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WO1994022485A1 - Targeted delivery of genes encoding cellular microsomal enzymes - Google Patents

Targeted delivery of genes encoding cellular microsomal enzymes Download PDF

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Publication number
WO1994022485A1
WO1994022485A1 PCT/US1994/003648 US9403648W WO9422485A1 WO 1994022485 A1 WO1994022485 A1 WO 1994022485A1 US 9403648 W US9403648 W US 9403648W WO 9422485 A1 WO9422485 A1 WO 9422485A1
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Prior art keywords
gene
cell
binding agent
molecular complex
complex
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PCT/US1994/003648
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French (fr)
Inventor
George Y. Wu
Catherine H. Wu
Namita Roy Chowdhury
Jayanta Roy Chowdhury
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University Of Connecticut
Albert Einstein College Of Medicine Of Yeshiva University
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Application filed by University Of Connecticut, Albert Einstein College Of Medicine Of Yeshiva University filed Critical University Of Connecticut
Priority to AU66245/94A priority Critical patent/AU6624594A/en
Publication of WO1994022485A1 publication Critical patent/WO1994022485A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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

Definitions

  • SER smooth endoplasmic reticula
  • Hepatic microsomal enzymes are responsible for biotransformation of many endogenous and exogenous lipophilic molecules (for example, bilirubin and steroid hormones).
  • liver microsomal enzymes are responsible for two types of biotrans ⁇ formation of these molecules.
  • the first type is the conversion of a molecule to a polar metabolite by oxidation, reduction or hydrolysis.
  • An important class of enzymes which catalyze biotransformations of the first type are the cytochromes P450 which catalyze oxidative reactions.
  • the second type ofbiotrans- formations are conjugations.
  • glucuronide conjugates are formed by hepatic glucuronyl transferases which use uridine diphosphate (UDP)-glucuronate as the donor of glucuronate to form the conjugate.
  • UDP uridine diphosphate
  • This enzyme glucuronidates bilirubin, converting it to the water soluble glucuronide to hasten its detoxification. Deficiency in this enzyme has been linked to forms of inherited jaundice.
  • This invention pertains to a soluble molecular complex for targeting a gene encoding a rr ⁇ crosomal enzyme to a specific cell in vivo and obtaining expression and function of the enzyme within the targeted cell.
  • the molecular complex comprises an expressible gene encoding a gene encoding the microsomal enzyme complexed to a carrier which is a conjugate of a cell-specific binding agent and a gene-binding agent.
  • 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, etc.) or it can be an antibody, or an analogue thereof, which specifi- cally binds a cellular surface structure which then mediates internalization of the bound complex.
  • the gene-binding component of the conjugate is a compound such as a polycation which stably complexes the gene under extracellular conditions and releases the gene under intracellular conditions so that it can function within the cell.
  • the complex of the gene and the carrier is stable and the carrier 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 gene expressed and the gene-encoded product accumulates within the transfected cell.
  • the soluble molecular complex of this invention can be used to specifically transfect cells in vivo or in vitro to provide for synthesis of a desired microsomal enzyme.
  • the method can be used to treat deficiencies in microsomal enzymes such as oxygenases and glucuronosyltransferases or to enhance normal levels of the enzymes.
  • the Figure shows serum bilirubin levels in rats receiving pSV3-hBUGT ⁇ , a vector fo expression of bilirubin-UDP-glucuronosyltransferase: Gunn rats were injected with colchicine (0.75 mg/kg, intraperi- toneally) 30 minutes before infusion of the carrier- DNA complex.
  • Three groups of results are shown: (a) Control rats: no treatment ( — O ); (b) colchicine only (MI ⁇ in); and (c) colchicine followed by DNA targeting ( — • — ). Blood wa collected from tail veins at time points indicated on the abscissa and serum bilirubin level was determined as described in the text.
  • a soluble, targetable molecular complex is used to selectively deliver a gene encodin a microsomal enzyme to a target cell or tissue in vivo or in vitro.
  • the molecular complex comprises the gene to be delivered complexed to a carrier made up of a binding agent specifi for the target cell and a gene-binding agent.
  • the complex is selectively taken up by the targe cell and the polyribonucleotide is produced therein.
  • the gene encodes the desired enzyme.
  • the gene comprises a DNA sequence encoding the enzyme in a form suitable for transcription and post-transcriptional processing by the target cell.
