JPH07500961A - Targeting viruses and cells for selective internalization by cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 40. Animals infected with a modified human virus but not susceptible to the native form of the virus. Animals that do not dye.

Specification Background of the invention: Targeting viruses and cells for selective internalization by cells Viruses are a natural and efficient means of introducing foreign genes into cells. Therefore, research on gene regulation in cadavers and in vitro and is used as a useful tool in gene therapy. However, most Viruses have broad cell specificity and infect a variety of cell types. That is, , induces the expression of foreign genes in various tissues, including Also includes undesirable items.

Viral infections usually involve interactions between the viral envelope and the plasma membrane of target cells. Therefore, it is mediated. In many cases, cellular receptors are linked to specific viral structures. Recognize and connect to this. For example, HIV (human immunodeficiency virus) Viruses) transfer their glycoprotein envelope via the receptor CD4 (T4). , coupled to the Helper 1973 sphere (Dalgleich, A.G., et al. ture 312 Near 63-767 (1984)). This kind of interaction It is known that species and organ specificity are determined by

In addition, some research has shown that by binding antibodies to viruses, virus-specific It has been demonstrated that it is possible to change gender. For example, anti-transferrin receptor -By binding antibodies to retroviruses, transferrin receptors can be activated. Retroviruses can be directed to human cells that have - However, in this place In this case, retrovirus binding and internalization to cells is induced, but infection and replication do not occur. No. targeting viral or other types of nucleic acid vectors containing foreign genes. Gene therapy is a means to induce virus infection and replication in target cells. It is useful.

Free-standing mold The present invention aims to direct viruses or cells to target cells in vivo (or live). Regarding the method for selectively internalizing it into target cells (in vitro), Concerning viruses and cells that have been modified to target them for selective internalization. do.

by introducing receptor-specific molecules onto the surface of viruses or cells. , the virus or Degenerates cells. That is, a modified protein that specifically binds to surface receptors on target cells. form viruses or cells. administering a modified virus or cell to an organism and the modified virus or cell to the receptor of the target cell of the organism, Combine selectively. As a result of receptor binding, modified viruses or cells is internalized by target cells.

Cellular receptors are receptors that mediate the uptake of bound ligands into cells. For example, the asialoglycoprotein receptor of hepatocytes, and the receptor specificity The child is a natural or artificial ligand for the receptor. receptor special The isomeric molecule can be attached to the surface of a virus or cell (e.g. viral envelope or cell membrane). will be introduced in In this case, chemically receptor-specific It is also possible to bind sexual molecules to the surface, or to treat the surface so that it can be recognized by receptors. The sea urchin molecules may be exposed.

Furthermore, using the method of the present invention, substances such as nucleic acids (genes) can be selectively delivered to target cells. Viral or cellular vectors can be generated for delivery.

For example, a foreign gene is selectively introduced into target cells and expressed. these This vector is applied to gene therapy, etc., which requires selective gene conversion of cells. Ru.

The method of the invention further alters the natural tropism of infectious agents such as viruses and bacteria. It provides a means to That is, the infectious agent is denatured and released in its native form. causes infection in cells that would not normally be infected. Using this method, you can Develop animal models of human diseases for which laboratory animal controls do not exist, and study those diseases. can conduct research. For example, environmental indicators (such as hepatitis or the AIDS virus) denaturing a tropic human pathogen and infecting a non-human host to destroy the pathogen and the disease. Generate an experimental system to study the disease.

1 Light” I Waki Ao Plate Figure 1 shows NIH3 treated separately with native or modified murine leukemia virus. In situ in T3, HepG2, and 5KHepl cells ( Figure 2 shows the expression of β-galactosidase (innaitu).

Figure 2 shows the internalization of modified Moloney murine leukemia virus biolabeled with AIES. It shows that

Figure 3 shows complexation with asialoorosomucoid using Sephadex G150. This is a chromatogram of the Psi2 virus.

Figure 4 shows that various cells were exposed to Psi2 virus-asialoglycoprotein conjugate. The β-galactosidase activity of the cases is shown.

X-type flatulence alliance Modify the surface of the virus or cell to become specific to the surface receptors of the target cell Direct viruses or cells to target cells by introducing molecules that bind to selective internalization. Cell surface receptors target viruses or mediate cellular internalization. Denatured viruses or cells are grown in vivo. ivO) binds to the receptor of the target cell and is taken up into the target cell.

According to the methods of the invention, viruses are modified to infect specific target cells. It can be done. Using this modified virus, foreign functional D By selectively delivering NA to target cells, new biological or imparts biochemical properties to target cells. Furthermore, the tropism of the virus can be denatured and changed. It is possible to infect cell types that would not normally be infected by the virus in its natural (unmodified) form. Can be targeted.

The method of the invention is applicable to various types of enveloped viruses. In this case, Viruses can be RNA viruses (retroviruses) or DNA viruses (hepatitis virus). virus, adenovirus, etc.), or even those with incomplete replication ability. Alternatively, it may be defective in structure or function. For example, essentially or completely Applicable to viral bodies that lack nucleotide nucleic acids (“empty” viral envelopes) It is.

The present invention is also applicable to cells, and can be applied to microorganisms and protozoa whose tropism can be changed. , cellular organisms such as trypanosomes, and mammalian cells are also applicable.

A receptor-specific molecule is a ligand for a surface receptor on a target cell, particularly a desired Alternatively, a cell surface receptor that mediates internalization of the ligand by its intracellular uptake action. - for example, a ligand for the asialoglycoprotein receptor of hepatocytes. .

