WO1993004701A1 - Apport cible de polynucleotides ou d'oligonucleotides a des cellules - Google Patents
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- WO1993004701A1 WO1993004701A1 PCT/US1992/007339 US9207339W WO9304701A1 WO 1993004701 A1 WO1993004701 A1 WO 1993004701A1 US 9207339 W US9207339 W US 9207339W WO 9304701 A1 WO9304701 A1 WO 9304701A1
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
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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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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3513—Protein; Peptide
Definitions
- Antisense oligonucleotides hold great promise as means of specifically inhibiting unwanted gene expression in cells. Improvements in the delivery of oligonucleotides to cells will enhance effectiveness. Naked antisense oligonucleotides can be taken up by cells non-specifically and at low efficiency. Some methods have been explored to increase uptake. Lemaitre et al. covalently coupled an oligonucleotide to polylysine and demonstrated inhibition of viral gene expression at several fold lower than DNA concentrations compared to mixtures of polylysine and antisense DNA (Lemaitre, M. et. al. Proc. Natl. Acad. Sci. USA 84:648-652). Although specific antiviral effects were shown, specific delivery was not demonstrated.
- This invention pertains to a soluble, targetable molecular complex for targeting poly- or oligonucleo ⁇ tides to a specific cell to inhibit the expression of a gene or genes.
- the molecular complex comprises a single-stranded poly- or oligonucleotide which hybridizes to an RNA transcript of the gene, complexed to a carrier which is a conjugate of a cell-specific binding agent and a poly- or oligonucleotide-binding agent.
- the complex is administered in a pharmaceutically acceptable solution in an amount sufficient to hybridize to and inhibit the function of the RNA transcript.
- the poly- or oligonucleotide can be DNA or RNA.
- an antisense oligodeoxy- nucleotide can be used which hybridizes to and inhibits the function of an RNA.
- the targeted RNA is typically a messenger RNA.
- the oligonucleotide can also be an RNA which has catalytic activity (a ribozyme) .
- the target for antisense or ribozyme- mediated inhibition can be a gene or genes of cellular origin (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus) .
- the cell-specific binding agent is specific for a cellular surface structure, typically a receptor, which mediates internalization of bound ligands by endocytosis, such as the asialoglycoprotein receptor of hepatocytes.
- the cell-specific binding agent can be a natural or synthetic ligand (for example, a protein, polypeptide, glycoprotein, carbohydrate, etc.) or it can be an antibody, or an analogue thereof, which specifically binds a cellular surface structure which then mediates internalization of the bound complex.
- the poly- or oligonucleotide-binding component of the conjugate is a compound such as a polycation which stably complexes the single-stranded poly- or oligonucleotide under " extracellular conditions and releases it under intracellular conditions so that it can function within the cell.
- the complex of the gene and the carrier can be used in vitro or in vivo to selectively deliver poly- or oligonucleotides to target cells.
- the complex is stable and soluble in physiological fluids. It can be administered in vivo where it is selectively taken up by the target cell via the surface-structure- mediated endocytotic pathway.
- the incorporated poly- or oligonucleotide hybridizes with its complementary RNA, thereby inhibiting function of the RNA and expression of the target gene or genes.
- Figure 1 shows the uptake of complexed antisense DNA by HepG2 and SK Hepl cells.
- Figure 2 shows the effect of complexed antisense DNA on hepatitis B virus surface antigen concentration in culture medium.
- FIG. 3 shows Southern blots of DNA extracted from medium and cells after 24 hrs of exposure to antisense DNA.
- a soluble, targetable molecular complex is used to selectively deliver a single-stranded poly- or oligonucleotide to a target cell or tissue in vivo to specifically inhibit gene expression.
- the molecular complex comprises the oligonucleotide to be delivered complexed to a carrier made up of a binding agent specific for the target cell and a DNA-binding agent. The complex is selectively taken up by the target cell and the oligonucleotide hybridizes to the RNA transcript which inhibits expression of the targeted gene(s) .
- the poly- or oligonucleotide is a single- stranded molecule which hybridizes to a specific RNA under intracellular conditions.
- the degree of complementarity required for appropriately specific hybridization to the target RNA sequence under intra ⁇ cellular conditions can be determined empirically.
- the oligonucleotide is an antisense oligodeoxynucleotide.