  • the gene is linked to appropriate genetic regulatory elements required for transcription of the gene by a cellular RNA polymerase and processing of the primary RNA transcript by cellular proteins into a stable form of RNA, such as mRNA.
  • RNA such as mRNA.
  • promoter and enhancer elements operable in the target cell, as well as other elements such as polyadenylation signals and splicing signals, which determine the internal and 3 '-end structure of the RNA.
  • the gene ca be contained in an expression vector such as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for transcription of the gene.
  • the gene should encode at least that portion of the protein required for enzymatic activity in the cell.
  • the gene should also include the membrane insertion signal and the membrane anchoring regions for insertion and anchoring of the enzyme in the SER.
  • the carrier component of the complex is a conjugate of a cell-specific binding agent and a gene-binding agent.
  • the cell-specific binding agent specifically binds a cellular surfac 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 endocytosis of a ligand.
  • the binding agent can be a natural or synthetic ligand which binds the receptor.
  • the ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan) or artificial carriers such as liposomes.
  • 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. For targeting hepatocytes, glycoproteins having exposed terminal carbohydrate groups such as asialoglyco- proteins (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed. Examples of asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus.
  • Such ligands can be formed by chemical (e.g., sulfuric acid) or enzymatic (e.g., neuraminidase) 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.
  • ligands For targeting the molecular complex to other cell surface receptors, other types of ligands can be used, such as mannose for macrophages (lymphoma), mannose-6-phosphate glycoproteins for fibroblasts (fibrosarcoma), intrinsic factor- vitamin B 12 and bile acids (See Kramer et aL (1992) J. Biol. Chem. 267: 18598-18604) 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.
  • ligand e.g., antigen
  • the gene-binding agent complexes the gene to be delivered. Complexation with the gene must be sufficiently stable in vivo to prevent significant uncoupling of the gene extracellularly prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene is released in functional form. For example, the complex can be labile in the acidic and enzyme rich environment of lysosomes. A noncovalent bond based on electrostatic attraction between the gene-binding agent and the expressible gene provides extracellular stability and is releasable under intracellular conditions.
  • Preferred gene-binding agents are poiycations that bind negatively charged polynucleotides.
  • Suitable poiycations 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 expressible gene include hydrogen bonding, hydrophobic bonding electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleo- tide, and strepavidin or avidin binding to polynucleotide containing biotinylated nucleotides.
  • the carrier can be formed by chemically linking the cell-specific binding agent and the gene-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. Commun. 101 ::599-606.
  • An alternative linkage is a disulfid bond.
  • the linkage reaction can be optimized for the particular cell-specific binding agent and gene- 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 gene-binding agent can be determined empirically. When poiycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the gene- binding agent. In general, this ratio ranges from about 10:1 to 1 :1, preferably about 5:1. Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., Waters AP-1 carboxymethyl functionalized column or AquaporeTM cation exchange, Rainin).
  • molecular sieve or ion exchange chromatography e.g., Waters AP-1 carboxymethyl functionalized column or AquaporeTM cation exchange, Rainin.
  • asialoorosomucoid-polylysine conjugate is formed with the crosslinking agent l-(3-dimethylaminopropyl)-3-ethyl carbodiimide. After dialysis, the conjugate is separated from unconjugated components by preparative acid-urea polyacrylamide gel electrophoresis (pH 4-5). The conjugate can be further purified on the carboxymethyl functionalized column. See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1993, the teachings of which are incorporated by reference herein.
  • the gene encoding the microsomal enzyme can be complexed to the carrier by a stepwise dialysis procedure.
  • the dialysis procedure begins with a 2M NaCl dialyzate and ends with a .15M NaCl solution.
  • the gradually decreasing NaCl concentration results in binding of the gene to the carrier.
  • dialysis may not be necessary; the gene and carrier are simply mixed and incubated.
  • the molecular complex can contain more than one copy of the same gene or one or more different genes.
  • the weight ratio of gene to the carrier is from about 1 :5 to 5:1, preferably about 1 :2 (approximate molar ratio 1 : 100 to 1 :200).
  • the appropriate ratio for a particular carrier and polynucleotide can be determined by the gel retardation assay described in U.S. Patent No. 5,166,320.
  • 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.
  • Cells can be transfected in vivo for transient production of the polyribonucleotide.
  • the gene can be administered repeatedly.
