Nucleic Acids Research, 2003, Vol. 31, No. 8 e46
© 2003 Oxford University Press
The hepatitis C virus internal ribosome entry site facilitates efficient protein synthesis in blood vessel endothelium during tumour angiogenesis
Grace T. Y. Chung,
Yoshihiro Yamada,
Richard Pannell,
Alan Forster and
Terence H. Rabbitts
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Yoshihiro Yamada, Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan
Received January 31, 2003; Revised and Accepted March 1, 2003
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ABSTRACT
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The development of gene delivery systems for therapeutic use
involves vectors (often retrovirus or adenovirus) which typically
encode one target protein, but the use of internal ribosome
entry sites (IRES) can confer the ability to express more than
one protein from bi- or polycistronic mRNAs. IRES elements can
display tissue-specific expression, so it is necessary to determine
suitable IRES for specific clinical applicability. Blood vessel
endothelial cells are important clinically since many different
conditions involve neo-vascularisation (angiogenesis). We have
demonstrated that the viral hepatitis C IRES element is a powerful
mediator of protein synthesis in angiogenesis, such as found
in solid tumours. Homologous recombination was used to introduce
IRES-lacZ sequences into the
Lmo2 gene, which is expressed in
endothelial cells. ß-Galactosidase expression was
determined during vascular remodelling in mouse embryos and
in sprouting endothelium during growth of solid tumours, and
showed that the hepatitis C IRES is used efficiently for protein
synthesis in endothelial cells. This IRES element can provide
the means to express two or more therapeutic genes in blood
vessel endothelium in clinical conditions, such as cancer, which
depend on angiogenesis.
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INTRODUCTION
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Specific expression of genes following delivery to target cells
is a key objective for molecular-based therapies, and blood
vessel endothelial cells are important targets in a variety
of clinical indications. The remodelling of established vasculature
(angiogenesis) occurs at specific times in normal conditions,
such as wound healing or menstruation, and in a number of conditions,
such as cancer, ischaemia, diabetic retinopathy and inflammatory
diseases (
1). In addition, neovascularisation, establishment
of collateral circulation in ischaemic diseases and formation
of granulation tissue in inflammatory diseases are a hallmark
of these pathological conditions (
1,
2). In cancer, angiogenesis
is important to support
in situ growth of primary solid tumours
and also in metastasis where tumour cells traverse the infiltrating
blood vessels (
3), enter the blood stream and eventually form
metastatic deposits in remote locations. The process of angiogenesis
has been advocated as an important point of therapeutic intervention
in cancer (
4), as depleting growing tumours of blood supply
can inhibit their growth. Many molecular targets will be confined
to intracellular locations which require access of molecular
therapeutics to the inside of the cell. This can be achieved
with the use of viral vectors such as adenovirus (
5) or lipid
formulations which carry DNA vectors across the plasma membrane
(
6). These impose restrictions in that they are usually designed
to express a single gene within the target cell. The use of
internal ribosome entry sites (IRES) in bicistronic mRNAs has
been a common strategy for dual gene expression, but these structural
elements in mRNA display developmental and cell type specificity
(
7,
8).
In view of the importance of gene expression in endothelial cells, we have assessed the potential of IRES sequences to achieve high level expression in sprouting endothelial cells in vivo. Using IRES-lacZ cassettes introduced into the Lmo2 gene [which is expressed in embryonic and tumour endothelial cells (9,10)], we have examined ß-galactosidase expression during vascular remodelling in embryos and during the growth of solid tumours. Our results show that the hepatitis C (HC) virus IRES, but not the encephalomyocarditis (EMC) IRES, is used efficiently for protein synthesis in endothelial cells, proving the means to co-express proteins in blood vessel endothelium in clinical conditions which depend on angiogenesis.
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MATERIALS AND METHODS
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Plasmid preparation and gene targeting
The plasmids for homologous recombination in the
Lmo2 gene were
based on pKO5tk, which has a unique
BamHI restriction site mutated
in exon 2 (
11). The HC-IRES-lacZ
Lmo2 knock-in targeting clone
was prepared by inserting an HC-IRES-LacZ-MC1neopA cassette
into the
BamHI site of pKO5tk. A 400 bp
BamHI fragment including
the HC-IRES (
12) was first cloned into the
BglII–
BamHI
sites of a modified pBSpt vector (pBspt-BGB4) to generate the
precursor pBSpt-HC-IRES with a unique
BamHI site into which
was cloned the
lacZ gene and pMC1-neo-pA (
13). The EMC-IRES
Lmo2 knock-in targeting clone was prepared by inserting the
lacZ gene fragment into pEMC IRES and addition of pMC1neo-pA,
and this cassette was cloned into pKO5tk. The in-frame fusion
of
lacZ with exon 2 of the
Lmo2 gene has been described previously,
as have the generation and characterisation of the germline
mouse carriers of the targeted allele (
14).
