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WO1992019749A1 - Apport cible de genes codant des recepteurs de surface cellulaire - Google Patents

Apport cible de genes codant des recepteurs de surface cellulaire Download PDF

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Publication number
WO1992019749A1
WO1992019749A1 PCT/US1992/003639 US9203639W WO9219749A1 WO 1992019749 A1 WO1992019749 A1 WO 1992019749A1 US 9203639 W US9203639 W US 9203639W WO 9219749 A1 WO9219749 A1 WO 9219749A1
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Prior art keywords
gene
cell
molecular complex
binding agent
receptor
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PCT/US1992/003639
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English (en)
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James M. Wilson
Mariann Grossman
Catherine H. Wu
Namita Roy Chowdhury
George Y. Wu
Jayanta Roy Chowdhury
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The Board Of Regents Acting For And On Behalf Of The University Of Michigan
University Of Connecticut
Albert Einstein College Of Medicine, A Division Of Yeshiva University
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Publication of WO1992019749A1 publication Critical patent/WO1992019749A1/fr

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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Familial hypercholesterolemia is an inherited disease in humans, caused by a deficiency of low-density lipoprotein (LDL) receptors. FH is associated with hypercholesterolemia and premature development of coronary heart disease. J.L. Goldstein and M.S. Brown in The Metabolic Basis of Inherited Disease (C.R. Scriver, A.L. Beaudet, W.S. Sly and D. Valle eds.) McGraw-Hill, New York, Sixth edition, pp. 1215-1250. Current therapies for FH primarily attempt to decrease serum LDL cholesterol by increasing hepatic expression of the LDL receptor. J.L. Goldstein and M.S.
  • ⁇ s vivo gene therapy in the liver may be limited, in part, by the morbidity of the invasive procedures used to harvest and transplant hepatocytes. Delivery of genes to hepatocytes and other cells in vivo would be of value in treating FH and other acquired and inherited diseases.
  • This invention pertains to a soluble molecular complex for targeting a gene encoding a cell surface receptor, such as the LDL receptor, to a specific cell in vivo and obtaining expression of the gene by the targeted cell.
  • the molecular complex comprises an expressible gene encoding a desired cell surface receptor complexed with 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, poly- peptide, glycoprotein, 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 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 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 is expressed and the gene-encoded receptor is processed and inserted into the cell membrane of the transfected cell.
  • the soluble molecular complex of this invention can be used to specifically transfect cells in vivo to provide for expression of a desired cell surface receptor. This selective transfection is useful for gene therapy and in other applications which require selective genetic alteration of cells to produce a desired surface receptor.
  • a normal gene can be targeted to a specific cell to correct or alleviate an inherited or acquired abnormality involving a cell surface receptor, such as familial hypercholesterolemia, caused in part by a defect in the LDL receptor gene.
  • FIG. 1 shows the structure of the LDL receptor expression vector p9-12alb(h)LDLR.
  • the vector contains the structural gene for human LDL receptor driven by rat albumin promoter and mouse albumin enhancer sequences.
  • Figure 2 shows that the LDLR-complex is primarily targeted to the liver. 125 I uptake by various organs was determined following injection of radiolabeled LDLR-complex.
  • Figure 3 shows the cellular distribution of the
  • LDLR-complex which indicates the complex is predominantly taken up by hepatocytes.
  • Figure 4A shows DNA blot hybridization analysis of total cellular DNA which indicates that the LDLR-complex remains intact extracellularly and delivers functional DNA.
  • Figure 4B shows RNase protection analysis which confirms the presence of recombinant human LDL receptor transcripts in the liver.
  • Figure 5 shows the results of a two treatment crossover study measuring total serum cholesterol to confirm the presence of human LDL receptor.
  • a soluble, targetable molecular complex is used to selectively deliver a gene encoding a cell surface receptor to a target cell or tissue n vivo.
  • the molecular complex comprises the receptor-encoding gene to be delivered complexed to a carrier made up of a binding agent specific for the target cell and a gene-binding agent.
  • the complex is selectively taken up by the target cell and the gene-encoded receptor is expressed, processed, and inserted into the cellular membrane.
  • the gene encodes the desired cell surface receptor.
  • the gene comprises a structural gene encoding the receptor in a form suitable for processing by the target cell.
