing
(52). Homologous recombination-based gene therapy has the potential
to develop into a powerful therapeutic modality for genetic
diseases. It can offer permanent expression and normal regulation
of corrected genes in appropriate cells or organs and probably can
be used for treating dominantly inherited diseases such as
polycystic kidney disease.
[0036] Genomic sequences function in regulation of gene
expression:
[0037] The efficient expression of therapeutic genes in target
cells or tissues is an important component of efficient and safe
gene therapy. The expression of genes is driven by the promoter
region upstream of the coding sequence, although regulation of
expression may be supplemented by farther upstream or downstream
DNA sequences or DNA in the introns of the gene. Since this
important information is embedded in the DNA, the description of
gene structure is crucial to the analysis of gene regulation.
Characterization of cell specific or tissue specific promoters, as
well as other tissue specific regulator elements enables the use of
such sequences to direct efficient cell specific, or developmental
stage specific gene expression. This information provides the basis
for targeting individual genes and for control of their expression
by exogenous agents, such as drugs. Identification of transcription
factors and other regulatory proteins required for proper gene
expression will point at new potential targets for modulating gene
expression, when so desired or required.
[0038] Efficient expression of many mammalian genes depends on the
-5 presence of at least one intron. The expression of mouse
thymidylate synthase (TS) gene, for example, is greatly influenced
by intron sequences.
[0039] The addition of almost any of the introns from the mouse TS
gene to an intronless TS minigene leads to a large increase in
expression (42). The involvement of intron 1 in the regulation of
expression was demonstrated for many other genes. In human factor
IX (hFIX), intron 1 is able to increase the expression level about
3 fold mare as compared to that of the hFIX cDNA (43). The
expression enhancing activity of intron 1 is due to efficient
functional splicing sequences, present in the precursor mRNA. By
being efficiently assembled into spliceosome complexes, transcripts
with is splicing sequences may be better protected in the nucleus
from random degradations, than those without such sequences
(44).
[0040] A forward-inserted intron 1-carrying hFIX expression
cassette suggested to be useful for directed gene transfer, while
for retroviral-mediated gene transfer system, reversely-inserted
intron 1-carrying hFIX expression cassette was considered (43).
[0041] A highly conserved cis-acting sequence element was
identified in the first intron of the mouse and rat c-Ha-ras, and
in the first exon of Ha- and Ki-ras genes of human, mouse and rat.
This cis-acting regulatory sequence confers strong transcription
enhancer activity that is differentially modulated by steroid
hormones in metastatic and nonmetastatic subpopulations.
Perturbations in the regulatory activities of such cis-acting
sequences may play an important role in governing oncogenic potency
of Ha-ras through transcriptional control mechanisms (45).
[0042] Intron sequences affect tissue specific, as well as
inducible gene expression. A 182 bp intron 1 DNA segment of the
mouse Col2a1 gene contains the necessary information to confer
high-level, temporally correct, chondrocyte expression on a
reporter gene in intact mouse embryos, while Col2a1 promoter
sequences are dispensable for chondrocyte expression (46). In
Col1A1 gene the intron plays little or no role in constitutive
expression of collagen in the skin, and in cultured cells derived
from the skin, however, in the lungs of young mice, intron deletion
results in decrease of expression to less than 50% (47).
[0043] A classical enhancer activity was shown in the 2 kb intron
fragment in bovine beta-casein gene. The enhancer activity was
largely dependent on the lactogenic hormones, especially prolactin.
It was suggested that several elements in the intron-1 of the
bovine beta-casein gene cooperatively interact not only with each
other but also with its promoter for hormonal induction (48).
[0044] Identification and characterization of regulatory elements
in genomic non-coding sequences, such as introns, provides a tool
for designing and constructing novel vectors for tissue specific,
hormone regulated or any other defined expression pattern, for gene
therapy. Such an expression cassette was developed, utilizing
regulatory elements from the human cytokeratin 18 (K18) gene,
including 5' genomic sequences and one of its introns. This
cassette efficiently expresses reporter genes, as well as the human
cystic fibrosis transmembrane conductance regulator (CFTR) gene, in
cultured lung epithelial cells (49).
[0045] Alternative Splicing:
[0046] Alternative splicing of pre mRNA is a powerful and versatile
regulatory mechanism that can effect quantitative control of gene
expression and functional diversification of proteins. It
contributes to major developmental decisions and also to a
fine-tuning of gene function. Genetic and biochemical approaches
have identified cis-acting regulatory elements and trans-acting
factors that control alternative splicing of specific mRNAs. This
mechanism results in the generation of variant isoforms of various
proteins from a single gene. These include cell surface molecules
such as CD44, receptors, cytokines such as VEGF and enzymes.
Products of alternatively spliced transcripts differ in their
expression pattern, substrate specificity and other biological
parameters.
[0047] The FGF receptor RNA undergoes alternative splicing which
results in the production of several isoforms, which exhibit
different ligand binding specificities. The alternative splicing is
regulated in a cell specific manner (53).
[0048] Alternative spliced mRNAs are often correlated with
malignancy. An increase in specific splice variant of tyrosinase
was identified in murine melanomas (54). Multiple splicing variants
of estrogen receptor are present in individual human breast tumors.
CD44 has various isoform, some are characteristic of malignant
tissues.
[0049] Identification of tumor specific alternative splice variants
provide new tool for cancer diagnostics. CD44 variants have been
used for detection of malignancy in urine samples from patients
with urothelial cancer by competitive RT-PCR (55). CD44 exon 6 was
suggested as prognostic indicator of metastasis in breast cancer
(56).
[0050] Different enzymes or polypeptides generated by alternative
splicing may have different function or catalytic specificity. The
identification and characterization of the enzyme forms, which are
involved in pathological processes, is crucial for the design of
appropriate and efficient drugs.
[0051] Modulation of gene Expression--Antisense Technology:
[0052] An antisense oligonucleotide (e.g., antisense
oligodeoxyribonucleotide) may bind its target nucleic acid either
by Watson-Crick base pairing or Hoogsteen and anti-Hoogsteen base
pairing (64). According to the Watson-Crick base pairing,
heterocyclic bases of the antisense oligonucleotide form hydrogen
bonds with the heterocyclic bases of target single-stranded nucleic
acids (RNA or single-stranded DNA), whereas according to the
Hoogsteen base pairing, the heterocyclic bases of -5 the target
nucleic acid are double-stranded DNA, wherein a third strand is
accommodated in the major groove of the B-form DNA duplex by
Hoogsteen and anti-Hoogsteen base pairing to form a triple helix
structure.
[0053] According to both the Watson-Crick and the Hoogsteen base
pairing models, antisense oligonucleotides have the potential to
regulate gene expression and to disrupt the essential functions of
the nucleic acids in cells. Therefore, antisense oligonucleotides
have possible uses in modulating a wide range of diseases in which
gene expression is altered.
[0054] Since the development of effective methods for chemically
synthesizing oligonucleotides, these molecules have been
extensively used in biochemistry and biological research and have
the potential use in medicine, since carefully devised
oligonucleotides can be used to control gene expression by
regulating levels of transcription, transcripts and/or
translation.
[0055] Oligodeoxyribonucleotides as long as 100 base pairs (bp) are
routinely synthesized by solid phase methods using commercially
available, fully automated synthesis machines. The chemical
synthesis of oligoribonucleotides, however, is far less routine.
Oligoribonucleotides are also much less stable than
oligodeoxyribonucleotides, a fact which has contributed to the more
prevalent use of oligodeoxyribonucleotides in medical and
biological research, directed at, for example, the regulation of
transcription or translation levels.
[0056] Gene expression involves few distinct and well regulated
steps. The first major step of gene expression involves
transcription of a messenger RNA (mRNA) which is an RNA sequence
complementary to the antisense (i.e., -) DNA strand, or, in other
words, identical in sequence to the DNA sense (i.e., +) strand,
composing the gene. In eukaryotes, transcription occurs in the cell
nucleus.
[0057] The second major step of gene expression involves
translation of a protein (e.g., enzymes, structural proteins,
secreted proteins, gene expression factors, etc.) in which the mRNA
interacts with ribosomal RNA complexes (ribosomes) and amino acid
activated transfer RNAs (tRNAs) to direct the synthesis of the
protein coded for by the mRNA sequence.
[0058] Initiation of transcription requires specific recognition of
a promoter DNA sequence located upstream to the coding sequence of
a gene by an RNA-synthesizing enzyme--RNA polymerase. This
recognition is preceded by sequence-specific binding of one or more
transcription factors to the promoter sequence. Additional proteins
which bind at or close to the promoter sequence may trans
upregulate transcription via cis elements known as enhancer
sequences. Other proteins which bind to or close to the promoter,
but whose binding prohibits the action of RNA polymerase, are known
as repressors.
[0059] There are also evidence that in some cases gene expression
is downregulated by endogenous antisense RNA repressors that bind a
complementary mRNA transcript and thereby prevent its translation
into a functional protein.
[0060] Thus, gene expression is typically upregulated by
transcription factors and enhancers and downregulated by
repressors.
[0061] However, in many disease situation gene expression is
impaired. In many cases, such as different types of cancer, for
various reasons the expression of a specific endogenous or
exogenous (e.g., of a pathogen such as a virus) gene is
upregulated. Furthermore, in infectious diseases caused by
pathogens such as parasites, bacteria or viruses, the disease
progression depends on expression of the pathogen genes, this
phenomenon may also be considered as far as the patient is
concerned as upregulation of exogenous genes.
[0062] Most conventional drugs function by interaction with and
modulation of one or more targeted endogenous or exogenous
proteins, e.g., enzymes. Such drugs, however, typically are not
specific for targeted proteins but interact with other proteins as
well. Thus, a relatively large dose of drug must be used to
effectively modulate a targeted protein.
[0063] Typical daily doses of drugs are from 10.sup.-5-10.sup.-1
millimoles per kilogram of body weight or 10.sup.-3-10 millimoles
for a 100 kilogram person. If this modulation instead could be
effected by interaction with and inactivation of mRNA, a dramatic
reduction in the necessary amount of drug could likely be achieved,
along with a corresponding reduction in side effects. Further
reductions could be effected if such interaction could be rendered
site-specific. Given that a functioning gene continually produces
mRNA, it would thus be even more advantageous if gene transcription
could be arrested in its entirety.
[0064] Given these facts, it would be advantageous if gene
expression could be arrested or downmodulated at the transcription
level.
[0065] The ability of chemically synthesizing oligonucleotides and
analogs thereof having a selected predetermined sequence offers
means for downmodulating gene expression. Three types of gene
expression modulation strategies may be considered.
[0066] At the transcription level, antisense or sense
oligonucleotides or analogs that bind to the genomic DNA by strand
displacement or the formation of a triple helix, may prevent
transcription (64).
[0067] At the transcript level, antisense oligonucleotides or
analogs that bind target mRNA molecules lead to the enzymatic
cleavage of the hybrid by intracellular RNase H (65). In this case,
by hybridizing to the targeted mRNA, the oligonucleotides or
oligonucleotide analogs provide a duplex hybrid recognized and
destroyed by the RNase H enzyme. Alternatively, such hybrid
formation may lead to interference with correct splicing (66). As a
result, in both cases, the number of the target mRNA intact
transcripts ready for translation is reduced or eliminated.
[0068] At the translation level, antisense oligonucleotides or
analogs that bind target mRNA molecules prevent, by steric
hindrance, binding of essential translation factors (ribosomes), to
the target mRNA, a phenomenon known in the art as hybridization
arrest, disabling the translation of such mRNAs (67).
[0069] Thus, antisense sequences, which as described hereinabove
may arrest the expression of any endogenous and/or exogenous gene
depending on their specific sequence, attracted much attention by
scientists and pharmacologists who were devoted at developing the
antisense approach into a new pharmacological tool (68).
[0070] For example, several antisense oligonucleotides have been
shown to arrest hematopoietic cell proliferation (69), growth (70),
entry into the S phase of the cell cycle (71), reduced survival
(72) and prevent receptor mediated responses (73). For use of
antisense oligonucleotides as antiviral agents the reader is
referred to reference 74.
[0071] For efficient in vivo inhibition of gene expression using
antisense oligonucleotides or analogs, the oligonucleotides or
analogs must fulfill the following requirements (i) sufficient
specificity in binding to the target sequence; (ii) solubility in
water; (iii) stability against intra- and extracellular nucleases;
(iv) capability of penetration through the cell membrane; and (v)
when used to treat an organism, low toxicity.
[0072] Unmodified oligonucleotides are impractical for use as
antisense sequences since they have short in vivo half-lives,
during which they are degraded rapidly by nucleases. Furthermore,
they are difficult to prepare in more than milligram quantities. In
addition, such oligonucleotides are poor cell membrane penetraters
(75).
[0073] Thus it is apparent that in order to meet all the above
listed requirements, oligonucleotide analogs need to be devised in
a suitable manner. Therefore, an extensive search for modified
oligonucleotides has been initiated.
[0074] For example, problems arising in connection with
double-stranded DNA (dsDNA) recognition through triple helix
formation have been diminished by a clever "switch back" chemical
linking, whereby a sequence of polypurine on one strand is
recognized, and by "switching back", a homopurine sequence on the
other strand can be recognized. Also, good helix formation has been
obtained by using artificial bases, thereby improving binding
conditions with regard to ionic strength and pH.
[0075] In addition, in order to improve half-life as well as
membrane penetration, a large number of variations in
polynucleotide backbones have been done, nevertheless with little
success.
[0076] Oligonucleotides can be modified either in the base, the
sugar or the phosphate moiety. These modifications include, for
example, the use of methylphosphonates, monothiophosphates,
dithiophosphates, phosphoramidates, phosphate esters, bridged
phosphorothioates, bridged phosphoramidates, bridged
methylenephosphonates, dephospho internucleotide analogs with
siloxane bridges, carbonate bridges, carboxymethyl ester bridges,
carbonate bridges, carboxymethyl ester bridges, acetamide bridges,
carbamate bridges, thioether bridges, sulfoxy bridges, sulfono
bridges, various "plastic" DNAs, o-anomeric bridges and borane
derivatives. For further details the reader is referred to
reference 76.
[0077] International patent application WO 89/12060 discloses
various building blocks for synthesizing oligonucleotide analogs,
as well as oligonucleotide analogs formed by joining such building
blocks in a defined sequence. The building blocks may be either
"rigid" (i.e., containing a ring structure) or "flexible" (i.e.
lacking a ring structure). In both cases, the building blocks
contain a hydroxy group and a mercapto group, through which the
building blocks are said to join to form oligonucleotide analogs.
The linking moiety in the oligonucleotide analogs is selected from
the group consisting of sulfide (--S--), sulfoxide (--SO--), and
sulfone (--SO.sub.2--). However, the application provides no data
supporting the specific binding of an oligonucleotide analog to a
target oligonucleotide.
[0078] International patent application WO 92/20702 describe an
acyclic oligonucleotide which includes a peptide backbone on which
any selected chemical nucleobases or analogs are stringed and serve
as coding characters as they do in natural DNA or RNA. These new
compounds, known as peptide nucleic acids (PNAs), are not only more
stable in cells than their natural counterparts, but also bind
natural DNA and RNA 50 to 100 times more tightly than the natural
nucleic acids cling to each other (77). PNA oligomers can be
synthesized from the four protected monomers containing thymine,
cytosine, adenine and guanine by Merrifield solid-phase peptide
synthesis. In order to increase solubility in water and to prevent
aggregation, a lysine amide group is placed at the C-terminal.
[0079] Thus, antisense technology requires pairing of messenger RNA
with an oligonucleotide to form a double helix that inhibits
translation. The concept of antisense-mediated gene therapy was
already introduced in 1978 for cancer therapy. This approach was
based on certain genes that are crucial in cell division and growth
of cancer cells. Synthetic fragments of genetic substance DNA can
achieve this goal. Such molecules bind to the targeted gene
molecules in RNA of tumor cells, thereby inhibiting the translation
of the genes and resulting in dysfunctional growth of these cells.
Other mechanisms has also been proposed. These strategies have been
used, with some success in treatment of cancers, as well as other
illnesses, including viral and other infectious diseases. Antisense
oligonucleotides are typically synthesized in lengths of 13-30
nucleotides. The life span of oligonucleotide molecules in blood is
rather short. Thus, they have to be chemically modified to prevent
destruction by ubiquitous nucleases present in the body.
Phosphorothioates are very widely used modification in antisense
oligonucleotide ongoing clinical trials (57). A new generation of
antisense molecules consist of hybrid antisense oligonucleotide
with a central portion of synthetic DNA while four bases on each
end have been modified with 2'O-methyl ribose to resemble RNA. In
preclinical studies in laboratory animals, such compounds have
demonstrated greater stability to metabolism in body tissues and an
improved safety profile when compared with the first-generation
unmodified phosphorothioate (Hybridon Inc. news). Dosens of other
nucleotide analogs have also been tested in antisense
technology.
[0080] RNA oligonucleotides may also be used for antisense
inhibition as they form a stable RNA-RNA duplex with the target,
suggesting efficient inhibition. However, due to their low
stability RNA oligonucleotides are typically expressed inside the
cells using vectors designed for this purpose. This approach is
favored when attempting to target a mRNA that encodes an abundant
and long-lived protein (57).
[0081] Recent scientific publications have validated the efficacy
of antisense compounds in animal models of hepatitis, cancers,
coronary artery restenosis and other diseases. The first antisense
drug was recently approved by the FDA. This drug Fomivirsen,
developed by Isis, is indicated for local treatment of
cytomegalovirus in patients with AIDS who are intolerant of or have
a contraindication to other treatments for CMV retinitis or who
were insufficiently responsive to previous treatments for CMV
retinitis (Pharmacotherapy News Network).
[0082] Several antisense compounds are now in clinical trials in
the United States. These include locally administered antivirals,
systemic cancer therapeutics. Antisense therapeutics has the
potential to treat many life-threatening diseases with a number of
advantages over traditional drugs. Traditional drugs intervene
after a disease-causing protein is formed. Antisense therapeutics,
however, block mRNA transcription/translation and intervene before
a protein is formed, and since antisense therapeutics target only
one specific mRNA, they should be more effective with fewer side
effects than current protein-inhibiting therapy.
