Note: Descriptions are shown in the official language in which they were submitted.
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SPECIFICATION
METHOD OF GENE TRANSFER USING GALACTOSYLATED HISTONE
TECHNICAL FIELD
The present invention relates to the field of introducing genetic material to
cultured cells and into cells of animals in vivo. In particular, the invention relates
5 to the use of receptor-targeted neoglycoprotein to carry genetic material into cells.
The invention also uses nuclear-localization signals(Gerace et al. Annu. Rev. Cell
Biol. 1988, 4:335-374), to facilitate the entry of genetic materials into cell nucleus
for gene expression. The present invention has a wide variety of applications, for
example, in treatment of genetic diseases, in study of cell biology, and in genetic
10 interventions of physiological and pathological processes.
BACKGROUND OF THE INVENTION
Many methods for gene transfer are currently available, including viral
vectors, liposomal vesicles, electroporation, calcium phosphate-DNA co-
precipitation, and receptor-mediated DNA delivery system. These methods have
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been used in various studies to introduce genes into cultured cells and into tissues
of intact animals. Although viral vectors have been widely used for somatic gene
therapy, they are not ideally suitable for gene delivery in situations where large
amount of DNA has to be delivered in vivo into a specific tissue. Because viral
5 vectors currently being used are not tissue-specific and are inefficient when used in
vivo, nearly all attempts of somatic gene therapy using viral vector have been ex-
vivo cell replacement gene transfer. This procedure involves in taking cells from a
patient, introducing a viral vector containing a therapeutic gene into these cultured
cells, and reintroducing the genetically altered cells into the patient. On the other
10 hand, using the receptor-mediated DNA delivery system, DNA can be simply
injected into the blood stream of intact animal or human and taken up only by a
specific cell type. The receptor-mediated gene transfer system is proven more
effective than viral vector in vivo.
Receptor-mediated endocytosis was first used as a mechanism for transfer
of genetic materials into animal cells by Wu and Wu(J. Biol. Chem. 1988, 263,
14621-14624). Wu et al have successfully introduced genes into hepatocytes and
hepatoma cells by packaging DNA with naturally-occurring glycoprotein
recognisable by asialoglycoprotein-receptor (ASGP receptor). The ligand used in
Wu's method is the orusomucoid isolated from blood, and is treated with
20 neurominidase to remove the sialic acid. As result, the asialoglycoprotein has
galactose as its terminal sugar and is capable of binding to ASGP receptor on
hepatocytes and on hepatoma cells. The binding of asialoglycoprotein and its
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ligands requires galactose and triggers endocytosis(Ashwell et al. Adv. Enzymol.41, 99-128) In order to carry DNA, asialoorusomucoid is linked with poly-L-lysine
and used to introduce genes into cells that express ASGP receptor on the cell
membrane (Wu et al. J. Biol. Chem. 1988, 263, 14621-14624).
Successful introduction and expression of reporter genes have been reported
in hepatoma cells(HepG2) and hepatocytes in vivo, using Wu's method. In their
early publications, Wu et al have introduced plasmid DNA containing the gene forchloramphenicol acetyltransferase (CAT) driven by a SV40 viral promoter (pSV-
CAT) into HepG2 cells and plasmid DNA containing the CAT gene driven by
mouse albumin promoter(pAlb-CAT) into hepatocytes of intact adult rats. The level
of gene expression in HepG2 cells was two times higher than that resulted from
calcium phosphate co-precipitation (Wu et al. Biochem. 1988, 27, 887-892).
After intravenous injection of packaged DNA, expression of CAT gene in the rat
livers was found to be transient but lasts up to 11 weeks if partial hepatectomy is
performed (J. Biol. Chem. 1989, 264, 16985-16987) . The gene for low density
lipoprotein receptor was also introduced with Wu's method into the liver of
Watanabe rabbits, an animal model for familial hypercholesterolemia. A partial
reduction (30%) of blood cholesterol was observed in the treated animals.
The efficacy of Wu's method have not been demonstrated by other
investigators. l have limited success with Wu's method. The expression of genes
introduced in vivo by Wu's method have been low and variable in my hand. lts
effectiveness as a gene transfer method was compared to that of galactosylated
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albumin, using both cultured cells and hepatocytes of intact adult rats. In both
cases, no significant difference in the level of transgene expression have been
observed .