  • the gene encodes appropriate signal sequences which direct processing and membrane insertion of the receptor.
  • the signal sequence may be the natural sequence of the receptor or exogenous sequences.
  • the structural gene is linked to appropriate genetic regulatory elements required for expression of the gene-encoded receptor by the target cell. These include a promoter and optionally an enhancer element operable in the target cell.
  • the gene can be contained in an expression vector such.as a plasmid or a transposable genetic element along with the genetic regulatory elements necessary for expression of the gene and production of the gene-encoded product.
  • 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 surface structure which mediates 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.
  • 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 such as lactose or arabinogalactan
  • other types of ligands can be used, such as mannose for macrophages, mannose-6- phosphate glycoproteins for fibroblasts, 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.
  • ligand e.g., antigen
  • the gene-binding agent complexes the gene to be delivered. Complexation with the gene must be sufficiently stable 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 gene provides extracellular stability and is releasable under intracellular conditions.
  • Preferred gene-binding agents are polycations that bind negatively charged polynucleotides.
  • Suitable polycations are polylysine, polyarginine, polyornithine, basic proteins such as histones, avidin, protamines and the like.
  • a preferred polycation is polylysine.
  • 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 polynucleotide, 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 G. Jung e_t .al. Biochem. Biophvs. Res. Commun. l£JL:599-606 (1981).
  • An alternative linkage is a disulfide 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.
  • the molar ratio of the components will vary with the size of the polycation and the size of the gene. 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 chromatography.
  • the gene encoding the desired cell surface receptor can be complexed to the carrier by a stepwise dialysis procedure. In a preferred method, for use with carriers made of polycations such as polylysine, the dialysis procedure begins with a 2M NaCl dialyzate and ends with a 0.15M NaCl solution. The gradually decreasing NaCl concentration results in binding of the DNA to the carrier.
  • the molecular complex can contain more than one copy of the same gene or one or more different genes.
  • the ratio of polynucleotide to the carrier is from about 1:5 to 5:1, preferably about 1:2.
  • 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 expression and production of the gene product. For prolonged expression and production, the gene can be administered repeatedly. Alternatively, the transfected target cell can be stimulated to replicate by surgical or pharmacological means to prolong expression 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.
  • the method of this invention can be used in gene therapy to selectively deliver a gene encoding a cell surface receptor to a target cell in vivo for expression.
  • a gene encoding a desired cell surface receptor can be targeted to a cell which normally expresses the receptor but which lacks the capacity to produce the receptor or produces an insuffient amount of the receptor because of an acquired or inherited defect.
  • a normal gene can be targeted to a specific cell to correct or alleviate a metabolic or genetic abnormality caused by an inherited or acquired defect in a corresponding endogenous gene encoding a cell surface receptor.
  • the gene can be delivered to a cell which does not normally express the surface receptor to confer a new function upon the cell.
  • the cell surface receptor can be a receptor for a natural ligand such as a metabolite, a hormone, a growth factor, a cytokine, an ion (ion transport protein), a virus, or a protozoan. It can also be a receptor that mediates cell-cell interaction.
  • a natural ligand such as a metabolite, a hormone, a growth factor, a cytokine, an ion (ion transport protein), a virus, or a protozoan.
  • It can also be a receptor that mediates cell-cell interaction.
  • In vivo gene transfer has several potential advantages over organ/cell transplantation in the treatment of metabolic diseases of the liver.
  • One advantage is that the therapeutic gene is expressed in situ in a cell and organ that has not been manipulated ⁇ x vivo.
  • the capacity of this approach to reconstitute hepatic gene expression is theoretically greater than the capacity of cellular therapies which are usually limited by the number of cells that will engraft
  • Familial hypercholesterolemia is an inherited disease in humans caused by a deficiency in the receptor for LDL.
  • the gene encoding the human LDL receptor is complexed to a conjugate of an asialoglycoprotein and a polycation.
  • the resulting soluble complex is administered parenterally to the individual afflicted with the LDL receptor deficiency in amounts sufficient to selectively transfect cells and to provide sufficient production of the cell surface receptor to attain normal levels of serum cholesterol.
  • DNA encoding human LDL receptor is described in U.S. Patent 4,745,060, the contents of which are incorporated herein by reference.
  • asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid (ASOR) coupled to polylysine, was used to form a soluble DNA complex capable of specifically targeting hepatocytes via asialoglycoprotein receptors present on these cells.
  • the DNA comprised a plasmid, p9-12alb(h)LDLR, containing the structural gene for the human LDL receptor driven by rat albumin promoter and mouse albumin enhancer elements.
  • WHHL Watanabe Heritable Hyperlipidemic
  • NZW Wild-type New Zealand White
  • Animals were maintained on a Purina laboratory rabbit chow and weighed 2-3 kg at the time of experi ⁇ mentation. Phlebotomies were performed at 3 to 4 PM. Experiments were conducted in accordance with the guidelines of the Committee on Use and Care of Animals from the University of Michigan and Albert Einstein College of Medicine. Construction of DNA Vector
  • a vector capable of expressing normal human LDL receptor was constructed (p9-12alb(h)LDLR) for in vivo gene transfer experiments in WHHL rabbits ( Figure 1) .
  • Transcriptional elements from the mouse albumin gene were used to drive expression of a full-length cDNA for the human LDL receptor.
  • Fragment A is an Xbal to Bglll fragment (3.6 kb) of plasmid MTEV.JT (provided by J.
  • Trill which contains several functional elements including a 231 base pair (bp) fragment of genomic DNA spanning the polyadenyl- ation signal of the bovine growth hormone gene, ⁇ -lactamase and the prokaryotic origin of replication from PUC 19, and a eukaryotic transcriptional unit expressing xanthine-guanine phosphoribosyltransferase (XGPRT) ; Fragment B includes sequences spanning an enhancer located 5' to the mouse albumin gene (-12 to -9 kb) excised on an Eco RV to Bglll fragment and fused in reverse orientation to sequences spanning the mouse albumin promoter (-282 to +21). L.E. Babiss e_£ ⁇ l- Proc. Natl.
  • the 5' end of the chimeric fragment is formed by the natural Bglll site of the albumin enhancer (-9 kb) while the 3' end of the fragment contains a synthetic Sail site attached to position +22 of the albumin promoter sequence; and Fragment C is a Sail to Xbal fragment (3.0 kb) derived from a previously published retroviral vector LTR-LDLR. J.M. Wilson st al. Proc. Natl. Acad. Sci. ££:4421 (1988).
  • the LTR-LDLR vector includes the entire cDNA for human LDL receptor (2.6 kb) along with 430 bp of additional 3' sequence derived from the retroviral genome (nucleotides 7816 to 8113 of the Moloney murine leukemia virus genome; see C. Van Beveren e_£ Si. in RNA Tumor Viruses (R. Weiss, N. Teich, H. Varmus, and J. Coffin eds.) Cold Spring Harbor Lab., Cold Spring Harbor N.Y., 2nd Ed., pp. 766-783 (1985) for numbering).
  • the 5' end of the LDL receptor cDNA was converted to a Sail site in preparation for this construction.
  • LDL receptor sequences in p9-12alb(h)LDLR were replaced with sequences encoding the prokaryotic gene chloroamphenicol acetyltransferase (CAT) to generate a vector called p9-12albCAT (provided by S. Camper).
  • CAT chloroamphenicol acetyltransferase
  • This vector was used as a negative control in metabolic experiments based on the assumption that CAT expression does not specifically affect intracellular hepatic cholesterol metabolism.
  • DNA/protein complexes were synthesized with either p9-2alb(h)LDLR (LDLR-complex) or p9-12albCAT (CAT-complex) (See below) . Construction of Gene Carrier and Gene Carrier Complexes
  • a high affinity ligand for the asialoglycoprotein receptor, asialoorosomucoid (ASOR) was covalently attached to polylysine to produce a gene carrier useful in the study of the organ and cellular distribution of DNA/protein complex uptake in vivo.
  • ASOR asialoorosomucoid
  • LDLR- complexes were synthesized with ASOR.
  • the gene carriers were prepared as described previously. G.Y. Wu fit al. J. Biol. Chem. 2£4.:16985 (1989).