- the antisense oligodeoxynucleotide can be a normal oligodeoxy ⁇ nucleotide or an analogue of an oligodeoxynucleotide (e.g., phosphorothioate oligonucleotides., in which one of the phosphate oxygens is replaced by a sulfur atom) sufficiently stable to reach the target in effective concentrations. See e.g., Stein, CA. and Cohen, J.S. (1988) Cancer Research 48:2659-2668.
- Antisense oligodeoxynucleotides can be prepared by standard synthetic procedures. The antisense oligonucleotides can be designed to operate by different mechanisms of gene inhibition.
- these mechanisms involve the hybridization of the oligonucleotide to a specific RNA sequence, typically a messenger RNA.
- the targeted sequence can be located in the coding region of the RNA or it can be a signal sequence required for processing or translation of the RNA.
- the targeted sequence can be a sequence normally found in an organism or a sequence found in a pathogenic organism but not in its host.
- the oligonucleotide may form a triple helix DNA structure, inhibiting transcription of the mRNA sequence.
- the oligonucleotide can be an RNA molecule which has catalytic activity, i.e., a ribozyme.
- Ribozymes are advantageous because they specifically cleave and, thus, destroy the targeted RNA sequence. Ribozymes are described in U.S. Patent No. 4,987,071.
- the carrier component of the complex is a conjugate of a cell-specific binding agent and an oligonucleotide-binding agent.
- the cell- specific binding agent specifically binds a cellular surface structure which mediates its internalization by, for example, the process of endocytosis.
- the surface structure can be a protein, polypeptide, carbohydrate, lipid or combination thereof. It is typically a surface receptor which mediates endo ⁇ cytosis of a ligand.
- the binding agent can be a natural or synthetic ligand which binds the receptor.
- the ligand can be a protein, polypeptide, carbohydrate, lipid or a combination thereof which has functional groups that are exposed sufficiently to be recognized by the cell surface structure.
- the binding agent can also be an antibody, or an analogue of an antibody such as a single chain antibody, which binds the cell surface structure.
- Ligands useful in forming the carrier will vary according to the particular cell to be targeted.
- glycoproteins having exposed terminal carbohydrate groups such as asialoglyco- protein (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed.
- asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
- Such ligands can be formed by chemical or enzymatic desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues.
- asialoglycoprotein ligands can be formed by coupling galactose terminal carbohydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive lactosamination.
- galactose terminal carbohydrates such as lactose or arabinogalactan
- non-galactose bearing proteins by reductive lactosamination.
- other types of ligands can be used, such as mannose for macrophages (lymphoma), mannose-6-phosphate glycoproteins for fibroblasts (fibrosarcoma) , intrinsic factor-vitamin B12 for enterocytes and insulin for fat cells.
- the cell-specific binding agent can be a receptor or receptor-like molecule, such as an antibody which binds a ligand (e.g., antigen) on the cell surface.
- Such antibodies can be produced by standard procedures.
- the poly- or oligonucleotide-binding agent complexes the oligonucleotide to be delivered. Complexation with the oligonucleotide must be sufficiently stable in vivo to prevent significant uncoupling of the oligonucleotide extracellularly prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the oligonucleo ⁇ tide is released in functional, hybridizable form.
- the complex can be labile in the acidic and enzyme rich environment of lysosomes.
- a non ⁇ covalent bond based on electrostatic attraction between the binding agent and the oligonucleotide provides extracellular stability and is releasable under intracellular conditions.
- Preferred poly- or oligonucleotide-binding agents are polycations that bind the negatively charged nucleic acid strands. These positively charged materials can bind noncovalently with the poly- or oligonucleotide to form a soluble, targetable molecular complex which is stable extracellularly but which releases the poly- or oligonucleotide as a functional (e.g., hybridizable) molecule intracellularly.
- Suitable polycations are polylysine, polyarginine, polyornithine, basic proteins such as histones, avidin, protamines and the like.
- a preferred polycation is polylysine (e.g., ranging from 3,800 to 60,000 daltons) .
- noncovalent bonds that can be used to releasably link the poly- or oligonucleotide include hydrogen bonding, hydrophobic bonding, electrostatic bonding alone or in combination such as, anti-poly- or oligonucleotide antibodies bound to poly- or oligonucleotide, and streptavidin or avidin binding to poly- or oligonucleotide containing biotinylated nucleotides.
- the carrier can be formed by chemically linking the cell-specific binding agent and the oligonucleo ⁇ tide-binding agent.