[0083] A second option for disrupting gene expression at the level
of transcription uses synthetic oligonucleotides capable of
hybridizing with double stranded DNA. A triple helix is formed.
Such oligonucleotides may prevent binding of transcription factors
to the gene's promoter and therefore inhibit transcription.
Alternatively, they may prevent duplex unwinding and, therefore,
transcription of genes within the triple helical structure.
[0084] Another approach is the use of specific nucleic acid
sequences to act as decoys for transcription factors. Since
transcription factors bind specific DNA sequences it is possible to
synthesize oligonucleotides that will effectively compete with the
native DNA sequences for available transcription factors in vivo.
This approach requires the identification of gene specific
transcription factor (57).
[0085] Indirect inhibition of gene expression was demonstrated for
matrix metalloproteinase genes (MMP-1, -3, and -9), which are
associated with invasive potential of human cancer cells. E1AF is a
transcription activator of MMP genes. Expression of E1AF antisense
RNA in HSC3AS cells showed decrease in mRNA and protein levels of
MMP-1, -3, and -9. Moreover, HSC3AS showed lower invasive potential
in vitro and in vivo. These results imply that transfection of
antisense inhibits tumor invasion by down-regulating MMP genes
(58).
[0086] Ribozymes:
[0087] Ribozymes are being increasingly used for the
sequence-specific inhibition of gene expression by the cleavage of
mRNAs encoding proteins of interest. The possibility of designing
ribozymes to cleave any specific target RNA has rendered them
valuable tools in both basic research and therapeutic applications.
In the therapeutics area, ribozymes have been exploited to target
viral RNAs in infectious diseases, dominant oncogenes in cancers
and specific somatic mutations in genetic disorders. Most notably,
several ribozyme gene therapy protocols for HIV patients are
already in Phase 1 trials (62). More recently, ribozymes have been
used for transgenic animal research, gene target validation and
pathway elucidation.
[0088] Several ribozymes are in various stages of clinical trials.
ANGIOZYME was the first chemically synthesized ribozyme to be
studied in human clinical trials. ANGIOZYME specifically inhibits
formation of the VEGF-r (Vascular Endothelial Growth Factor
receptor), a key component in the angiogenesis pathway. Ribozyme
Pharmaceuticals, Inc., as well as other firms have demonstrated the
importance of anti-angiogenesis therapeutics in animal models.
HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C
Virus (HCV) RNA, was found effective in decreasing Hepatitis C
viral RNA in cell culture assays (Ribozyme Pharmaceuticals,
Incorporated-WEB home page).
[0089] Gene Disruption in Animal Models:
[0090] The emergence of gene inactivation by homologous
recombination methodology in embryonic stem cells has
revolutionized the field of mouse genetics. The availability of a
rapidly growing number of mouse null mutants has represented an
invaluable source of knowledge on mammalian development, cellular
biology and physiology, and has provided many models for human
inherited diseases. Animal models are required for an effective
drug delivery development program and evaluation of gene therapy
approach. The improvement of the original knockout strategy, as
well as exploitation of exogenous enzymatic systems that are active
in the recombination process, has been considerably extended the
range of genetic manipulations that can be produced. Additional
methods have been developed to provide versatile research tools:
Double replacement method, sequential gene targeting, conditional
cell type specific gene targeting, single copy integration method,
inducible gene targeting, gene disruption by viral delivery,
replacing one gene with another, the so called knock-in method and
the induction of specific balanced chromosomal translocation. It is
now possible to introduce a point mutation as a unique change in
the entire genome, therefore allowing very fine dissection of gene
function in vivo. Furthermore, the advent of methods allowing
conditional gene targeting opens the way for analysis of
consequence of a particular mutation in a defined organ and at a
specific time during the life of the experimental animal (59).
[0091] DNA Vaccination:
[0092] Observations in the early 1990s that plasmid DNA could
directly transfect animal cells in vivo sparked exploration of the
use of DNA plasmids to induce immune response by direct injection
into animal of DNA encoding antigenic protein. When a DNA vaccine
plasmid enters the eukaryotic cell, the protein it encodes is
transcribed and translated within the cell. In the case of
pathogens, these proteins are presented to the immune system in
their native form, mimicking the presentation of antigens during a
natural infection. DNA vaccination is particularly useful for the
induction of T cell activation. It was applied for viral and
bacterial infectious diseases, as well as for allergy and for
cancer. The central hypothesis behind active specific immunotherapy
for cancer is that tumor cells express unique antigens that should
stimulate the immune system. The first DNA vaccine against tumor
was carcino-embrionic antigen (CEA). DNA vaccinated animals
expressed immunoprotection and immunotherapy of human
CEA-expressing syngeneic mouse colon and breast carcinoma (61). In
a mouse model of neuroblastoma, DNA immunization with HuD resulted
in tumor growth inhibition with no neurological disease (60).
Immunity to the brown locus protein, gp.sup.75 tyrosinase-related
protein-1, associated with melanoma, was investigated in a
syngeneic mouse model. Priming with human gp75 DNA broke tolerance
to mouse gp75. Immunity against mouse gp75 provided significant
tumor protection (60).
[0093] Glycosyl Hydrolases:
[0094] Glycosyl hydrolases are a widespread group of enzymes that
hydrolyze the o-glycosidic bond between two or more carbohydrates
or between a carbohydrate and a noncarbohydrate moiety. The
enzymatic hydrolysis of glycosidic bond occurs by using major one
or two mechanisms leading to overall retention or inversion of the
anomeric configuration. In both mechanisms catalysis involves two
residues: a proton donor and a nucleophile. Glycosyl hydrolyses
have been classified into 58 families based on amino acid
similarities. The glycosyl hydrolyses from families 1, 2, 5, 10,
17, 30, 35, 39 and 42 act on a large variety of substrates,
however, they all hydrolyze the glycosidic bond in a general acid
catalysis mechanism, with retention of the anomeric configuration.
The mechanism involves two glutamic acid residues, which are the
proton donors and the nucleophile, with an aspargine always
preceding the proton donor. Analyses of a set of known 3D
structures from this group revealed that their catalytic domains,
despite the low level of sequence identity, adopt a similar
(.alpha./.beta.) 8 fold with the proton donor and the nucleophile
located at the C-terminal ends of strands .beta.4 and .beta.7,
respectively. Mutations in the functional conserved amino acids of
lysosomal glycosyl hydrolases were identified in lysosomal storage
diseases.
[0095] Lysosomal glycosyl hydrolases including
.beta.-glucuronidase, .beta.-manosidase, .beta.-glucocerebrosidase,
.beta.-galactosidase and .alpha.-L iduronidase, are all
exo-glycosyl hydrolases, belong to the GH-A clan and share a
similar catalytic site. However, many endo-glucanases from various
organisms, such as bacterial and fungal xylenases and cellulases
share this catalytic domain.
[0096] Genomic Sequence of hpa Gene and its Implications:
[0097] It is well established that heparanase activity is
correlated with cancer metastasis. This correlation was
demonstrated at the level of enzymatic activity as well as the
levels of protein and hpa cDNA expression in highly metastatic
cancer cells as compared with non-metastatic cells. As such,
inhibition of heparanase activity is desirable, and has been
attempted by several means. The genomic region, encoding the hpa
gene and the surrounding, provides a new powerful tool for
regulation of heparanase activity at the level of gene expression:
Regulatory sequences may reside in noncoding regions both upstream
and downstream the transcribed region as well as in intron
sequences. A DNA sequence upstream of the transcription start site
contains the promoter region and potential regulatory elements.
Regulatory factors, which interact with the promoter region may be
identified and be used as potential drugs for inhibition of cancer,
metastasis and inflammation. The promoter region can be used to
screen for inhibitors of heparanase gene expression. Furthermore,
the hpa promoter can be used to direct cell specific, particularly
cancer cell specific, expression of foreign genes, such as
cytotoxic or apoptotic genes, in order to specifically destroy
cancer cells.
[0098] Cancer and yet unknown related genetic disorders may involve
rearrangements and mutations in the heparanase gene, either in
coding or non-coding regions. Such mutations may affect expression
level or enzymatic activity. The genomic sequence of hpa enables
the amplification of specific genomic DNA fragments, identification
and diagnosis of mutations.
[0099] There is thus a widely recognized need for, and it would be
highly advantageous to have genomic, cDNA and composite
polynucleotides encoding a polypeptide having heparanase activity,
vectors including same, genetically modified cells expressing
heparanase and a recombinant protein having heparanase activity, as
well as antisense oligonucleotides, constructs and ribozymes which
can be used for down regulation heparanase activity.
SUMMARY OF THE INVENTION
[0100] Cloning of the human hpa gene which encodes heparanase, and
expression of recombinant heparanase by transfected host cells is
reported herein, as well as downregulation of heparanase activity
by antisense technology.
[0101] A purified preparation of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion and
microsequencing. The YGPDVGQPR (SEQ ID NO:8) sequence revealed was
used to screen EST databases for homology to the corresponding back
translated DNA sequence. Two closely related EST sequences were
identified and were thereafter found to be identical. Both clones
contained an insert of 1020 bp which included an open reading frame
of 973 bp followed by a 27 bp of 3' untranslated region and a Poly
A tail. Translation start site was not identified.
[0102] Cloning of the missing 5' end of hpa was performed by PCR
amplification of DNA from placenta Marathon RACE cDNA composite
using primers selected according to the EST clones sequence and the
linkers of the composite. A 900 bp PCR fragment, partially
overlapping with the identified 3' encoding EST clones was
obtained. The joined cDNA fragment (hpa), 1721 bp long (SEQ ID
NO:9), contained an open reading frame which encodes a polypeptide
of 543 amino acids (SEQ ID NO:10) with a calculated molecular
weight of 61,192 daltons.
[0103] Cloning an extended 5' sequence was enabled from the human
SK-hep1 cell line by PCR amplification using the Marathon RACE. The
5' extended sequence of the SK-hep1 hpa cDNA was assembled with the
sequence of the hpa cDNA isolated from human placenta (SEQ ID
NO:9). The assembled sequence contained an open reading frame, SEQ
ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15,
a polypeptide of 592 amino acids with a calculated molecular weight
of 66,407 daltons.
[0104] The ability of the hpa gene product to catalyze degradation
of heparan sulfate in an in vitro assay was examined by expressing
the entire open reading frame of hpa in insect cells, using the
Baculovirus expression system. Extracts and conditioned media of
cells infected with virus containing the hpa gene, demonstrated a
high level of heparan sulfate degradation activity both towards
soluble ECM-derived HSPG and intact ECM. This degradation activity
was inhibited by heparin, which is another substrate of heparanase.
Cells infected with a similar construct containing no hpa gene had
no such activity, nor did non-infected cells. The ability of
heparanase expressed from the extended 5' clone towards heparin was
demonstrated in a mammalian expression system.
[0105] The expression pattern of hpa RNA in various tissues and
cell lines was investigated using RT-PCR. It was found to be
expressed only in tissues and cells previously known to have
heparanase activity.
[0106] A panel of monochromosomal human/CHO and human/mouse somatic
cell hybrids was used to localize the human heparanase gene to
human chromosome 4. The newly isolated heparanase sequence can be
used to identify a chromosome region harboring a human heparanase
gene in a chromosome spread.
[0107] A human genomic library was screened and the human locus
harboring the heparanase gene isolated, sequenced and
characterized. Alternatively spliced heparanase mRNAs were
identified and characterized. The human heparanase promoter has
been isolated, identified and positively tested for activity. The
mouse heparanase promoter has been isolated and identified as well.
Antisense heparanase constructs were prepared and their influence
on cells in vitro tested. A predicted heparanase active site was
identified. And finally, the presence of sequences hybridizing with
human heparanase sequences was demonstrated for a variety of
mammalians and for an avian.
[0108] According to one aspect of the present invention there is
provided an isolated nucleic acid comprising a genome,
complementary or composite polynucleotide sequence encoding a
polypeptide having heparanase catalytic activity.
[0109] According to further features in preferred embodiments of
the invention described below, the polynucleotide or a portion
thereof is hybridizable with SEQ ID NOs: 9, 13, 42, 43 or a portion
thereof at 68.degree. C. in 6.times.SSC, 1% SDS, 5.times. Denharts,
10% dextran sulfate, 100 .mu.g/ml salmon sperm DNA, and .sup.32p
labeled probe and wash at 68.degree. C. with 3.times.SSC and 0.1%
SDS.
[0110] According to still further features in the described
preferred embodiments the polynucleotide or a portion thereof is at
least 60% identical with SEQ ID NOs: 9, 13, 42, 43 or portions
thereof as determined using the Bestfit procedure of the DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty-12, gap extension penalty-4).
[0111] According to still further features in the described
preferred embodiments the polypeptide is as set forth in SEQ ID
NOs:10, 14, 44 or portions thereof.
[0112] According to still further features in the described
preferred embodiments the polypeptide is at least 60% homologous to
SEQ ID NOs:10, 14, 44 or portions thereof as determined with the
Smith-Waterman algorithm, using the Bioaccelerator platform
developed by Compugene (gapop: 10.0, gapext: 0.5, matrix:
blosum62).
[0113] According to additional aspects of the present invention
there are provided a nucleic acid construct (vector) comprising the
isolated nucleic acid described herein and a host cell comprising
the construct.
[0114] According to a further aspect of the present invention there
is provided an antisense oligonucleotide comprising a
polynucleotide or a polynucleotide analog of at least 10 bases
being hybridizable in vivo, under physiological conditions, with a
portion of a polynucleotide strand encoding a polypeptide having
heparanase catalytic activity.
[0115] According to an additional aspect of the present invention
there is provided a method of in vivo downregulating heparanase
activity comprising the step of in vivo administering the antisense
oligonucleotide herein described.
[0116] According to yet an additional aspect of the present
invention there is provided a pharmaceutical composition comprising
the antisense oligonucleotide herein described and a
pharmaceutically acceptable carrier.
[0117] According to still an additional aspect of the present
invention there is provided a ribozyme comprising the antisense
oligonucleotide described herein and a ribozyme sequence.
[0118] According to a further aspect of the present invention there
is provided an antisense nucleic acid construct comprising a
promoter sequence and a polynucleotide sequence directing the
synthesis of an antisense RNA sequence of at least 10 bases being
hybridizable in vivo, under physiological conditions, with a
portion of a polynucleotide strand encoding a polypeptide having
heparanase catalytic activity.
[0119] According to further features in preferred embodiments of
the invention described below, the polynucleotide strand encoding
the polypeptide having heparanase catalytic activity is as set
forth in SEQ ID NOs: 9, 13, 42 or 43.
[0120] According to still further features in the described
preferred embodiments the polypeptide having heparanase catalytic
activity is as set forth in SEQ ID NOs: 10, 14 or 44.
[0121] According to still a further aspect of the present invention
there is provided a method of in vivo downregulating heparanase
activity comprising the step of in vivo administering the antisense
nucleic acid construct herein described.
[0122] According to yet a further aspect of the present invention
there is provided a pharmaceutical composition comprising the
antisense nucleic acid construct herein described and a
pharmaceutically acceptable carrier.
[0123] According to a further aspect of the present invention there
is provided a nucleic acid construct comprising a polynucleotide
sequence functioning as a promoter, the polynucleotide sequence is
derived from SEQ ID NO:42 and includes at least nucleotides
2535-2635 thereof or from SEQ ID NO:43 and includes at least
nucleotides 320-420.
[0124] According to a further aspect of the present invention there
is provided a method of expressing a polynucleotide sequence
comprising the step of ligating the polynucleotide sequence into
the nucleic acid construct described above, downstream of the
polynucleotide sequence derived from SEQ ID NOs:42 or 43.
[0125] According to a further aspect of the present invention there
is provided a recombinant protein comprising a polypeptide having
heparanase catalytic activity.
[0126] According to further features in preferred embodiments of
the invention described below, the polypeptide includes at least a
portion of SEQ ID NOs:10, 14 or 44.
[0127] According to still further features in the described
preferred embodiments the protein is encoded by a polynucleotide
hybridizable with SEQ ID NOs: 9, 13, 42, 43 or a portion thereof at
68.degree. C. in 6.times.SSC, 1% SDS, 5.times. Denharts, 10%
dextran sulfate, 100 .mu.g/ml salmon sperm DNA, and .sup.32p
labeled probe and wash at 68.degree. C. with 3.times.SSC and 0.1%
SDS.
[0128] According to still further features in the described
preferred embodiments the protein is encoded by a polynucleotide at
least 60% identical with SEQ ID NOs: 9, 13, 42, 43 or portions
thereof as determined using the Bestfit procedure of the DNA
sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty-12, gap extension penalty-4).
[0129] According to a further aspect of the present invention there
is provided a pharmaceutical composition comprising, as an active
ingredient, the recombinant protein herein described.
[0130] According to a further aspect of the present invention there
is provided a method of identifying a chromosome region harboring a
heparanase gene in a chromosome spread comprising the steps of (a)
hybridizing the chromosome spread with a tagged polynucleotide
probe encoding heparanase; (b) washing the chromosome spread,
thereby removing excess of non-hybridized probe; and (c) searching
for signals associated with the hybridized tagged polynucleotide
probe, wherein detected signals being indicative of a chromosome
region harboring a heparanase gene.
[0131] According to a further aspect of the present invention there
is provided a method of in vivo eliciting anti-heparanase
antibodies comprising the steps of administering a nucleic acid
construct including a polynucleotide segment corresponding to at
least a portion of SEQ ID NOs:9, 13 or 43 and a promoter for
directing the expression of said polynucleotide segment in vivo.
Accordingly, there is provided also a DNA vaccine for in vivo
eliciting anti-heparanase antibodies comprising a nucleic acid
construct including a polynucleotide segment corresponding to at
least a portion of SEQ ID NOs:9, 13 or 43 and a promoter for
directing the expression of said polynucleotide segment in
vivo.