  • LDLR-Complex Targeting of the LDLR-Complex to the Liver Animals were anesthetized with ketamine HC1 (40 mg/kg) and xylazine (10 mg/kg) in preparation for the experiment. Radiolabeled LDLR-complexes were injected into the marginal ear vein of New Zealand White (NZW) or WHHL adult rabbits (2 to 3 kg) which were euthenized and exanguinated 10 minutes later. Individual organs were harvested and analyzed for incorporation of radioactivity (Figure 2) . DNA/protein complex was rapidly cleared from the plasma and primarily taken up by the liver (85% of total recovered radioactivity) after 10 minutes.
  • NZW New Zealand White
  • WHHL adult rabbits 2 to 3 kg
  • Organ distribution of uptake was shown to be independent of the phenotype of the recipient animal (NZW, heterozygous WHHL, and homozygous WHHL) and the dose of injected complex (from 0.17 mg to 4 mg of DNA in a complex) .
  • B Tracer quantities of ASOR labeled complex into a WHHL rabbit (heterozygous).
  • LDLR-Complex Although the hepatocyte is the predominant cell type in the liver and the likely target for gene transfer, nonparenchymal cells of the liver, such as endothelial cells and kupffer cells, could potential ⁇ ly serve as alternative targets.
  • rabbits were injected with radiolabeled LDLR-complex as described above. Ten minutes after injection, the portal vein was cannulated, and the liver was perfused with 154 mM NaCl for 5 minutes followed by Karnovsky's fixative for 10 minutes. R. St. Hilaire fit al * Proc. Natl. Acad. Sci. 80:3797 (1983).
  • Random blocks of liver were cut on ice to 2 - 5 mm ⁇ cubes and were kept in fresh fixative for 2 hours prior to transfer to a 0.2 M sodium bicarbonate, pH 7.4, overnight at room temperature. Blocks were embedded in Epox 812 (W.O. Dobbins III in Diagnostic Electron Microscopy (B.F. Trump and R.T. Jones eds.) Wiley, New York, Vol. 1, pp. 253-339) and 1 ⁇ m sections were cut and mounted on polylysine-coated slides which were dipped in NTB3 nuclear emulsion.
  • Panel B Analysis of liver tissue from a WHHL rabbit that was injected with saline. In each case, magnification is 400x. Radioactive signal, detected as silver grains, was 100-fold over that observed in unlabeled liver tissue ( Figure 3). The majority of this signal (>90%) was seen as discrete grains located over hepatocytes ( Figure 3A) .
  • Radioactive signal was infrequently seen over kupffer cells as aggregates or discrete grains (Figure 3A) .
  • This nonparenchymal cell uptake may represent larger forms of the DNA/protein complex that have been phagocytized by kupffer cells.
  • the intracellular fate of the recombinant gene was studied in WHHL rabbits after administration of unlabeled LDLR-complex. Animals were euthanized 10 minutes, 4 hours, and 24 hours after in vivo gene transfer and liver tissue was characterized with respect to the abundance and structural integrity of the recombinant gene as well as the level of recombinant derived RNA.
  • Samples include control WHHL DNA (10 ⁇ g) mixed with the equivalent of 100 copies/cell (750 pg; lane *100c'), 10 copies/cell (75 pg; lane '10c'), and 1 copy/cell (7.5 pg; lane , lc') of p9-12alb(h)LDLR plasmid DNA. Additional DNA samples (10 ⁇ g) are from WHHL rabbits injected with LDLR-complex and harvested 10 minutes (lane '10 min'), 4 hours (lane '4 hr"), and 24 hours (lane *24 hr') later. The filter was exposed to film for 45 minutes (top panel) and 24 hours (bottom panel) . Molecular weight markers are noted in base pairs along the left border.
  • liver tissue harvested 10 minutes after injection of the LDLR-complex demonstrated very high levels of the intact fragment along with some partially degraded plasmid DNA; comparison to plasmid controls indicated that this tissue contained approximately 5,000 to 10,000 copies of plasmid per cell.
  • This estimate of gene targeting is in agreement with the level of gene delivery to the liver expected from the amount of DNA injected (this estimate is based on the injection of 4 mg of plasmid (10 kb in size) into a 2-3 kg rabbit that has 2 - 4 x 10 10 hepatocytes).
  • Analysis of livers harvested at later time points revealed ongoing degradation of the plasmid and a progressive decline in the abundance of the intact plasmid from 100 copies/cell at 4 hours to 1 copy/cell at 24 hours.