Additionally, glycoproteins with a predetermined exposed terminal carbohydrate group can be used as receptor-specific molecules. It can also be used as When specifically targeting hepatocytes, asialo asialoglycoproteins such as rosomucoid and asialofetuin (galactose powder) End) ligands are preferred. Other galaxies used when targeting liver cells Examples of ctose-terminated carbohydrates include carbohydrates derived from natural glycoproteins. , especially containing or exposing a terminal galactose residue tri- or tetra-branched structures that are enzymatically treated to be Alternatively, naturally occurring plant carbohydrates such as arabinogalactan can be used. Ru.

Different carbohydrates are used depending on the type of receptor. For example, the macro file When targeting macrophages (macrophages) or endothelial cells, mannose, manno Su-6-phosphate or carbohydrates having these terminal structures are used.

Furthermore, using receptor ligands such as peptide hormones, we can target viruses and cells. It can also be directed to the corresponding receptor. Peptide hormones include insulin phosphorus, glucagon, gastrin polypeptide, and their respective receptors is included.

Or, conversely, antibodies, etc., that bind to ligands (e.g., antigens) on the cell surface. Using a receptor or receptor-like molecule as a receptor-specific molecule You can also do it. In this case, antibodies specific for cell surface receptors are prepared according to standard methods. is generated.

When a receptor-specific molecule is introduced onto the surface of a virus or cell, the same Recognition of this molecule is carried out by surface receptors. Receptor-specific molecules are For example, proteinaceous components, generally introduced onto the virus envelope or cell membrane, Alternatively, it binds (and is exposed on the main component) to other components.

Receptor-specific molecules may be introduced onto the virus or cell surface by other means. Can also be done. In this case, receptor-specific molecules can be chemically isolated from viruses or cells. It is desirable to bind it to the surface, for example, (asialoglycoprotein receptor The galactose moiety (which is a ligand for By amination or via iminomethoxyethyl derivatives, covalently attached to surface proteins of cells or cells. Alternatively, receptor specific Isomeric molecules are added to the surface of viruses or cells through biotin, avidin, etc. It may also be chemically bonded to the surface component. For example, biotinylated receptors specificity molecules via avidin or streptavidin, Binds to biotinylated components on the cell surface.

Alternatively, the virus or cell may be chemically treated to form a receptor on its surface. The protein specific molecules may be exposed. In this case, surface carbohydrate polymerization The end that specifically recognizes the receptor and binds to the receptor by cutting the body with an enzyme Desired carbohydrate residues (eg, galactose residues) are exposed as residues.

For example, a given carbohydrate polymer can be treated with neurominidase to Expose the terminal galactose residue with a tri-branched sequence.

Viruses or cells modified as described above saturate the target cell receptors. and in a sufficient amount to maximize cellular uptake. ivO). Usually in a physiologically acceptable vehicle, such as normal saline. It is encapsulated and administered parenterally (usually intravenously).

Using the method of the present invention, nucleic acids (DNA or RNA) can be transformed in vivo. o) Or selectively delivered to target cells in vitro and intracellularly It can be expressed in the body. The nucleic acids mentioned here include foreign genes, gene regulatory factors, or anti-sensory inhibitors of gene function. modified according to the method of the invention. The nucleic acid is inserted into a transfected viral vector and directed to target cells. Outpatient Transporting genes in vivo or ex vivo Retroviruses are suitable examples of viral vectors used to Can be mentioned. Target-directed viral vectors are transferred in vivo as described above. iv) Upon administration, target cells selectively take up this vector.

Furthermore, the methods of the invention can be used to alter the natural tropism of an infectious agent.

For example, an environmentally tropic (species-restricted) factor can be modified so that it is normally infective in its native form. This agent can also be used to infect species that are not present. Infection targets of infectious agents Develop new experimental systems to study human infectious diseases by setting up , to correct genetic defects in vivo, cells that target the corrected gene in vivo).

Specified pathogens such as hepatitis virus and human immunodeficiency virus (AIDS virus) The virus infects only human cells.6 Using the method of the present invention, such viruses can be The virus is denatured so that it can infect experimental animals such as rodents. For example, human liver A hepatitis virus that only infects liver cells can now also infect non-human liver cells. , can be denatured. for the asialoglycoprotein receptor of rodents By introducing a ligand (e.g. galactose) onto the surface of the hepatitis virus, Forms a modified hepatitis virus that infects rodent liver cells. This degenerative hepatitis virus Because the virus infects rodents, infected rodents or rodent cells are used to treat the liver. It is possible to obtain an experimental animal model for conducting research on inflammatory viruses.

The present invention will be explained in more detail according to the following examples.

Satoran example le A typical retrovirus with host characteristics similar to those of a torovirus. An experiment was conducted using the system. The virus used is produced in the ψcre cell line. environmentally tropic and replication-deficient, containing the gene for bacterial β-galactosidase Moloney murine leukemia virus, developed by Dr. James Wilson of the University of Michigan. (Wilson, J. M., Ram μ and Natl., 8). cad, Sci, USA 87:439-443 (1990)), usually Under these conditions, the virus infects only rodent cells (Wilson, J, M, et al. Proc, Natl, Acad, Sci, USA85:301 4-3018 (1988); Goud, B. et al. 163:251- 254 (1988)). Virus-producing cells were incubated with 10% heat-inactivated calf serum (G IBCO Research Institute: Grand Island, New York) The cells were cultured in G.E. Eagle's medium (GIBCO). as impure as possible from serum proteins In order to obtain the virus with less contamination, the virus-producing cells were incubated for an additional 3 days. The cells were cultured in Durbetzko's modified Eagle's medium without serum. Next, get The surface of the virus was denatured using the following two methods.