[0132] The present invention can be used to develop new drugs to
inhibit tumor cell metastasis, inflammation and autoimmunity. The
identification of the hpa gene encoding for heparanase enzyme
enables the production of a recombinant enzyme in heterologous
expression systems. Additional features, advantages, uses and
applications of the present invention in biological science and in
diagnostic and therapeutic medicine are described hereinafter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0133] The invention herein described, by way of example only, with
reference to the accompanying drawings, wherein:
[0134] FIG. 1 presents nucleotide sequence and deduced amino acid
sequence of hpa cDNA. A single nucleotide difference at position
799 (A to T) between the EST (Expressed Sequence Tag) and the PCR
amplified cDNA (reverse transcribed RNA) and the resulting amino
acid substitution (Tyr to Phe) are indicated above and below the
substituted unit, respectively. Cysteine residues and the poly
adenylation consensus sequence are underlined. The asterisk denotes
the stop codon TGA.
[0135] FIG. 2 demonstrates degradation of soluble sulfate labeled
HSPG substrate by lysates of High Five cells infected with pFhpa2
virus. Lysates of High Five cells that were infected with pFhpa2
virus (.cndot.) or control pF2 virus (.quadrature.) were incubated
(18 h. 37.degree. C.) with sulfate labeled ECM-derived soluble HSPG
(peak I). The incubation medium was then subjected to gel
filtration on Sepharose 6B. Low molecular weight HS degradation
fragments (peak II) were produced only during incubation with the
pFhpa2 infected cells, but there was no degradation of the HSPG
substrate (z,900 ) by lysates of pF2 infected cells.
[0136] FIGS. 3a-b demonstrate degradation of soluble sulfate
labeled HSPG substrate by the culture medium of pFhpa2 and pFhpa4
infected cells. Culture media of High Five cells infected with
pFhpa2 (3a) or pFhpa4 (3b) viruses (.cndot.), or with control
viruses (.quadrature.) were incubated (18 h, 37.degree. C.) with
sulfate labeled ECM-derived soluble HSPG (peak I, ). The incubation
media were then subjected to gel filtration on Sepharose 6B. Low
molecular weight HS degradation fragments (peak II) were produced
only during incubation with the hpa gene containing viruses. There
was no degradation of the HSPG substrate by the culture medium of
cells infected with control viruses.
[0137] FIG. 4 presents size fractionation of heparanase activity
expressed by pFhpa2 infected cells. Culture medium of pFhpa2
infected High Five cells was applied onto a 50 kDa cut-off
membrane. Heparanase activity (conversion of the peak I substrate,
() into peak II HS degradation fragments) was found in the high
(>50 kDa) (.cndot.), but not low (<50 kDa) (.smallcircle.)
molecular weight compartment.
[0138] FIGS. 5a-b demonstrate the effect of heparin on heparanase
activity expressed by pFhpa2 and pFhpa4 infected High Five cells.
Culture media of pFhpa2 (5a) and pFhpa4 (5b) infected High Five
cells were incubated (18 h, 37.degree. C.) with sulfate labeled
ECM-derived soluble HSPG (peak I, ) in the absence (.smallcircle.)
or presence () of 10 .mu.g/ml heparin. Production of low molecular
weight HS degradation fragments was completely abolished in the
presence of heparin, a potent inhibitor of heparanase activity (6,
7).
[0139] FIGS. 6a-b demonstrate degradation of sulfate labeled intact
ECM by virus infected High Five and Sf21 cells. High Five (6a) and
Sf21 (6b) cells were plated on sulfate labeled ECM and infected (48
h, 28.degree. C.) with pFhpa4 (.smallcircle.) or control pF1
(.quadrature.) viruses. Control non-infected Sf21 cells (R) were
plated on the labeled ECM as well. The pH of the cultured medium
was adjusted to 6.0-6.2 followed by 24 h incubation at 37.degree.
C. Sulfate labeled material released into the incubation medium was
analyzed by gel filtration on Sepharose 6B. HS degradation
fragments were produced only by cells infected with the hpa
containing virus.
[0140] FIGS. 7a-b demonstrate degradation of sulfate labeled intact
ECM by virus infected cells. High Five (7a) and Sf21 (7b) cells
were plated on sulfate labeled ECM and infected (48 h, 28.degree.
C.) with pFhpa4 (.smallcircle.) or control pF1 (.quadrature.)
viruses. Control non-infected Sf21 cells (R) were plate on labeled
ECM as well. The pH of the cultured medium was adjusted to 6.0-6.2,
followed by 48 h incubation at 28.degree. C. Sulfate labeled
degradation fragments released into the incubation medium was
analyzed by gel filtration on Sepharose 6B. HS degradation
fragments were produced only by cells infected with the hpa
containing virus.
[0141] FIGS. 8a-b demonstrate degradation of sulfate labeled intact
ECM by the culture medium of pFhpa4 infected cells. Culture media
of High Five (8a) and Sf21 (8b) cells that were infected with
pFhpa4 (.cndot.) or control pF1 (.quadrature.) viruses were
incubated (48 h, 37.degree. C., pH 6.0) with intact sulfate labeled
ECM. The ECM was also incubated with the culture medium of control
non-infected Sf21 cells (R). Sulfate labeled material released into
the reaction mixture was subjected to gel filtration analysis.
Heparanase activity was detected only in the culture medium of
pFhpa4 infected cells.
[0142] FIGS. 9a-b demonstrate the effect of heparin on heparanase
activity in the culture medium of pFhpa4 infected cells. Sulfate
labeled ECM was incubated (24 h, 37.degree. C., pH 6.0) with
culture medium of pFhpa4 infected High Five (9a) and Sf21 (9b)
cells in the absence (.cndot.) or presence (V) of 10 .mu.g/ml
heparin. Sulfate labeled material released into the incubation
medium was subjected to gel filtration on Sepharose 6B. Heparanase
activity (production of peak II HS degradation fragments) was
completely inhibited in the presence of heparin.
[0143] FIGS. 10a-b demonstrate purification of recombinant
heparanase on heparin-Sepharose. Culture medium of Sf21 cells
infected with pFhpa4 virus was subjected to heparin-Sepharose
chromatography. Elution of fractions was performed with 0.35-2 M
NaCl gradient (). Heparanase activity in the eluted fractions is
demonstrated in FIG. 10a (.smallcircle.). Fractions 15-28 were
subjected to 15% SDS-polyacrylamide gel electrophoresis followed by
silver nitrate staining. A correlation is demonstrated between a
major protein band (MW .about.63,000) in fractions 19-24 and
heparanase activity.
[0144] FIGS. 11a-b demonstrate purification of recombinant
heparanase on a Superdex 75 gel filtration column. Active fractions
eluted from heparin-Sepharose (FIG. 10a) were pooled, concentrated
and applied onto Superdex 75 FPLC column. Fractions were collected
and aliquots of each fraction were tested for heparanase activity
(c, FIG. 11a) and analyzed by SDS-polyacrylamide gel
electrophoresis followed by silver nitrate staining (FIG. 11b). A
correlation is seen between the appearance of a major protein band
(MW .about.63,000) in fractions 4-7 and heparanase activity.
[0145] FIGS. 12a-e demonstrate expression of the hpa gene by RT-PCR
with total RNA from human embryonal tissues (12a), human
extra-embryonal tissues (12b) and cell lines from different origins
(12c-e). RT-PCR products using hpa specific primers (I), primers
for GAPDH housekeeping gene (II), and control reactions without
reverse transcriptase demonstrating absence of genomic DNA or other
contamination in RNA samples (III). M-DNA molecular weight marker
VI (Boehringer Mannheim). For 12a: lane 1-neutrophil cells (adult),
lane 2-muscle, lane 3-thymus, lane 4-heart, lane 5-adrenal. For
12b: lane 1-kidney, lane 2-placenta (8 weeks), lane 3-placenta (11
weeks), lanes 4-7-mole (complete hydatidiform mole), lane
8-cytotrophoblast cells (freshly isolated), lane 9-cytotrophoblast
cells (1.5 h in vitro), lane 10-cytotrophoblast cells (6 h in
vitro), lane 11-cytotrophoblast cells (18 h in vitro), lane
12-cytotrophoblast cells (48 h in vitro). For 12c: lane 1-JAR
bladder cell line, lane 2-NCITT testicular tumor cell line, lane
3-SW-480 human hepatoma cell line, lane 4-HTR (cytotrophoblasts
transformed by SV40), lane 5-HPTLP-I hepatocellular carcinoma cell
line, lane 6-EJ-28 bladder carcinoma cell line. For 12d: lane
1-SK-hep-1 human hepatoma cell line, lane 2-DAMI human
megakaryocytic cell line, lane 3-DAMI cell line+PMA, lane 4-CHRF
cell line+PMA, lane 5-CHRF cell line. For 12e: lane 1-ABAE bovine
aortic endothelial cells, lane 2-1063 human ovarian cell line, lane
3-human breast carcinoma MDA435 cell line, lane 4-human breast
carcinoma MDA231 cell line.
[0146] FIG. 13 presents a comparison between nucleotide sequences
of the human hpa and a mouse EST cDNA fragment (SEQ ID NO:12) which
is 80% homologous to the 3' end (starting at nucleotide 1066 of SEQ
ID NO:9) of the human hpa. The aligned termination codons are
underlined.
[0147] FIG. 14 demonstrates the chromosomal localization of the hpa
gene. PCR products of DNA derived from somatic cell hybrids and of
genomic DNA of hamster, mouse and human of were separated on 0.7%
agarose gel following amplification with hpa specific primers. Lane
1-Lambda DNA digested with BstEII, lane 2-no DNA control, lanes
3-29, PCR amplification products. Lanes 3-5-human, mouse and
hamster genomic DNA, respectively. Lanes 6-29, human
monochromosomal somatic cell hybrids representing chromosomes 1-22
and X and Y, respectively. Lane 30-Lambda DNA digested with BstEII.
An amplification product of approximately 2.8 Kb is observed only
in lanes 5 and 9, representing human genomic DNA and DNA derived
from cell hybrid carrying human chromosome 4, respectively. These
results demonstrate that the hpa gene is localized in human
chromosome 4.
[0148] FIG. 15 demonstrates the genomic exon-intron structure of
the human hpa locus (top) and the relative positions of the lambda
clones used as sequencing templates to sequence the locus (below).
The vertical rectangles represent exons (E) and the horizontal
lines therebetween represent introns (I), upstream (U) and
downstream (D) regions. Continuous lines represent DNA fragments,
which were used for sequence analysis. The discontinuous line in
lambda 6 represent a region, which overlaps with lambda 8 and hence
was not analyzed. The plasmid contains a PCR product, which bridges
the gap between L3 and L6.
[0149] FIG. 16 presents the nucleotide sequence of the genomic
region of the hpa gene. Exon sequences appear in upper case and
intron sequences in lower case. The deduced amino acid sequence of
the exons is printed below the nucleotide sequence. Two predicted
transcription start sites are shown in bold.
[0150] FIG. 17 presents an alignment of the amino acid sequences of
human heparanase, mouse and partial sequences of rat homologues.
The human and the mouse sequences were determined by sequence
analysis of the isolated cDNAs. The rat sequence is derived from
two different EST clones, which represent two different regions (5'
and 3') of the rat hpa cDNA. The human sequence and the amino acids
in the mouse and rat homologues, which are identical to the human
sequence, appear in bold.
[0151] FIG. 18 presents a heparanase Zoo blot. Ten micrograms of
genomic DNA from various sources were digested with EcoRI and
separated on 0.7% agarose-TBE gel. Following electrophoresis, the
was gel treated with HCl and than with NaOH and the DNA fragments
were downward transferred to a nylon membrane (Hybond N+, Amersham)
with 0.4 N NaOH. The membrane was hybridized with a 1.6 Kb DNA
probe that contained the entire hpa cDNA. Lane order: H--Human;
M--Mouse; Rt--Rat; P--Pig; Cw--Cow; Hr--Horse; S--Sheep;
Rb--Rabbit; D--Dog; Ch--Chicken; F--Fish. Size markers (Lambda
BsteII) are shown on the left
[0152] FIG. 19 demonstrates the secondary structure prediction for
heparanase performed using the PHD server--Profile network
Prediction Heidelberg. H--helix, E--extended (beta strand), The
glutamic acid predicted as the proton donor is marked by asterisk
and the possible nucleophiles are underlined.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0153] The present invention is of a polynucleotide or nucleic
acid, referred to hereinbelow interchangeably as hpa, hpa cDNA or
hpa gene or identified by its SEQ ID NOs, encoding a polypeptide
having heparanase activity, vectors or nucleic acid constructs
including same and which are used for over-expression or antisense
inhibition of heparanase, genetically modified cells expressing
same, recombinant protein having heparanase activity, antisense
oligonucleotides and ribozymes for heparanase modulation, and
heparanase promoter sequences which can be used to direct the
expression of desired genes.
[0154] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not limited
in its application to the details of construction and the
arrangement of the components set forth in the following
description or illustrated in the drawings. The invention is
capable of other embodiments or of being practiced or carried out
in various ways. Also, it is to be understood that the phraseology
and terminology employed herein is for the purpose of description
and should not be regarded as limiting.
[0155] Cloning of the human and mouse hpa genes, cDNAs and genomic
sequence (for human), encoding heparanase and expressing
recombinant heparanase by transfected cells is reported herein.
These are the first mammalian heparanase genes to be cloned.
[0156] A purified preparation of heparanase isolated from human
hepatoma cells was subjected to tryptic digestion and
microsequencing.
[0157] The YGPDVGQPR (SEQ ID NO:8) sequence revealed was used to
screen EST databases for homology to the corresponding back
translated DNA sequences. Two closely related EST sequences were
identified and were thereafter found to be identical.
[0158] Both clones contained an insert of 1020 bp which includes an
open reading frame of 973 bp followed by a 3' untranslated region
of 27 bp and a Poly A tail, whereas a translation start site was
not identified.
[0159] Cloning of the missing 5' end was performed by PCR
amplification of DNA from placenta Marathon RACE cDNA composite
using primers selected according to the EST clones sequence and the
linkers of the composite.
[0160] A 900 bp PCR fragment, partially overlapping with the
identified 3' encoding EST clones was obtained. The joined cDNA
fragment (hpa), 1721 bp long (SEQ ID NO:9), contained an open
reading frame which encodes, as shown in FIG. 1 and SEQ ID NO:11, a
polypeptide of 543 amino acids (SEQ ID NO:10) with a calculated
molecular weight of 61,192 daltons.
[0161] A single nucleotide difference at position 799 (A to T)
between the EST clones and the PCR amplified cDNA was observed.
This difference results in a single amino acid substitution (Tyr to
Phe) (FIG. 1). Furthermore, the published EST sequences contained
an unidentified nucleotide, which following DNA sequencing of both
the EST clones was resolved into two nucleotides (G and C at
positions 1630 and 1631 in SEQ ID NO:9, respectively).
[0162] The ability of the hpa gene product to catalyze degradation
of heparan sulfate in an in vitro assay was examined by expressing
the entire open reading frame in insect cells, using the
Baculovirus expression system.
[0163] Extracts and conditioned media of cells infected with virus
containing the hpa gene, demonstrated a high level of heparan
sulfate degradation activity both towards soluble ECM-derived HSPG
and intact ECM, which was inhibited by heparin, while cells
infected with a similar construct containing no hpa gene had no
such activity, nor did non-infected cells.
[0164] The expression pattern of hpa RNA in various tissues and
cell lines was investigated using RT-PCR. It was found to be
expressed only in tissues and cells previously known to have
heparanase activity.
[0165] Cloning an extended 5' sequence was enabled from the human
SK-hep1 cell line by PCR amplification using the Marathon RACE. The
5' extended sequence of the SK-hep1 hpa cDNA was assembled with the
sequence of the hpa cDNA isolated from human placenta (SEQ ID
NO:9). The assembled sequence contained an open reading frame, SEQ
ID NOs: 13 and 15, which encodes, as shown in SEQ ID NOs:14 and 15,
a polypeptide of 592 amino acids, with a calculated molecular
weight of 66,407 daltons. This open reading frame was shown to
direct the expression of catalytically active heparanase in a
mammalian cell expression system. The expressed heparanase was
detectable by anti heparanase antibodies in Western blot
analysis.
[0166] A panel of monochromosomal human/CHO and human/mouse somatic
cell hybrids was used to localize the human heparanase gene to
human chromosome 4. The newly isolated heparanase sequence can
therefore be used to identify a chromosome region harboring a human
heparanase gene in a chromosome spread.
[0167] The hpa cDNA was then used as a probe to screen a a human
genomic library. Several phages were positive. These phages were
analyzed and were found to cover most of the hpa locus, except for
a small portion which was recovered by bridging PCR. The hpa locus
covers about 50,000 bp. The hpa gene includes 12 exons separated by
11 introns.
[0168] RT-PCR performed on a variety of cells revealed
alternatively spliced hpa transcripts.
[0169] The amino acid sequence of human heparanase was used to
search for homologous sequences in the DNA and protein databases.
Several human EST's were identified, as well as mouse sequences
highly homologous to human heparanase. The following mouse EST's
were identified AA177901, AA674378, AA67997, AA047943, AA690179,
AI122034, all sharing an identical sequence and correspond to amino
acids 336-543 of the human heparanase sequence. The entire mouse
heparanase cDNA was cloned, based on the nucleotide sequence of the
mouse EST's using Marathon cDNA libraries. The mouse and the human
hpa genes share an average homology of 78% between the nucleotide
sequences and 81% similarity between the deduced amino acid
sequences. hpa homologous sequences from rat were also uncovered
(EST's AI060284 and AI237828).
[0170] Homology search of heparanase amino acid sequence against
the DNA and the protein databases and prediction of its protein
secondary structure enabled to identify candidate amino acids that
participate in the heparanase active site.
[0171] Expression of hpa antisense in mammalian cell lines resulted
in about five fold decrease in the number of recoverable cells as
compared to controls.
[0172] Human Hpa cDNA was shown to hybridize with genomic DNAs of a
variety of mammalian species and with an avian.