  • Liver tissues were analyzed for the presence and abundance of recombinant human LDL receptor transcripts using a quantitative RNase protection assay. J.M. Wilson f it al. Proc. Natl. Acad. Sci.
  • RNA derived from the p9-12alb(h)LDLR vector was detected with an antisense RNA probe that is synthesized from the previously described vector 3Z-env. J.M. Wilson fit al. Proc. Natl. Acad. Sci. £7:8437 (1990).
  • This RNA probe is complementary to vector specific sequences in the 3* untranslated region of the recombinant transcript. RNase protection of the resulting duplex produces a protected fragment of 172 bp ( Figure 4B) .
  • Antisense RNA that specifically detects endogenous WHHL LDL receptor RNA was used as an internal control in each assay.
  • the transcription vector used to synthesize the RNA probe (3Z-WLDLR) was constructed in the following manner.
  • the Nar I site was converted to a Hind III site with synthetic linkers and the revised fragment was ligated with the Hind III to Sma I backbone fragment of pGEM3Zf(+) (Promega).
  • Endogenous WHHL LDL receptor RNA is detected with the 3Z-wLDLR probe in RNase protection assays as a 80 bp band.
  • RNA probes were gel purified prior to use. M.H. Finer in Methods in Molecular Biology. Vol. 7: Gene Transfer and Expression Techniques (E.J. Murray and J.M. Walker eds.) The Humana Press Inc., Clifton, NJ, pp. ⁇ _i5 (1990).
  • Total cellular RNA prepared from liver was hybridized with equal quantities of 3Z-env and 3Z-wLDLR probe (5x10-5 cpm of each probe per assay) and analyzed for protection to digestion with RNase A. J.M. Wilson fit al. Proc. Natl. Acad. Sci. £7:8437 (1990). Samples were electrophoresed through a 6% polyacrylamide/urea denaturing gel; a representative autoradiograph is presented. Radioactivity in the resulting bands was quantified with a Beta Scope 630 (Betagen, Waltham MA) . The following samples were analyzed.
  • Lane '3T3' contains RNA (100 ⁇ g) from a control WHHL supplemented with RNA (100 ng) from a fibroblast cell line (NIH3T3) that produces recombinant LDL receptor transcripts containing sequences complementary to the 3z-env probe.
  • RNA 100 ⁇ g
  • WHHL rabbit a untransfected WHHL rabbit
  • WHHL rabbits injected with LDLR-complex and harvested 10 minutes (lane "10 min*), 4 hours (lane '4 hr'), and 24 hours (lanes 24A and 24B, representing two different animals) .
  • the undigested probes (2 x 10 3 cpm/lane) were electrophoresed in lanes '3Z-env' and '3Z-WLDLR'.
  • Molecular weight markers ⁇ ATP labeled pBr322/X Hae III fragments
  • their corresponding sizes in base pairs are presented in the far right lane.
  • the closed arrow indicates the location of the 3Z-env protected band while the open arrow indicates the location of the 3Z-wLDLR protected band.
  • Endogenous LDL receptor RNA was specifically detected in RNase protection assays as a 80 bp protected fragment; no hybridization to human LDL receptor RNA was noted. The intensity of the resulting bands is proportional to the amount of RNA used in the initial hybridization suggesting that this assay can be used to quantify recombinant and endogenous transcripts.
  • Recombinant RNA was not detected in mock-transfected liver or in liver harvested 10 minutes after administration of the LDLR-complex. Livers removed 4 hours and 24 hours after in vivo gene transfer contained significant levels of the recombinant transcript. Quantitative analysis of the assay was used to estimate the abundance of vector- derived RNA relative to the endogenous LDL receptor transcript; levels of the recombinant LDL receptor transcript rose from undetectable at 10 minutes ( ⁇ 0.05% of endogenous), to 1.3% of endogenous at 4 hours, and 4.0% (24A) and 2.0% (24B) of endogenous at 24 hours.
  • WHHL rabbits were injected with LDLR-complex or CAT-complex and analyzed for changes in total serum cholesterol.
  • Six WHHL rabbits were entered into a protocol that involved a two treatment (injection of p9-12alb(h)LDLR (LDLR-complex) or p9-12albCAT (CAT-complex) plasmid; 5.0 g of total DNA in a complex/dose), two period, cross-over design, with repeated measurements of total serum cholesterol within each period (Figure 5).