  • the transfected target cell can be stimulated to replicate by surgical or pharmacological means to prolong the activity of the incorporated gene.
  • Drugs that disrupt translocation or fusion of endosomes to lysosomes such as colchicine or taxol can also be used to prolong expression. See U.S. Patent Application Serial No. 950,789, filed September 24, 1992, the teachings of which are incorporated by reference herein.
  • the molecular complex of this invention is adaptable for delivery of a wide range of microsomal enzymes to a specific cell or tissue.
  • Microsomal enzymes include cytochrome P45O proteins (monooxy- genases), flavin-containing monooxygenases, ketone reductase esterases and transferases such as glucuronyltransferases.
  • the complex is targeted to the liver by exploiting the hepatic asialoglycoprotein receptor system which allows for in vivo transfection of hepatocytes by the process of receptor-mediated endocytosis.
  • the method of the invention can be used to treat diseases related to the absence or deficiency of a microsomal enzyme.
  • the method can be used to treat jaundice resulting from the inherited deficiency of hepatic bilirubin-UDP-glucuronosyl- transferase - the microsomal transferase which catalyzes the glucuronidation of bilirubin.
  • DNA encoding two isoforms of the enzyme is described by Ritter, J.K. et al (1991) J. Biological Chem. 266_: 1043.
  • the method can also be used to increase normal levels of the enzymes. For example, the level of an enzyme involved in the metabolism of a drug might be increased to treat drug overdose.
  • the invention is illustrated further by the following examples. EXEMPLIFICATION
  • mice Male Sprague-Dawley rats (150-200 g) and bilirubin-UDP-glucuronosyltransferase (B-UGT) deficient Gunn rats (150-225 g) were obtained from a colony at the Albert Einstein College of Medicine. The rats were maintained on standard laboratory rat chow in a 12 hour light/dark cycle.
  • B-UGT bilirubin-UDP-glucuronosyltransferase
  • colchicine 0.5 mg/ml of dimethylsulfoxide
  • 0.75 mg/kg colchicine was used 30 minutes before administration of the carrier-DNA complex (see below).
  • a plasmid pSVK3-hBUGT ⁇ was used for transferring B-UGT activity to Gunn rat livers.
  • This construct was produced by cloning the full-length coding region of human liver B-UGT ! cDNA (Ritter, J.K. gt aL H991 J. Biol. Chem. 266:1043-1047 downstream to the SV40 promoter region of the pSVK3 backbone (Pharmacia, Uppsala, Sweden). The insert is followed by an SV40 splice site and an SV40 polyadenylation region.
  • OR was prepared from pooled human serum (Wu, G.Y. and Wu, C.H. (1988) J. Biol. Chem. 263:14621-14624: Whitehead, D.H. and Sammons, H.G. (1966) Biochim. Biophys. Acta 124:209-211) and dissolved in water.
  • An equal volume of 0.2 NH2SO4 was added to the OR solution and the resulting mixture heated at 80°C for 1 hour in a water bath to hydrolyse sialic acids from the protein.
  • the acidolysis mixture was removed from water bath, neutralized with NaOH, dialyzed against water for 2 days and then lyophilized.
  • Asialoorosomucoid was covalently linked with polylysine (Sigma, average molecular weight 59000) as previously described (Wu, G.Y. gt aL (1991) J. Biol. Chem. 266: 14338- 14342 and Wilson, J.M. et aL (1992) J. Biol. Chem. 262:11483-11489).
  • plasmid DNA 0.5 mg in 1 ml of 2M NaCl was added to the asialoorosomucoid-polylysine conjugate (containing 0.15 mg asialoorosomucoid) in 0.6 ml o 2M NaCl at 25°C.
  • the mixture was placed in a dialysis tubing (1.0 cm flat width) with an exclusion limit of 12000 to 14000 dalton and dialyzed successively at 4°C for 30 minutes against 1000 ml of each of the following concentrations of NaCl: 1.5 M, 1.0 M, 0.5 M, 0.25 M and 0.15 M. After the final dialysis, the complex was filtered through 0.45 ⁇ membranes for injection into rats.
  • Rats were anesthetized with ether and the right external jugular vein was exposed.