A) Chemically attach galactose residues to the virus.

B) Chemically attach the asialoglycoprotein to the virus.

A. Retrovirus lactosamine Gaud's method (198 8)), the virus was isolated from the culture medium and simultaneously injected into lactose. Will OmM Tris-C1, 150mM NaCl, 1mM to allow binding. Sucrose (Sigma: St. Louis, MO) was α-lactated in EDTA. VT at 4°C for 17 hours. 4 using i550-tar (Beckman Laboratories: Sun Ramon, CA). 0. Ultracentrifugation was performed at OOOrpm (LB-55: Beckman). Perform centrifugation Before denaturation, samples were adjusted to various pHs of 7,4-8,4 to find the optimal conditions for denaturation.

After centrifugation, protein 3. Contains Omg (0.1 mg of virus-derived RNA). The final fraction of the lactose gradient was prepared as described above (C + oud, B et al. 16 Shadow 251-254 (1988)), Sodium Cyanoborohydride (Sig (ma) was reacted. After dialysis with minimum specific medium for 24 hours at 4°C, the test The sample was filtered through a 0.45μ filter (Gelman Sciences, Inc., Missouri). It was sterilized by passing it through a tube arbor. Before exposing the virus to cells, follow the manufacturer's instructions. As shown, protein assay method (Bio-Rad: Los Angeles, CA) After quantifying the virus present in the sample and performing RNA extraction, A test method (Munro, H, N, & Fleck, A, Meth, B A 10% solution was prepared by adding ioche. Samples except for stability experiments All were used immediately after preparation. NIH3T3 murine fibroblasts Viability of denatured virus samples was tested using limiting dilution virus stocks. by transfection assay (Danos, O, & Mulligan, R, C, Proc, Natl, Acad, Sci.

USA85:6460-6464 (1988)), stain positive cells with X-gal. (Sanes, J. R., et al. EMBOJ, 5:31 33-3242 (1986)).

To examine intracellular uptake, 10 μCi/ml of 265-methionine (American Rusham: Arlington Heights, IL) 50% serum free and 50% serum free Viruses were grown in 0% serum and methionine-free Durbetzko's modified Eagle's medium. Producer cells (5×10 6 cells) were cultured for 3 days to biosynthetically label the virus.

Isolate the virus from the supernatant, denature the virus as described above, and remove the virus from the minimal native medium. Dialysis was performed using the patient's body.

Faint and fallen leader We evaluated the effect of chemical denaturation on virus infection specificity. In this experiment, the following Several cell lines were used. Wistar Research, Philadelphia, Pennsylvania Human liver cancer cell line HepG2 obtained from B. B. Knowles: Asialo Glycoprotein receptor (+) (Schwartz, A, L, et al.) Lilith 256:8878-8881 (1981)), Bronk, New York. Entered from Albert Einstein College of Medicine, A., 5hafritz. Handcrafted 5KHe pl: Receptor (-), rat liver cancer cell line MOrriS 7777: Receptor (-) (Nu, G, Y, et al. L-Biol, Chem , 263 24719-4723 (1988)), and murine fibroblasts. NIH3T3: asialoglycoprotein receptor () (Goud, f3 ．． et al.Viro1o■16 Shadow 251-254 (198g)). last two cells The series is from the American Type Culture Collection (Rock, Maryland). Purchased from Bill. All cells were 10% heat inactivated at 37°C under 5% CO2. Stored in Eagle Minimum Specific Medium supplemented with fetal bovine serum.

Determination of viral sensation and Noh gene To examine whether the virus maintains its infectivity and functionality even after chemical denaturation. Two types of human cell lines and two types of sawtooth cell lines were infected with modified and unmodified viruses. The β-galactosidase activity of the cells was measured. First of all, 60 Falcon sauna, New Jersey, on a plastic plate of mm. 0.5-2.0x10G/ml of target cells It was applied at a density of In Durbetzko's modified Eagle's medium, the same amount, i.e. 16.7 μg RNA (0.5 mg viral protein) of denatured virus and intact virus. Denatured virus was added to the culture medium and exposed to cells for 5 days at 37°C under 5% COa. did.

Gorman's method (Gorman, C, Takumishi μμEsara 2nd edition, Glover, D, M, IRL Press, Washington DC, C, pp157-158 (1986)), by measuring the expression of foreign genes, Reduced lactosidase activity. This operation will be briefly explained.

First, a monolayer of cells (approximately 10 cells/60 mm dish) was washed with phosphate buffered saline. and dissolved. Add 0.1 ml of the solution to 0-nitrophenyl-galactopyranosyl (ONPG: Sigma) and the reaction was terminated by adding Na2COa. Afterwards, β-galactosidase activity was measured based on absorbance at 420 nm. quantitative The results are shown using purified Escherichia coli β-galactosidase (Sigma) as a standard. Twoton and Coffin's method (Norton, P. A. & Coffin, J, M, Mo1. Ce11. Biol, 5:281-290 (198 5)), expressed in U/mg of cellular protein. Proteins in cell samples The protein concentration was determined using the Bio-Rad Protein Assay Kit (Bio-Rad Protein Assay Kit) according to the manufacturer's instructions. -Rad). 100-fold molar excess of natural asialoglycoprotein The virus is added to the cell culture medium along with Asiaroorosomucoid, a type of Anti-effects were investigated. Asialoorosomucoids are described in the literature (Whitehead, D. , H, & Sammona, HjC, Biochim, Bio h a, A cta 124:209-211 (1966)), Desialylation of somcoid (Oka, J.A. & Weigel, P. , H, L” Kyohei, Chem, 25Jl2 10253-10262 (198 3)). Untreated background corresponding to enzyme activity Measurements were made using cells, and background values were subtracted from the values of virus-treated samples.