  • the DNA/protein complex was injected into the marginal ear vein over a 2 minute time period.

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Abstract

Complexes moléculaires permettant de cibler in vivo un gène codant un récepteur de surface cellulaire sur une cellule spécifique, et de produire l'expression du gène et l'insertion du récepteur codé par le gène dans la membrane de la cellule. On transforme sous forme de complexe un gène pouvant être exprimé codant un récepteur de surface cellulaire prédéterminé en le combinant avec un vecteur d'agent de liaison à spécificité cellulaire et un agent de liaison du gène. L'agent de liaison à spécificité cellulaire est spécifique contre une structure de surface cellulaire qui assure l'internalisation des ligands par endocytose. Le récepteur d'asialoglycoprotéine des hépatocytes en est un exemple. L'agent de liaison du gène est un composé tel qu'un polycation qui transforme en complexe de manière stable le gène dans des conditions extracellulaires et libère ledit gène dans des conditions intracellulaires de manière à ce qu'il fonctionne à l'intérieur d'une cellule. Le complexe moléculaire est stable et soluble dans des fluides physiologiques et on peut l'utiliser dans la thérapie génétique pour transfecter sélectivement des cellules in vivo afin d'assurer la production, l'insertion membranaire et le fonctionnement d'un récpteur de surface cellulaire.
PCT/US1992/003639 1991-05-03 1992-05-01 Apport cible de genes codant des recepteurs de surface cellulaire WO1992019749A1 (fr)

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Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993025673A1 (fr) * 1992-06-04 1993-12-23 The Regents Of The University Of California Therapie genique in vivo a l'aide d'une sequence significative sans intron
WO1995006723A1 (fr) * 1993-09-01 1995-03-09 Boehringer Mannheim Gmbh Procede de marquage de cellules eucariotes par utilisation d'un recepteur de surface cellulaire en tant que marqueur
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US6806084B1 (en) 1992-06-04 2004-10-19 The Regents Of The University Of California Methods for compositions for in vivo gene delivery
US6875588B2 (en) 2001-11-30 2005-04-05 Avigenics, Inc. Ovomucoid promoter and methods of use
WO2006081331A2 (fr) 2005-01-25 2006-08-03 Prolexys Pharmaceuticals, Inc. Erastine et proteines de liaison d'erastine, et utilisations de celles-ci
US7135562B2 (en) 2002-03-14 2006-11-14 University Of Cincinnati Avian iFABP gene expression controlling region
US7176300B2 (en) 2001-03-30 2007-02-13 Avigenics, Inc. Avian lysozyme promoter
US7255874B1 (en) 2001-12-21 2007-08-14 Closure Medical Corporation Biocompatible polymers and adhesives: compositions, methods of making and uses related thereto
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US7323297B1 (en) 1992-04-03 2008-01-29 The Regents Of The University Of California Stabilized polynucleotide complexes and methods
US7335761B2 (en) 2001-11-30 2008-02-26 Avigenics, Inc. Avian gene expression controlling regions
US7541512B2 (en) 2001-03-30 2009-06-02 Synageva Biopharma Corp. Avians containing a lysozyme promoter transgene
US7550561B1 (en) 1991-05-16 2009-06-23 Cold Spring Harbor Laboratory p16INK4 polypeptides
US7550650B2 (en) 2001-09-18 2009-06-23 Synageva Biopharma Corp. Production of a transgenic avian by cytoplasmic injection
US7691632B2 (en) 1993-11-18 2010-04-06 Cold Spring Harbor Laboratory Kit for detecting the level of cyclin-dependent kinase inhibitor P16 gene expression
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US5521291A (en) * 1991-09-30 1996-05-28 Boehringer Ingelheim International, Gmbh Conjugates for introducing nucleic acid into higher eucaryotic cells
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US5922859A (en) * 1992-02-01 1999-07-13 Boehringer Ingelheim International Gmbh Complexes containing nucleic acid which can be taken-up by endocytosis into higher eukaryotic cells
US7323297B1 (en) 1992-04-03 2008-01-29 The Regents Of The University Of California Stabilized polynucleotide complexes and methods
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