  • B-UGT activity in the liver microsomal fractions was determined using UDP- [1 4 C]glucuronic acid as substrate (5 mM, 0.2 ⁇ Ci/ ⁇ mol) (Jansen, P.L.M. ej al (1977) J. Biol. Chem. 252:2710-2716). Blanks contained no bilirubin. Bilirubin and its glucuronides were extracted and separated by thin-layer chromatography (Chowdhury, R. et aL (1979) J. Biol. Chem. 254:8336-8339) and detected by autoradiography using authentic bilirubin monoglucuronide and diglucuronide as standard. The silica gel containing the bilirubin monoglucuronide and diglucuronide bands were collected by scraping, the pigments were extracted in methanol and radioactivity was determined after appropriate quench correction.
  • Control Gunn rat bile contained no detectable bilirubin monoglucuronide or diglucuronide.
  • bile pigments were analyzed at day 5, day 14 and day 60 after DNA administration.
  • bilirubin monoglucuronide and diglucuronide were detected in bile.
  • Bilirubin glucuronides accounted for 9 to 15% of total pigment excretion in bile.

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Abstract

Molecular complexes for targeting a gene encoding a microsomal enzyme selective for delivery to a cell in vivo or in vitro obtaining production of the enzyme within the targeted cell are disclosed. An expressible gene encoding a desired enzyme is complexed to a conjugate of a cell-specific binding agent and a gene-binding agent. The cell-specific binding agent is specific for a cellular surface structure which mediates internalization of ligands by endocytosis. An example is the asialoglycoprotein receptor of hepatocytes. The gene-binding agent is a compound such as a polycation which stably complexes the gene under extracellular conditions and releases the gene under intracellular conditions so that it can function whithin a cell. The molecular complex is stable and soluble in physiological fluids and can be used in antisense gene therapy to selectively transfect cells in vivo to treat inherited or acquired deficiencies in cellular microsomal enzymes.

Description

TARGETED DFXIYl RY OF GENES ENCODING CELLULAR MICROSOMAL ENZYMES
Government Support
The work leading to this invention was supported, in part, by research grants from the United States government.
Background of the Invention
Cellular microsomal enzymes are involved in many critical metabolic pathways. These enzymes are associated with smooth endoplasmic reticula (SER) of cells. Microsomes are formed by the breakup of the SER when cells are homogenized.
Hepatic microsomal enzymes are responsible for biotransformation of many endogenous and exogenous lipophilic molecules (for example, bilirubin and steroid hormones). In general, liver microsomal enzymes are responsible for two types of biotrans¬ formation of these molecules. The first type is the conversion of a molecule to a polar metabolite by oxidation, reduction or hydrolysis. An important class of enzymes which catalyze biotransformations of the first type are the cytochromes P450 which catalyze oxidative reactions. The second type ofbiotrans- formations are conjugations. In these reactions an endogenous molecule such as a glucuronate or sulfate is coupled to the molecule (often subsequent to it having undergone a biotransformation of the first type). For example, glucuronide conjugates are formed by hepatic glucuronyl transferases which use uridine diphosphate (UDP)-glucuronate as the donor of glucuronate to form the conjugate. This enzyme glucuronidates bilirubin, converting it to the water soluble glucuronide to hasten its detoxification. Deficiency in this enzyme has been linked to forms of inherited jaundice.
Summary of the Invention
This invention pertains to a soluble molecular complex for targeting a gene encoding a rrμcrosomal enzyme to a specific cell in vivo and obtaining expression and function of the enzyme within the targeted cell. The molecular complex comprises an expressible gene encoding a gene encoding the microsomal enzyme complexed to a carrier which is a conjugate of a cell-specific binding agent and a gene-binding agent.
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, etc.) or it can be an antibody, or an analogue thereof, which specifi- cally binds a cellular surface structure which then mediates internalization of the bound complex. The gene-binding component of the conjugate is a compound such as a polycation which stably complexes the gene under extracellular conditions and releases the gene under intracellular conditions so that it can function within the cell.
The complex of the gene and the carrier is stable and the carrier 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 gene expressed and the gene-encoded product accumulates within the transfected cell.
The soluble molecular complex of this invention can be used to specifically transfect cells in vivo or in vitro to provide for synthesis of a desired microsomal enzyme. The method can be used to treat deficiencies in microsomal enzymes such as oxygenases and glucuronosyltransferases or to enhance normal levels of the enzymes.