- the linkage is typically covalent.
- a preferred linkage is a peptide bond. This can be formed with a water soluble carbodiimide as described by Jung, G. et al. (1981) Biochem. Biophys. Res. Cornmun. 101:599-606.
- An alternative linkage is a disulfide bond.
- the linkage reaction can be optimized for the particular cell-specific binding agent and oligonucleotide-binding agent used to form the carrier. Reaction conditions can be designed to maximize linkage formation but to minimize the formation of aggregates of the carrier components.
- the optimal ratio of cell-specific binding agent to poly- or oligonucleotide-binding agent can be determined empirically. When polycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the poly- or oligonucleotide.
- the ratio can range from 2:1 to 1:2 by weight (asialoorosomucoid:oligonucleotide) can be used.
- Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography.
- the conjugate is purified on a high pressure liquid cation exchange column (AquaporeTM cation-exchange, Rainin) with stepwise elution with 0.1 M sodium acetate, pH 5.0, 2.5, 2.25 and 2.0.
- the poly- or oligonucleo ⁇ tide and carrier are mixed and incubated under conditions conducive to complexation.
- the poly- or oligonucleotide and carrier can be mixed at the appropriate ratio in 2 M NaCl and the solution can be diluted to 0.15 M and filtered to provide an administrable composition.
- the molecular complex can contain more than one copy of the nucleic acid strand.
- the amount of poly- or oligonucleotide should not exceed that required to maintain solubility of the resulting complex.
- the preferred weight or molar ratio of carrier to poly- or oligonucleotide for the construct employed can be determined by routine experimentation,
- the molecular complex of this invention can be administered parenterally. Preferably, it is injected intravenously.
- the complex is administered in solution in a physiologically acceptable vehicle.
- the molecular complex of this invention can be used to target delivery of a wide range of poly- and oligonucleotide for specific hybridization usually to an RNA target.
- the target can also be DNA by triplex formation.
- the oligonucleotide can be directed against translation of a gene or genes of cellular origin (e.g., a cellular oncogene) or of noncellular origin (e.g., a viral oncogene or the genes of an infecting pathogen such as a virus or a parasite such as malaria, trypanosome, lysteria or mycoplasma) .
- the antisense can be directed against transcription of target genes.
- the method of this invention can be used to treat hepatitis infection.
- the complex can be used to deliver an antisense oligo ⁇ deoxynucleotide specifically to liver cells to block production of hepatitis virus.
- One strategy is to take advantage of the fact that, because of its compact nature, the human hepatitis B virus has only one polyadenylation signal (nucleotides 1903-1923) . The signal is common to all hepatitis B viral-derived mRNA and is different from the mammalian signal. As a result, antisense oligodeoxynucleotides comple ⁇ mentary to this region can block viral protein synthesis.
- the antisense strand is complexed to a carrier comprising a ligand for the hepatic asialoglycoprotein receptor and a polycationic protein such as polylysine to provide a soluble molecular complex targetable to the liver.
- the method of this invention can be used to alter the expression of a gene of cellular origin.
- This method may be useful in the treatment of diseases characterized by abnormal biosynthesis, especially overexpression, of normal or abnormal cellular proteins.
- a targetable, soluble DNA carrier was prepared by coupling asialoorosomucoid to poly L-lysine
- a 21-mer oligodeoxynucleotide, complementary to a portion of the human hepatitis B virus (ayw subtype) (Hirschman, S.Z. et al. (1980) Proc. Natl. Acad. Sci. USA
- nucleotides 1903-1923 See SEQUENCE ID NO. 1 - TTTATAAGGGTCGATGTCCAT) of the viral genome, was synthesized with phosphorothioate linkages on an automated nucleotide synthesizer (Applied Biosystems) (Matsukura, M. et al. (1987) Proc. Natl. Acad. Sci. USA 84:7705-7710). As a control, a random 21-mer sequence was prepared in an identical fashion. The purity of oligonucleotides was determined by electrophoresis through 15% polyacrylamide gels stained with ethidium bromide.
- Antisense DNA was titrated with conjugate to form a soluble complex using an agarose gel retardation system as described previously (Wu, G.Y. and Wu, CH. (1987) J. Biol. Chem. 262:4429-4432) and a conjugate to DNA ratio of 1.6:1 by weight (asialoorosomucoid: DNA) was selected.