All measurements were performed three times, and the results are average ± S, E.

It was shown in

From Table 1, the following results are derived. That is, as expected from the environmental tropism of the virus. Uniformly, the native virus is able to develop the enzyme in human HepG2 or 5KHepl cells. It showed no activity. In addition, the modified virus has 5KHe p1: asialosaccharide Protein receptor (-) does not show β-galactosidase activity in cells However, in human HepG2: asialoglycoprotein receptor (+) cells, 7 High β-galactosidase activity of 1.2±4.8 U/mg cellular protein 6.1. = By adding a large molar excess of asialoorosomucoid , this enzyme activity was completely inhibited. This is a transfection with a modified virus. The idea is that the action is mediated by the asialoglycoprotein receptor. It supports the idea. As expected from their environmental tropism, exposure to native virus Morr+57777 rat cells had a high β-galactin content of 50.6±5.2. It showed tosidase activity. Interestingly, this same cell was treated with the same amount of denatured virus. When exposed to , its β-galactosidase activity was significantly lower. similar trends However, it was also observed in murine NIH3T3 cells, which are naturally highly active. That is, the unaltered Wi The value of enzyme activity when exposed to virus is 56.7 ± 1.8, which is 56.7 ± 1.8 when exposed to modified virus. The value of 27°0±0. It was more than twice that of 9.

The binding reaction that binds lactose to proteins is accelerated under alkaline conditions. It is known that (Schwartz, B., A. & Gray, G. R, Arch, Biochetn, Dankutou 13 = 181 + 542-549 (1977)). However, such conditions are harmful to viruses. So Here, we determined the optimal pH to denature the vector while retaining its functionality. various By administering the virus denatured at a certain pH to HepG2 cells, β-galactosidase activity was measured. As shown in Table 2, the enzyme activity was denatured at pH 7.4. From the value 50.3 ± 1.2 for viruses, it is effective against viruses denatured at pH 8.0. The value increased to 71.2 ± 4.8, but it was not treated with the virus denatured at pH 8.4. In the treated cells, the enzyme activity was significantly reduced to 25.1±2.4.

- Confirm the histochemical and colorimetric results for galactosidase measurement and Determining the cell fraction that expresses the β-galactosidase gene when exposed to To do this, we used the method of Sanes et al. 86)), β-galactosidase in situ Activity was measured by histochemical staining. This operation will be briefly explained. First, 0. Cultured cells in a 35 mm dish containing 5-IXIO" cells were added in equal amounts, i.e. Denaturation of 8.4 μg of virus-derived RNA (0.3 mg of virus-derived protein) The cells were treated with virus or native virus for 5 days. Cells were treated with 0.5% glutamate. After fixation with Ruraldehyde (Sigma) phosphate buffered saline, 1 mM MgCl2 solution was added. 4-CI-5-Br-3-indolyl at 1 mg/ml. -β-galactosidase (X-gal) (BRL: Washington, D.C.), 5mM Soaked in phosphate buffered saline containing potassium ferricyanide, 1mM MgCl, Ta.

After incubation for 1 hour at 37°C, the dishes were washed with phosphate-buffered saline and the reaction Terminate the cells, count the positive (blue) cells using a light microscope, and compare the results to 10 It is expressed as the percentage of positive cells for the high power region.

Figure 1 shows the β-galactosidase activity of cells treated with various virus samples. The results of in situ (in 5 itu) staining are shown. As shown in panel B, unmodified As a result of staining virus-treated sawtooth NIH3T3 cells with X-gal, 12 ．． 6% were positive for β-galactosidase activity. In this case, untreated NIH3T The background staining values for 3 cells were at undetectable levels as shown in panel A. Met. We also exposed cells to modified virus under the same conditions as shown in panel C. When tested, only 3.6% were positive. Regarding human 5K Hapl cells , in the untreated case shown in panel H, and in the case exposed to native virus shown in Banerue. Also, when exposed to the modified virus shown in panel J, the staining value was , the level was undetectable. treated with native virus as shown in panel E. Similarly, HepG2: asialoglycoprotein receptor (10) cells showed significant The modified virus shown in panel F did not show β-galactosidase activity. Significant staining was observed in treated HepG2:receptor (+) cells.

As a result of counting using a microscope, 36.4% of HepG2 cells had no detectable enzyme. It had activity. When a 100-fold molar excess of asialoorosomucoid was added , intracellular uptake by the asialoglycoprotein receptor, as shown in panel G. A competitive reaction against Sul occurred, and color development was completely suppressed. This is a transfer Involvement of asialoglycoprotein receptor in the process of It is something.