Brief Description of the Figure
The Figure shows serum bilirubin levels in rats receiving pSV3-hBUGTι , a vector fo expression of bilirubin-UDP-glucuronosyltransferase: Gunn rats were injected with colchicine (0.75 mg/kg, intraperi- toneally) 30 minutes before infusion of the carrier- DNA complex. Three groups of results are shown: (a) Control rats: no treatment ( — O ); (b) colchicine only (MI ♦ in); and (c) colchicine followed by DNA targeting ( — • — ). Blood wa collected from tail veins at time points indicated on the abscissa and serum bilirubin level was determined as described in the text.
Detailed Description of the Invention
A soluble, targetable molecular complex is used to selectively deliver a gene encodin a microsomal enzyme to a target cell or tissue in vivo or in vitro. The molecular complex comprises the gene to be delivered complexed to a carrier made up of a binding agent specifi for the target cell and a gene-binding agent. The complex is selectively taken up by the targe cell and the polyribonucleotide is produced therein.
The gene, generally in the form of DNA, encodes the desired enzyme. Typically, the gene comprises a DNA sequence encoding the enzyme in a form suitable for transcription and post-transcriptional processing by the target cell. For example, the gene is linked to appropriate genetic regulatory elements required for transcription of the gene by a cellular RNA polymerase and processing of the primary RNA transcript by cellular proteins into a stable form of RNA, such as mRNA. These include promoter and enhancer elements operable in the target cell, as well as other elements such as polyadenylation signals and splicing signals, which determine the internal and 3 '-end structure of the RNA. The gene ca be contained in an expression vector such as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for transcription of the gene. The gene should encode at least that portion of the protein required for enzymatic activity in the cell. The gene should also include the membrane insertion signal and the membrane anchoring regions for insertion and anchoring of the enzyme in the SER.
The carrier component of the complex is a conjugate of a cell-specific binding agent and a gene-binding agent. The cell-specific binding agent specifically binds a cellular surfac 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 endocytosis of a ligand. Thus, the binding agent can be a natural or synthetic ligand which binds the receptor. The ligand can be a protein, polypeptide, glycoprotein or glycopeptide which has functional groups that are exposed sufficiently to be recognized by the cell surface structure. It can also be a component of a biological organism such as a virus, cells (e.g., mammalian, bacterial, protozoan) or artificial carriers such as liposomes.
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. For targeting hepatocytes, glycoproteins having exposed terminal carbohydrate groups such as asialoglyco- proteins (galactose-terminal) can be used, although other ligands such as polypeptide hormones may also be employed. Examples of asialoglycoproteins include asialoorosomucoid, asialofetuin and desialylated vesicular stomatitis virus. Such ligands can be formed by chemical (e.g., sulfuric acid) or enzymatic (e.g., neuraminidase) desialylation of glycoproteins that possess terminal sialic acid and penultimate galactose residues. Alternatively, asialoglycoprotein ligands can be formed by coupling galactose terminal carbohydrates such as lactose or arabinogalactan to non-galactose bearing proteins by reductive lactosamination. For targeting the molecular complex to other cell surface receptors, other types of ligands can be used, such as mannose for macrophages (lymphoma), mannose-6-phosphate glycoproteins for fibroblasts (fibrosarcoma), intrinsic factor- vitamin B 12 and bile acids (See Kramer et aL (1992) J. Biol. Chem. 267: 18598-18604) for enterocytes and insulin for fat cells. Alternatively, 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 gene-binding agent complexes the gene to be delivered. Complexation with the gene must be sufficiently stable in vivo to prevent significant uncoupling of the gene extracellularly prior to internalization by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene is released in functional form. For example, the complex can be labile in the acidic and enzyme rich environment of lysosomes. A noncovalent bond based on electrostatic attraction between the gene-binding agent and the expressible gene provides extracellular stability and is releasable under intracellular conditions. Preferred gene-binding agents are poiycations that bind negatively charged polynucleotides. These positively charged materials can bind noncovalently with the gene to form a soluble, targetable molecular complex which is stable extracellularly but releasable intracellular ly. Suitable poiycations 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). Other noncovalent bonds that can be used to releasably link the expressible gene include hydrogen bonding, hydrophobic bonding electrostatic bonding alone or in combination such as, anti-polynucleotide antibodies bound to polynucleo- tide, and strepavidin or avidin binding to polynucleotide containing biotinylated nucleotides.
The carrier can be formed by chemically linking the cell-specific binding agent and the gene-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. Commun. 101 ::599-606. An alternative linkage is a disulfid bond.