Generation and analysis of gene targeted mice
Embryonic stem (ES) cells (CCB) were transfected and selected for G418 resistance and gancyclovir sensitivity as described (11) and targeted clones characterised by Southern filter hybridisation using two external probes, A and B (Fig. 1A). Targeted clones were injected into C57Bl6 blastocysts, and chimaeric mice were generated, from which germline transmission was obtained by breeding male chimeras with C57Bl6 females. Timed matings were set up between heterozygous mice carrying one of the three Lmo2 knock-in alleles and wild-type C57Bl6 mice. At the appropriate times, the pregnant females were euthanased, embryos removed and whole mount stained with X-gal to detect ß-galactosidase as described (10). Post-fixed embryos (10% formalin) were sectioned after wax embedding. Sections (4 µM) were mounted on microscope slides and counter-stained with haematoxylin and eosin. Detection of the endothelial marker PECAM (CD31) was carried out using MEC13.3 anti-CD31 antibody (Pharmingen) by the avidin– biotin-conjugated peroxidase method as described (15).
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Figure 1. Activity of IRES elements in mouse embryo vascular endothelium. (A) Constructs for homologous recombination. Top line: the partial restriction map of the mouse Lmo2 gene shows the location of Lmo2 exons 2 and 3, together with the two probes (A and B) used to detect homologous recombination (10). Middle line: a map of the targeting vector pKO5tk (10) which has a BamHI restriction site introduced within exon 2 to facilitate cloning of exogenous elements into Lmo2. Bottom line: the maps of the lacZ gene insertions cloned in the exon 2 BamHI site for Lmo2-lacZ (in-frame lacZ fusion with the 5' end of Lmo2) (14), HC-IRES and EMC-IRES. (B) Whole-mount X-gal staining of mouse embryos at embryonic stages E9.5, E10.5 and E12.5 showing expression of ß-galactosidase from the Lmo2 gene in de novo capillary formation (vasculogenesis) and endothelial remodelling (angiogenesis) during mouse embryo development. Wt = wild-type C57Bl6
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Tumour endothelial cell analysis
Lewis lung carcinoma cells were injected into both flanks of
mice from each of the
Lmo2 knock-in mouse lines or C57Bl6 controls
(about 10
6 cells per site). When primary site solid tumours
reached
1 cm size, the recipient mice were euthanased, tumours
resected and whole-mount X-gal staining carried out as for the
embryos. After post-fixation in 10% formalin, sections were
prepared from wax-embedded specimens, and 4 µM sections
were mounted and counter-stained with haematoxylin and eosin.
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RESULTS
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Efficiency of HC-IRES in vascular endothelium during embryogenesis
A range of IRES elements potentially could be used to express
proteins in endothelial cells (
16). We compared the ability
of the HC virus IRES and EMC virus IRES to facilitate protein
synthesis in blood vessel endothelial cells during embryonic
development. The
Lmo2 gene is expressed in and is necessary
for sprouting endothelium in embryogenesis (
9) and tumour growth
(
10). We chose this gene as a test situation for expressing
bicistronic mRNA species in endothelial cells
in vivo since
the mouse
Lmo2 gene is amenable to gene targeting in ES cells
(
11). We have created two lines of mice in which the expression
of
lacZ is controlled from an IRES element in the mRNA, namely
HC-IRES and
EMC-IRES lines, respectively (Fig.
1A). In addition,
we compared the
Lmo2-lacZ mouse line in which an in-frame fusion
has been made between the
lacZ gene and
Lmo2 (
9). Timed matings
were established for the three lines and embryos were whole
mount stained with X-gal to detect ß-galactosidase
activity at embryonic day E9.5, 10.5 and 12.5 (Fig.
1B). As
previously reported (
9), the developing vasculature of the
Lmo2-lacZ embryos expresses the
Lmo2 gene which can be detected readily
via the ß-galactosidase reporter. No ß-galactosidase
activity was detected in wild-type embryo littermates (Fig.
1B). In the developing
Lmo2-lacZ embryos, ß-galactosidase
is widely expressed in blood vessels, being found in whole body
developing vasculature which coincides with expression of the
pan-endothelial marker PECAM/CD31, detected with anti-CD31 antibodies
in histological sections of embryos at E10.5 (Fig.
2, top panels).
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Figure 2. Histology of Lmo2-lacZ knock-in E10.5 embryos shows co-expression of ß-galactosidase and the pan-endothelial marker CD31. E10.5 embryo specimens were whole mount stained with X-gal (Fig. 2), sectioned (4 µM), and counter-stained with haematoxylin and eosin. CD31 protein expression was detected in serial sections using anti-CD31 antibody and peroxidase. The montage shows embryo sections from each indicated Lmo2 knock-in mouse line, or wild-type (wt) controls stained only with X-gal (left) or co-stained with X-gal and anti-CD31 (right). Arrowheads indicate endothelial cells lining the blood vessel walls.
