CA2092323A1 - Targeting viruses and cells for selective internalization by cells - Google Patents
Targeting viruses and cells for selective internalization by cellsInfo
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- CA2092323A1 CA2092323A1 CA 2092323 CA2092323A CA2092323A1 CA 2092323 A1 CA2092323 A1 CA 2092323A1 CA 2092323 CA2092323 CA 2092323 CA 2092323 A CA2092323 A CA 2092323A CA 2092323 A1 CA2092323 A1 CA 2092323A1
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- virus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
- C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
- C12N2740/13041—Use of virus, viral particle or viral elements as a vector
- C12N2740/13045—Special targeting system for viral vectors
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
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- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
- C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
- C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
- C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins
Abstract
Viruses or cells are targeted for selective internalization into a target in vivo. A molecule specific for a receptor on the surface of the target cell is introduced onto the surface of the virus or cell. The modified virus or cell binds the receptor in vivo) and is internalized by the target cell. The method provides vectors for selective delivery of nucleic acids to specific cell types in vivo and a means to alter the tropism of an infectious agent.
Description
~-)92/()6180 PCr/US~)l/0~103 2 3 ~ 3 TAP~GETIMC VIRUSES AND CELLS FOR
SELECTIVE INTERNALIZATION BY CELLS
Back~round of the Invention Viruses represent a natural and efficient means 05 for the introduction of foreign genes into cells.
For this reason, they are useful tools for the study of genes, and gene regulation n vitro and ~or gene therapy. However, most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene e~pression in many tissues, some of which may be undesirable, especially for clinical applications.
Generally, viral infection is mediated by interactions between viral enve].opes and plasma -~
15 membranes of target cells. In many cases, specific -~
viral structures are recognized and bound by ~-cellular receptors. For e~ample, HIV employs envelope glycoproteins to bind l:o helper T
lymphocytes via CD4 (T4) receptors. Dalgleich, A.G., et al. Nature ~ 763-767 (1984). These interactions have been shown to be responsible for the observed species and organ specificity.
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Some investigators have shown that virus specificity can be redirected by attaching antibodies to viruses. For e~ample, Goud, B., ~t al. ViroloqY 163:251-254 (1988) linked 05 anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor. However, while binding and internalization occurred, infection and replication did not. A means for targeting viral or other types of nucleic acid vectors containing foreign genes to a target cell and obtaining infection and replication of the virus would be useful in gene therapy.
Summarv of the Invention The invention pertains to a method of targeting a virus or a cell to a target cell for select:ive internalization in YiVo (or in _~EQ) by the cell and to modified viruses and cells which are targeted for selective internalization by a target cell.
A virus or cell is targeted to the target cell for internalization by introducing a receptor- -specific molecule onto the surface of the virus or cell to produce a modified virus or cell which specifically binds to a receptor on the surface of 25 the target cell. The modified virus or cell can be administered to an organism where it binds selectively to the receptor of the target cell. The receptor-binding results in internalization by the target cell.
. , , , ~ , , WO92/0618~) 2 U 9 i~ rlUs~ 7l(~3 The cellular receptor can be a receptor which mediates endocytosis of a bound ligand such as the asialoglycoprotein receptor of hepatocytes and the receptor-specific molecule can be a natural or 05 synthetic ligand for the receptor. The receptor-specific molecule can be introduced onto the surface of the virus or cell (e.g., onto a viral envelope or cellular rnembrane) by chemically couplin~ it, either directly or through bridging lO agents, to the surface or by treating the surface to e~pose the molecule for receptor recognition.
The method of this invention can be used to produce viral or cellular vectors for selective delivery of material such as nucleic acid ~genes) to 15 a target cell. For example, exogenous genes can be incorporated and expressed selectively in a target cell. These vectors can be used in gene therapy and in other applications which call for selective genetic alteration of cells.
The method also provides a means for altering the natural tropism of an infective agent such as a virus or bacterium. An infective agent can be modified so that it will infect ia cell which, in unmodified form, it would not normally infect. In 25 this way, animal mod~ls of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases. For example, an ecotropic human pathogen (such as the hepatitis or AIDS virus) can be modified to infect a 30 non-human host to produce an e~perimental system for study of the pathogen and the disease.
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W092/06lX0 ~ ~ PCr/US9l/07l0 ~ -4-Brief DescriPtion of the Fiaures Figure 1 shows in situ B-galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified 05 murine leukemia virus.
Figure 2 shows internalization of 35S-biolabeled modified Moloney murine leukemia virus.
Figure 3 shows a chromatogram of asialooro-10 mucoid-complexed Psi2 virus on Sephadex G150.
Figure 4 shows the B-galactosidase activity of various cells exposed to Psi2 virus-asialoglyco-protein conjugate.
Detailed Descri~tion of the Invention A virus or cell is targeted for selective internalization into a target cell by modify;ng the surface of the virus or cell to lntroduce a molecule which specifically binds to a surface receptor of the target cell. The cellular surface receptor is 20 one which will mediate internali;zation of the targeted virus or cell. The modified virus or cell binds to the receptor of the target cell in vivo and is internalized by the cell.
According to the method of this invention, 25 viruses can be modified to infect specific target cells. Such modified viruses can be used to selectively deliver exogenous, functional DNA to a target cell in order confer a new biological or biochemical property upon the cell or to abrogaté an 30 existing property. In addition, the tropism of a virus can be altered or redirected to targat infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form.
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WO 92/06180 PCT/US91/1371(~3 - 5 - ~ ~3~ .J~
A variety of different enveloped viruses can be targeted by the method of this invention. The viruses can be RNA (retroviruses) or DNA viruses (e.g., hepatitis virus, adenovirus). The virus can 05 be replication defective or otherwise defective in structure or function. For e~ample, viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted.
10The present method al50 provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose -tropism can be altered. In addition, mammalian cells can be targeted.
15The receptor-specific molecule can be a ligand for the surface receptor of the target sell.
Preferably, the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as 20 the asialoglycoprotein receptor of hepatocytes.
Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules. For specific targeting to hepatocytes, asialoglycoprotein (galactose-terminal~ ligands are 25 preferred. Examples of asialoglycoproteins include ~ -asialoorosomucoid or asialofetuin. Other useful galactose-terminal carbohydrates for hepatocyte ~
targeting include carbohydrate trees obtained from ~;
natural glycoproteins, especially tri- and 30 tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to espose terminal galactose residues~ In addition, naturally occurring plant carbohydrates such as arabinogalactan can be used.
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WO92/06180 ~ Pcr/us9l/n71 For targeting other receptors, other types of carbohydrates can be used. For example, mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to 05 target macrophages or endothelial cells.
Other receptor ligands such as peptide hormones could also be used to target viruses or cells to corresponding receptors. These include insulin, glucagon, gastrin polypeptides and their respective l0 receptors.
Alternatively, the receptor-specific molecule can be a receptor or receptor-like molecule, such as an antibody, which binds a ligand (e.g., antigen) on the cell surface. Antibodies specific for cellular lS surface receptors can be produced by standard procedures.
The receptor-specific molecule is introduced onto the surface of the virus or cell so that it will be recognized by the cognate cellular surface 20 receptor. For esample, the receptor-specific molecule can be introduced onto t]he envelope of a virus or the membrane of a cell. In general, the molecule will be coupled to (or exposed on~ a proteinaceous component of the surface but other 25 components may be used.
The receptor-specific molecule can be introduced onto the surface of the virus or cell by different means. Preferably, the receptor-specific molecule is chemically coupled to the surface. For 30 example, galackose moieties (ligand for the asialoglycoprotein receptor) can be covalently coupled to viral or cellular surface proteins by lactosamination, reductive amination, or via -: , , ,: . . . .
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WO92/0618() 7 2 ~ ~ 2 3 2 3 PCr/US~)''0~103 iminomethoxyethyl derivatives. In other embodiments, the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as 05 biotin and avidin. For instance, a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
Alternatively, the virus or cell can be lO chemically treated to expose a receptor-specific molecule on the surface. Surface polycarbohydrates can be enzymatically cleaved to expose desired carbohydrate residues (e.g., galactose residues) as terminal residues for specific receptor recognition 15 and bindin~. For e~ample, neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
The modified virus or cell .Ls administered n 20 vivo, generally in an amount suf~icient to saturate receptors of the target cell and thereby maximize uptake by the cell. They can be administered parenterally (typically intravenously) in a physiologically acceptable vehicle such as normal 25 saline.
The method of this invention can be used to selectively deliver nucleic acid (DNA or RNA) to a target cell in vivo (or in yitro~ so that it is expressed in the cell. The nucleic acid can be an 30 exogenous gene, a genetic regulatory element or an antisense inhibitor of gene function. The nucleic acid is incorporated into a viral vector which has been modified, according to the method of this '': ,: '' ' ~
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invention, to target it to the cell. Preferred viral vectors for delivery of foreign genes in VlVO
(or ex vivo) are retroviruses. The targeted viral vector is administered in v v~, as described, where 05 it is selectively taken up by the target cell The method of this invention can be used to alter the natural tropism of an infectious agent.
Ecotropic (species-restricted) agents can be made to infect species which they normally, in unmodified lO form, do not infect. The ability to target the infectivity of an infectious agent can be used to develop new experimental systems for the study of human infectious diseases to produce cells that can correct genetic defects in vivo, or target a 15 corrective gene n vivo.
Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells. By the method of thi.s invention, such viruses can be modified to enable them to infect 20 e~perimental animals such as rodents. For example, the hepatitis virus which infects only human liver cells, can be modified so that it will infect non-human liver cells. To deuelop rodent models of hepatitis, for example, a ligand for rodent 25 asialoglycoprotein receptor (e.g., galactose) can be introduced onto the surface of the hepatitis virus.
