+

WO1999015687A1 - Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite - Google Patents

Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite Download PDF

Info

Publication number
WO1999015687A1
WO1999015687A1 PCT/US1998/020154 US9820154W WO9915687A1 WO 1999015687 A1 WO1999015687 A1 WO 1999015687A1 US 9820154 W US9820154 W US 9820154W WO 9915687 A1 WO9915687 A1 WO 9915687A1
Authority
WO
WIPO (PCT)
Prior art keywords
polynucleotide
molecular complex
dna
complex
asor
Prior art date
Application number
PCT/US1998/020154
Other languages
English (en)
Inventor
George Y. Wu
Catherine H. Wu
Original Assignee
University Of Connecticut
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Connecticut filed Critical University Of Connecticut
Priority to AU96677/98A priority Critical patent/AU9667798A/en
Publication of WO1999015687A1 publication Critical patent/WO1999015687A1/fr

Links

Classifications

    • 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
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a broad variety of therapeutic polynucleotides have been delivered to cells by receptor-mediated endocytosis.
  • the polynucleotide When linked to a target cell-specific ligand, the polynucleotide is cointernalized by the target cell along with the ligand. Once inside the cell, the polynucleotide is released in functional form, for example, as an expressible gene or as an antisense construct which inhibits expression of an endogenous gene.
  • the plasmid DNA the plasmid is maintained in the target cell as a non- replicating episome without integrating into the cell's genome (Wilson et al. (1992) J Biol. Chem 267(16):! 1483-11489).
  • the system developed by Wu et al. employs a soluble polynucleotide-carrier complex made up of a gene or other polynucleotide electrostatically linked to a bifunetional carrier molecule.
  • the carrier molecule comprised of a polycation-ligand conjugate, serves the dual function of linking the gene (via the polycation moiety, e.g., polylysine) and binding to the target cell (via the ligand, e.g., an asialoglycoprotein), resulting in internalization of the carrier molecule by the cell.
  • Wu et al. linked the gene to the carrier in a step- down dialysis from a high salt solution, thereby slowly complexing the negatively charged DNA and the positively charged polycation-ligand carrier.
  • Wu et al. recognized that the method used to form the complex must result in a structure which (a) is soluble in solution so that it can easily pass through physiological barriers when administered in vivo to reach target cells or tissues, (b) is stable extracellularly so that the polynucleotide remains linked to the carrier, and (c) releases the polynucleotide in functional form under intracellular conditions, e.g., so that the polynucleotide is expressed.
  • the present invention provides a novel muticomponent polynucleotide complex for efficient targeted delivery of polynucleotides to cells.
  • the complex comprises a bifunetional carrier component linked (e.g., complexed) to a polynucleotide.
  • the bifunetional carrier component serves the dual function of binding to the polynucleotide and binding to a cell surface receptor.
  • the complex can further include additional components to increase transfection efficiency and/or expression of the polynucleotide once internalized into a cell.
  • the complex is a "multicomponent complex" because it contains multiple components, in addition to its constituent polynucleotide, which serve to increase transfection efficiency, increase expression and/or target the polynucleotide to extracellular or intracellular components.
  • the complex comprises a polynucleotide and a ligand which binds to a component on the surface of a cell so that the polynucleotide is targeted to and internalized by the cell.
  • the complex comprises a polynucleotide and an endosomolytic agent, such as a peptide from vesicular stomatitis virus (VSV), to enhance endosomal escape once the complex is internalized (e.g., endocytosed) by a cell.
  • VSV vesicular stomatitis virus
  • the complex comprises a polynucleotide and a nuclear targeting agent, such as a peptide derived from the Simian Virus-40 (SV40), to direct the complex to the nucleus (e.g., where the polynucleotide is expressed) once the complex is internalized into a cell.
  • a nuclear targeting agent such as a peptide derived from the Simian Virus-40 (SV40)
  • SV40 Simian Virus-40
  • all or various combinations of the aforementioned components are combined into a single, multicomponent polynucleotide complex (see e.g., Figure 1) which can be used to transfect a variety of cells either in vitro or in vivo in a highly efficient and specific manner.
  • Figure 1 is a schematic representation of a multicomponent polynucleotide complex containing (1) a combination cell targeting and DNA binding component (i.e., a bifunetional binding component), (2) DNA, (3) an endosomal disruption component, and (4) a nuclear targeting component.
  • a combination cell targeting and DNA binding component i.e., a bifunetional binding component
  • Figure 2 is a gel showing semiquantitative competitive PCR analysis of episomal DNA extracted from isolated liver cell nuclei 24 hours after injection of free DNA or DNA complexed with the various protein carriers.
  • Panel A is a negative control;
  • panel B contains free DNA;
  • panel C contains complex made up of AsOR-lysine-DNA;
  • panel D contains complex made up of AsOR-lysine-VSV-DNA,
  • panel E contains complex made up of AsOR-Lysine-VSV-NP-DNA.
  • Each PCR reaction contained the same amount of extracted episomal DNA (5 ⁇ l of a total of 100 ⁇ l) and an increasing amount of competitive template from lane 1 to lane 8 of 0, 0.25, 1.25, 2.5, 12.5, 25, 125, 250 fg, respectively.