[0173] The human and mouse hpa promoters were identified and the
human promoter was tested positive in directing the expression of a
reporter gene.
[0174] Thus, according to the present invention there is provided
an isolated nucleic acid comprising a genomic, complementary or
composite polynucleotide sequence encoding a polypeptide having
heparanase catalytic activity.
[0175] The phrase "composite polynucleotide sequence" refers to a
sequence which includes exonal sequences required to encode the
polypeptide having heparanase activity, as well as any number of
intronal sequences. The intronal sequences can be of any source and
typically will include conserved splicing signal sequences. Such
intronal sequences may further include cis acting expression
regulatory elements.
[0176] The term "heparanase catalytic activity" or its equivalent
term "heparanase activity" both refer to a mammalian
endoglycosidase hydrolyzing activity which is specific for heparan
or heparan sulfate proteoglycan substrates, as opposed to the
activity of bacterial enzymes (heparinase I, II and III) which
degrade heparin or heparan sulfate by means of .beta.-elimination
(37).
[0177] According to a preferred embodiment of the present invention
the polynucleotide or a portion thereof is hybridizable with SEQ ID
NOs: 9, 13, 42, 43 or a portion thereof at 68.degree. C. in
6.times.SSC, 1% SDS, 5.times. Denharts, 10% dextran sulfate, 100
.mu.g/ml salmon sperm DNA, and .sup.32p labeled probe and wash at
68.degree. C. with 3, 2, 1, 0.5 or 0.1.times.SSC and 0.1% SDS.
[0178] According to another preferred embodiment of the present
invention the polynucleotide or a portion thereof is at least 60%,
preferably at least 65%, more preferably at least 70%, more
preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, most
preferably, 95-100% identical with SEQ ID NOs: 9, 13, 42, 43 or
portions thereof as determined using the Bestfit procedure of the
DNA sequence analysis software package developed by the Genetic
Computer Group (GCG) at the university of Wisconsin (gap creation
penalty-12, gap extension penalty-4--which are the default
parameters).
[0179] According to another preferred embodiment of the present
invention the polypeptide encoded by the polynucleotide sequence is
as set forth in SEQ ID NOs:10, 14, 44 or portions thereof having
heparanase catalytic activity. Such portions are expected to
include amino acids Asp-Glu 224-225 (SEQ ID NO:10), which can serve
as proton donors and glutamic acid 343 or 396 which can serve as a
nucleophile.
[0180] According to another preferred embodiment of the present
invention the polypeptide encoded by the polynucleotide sequence is
at least 60%, preferably at least 65%, more preferably at least
70%, more preferably at least 75%, more preferably at least 80%,
more preferably at least 85% more preferably at least 90%, most
preferably, 95-100% homologous (both similar and identical acids)
to SEQ ID NOs:10, 14, 44 or portions thereof as determined with the
Smith-Waterman algorithm, using the Bioaccelerator platform
developed by Compugene (gapop: 10.0, gapext: 0.5, matrix: blosum62,
see also the description to FIG. 17).
[0181] Further according to the present invention there is provided
a nucleic acid construct comprising the isolated nucleic acid
described herein. The construct may and preferably further include
an origin of replication and trans regulatory elements, such as
promoter and enhancer sequences.
[0182] The construct or vector can be of any type. It may be a
phage which infects bacteria or a virus which infects eukaryotic
cells. It may also be a plasmid, phagemid, cosmid, bacmid or an
artificial chromosome.
[0183] Further according to the present invention there is provided
a host cell comprising the nucleic acid construct described herein.
The host cell can be of any type. It may be a prokaryotic cell, an
eukaryotic cell, a cell line, or a cell as a portion of an
organism. The polynucleotide encoding heparanase can be permanently
or transiently present in the cell. In other words, genetically
modified cells obtained following stable or transient transfection,
transformation or transduction are all within the scope of the
present invention. The polynucleotide can be present in the cell in
low copy (say 1-5 copies) or high copy number (say 5-50 copies or
more). It may be integrated in one or more chromosomes at any
location or be present as an extrachromosomal material.
[0184] The present invention is further directed at providing a
heparanase over-expression system which includes a cell
overexpressing heparanase catalytic activity. The cell may be a
genetically modified host cell transiently or stably transfected or
transformed with any suitable vector which includes a
polynucleotide sequence encoding a polypeptide having heparanase
activity and a suitable promoter and enhancer sequences to direct
over-expression of heparanase. However, the overexpressing cell may
also be a product of an insertion (e.g., via homologous
recombination) of a promoter and/or enhancer sequence downstream to
the endogenous heparanase gene of the expressing cell, which will
direct over-expression from the endogenous gene.
[0185] The term "over-expression" as used herein in the
specification and claims below refers to a level of expression
which is higher than a basal level of expression typically
characterizing a given cell under otherwise identical
conditions.
[0186] According to another aspect the present invention provides
an antisense oligonucleotide comprising a polynucleotide or a
polynucleotide analog of at least 10, preferably 11-15, more
preferably 16-17, more preferably 18, more preferably 19-25, more
preferably 26-35, most preferably 35-100 bases being hybridizable
in vivo, under physiological conditions, with a portion of a
polynucleotide strand encoding a polypeptide having heparanase
catalytic activity. The antisense oligonucleotide can be used for
downregulating heparanase activity by in vivo administration
thereof to a patient. As such, the antisense oligonucleotide
according to the present invention can be used to treat types of
cancers which are characterized by impaired (over) expression of
heparanase, and are dependent on the expression of heparanase for
proliferating or forming metastases.
[0187] The antisense oligonucleotide can be DNA or RNA or even
include 10 nucleotide analogs, examples of which are provided in
the Background section hereinabove. The antisense oligonucleotide
according to the present invention can be synthetic and is
preferably prepared by solid phase synthesis. In addition, it can
be of any desired length which still provides specific base pairing
(e.g., 8 or 10, preferably more, nucleotides long) and it can
include mismatches that do not hamper base pairing under
physiological conditions.
[0188] Further according to the present invention there is provided
a pharmaceutical composition comprising the antisense
oligonucleotide herein described and a pharmaceutically acceptable
carrier. The carrier can be, for example, a liposome loadable with
the antisense oligonucleotide.
[0189] According to a preferred embodiment of the present invention
the antisense oligonucleotide further includes a ribozyme sequence.
The ribozyme sequence serves to cleave a heparanase RNA molecule to
which the antisense oligonucleotide binds, to thereby downregulate
heparanase expression.
[0190] Further according to the present invention there is provided
an antisense nucleic acid construct comprising a promoter sequence
and a polynucleotide sequence directing the synthesis of an
antisense RNA sequence of at least 10 bases being hybridizable in
vivo, under physiological conditions, with a portion of a
polynucleotide strand encoding a polypeptide having heparanase
catalytic activity. Like the antisense oligonucleotide, the
antisense construct can be used for downregulating heparanase
activity by in vivo administration thereof to a patient. As such,
the antisense construct, like the antisense oligonucleotide,
according to the present invention can be used to treat types of
cancers which are characterized by impaired (over) expression of
heparanase, and are dependent on the expression of heparanase for
proliferating or forming metastases.
[0191] Thus, further according to the present invention there is
provided a pharmaceutical composition comprising the antisense
construct herein described and a pharmaceutically acceptable
carrier. The carrier can be, for example, a liposome loadable with
the antisense construct.
[0192] Formulations for topical administration may include, but are
not limited to, lotions, ointments, gels, creams, suppositories,
drops, liquids, sprays and powders. Conventional pharmaceutical
carriers, aqueous, powder or oily bases, thickeners and the like
may be necessary or desirable. Coated condoms, stents, active pads,
and other medical devices may also be useful. Compositions for oral
administration include powders or granules, suspensions or
solutions in water or non-aqueous media, sachets, capsules or
tablets. Thickeners, diluents, flavorings, dispersing aids,
emulsifiers or binders may be desirable. Formulations for
parenteral administration may include, but are not limited to,
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives.
[0193] Dosing is dependent on severity and responsiveness of the
condition to be treated, but will normally be one or more doses per
day, week or month with course of treatment lasting from several
days to several months or until a cure is effected or a diminution
of disease state is achieved. Persons ordinarily skilled in the art
can easily determine optimum dosages, dosing methodologies and
repetition rates.
[0194] Further according to the present invention there is provided
a nucleic acid construct comprising a polynucleotide sequence
functioning as a promoter, the polynucleotide sequence is derived
from SEQ ID NO:42 and includes at least nucleotides 2135-2635,
preferably 2235-2635, more preferably 2335-2635, more preferably
2435-2635, most preferably 2535-2635 thereof, or SEQ ID NO:43 and
includes at least nucleotides 1-420, preferably 120-420, more
preferably 220-420, most preferably 320-420, thereof. These
nucleotides are shown in the example section that follows to direct
the synthesis of a reporter gene in transformed cells. Thus,
further according to the present invention there is provided a
method of expressing a polynucleotide sequence comprising the step
of ligating the polynucleotide sequence downstream to either of the
promoter sequences described herein. Heparanase promoters can be
isolated from a variety of mammalian an other species by cloning
genomic regions present 5' to the coding sequence thereof. This can
be readily achievable by one ordinarily skilled in the art using
the heparanase polynucleotides described herein, which are shown in
the Examples section that follows to participate in efficient cross
species hybridization.
[0195] Further according to the present invention there is provided
a recombinant protein comprising a polypeptide having heparanase
catalytic activity. The protein according to the present invention
include modifications known as post translational modifications,
including, but not limited to, proteolysis (e.g., removal of a
signal peptide and of a pro- or preprotein sequence), methionine
modification, glycosylation, alkylation (e.g., methylation),
acetylation, etc. According to preferred embodiments the
polypeptide includes at least a portion of SEQ ID NOs:10, 14 or 44,
the portion has heparanase catalytic activity. According to
preferred embodiments of the present invention the protein is
encoded by any of the above described isolated nucleic acids.
Further according to the present invention there is provided a
pharmaceutical composition comprising, as an active ingredient, the
recombinant protein described herein.
[0196] The recombinant protein may be purified by any conventional
protein purification procedure close to homogeneity and/or be mixed
with additives. The recombinant protein may be manufactured using
any of the genetically modified cells described above, which
include any of the expression nucleic acid constructs described
herein. The recombinant protein may be in any form. It may be in a
crystallized form, a dehydrated powder form or in solution. The
recombinant protein may be useful in obtaining pure heparanase,
which in turn may be useful in eliciting anti-heparanase
antibodies, either poly or monoclonal antibodies, and as a
screening active ingredient in an anti-heparanase inhibitors or
drugs screening assay or system.
[0197] Further according to the present invention there is provided
a method of identifying a chromosome region harboring a human
heparanase gene in a chromosome spread. the method is executed
implementing the following method steps, in which in a first step
the chromosome spread (either interphase or metaphase spread) is
hybridized with a tagged polynucleotide probe encoding heparanase.
The tag is preferably a fluorescent tag. In a second step according
to the method the chromosome spread is washed, thereby excess of
non-hybridized probe is removed. Finally, signals associated with
the hybridized tagged polynucleotide probe are searched for,
wherein detected signals being indicative of a chromosome region
harboring the human heparanase gene. One ordinarily skilled in the
art would know how to use the sequences disclosed herein in
suitable labeling reactions and how to use the tagged probes to
detect, using in situ hybridization, a chromosome region harboring
a human heparanase gene.
[0198] Further according to the present invention there is provided
a method of in vivo eliciting anti-heparanase antibodies comprising
the steps of administering a nucleic acid construct including a
polynucleotide segment corresponding to at least a portion of SEQ
ID NOs:9, 13 or 43 and a promoter for directing the expression of
said polynucleotide segment in vivo. Accordingly, there is provided
also a DNA vaccine for in vivo eliciting anti-heparanase antibodies
comprising a nucleic acid construct including a polynucleotide
segment corresponding to at least a portion of SEQ ID NOs:9, 13 or
43 and a promoter for directing the expression of said
polynucleotide segment in vivo. The vaccine optionally further
includes a pharmaceutically acceptable carrier, such as a virus,
liposome or an antigen presenting cell. Alternatively, the vaccine
is employed as a naked DNA vaccine
[0199] The present invention can be used to develop treatments for
various diseases, to develop diagnostic assays for these diseases
and to provide new tools for basic research especially in the
fields of medicine and biology.
[0200] Specifically, the present invention can be used to develop
new drugs to inhibit tumor cell metastasis, inflammation and
autoimmunity. The identification of the hpa gene encoding for the
heparanase enzyme enables the production of a recombinant enzyme in
heterologous expression systems.
[0201] Furthermore, the present invention can be used to modulate
bioavailability of heparin-binding growth factors, cellular
responses to heparin-binding growth factors (e.g., bFGF, VEGF) and
cytokines (e.g., IL-8), cell interaction with plasma lipoproteins,
cellular susceptibility to viral, protozoa and some bacterial
infections, and disintegration of neurodegenerative plaques.
Recombinant heparanase offers a potential treatment for wound
healing, angiogenesis, restenosis, atherosclerosis, inflammation,
neurodegenerative diseases (such as, for example,
Genstmann-Straussler Syndrome, Creutzfeldt-Jakob disease, Scrape
and Alzheimer's disease) and certain viral and some bacterial and
protozoa infections. Recombinant heparanase can be used to
neutralize plasma heparin, as a potential replacement of
protamine.
[0202] As used herein, the term "modulate" includes substantially
inhibiting, slowing or reversing the progression of a disease,
substantially ameliorating clinical symptoms of a disease or
condition, or substantially preventing the appearance of clinical
symptoms of a disease or condition. A "modulator" therefore
includes an agent which may modulate a disease or condition.
Modulation of viral, protozoa and bacterial infections includes any
effect which substantially interrupts, prevents or reduces any
viral, bacterial or protozoa activity and/or stage of the virus,
bacterium or protozoon life cycle, or which reduces or prevents
infection by the virus, bacterium or protozoon in a subject, such
as a human or lower animal.
[0203] As used herein, the term "wound" includes any injury to any
portion of the body of a subject including, but not limited to,
acute conditions such as thermal burns, chemical burns, radiation
burns, burns caused by excess exposure to ultraviolet radiation
such as sunburn, damage to bodily tissues such as the perineum as a
result of labor and childbirth, including injuries sustained during
medical procedures such as episiotomies, trauma-induced injuries
including cuts, those injuries sustained in automobile and other
mechanical accidents, and those caused by bullets, knives and other
weapons, and post-surgical injuries, as well as chronic conditions
such as pressure sores, bedsores, conditions related to diabetes
and poor circulation, and all types of acne, etc.
[0204] Anti-heparanase antibodies, raised against the recombinant
enzyme, would be useful for immunodetection and diagnosis of
micrometastases, autoimmune lesions and renal failure in biopsy
specimens, plasma samples, and body fluids. Such antibodies may
also serve as neutralizing agents for heparanase activity.
[0205] The genomic heparanase sequences described herein can be
used to construct knock-in and knock-out constructs. Such
constructs include a fragment of 10-20 Kb of a heparanase locus and
a negative and a positive selection markers and can be used to
provide heparanase knock-in and knock-out animal models by methods
known to the skilled artisan. Such animal models can be used for
studying the function of heparanase in developmental processes, and
in normal as well as pathological processes. They can also serve as
an experimental model for testing drugs and gene therapy protocols.
The complementary heparanase sequence (cDNA) can be used to derive
transgenic animals, overexpressing heparanase for same.
Alternatively, if cloned in the antisense orientation, the
complementary heparanase sequence (cDNA) can be used to derive
transgenic animals under-expressing heparanase for same.
[0206] The heparanase promoter sequences described herein and other
cis regulatory elements linked to the heparanase locus can be used
to regulated the expression of genes. For example, these promoters
can be used to direct the expression of a cytotoxic protein, such
as TNF, in tumor cells. It will be appreciated that heparanase
itself is abnormally expressed under the control of its own
promoter and other cis acting elements in a variety of tumors, and
its expression is correlated with metastasis. It is also abnormally
highly expressed in inflammatory cells. The introns of the
heparanase gene can be used for the same purpose, as it is known
that introns, especially upstream introns include cis acting
element which affect expression. A heparanase promoter fused to a
reporter protein can be used to study/monitor its activity.
[0207] The polynucleotide sequences described herein can also be
used to provide DNA vaccines which will elicit in vivo anti
heparanase antibodies. Such vaccines can therefore be used to
combat inflammatory and cancer.
[0208] Antisense oligonucleotides derived according to the
heparanase sequences described herein, especially such
oligonucleotides supplemented with ribozyme activity, can be used
to modulate heparanase expression. Such oligonucleotides can be
from the coding region, from the introns or promoter specific.
Antisense heparanase nucleic acid constructs can similarly
function, as well known in the art.
[0209] The heparanase sequences described herein can be used to
study the catalytic mechanism of heparanase. Carefully selected
site directed mutagenesis can be employed to provide modified
heparanase proteins having modified characteristics in terms of,
for example, substrate specificity, sensitivity to inhibitors,
etc.
[0210] While studying heparanase expression in a variety of cell
types alternatively spliced transcripts were identified. Such
transcripts if found characteristic of certain pathological
conditions can be used as markers for such conditions. Such
transcripts are expected to direct the synthesis of heparanases
with altered functions.
[0211] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0212] Generally, the nomenclature used herein and the laboratory
procedures in recombinant DNA technology described below are those
well known and commonly employed in the art. Standard techniques
are used for cloning, DNA and RNA isolation, amplification and
purification. Generally enzymatic reactions involving DNA ligase,
DNA polymerase, restriction endonucleases and the like are
performed according to the manufacturers' specifications. These
techniques and various other techniques are generally performed
according to Sambrook et al., Molecular Cloning--A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
(1989), which is incorporated herein by reference. Other general
references are provided throughout this document. The procedures
therein are believed to be well known in the art and are provided
for the convenience of the reader. All the information contained
therein is incorporated herein by reference.