This yields a modified hepatitis virus which will infect rodent liver cells. This modified hepatitis virus which can infect a rodent and the infected 30 rodent or rodent cells, provides an experimental animal system for study of the hepatitis virus.
WO92/0618(~ 2 3 9 2 3 2 ~ P~l/US91/07103 The invention is illustrated further by the following e~amples.
Chemical Modification and Alteration of Host Cell 05 SPecifi~i~Y of a Retrovirus A model retroviral system was used. The virus, an ecotropic, replication-defective, Moloney murine leukemia virus containing the gene for bacterial ~-galactosidase produced in a ~ cre cell line was lO kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., et al. Proc. Natl. Acad.
Sci. USA 87:439-443 ~l990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M., et al. Proc. Natl. Acac~ Sci.
15 ~ 3014-3018 (198R); Goud, B., et al. ViFoloqy 163:251-254 (198B). The producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO
Laboratories, Grand Island, NY) ~upplemented with lO% heat-inactivated calf serum (GIBCO). To prepare 20 virus with as little contamination as possible from serum proteins, producer cells wlere cultured in serum-free Dulbecco's modified Eagle's medium for 3 days. Using this viral preparation, two strategies were developed for the modification of the surface 25 of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus.
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A. LACTOSAMINATION OF RETROVIRUSE:S
Virus was isolated from the culture medium according to the method of Goud, B., et al. Virolpqy 163:251-254 (1988), but modified to permit coupling 05 of lactose during the isolation procedure. In brief, virus-containing medium was applied on a 10-20% sugar gradient in which a-lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was 10 ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4C for 17 hours. Samples were adjusted to various pHs, 7.4-8.4, prior to centrifugation in order to determine optimal conditions for 15 modification. After centrifugation, the bottom fraction of the lactose gradient containing 3.0 mg of protein (0.1 mg viral RNA) was reacted with sodium cyanoborohydride (Sigma) as described previously. Goud, B., et ~11 Viroloq~ 163:251-254 20 (1988). Following dialysis against minimum essential medium at 4C for 24 hours, the samples were sterilized by passage through 0.45~ filters (Gelman Science Co., Ann Arbor, MI). Quantitation of the amount of virus present in samples prior to 25 exposure to cells was determined by protein assay (Bio-Rad, Los Angeles, CA) according to the manufacturer's instructions, and confirmed by RNA
assay (Nunro, H.N. and Fleck, A. Meth. Biochem.
Anal. 1~:113-176 (1966)) after RNA e~traction.
30 Chomczynski, P. and Sacci, N., Anal. Biochem, 162:156-159 (19~7). Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used , W092/06l80 ~ 3 2 ~
immediately after preparation. Viability of unmodified virus preparations was determined by transfection assays in NIH 3T3 mouse fibroblasts using limiting dilutions of viral stock (Danos, 0.
05 and Mulligan, R.C. Proc. Natl. Acad. Sci. USA
~5:6460-6464 (1988)) and quantitated by determination of positive cells stained with X-gal.
Sanes, J.R., et al. EMBO J. 5:3133-3142 (1986).
For uptake studies, virus was biosynthetically ?
10 labeled by incubation of producer cells (5.0 x 106 cells) in 50% serum-free and 50% serum- and methionine-free Dulbecco's modified Eagle's medium containing 10 ~Ci/ml 35S-methionine (Amersham, Arlington Heights, IL) for 3 days. Virus was 15 isolated from supernatants and modified as described abo~e followed by dialysis against minimum essential medium.
Cells and Cell Culture To evaluate the effects of chemical 20 modification on viral infection specificity, several cell lines were employed: human hepatoma cell lines, HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., et al. J. Biol. Chem. 256:8878-8881 ~1981)) obtained from B.B. Knowles, Wistar Institute, 25 Philadelphia, PA; and SK Hepl, receptor (-) from D.A. Shafritz, Albert Einstein College, of Medicine, Bronx, NY; a rat hepatoma cell line, Morris 7777, receptor (-) (Wu. G.Y., et al. ~. Biol. Chem. 263: -4719-4723 (1988)); and a murine fibroblast cell line 30 NIH 3T3 (Goud, B., ç~ al. YiDQlQgY L~:251-254 (1988)) which is also asialoglycoprotein receptor (-). The latter two cell lines were purchased from .
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r~3 WV92/06l80 ~ 12- PCT/US9l/0710 American Type Culture Collection tRockville, MD).
All were maintained in Eagle's minimum essential medium supplemented with lO~o heat inactivated fetal bovine serum at 37C under 5% C02.
05 Assays for Viral Infection and Functio~al Gene ExPression In order to determine whether virus remained infectious and functional after chemical modification, the two human and two rodent cell lines were exposed to modified and unmodified virus followed by measurement of cellular ~-galactosidase activity. Target cells were plated at a density of O.5-2.0 x 105 cells/ml in 60 mm plastic dishes (Falcon Scientific Co, Lincoln Park, NJ). Equal 15 amounts, 16.7 ~g RNA, (0.5 mg viral protein) of modified and unmodified virus, in Dulbecco's modified Eagle's medium were added to the culture medium and e2posed to cells for 5 days at 37C under 5% CO2. Cells were assayed for B-galactosidase 20 activity as a measure of foreign gene expression accor~ing to the method of Gorman (Gorman, C. DNA
Clonin~, vol. 2 eds, Glover, D.M. IRL Press, Washington D.C~ pp 157-158 (1986)). In brief, cell monolayers (approximately lx106 cells/60 mm dish) 25 were washed with phosphate buffered saline, then `~:
lysed. The lysate, 0.1 ml, was reacted with o-nitrophenyl-galactopyranoside (ONPG, Sigma) and -B-galactosidase activity quantitated by absorbance at 420 nm after addition of Na2CO3 to terminate the 30 reaction. Results were expressed in U/mg of cellular protein according to the method by Norton, : . . . .
':
~. , WO9~/061X() -13- 2 0 ~ 2 3 2Pcr/us91/07l03 P.A. and Coffin, J.M. Mol. Cell._Biol. 5:281-290 (1985), using purified E. c~li B-galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were 05 determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions. For competition experiments, virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, 10 asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. ~ h~l. Chem. 258:
10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sammons, ~.G.
Biochim. BioPhvs. A~a 124:209-211 (1966).
15 Background enzyme activity was determined in corresponding untreated cells and subtracted from the values of viral-treated samples. All assays were performed in triplicate and the results expressed as mea~s + S.E.
Table 1 shows that unmodified virus did not produce enz~matic activity in human HepG2 or SK Hepl~;, cells as e~pected from the ecotropism of the virus. ~
Also, modified virus did n~t produce B-galactosidase ~ -activity in SK Hepl, asialoglycoprotein receptor (-) 25 cellsO However, modified virus did produce high B-galactosidase activity, 71.2 + 4.8U/mg of cellular protein, in human HepG2, asialoglycoprotein receptor (+) cells. Furthermore, this enzymatic activity was completely suppressed by addition of a large molar 30 e~cess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors. As e~pected from the ecotropism, B-galactosid3se . . , .. ., , ", .. . . . .
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~ " ~ ' , W092/06l80 ~ 'CrlUS~ )71~3 activity was high, 50.6 + 5.2. in Morris 7777 rat cells after exposure to unmodified virus.
Interestingly, B-galactosidase activity in these same cells was significantly lower when exposed to 05 the same amount of modified virus. The same tendency was seen in originally susceptible murine NIH 3T3 cell as enzymatic activity after exposure to unmodified virus, 56.7 + 1.8, was more than double that following exposure to modified virus 27.0 + 0.9.
The coupling reaction lin~ing lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. ~rch.
Biochem. Biophys. lBl:542-549 (1977~. However, such conditions could be detrimental to the virus. To 15 determine the optimal pH that results in modified, yet functional vectors, virus modified at different pHs were administered to HepG2 cells, and B-galactosidase activity measured. Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for 20 virus modified at pH 7.4; to 71.2 + 4.8, for virus - -~
modified at pH 8Ø However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
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w09t/06lxo -15- 2 3 9 ~ 3 2 3 Pcr~us91~o7lo3 Table 1 Cellular ~-Galactosidase ActivitY
Followina ExPosure to Viral Pre~arations~
~-Galactosidase Activity*
Mean i S.E. (U/mg) ,, :
____________________________________________~________ os AsG Unmodified Modified Modified :~-Receptor Virus Virus Virus + -Status ASOR~*
__________ ________________ _________________ _______ :~
Cell hine .:
(Source) .
______ __ , 10 HepG2 (huma~) ~+) 108 + 1.9 71.2 + 4.8 2.9 + 1.1 SK Hepl ; ~(human) ~-) 1.7 + 3.4 0.8 + 4.6 1.6 + 2.5 .: -Morris 7777 15 (rat) (-) 50.6 + 5.2 16.3 + 4.4 15.7 + 4.7 NIH 3T3 ~ -(mouse) ( ) 52.1 + 4.9 15.4 + 1.1 16.3 ~ 3.9 ~ .. -~., ~ -, .
+ virus was modified at pH 8.0 then incubated ~- with cells for 5 days. -20 * calculated as the difference in activity between :~
treated and,untreated cells.
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** asialoorosomucoid (ASOR) in 100-fold molar e~cess.
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WO 92/06l8n ~ Pcr/usl)l/n7l0~-~C~ -16-Table 2 Effect of the PH Durinq Modification of Viral ~ransfection in HePG2 Cells Specific ~-Galactosidase Activity (Mean ~ S.E. U/mg protein)*
. ' 05 Modified Virus Modified Virus + Asialoorosomucoid** -' PH
7.0 50.3 ~ 1.2 6.4 + 1.9 :
8.0 71.8 + 4.1 4.9 + 0.4 10 8.425.1 ~ 2.4 0.0 + 1.6 after 5 days of exposure to modified virus.
