CA2090355C - Method of gene transfer using galactosylated histones - Google Patents

Abstract

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 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 interventions of physiological and pathological processes. The present invention is a effective method for introducing and expressing genes in hepatoma cells and hepatocytes in vivo. Its design has several novel features. First, this invention uses a simple coupling reagent (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) to chemically 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, the invention uses a protein which is a natural DNA-binding protein; Three, this invention uses a 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 engineering-design point of view.

Description

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 20qo3ss 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 209035~

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 -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 .

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 -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 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

~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 20~0355 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 20qo35~

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, 20~0355 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, 20~0355 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 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 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 20qo3ss 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.

Claims (12)

1. A process for the transfer of DNA into mammalian cells comprising the steps of:

a. chemically or enzymatically glycosylating histones or other DNA binding proteins which contain nuclear localization signal(NLS), resulting in glycosylated histone(s) or other glycosylated DNA binding proteins;

b. conjugating noncovalently the glycosylated histones or other said proteins to DNA by mixing the glycosylated histone or glycosylated DNA binding protein at a ratio of said protein/DNA between 1 and 4, in 2 M NaCl solution, and incubating the mixture for one hour before dialysing said mixture against 0.15 M NaCl solution for two days at 4 °C, producing a protein-DNA complex;

c. introducing said protein-DNA complex into cells in vitro;

d. delivering said protein-DNA complex to the cell nucleus by NLS-facilitated processes or by simple diffusion across the cell nuclear membranes.

2. The process of Claim 1 in which galactose residues are linked covalently or noncovalently to histone 1 or other said proteins, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) or using any other method.

3. The process of Claim 1 in which galactosylated histone-DNA complexes are formed in step b, which are used to carry DNA and to bind to asialoglycoprotein receptor on cell membrane surface.

4. The process of Claim 3 in which said complexes enter cells expressing asialoglycoprotein receptor, selected from the group consisting of HepG2 cells, animal hepatocytes, or human hepatocytes.

5. The process of Claim 1 in which mannose is linked to histones or other said proteins at step a, to form complexes at step b, which can bind to mannose receptors.

6. The process of Claim 1 in which said complexes are used to transfer genes into cells expressing targeted receptors.

7. The process of Claim 1 in which binding to the cell surface receptor and DNA, and traversing the cell nuclear membranes are the functions of the glycosylated histones.

8. The process of Claim 1 in which Histone 1 or other said proteins are used to form complexes at step b which carry DNA across membranes of the cell nucleus.

9. The process of Claim 1 in which facilitated transport is characterized as movement of molecules into the cell nucleus in a manner other than simple diffusion, said facilitated transport having all or one of the following features, requirements for one or more receptors and energy, temperature dependence, and requirement for nuclear location signals defined as amino acid sequences which direct protein, DNA or any other molecules into cell nucleus.

10. The process of Claim 1 in which said DNA is any gene, plasmid, bacteriophage, viral vectors, and yeast vectors.

11. The process of Claim 1 in which said histones or other DNA binding proteins are natural nuclear proteins or synthetic fragments of these nuclear proteins, and are used for the purpose of prolonging the survival of the introduced DNA in the cell nucleus.

12. A process according to Claim 11, wherein natural nuclear proteins are isolated histones, non-histone chromosomal proteins, transcription factors, and other proteins found in the cell nucleus.