The current invention is a highly effective and unique method with a
5 mechanism for DNA entry into cell nucleus that have never been used by any other
gene transfer methods. Compared with Wu's method, the present invention has
the following advantages and additional unique features:
a. The present invention is 10 times more effective than Wu's method,
judged by the expression level of CAT gene introduced under identical
1 0 conditions.
b. Histone 1 used in the present invention is a Iysine rich protein and is
capable of binding to DNA naturally, whereas asialoorusomucoid used in
Wu's method has to be coupled to poly-lysine in order to bind to DNA.
c. The galactosylated histone 1 has more galactose residues per molecule
than that on the asialoorusomucoid molecule. Therefore, it has higher
affinity for the asialoglycoprotein receptor than asialoorusomucoid.
d. Histone 1 is actively transported in a facilitated process into nucleus
where gene transcription occurs. No such mechanism exists for
asialoorusomucoid .
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e. The present invention is simple and effective because histone 1 posses
multiple functions and requires minimum modification.
f. The present invention can be easily adapted to targeting other cell surface
receptors. For example, if mannose is added to histone 1, the mannose
receptor on kupffer cells and on macrophages can be targeted using the
present invention.
g. Nuclear proteins such as histones are not degraded rapidly as non-nuclear
proteins are(Yamaizumi, M., Uchida, T., Okada, Y., Furusawa, M. and
Mitsui, H.(1978) Rapid transfer of non-histone chromosomal proteins to the
nucleus of living cells. Nature. 273, 782-784). Therefore, DNA carriers
made from nuclear proteins would prolong the survival of the introduced
DNA as compared with DNA introduced by non-nuclear proteins.
h. High level, long-term gene expression has been achieved when partial
hepatectomy is performed together with gene delivery. In addition to
observing high CAT gene expression, I also be able to get positive beta-
galactosidase staining in liver sections obtained from rats 3 months after
DNA injection. No such data have been reported in the literature. The
Positive beta-galactosidase stain in liver sections is more difficult to obtain
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because higher level expression of this gene is required when compared to
the detection of other commonly used reporter genes such as CAT and
luciferase.
Applications of the present invention is obvious in the treatment of liver-
5 specific genetic diseases (for example, haemophilia, familial hypercholesterolemia,
and Gausher's disease). Other biological and therapeutic applications are also
feasible in cases when gene transfer is required. For example, bovine growth
hormone gene can be introduced into livers of dairy cows to increase milk
production. Currently, Some of dairy cows are directly injected with growth
10 hormone which is less cost effective than somatic gene transfer.
SUMMARY OF THE INVENTION
The present invention is the most effect method for introducing and
expressing genes in hepatoma cells and hepatocytes in vivo. It is, on the average,
10 times more effective in gene transferring (range from 8 to 14 times) than the
15 best hepatocyte-targeted gene delivery system using asialoorusomucoid(Wu et al.
Biochem. 1988, 27, 887-892). Its design has several novel features which are not
seen in any other gene transfer systems. First, this invention uses a simple
coupling reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) to chemically
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add galactose to a protein, histone, in a one step reaction. The galactosylated
protein (for example, albumin, histone and any other protein) has high affinity for
the ASGP receptor; Second, this invention uses a chemically modified protein
(galactosylated histone 1 ) as DNA carrier to bind to the asialoglycoprotein receptor
5 instead of using the receptor's natural ligand. The galactosylated histone has more
galactose residues than the asialoorusomucoid, and therefore has higher affinity for
the ASGP receptor; Three, the invention uses a protein which is a natural DNA-
binding protein, whereas other similar methods have to link poly-lysine to a protein
of choice in order to form DNA-protein complex; Four, this invention uses a
10 protein which crosses the membranes of cell nucleus by a facilitated process in
addition to simple diffusion. This feature contributes greatly to the high level
expression of the introduced foreign genes; Last, the galactosylated histone 1 is
the physical embodiment of all the features in this invention. This means this gene
delivery system has only one component and is simple, reliable, and efficient from
15 engineering-design point of view.
It is the objective of this invention to deliver effectively genes into liver cells
and to achieve high level expression of the introduced genes. The present invention
uses the galactosylated histone 1 to bind to the ASGP receptor and transfers DNA
into the targeted cells through the receptor-mediated endocytosis. The potential
20 applications of this method and its clinical significance lie in part with its intended
target, the liver, and its effectiveness of application in vivo. Many liver-specific
genetic diseases, as well as non-liver specific disorders, can be treated with
X
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~corrective genes introduced by this method. Application of this method in vivo is
so simple and effective that it makes the ideal method for gene therapy in vivo.For example, DNA packaged by the galactosylated histone 1 can be injected into ablood vessel (e.g the tail vein of a rat) and is taken up only by the hepatocytes.
Another objective of this invention is to make proteins of choice capable of
binding to specific receptors in a simple step. Before present invention, DNA carrier
in the receptor-mediated gene transfer is limited to the proteins which are natural
ligands for the specific receptor. Present invention offers the possibility of making
any protein recognisable by the ASGP receptor or by some other receptor. For
example, the mannose receptor on the Kupffer cells of liver may be targeted by
adding mannose to any protein of choice.