** B-galactosidase activity of samples treated with modified virus plus a 100-fold molar e~cess of asialoorosomucoid.
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WO92/061~0 PCr/lJS~l/07lU3 -17~ 2 ~ ~ 3 Histochemical Staininq to Demons~5 B-Galactosidase Activity To confirm the colorimetric results, and to determine the fraction of cells that expressed the 05 ~-galactosidase gene after exposure to viral samples, histochemical staining of in ~
B-galactosidase activity was performed according to the method of Sanes et al. EMBO J. 5:3133-3142 (1986). In brief, cultured cells in 35 mm dishes containing 0.5 -1 x 1o6 cells treated for 5 days with equal amounts, 8.4 ~g of viral RNA (0.3 mg viral protein), of modified or unmodified virus.
Cells were fixed in 0.5% glutaraldehyde ~Sigma), phosphate buffered saline, then incubated with 1 mM
MgC12, phosphate buffered saline, and overlaid with lmg/ml 4-Cl-5-Br-3-indoylyl-~-galactosidase ~X-gal) (BRL, Washington, D.C.), 5 ~M potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM
MgC12 in phosphate buffered saline. After incubation at 37C for l hour, the dishes were washed in phosphate buffered saline to quench the reaction and evaluated by counting positive (blue) cells under a light microscope a,nd the results expressed as the percent of positive/10 high power fields.
In situ staining for ~-galactosidase activity -in cells treated with various viral preparations is shown in Figure l. In rodent NIH 3T3 cells treated with unmodified virus, panel B, 12.6% were positive for ~-galactosidase activity using the X-gal stain.
Background staining in untreated NIH 3T3 cells was not detectable, panel A. After e~posure to modified virus, only 3.6% were positive under otherwise .,, , ~ , .
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identical conditions, panel C. Human SK Hepl cells that were untreated, panel H, or exposed to either unmodified virus, panel I, or modified virus, panel J, failed to develop detectable staining.
05 Similarly, HepG2, asialoglycoprotein receptor (~) cells treated with unmodified virus, panel E, did not develop evidence of significant B-galactosidase ;
activity. However, HepG2, receptor (+) cells treated with modified virus, panel F, did develop substantial staining. Microscopic counting revealed that 36.4 ~ of the HepG2, cells possessed detectable marker enzyme. The observed color development was completely suppressed by addition of a 100-fold molar excess asialoorosomucoid to compete for uptake by asialoglycoprotein receptors, panel G, indicating involvement of asialoglycoprotein receptors in the transfection process.
Assavs for Cellular Uptake_o~ Virus To determine whether the modified virus was actually taken up by cells and, if so, whether asialoylycoprotein receptors were involved, HepG2, SK Hepl and Morris 7777 cells, 5.0x105 cells/35 mm -dish, were incubated at 37C in serum-free Dulbecco's modified Eagle's medium containing 35S-biolabeled, modified virus, 3.3 ~g viral RNA
(98 ~g viral protein) (Watanabe, N., et al. Cancer Immunol. Immunother. 28:157-163 (1989)~ with a specific activity of 6.1~105 cpm/mg viral RNA. At various times, medium was removed, and cells were chilled to 4~C, washed with ice-cold minimum essential medium containing lmg/ml bovine serum albumin. Surface-bound radioactivity was stripped : ~ .
.
. ' . . ' ,~ . , W0~2/06l80 ~'Cr/US'JI/~ 03 ~ 03~3~3 with cold 0.5 ml phosphate buffered saline, pH /.2 containing 0.4% trypsin, 0.02% EDTA and separated from cells by centrifugation. The cell pellet was solubilized in 0.2 N NaOH and Poly-Fluor (Packard, 05 Chicago, IL), and trypsin-EDTA resistant (internalized) radioactivity was measured by scintillat.ion counting (TRI-CARB 4530, Packard).
Schwartz, A.L., e~ al J. Biol. Chem. 256:8878-8881 (1981). Non-specific uptake was measured in the presence of a 100-fold molar excess of asialoorosomucoid, and specific uptake calculated as the difference between total and non-specific measurements. All assays were per~ormed in triplicate and the results expressed as means + S.E.
in terms of ng viral RNA/105 cells as a function of time.
Figure 2 shows that, of the two human and one rodent cell lines, only the human HepG2 asialoglycoprotein receptor (+) cells demonstrated significant specific uptake of labeled virus.
Counts resistant to EDTA and tryE~sin, increased as a function of time and continued to rise lïnearly through 120 minutes of incubation at a rate of approximately 800 ng viral protein/hr/105 cells.
These data further support the notion that the observed expression of the galactosidase gene by modified virus was in fact due to internalization of the virus by asialoglycoprotein receptors.
StabilitY of Modified Virus To assess the stability of modified virus, samples of freshly prepared sterile, modified virus were incubated in serum-free Dulbecco's modified , , . ".
, , ~ ', WO92/06l80 c~ Pcr/ us~ 1/071 ~ 20-Eagle's medium at 4~C and 25C. At various times, samples were added to the medium of ~epG2 cells and incubated for 5 days. Cells were then assayed for B-galactosidase activity by colorimetric assay as 05 described above. ~11 assays were performed in triplicate and the results expressed as means + S.E.
in terms of U/mg cell protein normalized for the amount of virus added as a function of time of incubation. Table 3 shows that enz~matic activity at both 4C and 25C, decreased with time to approximately 50% of original activity after 48 hours.
Table 3 Stability of Modifiçd Virus '~
Specific Activity Temperature Time of (Mean ~ S.E.
Incubation U/mg protein)*
4C 0 hr 50.3 + 1.2 24 hr 42.0 + 5.6 4B hr 22.6 + 2.5 25C 24 hr 37.1 + 2.8 48 hr 17.6 ~ 1.1 * after 5 days of exposure to modified virus Specific B-galactosidase activity was calculated as the difference between samples treated with virus alone, and samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid.
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. ' . , , WO92/06180 YCT/~S91/07~J3 The coupling of lactose to proteins to target artiicial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino 05 groups to re~der the bonds irreversible. Treatment of viruses with aldehydes is not always similarly benign. For exarnple formaldehyde has been used to inaçtivate viruses in the production of vaccines.
Buynak, E.B., et al. ~. Am. Med. Assoc. 235:
2832-2834 (1976). The data presented here indicate that under the conditions described, the modification process results not only in altered specificity of infection, but also results in preservation of viral gene expression. Furthermore, the data indicate that the production of modified yet functional virus increased with increasing pH of ~-the modification reaction up to a limit of ;
appro~imately 8.0, beyond which the function o the virus became compromised.
Many retroviruses have been shown to enter cells normally via endocytosis and are thought to introduce their genetic material during an acidification step in the pathway. Andersen, K.B.
and Nexo, P.A. ViroloaY 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., et al. Biochim. Biophys.
Acta 58S:71-84 ~1979)), early in the internalization process, endosomal endocytotic compartments are acidified prior to fusion with lysosomes. Tycko, B.
and Maxfield, R.F. ÇÇ11 28:643-651 (1982). This period of acid exposure may be analogous to the . . .
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natural route of entry for some viruses (Nussbaum, O., and Loyter, A. FEBS Lett. 22~:61-67 (1987)) and may provide the requisite conditions for acid-mediated fusion of the viral envelope of endosomal 05 membrane prior to destruction of the virus.
Helenius, A. Biol. Cell Sl:181-186 (1984). The fact that modified virus is still able to introduce its genome into target cells suggests that the process of chemical modification did not abolish the function of those elements of the virus.
B. VIRUS-ASIALOGLYCOPROTEIN CONJUGATES
Crude preparations of virus obtained by low-speed centrifugation of medium from producer BAG
cells followed by high-speed centrifugation through a discontinuous sucrose gradient as described previously was dialyzed against 0.9~ saline, pH 7.5, at 4C. After dialysis, NHS-LC-biotin (Pierce Chemical Co., Rockville, IL) was reacted with the virus ~0.1 mg/ml of virus) at room temperature for four hours. The sample was then dialyzed against 0.9~ saline, pH 7.5 at 4C. Asialoorosomucoid (AsOR) was obtained by desialylation of serum orosomucoid oriyinally derived from pooled human serum. Whitehead, D.H. and Sammons, H.P. Bioçhem.
Biophys. Acta 124:209 (1966). AsOR 0.1 mg was added to 1.0 mg of virus, thoroughly mixed and then 1.0 mg -~
of avidin per mg of virus was added and allowed to incubate at room temperature for four hours. The complex was then dialyzed against Modified Eagle's 30 Medium. Comple~ed virus was purified on a Sephadex G150 molecular sieve column. To determine conditions for purification, a viral complex was .. ..
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prepared in which asialoorosomucoid was labeled with 125I. Figure 3 shows that asialoorGsomucoid alone was eluted from the column beginning at fraction number 32. Avidin, as detected by its optical 05 density at 280 nm, eluted slightly later beginning at tube 33. However, unlabeled virus alone was much larger than either of the other two proteins and was eluted earlier with a peak at tube 29. The column was able to completely resolve virus from asialoorosomucoid and avidin. Figure 3 also shows that this virus complexed with 125I-labeled AsO~
mediated by biotin-avidin bonds, the radioactivity from the AsOR moved to the same position as expected for the intact virus, namely with a peak at tube 29. These data indicate that some labeled asialoorosomucoid was bound by the virus and ^ -migrated with it through the column.