[0213] The following protocols and experimental details are
referenced in the Examples that follow:
[0214] Purification and characterization of heparanase from a human
hepatoma cell line and human placenta: A human hepatoma cell line
(Sk-hep-1) was chosen as a source for purification of a human
tumor-derived heparanase. Purification was essentially as described
in U.S. Pat. No. 5,362,641 to Fuks, which is incorporated by
reference as if fully set forth herein. Briefly, 500 liter,
5.times.10.sup.11 cells were grown in suspension and the heparanase
enzyme was purified about 240,000 fold by applying the following
steps: (i) cation exchange (CM-Sephadex) chromatography performed
at pH 6.0, 0.3-1.4 M NaCl gradient; (ii) cation exchange
(CM-Sephadex) chromatography performed at pH 7.4 in the presence of
0.1% CHAPS. 0.3-1.1 M NaCl gradient; (iii) heparin-Sepharose
chromatography performed at pH 7.4 in the presence of 0.1% CHAPS,
0.35-1.1 M NaCl gradient; (iv) ConA-Sepharose chromatography
performed at pH 6.0 in buffer containing 0.1% CHAPS and 1 M NaCl,
elution with 0.25 M o-methyl mannoside; and (v) HPLC cation
exchange (Mono-S) chromatography performed at pH 7.4 in the
presence of 0.1% CHAPS, 0.25-1 M NaCl gradient.
[0215] Active fractions were pooled, precipitated with TCA and the
precipitate subjected to SDS polyacrylamide gel electrophoresis
and/or tryptic digestion and reverse phase HPLC. Tryptic peptides
of the purified protein were separated by reverse phase HPLC (C8
column) and homogeneous peaks were subjected to amino acid sequence
analysis.
[0216] The purified enzyme was applied to reverse phase HPLC and
subjected to N-terminal amino acid sequencing using the amino acid
sequencer (Applied Biosystems).
[0217] Cells: Cultures of bovine corneal endothelial cells (BCECs)
were established from steer eyes as previously described (19, 38).
Stock cultures were maintained in DMEM (1 g glucose/liter)
supplemented with 10% newborn calf serum and 5% FCS. bFGF (1 ng/ml)
was added every other day during the phase of active cell growth
(13, 14).
[0218] Preparation of dishes coated with ECM: BCECs (second to
fifth passage) were plated into 4-well plates at an initial density
of 2.times.10.sup.5 cells/ml, and cultured in sulfate-free Fisher
medium plus 5% dextran T-40 for 12 days. Na.sub.2.sup.35SO.sub.4
(25 .mu.Ci/ml) was added on day 1 and 5 after seeding and the
cultures were incubated with the label without medium change. The
subendothelial ECM was exposed by dissolving (5 min., room
temperature) the cell layer with PBS containing 0.5% Triton X-100
and 20 mM NH.sub.4OH, followed by four washes with PBS. The ECM
remained intact, free of cellular debris and firmly attached to the
entire area of the tissue culture dish (19, 22).
[0219] To prepare soluble sulfate labeled proteoglycans (peak I
material), the ECM was digested with trypsin (25 .mu.g/ml, 6 h,
37.degree. C.), the digest was concentrated by reverse dialysis and
the concentrated material was applied onto a Sepharose 6B gel
filtration column. The resulting high molecular weight material
(Kav<0.2, peak I) was collected. More than 80% of the labeled
material was shown to be composed of heparan sulfate proteoglycans
(11, 39).
[0220] Heparanase activity: Cells (1.times.10.sup.6/35-mm dish),
cell lysates or conditioned media were incubated on top of
.sup.35S-labeled ECM (18 h, 37.degree. C.) in the presence of 20 mM
phosphate buffer (pH 6.2). Cell lysates and conditioned media were
also incubated with sulfate labeled peak I material (10-20 .mu.l).
The incubation medium was collected, centrifuged (18,000.times.g,
4.degree. C., 3 min.), and sulfate labeled material analyzed by gel
filtration on a Sepharose CL-6B column (0.9.times.30 cm). Fractions
(0.2 ml) were eluted with PBS at a flow rate of 5 ml/h and counted
for radioactivity using Bio-fluor scintillation fluid. The excluded
volume (V.sub.O) was marked by blue dextran and the total included
volume (V.sub.t) by phenol red. The latter was shown to comigrate
with free sulfate (7, 11, 23). Degradation fragments of HS side
chains were eluted from Sepharose 6B at 0.5<Kav<0.8 (peak II)
(7, 11, 23). A nearly intact HSPG released from ECM by
trypsin--and, to a lower extent, during incubation with PBS
alone--was eluted next to V.sub.O (Kav<0.2, peak I). Recoveries
of labeled material applied on the columns ranged from 85 to 95% in
different experiments (11). Each experiment was performed at least
three times and the variation of elution positions (Kav values) did
not exceed +/-15%.
[0221] Cloning of hpa cDNA: cDNA clones 257548 and 260138 were
obtained from the I.M.A.G.E Consortium (2130 Memorial Parkway SW,
Hunstville, Ala. 35801). The cDNAs were originally cloned in EcoRI
and NotI cloning sites in the plasmid vector pT3T7D-Pac. Although
these clones are reported to be somewhat different, DNA sequencing
demonstrated that these clones are identical to one another.
Marathon RACE (rapid amplification of cDNA ends) human placenta
(poly-A) cDNA composite was a gift of Prof. Yossi Shiloh of Tel
Aviv University. This composite is vector free, as it includes
reverse transcribed cDNA fragments to which double, partially
single stranded adapters are attached on both sides. The
construction of the specific composite employed is described in
reference 39a.
[0222] Amplification of hp3 PCR fragment was performed according to
the protocol provided by Clontech laboratories. The template used
for amplification was a sample taken from the above composite. The
primers used for amplification were:
[0223] First step: 5'-primer: AP1:
5'-CCATCCTAATACGACTCACTATAGGGC-3', SEQ ID NO:1; 3'-primer: HPL229:
5'-GTAGTGATGCCATGTAACTGAATC-3', SEQ ID NO:2.
[0224] Second step: nested 5'-primer: AP2:
5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:3; nested 3'-primer:
HPL171: 5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:4. The HPL229 and
HPL171 were selected according to the sequence of the EST clones.
They include nucleotides 933-956 and 876-897 of SEQ ID NO:9,
respectively.
[0225] PCR program was 94.degree. C.-4 min., followed by 30 cycles
of 94.degree. C.-40 sec., 62.degree. C.-1 min., 72.degree. C.-2.5
min. Amplification was performed with Expand High Fidelity
(Boehringer Mannheim). The resulting ca. 900 bp hp3 PCR product was
digested with BfrI and PvuII. Clone 257548 (phpa1) was digested
with EcoRI, followed by end filling and was then further digested
with BfrI. Thereafter the PvuII-BfrI fragment of the hp3 PCR
product was cloned into the blunt end-BfrI end of clone phpa1 which
resulted in having the entire cDNA cloned in pT3T7-pac vector,
designated phpa2.
[0226] RT-PCR: RNA was prepared using TR1-Reagent (Molecular
research center Inc.) according to the manufacturer instructions.
1.25 .mu.g were taken for reverse transcription reaction using
MuMLV Reverse transcriptase (Gibco BRL) and Oligo (dT).sub.15
primer, SEQ ID NO:5, (Promega). Amplification of the resultant
first strand cDNA was performed with Taq polymerase (Promega). The
following primers were used:
1 HPU-355: 5'-TTCGATCCCAAGAAGGAATCAAC-3',, SEQ ID NO: 6 nucleotides
372-394 in. SEQ ID NOs: 9 or 11 HPL-229:
5'-GTAGTGATGCCATGTAACTGAATC-3',, SEQ ID NO: 7 nucleotides 933-956
in. SEQ ID NOs: 9 or 11
[0227] PCR program: 94.degree. C.-4 min., followed by 30 cycles of
94.degree. C.-40 sec., 62.degree. C.-1 min., 72.degree. C.-1
min.
[0228] Alternatively, total RNA was prepared from cell cultures
using Tri-reagent (Molecular Research Center, Inc.) according to
the manufacturer recommendation. Poly A+ RNA was isolated from
total RNA using mRNA separator (Clontech). Reverse transcription
was performed with total RNA using Superscript II (GibcoBRL). PCR
was performed with Expand high fidelity (Boehringer Mannheim).
Primers used for amplification were as follows:
2 Hpu-685, 5'-GAGCAGCCAGGTGAGCCCAAGAT-3', SEQ ID NO: 24 Hpu-355,
5'-TTCGATCCCAAGAAGGAATCAAC-3', SEQ ID NO: 25 Hpu 565,
5'-AGCTCTGTAGATGTGCTATACAC-3', SEQ ID NO: 26 Hpl 967,
5'-TCAGATGCAAGCAGCAACTTTGGC-3', SEQ ID NO: 27 Hpl 171,
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO: 28 Hpl 229,
5'-GTAGTGATGCCATGTAACTGAATC-3- ', SEQ ID NO: 29
[0229] PCR reaction was performed as follows: 94.degree. C. 3
minutes, followed by 32 cycles of 94.degree. C. 40 seconds.
64.degree. C. 1 minute. 72.degree. C. 3 minutes, and one cycle
72.degree. C., 7 minutes.
[0230] Expression of recombinant heparanase in insect cells: Cells,
High Five and Sf21 insect cell lines were maintained as monolayer
cultures in SF90010II-SFM medium (GibcoBRL).
[0231] Recombinant Baculovirus: Recombinant virus containing the
hpa gene was constructed using the Bac to Bac system (GibcoBRL).
The transfer vector pFastBac was digested with SalI and NotI and
ligated with a 1.7 kb fragment of phpa2 digested with XhoI and
NotI. The resulting plasmid was designated pFasthpa2. An identical
plasmid designated pFasthpa4 was prepared as a duplicate and both
independently served for further experimentations. Recombinant
bacmid was generated according to the instructions of the
manufacturer with pFasthpa2, pFasthpa4 and with pFastBac. The
latter served as a negative control. Recombinant bacmid DNAs were
transfected into Sf21 insect cells. Five days after transfection
recombinant viruses were harvested and used to infect High Five
insect cells, 3.times.10.sup.6 cells in T-25 flasks. Cells were
harvested 2-3 days after infection. 4.times.10.sup.6 cells were
centrifuged and resuspended in a reaction buffer containing 20 mM
phosphate citrate buffer, 50 mM NaCl. Cells underwent three cycles
of freeze and thaw and lysates were stored at -80.degree. C.
Conditioned medium was stored at 4.degree. C.
[0232] Partial purification of recombinant heparanase: Partial
purification of recombinant heparanase was performed by
heparin-Sepharose column chromatography followed by Superdex 75
column gel filtration. Culture medium (150 ml) of Sf21 cells
infected with pFhpa4 virus was subjected to heparin-Sepharose
chromatography. Elution of 1 ml fractions was performed with 0.35-2
M NaCl gradient in presence of 0.1% CHAPS and 1 mM DTT in 10 mM
sodium acetate buffer, pH 5.0. A 25 .mu.l sample of each fraction
was tested for heparanase activity. Heparanase activity was eluted
at the range of 0.65-1.1 M NaCl (fractions 18-26, FIG. 10a). 5
.mu.l of each fraction was subjected to 15% SDS-polyacrylamide gel
electrophoresis followed by silver nitrate staining. Active
fractions eluted from heparin-Sepharose (FIG. 10a) were pooled and
concentrated (.times.6) on YM3 cut-off membrane. 0.5 ml of the
concentrated material was applied onto 30 ml Superdex 75 FPLC
column equilibrated with 10 mM sodium acetate buffer, pH 5.0,
containing 0.8 M NaCl, 1 mM DTT and 0.1% CHAPS. Fractions (0.56 ml)
were collected at a flow rate of 0.75 ml/min. Aliquots of each
fraction were tested for heparanase activity and were subjected to
SDS-polyacrylamide gel electrophoresis followed by silver nitrate
staining (FIG. 11b).
[0233] PCR amplification of genomic DNA: 94.degree. C. 3 minutes,
followed by 32 cycles of 94.degree. C. 45 seconds, 64.degree. C. 1
minute, 68.degree. C. 5 minutes, and one cycle at 72.degree. C., 7
minutes. Primers used for amplification of genomic DNA
included:
3 GHpu-L3 5'-AGGCACCCTAGAGATGTTCCAG-3', SEQ ID NO: 30 GHpl-L6
5'-GAAGATTTCTGTTTCCATGACGTG-3',. SEQ ID NO: 31
[0234] Screening of genomic libraries: A human genomic library in
Lambda phage EMBLE3 SP6/T7 (Clontech, Paulo Alto, Calif.) was
screened. 5.times.10.sup.5 plaques were plated at 5.times.10.sup.4
pfu/plate on NZCYM agar/top agarose plates. Phages were absorbed on
nylon membranes in duplicates (Qiagen). Hybridization was performed
at 65.degree. C. in 5.times.SSC, 5.times. Denhart's, 10% dextran
sulfate, 100 .mu.g/ml Salmon sperm, .sup.32p labeled probe (106
cpm/ml). A 1.6 kb fragment, containing the entire hpa cDNA was
labeled by random priming (Boehringer Mannheim). Following
hybridization membranes were washed once with 2.times.SSC, 0.1% SDS
at 65.degree. C. for 20 minutes, and twice with 0.2.times.SSC, 0.1%
SDS at 65.degree. C. for 15 minutes. Hybridizing plaques were
picked, and plated at 100 pfu/plate. Hybridization was performed as
above and single isolated positive plaques were picked.
[0235] Phage DNA was extracted using a Lambda DNA extraction kit
(Qiagen). DNA was digested with XhoI and EcoRI, separated on 0.7%
agarose gel and transferred to nylon membrane Hybond N+ (Amersham).
Hybridization and washes were performed as above.
[0236] cDNA Sequence analysis: Sequence determinations were
performed with vector specific and gene specific primers, using an
automated DNA sequencer (Applied Biosystems, model 373A). Each
nucleotide was read from at least two independent primers.
[0237] Genomic sequence analysis: Large-scale sequencing was
performed by Commonwealth Biotechnology Incorporation.
[0238] Isolation of mouse hpa: Mouse hpa cDNA was amplified from
either Marathon ready cDNA library of mouse embryo or from mRNA
isolated from mouse melanoma cell line BL6, using the Marathon RACE
kit from Clontech. Both procedures were performed according to the
manufacturer's recommendation.
[0239] Primers used for PCR amplification of mouse hpa:
4 Mhpl773 5'-CCACACTGAATGTAATACTGAAGTG-3', SEQ ID NO: 32 MHpl736
5'-CGAAGCTCTGGAACTCGGCAAG-3', SEQ ID NO: 33 MHp183
5'-GCCAGCTGCAAAGGTGTTGGAC-3', SEQ ID NO: 34 Mhpl152
5'-AACACCTGCCTCATCACGACTTC-3', SEQ ID NO: 35 Mhpl114
5'-GCCAGGCTGGCGTCGATGGTGA-3', SEQ ID NO: 36 MHpl103
5'-GTCGATGGTGATGGACAGGAAC-3', SEQ ID NO: 37 Apl
5'-GTAATACGACTCACTATAGGGC-3',- SEQ ID NO: 38
[0240] (Genome walker)
[0241] Ap2 5'-ACTATAGGGCACGCGTGGT-3', SEQ ID NO:39-(Genome
walker)
[0242] Ap1 5'-CCATCCTAATACGACTCACTATAGGGC-3', SEQ ID
NO:40-(Marathon RACE)
[0243] Ap2 5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:41-(Marathon
RACE)
[0244] Southern analysis of genomic DNA: Genomic DNA was extracted
from animal or from human blood using Blood and cell culture DNA
maxi kit (Qiagene). DNA was digested with EcoRI, separated by gel
electrophoresis and transferred to a nylon membrane Hybond N+
(Amersham). Hybridization was performed at 68.degree. C. in
6.times.SSC, 1% SDS, 5.times. Denharts, 10% dextran sulfate, 100
.mu.g/ml salmon sperm DNA, and .sup.32p labeled probe. A 1.6 kb
fragment, containing the entire hpa cDNA was used as a probe.
Following hybridization, the membrane was washed with 3.times.SSC,
0.1% SDS, at 68.degree. C. and exposed to X-ray film for 3 days.
Membranes were then washed with 1.times.SSC, 0.1% SDS, at
68.degree. C. and were reexposed for 5 days.
[0245] Construction of hpa promoter-GFP expression vector. Lambda
DNA of phage L3, was digested with SacI and BglII, resulting in a
1712 bp fragment which contained the hpa promoter (877-2688 of SEQ
ID NO:42). The pEGFP-1 plasmid (Clontech) was digested with BglII
and SacI and ligated with the 1712 bp fragment of the hpa promoter
sequence. The resulting plasmid was designated phpEGL. A second hpa
promoter-GFP plasmid was constructed containing a shorter fragment
of the hpa promoter region: phpEGL was digested with HindIII, and
the resulting 1095 bp fragment (nucleotides 1593-2688 of SEQ ID
NO:42) was ligated with HindIII digested pEGFP-1. The resulting
plasmid was designated phpEGS.
[0246] Computer analysis of sequences: Homology searches were
performed using several computer servers, and various databases.
Blast 2.0 service, at the NCBI server was used to screen the
protein database swplus and DNA databases such as GenBank, EMBL,
and the EST databases. Blast 2.0 search was performed using the
basic search option of the NCBI server. Sequence analysis and
alignments were done using the DNA sequence analysis software
package developed by the Genetic Computer Group (GCG) at the
university of Wisconsin. Alignments of two sequences were performed
using Bestfit (gap creation penalty-12, gap extension penalty-4).
Protein homology search was performed with the Smith-Waterman
algorithm, using the Bioaccelerator platform developed by
Compugene. The protein database swplus was searched using the
following parameters: gapop: 10.0, gapext: 0.5, matrix: blosum62.
Blocks homology was performed using the Blocks WWW server developed
at Fred Hutchinson Cancer Research Center in Seattle, Wash., USA.