Another objective of this invention is to pioneer the use of a facilitated
process for DNA to enter cell nucleus. Histone 1 is a natural DNA-binding protein
which contains nuclear localization signal and crosses the double membrane of cell
nucleus by a facilitated process(Breuver et al. 1990, Cell, 60, 999-1008). This
gives the present invention great advantage over other methods since entry into
nucleus is obligatory for gene expression and for possible integration of the foreign
genes into the cell genome. No other methods have this unique feature.
Another objective of this invention is to select and use a protein which has
all or most desired features for efficient gene transfer, thus eliminating the need for
extensive modification and additional components. The choice, histone 1, is a
natural DNA-binding protein and is actively transported into the nucleus. It also
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contains the so-called nuclear localization signal. Only modification it needed is the
galactosylation. The galactosylated histone 1 is proven to be the best DNA carrier
in part because it has multiple functions and is simple.
Yet another objective is to achieve high level, long-term expression of the
5 introduced genes in host cells. High level and long-term expression of genes
transferred by the present invention has been achieved in partially hepatectomized
rats.
DISCLOSURE OF INVENTION
The current invention is a gene delivery system targeting at cells expressing
10 asialoglycoprotein receptor. To best understand the design and the operation
procedure of this invention, the major functional components of this system will be
discussed first, followed by the description of operational steps in routine
applications.
1. Components of the gene delivery system:
The core to the present invention is a carrier molecule which has four major
functions, namely, receptor binding, DNA binding, escape from endosome, and
facilitated entry into nucleus. These four functions can be carried out by four
components of the system. As a ideal embodiment of this system design, histone
1 posses three of the four functions naturally (DNA binding, escape from
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endosome and facilitated entry into cell nucleus) and is galactosylated in a simple
step to acquire the fourth function (receptor binding).
1) synthesis of a ligand capable of binding to the asialoglycoprotein receptor
and DNA- The first component to be prepared is a molecule which binds to the
5 ASGP receptor and which can be further made capable of binding to DNA or RNA.
Often, the binding between proteins or peptides and their receptors requires sugar
molecules. Galactose is chemically linked to histone 1 or any other protein in order
for the modified proteins to bind to ASGP receptor. The reaction for
galactosylation of histone or any other proteins is carried out in a mixture
consisting of 2 mg protein, 100 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC), 60 mg galactose, and 0.8 mg poly-L-lysine (omitted when the protein is
histone 1), in 2 ml water at pH 7.5. The reaction mixture is incubated in the dark
at room temperature for 48 hours. The galactosylated histone 1 is ready to be
used in gene transfer experiments and can be stored at -20 C for many months.
2) Component which disrupts endosome- When internalized through the
receptor-mediated endocytosis, the DNA-protein complex is enclosed in
endosomes. These endosome will fuse with Iysosomes and the contents of
endosome are digested by Iysosomal enzymes. Only the DNA-protein complex
which escapes from endosome can enter the cell nucleus. Many kinds of
20 molecules disrupt endosome membrane or prevent the fusion of endosome with
Iysosomes. While the exact mechanism for such action is not known, many
molecules have been identified for having such property, including, for example,
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hemagglutinin protein of influenza virus(Wagner et al. Proc. Natl. Acad. Sci. USA.
1992, 89: 7934-7938), cytochalasin B and Bredfeldin A. Cytochalasin B and
Bredfeldin A have been used in my transfection experiments in cell culture.
Cytochalasin B, at the concentration of 30 to 50 uM, increases gene expression in
5 cell culture by two-fold.
3) nuclear localization signal(NLS)- The nuclear localization signals are amino
acid sequences that direct proteins to the nucleus. The amino acids that are
common to many nuclear localization signals have been reviewed by Gerace and
Burke( 1988, Annu. Rev. Cell Biol. 4, 335-374). Examples of nuclear localization
signal-containing proteins are histones(Moreland et al., 1987, Mol. Cell Biol., 9,
384-389), high mobility group 1 chromosomal nonhistone protein(Tsuneoka et al.,
1986, J. Biol. Chem. 261, 1829-1834), and SV40 large T antigen(Geoldfarg et al.,
1986, Nature, 322, 641-644). The nuclear import of many these proteins has been
shown to be saturable and hence receptor mediated(Geoldfarg et al., 1986,
Nature, 322, 641 -644). Putative receptors for the NLSs have been identified both
in the cytoplasm and in association with the nuclear envelope (Yoneda et al.,
1988, 242, 275-278). The present invention takes advantage of these NLS
containing proteins to transport DNA into cell nucleus. In particular, histone
1 (obtained from Sigma company as fraction V-S) is used because it is commercially
20 available, naturally bound to DNA, and is proven highly effective in my gene
transfer experiments. The NLS domain, located near the C-terminus, contains an
about 110 amino acid rich in Iysines and prolines (Ven Holde, 1989, Chromatin,
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New York: Springer-Verlag). A proteolytic fragment from calf thymus H1 that
contains this domain accumulated in oocyte nuclei(Dingwall and Allan, 1984,
EMBO J. 3, 1933-1937).