- antisense DNA was end-labeled with 32 P (Sambrook, J. et al. (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, 2nd ed., vol. 2, pg. 11.31). DNA alone, or in the form of a complex was added to medium of HepG2 and SK Hepl cells to make 50 ⁇ M solutions with respect to added antisense DNA. Uptake was determined as described previously for asialoglycoproteins (Schwartz, A.L. et al. (1981) J. Biol. Chem. 256:8878-8881).
- HepG2 2.2.15 cells were seeded six days pre-confluence and incubated at 37°C in medium containing antisense DNA alone, complexed antisense DNA, complexed random DNA, or medium alone. All media containing added DNA were initially 50 ⁇ M with respect to DNA. At daily intervals, 50 ⁇ l of medium was sampled and assayed for hepatitis B surface antigen by an ELISA (Abbott) method as described by the manufacturer, modified for quantitation using serially diluted standard surface antigen (CalBiochem) which produced a linear response within the range of antigen levels found in the samples. Cell number was determined by microscopic counting cells stained with trypan blue. All points were determined in triplicate and the results of four experiments are shown as means ⁇ SEM expressed as ⁇ g/ml/10 6 cells. Effect of Antisense DNA on Protein Secretion
- HepG2 2.2.15 cells were incubated with 50 ⁇ M complexed DNA as described in Figure 2 except that 10 ⁇ Ci [ 35 S]-methionine (Amersham), specific activity 1000 Ci/mmole, was added to label newly synthesized proteins. After 24 hrs, medium was moved, cells washed with phosphate buffered saline and lysed with 1% sodium dodecyl sulfate which was subsequently removed with triton X-100. Both media and cell lysates were treated with a specific rabbit anti- surface antigen antibody (DAKO) and precipitated with protein-A sepharose (Sigma) . Precipitates were scintillation counted and each point assayed in triplicate. Total cell protein was determined by colorimetric assay (Bio-Rad) . The results of three experiments are shown as means ⁇ SEM expressed as cpm/mg cell protein.
- HBV DNA was identified by Southern blot using an EcoRI-Bglll fragment of the HBV genome (nucleotide 0 to 1982) as a probe labeled with 32 P and exposed to x-ray film as described previously (Sambrook, J. et al. (1989) Molecular Cloning - A
- FIG. 1 shows uptake of complexed antisense DNA by HepG2 and SK Hepl cells.
- a targetable, soluble DNA carrier was prepared by coupling asialooroso ⁇ mucoid to poly L-lysine using a water soluble carbodiimide as described previously (Wu, G.Y.
- Figure 2 shows the effect of complexed antisense DNA on hepatitis B virus surface antigen concentration in culture medium.
- HepG2 2.2.15 cells were incubated at 37°C in medium containing antisense DNA alone, complexed antisense DNA, complexed random DNA, or medium alone. All media containing added DNA were initially 50 ⁇ M with respect to DNA.
- medium was sampled and assayed for the presence of hepatitis B surface antigen by an ELISA (Abbott) method as described by the manufacturer, modified as described in Materials and Methods.
- Cell number was determined by microscopically counting cells stained with trypan blue. All points were determined in triplicate and the results of four experiments are shown as means £ SEM expressed as ⁇ g/ml/10 6 cells.
- Figure 2 shows that in untreated control cells, hepatitis B viral surface antigen steadily increased in concentration in their media, rising from 1 ⁇ g/ml/10 6 cells on the first day to 5.5 ⁇ g/ml/10 6 cells by the seventh day. Exposure of cells to antisense DNA alone had no significant effect until the 3rd day at which time surface antigen concen ⁇ tration was 30% lower than untreated controls. Nevertheless, surface antigen concentrations in the presence of antisense DNA alone continued to rise steadily throughout the 7 days of exposure. However, treatment with complexed antisense DNA resulted in an 80% inhibition after the 1st day and 95% inhibition by the 7th day compared to untreated controls. There was no significant increase in surface antigen concentration after the first 24 hrs. Complexed random DNA of the same size had no effect on antigen concentrations at any time point under identical conditions.
- FIG. 3 shows Southern blots of DNA extracted from medium and cells after 24 hrs exposure to antisense DNA. Cells were incubated as described for Figure 2 with 50 ⁇ M antisense DNA alone, or in the form of complexes as described for Figure 2. After 24 hrs, medium was removed and DNA, extracted from the medium (Sells, M.A. et al. (1987) Proc. Natl. Acad. Sci. USA M 1005-1009) and from the cell layer (Hirt, B.J. (1967) Mol. Biol. 26:365-371).