In order to determine whether this complex could be used to target gene e~pression specifically to asialoglycoprotein receptor (+) cells, conjugated virus was incubated for 10 days with each of five cell lines: Hep G2, receptor (~); Huh-7, receptor .
(+); SK Hepl, human hepatoma receptor (-); Mahlavu, receptor (-~ and Morris 7777, rat hepatoma receptor (-) cells. Figure 4, lane 1 shows that Hep G2 receptor ~+) cells treated with conjugate had beta-galactosidase activity at a level of 2.3 units/mg of cell protein which is approximately 50%
of the activity of the producer cell line, BAG shown in lane 11. Hep G2 cells without treatment were at a level of 1.81 units/mg. Huh-7 receptor (~) cells treated with conjugate had higher levels of beta-galactosidase, 3.8 units/mg as shown in lane 3 ';
~092/06180 ~3 ~CI/US')l/07103 ~c~3 -24-cl~
compared to those cells treated with biotinylated virus without asialoorosomucoid present in a complex shown in lane 4. This was similar to the levels obtained from these cells that were not treated at 05 all as seen in lane 5. Lane 6 shows that Mahlavu receptor (-) cells treated with conjugate did not have any significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 7. Similarly lanes 8 and 9 show that Morris 7777 cells treated with other conjugate or biotinylated virus without asialoorosomucoid, lanes 8 and 9 respectively, showed no significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 10. SK HEPL
cells responded similarly to the receptor (-) Morris 7777 cells.
In the staining procedure described in Example ~;
1, Hep G2 cells treated with the conjugated virus produced a bluish coloration as did the Huh-7 cells treated similarly. However, cells that did not receive treatment had no staining. ~-~XAMPLE 2 Chemical Modification and ~lteration of Host Cell S~ecificit~ of He~atitis B Virus (HBV) Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities. 1~ vitro, the virus is also very fastidious as evidenced by the fact that human hepatocytes or hepatoma cells in culture cannot be infected by HBV without unusual and highly artificial conditions such as high concentrations of corticosteroids.
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WO92/061~0 PC'r/US')1/071~3 -25- 20~232.3 Cells and Cell Cult~re Hepatitis B virus (HBV) was obtained from Hep G2 producer cells chronically infected with HBV
as described by Sells et al. Proc. Natl. A~ad. Sci.
05 84:1005-1009 (1987), and maintained in Dulbecco's modified Eagle's medium (MEM) containing G418 as 380 mg/ml, supplemented with 10% heat inactivated fetal bovine serum. To test the infectivity and specificity of unmodified and modified HBV, two human cell lines were cultured. Huh7 human hepatoma cell line which possesses asialoglycoprotein receptors and IMR-90 fibroblasts which do not possess asialoglycoprotein receptors were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS). , Isolati~n of HBV
HepG2 cells were cultured in serum free media for three days. The medium was centrifuged at 2000 rpm to remove debris and the supernatant applied on 10-20% lactose gradient, pH 7.4, 8.0 or 8.4, and ultracentrifuged at 40000 rpm in VTi55 rotor at 49C
for 16 hours to pellet and isolate the YiruS.
Chemical Modification of HBV
HBV obtained (3.0 mg of protein~ was lactosaminated in a similar fashion to that described in Example 1 using 10 mg of sodium cyanoborohydride for 3 hours at 25C. The modified virus was sterilized by filtration through 0.45 ~m 30 membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% F3S.
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Infection of Cells with Unmodi~ied and Modified_~Y
Huh7 and IMR-90 cells were plated at 25-50~
confluence in 35 or lO0 mm diameter plastic dishes.
Cell medium was removed and replaced with medium 05 containing modified or unmodified virus and incubated at 37C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of H~V
DNA and medium analyzed for the presence of hepatitis B surface antlgen (HBsAg).
DetectiQn of Tarqeted HBV DNA in Huh7 Cells Treated with Modified an~ ~nm~dified H~V
DNA was e~tracted fro~ cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 3:2303-2312 (1976), in which the cells were washed twice with lO ml of cold Tris-buffered saline (TBS), scraped off into TBS and centri~uged at 200 rpm. The cell pellet was resuspended in lO mM
Tris-HCl, pH 8.0, 1 mM EDTA, pH 3.0, was addeid to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA
was isolateid by ethanol precipit~tion after phenol extraction. The DNA was analyzed by Southern blot using a y32P-ATP labeled cDNA probe specific for HBV
sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genoime, obtained from Dr.
Jake Liang, Massachusetts General Hospital).
The Southern blot showed no hybridizable sequences when probed with our cDNA probe specific 30 for HBV. This confirms the previous finding that Huh7 cells, even though of human origin, cannot be infeicted by unmodified HBV under the conditions of . , , , . - . ,. , :.. .. .
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, WO 92/0~,l8n ~cr/U.~91/n7.~03 2 ~ J 2 3 routine cell culture. In addition, the data indicate that the washing procedures eliminate any detectable non-specifically bound HBV DNA on these cells. However, treatment of the Huh7 cells with 05 modified HBV for as little as one day of incubation resulted in a strong signal of hybridizable bands on the Southern blot corresponding to those expected for the plasmid sequences. IMR-90, asialoglycoprotein (-) cells did not produce hybridizable sequences under any conditions.
Detection of HBsAa in the SuPernatant of Huh7 and IMR-90 Cells ExPosed t~_Unmodified or Modifi~d ~BV
Medium from Huh7 and IMR-90 cells was incubated with modified or unmodified HBV as described above and at various intervals was assayed for HBsAg by an enzyme immunoassay kit (Auszyme Monoclonal). The conditions were those recommended by the manufacturer, Abbott Labs.
As shown in Table 4, the background color absorbance was approximately 0.1:21 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. UnmodiEied HBV did not result in significant production of HBsAg.
Absorbance here was approximately 0.180. Similarly, the color absorbance r-flecting HBV levels in IMR-90 cells did not exceed 0.110. However, Huh7 cells treated with modified HBV released HBsAg into their supernatants, with absorbance ranging from 0.760 to 0.865.
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Table 4 Levels of Hepatitis B Surface Antigen (HBsAg) in Culture Medium as Determined by Auszyme Assay (Absorbance Units) 05 Cells IMR- 90 ~1~1h7 DaY Modified Untreated Unmodified Modified HBV HBV HBV
1 .121 ~ .05q .135 ~ .017 .850 ~ .010 3 .186 ~ .036 .700 ~ .012 .171 + .010 .865 + .053 7 .110 + .023 .764 ' .067 Equivalents Those skilled in the art will recognize, or be ablé to ascertain using no more than routine e~perimentation, numerous eguivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following 20 claims.
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SELECTIVE INTERNALIZATION BY CELLS
Back~round of the Invention Viruses represent a natural and efficient means 05 for the introduction of foreign genes into cells.
For this reason, they are useful tools for the study of genes, and gene regulation n vitro and ~or gene therapy. However, most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene e~pression in many tissues, some of which may be undesirable, especially for clinical applications.
Generally, viral infection is mediated by interactions between viral enve].opes and plasma -~
15 membranes of target cells. In many cases, specific -~
viral structures are recognized and bound by ~-cellular receptors. For e~ample, HIV employs envelope glycoproteins to bind l:o helper T
lymphocytes via CD4 (T4) receptors. Dalgleich, A.G., et al. Nature ~ 763-767 (1984). These interactions have been shown to be responsible for the observed species and organ specificity.
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Some investigators have shown that virus specificity can be redirected by attaching antibodies to viruses. For e~ample, Goud, B., ~t al. ViroloqY 163:251-254 (1988) linked 05 anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor. However, while binding and internalization occurred, infection and replication did not. A means for targeting viral or other types of nucleic acid vectors containing foreign genes to a target cell and obtaining infection and replication of the virus would be useful in gene therapy.
Summarv of the Invention The invention pertains to a method of targeting a virus or a cell to a target cell for select:ive internalization in YiVo (or in _~EQ) by the cell and to modified viruses and cells which are targeted for selective internalization by a target cell.
A virus or cell is targeted to the target cell for internalization by introducing a receptor- -specific molecule onto the surface of the virus or cell to produce a modified virus or cell which specifically binds to a receptor on the surface of 25 the target cell. The modified virus or cell can be administered to an organism where it binds selectively to the receptor of the target cell. The receptor-binding results in internalization by the target cell.
. , , , ~ , , WO92/0618~) 2 U 9 i~ rlUs~ 7l(~3 The cellular receptor can be a receptor which mediates endocytosis of a bound ligand such as the asialoglycoprotein receptor of hepatocytes and the receptor-specific molecule can be a natural or 05 synthetic ligand for the receptor. The receptor-specific molecule can be introduced onto the surface of the virus or cell (e.g., onto a viral envelope or cellular rnembrane) by chemically couplin~ it, either directly or through bridging lO agents, to the surface or by treating the surface to e~pose the molecule for receptor recognition.
The method of this invention can be used to produce viral or cellular vectors for selective delivery of material such as nucleic acid ~genes) to 15 a target cell. For example, exogenous genes can be incorporated and expressed selectively in a target cell. These vectors can be used in gene therapy and in other applications which call for selective genetic alteration of cells.
The method also provides a means for altering the natural tropism of an infective agent such as a virus or bacterium. An infective agent can be modified so that it will infect ia cell which, in unmodified form, it would not normally infect. In 25 this way, animal mod~ls of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases. For example, an ecotropic human pathogen (such as the hepatitis or AIDS virus) can be modified to infect a 30 non-human host to produce an e~perimental system for study of the pathogen and the disease.
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W092/06lX0 ~ ~ PCr/US9l/07l0 ~ -4-Brief DescriPtion of the Fiaures Figure 1 shows in situ B-galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified 05 murine leukemia virus.