7 Luz Lily Minori Whether or not the modified virus is actually taken up by cells, and if it is taken up. If so, is the asialoglycoprotein receptor involved in uptake? , and the concentration of HepG2.5KHe in Asahi 5.0 x 106 pieces/35 mm dish. pl, and Morris7777 cells, 6. Will of lX10’cpm/mg Virus-derived denatured virus biolabeled with 265 and has specific activity of SRNA. Contains 3.3μg of RNA (98μg of virus-derived protein), serum-free Culture was carried out at 37°C using Dulbecco's modified Eagle's medium (Watanabe , N, et al. Cancer rmmunol, Immunother, 28: 157-163 (1989)), after a predetermined period of time, the medium is removed and the cells was cooled at 4°C and the cooled solution containing 1 mg/ml bovine serum albumin was added. % specific medium. The radioactive substance bound to the surface was removed with 0.4% trypsin, 0. Remove with 0.5 ml of chilled phosphate buffered saline, pH 7.2, containing 2% EDTA. and separated from the cells by centrifugation. Cell pellet with 0.2N NaO H and XPoly-Fluor (Patsu Card: Chicago, IL). , tryfusin-EDTA resistant (internalized) radioactivity was measured using a scintillation counter (T RI-CARB4530: Paracard) (Schwartz, A. L., et al. L.J. Sharp: 8878-8881 (1981)). Also, non-specific uptake was measured in the presence of a 100-fold molar excess of asialoorosomucoid. , specific uptake was calculated as the difference between 1 measurement and non-specific measurement. measurement All measurements were performed in triplicate, and the measurement results showed that the virus-derived RNA Expressed as mean value of /10' cells±S, E.

As shown in Figure 2, among two human cell lines and one sawtooth cell line, human HepG2: Only asialoglycoprotein receptor (+) cells showed significant labeling. showed specific uptake of the virus. EDTA and trypsin resistance counts are Rise linearly with increasing incubation time; during incubation Approximately 800 ng viral protein/hour/106 cells when the time is 120 minutes. reached. These data indicate that the expression of the galactosidase gene by the degenerated virus is In fact, this is due to internalization of the virus by the asialoglycoprotein receptor. This supports the concept that

Stability of production The stability of the modified virus was measured. First, a freshly prepared sterile denatured virus sample is The samples were cultured in serum-free Dulbecco's modified Eagle's medium at 4°C and 25°C. did. After the predetermined time(s) have elapsed, the sample is added to the HepG2 cell culture medium and further After culturing for 5 days, the β-galactosidase activity of the cells was determined by the colorimetric assay described above. It was measured. All measurements were performed in triplicate and the results were expressed as a function of incubation time. The mean value of 0.7 mg cellular protein, normalized by the amount of virus added, as It is shown as a value of ±S, E. Table 3 shows the enzyme activity at 4°C and 25°C.

As is clear from the table, the enzyme activity decreased with incubation time and 48 After hours, the activity decreased to about 50% of the initial activity.

Binding of lactose-proteins to the target artificial asialoglycoproteins This is based on the specificity of sodium hydride, and the combination of an alde7do group and an amino group. The Schiff bases formed in between are reduced, making the bond irreversible. However, c Treating viruses with aldehydes does not always have a positive effect.

For example, formaldehyde is used to inactivate viruses during the vaccine manufacturing process. (Buynak, E. B. et al., Am, Med. As5oc, 235:2832-2834 (1976)). shown here Based on the data, it is possible to cause the virus to denature under the conditions described. This not only changes infection specificity but also maintains virus-derived gene expression. This will lead to the result that I understand that. Furthermore, it retains its functionality even after denaturation. The production of denatured virus increases as the pH of the denaturation reaction increases up to about pH 8. However, it has been shown that at higher pH, the functionality of the virus decreases. It will be done.

Most retroviruses normally enter cells by intracellular uptake. It is known that genetic material is introduced during the acidification process in this pathway. It is believed that (Andersen, K., B. & Nexo, P. A. Watashidan 125:85-98 (1983)). transports the ligand to the lysosome By transporting the asialoglycoprotein into the cell, it is ultimately degraded. (Tolleshaug, H, et al. 4■ Engineering Acta 585 Near 1-8 4 (1979)), and at the early stage of the internalization process, prior to lysosome lysis, e.g. The intracellular uptake compartment of the endosome is acidified (Tycko, B. & M. axfield, R.F. Cell: 643-651 (1982)).

Acid exposure during this period is similar to the natural entry route for some viruses ( Nugsbaum O, & Loyter, A, FEBS Lett, 221:61-67 (1987)), prior to viral degradation, endosomal membrane provides the necessary conditions for acid-mediated thawing of the viral envelope ( Helenia, A, Biol, Cel] 51:181-186 ( (1984)), a modified virus retains the ability to introduce its genome into target cells. The fact that these factors are lost due to the process of chemical denaturation means that the functions of these factors in the virus are lost. This shows that there are no.

B, Rousseau asialo protein, 'low speed medium from virus-producing BAG cells Centrifuge and then high-speed centrifugation through a discontinuous sucrose gradient as previously described. The unpurified sample of the virus obtained by the separation was incubated at pH 7, 5, and 0.5°C at 4°C. Dialysis was performed in 9% saline. After dialysis, NH5-LC-biotin (Pierce Chem. Cal Inc. (Rockville, IL) was incubated for 4 hours at room temperature with the virus (0, Im g/ml) and the sample was again incubated at pH 7,5,0. 9% saline solution Serum orosomucoid derived from human serum dialyzed and pooled in Asahi was desialylated. Asiaroorosomucoid (AsOR) was obtained (Whitehead, D. H, & Sammons, H, P, Biochem, Biohs, Acta 124:20X (+9 66)), As0RO, 1 mg was added to virus 1. After adding Omg and mixing thoroughly Add avidin at a ratio of 1.0 mg to 1 mg of virus and incubate for 4 hours at room temperature. fed. After dialysis of the complex using modified Eagle's medium, Sephadex G150 The complex virus was purified using a molecular sieve column. here , a +26I-labeled virus complex of asialoorosomucoid was prepared and purified. I met the conditions. From the graph in Figure 3, it can be seen that in fraction numbers 32 and onwards, asialium is removed from the column. It can be seen that only rosomucoid is eluted. Also, the absorption at 280 nm Elution of avidin detected by photointensity was slightly observed after fraction number 33. It will be done.