- Total cell protein was determined by colorimetric assay (Bio-Rad) .
- DNA extracted from equal volumes of medium and from approximately equal numbers of cells were applied on an agarose gel, HBV DNA was identified by Southern blot using an EcoRI-Bglll fragment of the HBV genome as a probe labeled with 32 P and exposed to x-ray film (Sambrook, J. et al. (1989) Molecular Cloning - A
- Figure 3 lanes 1 and 5 show that untreated cells produced bands at positions expected for relaxed circular and single-stranded linear viral replicative DNA forms. Other minor bands are present, at 2.3 kb for example, as described previously for this cell line (Sells, M.A. et al. (1987) Proc. Natl. Acad. Sci. USA rl005-1009) .
- Lanes 2 and 6 show that treatment of cells with complexed antisense DNA decreased the amount of all viral DNA forms in the medium by approximately 80% compared to untreated cells (lanes 1 and 5).
- Complexed random DNA lanes 3 and 7, had no detectable effect on the levels of HBV DNA under identical conditions.
- Agrawal et al. administered infectious virus (HIV) together with antisense oligonucleotides in a non-targeted manner to cell media (Agrawal, S. et al. (1989) Proc. Natl.
- hepatitis B virus in vivo, persistent production of hepatitis B virus is usually due to the presence of unintegrated viral DNA (Shafritz, D. et al. (1981) N. Eng. J. Med. 305:1067-1073). Integration of the viral genome into that of the host is usually associated with a cessation of production of complete viral particles (Ganem, D. (1982) Rev. Infect. Pis. 4:1026). Whether targeted antisense delivery can be effective in the presence of an infection generated by unintegrated viral DNA remains to be seen.
- asialoglycoprotein uptake in hepatitis virus-infected HepG2 cells was found to be not substantially different from non-infected HepG2 cells (data not shown) , indicating that infection by the virus did not alter the receptor activity in these cells.
- targeted delivery of antisense oligonucleotides may be generally applicable to naturally infected hepatocytes, that are otherwise normal, via asialoglycoprotein receptors. It should be noted that the oligonucleotides used in this current work were linked together by phosphorothioate bonds. These linkages are less susceptible to nuclease degradation than normal phosphodiester bonds.
- Confluent 35mm dishes of 3T3-AsGR were treated with ⁇ l(I) antisense DNA alone (1.3 ⁇ M or 2.7 ⁇ M) or varying concentrations of antisense DNA complex (1.3 ⁇ M, 1.7 ⁇ M, or 2.7 ⁇ M antisense) for 12-16 hours (overnight) at 37°C in DMEM and 10% FBS.
- the medium was removed and replaced with labeling medium containing 5 ⁇ Ci/ml of [ 3 H]-proline, 50 ⁇ g/ml
- L-ascorbate L-ascorbate and varying concentrations of antisense DNA or complexes. Cells were incubated for 4 hours at 37°C in the labeling medium. Newly synthesized procollagens and other proteins were determined by bacterial collagenase digestion (Peterkofsky, B. and Diegelmann, Biochemistry 05 10.:988-994 (1971)) .
- the labeling medium was removed and set aside.
- Buffer containing protease inhibitors (.5 M Tris, pH 7.4, .4 mM NEM, .2 mM PMSF, 2.5 ⁇ i EDTA) was added to 0 the cell layer and the cell layer was removed and pooled with the supernatant. The entire mixture was homogenized using a Dounce homogenizer and ice cold TCA was added to a final concentration of 15%.
- TCA precipitable proteins were digested with bacterial 5 collagenase (Form III, Advanced Biofactures, Lynbrook, NY) at 37°C for 2 hours followed by precipitation with TCA-tannic acid.
- the collagenase-sensitive radioactivity in the supernatant was separated by centrifugation to measure newly synthesized 0 procollagen production.
- the collagenase-resistant precipitated radioactivity was used to calculate non-collagenous protein production. All assays were normalized to equal numbers of cells.
- Antisense DNA was titrated with conjugate to form a soluble complex using an agarose gel retardation system as described previously (Wu, G.Y. and Wu, CH. (1987) J. Biol. Chem. 262:4429-4432) and a conjugate to DNA ratio of 2:1 by weight (asialoorosomucoid-pplylysine (AsOR-PL) : DNA) was selected.