Figure 2 shows internalization of 35S-biolabeled modified Moloney murine leukemia virus.
Figure 3 shows a chromatogram of asialooro-10 mucoid-complexed Psi2 virus on Sephadex G150.
Figure 4 shows the B-galactosidase activity of various cells exposed to Psi2 virus-asialoglyco-protein conjugate.
Detailed Descri~tion of the Invention A virus or cell is targeted for selective internalization into a target cell by modify;ng the surface of the virus or cell to lntroduce a molecule which specifically binds to a surface receptor of the target cell. The cellular surface receptor is 20 one which will mediate internali;zation of the targeted virus or cell. The modified virus or cell binds to the receptor of the target cell in vivo and is internalized by the cell.
According to the method of this invention, 25 viruses can be modified to infect specific target cells. Such modified viruses can be used to selectively deliver exogenous, functional DNA to a target cell in order confer a new biological or biochemical property upon the cell or to abrogaté an 30 existing property. In addition, the tropism of a virus can be altered or redirected to targat infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form.
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A variety of different enveloped viruses can be targeted by the method of this invention. The viruses can be RNA (retroviruses) or DNA viruses (e.g., hepatitis virus, adenovirus). The virus can 05 be replication defective or otherwise defective in structure or function. For e~ample, viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted.
10The present method al50 provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose -tropism can be altered. In addition, mammalian cells can be targeted.
15The receptor-specific molecule can be a ligand for the surface receptor of the target sell.
Preferably, the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as 20 the asialoglycoprotein receptor of hepatocytes.
Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules. For specific targeting to hepatocytes, asialoglycoprotein (galactose-terminal~ ligands are 25 preferred. Examples of asialoglycoproteins include ~ -asialoorosomucoid or asialofetuin. Other useful galactose-terminal carbohydrates for hepatocyte ~
targeting include carbohydrate trees obtained from ~;
natural glycoproteins, especially tri- and 30 tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to espose terminal galactose residues~ In addition, naturally occurring plant carbohydrates such as arabinogalactan can be used.
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WO92/06180 ~ Pcr/us9l/n71 For targeting other receptors, other types of carbohydrates can be used. For example, mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to 05 target macrophages or endothelial cells.
Other receptor ligands such as peptide hormones could also be used to target viruses or cells to corresponding receptors. These include insulin, glucagon, gastrin polypeptides and their respective l0 receptors.
Alternatively, the receptor-specific molecule can be a receptor or receptor-like molecule, such as an antibody, which binds a ligand (e.g., antigen) on the cell surface. Antibodies specific for cellular lS surface receptors can be produced by standard procedures.
The receptor-specific molecule is introduced onto the surface of the virus or cell so that it will be recognized by the cognate cellular surface 20 receptor. For esample, the receptor-specific molecule can be introduced onto t]he envelope of a virus or the membrane of a cell. In general, the molecule will be coupled to (or exposed on~ a proteinaceous component of the surface but other 25 components may be used.
The receptor-specific molecule can be introduced onto the surface of the virus or cell by different means. Preferably, the receptor-specific molecule is chemically coupled to the surface. For 30 example, galackose moieties (ligand for the asialoglycoprotein receptor) can be covalently coupled to viral or cellular surface proteins by lactosamination, reductive amination, or via -: , , ,: . . . .
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WO92/0618() 7 2 ~ ~ 2 3 2 3 PCr/US~)''0~103 iminomethoxyethyl derivatives. In other embodiments, the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as 05 biotin and avidin. For instance, a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
Alternatively, the virus or cell can be lO chemically treated to expose a receptor-specific molecule on the surface. Surface polycarbohydrates can be enzymatically cleaved to expose desired carbohydrate residues (e.g., galactose residues) as terminal residues for specific receptor recognition 15 and bindin~. For e~ample, neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
The modified virus or cell .Ls administered n 20 vivo, generally in an amount suf~icient to saturate receptors of the target cell and thereby maximize uptake by the cell. They can be administered parenterally (typically intravenously) in a physiologically acceptable vehicle such as normal 25 saline.
The method of this invention can be used to selectively deliver nucleic acid (DNA or RNA) to a target cell in vivo (or in yitro~ so that it is expressed in the cell. The nucleic acid can be an 30 exogenous gene, a genetic regulatory element or an antisense inhibitor of gene function. The nucleic acid is incorporated into a viral vector which has been modified, according to the method of this '': ,: '' ' ~
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invention, to target it to the cell. Preferred viral vectors for delivery of foreign genes in VlVO
(or ex vivo) are retroviruses. The targeted viral vector is administered in v v~, as described, where 05 it is selectively taken up by the target cell The method of this invention can be used to alter the natural tropism of an infectious agent.
Ecotropic (species-restricted) agents can be made to infect species which they normally, in unmodified lO form, do not infect. The ability to target the infectivity of an infectious agent can be used to develop new experimental systems for the study of human infectious diseases to produce cells that can correct genetic defects in vivo, or target a 15 corrective gene n vivo.
Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells. By the method of thi.s invention, such viruses can be modified to enable them to infect 20 e~perimental animals such as rodents. For example, the hepatitis virus which infects only human liver cells, can be modified so that it will infect non-human liver cells. To deuelop rodent models of hepatitis, for example, a ligand for rodent 25 asialoglycoprotein receptor (e.g., galactose) can be introduced onto the surface of the hepatitis virus.
This yields a modified hepatitis virus which will infect rodent liver cells. This modified hepatitis virus which can infect a rodent and the infected 30 rodent or rodent cells, provides an experimental animal system for study of the hepatitis virus.
WO92/0618(~ 2 3 9 2 3 2 ~ P~l/US91/07103 The invention is illustrated further by the following e~amples.
Chemical Modification and Alteration of Host Cell 05 SPecifi~i~Y of a Retrovirus A model retroviral system was used. The virus, an ecotropic, replication-defective, Moloney murine leukemia virus containing the gene for bacterial ~-galactosidase produced in a ~ cre cell line was lO kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., et al. Proc. Natl. Acad.
Sci. USA 87:439-443 ~l990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M., et al. Proc. Natl. Acac~ Sci.
15 ~ 3014-3018 (198R); Goud, B., et al. ViFoloqy 163:251-254 (198B). The producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO
Laboratories, Grand Island, NY) ~upplemented with lO% heat-inactivated calf serum (GIBCO). To prepare 20 virus with as little contamination as possible from serum proteins, producer cells wlere cultured in serum-free Dulbecco's modified Eagle's medium for 3 days. Using this viral preparation, two strategies were developed for the modification of the surface 25 of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus.
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A. LACTOSAMINATION OF RETROVIRUSE:S
Virus was isolated from the culture medium according to the method of Goud, B., et al. Virolpqy 163:251-254 (1988), but modified to permit coupling 05 of lactose during the isolation procedure. In brief, virus-containing medium was applied on a 10-20% sugar gradient in which a-lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was 10 ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4C for 17 hours. Samples were adjusted to various pHs, 7.4-8.4, prior to centrifugation in order to determine optimal conditions for 15 modification. After centrifugation, the bottom fraction of the lactose gradient containing 3.0 mg of protein (0.1 mg viral RNA) was reacted with sodium cyanoborohydride (Sigma) as described previously. Goud, B., et ~11 Viroloq~ 163:251-254 20 (1988). Following dialysis against minimum essential medium at 4C for 24 hours, the samples were sterilized by passage through 0.45~ filters (Gelman Science Co., Ann Arbor, MI). Quantitation of the amount of virus present in samples prior to 25 exposure to cells was determined by protein assay (Bio-Rad, Los Angeles, CA) according to the manufacturer's instructions, and confirmed by RNA
assay (Nunro, H.N. and Fleck, A. Meth. Biochem.
Anal. 1~:113-176 (1966)) after RNA e~traction.
30 Chomczynski, P. and Sacci, N., Anal. Biochem, 162:156-159 (19~7). Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used , W092/06l80 ~ 3 2 ~
immediately after preparation. Viability of unmodified virus preparations was determined by transfection assays in NIH 3T3 mouse fibroblasts using limiting dilutions of viral stock (Danos, 0.
05 and Mulligan, R.C. Proc. Natl. Acad. Sci. USA
~5:6460-6464 (1988)) and quantitated by determination of positive cells stained with X-gal.
Sanes, J.R., et al. EMBO J. 5:3133-3142 (1986).
For uptake studies, virus was biosynthetically ?
10 labeled by incubation of producer cells (5.0 x 106 cells) in 50% serum-free and 50% serum- and methionine-free Dulbecco's modified Eagle's medium containing 10 ~Ci/ml 35S-methionine (Amersham, Arlington Heights, IL) for 3 days. Virus was 15 isolated from supernatants and modified as described abo~e followed by dialysis against minimum essential medium.
Cells and Cell Culture To evaluate the effects of chemical 20 modification on viral infection specificity, several cell lines were employed: human hepatoma cell lines, HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., et al. J. Biol. Chem. 256:8878-8881 ~1981)) obtained from B.B. Knowles, Wistar Institute, 25 Philadelphia, PA; and SK Hepl, receptor (-) from D.A. Shafritz, Albert Einstein College, of Medicine, Bronx, NY; a rat hepatoma cell line, Morris 7777, receptor (-) (Wu. G.Y., et al. ~. Biol. Chem. 263: -4719-4723 (1988)); and a murine fibroblast cell line 30 NIH 3T3 (Goud, B., ç~ al. YiDQlQgY L~:251-254 (1988)) which is also asialoglycoprotein receptor (-). The latter two cell lines were purchased from .