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The levels of ß-galactosidase reporter expression
in the knock-in mouse lines with the
Lmo2-HC-IRES-lacZ gene
were less than in the direct
lacZ gene knock-in
Lmo2 (Fig.
1B),
but the detectable ß-galactosidase in the blood vessel
endothelial cells in the
EMC-lacZ mice was very low and indeed
virtually undetectable at embryonic day E10.5 (Figs
1B and
2).
This suggests that the EMC virus IRES is unsuitable for endothelial
expression
in vivo. The HC-IRES, on the other hand, yielded
readily detectable levels of ß-galactosidase activity.
By embryonic day E12.5, profound levels of endothelial expression
had occurred, indicating that the HC-IRES was used efficiently
by the protein synthesis machinery of endothelial cells of mouse
embryos.
The HC-IRES mediates endothelial protein synthesis in tumour angiogenesis
Angiogenesis is a target of cancer therapy (4,17,18), requiring targeting of anti-endothelial reagents to these specific cells. The efficacy of the HC-IRES in tumour blood vessels was tested using the lacZ knock-in mouse lines to support growth of tumour grafts which become vascularised by sprouting of existing blood vessels from the host. Lewis lung carcinoma cells were injected subcutaneously into the Lmo2-lacZ and HC-IRES mice (and C57Bl6 wild-type controls), and solid tumours were allowed to develop in situ at the site of injection. As the vascularisation of these tumours is contributed by the recipient mouse, the blood vessel endothelium would be expected to express the Lmo2-based lacZ reporter (Fig. 3A). This was analysed by staining isolated tumours with X-gal and histological sectioning to examine endothelial expression. Figure 3B shows a comparison of sections made from X-gal-stained tumours of the three sources, showing that the Lmo2-lacZ and HC-IRES-transplanted tumours had comparable levels of ß-galactosidase activity in this situation. The HC-IRES therefore has significant activity in the developing vasculature of tumours.
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Figure 3. Expression of ß-galactosidase from HC IRES in Lewis lung solid tumours. Lewis lung carcinoma cells were implanted subcutaneously in the Lmo2 HC-IRES knock-in mouse line and Lmo2-lacZ or wild-type (wt) controls. (A) The vasculature of the tumours growing in the recipient mice comes from the latter, and therefore the endothelial cells will be expressing the Lmo2 reporter of the recipient. In the case of Lmo2-lacZ and HC-IRES mouse lines, the expression of ß-galactosidase is detected using X-gal substrate. (B) After tumour growth, solid tumours were whole mount stained with X-gal, and 4 µM sections made for examination of tumour vascular endothelium which forms by sprouting of the existing endothelium from recipient mice. Arrowheads indicate endothelial cells lining the blood vessel walls.
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DISCUSSION
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IRES have evolved in viruses and allow viruses to express more
than one gene per mRNA, and the cell types in which this activity
occurs depends on the virus. IRES elements bind to cellular
protein factors, and these can be cell type specific, lending
an internal degree of specificity to the system. The corollary
is that, as a therapeutic or molecular biology methodology,
use of IRES elements in the design of vectors for
in vivo genes
must take cell specificity into account. Thus, if the objective
is targeting protein synthesis within endothelial cells, a suitably
efficient IRES must be employed. Our data show that for blood
vessel endothelial expression, the HC-IRES is used efficiently.
There are a number of important clinical indications where angiogenesis is an important consequence. Neovascularisation occurs around malignant tumours in order to supply enough oxygen and CO2 exchange for rapidly dividing cells (18). In chronic inflammatory diseases such as rheumatoid arthritis, sustained inflammation results in the formation of vascular-rich granulation tissues in the synovial membrane (1). Thus, in these circumstances, preventing blood vessel remodelling and neovascularisation is a potential therapeutic approach (2,4, 17). In circumstances where gene delivery is envisaged as a means of introducing proteins into target endothelial cells for therapy, the HC-IRES element could prove invaluable. In anti-angiogenesis therapies, a virus or other expression vector could encode therapeutic proteins [such as intracellular antibody fragments (19)] to two distinct intracellular targets, adding efficacy to the desired therapeutic effect. Alternatively, in solid tumour therapy, intracellular protein targets of angiogenesis, such as LMO2 (10), could be tackled by introduction of vectors encoding two blocking reagents aimed at prohibiting the function of the target protein in distinct ways [e.g. using an intracellular antibody fragment and a peptide aptamer (20)]. Methods for delivery of vectors to specific cells in vivo are becoming more effective, and specific ways of putting vectors into endothelial cells have been reported (21). Combining these delivery methods with the ability to express efficiently two or more proteins which can combat the function of specific targets is a possible approach to anti-angiogenesis therapies.
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ACKNOWLEDGEMENTS
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We are indebted to Dr Jonathan Karn for the crucial suggestion
of using the hepatitis IRES for this work, and to Dr Nancy Spandidos
for the pBspt-BGB4 vector. Y.Y. was partly funded by the National
Foundation for Cancer Research.
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ABSTRACT
INTRODUCTION