Secondary structure prediction was performed using the PHD
server--Profile network Prediction Heidelberg. Fold recognition
(threading) was performed using the UCLA-DOE structure prediction
server. The method used for prediction was gonnet+predss. Alignment
of three sequences was performed using the pileup application (gap
creation penalty-5, gap extension penalty-1). Promoter analysis was
performed using TSSW and TSSG programs (BCM Search Launcher Human
Genome Center, Baylor College of Medicine, Houston Tex.).
Example 1
Cloning of Human hpa cDNA
[0247] Purified fraction of heparanase isolated from human hepatoma
cells (SK-hep-1) was subjected to tryptic digestion and
microsequencing. EST (Expressed Sequence Tag) databases were
screened for homology to the back translated DNA sequences
corresponding to the obtained peptides. Two EST sequences
(accession Nos. N41349 and N45367) contained a DNA sequence
encoding the peptide YGPDVGQPR (SEQ ID NO:8). These two sequences
were derived from clones 257548 and 260138 (I.M.A.G.E Consortium)
prepared from 8 to 9 weeks placenta cDNA library (Soares). Both
clones which were found to be identical contained an insert of 1020
bp which included an open reading frame (ORF) of 973 bp followed by
a 3' untranslated region of 27 bp and a Poly A tail. No translation
start site (AUG) was identified at the 5' end of these clones.
[0248] Cloning of the missing 5' end was performed by PCR
amplification of DNA from a placenta Marathon RACE cDNA composite.
A 900 bp fragment (designated hp3), partially overlapping with the
identified 3' encoding EST clones was obtained.
[0249] The joined cDNA fragment, 1721 bp long (SEQ ID NO:9),
contained an open reading frame which encodes, as shown in FIG. 1
and SEQ ID NO:11, a polypeptide of 543 amino acids (SEQ ID NO:10)
with a calculated molecular weight of 61,192 daltons. The 3' end of
the partial cDNA inserts contained in clones 257548 and 260138
started at nucleotide G.sup.721 of SEQ ID NO:9 and FIG. 1.
[0250] As further shown in FIG. 1, there was a single sequence
discrepancy between the EST clones and the PCR amplified sequence,
which led to an amino acid substitution from Tyr.sup.246 in the EST
to Phe.sup.246 in the amplified cDNA. The nucleotide sequence of
the PCR amplified cDNA fragment was verified from two independent
amplification products. The new gene was designated hpa.
[0251] As stated above, the 3' end of the partial cDNA inserts
contained in EST clones 257548 and 260138 started at nucleotide 721
of hpa (SEQ ID NO:9). The ability of the hpa cDNA to form stable
secondary structures, such as stem and loop structures involving
nucleotide stretches in the vicinity of position 721 was
investigated using computer modeling. It was found that stable stem
and loop structures are likely to be formed involving nucleotides
698-724 (SEQ ID NO:9). In addition, a high GC content, up to 70%,
characterizes the 5' end region of the hpa gene, as compared to
about only 40% in the 3' region. These findings may explain the
immature termination and therefore lack of 5' ends in the EST
clones.
[0252] To examine the ability of the hpa gene product to catalyze
degradation of heparan sulfate in an in vitro assay the entire open
reading frame was expressed in insect cells, using the Baculovirus
expression system. Extracts of cells, infected with virus
containing the hpa gene, demonstrated a high level of heparan
sulfate degradation activity, while cells infected with a similar
construct containing no hpa gene had no such activity, nor did
non-infected cells. These results are further demonstrated in the
following Examples.
Example 2
Degradation of Soluble ECM-Derived HSPG
[0253] Monolayer cultures of High Five cells were infected (72 h,
28.degree. C.) with recombinant Bacoluvirus containing the pFasthpa
plasmid or with control virus containing an insert free plasmid.
The cells were harvested and lysed in heparanase reaction buffer by
three cycles of freezing and thawing. The cell lysates were then
incubated (18 h, 37.degree. C.) with sulfate labeled, ECM-derived
HSPG (peak I), followed by gel filtration analysis (Sepharose 6B)
of the reaction mixture.
[0254] As shown in FIG. 2, the substrate alone included almost
entirely high molecular weight (Mr) material eluted next to V.sub.O
(peak I, fractions 5-20, Kav<0.35). A similar elution pattern
was obtained when the HSPG substrate was incubated with lysates of
cells that were infected with control virus. In contrast,
incubation of the HSPG substrate with lysates of cells infected
with the hpa containing virus resulted in a complete conversion of
the high Mr substrate into low Mr labeled degradation fragments
(peak II, fractions 22-35, 0.5<Kav<0.75). Fragments eluted in
peak II were shown to be degradation products of heparan sulfate,
as they were (i) 5- to 6-fold smaller than intact heparan sulfate
side chains (Kav approx. 0.33) released from ECM by treatment with
either alkaline borohydride or papain; and (ii) resistant to
further digestion with papain or chondroitinase ABC, and
susceptible to deamination by nitrous acid (6, 11). Similar results
(not shown) were obtained with Sf21 cells. Again, heparanase
activity was detected in cells infected with the hpa containing
virus (pFhpa), but not with control virus (pF). This result was
obtained with two independently generated recombinant viruses.
Lysates of control not infected High Five cells failed to degrade
the HSPG substrate.
[0255] In subsequent experiments, the labeled HSPG substrate was
incubated with medium conditioned by infected High Five or Sf21
cells.
[0256] As shown in FIGS. 3a-b, heparanase activity, reflected by
the conversion of the high Mr peak I substrate into the low Mr peak
II which represents HS degradation fragments, was found in the
culture medium of cells infected with the pFhpa2 or pFhpa4 viruses,
but not with the control pF1 or pF2 viruses. No heparanase activity
was detected in the culture medium of control non-infected High
Five or Sf21 cells.
[0257] The medium of cells infected with the pFhpa4 virus was
passed through a 50 kDa cut off membrane to obtain a crude
estimation of the molecular weight of the recombinant heparanase
enzyme. As demonstrated in FIG. 4, all the enzymatic activity was
retained in the upper compartment and there was no activity in the
flow through (<50 kDa) material. This result is consistent with
the expected molecular weight of the hpa gene product.
[0258] In order to further characterize the hpa product the
inhibitory effect of heparin, a potent inhibitor of heparanase
mediated HS degradation (40) was examined.
[0259] As demonstrated in FIGS. 5a-b, conversion of the peak I
substrate into peak II HS degradation fragments was completely
abolished in the presence of heparin.
[0260] Altogether, these results indicate that the heparanase
enzyme is expressed in an active form by insect cells infected with
Baculovirus containing the newly identified human hpa gene.
Example 3
Degradation of HSPG in Intact ECM
[0261] Next, the ability of intact infected insect cells to degrade
HS in intact, naturally produced ECM was investigated. For this
purpose, High Five or Sf21 cells were seeded on metabolically
sulfate labeled ECM followed by infection (48 h, 28.degree. C.)
with either the pFhpa4 or control pF2 viruses. The pH of the medium
was then adjusted to pH 6.2-6.4 and the cells further incubated
with the labeled ECM for another 48 h at 28.degree. C. or 24 h at
37.degree. C. Sulfate labeled material released into the incubation
medium was analyzed by gel filtration on Sepharose 6B.
[0262] As shown in FIGS. 6a-b and 7a-b, incubation of the ECM with
cells infected with the control pF2 virus resulted in a constant
release of labeled material that consisted almost entirely
(>90%) of high Mr fragments (peak I) eluted with or next to
V.sub.O. It was previously shown that a proteolytic activity
residing in the ECM itself and/or expressed by cells is responsible
for release of the high Mr material (6). This nearly intact HSPG
provides a soluble substrate for subsequent degradation by
heparanase, as also indicated by the relatively large amount of
peak I material accumulating when the heparanase enzyme is
inhibited by heparin (6, 7, 12, FIG. 9). On the other hand,
incubation of the labeled ECM with cells infected with the pFhpa4
virus resulted in release of 60-70% of the ECM-associated
radioactivity in the form of low Mr sulfate-labeled fragments (peak
II, 0.5<Kav<0.75), regardless of whether the infected cells
were incubated with the ECM at 28.degree. C. or 37.degree. C.
Control intact non-infected Sf21 or High Five cells failed to
degrade the ECM HS side chains.
[0263] In subsequent experiments, as demonstrated in FIGS. 8a-b,
High Five and Sf21 cells were infected (96 h, 28.degree. C.) with
pFhpa4 or control pF1 viruses and the culture medium incubated with
sulfate-labeled ECM. Low Mr HS degradation fragments were released
from the ECM only upon incubation with medium conditioned by pFhpa4
infected cells. As shown in FIG. 9, production of these fragments
was abolished in the presence of heparin. No heparanase activity
was detected in the culture medium of control, non-infected cells.
These results indicate that the heparanase enzyme expressed by
cells infected with the pFhpa4 virus is capable of degrading HS
when complexed to other macromolecular constituents (i.e.
fibronectin, laminin, collagen) of a naturally produced intact ECM,
in a manner similar to that reported for highly metastatic tumor
cells or activated cells of the immune system (6, 7).
Example 4
Purification of Recombinant Human Heparanase
[0264] The recombinant heparanase was partially purified from
medium of pFhpa4 infected Sf21 cells by Heparin-Sepharose
chromatography (FIG. 10a) followed by gel filtration of the pooled
active fractions over an FPLC Superdex 75 column (FIG. 11a). A
.about.63 kDa protein was observed, whose quantity, as was detected
by silver stained SDS-polyacrylamide gel electrophoresis,
correlated with heparanase activity in the relevant column
fractions (FIGS. 10b and 11b, respectively). This protein was not
detected in the culture medium of cells infected with the control
pF1 virus and was subjected to a similar fractionation on
heparin-Sepharose (not shown).
Example 5
Expression of the Human hpa cDNA in Various Cell Types, Organs and
Tissues
[0265] Referring now to FIGS. 12a-e, RT-PCR was applied to evaluate
the expression of the hpa gene by various cell types and tissues.
For this purpose, total RNA was reverse transcribed and amplified.
The expected 585 bp long cDNA was clearly demonstrated in human
kidney, placenta (8 and 11 weeks) and mole tissues, as well as in
freshly isolated and short termed (1.5-48 h) cultured human
placental cytotrophoblastic cells (FIG. 12a), all known to express
a high heparanase activity (41). The hpa transcript was also
expressed by normal human neutrophils (FIG. 12b). In contrast,
there was no detectable expression of the hpa mRNA in embryonic
human muscle tissue, thymus, heart and adrenal (FIG. 12b). The hpa
gene was expressed by several, but not all, human bladder carcinoma
cell lines (FIG. 12c), SK hepatoma (SK-hep-1), ovarian carcinoma
(OV 1063), breast carcinoma (435, 231), melanoma and megakaryocytic
(DAMI, CHRF) human cell lines (FIGS. 12d-e).
[0266] The above described expression pattern of the hpa transcript
was determined to be in a very good correlation with heparanase
activity levels determined in various tissues and cell types (not
shown).
Example 6
Isolation of an Extended 5' End of hpa cDNA from Human SK-hep1 Cell
Line
[0267] The 5' end of hpa cDNA was isolated from human SK-hep1 cell
line by PCR amplification using the Marathon RACE (rapid
amplification of cDNA ends) kit (Clontech). Total RNA was prepared
from SK-hep1 cells using the TR1-Reagent (Molecular research center
Inc.) according to the manufacturer instructions. Poly A+ RNA was
isolated using the mRNA separator kit (Clonetech).
[0268] The Marahton RACE SK-hep1 cDNA composite was constructed
according to the manufacturer recommendations. First round of
amplification was performed using an adaptor specific primer AP1:
5'-CCATCCTAATACG ACTCACTATAGGGC-3', SEQ ID NO:1, and a hpa specific
antisense primer hpl-629: 5'-CCCCAGGAGCAGCAGCATCAG-3', SEQ ID
NO:17, corresponding to nucleotides 119-99 of SEQ ID NO:9. The
resulting PCR product was subjected to a second round of
amplification using an adaptor specific nested primer AP2:
5'-ACTCACTATAGGGCTCGAGCGGC-3', SEQ ID NO:3, and a hpa specific
antisense nested primer hpl-666 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ ID
NO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The PCR
program was as follows: a hot start of 94.degree. C. for 1 minute,
followed by 30 cycles of 90.degree. C.-30 seconds, 68.degree. C.-4
minutes. The resulting 300 bp DNA fragment was extracted from an
agarose gel and cloned into the vector pGEM-T Easy (Promega). The
resulting recombinant plasmid was designated pHPSK1.
[0269] The nucleotide sequence of the pHPSK1 insert was determined
and it was found to contain 62 nucleotides of the 5' end of the
placenta hpa cDNA (SEQ ID NO:9) and additional 178 nucleotides
upstream, the first 178 nucleotides of SEQ ID NOs:13 and 15.
[0270] A single nucleotide discrepancy was identified between the
SK-hep1 cDNA and the placenta cDNA. The "T" derivative at position
9 of the placenta cDNA (SEQ ID NO:9), is replaced by a "C"
derivative at the corresponding position 187 of the SK-hep1 cDNA
(SEQ ID NO:13).
[0271] The discrepancy is likely to be due to a mutation at the 5'
end of the placenta cDNA clone as confirmed by sequence analysis of
sevsral additional cDNA clones isolated from placenta, which like
the SK-hep1 cDNA contained C at position 9 of SEQ ID NO:9.
[0272] The 5' extended sequence of the SK-hep1 hpa cDNA was
assembled with the sequence of the hpa cDNA isolated from human
placenta (SEQ ID NO:9). The assembled sequence contained an open
reading frame which encodes, as shown in SEQ ID NOs:14 and 15, a
polypeptide of 592 amino acids with a calculated molecular weight
of 66,407 daltons. The open reading frame is flanked by 93 bp 5'
untranslated region (UTR).
Example 7
Isolation of the Upstream Genomic Region of the hpa Gene
[0273] The upstream region of the hpa gene was isolated using the
Genome Walker kit (Clontech) according to the manufacturer
recommendations. The kit includes five human genomic DNA samples
each digested with a different restriction endonuclease creating
blunt ends: EcoRV, ScaI, DraI, PvuII and SspI.
[0274] The blunt ended DNA fragments are ligated to partially
single stranded adaptors. The Genomic DNA samples were subjected to
PCR amplification using the adaptor specific primer and a gene
specific primer. Amplification was performed with Expand High
Fidelity (Boehringer Mannheim).
[0275] A first round of amplification was performed using the ap1
primer: 5'-G TAATACGACTCACTATAGGGC-3', SEQ ID NO:19, and the hpa
specific antisense primer hpl-666: 5'-AGGCTTCGAGCGCAGCAGCAT-3', SEQ
ID NO:18, corresponding to nucleotides 83-63 of SEQ ID NO:9. The
PCR program was as follows: a hot start of 94.degree. C.-3 minutes,
followed by 36 cycles of 94.degree. C.-40 seconds, 67.degree. C.-4
minutes.
[0276] The PCR products of the first amplification were diluted
1:50. One .mu.l of the diluted sample was used as a template for a
second amplification using a nested adaptor specific primer ap2:
5'-ACTATAGGGCACGCGTGGT-3', SEQ ID NO:20, and a hpa specific
antisense primer hpl-690, 5'-CTTGGGCTCACC TGGCTGCTC-3', SEQ ID
NO:21, corresponding to nucleotides 62-42 of SEQ ID NO:9. The
resulting amplification products were analyzed using agarose gel
electrophoresis. Five different PCR products were obtained from the
five amplification reactions. A DNA fragment of approximately 750
bp which was obtained from the SspI digested DNA sample was gel
extracted. The purified fragment was ligated into the plasmid
vector pGEM-T Easy (Promega). The resulting recombinant plasmid was
designated pGHP6905 and the nucleotide sequence of the hpa insert
was determined.
[0277] A partial sequence of 594 nucleotides is shown in SEQ ID
NO:16. The last nucleotide in SEQ ID NO:13 corresponds to
nucleotide 93 in SEQ ID:13. The DNA sequence in SEQ ID NO:16
contains the 5' region of the hpa cDNA and 501 nucleotides of the
genomic upstream region which are predicted to contain the promoter
region of the hpa gene.
Example 8
Expression of the 592 Amino Acids HPA Polypeptide in a Human 293
Cell Line
[0278] The 592 amino acids open reading frame (SEQ ID NOs:13 and
15) was constructed by ligation of the 110 bp corresponding to the
5' end of the SK-hep1 hpa cDNA with the placenta cDNA. More
specifically the Marathon RACE-PCR amplification product of the
placenta hpa DNA was digested with SacI and an approximately 1 kb
fragment was ligated into a SacI-digested pGHP6905 plasmid. The
resulting plasmid was digested with EarI and AatII. The EarI sticky
ends were blunted and an approximately 280 bp EarI/blunt-AatII
fragment was isolated. This fragment was ligated with pFasthpa
digested with EcoRI which was blunt ended using Klenow fragment and
further digested with AatII. The resulting plasmid contained a 1827
bp insert which includes an open reading frame of 1776 bp, 31 bp of
3' UTR and 21 bp of 5' UTR. This plasmid was designated
pFastLhpa.
[0279] A mammalian expression vector was constructed to drive the
expression of the 592 amino acids heparanase polypeptide in human
cells. The hpa cDNA was excised prom pFastLhpa with BssHII and
NotI. The resulting 1850 bp BssHII-NotI fragment was ligated to a
mammalian expression vector pSI (Promega) digested with MluI and
NotI. The resulting recombinant plasmid, pSIhpaMet2 was transfected
into a human 293 embryonic kidney cell line.