The unlabeled virus itself, compared to either of the other two proteins, It is quite large and eluted early, and its peak is present in fraction number 29. this The column completely separates the virus from asialoorosomucoid and avidin. I was able to do it. Furthermore, from Figure 3, it is clear that this virus mediates biotin-avidin binding. As a result, it forms a complex with AsOR labeled with 126I, and the radioactivity from AsOR moved to the same position as expected for untreated virus, fraction number 29. It can be seen that there is a peak at These data are labeled Asiaro A portion of orosomucoid binds to the virus and moves along with the virus through the column. It shows that you are doing it.

This complex is then used to specifically target gene expression to asialoglycoprotein. We investigated whether it is possible to set the quality receptor (+) in cells. Conjugate U virus, HepG2: receptor (+), Huh-7: receptor (+), 5 KHe p1: Human liver cancer receptor (-)t Mahlavu: Receptor (-), and Morris7777: 5 types of rat liver cancer receptor (-) cells with each of the cell lines for 10 days. In FIG. 4, it is shown in lane 1.

HepG2: receptor (+) cells treated with military body, 2.3 units/cell It had β-galactosidase activity at the level of 1 mg of protein. This value is , corresponding to about 50% of the activity of the virus-producing cell line BAG shown in lane 11. Ma The activity level of untreated HepG2 cells was 1.81 units/mg. . Huh-7:receptor (+) cells treated with the conjugate (lane 3) Will biotinylated in the absence of asialoorosomucoid Higher levels of β- The galactosidase activity value was 3.8 units/mg. The activity value of lane 4 is The activity level was not significantly different from that when the same type of cells were not treated at all (lane 5). . Mahlavu:receptor (-) cells treated with the conjugate shown in lane 6 , significant β-galactosidase activity compared to the same type of untreated cells shown in lane 7. did not show gender. Similarly, lanes 8 and 9 have FIA Morr Treatment of 1s7777 cells with military agents or absence of asialoorosomucoid The results of treatment with biotinylated virus are shown below. lane 8 and Lane 9 shows significant differences compared to the same type of untreated cells shown in lane 10. It did not show any significant β-galactosidase activity. 5K Hepl cells are receptor -(-)M.

It showed the same tendency as rris7777 cells.

In the staining process described in Example 1, HepG2 cells treated with military service virus showed the same blue coloration as similarly treated Huh-7 cells, but The cells that were treated did not develop any color.

Last example Hepatitis B virus (HBV) chemically inhibits the raw silk characteristics of hepatitis B virus (HBV). Viruses are human pathogens with very narrow host (species) and organ (liver) specificity. be. Unless there are unusual and highly artificial conditions, such as high concentrations of corticosteroids, Human hepatocytes or hepatoma cells in culture cannot be infected with HBV. When culturing viruses in vitro, as in the case of culturing viruses from seeds, There are also very strict conditions.

However, there are many people who have fallen into trouble. Selles et al.'s method (Selles et al. Proc, Natl, Acad, Sci , 84:1005-1009 (1987)). Hepatitis B virus (HBV) was extracted from the infected HepG2-producing cells and 0418 was added at a concentration of 380 mg 7 ml with 0% heat-inactivated calf serum. The cells were maintained in Durbetzko's Modified Eagle's Medium (MEM) containing at least 100% of the total volume. Two types of human figures The cell lines were cultured to examine the infectivity and specificity of native and modified HBV.

Huh7 human hepatoma cell line with asialoglycoprotein receptor and IMR-9σ fibroblasts, which do not have sialoglycoprotein receptors, were Stored in Durbetzko's modified Eagle's minimal specific medium supplemented with fetal bovine serum (FBS). Ta.

Dandan yΩ prisoner HepG2 cells were cultured for 3 days in serum-free medium. media at 200°r Centrifuge at pH 7, 4, 8, 0, or 8° to remove tissue debris. At 4, the supernatant was treated with a 10-20% lactose gradient. After that, VTi50 - Ultracentrifuge the supernatant at 4°C and 40,000 rpm for 16 hours. The virus was isolated by pelleting.

Dandan yΩ main productivity The obtained HBV (protein 3.0mg) was prepared according to the same method as in Example 1. , using 10 mg of sodium cyanoborohydride at 25°C for 3 hours. It was converted into cutosamine. Filtering the modified virus through a 0.45 μm membrane After sterilization, a membrane with a molecular weight exclusion limit of 12 to 14,000 is The solution was dialyzed with MEM and further dialyzed with MEM supplemented with ]0% FBS.

-Skinny feeling due to HBV Huh7 and IMR-90 cells were incubated at concentrations of 25-50% at 35 or 100 It was smeared onto a plastic dish with a diameter of mm. Remove cell culture medium and remove denatured or The medium was replaced with a medium containing native virus and cultured at 37°C. Wash the cells and Replace the medium with new medium every 3 days and regularly measure the HBV-DNA present in the cells. did. Furthermore, hepatitis B surface antigen (HBsAg) present in the medium was measured.

e@ of target HBV-DNA in Huh7 cells treated with intact and sexually active HBV, 3:Z303-'231z (1976)), DNA was extracted from the cells.