2. Operation procedure
1) preparation of protein-DNA complex- The galactosylated H1 is mixed
with plasmid DNA in 2 M NaCI at a weight ratio of protein /DNA between 1 and 4.
The DNA concentration in this mixture should be about 1 ug/1 Oul. The mixture isincubated at room temperature for one hour, and dialysed against 0.15 M NaCI fortwo days at 4 C using a dialysis tubing with molecular weight cut off of 3500 Da.
The dialysed protein-DNA complex is stored on ice before use.
2) gene transfer and expression in cell culture- The galactosylated histone 1
is designed to deliver DNA into cells expressing the ASGP receptor. The HepG2
cells, a human hepatoma cell line with ASGP receptor, were used in the in vitro
transfection experiments. The cells were grown in 75 mm flasks in Dulbecco's
Minimum Essential Medium (DMEM), supplemented with 10 % fetal calf serum.
When cells reach 50% confluence, the dialysed DNA-protein conjugate prepared as
described above is directly added to the cell culture medium along with 30 ul 1 M
CaCI2. 10 to 20 ug DNA can be used per flask. Cells can be harvested 2 to 3 daysafter the transfection and analyzed for gene products. Common reporter genes areused to test the transfection efficiency and to optimize the conditions of
transfection. For example, mammalian expression vectors for chloramphenicol
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acetyltransferase(CAT) and beta-galactosidase, both driven by the Rous Sarcoma
Virus Promoter, have been used in initial experiments to aid the development of the
present invention.
3) gene transfer and expression in intact animals or human- The primary
5 hepatocytes(liver cells) of many animals, including human, express ASGP receptor
and therefore can be targeted for introduction of foreign genes by the present
invention. For example, the dialysed galactosylated histone 1-DNA complex(about
200 ug DNA) can be injected into the tail vein of adult rats. To assess the
expression level of the foreign gene, blood samples and or liver samples can be
10 taken for analysis. If the expected gene product is a secretory protein such as the
bovine growth hormone used in my experiment, the protein can be detected in
blood. If the expected gene product is not
a secretory protein, liver samples have to be taken a few days after the DNA
injection, and analyzed for the expected gene product. Partial hepatectomy can
15 be performed on animals at the time DNA injection in order to achieve long-term
gene expression. For example, rats can be injected with 150 ug DNA-protein
conjugate through the tail vein. On the following day, partial hepatectomy is
performed on these injected rats. Typically, 30% to 50% of the liver is removed
and additional 150 ug DNA is injected into the portal vein before the surgical
20 closure of abdomen.
Although the gene transfer experiments and genes used as described above
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are limited, it is not intended to imply the scope of the present invention is limited
as such.
According to the present invention virtually any gene may be transferred into
the targeted cells. Using the present invention, no special modification or
5 subcloning of a simple gene expression vector is necessary. But this is not the
case for viral vectors. Genes need to be subcloned into a viral vector of choice
before they can be transferred into cells. These extra cloning steps is often costly
in term of time and resource. Also, there is limit to the size of the gene that can
be cloned into a viral vector. The present invention can deliver genes of any size.
According to the present invention liver is targeted organ. The liver is one of
the ideal target organs for gene therapy. Many liver specific-genetic diseases can
be treated by gene therapy using the present invention. But application of the
present invention is not limited for liver-specific diseases. For example, any
therapeutic or nontherapeutic application which requires the intended gene product
15 be present in blood can be achieved by present invention. In such scenarios,
genes are to be delivered and expressed in liver cells; and the gene product is
secreted into blood stream.
According to the present invention other cell surface receptors and therefore
other tissues can be targeted by the present invention. Only the receptor
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recognition domain needs to be changed for the present invention to target another
receptor. For example, Mannose can be added to the histone 1 in order to target
the mannose receptor on the cell surface of macrophage.
According to the present invention other NLS containing proteins can be
5 used as DNA carriers. Since these proteins provide facilitated mechanisms for DNA
entry into cell nucleus, any NLS-containing proteins or synthetic peptides having
NLS should be considered an integral part of the present invention when they are
used in transferring materials into cell nucleus.
