EP0553235A1 - Targeting viruses and cells for selective internalization by cells - Google Patents

Targeting viruses and cells for selective internalization by cells

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Publication number
EP0553235A1
EP0553235A1 EP91919436A EP91919436A EP0553235A1 EP 0553235 A1 EP0553235 A1 EP 0553235A1 EP 91919436 A EP91919436 A EP 91919436A EP 91919436 A EP91919436 A EP 91919436A EP 0553235 A1 EP0553235 A1 EP 0553235A1
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Prior art keywords
virus
cell
modified
receptor
cells
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French (fr)
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EP0553235A4 (enrdf_load_stackoverflow
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George Y. Wu
Catherine H. Wu
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University of Connecticut
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University of Connecticut
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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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
    • C—CHEMISTRY; METALLURGY
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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

Definitions

  • Viruses represent a natural and efficient means for the introduction of foreign genes into cells.
  • viruses are useful tools for the study of genes, and gene regulation in vitro and for gene therapy.
  • most viruses have broad cell specificity and can infect a wide variety of cell types. This can lead to foreign gene expression in many tissues, some of which may be undesirable, especially for clinical applications.
  • viral infection is mediated by interactions between viral envelopes and plasma membranes of target cells.
  • specific viral structures are recognized and bound by cellular receptors.
  • HIV employs envelope glycoproteins to bind to helper T lymphocytes via CD4 (T4) receptors.
  • T4 CD4
  • virus specificity can be redirected by attaching antibodies to viruses.
  • Goud, B., et aL. Virolo ⁇ v 161:251-254 (1988) linked anti-transferrin receptor antibodies to obtain delivery of a retrovirus to human cells bearing the transferrin receptor.
  • 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.
  • the invention pertains to a method of targeting a virus or a cell to a target cell for selective internalization in vivo (or i vitro) 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 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.
  • 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 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 membrane) by chemically coupling it, either directly or through bridging agents, to the surface or by treating the surface to expose 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 a target cell.
  • 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 a cell which, in unmodified form, it would not normally infect. In this way, animal models of human diseases which do not have adequate experimental animal counterparts can be developed for study of the diseases.
  • an ecotropic human pathogen such as the hepatitis or AIDS virus
  • Figure 1 shows in situ ⁇ -galactosidase expression in NIH 3T3, HepG2 and SK Hepl cells treated separately with unmodified or modified murine leukemia virus.
  • Figure 2 shows internalization of 3 5 S-biolabeled modified Moloney murine leukemia virus.
  • Figure 3 shows a chromatogram of asialooro- mucoid-complexed Psi2 virus on Sephadex G150.
  • Figure 4 shows the ⁇ -galactosidase activity of various cells exposed to Psi2 virus-asialoglyco- protein conjugate.
  • a virus or cell is targeted for selective internalization into a target cell by modifying the surface of the virus or cell to introduce a molecule which specifically binds to a surface receptor of the target cell.
  • the cellular surface receptor is one which will mediate internalization 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.
  • 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 abrogate an existing property.
  • the tropism of a virus can be altered or redirected to target infectivity to a cell type or types not normally infected by the virus in natural (or unaltered) form.
  • 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 be replication defective or otherwise defective in structure or function.
  • viral particles either essentially or completely devoid of genomic nucleic acid (e.g., "empty" viral envelope) can also be targeted.
  • the present method also provides a means of targeting cells. These include cellular organisms such as bacteria, protozoa or trypanosomes whose tropism can be altered.
  • mammalian cells can be targeted.
  • the receptor-specific molecule can be a ligand for the surface receptor of the target cell.
  • the molecule is a ligand for a cellular surface receptor which mediates internalization of the ligand by the process of endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
  • Glycoproteins having certain exposed terminal carbohydrate groups can be used as receptor-specific molecules.
  • asialoglycoprotein (galactose-terminal) ligands are preferred.
  • asialoglycoproteins include asialoorosomucoid or asialofetuin.
  • Other useful galactose-terminal carbohydrates for hepatocyte targeting include carbohydrate trees obtained from natural glycoproteins, especially tri- and tetra-antennary structures that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues.
  • naturally occurring plant carbohydrates such as arabinogalactan can be used.
  • other types of carbohydrates can be used.
  • mannose and mannose-6 phosphate or carbohydrates having these terminal carbohydrate structures could used to target macrophages or endothelial cells.