[0280] Transient expression of the 592 amino-acids heparanase was
examined by western blot analysis and the enzymatic activity was
tested using the gel shift assay. Both these procedures are
described in length in U.S. patent application Ser. No. 09/071,739,
filed May 1, 1998, which is incorporated by reference as if fully
set forth herein. Cells were harvested 3 days following
transfection. Harvested cells were re-suspended in lysis buffer
containing 150 mM NaCl, 50 mM Tris pH 7.5, 1% Triton X-100, 1 mM
PMSF and protease inhibitor cocktail (Boehringer Mannheim). 40
.mu.g protein extract samples were used for separation on a
SDS-PAGE. Proteins were transferred onto a PVDF Hybond-P membrane
(Amersham). The membrane was incubated with an affinity purified
polyclonal anti heparanase antibody, as described in U.S. patent
application Ser. No. 09/071,739. A major band of approximately 50
kDa was observed in the transfected cells as well as a minor band
of approximately 65 kDa. A similar pattern was observed in extracts
of cells transfected with the pShpa as demonstrated in U.S. patent
application Ser. No. 09/071,739. These two bands probably represent
two forms of the recombinant heparanase protein produced by the
transfected cells. The 65 kDa protein probably represents a
heparanase precursor, while the 50 kDa protein is suggested herein
to be the processed or mature form.
[0281] The catalytic activity of the recombinant protein expressed
in the pShpaMet2 transfected cells was tested by gel shift assay.
Cell extracts of transfected and of mock transfected cells were
incubated overnight with heparin (6 .mu.g in each reaction) at
37.degree. C., in the presence of 20 mM phosphate citrate buffer pH
5.4, 1 mM CaCl.sub.2, 1 mM DTT and 50 mM NaCl. Reaction mixtures
were then separated on a 10% polyacrylamide gel. The catalytic
activity of the recombinant heparanase was clearly demonstrated by
a faster migration of the heparin molecules incubated with the
transfected cell extract as compared to the control. Faster
migration indicates the disappearance of high molecular weight
heparin molecules and the generation of low molecular weight
degradation products.
Example 9
Chromosomal Localization of the hpa Gene
[0282] Chromosomal mapping of the hpa gene was performed utilizing
a panel of monochromosomal human/CHO and human/mouse somatic cell
hybrids, obtained from the UK HGMP Resource Center (Cambridge,
England).
[0283] 40 ng of each of the somatic cell hybrid DNA samples were
subjected to PCR amplification using the hpa primers: hpu565
5'-AGCTCTGTAGATGTGC TATACAC-3', SEQ ID NO:22, corresponding to
nucleotides 564-586 of SEQ ID NO:9 and an antisense primer hpl171
5'-GCATCTTAGCCGTCTTTCTTCG-3', SEQ ID NO:23, corresponding to
nucleotides 897-876 of SEQ ID NO:9.
[0284] The PCR program was as follows: a hot start of 94.degree.
C.-3 minutes, followed by 7 cycles of 94.degree. C.-45 seconds,
66.degree. C.-1 minute, 68.degree. C.-5 minutes, followed by 30
cycles of 94.degree. C.-45 seconds, 62.degree. C.-1 minute,
68.degree. C.-5 minutes, and a 10 minutes final extension at
72.degree. C.
[0285] The reactions were performed with Expand long PCR
(Boehringer Mannheim). The resulting amplification products were
analyzed using agarose gel electrophoresis. As demonstrated in FIG.
14, a single band of approximately 2.8 Kb was obtained from
chromosome 4, as well as from the control human genomic DNA. A 2.8
kb amplification product is expected based on amplification of the
genomic hpa clone (data not shown). No amplification products were
obtained neither in the control DNA samples of hamster and mouse
nor in somatic hybrids of other human chromosome.
Example 10
Human Genomic Clone Encoding Heparanase
[0286] Five plaques were isolated following screening of a human
genomic library and were designated L3-1, L5-1, L8-1, L10-1 and
L6-1. The phage DNAs were analyzed by Southern hybridization and by
PCR with hpa specific and vector specific primers. Southern
analysis was performed with three fragments of hpa cDNA: a
PvuII-BamHI fragment (nucleotides 32-450., SEQ ID NO:9), a
BamHI-NdeI fragment (nucleotides 451-1102, SEQ ID NO:9) and an
NdeI-XhoI fragment (nucleotides 1103-1721, SEQ ID NO:9).
[0287] Following Southern analysis, phages L3, L6, L8 were selected
for further analysis. A scheme of the genomic region and the
relative position of the three phage clones is depicted in FIG. 15.
A 2 kb DNA fragment containing the gap between phages L6 and L3 was
PCR amplified from human genomic DNA with two gene specific primers
GHpuL3 and GHplL6. The PCR product was cloned into the plasmid
vector pGEM-T-easy (Promega).
[0288] Large scale DNA sequencing of the three Lambda clones and
the amplified fragment was performed with Lambda purified DNA by
primer walking. A nucleotide sequence of 44,898 bp was analyzed
(FIG. 16, SEQ ID NO:42). Comparison of the genomic sequence with
that of hpa cDNA revealed 12 exons separated by 11 introns (FIGS.
15 an 16). The genomic organization of the hpa gene is depicted in
FIG. 15 (top). The sequence include the coding region from the
first ATG to the stop codon which spans 39,113 nucleotides, 2742
nucleotides upstream of the first ATG and 3043 nucleotides
downstream of the stop codon. Splice site consensus sequences were
identified at exon/intron junctions.
Example 11
Alternative Splicing
[0289] Several minor RT-PCR products were obtained from various
cell types, following amplification with hpa specific primers. Each
one found to contain a deletion of one or two exons. Some of these
PCR products contain ORFs, which encode potential shorter
proteins.
[0290] Table 1 below summarizes the alternative spliced products
isolated from various cell lines.
[0291] Fragments of similar sizes were obtained following
amplification with two cell lines, placenta and platelets.
5 Cell type Nucleotides deleted Exons deleted ORF Platelets
1047-1267 8, 9 + Platelets 1154-1267 9 - Platelets 289-435, 562-735
2, 4 - Sk-hep1, platelets, Zr75 562-735 4 + Sk-hep1 (hepatoma)
561-904 4, 5 - Zr75 (breast carcinoma) 96-203 1 (partial) +
Example 12
Mouse and Rat hpa
[0292] EST databases were screened for sequences homologous to the
hpa gene. Three mouse EST's were identified (accession No.
Aa177901, from mouse spleen, Aa067997 from mouse skin, Aa47943 from
mouse embryo), assembled into a 824 bp cDNA fragment which contains
a partial open reading frame (lacking a 5' end) of 629 bp and a 3'
untranslated region of 195 bp (SEQ ID NO:12). As shown in FIG. 13,
the coding region is 80% similar to the 3' end of the hpa cDNA
sequence. These EST's are probably cDNA fragments of the mouse hpa
homolog that encodes for the mouse heparanase. Searching for
consensus protein domains revealed an amino terminal homology
between the heparanase and several precursor proteins such as
Procollagen Alpha 1 precursor, Tyrosine-protein kinase-RYK,
Fibulin-1, Insulin-like growth factor binding protein and several
others. The amino terminus is highly hydrophobic and contains a
potential trans-membrane domain. The homology to known signal
peptide sequences suggests that it could function as a signal
peptide for protein localization.
[0293] The amino acid sequence of human heparanase was used to
search for homologous sequences in the DNA and protein databases.
Several human EST's were identified, as well as mouse sequences
highly homologous to human heparanase. The following mouse EST's
were identified AA177901, AA674378, AA67997, AA047943, AA690179,
AI122034, all sharing an identical sequence and correspond to amino
acids 336-543 of the human heparanase sequence. The entire mouse
heparanase cDNA was cloned, based on the nucleotide sequence of the
mouse EST's.
[0294] PCR primers were designed and a Marathon RACE was performed
using a Marathon cDNA library from 15 days mouse embryo (Clontech)
and from BL6 mouse melanoma cell line. The mouse hpa homologous
cDNA was isolated following several amplification steps. A 1.1 kb
fragment was amplified from mouse embryo Marathon cDNA library. The
first cycle of amplification was performed with primers mhpl773 and
Ap1 and the second cycle with primers mhpl736 and AP2. A 1.1 kb
fragment was then amplified from BL6 Marathon cDNA library. The
first cycle of amplification was performed with the primers mhpl152
and Ap1, and the second with mhpl83 and AP2. The combined sequence
was homologous to nucleotides 157-1702 of the human hpa cDNA, which
encode amino acids 33-543. The 5' end of the mouse hpa gene was
isolated from a mouse genomic DNA library using the Genome Walker
kit (Clontech). An 0.9 kb fragment was amplified from a DraI
digested Genome walker DNA library. The first cycle of
amplification was performed with primers mhpl114 and Ap1 and the
second with primers mhpl103 and AP2. The assembled sequence (SEQ ID
NOs:43, 45) is 2396 nucleotides long. It contains an open reading
frame of 1605 nucleotides, which encode a polypeptide of 535 amino
acids (SEQ ID NOs:44, 45), 196 nucleotides of 3' untranslated
region (UTR), and an upstream sequence which includes the promoter
region and the 5'-UTR of the mouse hpa cDNA. According to two
promoter predicting programs TSSW and TSSG, the transcription start
site is localized to nucleotide 431 of SEQ ID NOs:43, 45, 163
nucleotides upstream of the first ATG codon. The 431 upstream
genomic sequence contains the promoter region. A TATA box is
predicted at position 394 of SEQ ID NOs:43, 45. The mouse and the
human hpa genes share an average homology of 78% between the
nucleotide sequences and 81% similarity between the deduced amino
acid sequences.
[0295] Search for hpa homologous sequences, using the Blast 2.0
server revealed two EST's from rat: AI060284 (385 nucleotides, SEQ
ID NO:46) which is homologous to the amino terminus (68% similarity
to amino acids 12-136) of human heparanase and AI237828 (541
nucleotides, SEQ ID NO:47) which is homologous to the carboxyl
terminus (81% similarity to amino acids 500-543) of human
heparanase, and contains a 3'-UTR. A comparison between the human
heparanase and the mouse and rat homologous sequences is
demonstrated in FIG. 17.
Example 13
Prediction of Heparanase Active Site
[0296] Homology search of heparanase amino acid sequence against
the DNA and the protein databases revealed no significant
homologies. The protein secondary structure as predicted by the PHD
program consists of alternating alpha helices and beta sheets. The
fold recognition server of UCLA predicted alpha/beta barrel
structure, with under-threshold confidence.
[0297] Five of 15 proteins, which were predicted to have most
similar folds, were glycosyl hydrolases from various organisms:
1xyza--xylanase from Clostridium Thermocellum,
lpbga--6-phospho-beta-.delta.-galactosidas- e from Lactococcus
Lactis, lamy--alpha-amylase from Barley, lecea--endocellulase from
Acidothermus Cellulolyticus and lqbc--hexosaminidase alpha chain,
glycosyl hydrolase.
[0298] Protein homology search using the bioaccelerator pulled out
several proteins, including glycosyl hydrolyses such as
beta-fructofuranosidase from Vicia faba (broad bean) and from
potato, lactase phlorizin hydrolase from human, xylanases from
Clostridium thermocellum and from Streptomyces halstedii and
cellulase from Clostridium thermocellum. Blocks 9.3 database pulled
out the active site of glycosyl hydrolases family five, which
includes cellulases from various bacteria and fungi. Similar active
site motif is shared by several lysosomal acid hydrolases (63) and
other glycosyl hydrolases. The common mechanism shared by these
enzymes involves two glutamic acid residues, a proton donor and a
nucleophile.
[0299] Despite the lack of an overall homology between the
heparanase and other glycosyl hydolases, the amino acid couple
Asp-Glu (NE), which is characteristic of the proton donor of
glycosyl hydrolyses of the GH-A clan, was found at positions
224-225 of the human heparanase protein sequence. As in other clan
members, this NE couple is located at the end of a .beta.
sheet.
[0300] Considering the relative location of the proton donor and
the predicted secondary structure, the glutamic acid that functions
as nucleophile is most likely located at position 343, or at
positon 396. Identification of the active site and the amino acids
directly involved in hydrolysis opens the way for expression of the
defined catalytic domain. In addition, it will provide the tools
for rational design of enzyme activity either by modification of
the microenviroment or catalytic site itself.
Example 14
Expression of hpa Antisense in Mammalian Cell Lines
[0301] A mammalian expression vector Hpa2Kepcdna3 was constructed
in order to express hpa antisense in mammalian cells. hpa cDNA (1.7
kb EcoRI fragment) was cloned into the plasmid pCDNA3 in 3'>5'
(antisense) orientation. The construct was used to transfect
MBT2-T50 and T24P cell lines. 2.times.10.sup.5 cells in 35 mm
plates were transfected using the Fugene protocol (Boehringer
Mannheim). 48 hours after transfection cells were trypsinized and
seeded in six well plates. 24 hours later G418 was added to
initiate selection. The number of colonies per 35 mm plate
following 3 weeks:
6 Antisense No insert T24P 15 60 MBT-T50 1 6
[0302] The lower number of colonies obtained after transfection
with hpa antisense, as compared with the control plasmid suggests
that the introduction of hpa antisense interfere with cell growth.
This experiment demonstrates the use of complementary antisense hpa
DNA sequence to control heparanase expression in cells. This
approach may be used to inhibit expression of heparanase in vivo,
in, for example, cancer cells and in other pathological processes
in which heparanase is involved.
Example 15
Zoo Blot
[0303] Hpa cDNA was used as a probe to detect homologous sequences
in human DNA and in DNA of various animals. The autoradiogram of
the Southern analysis is presented in FIG. 18. Several bands were
detected in human DNA, which correlated with the accepted pattern
according to the genomic hpa sequence. Several intense bands were
detected in all mammals, while faint bands were detected in
chicken. This correlates with the phylogenetic relation between
human and the tested animals. The intense bands indicate that hpa
is conserved among mammals as well as in more genetically distant
organisms. The multiple bands patterns suggest that in all animals,
like in human, the hpa locus occupy large genomic region.
Alternatively, the various bands could represent homologous
sequences and suggest the existence of a gene family, which can be
isolated based on their homology to the human hpa reported herein.
This conservation was actually found, between the isolated human
hpa cDNA and the mouse homologue.
Example 16
Characterization of the hpa Promoter
[0304] The DNA sequence upstream of the hpa first ATG was subjected
to computational analysis in order to localize the predicted
transcription start site and to identify potential transcription
factors binding sites. Recognition of human PolII promoter region
and start of transcription were predicted using the TSSW and TSSG
programs. Both programs identified a promoter region upstream of
the coding region. TSSW pointed at nucleotide 2644 and TSSG at 2635
of SEQ ID NO:42. These two predicted transcription start sites are
located 4 and 13 nucleotides upstream of the longest hpa cDNA
isolated by RACE.
[0305] A hpa promoter-GFP reporter vector was constructed in order
to investigate the regulation of hpa transcription. Two constructs
were made, containing 1.8 kb and 1.1 kb of the hpa promoter region.
The reporter vector was transfected into T50-mouse bladder
carcinoma cells. Cells transfected with both constructs exhibited
green fluorescence, which indicated the promoter activity of the
genomic sequence upstream of the hpa-coding region. This reporter
vector, enables the monitoring of hpa promoter activity, at various
conditions and in different cell types and to characterize the
factors involved regulation of hpa expression.
[0306] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
LIST OF REFERENCES
[0307] 1. Wight, T. N., Kinsella, M. G., and Qwarnstromn, E. E.
(1992). The role of proteoglycans in cell adhesion, migration and
proliferation. Curr. Opin. Cell Biol., 4, 793-801.
[0308] 2. Jackson, R. L., Busch, S. J., and Cardin, A. L. (1991).
Glycosaminoglycans: Molecular properties, protein interactions and
role in physiological processes. Physiol. Rev., 71, 481-539.
[0309] 3. Wight, T. N. (1989). Cell biology of arterial
proteoglycans. Arteriosclerosis, 9, 1-20.
[0310] 4. Kjellen, L. and Lindahl, U. (1991). Proteoglycans:
structures and interactions. Annu. Rev. Biochem., 60, 443-475.
[0311] 5. Ruoslahti, E., and Yamaguchi, Y. (1991). Proteoglycans as
modulators of growth factor activities. Cell, 64, 867-869.
[0312] 6. Vlodavsky, I., Eldor, A., Haimovitz-Friedman, A.,
Matzner, Y., Ishai-Michaeli, R., Levi, E., Bashkin, P., Lider, O.,
Naparstek, Y., Cohen, I. R., and Fuks, Z. (1992). Expression of
heparanase by platelets and circulating cells of the immune system:
Possible involvement in diapedesis and extravasation. Invasion
& Metastasis, 12, 112-127.
[0313] 7. Vlodavsky, I., Mohsen, M., Lider, O., Ishai-Michaeli, R.,
Ekre, H.-P., Svahn, C. M., Vigoda, M., and Peretz, T. (1995).
Inhibition of tumor metastasis by heparanase inhibiting species of
heparin. Invasion & Metastasis, 14, 290-302.
[0314] 8. Nakajima, M., Irimura, T., and Nicolson, G. L. (1988).
Heparanase and tumor metastasis. J. Cell. Biochem., 36,
157-167.
[0315] 9. Nicolson, G. L. (1988). Organ specificity of tumor
metastasis: Role of preferential adhesion, invasion and growth of
malignant cells at specific secondary sites. Cancer Met. Rev., 7,
143-188.
[0316] 10. Liotta, L. A., Rao, C. N., and Barsky, S. H. (1983).
Tumor invasion and the extracellular matrix. Lab. Invest., 49,
639-649.
[0317] 11. Vlodavsky, I., Fuks, Z., Bar-Ner, M., Ariav, Y., and
Schirrmacher, V. (1983). Lymphoma cell mediated degradation of
sulfated proteoglycans in the subendothelial extracellular matrix:
Relationship to tumor cell metastasis. Cancer Res., 43,
2704-2711.
[0318] 12. Vlodavsky, 1., Ishai-Michaeli, R., Bar-Ner, M., Fridman,
R., Horowitz, A. T., Fuks, Z. and Biran, S. (1988). Involvement of
heparanase in tumor metastasis and angiogenesis. Is. J. Med., 24,
464-470.
[0319] 13. Vlodavsky, I., Liu, G. M., and Gospodarowicz, D. (1980).
Morphological appearance, growth behavior and migratory activity of
human tumor cells maintained on extracellular matrix vs. plastic.
Cell, 19, 607-616.