The linkage reaction can be optimized for the particular cell-specific binding agent and gene- 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 gene-binding agent can be determined empirically. When poiycations are used, the molar ratio of the components will vary with the size of the polycation and the size of the gene- binding agent. In general, this ratio ranges from about 10:1 to 1 :1, preferably about 5:1. Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., Waters AP-1 carboxymethyl functionalized column or Aquapore™ cation exchange, Rainin).
In one embodiment, asialoorosomucoid-polylysine conjugate is formed with the crosslinking agent l-(3-dimethylaminopropyl)-3-ethyl carbodiimide. After dialysis, the conjugate is separated from unconjugated components by preparative acid-urea polyacrylamide gel electrophoresis (pH 4-5). The conjugate can be further purified on the carboxymethyl functionalized column. See U.S. Patent Application Serial No. 08/043,008, filed on April 5, 1993, the teachings of which are incorporated by reference herein.
The gene encoding the microsomal enzyme can be complexed to the carrier by a stepwise dialysis procedure. In a preferred method, for use with carriers made of poiycations such as polylysine, the dialysis procedure begins with a 2M NaCl dialyzate and ends with a .15M NaCl solution. The gradually decreasing NaCl concentration results in binding of the gene to the carrier. In some instances, particularly when concentrations of the gene and carrier are low, dialysis may not be necessary; the gene and carrier are simply mixed and incubated. The molecular complex can contain more than one copy of the same gene or one or more different genes. Preferably, the weight ratio of gene to the carrier is from about 1 :5 to 5:1, preferably about 1 :2 (approximate molar ratio 1 : 100 to 1 :200). The appropriate ratio for a particular carrier and polynucleotide can be determined by the gel retardation assay described in U.S. Patent No. 5,166,320.
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.
Cells can be transfected in vivo for transient production of the polyribonucleotide. For prolonged production, the gene can be administered repeatedly. Alternatively, the transfected target cell can be stimulated to replicate by surgical or pharmacological means to prolong the activity of the incorporated gene. See, for example, U.S. Patent Application Serial No. 588,013, filed September 25, 1990, the teachings of which are incorporated by reference herein. Drugs that disrupt translocation or fusion of endosomes to lysosomes such as colchicine or taxol can also be used to prolong expression. See U.S. Patent Application Serial No. 950,789, filed September 24, 1992, the teachings of which are incorporated by reference herein. Delivery of the gene can be enhanced by coupling the conjugate to a virus such as adenovirus. See U.S. Patent Application Serial No. 950,453, filed September 24, 1992 the teachings of which are incorporated by reference herein. The molecular complex of this invention is adaptable for delivery of a wide range of microsomal enzymes to a specific cell or tissue. Microsomal enzymes include cytochrome P45O proteins (monooxy- genases), flavin-containing monooxygenases, ketone reductase esterases and transferases such as glucuronyltransferases. In a preferred embodiment, the complex is targeted to the liver by exploiting the hepatic asialoglycoprotein receptor system which allows for in vivo transfection of hepatocytes by the process of receptor-mediated endocytosis.
The method of the invention can be used to treat diseases related to the absence or deficiency of a microsomal enzyme. For example, the method can be used to treat jaundice resulting from the inherited deficiency of hepatic bilirubin-UDP-glucuronosyl- transferase - the microsomal transferase which catalyzes the glucuronidation of bilirubin. DNA encoding two isoforms of the enzyme is described by Ritter, J.K. et al (1991) J. Biological Chem. 266_: 1043. The method can also be used to increase normal levels of the enzymes. For example, the level of an enzyme involved in the metabolism of a drug might be increased to treat drug overdose. The invention is illustrated further by the following examples. EXEMPLIFICATION
Animals
Male Sprague-Dawley rats (150-200 g) and bilirubin-UDP-glucuronosyltransferase (B-UGT) deficient Gunn rats (150-225 g) were obtained from a colony at the Albert Einstein College of Medicine. The rats were maintained on standard laboratory rat chow in a 12 hour light/dark cycle.
Colchicine Administration In experiments to determine the effect of colchicine on hepatic microtubules, colchicine (0.5 mg/ml of dimethylsulfoxide) in doses of 0.25 mg to 1.0 mg per kg was injected intraperitoneally into Sprague-Dawley rats. For gene targeting experiments, 0.75 mg/kg colchicine was used 30 minutes before administration of the carrier-DNA complex (see below).