  • 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 receptors.
  • 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.
  • a ligand e.g., antigen
  • Antibodies specific for cellular 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 receptor.
  • the receptor-specific molecule can be introduced onto the envelope of a virus or the membrane of a cell.
  • the molecule will be coupled to (or exposed on) a proteinaceous component of the surface but other components may be used.
  • the receptor-specific molecule can be introduced onto the surface of the virus or cell by different means.
  • the receptor-specific molecule is chemically coupled to the surface.
  • galactose moieties ligand for the asialoglycoprotein receptor
  • the receptor-specific molecule can be chemically coupled to components of the surface of the virus or cell through bridging agents such as biotin and avidin.
  • a biotinylated receptor-specific molecule can be linked through avidin or streptavidin to a biotinylated surface component of the virus or cell.
  • the virus or cell can be 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 and binding.
  • desired carbohydrate residues e.g., galactose residues
  • neurominidase treatment of certain polycarbohydrates leaves exposed terminal galactose residues in a tri- or tetra-antennary arrangement.
  • the modified virus or cell is administered in vivo, generally in an amount sufficient 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 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 vitro) so that it is expressed in the cell.
  • the nucleic acid can be an 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 invention, to target it to the cell.
  • Preferred viral vectors for delivery of foreign genes in vivo (or ex vivo) are retroviruses.
  • the targeted viral vector is administered in vivo, as described, where i 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 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 corrective gene in vivo.
  • Certain pathogenic viruses such as hepatitis virus or human immunodeficiency virus infect only human cells.
  • such viruses can be modified to enable them to infect experimental animals such as rodents.
  • the hepatitis virus which infects only human liver cells can be modified so that it will infect non-human liver cells.
  • a ligand for rodent asialoglycoprotein receptor e.g., galactose
  • galactose asialoglycoprotein receptor
  • This modified hepatitis virus which can infect a rodent and the infected rodent or rodent cells provides an experimental animal system for study of the hepatitis virus.
  • the invention is illustrated further by the following examples.
  • 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 ⁇ ere cell line was kindly provided by Dr. James Wilson, University of Michigan. Wilson, J.M., e_£ al. Proc. Natl. Acad. Sci. USA 87:439-443 (1990). Under normal circumstances, this virus infects only rodent cells. Wilson, J.M. , e_£ al. Proc. Natl. Acad. Sci. USA £5_:3014-3018 (1988); Goud, B., et al. Virology JL__3.:251-254 (1988).
  • the producer cells were grown in Dulbecco's modified Eagle's medium (GIBCO Laboratories, Grand Island, NY) supplemented with 10% heat-inactivated calf serum (GIBCO) .
  • Dulbecco's modified Eagle's medium GIBCO Laboratories, Grand Island, NY
  • heat-inactivated calf serum GIBCO
  • producer cells were cultured in serum-free Dulbecco's modified Eagle's medium for 3 days.
  • two strategies were developed for the modification of the surface of the harvested virus: A) chemical coupling of galactose residues to the virus and B) chemical coupling of an asialoglycoprotein to the virus.
  • virus-containing medium was applied on a 10-20% sugar gradient in which ⁇ -lactose was substituted for sucrose (Sigma, St. Louis, MO) in 10 mM Tris-Cl, 150 mM NaCl, 1 mM EDTA, and was ultracentrifuged (LB-55, Beckman Instruments, San Ramon, CA) at 40,000 rpm in VTi 55 rotor (Beckman) at 4°C for 17 hours.
  • Fetal bovine serum (GIBCO) was added subsequently to make a 10% solution. Except for stability experiments, all samples were used 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. and Mulligan, R.C. Proc. Natl. Acad. Sci. USA &_>: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).
  • virus was biosynthetically labeled by incubation of producer cells (5.0 x 10 6 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 isolated from supernatants and modified as described above followed by dialysis against minimum essential medium.
  • human hepatoma cell lines HepG2, asialoglycoprotein receptor (+) (Schwartz, A.L., e ⁇ al. J. Biol. Chem. 25.6:8878-8881 (1981)) obtained from B.B. Knowles, Wistar Institute, 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., ej al. J. Biol. Chem.
  • NIH 3T3 murine fibroblast cell line NIH 3T3 (Goud, B., et aj___. Virology 163.:251-254 (1988)) which is also asialoglycoprotein receptor (-) .
  • the latter two cell lines were purchased from American Type Culture Collection (Rockville, MD) . All were maintained in Eagle's minimum essential medium supplemented with 10% heat inactivated fetal bovine serum at 37°C under 5% C ⁇ 2-
  • RNA 0.5 mg viral protein
  • Dulbecco's modified Eagle's medium were added to the culture medium and exposed to cells for 5 days at 37°C under 5% CO2.
  • Results were expressed in U/mg of cellular protein according to the method by Norton, P.A. and Coffin, J.M. Mol. Cell. Biol. 1:281-290 (1985), using purified E___ coli ⁇ -galactosidase (Sigma) activity as a standard. Protein concentrations of the cellular samples were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) following the manufacturer's instructions.