This method will be briefly explained. First, cells were soaked in 10 ml of chilled Tris-buffered saline (TB). After washing twice with S), it was scraped into TBS and centrifuged at 20 Orpm. . The resulting cell pellet was treated with 10 mM Tris-HCl, pH 8.0 and After resuspension in 1mM EDTA of O, 20mg/ml RNase, 0.5% S The same buffer containing DS was added plus treatment with proteinase. Phenol extraction Thereafter, cellular DNA was isolated by ethanol precipitation. The obtained DNA was transferred to HB V sequence (obtained from Dr. Sheikh Lien of Massachusetts General Hospital) In addition, the BamHI restriction fragment of plasmid adwHTD carrying the HBV genome was Southern analysis using a γ”P-ATP-labeled cDNA probe specific for ・Measure by plotting method When investigated using an HBV-specific cDNA probe, Southern blot analysis showed that No hybridizable sequences were found. Under normal cell culture conditions, this This confirms the above report that human-derived Huh7 cells are not infected with native HBV. It is shown that. On the other hand, by treating Huh7 cells with denatured HBV, even for just one day, When cultured, Southern blotting as expected for the plasmid sequence , a signal in the region where strong hybridization is possible is detected. IMR-90: Asiaro Glycoprotein (−) cells do not contain hybridizable sequences under any conditions. There was no production.

In the supernatant liquid of Huh7 and IMR-90 microorganisms exposed to virgin or hepatic HBV. Output of HBsA in Culture media for Huh7 and IMR-90 cells were prepared as denatured or undenatured as described above. After culturing with HBV and after a predetermined period of time, enzyme immunoassay kit (Auszume HBsAg was measured using the following method (Monoclonal). Measurement conditions are manufacturing The recommendations of the vendor Abbo t-Labs were followed.

As shown in Table 4, the background color absorbance was , about 0.121, and no particularly significant difference was observed between the 1st and 7th day.

Native HBV does not produce significant amounts of HBsAg and has an absorbance of approximately 0.180. It was. Similarly, in IMR-90 cells, the color absorbance reflecting the HBV level was , was less than 0.110, but Huh7 cells treated with denatured HBV HBsAg was released and its absorbance was between 0.760 and 0.865.

Takasato example It will be obvious to those skilled in the art that the method of the present invention described above goes beyond the gist of the invention. It is possible to make various changes and modifications within the scope of , covered by the claims below.

Table 1 -Galactosidase activity β-galactosidase activity of mucus exposed to Rusu Average value ± S, E, (U/mg) AsG Undenatured Denatured Denatured Receptor virus virus virus + status ASOR-.

cell line (source) He pG2 (+) 1.8f1.9 71.2±4.8 2.9±1.1( human) SK Hepl (-) 1.7±3.4 0.8±4.6 1.6±2.5( human) Morr 1s7777 (-) 50.6±5.2 16.3±4.4 15 ．． 7±4.7 (rat) NIH3T3 (-) 52.1±4.9 15.4±1.1 16.3±3. 9 (rat) 12. After denaturing the virus at pH 8.0, incubate it with cells for 5 days. I started.

*: Calculated as the difference in activity between treated and untreated cells.

**: 100-fold molar excess of Asialoorosomucoid (ASOR) AsG: asialoglycoprotein Table 2 In He G2 cells, rus transfection was carried out twice. Ωkon! Specific β-galactosidase activity (Above average values S, E, U/mg protein) Denatured virus Denatured virus + Asialoorosomucoid°.

H 7,050,3±1.2 6.4±1.98.0 71.8±4.1 4.9± 0.48.4 25.1±2.4 0.0±1.6*: 5 days for degenerated virus after exposure **: Test treated with modified virus + 100-fold molar excess of asialoorosomucoid. β-galactosidase activity in the body Table 3 Hisoren redundancy caused by childbirth Temperature Incubation time Specific activity (average value + S, E.

U/mg protein) 4℃ 0 hours 50.3±1.2 24 hours 42.0±5.6 48 hours 22.6±2.5 25℃ 24 hours 37.1±2.8 48 hours 17.6±1.1 *: After exposure to denatured virus for 5 days.

Specific β-galactosidase activity was determined between samples treated with virus alone and samples treated with denatured virus. and samples treated with a 100-fold molar excess of asialoorosomucoid. is calculated.

Person A Hepatitis B surface antigen (HBsA) in culture medium determined by Auszyme assay. g) level (absorbance unit) cell 1, 121±, 054. 135±, 017. 850±, 0103. 18 6±, 036. 700±, 0I25, 171±, 010. 865±, 05 37. 110±, 023. 764±, o67 blue dextran MuL V (OD2Bo) epG2 1. Conjugate 2.29 soil 0.16 2. Unsolved’!　1.81±0.01 Mohlavu 6. Conjugate 1:04 Sat 0.13 7, Not whts O, 89 Sat 0.09C7777 8. Conjugate 0.75 soil 0.01 s, Bio-MLV O, 99 Sat 0.3810 Song MA 0.80±0.07 AG 10. Oh, l! 5.00 Sat 0.08 Copy and translation of written amendment) Submission (Article 184-8 of the Patent Law) March 31, 1993

Claims (40)