Plasmids
For transferring B-UGT activity to Gunn rat livers, a plasmid pSVK3-hBUGTι was used. This construct was produced by cloning the full-length coding region of human liver B-UGT ! cDNA (Ritter, J.K. gt aL H991 J. Biol. Chem. 266:1043-1047 downstream to the SV40 promoter region of the pSVK3 backbone (Pharmacia, Uppsala, Sweden). The insert is followed by an SV40 splice site and an SV40 polyadenylation region.
Asialoorosomucoid
OR was prepared from pooled human serum (Wu, G.Y. and Wu, C.H. (1988) J. Biol. Chem. 263:14621-14624: Whitehead, D.H. and Sammons, H.G. (1966) Biochim. Biophys. Acta 124:209-211) and dissolved in water. An equal volume of 0.2 NH2SO4 was added to the OR solution and the resulting mixture heated at 80°C for 1 hour in a water bath to hydrolyse sialic acids from the protein. The acidolysis mixture was removed from water bath, neutralized with NaOH, dialyzed against water for 2 days and then lyophilized. The thiobarbituric acid assay of Warren was then used to verify desialylation of the OR (Warren, L. ri959) J. Biol. Chem. 234:1971-1975 Also see generally Warren, L. (1959) J. Clin. Invest. 2^:755-761 and Schmid, K. et aL (1967) Biochem. J. H)4.361-368.
Synthesis of the ASGP-Polylysine Conjugate and Formation of the DNA- Carrier Protein Complex
Asialoorosomucoid was covalently linked with polylysine (Sigma, average molecular weight 59000) as previously described (Wu, G.Y. gt aL (1991) J. Biol. Chem. 266: 14338- 14342 and Wilson, J.M. et aL (1992) J. Biol. Chem. 262:11483-11489). To form targetable carrier-DNA complexes, plasmid DNA, 0.5 mg in 1 ml of 2M NaCl was added to the asialoorosomucoid-polylysine conjugate (containing 0.15 mg asialoorosomucoid) in 0.6 ml o 2M NaCl at 25°C. The mixture was placed in a dialysis tubing (1.0 cm flat width) with an exclusion limit of 12000 to 14000 dalton and dialyzed successively at 4°C for 30 minutes against 1000 ml of each of the following concentrations of NaCl: 1.5 M, 1.0 M, 0.5 M, 0.25 M and 0.15 M. After the final dialysis, the complex was filtered through 0.45 μ membranes for injection into rats.
Administration of the DNA-Carrier Complex and Partial Hepatectomy
Rats were anesthetized with ether and the right external jugular vein was exposed. The soluble complex containing 22 pmol of the DNA in a volume of 0.5 ml, was infused into the right external jugular vein, the vein was ligated and the incision was closed.
Assay of B-UGT Activity
B-UGT activity in the liver microsomal fractions was determined using UDP- [14C]glucuronic acid as substrate (5 mM, 0.2 μCi/μmol) (Jansen, P.L.M. ej al (1977) J. Biol. Chem. 252:2710-2716). Blanks contained no bilirubin. Bilirubin and its glucuronides were extracted and separated by thin-layer chromatography (Chowdhury, R. et aL (1979) J. Biol. Chem. 254:8336-8339) and detected by autoradiography using authentic bilirubin monoglucuronide and diglucuronide as standard. The silica gel containing the bilirubin monoglucuronide and diglucuronide bands were collected by scraping, the pigments were extracted in methanol and radioactivity was determined after appropriate quench correction.
Analysis of Serum Bilirubin and Pigments Excreted in Bile Serum bilirubin levels were determined as previously described (Trotman, B.W. et aL
(1982) Anal. Biochem. 121 : 175-180). For bile pigment analysis, rats were placed under ether anesthesia and bile ducts were cannulated with PE-10 cannulae. The rats were placed in a restraining cage and allowed to regain consciousness and normal body temperature before collecting bile. Bile was analyzed by high pressure liquid chromatography (Spivak, W. and Carey, M.C. f!985) Biochem. J. 225:787-795.