[0320] 14. Gospodarowicz, D., Delgado, D., and Vlodavsky, I.
(1980). Permissive effect of the extracellular matrix on cell
proliferation in-vitro. Proc. Natl. Acad. Sci. USA., 77,
4094-4098.
[0321] 15. Bashkin, P., Doctrow, S., Klagsbrun, M., Svahn, C. M.,
Folkman, J., and Vlodavsky, I. (1989). Basic fibroblast growth
factor binds to subendothelial extracellular matrix and is released
by heparitinase and heparin-like molecules. Biochemistry, 28,
1737-1743.
[0322] 16. Parish, C. R., Coombe, D. R., Jakobsen, K. B., and
Underwood, P. A. (1987). Evidence that sulphated polysaccharides
inhibit tumor metastasis by blocking tumor cell-derived heparanase.
Int. J. Cancer, 40, 511-517.
[0323] 16a. Vlodavsky, I., Hua-Quan Miao., Benezra, M., Lider, O.,
Bar-Shavit, R., Schmidt, A., and Peretz, T. (1997). Involvement of
the extracellular matrix, heparan sulfate proteoglycans and heparan
sulfate degrading enzymes in angiogenesis and metastasis. In: Tumor
Angiogenesis. Eds. C. E. Lewis, R. Bicknell & N. Ferrara.
Oxford University Press, Oxford UK, pp. 125-140.
[0324] 17. Burgess, W. H., and Maciag, T. (1989). The
heparin-binding (fibroblast) growth factor family of proteins.
Annu. Rev. Biochem., 58, 575-606.
[0325] 18. Folkman, J., and Klagsbrun, M. (1987). Angiogenic
factors. Science, 235, 442-447.
[0326] 19. Vlodavsky, I., Folkman. J., Sullivan, R., Fridman, R.,
Ishai-Michaelli, R., Sasse, J., and Klagsbrun, M. (1987).
Endothelial cell-derived basic fibroblast growth factor: Synthesis
and deposition into subendothelial extracellular matrix. Proc.
Natl. Acad. Sci. USA, 84, 2292-2296.
[0327] 20. Folkman, J., Klagsbrun, M. Sasse. J., Wadzinski, M.,
Ingber, D., and Vlodavsky, I. (1980). A heparin-binding angiogenic
protein--basic fibroblast growth factor--is stored within basement
membrane. Am. J. Pathol., 130, 393-400.
[0328] 21. Cardon-Cardo, C., Vlodavsky, I., Haimovitz-Friedman, A.,
Hicklin, D., and Fuks, Z. (1990). Expression of basic fibroblast
growth factor in normal human tissues. Lab. Invest., 63,
832-840.
[0329] 22. Ishai-Michaeli, R., Svahn, C.-M., Chajek-Shaul, T.,
Korner, G., Ekre, H.-P., and Vlodavsky, I. (1992). Importance of
size and sulfation of heparin in release of basic fibroblast factor
from the vascular endothelium and extracellular matrix.
Biochemistry, 31, 2080-2088.
[0330] 23. Ishai-Michaeli, R., Eldor, A., and Vlodavsky, I. (1990).
Heparanase activity expressed by platelets, neutrophils and
lymphoma cells releases active fibroblast growth factor from
extracellular matrix. Cell Reg., 1, 833-842.
[0331] 24. Vlodavsky, I., Bar-Shavit, R., Ishai-Michaeli, R.,
Bashkin. P., and Fuks, Z. (1991). Extracellular sequestration and
release of fibroblast growth factor: a regulatory mechanism? Trends
Biochem. Sci., 16, 268-271.
[0332] 25. Vlodavsky, I., Bar-Shavit, R., Korner, G., and Fuks, Z.
(1993). Extracellular matrix-bound growth factors, enzymes and
plasma proteins. In Basement membranes: Cellular and molecular
aspects (eds. D. H. Rohrbach and R. Timpl), pp327-343. Academic
press Inc., Orlando, Fla.
[0333] 26. Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and
Ornitz, D. M. (1991). Cell surface, heparin-like molecules are
required for binding of basic fibroblast growth factor to its high
affinity receptor. Cell, 64, 841-848.
[0334] 27. Spivak-Kroizman, T., Lemmon, M. A., Dikic, I., Ladbury,
J. E., Pinchasi, D., Huang, J., Jaye, M., Crumley, G.,
Schlessinger, J., and Lax, I. (1994). Heparin-induced
oligomerization of FGF molecules is responsible for FGF receptor
dimerization, activation, and cell proliferation. Cell, 79,
1015-1024.
[0335] 28. Ornitz, D. M., Herr, A. B., Nilsson, M., West, a., J.,
Svahn,
[0336] C.-M., and Waksman, G. (1995). FGF binding and FGF receptor
activation by synthetic heparan-derived di- and trisaccharides.
Science, 268, 432-436.
[0337] 29. Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld,
G. (1992). Cell surface associated heparin-like molecules are
required for the binding of vascular endothelial growth factor
(VEGF) to its cell surface receptors. J. Biol. Chem., 267,
6093-6098.
[0338] 30. Lider, O., Baharav, E., Mekori, Y., Miller, T.,
Naparstek, Y., Vlodavsky, I., and Cohen, I. R. (1989). Suppression
of experimental autoimmune diseases and prolongation of allograft
survival by treatment of animals with heparinoid inhibitors of T
lymphocyte heparanase. J. Clin. Invest., 83, 752-756.
[0339] 31. Lider, O., Cahalon, L., Gilat, D., Hershkovitz, R.,
Siegel, D., Margalit. R., Shoseyov, O., and Cohn, I. R. (1995). A
disaccharide that inhibits tumor necrosis factor o is formed from
the extracellular matrix by the enzyme heparanase. Proc. Natl.
Acad. Sci. USA., 92, 5037-5041.
[0340] 31a. Rapraeger, A., Krufka, A., and Olwin, B. R. (1991).
Requirement of heparan sulfate for bFGF-mediated fibroblast growth
and myoblast differentiation. Science, 252, 1705-1708.
[0341] 32. Eisenberg, S., Sehayek. E., Olivecrona, T., and
Vlodavsky, I. (1992). Lipoprotein lipase enhances binding of
lipoproteins to heparan sulfate on cell surfaces and extracellular
matrix. J. Clin. Invest., 90, 2013-2021.
[0342] 33. Shieh, M-T., Wundunn, D., Montgomery, R. I., Esko, J.
D., and Spear. P. G. J. (1992). Cell surface receptors for herpes
simplex virus are heparan sulfate proteoglycans. J. Cell Biol.,
116, 1273-1281.
[0343] 33a. Chen, Y., Maguire, T., Hileman, R. E., Fromm, J. R.,
Esko, J. D., Linhardt, R. J., and Marks, R. M. (1997). Dengue virus
infectivity depends on envelope protein binding to target cell
heparan sulfate. Nature Medicine 3, 866-871.
[0344] 33b. Putnak, J. R., Kanesa-Thasan, N., and Innis, B. L.
(1997). A putative cellular receptor for dengue viruses. Nature
Medicine 3, 828-829.
[0345] 34. Narindrasorasak, S., Lowery, D., Gonzalez-DeWhitt, P.,
Poorman, R. A., Greenberg, B., Kisilevsky, R. (1991). High affinity
interactions between the Alzheimer's beta-amyloid precursor protein
and the basement membrane form of theparan sulfate proteoglycan. J.
Biol. Chem., 266, 12878-83.
[0346] 35. Ross, R. (1993). The pathogenesis of atherosclerosis: a
perspective for the 1990s. Nature (Lond.)., 362:801-809.
[0347] 36. Zhong-Sheng, J., Walter, J., Brecht, R., Miranda, D.,
Mahmood Hussain, M., Innerarity, T. L. and Mahley, W. R. (1993).
Role of heparan sulfate proteoglycans in the binding and uptake of
apolipoprotein E-enriched remnant lipoproteins by cultured cells.
J. Biol. Chem., 268, 10160-10167.
[0348] 37. Ernst, S., Langer, R., Cooney, Ch. L., and Sasisekharan,
R. (1995). Enzymatic degradation of glycosaminoglycans. Critical
Reviews in Biochemistry and Molecular Biology, 30(5), 387-444.
[0349] 38. Gospodarowicz, D., Mescher, A L., Birdwell, C R. (1977).
Stimulation of corneal endothelial cell proliferation in vitro by
fibroblast and epidermal growth factors. Exp Eye Res 25, 75-89.
[0350] 39. Haimovitz-Friedman, A., Falcone, D. J., Eldor, A.,
Schirrmacher, V. Vlodavsky, I., and Fuks, Z. (1991) Activation of
platelet heparitinase by tumor cell-derived factors. Blood, 78,
789-796.
[0351] 39a. Savitsky, K., Platzer, M., Uziel, T., Gilad, S.,
Sartiel, A., Rosental, A., Elroy-Stein, O., Siloh, Y. and Rotman,
G. (1997). Ataxia-telangiectasia: structural diversity of
untranslated sequences suggests complex post-translational
regulation of ATM gene expression. Nucleic Acids Res. 25(9),
1678-1684.
[0352] 40. Bar-Ner, M., Eldor, A., Wasserman, L., Matzner, Y., and
Vlodavsky, I. (1987). Inhibition of heparanase mediated degradation
of extracellular matrix heparan sulfate by modified and
non-anticoagulant heparin species. Blood, 70, 551-557.
[0353] 41. Goshen, R., Hochberg, A., Korner, G., Levi, E.,
Ishai-Michaeli, R., Elkin, M., de Grot, N., and Vlodavsky, I.
(1996). Purification and characterization of placental heparanase
and its expression by cultured cytotrophoblasts. Mol. Human
Reprod., 2, 679-684.
[0354] 42. Korb M., Ke Y. and Johnson L. F. (1993) Stimulation of
gene expression by introns: conversion of an inhibitory intron to a
stimulatory intron by alteration of the splice donor sequence.
Nucleic Acids Res., 25;21 (25):5901-8.
[0355] 43. Zheng B., Qiu X. Y., Tan M., Xing Y. N., Lo D., Xue J.
L. and Qiu X. F. (1997) Increment of hFIX expression with
endogenous intron 1 in vitro. Cell Res., 7(1):21-29.
[0356] 44. Kurachi S., Hitomi Y., Furukawa M. and Kurachi K. (1995)
Role of intron I in expression of the human factor IX gene. J.
Biol. Chem. 10, 270(10):5276-5281.
[0357] 45. Shekhar P. V. and Miller F. R. (1994-5) Correlation of
differences in modulation of ras expression with metastatic
competence of mouse mammary tumor subpopulations. Invasion
Metastasis, 14(1-6):27-37.
[0358] 46. Zhou G., Garofalo S., Mukhopadhyay K., Lefebvre V.,
Smith C. N., Eberspaecher H. and de Crombrugghe B. (1995) A 182 bp
fragment of the mouse pro alpha 1(II) collagen gene is sufficient
to direct chondrocyte expression in transgenic mice. J. Cell Sci.,
108 (Pt 12):3677-3684.
[0359] 47. Hormuzdi S. G., Penttinen R., Jaenisch R. and Bornstein
P. (1998) A gene-targeting approach identifies a function for the
first intron in expression of the alpha1(I) collagen gene. Mol.
Cell, 18(6):3368-3375.
[0360] 48. Kang Y. K., Lee C. S., Chung A. S. and Lee K. K. (1998)
Prolactin-inducible enhancer activity of the first intron of the
bovine beta-casein gene. Mol. Cells, 30;8(3):259-265.
[0361] 49. Chow Y. H., O'Brodovich H., Plumb J., Wen Y., Sohn K.
J., Lu Z., Zhang F., Lukacs G. L., Tanswell A. K., Hui C. C.,
Buchwald M. and Hu J. (1997) Development of an epithelium-specific
expression cassette with human DNA regulatory elements for
transgene expression in lung airways. Proc. Natl. Acad. Sci. USA,
23;94(26):14695-14700.
[0362] 50. Gottschalk U. and Chan S. (1998) Somatic gene therapy.
Present situation and future perspective. Arzneimittelforschung,
48(11):1111-1120.
[0363] 51. Ye S. Cole-Strauss A. C. Frank B. and Kmiec E. B. (1998)
Targeted gene correction: a new strategy for molecular medicine.
Mol. Med. Today, 4(10):431-437.
[0364] 52. Lai L., and Lien Y. (1999) Homologous recombination
based gene therapy. Exp. Nephrol., 7(1):11-14.
[0365] 53. Yazaki N., Fujita H., Ohta M., Kawasaki T. and Itoh N.
(1993) The structure and expression of the FGF receptor-1 mRNA
isoforms in rat tissues. Biochim. Biophys. Acta.,
20;1172(1-2):37-42.
[0366] 54. Le Fur N., Kelsall S. R., Silvers W. K. and Mintz B.
(1997) Selective increase in specific alternative splice variants
of tyrosinase in murine melanomas: a projected basis for
immunotherapy. Proc. Natl. Acad. Sci. USA, 13;94(10):5332-5337.
[0367] 55. Miyake H., Okamoto I., Hara I., Gohji K., Yamanaka K.,
Arakawa S., Kamidono S. and Saya H. (1998) Highly specific and
sensitive detection of malignancy in urine samples from patients
with urothelial cancer by CD44v8-10/CD44v10 competitive RT-PCR.
Int. J. Cancer, 18;79(6):560-564.
[0368] 56. Guriec N., Marcellin L., Gairard B., Calderoli H., Wilk
A., Renaud R., Bergerat J. P. and Oberling F. (1996) CD44 exon 6
expression as a possible early prognostic factor in primary node
negative breast carcinoma. Clin. Exp. Metastasis,
14(5):434-439.
[0369] 57. Gewirtz A. M., Sokol D. L. and Ratajczak M. Z. (1998)
Nucleic acid therapeutics: state of the art and future prospects.
Blood, 1;92(3):712-736.
[0370] 58. Hida K., Shindoh M., Yasuda M., Hanzawa M., Funaoka K.,
Kohgo T., Amemiya A., Totsuka Y., Yoshida K. and Fujinaga K (1997)
Antisense E1AF transfection restrains oral cancer invasion by
reducing matrix metalloproteinase activities. Am. J. Pathol.
150(6):2125-2132.
[0371] 59. Shastry B. S. (1998) Gene disruption in mice: models of
development and disease. Mol. Cell. Biochem. 1998
April;181(1-2):163-179.
[0372] 60. Carpentier A. F. Rosenfeld M. R. Delattre J. Y., Whalen
R. G., Posner J. B. and Dalmau J. (1998) DNA vaccination with HuD
inhibits growth of a neuroblastoma in mice. Clin. Cancer Res.,
4(11):2819-2824.
[0373] 61. Lai W. C. and Bennett M. (1998) DNA vaccines. Crit. Rev.
Immunol., 18(5):449-484.
[0374] 62. Welch P. J., Barber J. R., and Wong-Staal F. (1998)
Expression of ribozymes in gene transfer systems to modulate target
RNA levels. Curr. Opin. Biotechnol., 9(5):486-496.
[0375] 63. Durand P., Lehn P., Callebaunt I., Fabrega S., Henrissat
B. and Mornon J. P. (1997) Active-site motifs of lysosomal acid
hydrolyses: invariant features of clan GH-A glycosyl hydrolases
deduced from hydrophobic cluster analysis. Glycobiology,
7(2):277-284.
[0376] 64. Thuong and Helene (1993) Sequence specific recognition
and modification of double helical DNA by oligonucleotides Angev.
Chem. Int. Ed. Engl. 32:666
[0377] 65. Dash P., Lotan I., Knapp M., Kandel E. R. and Goelet P.
(1987) Selective elimination of mRNAs in vivo: complementary
oligodeoxynucleotides promote RNA degradation by an RNase H-like
activity. Proc. Natl. Acad. Sci. USA, 84:7896.
[0378] 66. Chiang M. Y., Chan H., Zounes M. A., Freier S. M. Lima
W. F. and Bennett C. F. (1991) Antisense oligonucleotides inhibit
intercellular adhesion molecule 1 expression by two distinct
mechanisms. J. Biol. Chem. 266:18162-71.
[0379] 67. Paterson Paterson B. M, Roberts B. E and Kuff E L.
(1977) Structural gene identification and mapping by DNA-mRNA
hybrid-arrested cell-free translation. Proc. Natl. Acad. Sci. USA,
74:4370.
[0380] 68. Cohen (1992) Oligonucleotide therapeutics. Trends in
Biotechnology, 10:87.
[0381] 69. Szczylik et al (1991) Selective inhibition of leukemia
cell proliferation by BCR-ABL antisense oligodeoxynucleotides.
Science 253:562.
[0382] 70. Calabretta et al. (1991) Normal and leukemic
hematopoietic cell manifest differential sensitivity to inhibitory
effects of c-myc antisense oligodeoxynucleotides: an in vitro study
relevant to bone marrow purging. Proc. Natl. Acad. Sci. USA
88:2351.
[0383] 71. Heikhila et al. (1987) A c-myc antisense
oligodeoxynucleotide inhibits entry into S phase but not progress
from G(0) to G(1). Nature, 328:445.
[0384] 72. Reed et al. (1990) Antisense mediated inhibition of BCL2
prooncogene expression and leukemic cell growth and survival:
comparison of phosphodiester and phosphorothioate
oligodeoxynucleotides. Cancer Res. 50:6565.
[0385] 73. Burch and Mahan (1991) Oligodeoxynucleotides antisense
to the interleukin I receptor m RNA block the effects of
interleukin I in cultured murine and human fibroblasts and in mice.
J. Clin. Invest. 88:1190.
[0386] 74. Agrawal (1992) Antisense oligonucleotides as antiviral
agents. TIBTECH 10:152.
[0387] 75. Uhlmann et al. (1990) Chem. Rev. 90:544.
[0388] 76. Cook (1991) Medicinal chemistry of antisense
oligonucleotides--future opportunities. Anti-Cancer Drug Design
6:585.
[0389] 77. Biotechnology research news (1993) Can DNA mimics
improve on the real thing? Science 262:1647.
* * * * *