  • virus was added to the cell media together with a 100-fold molar excess of a natural asialoglycoprotein, asialoorosomucoid, prepared by desialylation (Oka, J.A., and Weigel, P.H. J. Biol. Chem. 258: 10253-10262 (1983)) of orosomucoid as previously described by Whitehead., D.H., and Sam ons, H.G. Biochim. Biophys. Acta 124:209-211 (1966). 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 means + S.E.
  • Table 1 shows that unmodified virus did not produce enzymatic activity in human HepG2 or SK Hepl cells as expected from the ecotropism of the virus. Also, modified virus did not produce ⁇ -galactosidase activity in SK Hepl, asialoglycoprotein receptor (-) cells. However, modified virus did produce high ⁇ -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 excess of asialoorosomucoid, supporting the notion that the transfection by modified virus was, in fact, mediated by asialoglycoprotein receptors.
  • ⁇ -galactosidase activity was high, 50.6 ⁇ 5.2. in Morris 7777 rat cells after exposure to unmodified virus.
  • ⁇ -galactosidase activity in these same cells was significantly lower when exposed to 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 linking lactose to protein has been shown to be enhanced under alkaline conditions. Schwartz, B.A. and Gray, G.R. Arch. Biochem. Biophys. 181:542-549 (1977).
  • virus modified at different pHs were administered to He ⁇ G2 cells, and ⁇ -galactosidase activity measured.
  • Table 2 shows that enzymatic activity rose from 50.3 + 1.2, for virus modified at pH 7.4; to 71.2 + 4.8, for virus modified at pH 8.0. However, activity was significantly lower, 25.1 + 2.4, in cells treated with virus modified at pH 8.4.
  • + virus was modified at pH 8.0 then incubated with cells for 5 days. * calculated as the difference in activity between treated and untreated cells.
  • HepG2, SK Hepl and Morris 7777 cells were incubated at 37°C in serum-free Dulbecco's modified Eagle's medium containing 3 5s-biolabeled, modified virus, 3.3 ⁇ g viral RNA (98 ⁇ g viral protein) (Watanabe, N., e£ al. Cancer Immunol. Immunother. 28:157-163 (1989)) with a specific activity of 6.1x10 ⁇ cpm/mg viral RNA.
  • 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.
  • Specific ⁇ -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.
  • the coupling of lactose to proteins to target artificial asialoglycoproteins is based on the specificity of sodium cyanoborohydride to reduce Schiff's bases formed between aldehyde and amino groups to render the bonds irreversible.
  • Treatment of viruses with aldehydes is not always similarly benign. For example formaldehyde has been used to inactivate viruses in the production of vaccines. Buynak, E.B., et al. J. 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 approximately 8.0, beyond which the function of 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. Virology 125:85-98 (1983). Although the asialoglycoprotein endocytotic pathway is ultimately degradative with delivery of ligands to lysosomes (Tolleshaug, H., ei al. Biochim.
  • 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 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 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.
  • 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.
  • Hepatitis B virus is a human pathogen that possesses very narrow host (species) and organ (liver) specificities, in 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.
  • Hepatitis B virus was obtained from Hep G2 producer cells chronically infected with HBV as described by Sells et. ai. Proc. Natl. Acad. Sci. :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.
  • MEM Dulbecco's modified Eagle's medium
  • 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) .
  • FBS fetal bovine serum
  • He ⁇ G2 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 4°C for 16 hours to pellet and isolate the virus.
  • HBV 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 25°C.
  • the modified virus was sterilized by filtration through 0.45 ⁇ m membranes and then dialyzed against MEM through membranes with a 12-14000 molecular weight exclusion limit followed by dialysis against MEM plus 10% FBS.
  • Huh7 and IMR-90 cells were plated at 25-50% confluence in 35 or 100 mm diameter plastic dishes. Cell medium was removed and replaced with medium containing modified or unmodified virus and incubated at 37°C. Cells were washed and changed to fresh medium every three days and at regular intervals cells were studied for the presence of HBV DNA and medium analyzed for the presence of hepatitis B surface antigen (HBsAg) .
  • HBsAg hepatitis B surface antigen
  • DNA was extracted from cells according to the method by Blin, N. and Stafford, D.W. Nucleic Acid Res. 2:2303-2312 (1976), in which the cells were washed twice with 10 ml of cold Tris-buffered saline (TBS), scraped off into TBS and centrifuged at 200 rpm. The cell pellet was resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, was added to the same buffer containing 20 mg/ml RNase, 0.5% SDS, and then treated with proteinase K. Cellular DNA was isolated by ethanol precipitation after phenol extraction.
  • TBS cold Tris-buffered saline
  • the DNA was analyzed by Southern blot using a ⁇ 32 P-ATP labeled cDNA probe specific for HBV sequences (a Bam HI restriction fragment of plasmid adw HTD carrying the HBV genome, obtained from Dr. Jake Liang, Massachusetts General Hospital).
  • the background color absorbance was approximately 0.121 in untreated Huh7 cells and there was no significant difference between day 1 and day 7. Unmodified HBV did not result in significant production of HBsAg. Absorbance here was approximately 0.180. Similarly, the color absorbance reflecting 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. Table 4
  • HBsAg Hepatitis B Surface Antigen

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