[Claims] 1. targeting of viruses or cells for the purpose of internalization into target cells. A method for determining the surface receptor of the target cell on the surface of the virus or cell. By introducing a molecule that binds to the receptor, it binds to the receptor and becomes active in the living body. Expressing the nucleic acid that is selectively internalized and transported by the cells in vivo A method characterized by producing a modified virus or cell that causes 2. the virus or cell is a bacterium, a protozoan, or a mammalian cell; 2. A method according to claim 1, characterized in that: 3. When the virus or cell is in its native form, it usually enters the target cell. 2. The method according to claim 1, characterized in that the method is not localized. 4. The virus or cell is a human pathogen and the target cell is a non-human pathogen. 4. The method according to claim 3, wherein the cell is a foreign cell. 5. A method for internalizing viruses or viral components into target cells. on the surface of the virus or viral component that binds to the receptor of the target cell. By introducing a molecule that binds to the receptor and selectively binds to the cell. producing modified viruses or viral components that can be internalized into cells. How to characterize it. 6. 6. The method of claim 5, wherein the virus is infectious. 7. characterized in that the replication ability of the virus or viral component is incomplete; 6. The method according to claim 5. 8. 6. The method according to claim 5, wherein the virus is a retrovirus. Method. 9. Said virus or viral component, in its native form, infects said cell. 6. The method according to claim 5, characterized in that: 10. The virus is a human pathogen and the target cell is a non-human cell. 10. The method according to claim 9, characterized in that: 11. 6. The virus according to claim 5, wherein the virus is a pathogen of hepatocytes. the method of. 12. Claim 10, wherein the virus is a hepatitis virus. the method of. 13. said receptor mediates intracellular uptake of said molecule by said cell 13. The method according to claim 12, characterized in that: . 14. the receptor is an asialoglycoprotein receptor; The molecules introduced onto the surface of the virus or virus component are the asialoglycoproteins. is a ligand for proteins, and the target cell has an asialoglycoprotein receptor. 14. The method according to claim 13, further comprising: -. 15. The ligand for the asialoglycoprotein receptor is galactone. or N-acetylgalactosamine, which acts as an asialoglycoprotein receptor. 14. The target cell having - is a hepatocyte. Method. 16. The molecules bond to the surface of the virus or virus components by chemical bonding. Method according to claim 5, characterized in that it is introduced above. 17. Targeting cells with asialoglycoprotein receptors for viral infection on the surface of the virus in a manner that targets the asialoglycoprotein receptor. cells with asialoglycoprotein receptors can be A method characterized by producing a modified virus that infects cells. 18. The ligand for the asialoglycoprotein receptor is lactose. The method according to claim 17, characterized in that it is alternatively galactose. 19. The cell having the asialoglycoprotein receptor is a hepatocyte. 18. The method according to claim 17, characterized in that: 20. the virus is a human pathogen and the cell is a non-human cell; 18. The method according to claim 17, characterized in that: 21. 21. The method according to claim 20, wherein the virus is a hepatitis virus. the method of. 22. The surface of cells that are not normally infected by the native form of the virus. A modified virus or a component thereof that has molecules on its surface that bind to the component, A degenerative virus that binds to the cell and is internalized inside the cell. Rus or its components. 23. A cell surface component of the cell is a receptor that mediates intracellular uptake by the cell. The modified virus according to claim 22, which is a scepter. 24. the receptor is an asialoglycoprotein receptor, and the molecule is characterized in that it is a ligand for the asialoglycoprotein receptor. The modified virus according to claim 23. 25. 23. The modified virus according to claim 22, which is a human pathogen. 26. The modified virus according to claim 25, which is a hepatitis virus. 27. modified containing lactose or galactose-terminated carbohydrates on its surface hepatitis virus. 28. A method of introducing a nucleic acid into a cell, comprising: a) a molecule that binds to a surface component of the cell; A virus consisting of a modified virus or viral component that has on its surface Incorporating the nucleic acid into the virus vector, b) bringing said viral vector into contact with a cell; A method characterized by internalizing the introduced nucleic acid into the cell and expressing the introduced nucleic acid. 29. In native form, the virus or its components usually enter the cell. 29. The method of claim 28, wherein the method is non-infectious. 30. 29. The method according to claim 28, wherein the nucleic acid is an expressible gene. Method. 31. Claim 28, wherein the virus is a retrovirus. How to put it on. 32. The molecules introduced onto the surface of the virus or viral components are is a derivative of tosose, and the cell surface component is an asialoglycoprotein receptor. and the cell has an asialoglycoprotein receptor. 29. A method according to claim 28, characterized in that: 33. The cell having an asialoglycoprotein receptor is a hepatocyte. 33. The method of claim 32, characterized in that: 34. Human viruses that, in their native form, cannot normally infect animal cells. A method of infecting animal cells with a virus that binds to the animal cells and infects the cells. a modified animal cell having molecules on its surface that bind to surface components of the animal cell, such that by forming a human virus and contacting said modified virus with said cell. , binding the modified virus to the cell to infect the cell. How to make it a sign. 35. Claim 34, characterized in that the human virus is a human pathogen. How to put it on. 36. The animal cell and the modified virus come into contact in vivo. 35. A method according to claim 34, characterized in that: . 37. The animal cell and the modified virus come into contact in vitro. 35. A method according to claim 34, characterized in that: 38. An animal cell that is infected with a modified human virus, and the native form of said virus are uninfected animal cells. 39. Claim 38, characterized in that it contains hepatocytes infected with hepatitis virus. animal cells. 40. Animals infected with a modified human virus that are not susceptible to the native form of the virus. Animals that do not dye.