B-UGT Activity
Control Gunn rat livers had no B-UGT activity. Gunn rats that received carrier- pSK3-hBUGT complex, 30 minutes after the injection of colchicine, B-UGT activity was detected in the liver for up to 6 weeks (last time point of determination). The enzyme activity ranged from 3 to 5% of the activity in the normal Wistar rat liver. In Gunn rats that received the carrier-DNA complex, but no colchicine, hepatic B-UGT activity was detectable only 24 hours after administration of the DNA. Excretion of Bilirubin Glucuronides in Bile
Control Gunn rat bile contained no detectable bilirubin monoglucuronide or diglucuronide. In three rats that received colchicine followed by the carrier- DNA complex, bile pigments were analyzed at day 5, day 14 and day 60 after DNA administration. In all cases, bilirubin monoglucuronide and diglucuronide were detected in bile. Bilirubin glucuronides accounted for 9 to 15% of total pigment excretion in bile.
Serum Bilirubin Levels
In untreated Gunn rats and Gunn rats that received colchicine only, there was no significant decrease in serum bilirubin levels (See Figure 1 ). In rats that received the carrier- pSK3-hBUGT complex, but no colchicine, there was also no significant decrease in serum bilirubin levels. In contrast, when the carrier-DNA complex was administered 30 minutes after the injection of colchicine, 0.75 mg/kg, serum bilirubin concentration declined progressively for at least 5 weeks to 45% of the pretreatment level. After this period the bilirubin level gradually increased to 70% of the pretreatment level in 60 days. In another rat the serum bilirubin level decreased to 30% of the pretreatment level in 35 days and remained at that level for at least 60 days (duration of this experiment).
Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. A soluble molecular complex for targeting a gene encoding a cellular microsomal enzyme to a specific cell, the complex comprising an expressible gene encoding the enzyme complexed with a carrier comprising a cell-specific binding agent and a gene-binding agent.
2. A soluble molecular complex, wherein the microsomal enzyme is a cytochrome P450 or a glucuronosyltransferase.
3. A soluble complex of claim 1, wherein the gene encodes bilirubin-UDP- glucuronosyltransferase.
4. A soluble molecular complex of claim 1, wherein the gene-binding agent is a polycation.
5. A soluble molecular complex of claim 4, wherein the polycation is polylysine.
6. A soluble molecular complex of claim 1 , wherein the cell-specific binding agent binds a surface receptor of the cell which mediates endocytosis.
7. A soluble molecular complex of claim 1 , wherein the cell-specific binding agent is a ligand for an asialoglycoprotein receptor.
8. A soluble molecular complex of claim 7, wherein the ligand is an asialoglycoprotein and the targeted cell is a hepatocyte.
9. A soluble molecular complex of claim 1, wherein the expressible gene is complexed with the gene-binding agent by a noncovalent bond.
10. A therapeutic composition comprising a solution of the molecular complex of claim 1 in a physiologically acceptable vehicle.
11. A soluble molecular complex for targeting a gene encoding a microsomal enzyme to a hepatocyte, the complex comprising an expressible gene encoding the microsomal enzyme complexed with a carrier of a ligand for the asialoglycoprotein receptor and a polycation.
12. A soluble molecular complex of claim 11, wherein the enzyme is bilirubin-UDP- glucuronosyl- transferase.
13. A soluble molecular complex of claim 11 , wherein the polycation is polylysine.
14. A soluble molecular complex of claim 11 , wherein the gene is contained in an expression vector along with genetic regulatory elements necessary for expression of the gen by the hepatocyte.
15. A soluble molecular complex of claim 14, wherein the expression vector is a plasmid or viral DNA.
16. A therapeutic composition comprising a solution of the molecular complex of claim 11 , in a physiological vehicle.
17. A method of delivering a gene encoding a microsomal enzyme to a cell of an organism, comprising administering to an organism a soluble molecular complex comprising an expressible gene encoding the enzyme complexed with a carrier comprising a cell-specific binding agent and a gene-binding agent.
18. A method of claim 17, wherein the gene encodes bilirubin-UDP- glucuronosyltransferase.
19. A method of claim 17, wherein the gene-binding agent is a polycation.
20. A method of claim 19, wherein the polycation is polylysine.
21. A method of claim 17, wherein the cell-specific binding agent binds a surface recepto of the cell which mediates endocytosis.
22. A method of claim 21, wherein the cell-specific binding agent is a ligand for an asialoglycoprotein receptor.
23. A method of claim 22, wherein the ligand is an asialoglycoprotein and the targeted cell is a hepatocyte.
24. A method of claim 17, wherein the molecular complex is administered intravenously.
25. A method of claim 1 , wherein colchicine is administered in conjunction with the molecular complex.
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