- cells were incubated with [ 125 I]-AsOR at 37°C and at regular time intervals (0.5, 1, 2, or 4 hours), dishes were chilled to 4°C, washed three times with cold 10 mM EDTA-phosphate buffered saline and the cell layers solubilized with 0.1 NaOH and gamma counted.
- RNA samples 60 ⁇ g were heat denatured in 50% formamide, 2x formaldehyde running buffer, 7% formaldehyde at 55°C for 15 minutes (Sambrook, J., et al. Molecular Cloning 7.43, (1989)). Samples were run on formaldehyde gels (1% agarose, 2.2 M formaldehyde) for 4 hours at 100 mvolts on ice. Molecular weight and control RNAs were stained in 0.1 ammonium acetate and 0.5 ⁇ g/ml ethidium bromide and photographed with a fluorescent ruler.
- RNA was transferred onto a nitrocellulose filter under vacuum for one hour and cross-linked to the filter by exposure to ultraviolet irradiation using a Stratalinker. Conditions for prehybridization and hybridization were the same as in the slot blot studies.
- the 3T3-ASGR cells do not normally possess asialoglycoprotein receptors, they were transfected with the asialoglycoprotein receptor genes and were found to have a Kg of 1.5 x 10 -9 M, a binding saturation with [ 125 I]-AsOR at 1.8 ⁇ g/ml, and an uptake rate of [ 12 5 ⁇ ]-AsOR of 1.8 pmole/million cells/hr.
- the number of asialoglycoprotein receptors per cell was 250,000.
- the 3T3-AsGR cells displayed a linear uptake of ⁇ l(I) antisense DNA-AsOR-PL complex up to 4 hours at a rate of 18.2 pmole of DNA/million cells/hr. In contrast, the rate of uptake of labeled ⁇ l(I) antisense DNA alone was 0.15 pmole DNA/million cells/hr. Uptake of AsOR in a 300x excess by weight competed with the uptake of ⁇ l(I) antisense DNA complex. Effect of ⁇ l(I) antisense DNA complex on collagen synthesis ⁇ l(I) antisense DNA complexes inhibited collagen production by 3T3-AsGR cells. This inhibition was specific for collagen production and was dependent on the concentration of ⁇ l(I) antisense DNA in the complex. This result is set forth in the following table.
Abstract
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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EP92920863A EP0666923A1 (fr) | 1991-09-05 | 1992-09-04 | Apport cible de polynucleotides ou d'oligonucleotides a des cellules |
AU26780/92A AU681997B2 (en) | 1991-09-05 | 1992-09-04 | Targeted delivery of poly- or oligonucleotides to cells |
KR1019940700741A KR100252547B1 (ko) | 1991-09-05 | 1992-09-04 | 폴리-또는 올리고누클레오티드의 세포로의 표적화된 전달 |
JP5505335A JPH07500820A (ja) | 1991-09-05 | 1992-09-04 | ポリヌクレオチド又はオリゴヌクレオチドの細胞への標的を定めた送達 |
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US75508391A | 1991-09-05 | 1991-09-05 | |
US07/755,083 | 1991-09-05 | ||
US78811991A | 1991-11-04 | 1991-11-04 | |
US07/788,119 | 1991-11-04 | ||
US86400392A | 1992-04-03 | 1992-04-03 | |
US07/864,003 | 1992-04-03 |
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WO1993004701A1 true WO1993004701A1 (fr) | 1993-03-18 |
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PCT/US1992/007339 WO1993004701A1 (fr) | 1991-09-05 | 1992-09-04 | Apport cible de polynucleotides ou d'oligonucleotides a des cellules |
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EP (1) | EP0666923A1 (fr) |
JP (1) | JPH07500820A (fr) |
KR (1) | KR100252547B1 (fr) |
AU (1) | AU681997B2 (fr) |
CA (1) | CA2116107A1 (fr) |
WO (1) | WO1993004701A1 (fr) |
Cited By (94)
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CA2116107A1 (fr) | 1993-03-18 |
EP0666923A4 (fr) | 1995-01-20 |
JPH07500820A (ja) | 1995-01-26 |
EP0666923A1 (fr) | 1995-08-16 |
KR100252547B1 (ko) | 2000-09-01 |
AU2678092A (en) | 1993-04-05 |
AU681997B2 (en) | 1997-09-18 |
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