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r~3 WV92/06l80 ~ 12- PCT/US9l/0710 American Type Culture Collection tRockville, MD).
All were maintained in Eagle's minimum essential medium supplemented with lO~o heat inactivated fetal bovine serum at 37C under 5% C02.
05 Assays for Viral Infection and Functio~al Gene ExPression In order to determine whether virus remained infectious and functional after chemical modification, the two human and two rodent cell lines were exposed to modified and unmodified virus followed by measurement of cellular ~-galactosidase activity. Target cells were plated at a density of O.5-2.0 x 105 cells/ml in 60 mm plastic dishes (Falcon Scientific Co, Lincoln Park, NJ). Equal 15 amounts, 16.7 ~g RNA, (0.5 mg viral protein) of modified and unmodified virus, in Dulbecco's modified Eagle's medium were added to the culture medium and e2posed to cells for 5 days at 37C under 5% CO2. Cells were assayed for B-galactosidase 20 activity as a measure of foreign gene expression accor~ing to the method of Gorman (Gorman, C. DNA
Clonin~, vol. 2 eds, Glover, D.M. IRL Press, Washington D.C~ pp 157-158 (1986)). In brief, cell monolayers (approximately lx106 cells/60 mm dish) 25 were washed with phosphate buffered saline, then `~:
lysed. The lysate, 0.1 ml, was reacted with o-nitrophenyl-galactopyranoside (ONPG, Sigma) and -B-galactosidase activity quantitated by absorbance at 420 nm after addition of Na2CO3 to terminate the 30 reaction. Results were expressed in U/mg of cellular protein according to the method by Norton, : . . . .
':
~. , WO9~/061X() -13- 2 0 ~ 2 3 2Pcr/us91/07l03 P.A. and Coffin, J.M. Mol. Cell._Biol. 5:281-290 (1985), using purified E. c~li B-galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were 05 determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions. For competition experiments, virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, 10 asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. ~ h~l. Chem. 258:
10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sammons, ~.G.
Biochim. BioPhvs. A~a 124:209-211 (1966).
15 Background enzyme activity was determined in corresponding untreated cells and subtracted from the values of viral-treated samples. All assays were performed in triplicate and the results expressed as mea~s + S.E.
Table 1 shows that unmodified virus did not produce enz~matic activity in human HepG2 or SK Hepl~;, cells as e~pected from the ecotropism of the virus. ~
Also, modified virus did n~t produce B-galactosidase ~ -activity in SK Hepl, asialoglycoprotein receptor (-) 25 cellsO However, modified virus did produce high B-galactosidase activity, 71.2 + 4.8U/mg of cellular protein, in human HepG2, asialoglycoprotein receptor (+) cells. Furthermore, this enzymatic activity was completely suppressed by addition of a large molar 30 e~cess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors. As e~pected from the ecotropism, B-galactosid3se . . , .. ., , ", .. . . . .
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~ " ~ ' , W092/06l80 ~ 'CrlUS~ )71~3 activity was high, 50.6 + 5.2. in Morris 7777 rat cells after exposure to unmodified virus.
Interestingly, B-galactosidase activity in these same cells was significantly lower when exposed to 05 the same amount of modified virus. The same tendency was seen in originally susceptible murine NIH 3T3 cell as enzymatic activity after exposure to unmodified virus, 56.7 + 1.8, was more than double that following exposure to modified virus 27.0 + 0.9.
The coupling reaction lin~ing lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. ~rch.
Biochem. Biophys. lBl:542-549 (1977~. However, such conditions could be detrimental to the virus. To 15 determine the optimal pH that results in modified, yet functional vectors, virus modified at different pHs were administered to HepG2 cells, and B-galactosidase activity measured. Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for 20 virus modified at pH 7.4; to 71.2 + 4.8, for virus - -~
modified at pH 8Ø However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
, .
w09t/06lxo -15- 2 3 9 ~ 3 2 3 Pcr~us91~o7lo3 Table 1 Cellular ~-Galactosidase ActivitY
Followina ExPosure to Viral Pre~arations~
~-Galactosidase Activity*
Mean i S.E. (U/mg) ,, :
____________________________________________~________ os AsG Unmodified Modified Modified :~-Receptor Virus Virus Virus + -Status ASOR~*
__________ ________________ _________________ _______ :~
Cell hine .:
(Source) .
______ __ , 10 HepG2 (huma~) ~+) 108 + 1.9 71.2 + 4.8 2.9 + 1.1 SK Hepl ; ~(human) ~-) 1.7 + 3.4 0.8 + 4.6 1.6 + 2.5 .: -Morris 7777 15 (rat) (-) 50.6 + 5.2 16.3 + 4.4 15.7 + 4.7 NIH 3T3 ~ -(mouse) ( ) 52.1 + 4.9 15.4 + 1.1 16.3 ~ 3.9 ~ .. -~., ~ -, .
+ virus was modified at pH 8.0 then incubated ~- with cells for 5 days. -20 * calculated as the difference in activity between :~
treated and,untreated cells.
. .
** asialoorosomucoid (ASOR) in 100-fold molar e~cess.
AsG, Asialoglycoprotein . - ~ .- - -,, . , , ~ . . . , . .:
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WO 92/06l8n ~ Pcr/usl)l/n7l0~-~C~ -16-Table 2 Effect of the PH Durinq Modification of Viral ~ransfection in HePG2 Cells Specific ~-Galactosidase Activity (Mean ~ S.E. U/mg protein)*
. ' 05 Modified Virus Modified Virus + Asialoorosomucoid** -' PH
7.0 50.3 ~ 1.2 6.4 + 1.9 :
8.0 71.8 + 4.1 4.9 + 0.4 10 8.425.1 ~ 2.4 0.0 + 1.6 after 5 days of exposure to modified virus.
** B-galactosidase activity of samples treated with modified virus plus a 100-fold molar e~cess of asialoorosomucoid.
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WO92/061~0 PCr/lJS~l/07lU3 -17~ 2 ~ ~ 3 Histochemical Staininq to Demons~5 B-Galactosidase Activity To confirm the colorimetric results, and to determine the fraction of cells that expressed the 05 ~-galactosidase gene after exposure to viral samples, histochemical staining of in ~
B-galactosidase activity was performed according to the method of Sanes et al. EMBO J. 5:3133-3142 (1986). In brief, cultured cells in 35 mm dishes containing 0.5 -1 x 1o6 cells treated for 5 days with equal amounts, 8.4 ~g of viral RNA (0.3 mg viral protein), of modified or unmodified virus.
Cells were fixed in 0.5% glutaraldehyde ~Sigma), phosphate buffered saline, then incubated with 1 mM
MgC12, phosphate buffered saline, and overlaid with lmg/ml 4-Cl-5-Br-3-indoylyl-~-galactosidase ~X-gal) (BRL, Washington, D.C.), 5 ~M potassium ferricyanide, 5 mM potassium ferrocyanide and 2 mM
MgC12 in phosphate buffered saline. After incubation at 37C for l hour, the dishes were washed in phosphate buffered saline to quench the reaction and evaluated by counting positive (blue) cells under a light microscope a,nd the results expressed as the percent of positive/10 high power fields.
In situ staining for ~-galactosidase activity -in cells treated with various viral preparations is shown in Figure l. In rodent NIH 3T3 cells treated with unmodified virus, panel B, 12.6% were positive for ~-galactosidase activity using the X-gal stain.
Background staining in untreated NIH 3T3 cells was not detectable, panel A. After e~posure to modified virus, only 3.6% were positive under otherwise .,, , ~ , .
.
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identical conditions, panel C. Human SK Hepl cells that were untreated, panel H, or exposed to either unmodified virus, panel I, or modified virus, panel J, failed to develop detectable staining.
05 Similarly, HepG2, asialoglycoprotein receptor (~) cells treated with unmodified virus, panel E, did not develop evidence of significant B-galactosidase ;
activity. However, HepG2, receptor (+) cells treated with modified virus, panel F, did develop substantial staining. Microscopic counting revealed that 36.4 ~ of the HepG2, cells possessed detectable marker enzyme. The observed color development was completely suppressed by addition of a 100-fold molar excess asialoorosomucoid to compete for uptake by asialoglycoprotein receptors, panel G, indicating involvement of asialoglycoprotein receptors in the transfection process.
Assavs for Cellular Uptake_o~ Virus To determine whether the modified virus was actually taken up by cells and, if so, whether asialoylycoprotein receptors were involved, HepG2, SK Hepl and Morris 7777 cells, 5.0x105 cells/35 mm -dish, were incubated at 37C in serum-free Dulbecco's modified Eagle's medium containing 35S-biolabeled, modified virus, 3.3 ~g viral RNA
(98 ~g viral protein) (Watanabe, N., et al. Cancer Immunol. Immunother. 28:157-163 (1989)~ with a specific activity of 6.1~105 cpm/mg viral RNA. At various times, medium was removed, and cells were chilled to 4~C, washed with ice-cold minimum essential medium containing lmg/ml bovine serum albumin. Surface-bound radioactivity was stripped : ~ .
.
. ' . . ' ,~ . , W0~2/06l80 ~'Cr/US'JI/~ 03 ~ 03~3~3 with cold 0.5 ml phosphate buffered saline, pH /.2 containing 0.4% trypsin, 0.02% EDTA and separated from cells by centrifugation. The cell pellet was solubilized in 0.2 N NaOH and Poly-Fluor (Packard, 05 Chicago, IL), and trypsin-EDTA resistant (internalized) radioactivity was measured by scintillat.ion counting (TRI-CARB 4530, Packard).
Schwartz, A.L., e~ al J. Biol. Chem. 256:8878-8881 (1981). Non-specific uptake was measured in the presence of a 100-fold molar excess of asialoorosomucoid, and specific uptake calculated as the difference between total and non-specific measurements. All assays were per~ormed in triplicate and the results expressed as means + S.E.
in terms of ng viral RNA/105 cells as a function of time.
Figure 2 shows that, of the two human and one rodent cell lines, only the human HepG2 asialoglycoprotein receptor (+) cells demonstrated significant specific uptake of labeled virus.
Counts resistant to EDTA and tryE~sin, increased as a function of time and continued to rise lïnearly through 120 minutes of incubation at a rate of approximately 800 ng viral protein/hr/105 cells.
These data further support the notion that the observed expression of the galactosidase gene by modified virus was in fact due to internalization of the virus by asialoglycoprotein receptors.
StabilitY of Modified Virus To assess the stability of modified virus, samples of freshly prepared sterile, modified virus were incubated in serum-free Dulbecco's modified , , . ".
, , ~ ', WO92/06l80 c~ Pcr/ us~ 1/071 ~ 20-Eagle's medium at 4~C and 25C. At various times, samples were added to the medium of ~epG2 cells and incubated for 5 days. Cells were then assayed for B-galactosidase activity by colorimetric assay as 05 described above. ~11 assays were performed in triplicate and the results expressed as means + S.E.
in terms of U/mg cell protein normalized for the amount of virus added as a function of time of incubation. Table 3 shows that enz~matic activity at both 4C and 25C, decreased with time to approximately 50% of original activity after 48 hours.
Table 3 Stability of Modifiçd Virus '~
Specific Activity Temperature Time of (Mean ~ S.E.
Incubation U/mg protein)*
4C 0 hr 50.3 + 1.2 24 hr 42.0 + 5.6 4B hr 22.6 + 2.5 25C 24 hr 37.1 + 2.8 48 hr 17.6 ~ 1.1 * after 5 days of exposure to modified virus Specific B-galactosidase activity was calculated as the difference between samples treated with virus alone, and samples treated with modified virus plus a 100-fold molar excess of asialoorosomucoid.
:, ~ -, . ~
. ' . , , WO92/06180 YCT/~S91/07~J3 The coupling of lactose to proteins to target artiicial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino 05 groups to re~der the bonds irreversible. Treatment of viruses with aldehydes is not always similarly benign. For exarnple formaldehyde has been used to inaçtivate viruses in the production of vaccines.
Buynak, E.B., et al. ~. Am. Med. Assoc. 235:
2832-2834 (1976). The data presented here indicate that under the conditions described, the modification process results not only in altered specificity of infection, but also results in preservation of viral gene expression. Furthermore, the data indicate that the production of modified yet functional virus increased with increasing pH of ~-the modification reaction up to a limit of ;
appro~imately 8.0, beyond which the function o the virus became compromised.
Many retroviruses have been shown to enter cells normally via endocytosis and are thought to introduce their genetic material during an acidification step in the pathway. Andersen, K.B.
and Nexo, P.A. ViroloaY 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., et al. Biochim. Biophys.
Acta 58S:71-84 ~1979)), early in the internalization process, endosomal endocytotic compartments are acidified prior to fusion with lysosomes. Tycko, B.
and Maxfield, R.F. ÇÇ11 28:643-651 (1982). This period of acid exposure may be analogous to the . . .
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~3 -22~
natural route of entry for some viruses (Nussbaum, O., and Loyter, A. FEBS Lett. 22~:61-67 (1987)) and may provide the requisite conditions for acid-mediated fusion of the viral envelope of endosomal 05 membrane prior to destruction of the virus.
Helenius, A. Biol. Cell Sl:181-186 (1984). The fact that modified virus is still able to introduce its genome into target cells suggests that the process of chemical modification did not abolish the function of those elements of the virus.
B. VIRUS-ASIALOGLYCOPROTEIN CONJUGATES
Crude preparations of virus obtained by low-speed centrifugation of medium from producer BAG
cells followed by high-speed centrifugation through a discontinuous sucrose gradient as described previously was dialyzed against 0.9~ saline, pH 7.5, at 4C. After dialysis, NHS-LC-biotin (Pierce Chemical Co., Rockville, IL) was reacted with the virus ~0.1 mg/ml of virus) at room temperature for four hours. The sample was then dialyzed against 0.9~ saline, pH 7.5 at 4C. Asialoorosomucoid (AsOR) was obtained by desialylation of serum orosomucoid oriyinally derived from pooled human serum. Whitehead, D.H. and Sammons, H.P. Bioçhem.
Biophys. Acta 124:209 (1966). AsOR 0.1 mg was added to 1.0 mg of virus, thoroughly mixed and then 1.0 mg -~
of avidin per mg of virus was added and allowed to incubate at room temperature for four hours. The complex was then dialyzed against Modified Eagle's 30 Medium. Comple~ed virus was purified on a Sephadex G150 molecular sieve column. To determine conditions for purification, a viral complex was .. ..
~' W~92/06l80 ~23- ~'Cr/~S')l/0~l03 239~3~
prepared in which asialoorosomucoid was labeled with 125I. Figure 3 shows that asialoorGsomucoid alone was eluted from the column beginning at fraction number 32. Avidin, as detected by its optical 05 density at 280 nm, eluted slightly later beginning at tube 33. However, unlabeled virus alone was much larger than either of the other two proteins and was eluted earlier with a peak at tube 29. The column was able to completely resolve virus from asialoorosomucoid and avidin. Figure 3 also shows that this virus complexed with 125I-labeled AsO~
mediated by biotin-avidin bonds, the radioactivity from the AsOR moved to the same position as expected for the intact virus, namely with a peak at tube 29. These data indicate that some labeled asialoorosomucoid was bound by the virus and ^ -migrated with it through the column.
In order to determine whether this complex could be used to target gene e~pression specifically to asialoglycoprotein receptor (+) cells, conjugated virus was incubated for 10 days with each of five cell lines: Hep G2, receptor (~); Huh-7, receptor .
(+); SK Hepl, human hepatoma receptor (-); Mahlavu, receptor (-~ and Morris 7777, rat hepatoma receptor (-) cells. Figure 4, lane 1 shows that Hep G2 receptor ~+) cells treated with conjugate had beta-galactosidase activity at a level of 2.3 units/mg of cell protein which is approximately 50%
of the activity of the producer cell line, BAG shown in lane 11. Hep G2 cells without treatment were at a level of 1.81 units/mg. Huh-7 receptor (~) cells treated with conjugate had higher levels of beta-galactosidase, 3.8 units/mg as shown in lane 3 ';
~092/06180 ~3 ~CI/US')l/07103 ~c~3 -24-cl~
compared to those cells treated with biotinylated virus without asialoorosomucoid present in a complex shown in lane 4. This was similar to the levels obtained from these cells that were not treated at 05 all as seen in lane 5. Lane 6 shows that Mahlavu receptor (-) cells treated with conjugate did not have any significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 7. Similarly lanes 8 and 9 show that Morris 7777 cells treated with other conjugate or biotinylated virus without asialoorosomucoid, lanes 8 and 9 respectively, showed no significant beta-galactosidase activity compared to those same cells that were untreated shown in lane 10. SK HEPL
cells responded similarly to the receptor (-) Morris 7777 cells.
In the staining procedure described in Example ~;
1, Hep G2 cells treated with the conjugated virus produced a bluish coloration as did the Huh-7 cells treated similarly. However, cells that did not receive treatment had no staining. ~-~XAMPLE 2 Chemical Modification and ~lteration of Host Cell S~ecificit~ of He~atitis B Virus (HBV) Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities. 1~ vitro, the virus is also very fastidious as evidenced by the fact that human hepatocytes or hepatoma cells in culture cannot be infected by HBV without unusual and highly artificial conditions such as high concentrations of corticosteroids.
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WO92/061~0 PC'r/US')1/071~3 -25- 20~232.3 Cells and Cell Cult~re Hepatitis B virus (HBV) was obtained from Hep G2 producer cells chronically infected with HBV
as described by Sells et al. Proc. Natl. A~ad. Sci.
05 84:1005-1009 (1987), and maintained in Dulbecco's modified Eagle's medium (MEM) containing G418 as 380 mg/ml, supplemented with 10% heat inactivated fetal bovine serum. To test the infectivity and specificity of unmodified and modified HBV, two human cell lines were cultured. Huh7 human hepatoma cell line which possesses asialoglycoprotein receptors and IMR-90 fibroblasts which do not possess asialoglycoprotein receptors were maintained in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal bovine serum (FBS). , Isolati~n of HBV
HepG2 cells were cultured in serum free media for three days. The medium was centrifuged at 2000 rpm to remove debris and the supernatant applied on 10-20% lactose gradient, pH 7.4, 8.0 or 8.4, and ultracentrifuged at 40000 rpm in VTi55 rotor at 49C
for 16 hours to pellet and isolate the YiruS.
Chemical Modification of HBV
HBV obtained (3.0 mg of protein~ was lactosaminated in a similar fashion to that described in Example 1 using 10 mg of sodium cyanoborohydride for 3 hours at 25C. The modified virus was sterilized by filtration through 0.45 ~m 30 membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% F3S.
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Infection of Cells with Unmodi~ied and Modified_~Y
Huh7 and IMR-90 cells were plated at 25-50~
confluence in 35 or lO0 mm diameter plastic dishes.
Cell medium was removed and replaced with medium 05 containing modified or unmodified virus and incubated at 37C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of H~V
DNA and medium analyzed for the presence of hepatitis B surface antlgen (HBsAg).
DetectiQn of Tarqeted HBV DNA in Huh7 Cells Treated with Modified an~ ~nm~dified H~V
DNA was e~tracted fro~ cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 3:2303-2312 (1976), in which the cells were washed twice with lO ml of cold Tris-buffered saline (TBS), scraped off into TBS and centri~uged at 200 rpm. The cell pellet was resuspended in lO mM
Tris-HCl, pH 8.0, 1 mM EDTA, pH 3.0, was addeid to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA
was isolateid by ethanol precipit~tion after phenol extraction. The DNA was analyzed by Southern blot using a y32P-ATP labeled cDNA probe specific for HBV
sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genoime, obtained from Dr.
Jake Liang, Massachusetts General Hospital).
The Southern blot showed no hybridizable sequences when probed with our cDNA probe specific 30 for HBV. This confirms the previous finding that Huh7 cells, even though of human origin, cannot be infeicted by unmodified HBV under the conditions of . , , , . - . ,. , :.. .. .
... ..
.
, WO 92/0~,l8n ~cr/U.~91/n7.~03 2 ~ J 2 3 routine cell culture. In addition, the data indicate that the washing procedures eliminate any detectable non-specifically bound HBV DNA on these cells. However, treatment of the Huh7 cells with 05 modified HBV for as little as one day of incubation resulted in a strong signal of hybridizable bands on the Southern blot corresponding to those expected for the plasmid sequences. IMR-90, asialoglycoprotein (-) cells did not produce hybridizable sequences under any conditions.
Detection of HBsAa in the SuPernatant of Huh7 and IMR-90 Cells ExPosed t~_Unmodified or Modifi~d ~BV
Medium from Huh7 and IMR-90 cells was incubated with modified or unmodified HBV as described above and at various intervals was assayed for HBsAg by an enzyme immunoassay kit (Auszyme Monoclonal). The conditions were those recommended by the manufacturer, Abbott Labs.
As shown in Table 4, the background color absorbance was approximately 0.1:21 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. UnmodiEied HBV did not result in significant production of HBsAg.
Absorbance here was approximately 0.180. Similarly, the color absorbance r-flecting HBV levels in IMR-90 cells did not exceed 0.110. However, Huh7 cells treated with modified HBV released HBsAg into their supernatants, with absorbance ranging from 0.760 to 0.865.
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Table 4 Levels of Hepatitis B Surface Antigen (HBsAg) in Culture Medium as Determined by Auszyme Assay (Absorbance Units) 05 Cells IMR- 90 ~1~1h7 DaY Modified Untreated Unmodified Modified HBV HBV HBV
1 .121 ~ .05q .135 ~ .017 .850 ~ .010 3 .186 ~ .036 .700 ~ .012 .171 + .010 .865 + .053 7 .110 + .023 .764 ' .067 Equivalents Those skilled in the art will recognize, or be ablé to ascertain using no more than routine e~perimentation, numerous eguivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following 20 claims.
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Claims (40)
1. A method of targeting a virus or a cell for internalization into a target cell, comprising introducing onto the surface of the virus, or cell, a molecule which binds to a surface receptor of the target cell to produce a modified virus or cell which binds to the receptor, is internalized selectively by the cell in vivo and expresses the delivered nucleic acid.
2. The method of claim 1, wherein the virus or cell is a bacterium, a protozoan or a mammalian cell.
3. The method of claim 1, wherein the virus or cell, in unmodified form, is not normally internalized by the target cell.
4. The method of claim 3, wherein the virus or cell is a human pathogen and the target cell is a nonhuman cell.
5. A method of targeting the internalization of a virus or viral component into a target cell, comprising introducing onto the surface of the virus or viral component a molecule which binds to a receptor of the target cell to produce a modified virus or viral component which binds to the receptor and is internalized selectively by the cell.
6. The method of claim 5, wherein the virus is infective.
7. The method of claim 5, wherein the virus or viral component is replication defective.
8. The method of claim 5, wherein the virus is a retrovirus.
9. The method of claim 5, wherein the virus, or viral component, in unmodified form, does not infect the cell.
10. The method of claim 9, wherein the virus is a human pathogen and the target cell is a nonhuman cell.
11. The method of claim 5, wherein the virus is a pathogen for hepatocytes.
12. The method of claim 10, wherein the virus is a hepatitis virus.
13. The method of claim 12, wherein the receptor mediates endocytosis of the molecule by the cell.
14. The method of claim 13, wherein the receptor is an asialoglycoprotein receptor, the molecule introduced onto the surface of the virus, or viral component, is a ligand for the asialoglycoprotein receptor and the targeted cell bears an asialoglycoprotein receptor.
15. The method of claim 13, wherein the ligand for the asialoglycoprotein receptor is galactose or N-acetyl galactosamine and the target cell bearing an asialoglycoprotein receptor is an hepatocyte.
16. The method of claim 5, wherein the molecule is introduced onto the surface of the virus or viral component by chemical coupling.
17. A method of targeting the infectivity of a virus to a cell bearing an asialoglycoprotein receptor, comprising introducing onto the surface of the virus a ligand for the asialoglycoprotein receptor to produce a modified virus which infects a cell bearing asialoglycoprotein receptor.
18. The method of claim 17, wherein the ligand for the asialoglycoprotein receptor is lactose or galactose.
19. The method of claim 17, wherein the cell bearing the asialoglycoprotein receptor is an hepatocyte.
20. The method of claim 17, wherein the virus is a human pathogen and the cell is a non-human cell.
21. The method of claim 20, wherein the virus is hepatitis virus.
22. A modified virus, or component thereof, having on its surface a molecule which binds to a surface component of a cell which is not normally infectable by the virus in its unmodified form, the modified virus, or component thereof, being capable of binding to and being internalized by the cell.
23. The modified virus of claim 22, wherein the cellular surface component of the cell is a receptor which mediates endocytosis by the cell.
24. The modified virus of claim 23, wherein the receptor is an asialoglycoprotein receptor and the molecule is a ligand for the asialoglyco-protein receptor.
25. The modified virus of claim 22, which is a human pathogen.
26. The modified virus of claim 25, which is a hepatitis virus.
27. Modified hepatitis virus containing lactose or galactose terminal carbohydrates on its surface.
28. A method of introducing nucleic acid into a cell, comprising:
a) incorporating the nucleic acid into a viral vector comprising a modified virus, or viral component, containing a molecule on its surface which binds to a surface component of the cell; and b) contacting the viral vector and the cell under conditions which allow the vector to become internalized by the cell and expresses the introduced nucleic acid.
a) incorporating the nucleic acid into a viral vector comprising a modified virus, or viral component, containing a molecule on its surface which binds to a surface component of the cell; and b) contacting the viral vector and the cell under conditions which allow the vector to become internalized by the cell and expresses the introduced nucleic acid.
29. The method of claim 28, wherein the virus, or component thereof, in unmodified form, does not ordinarily infect the cell.
30. The method of claim 28, wherein the nucleic acid is an expressible gene.
31. The method of claim 28, wherein the virus is a retrovirus.
32. The method of claim 28, wherein the molecule introduced onto the surface of a virus or viral component is a galactose derivative, the cellular surface component is a ligand for the asialoglycoprotein receptor and the cell bears an asialoglycoprotein receptor.
33. The method of claim 32, wherein the cell bearing an asialoglycoprotein receptor is an hepatocyte.
34. A method of infecting an animal cell with a human virus that, in unmodified form, does not normally infect the animal cell, comprising providing a modified human virus having on its surface a molecule which binds to a surface component of the animal cell, the modified human virus being capable of binding to and infecting the animal cell and contacting the modified virus and the cell under conditions which allow the modified virus to bind to and infect the cell.
35. The method of claim 34, wherein the human virus is a human pathogen.
36. The method of claim 34, wherein the animal cell and the modified virus are contacted in vivo.
37. The method of claim 34, wherein the animal cell and the modified virus are contacted in vitro.
38. An animal cell infected with a modified human virus, the cell being uninfectable by the virus in unmodified form.
39. The animal cell of claim 38, comprising an hepatocyte infected with hepatitis virus.
40. An animal infected with a modified human virus, the animal being uninfectable by the virus in unmodified form.
,
,
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| US59095690A | 1990-10-01 | 1990-10-01 | |
| US590,956 | 1990-10-01 | ||
| US72270091A | 1991-06-28 | 1991-06-28 | |
| US722,700 | 1991-06-28 |
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| CA2092323A1 true CA2092323A1 (en) | 1992-04-02 |
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|---|---|---|---|
| CA 2092323 Abandoned CA2092323A1 (en) | 1990-10-01 | 1991-09-27 | Targeting viruses and cells for selective internalization by cells |
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| EP (1) | EP0553235A1 (en) |
| JP (1) | JPH07500961A (en) |
| AU (1) | AU660629B2 (en) |
| CA (1) | CA2092323A1 (en) |
| WO (1) | WO1992006180A1 (en) |
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| ATE152169T1 (en) * | 1988-02-05 | 1997-05-15 | Whitehead Biomedical Inst | MODIFIED HEPATOCYTES AND THEIR APPLICATION |
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- 1991-09-27 JP JP3517570A patent/JPH07500961A/en active Pending
- 1991-09-27 WO PCT/US1991/007103 patent/WO1992006180A1/en not_active Application Discontinuation
- 1991-09-27 CA CA 2092323 patent/CA2092323A1/en not_active Abandoned
- 1991-09-27 EP EP91919436A patent/EP0553235A1/en not_active Withdrawn
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| EP0553235A4 (en) | 1994-02-09 |
| EP0553235A1 (en) | 1993-08-04 |
| AU660629B2 (en) | 1995-07-06 |
| AU8860391A (en) | 1992-04-28 |
| WO1992006180A1 (en) | 1992-04-16 |
| JPH07500961A (en) | 1995-02-02 |
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