  • After digestion with Kpn I PCR product amplified from wildtype luciferase DNA appears as a band at 1538 bp,
  • PCR product amplified from competitive template appears as bands at 596 and 942 bp
  • the present invention is based on the discovery that transfection efficiency of targeted complexes containing polynucleotides linked to cell-binding agents can be increased by modifying and/or adding components to the complex to (a) increase or maintain solubility of the complex in solvent (e.g., under physiological conditions), (b) facilitate dissociation of the polynucleotide from the complex following internalization into a cell, (c) prevent endosomal degradation of the complex by facilitating its release from endosomes under intracellular conditions, (d) increase expression levels by targeting the polynucleotide of the complex to the nucleus of the cell where it can be expressed after it is internalized by the cell, and/or (e) target the polynucleotide to particular cell types so that the polynucleotide is efficiently taken up by cells.
  • polynucleotide binding agent which exhibits greater solubility following complexation with a polynucleotide than do conventional polynucleotide binding agents (e.g., polybasic amino acids, such as polylysine, polyarginine, polyornithine etc.).
  • polynucleotide binding agent is also recognized by a cell surface receptor and, therefore, is bifunetional in that it serves as both a polynucleotide binding agent and a cell binding agent (i.e., a cellular ligand).
  • Improved polynucleotide binding agents of the invention exhibit a lower tendency to precipitate polynucleotide at electroneutrality.
  • polybasic amino acids e.g., polylysine
  • polynucleotide binding agents of the present invention contain intervening polar groups which disperse the density of the positive charge.
  • the polynucleotide binding agent comprises a protein, preferably a cellular ligand, which has been modified to contain one or more non-naturally occurring amino acid residues.
  • the non-naturally occurring amino acid residue is a positively charged residue, such as lysine, which is capable of interacting (e.g., electrostatically) with the polynucleotide.
  • non-naturally occurring amino acid residue is a negatively charged amino acid residue which improves the solubility of the molecular complex.
  • non-naturally occurring means that the amino acid residue is not found in the protein in its wild-type form (i.e., naturally-occurring, unmodified form) at the particular position (i.e., base) in which it exists in the modified protein.
  • the polynucleotide binding agent comprises a protein which is chemically reacted with an agent which converts one or more negatively charged amino acids into positively charged residues. This generally results in the modified positively charged residues being interdispersed with other (e.g., neutral and negatively charged) amino acid residues which naturally occur within the protein, so that clustering of positively charged residues does not occur and polar groups are available to maintain solubility of the ligand when complexed with a polynucleotide.
  • this is achieved by modifying (e.g., cross-linking) a cellular ligand, such as an asialoglycoprotein (e.g., asialoorosomucoid (ASOR)) with lysine methyl ester to obtain a methylysine ester derivative (e.g., ASOR-methylysine).
  • a cellular ligand such as an asialoglycoprotein (e.g., asialoorosomucoid (ASOR)) with lysine methyl ester to obtain a methylysine ester derivative (e.g., ASOR-methylysine).
  • ASOR asialoorosomucoid
  • Other suitable ligands which can be similarly modified using standard cross- linking agents include other proteins, polypeptide, carbohydrates and lipids.
  • the ligand can be any natural or synthetic ligand which binds a cell surface receptor.
  • the ligand can be a protein, polypeptide, glycoprotein, glycopeptide or glycolipid which has functional groups that are exposed sufficiently to be recognized by the cell surface component once modified to contain dispersed positive charges which interact with negatively charged polynucleotides.
  • the ligand can also be a component of a biological organism such as a virus or cells (e.g., mammalian, bacterial, protozoan).
  • the ligand can comprise an antibody, antibody fragment (e.g., an F(ab')2 fragment) or analogues thereof (e.g., single chain antibodies) which binds the cell surface component (see e.g., Chen et al. (1994) FEBS Letters 338:167-169. Ferkol et al.
  • Ligands useful in forming bifunetional carrier components of the invention i.e., polycationic polynucleotide binding agents
  • proteins and synthetic ligands containing galactose-terminal carbohydrates such as carbohydrate trees (e.g., obtained from natural glycoproteins), can be used.
  • glycoproteins that either contain terminal galactose residues or can be enzymatically treated to expose terminal galactose residues (e.g., by chemical or enzymatic desialylation) can be used.
  • the ligand is an asialoglycoprotein, such as asialoorosomucoid, asialofetuin or desialylated vesicular stomatitis virus.
  • the ligand is a synthetic triantennary carbohydrate ligand containing positively charged groups dispersed among polar moieties which can interact with solvent.
  • Suitable ligands for targeting hepatocytes can be prepared by chemically coupling galactose-terminal carbohydrates (e.g., galactose, mannose, lactose, arabinogalactan etc.) to nongalactose-bearing proteins or polypeptides (e.g., polycations) by, for example, reductive lactosamination.
  • galactose-terminal carbohydrates e.g., galactose, mannose, lactose, arabinogalactan etc.
  • polypeptides e.g., polycations
  • the bifunetional carrier component of the complex can comprise other types of ligands.
  • mannose can be used to target Kupffer cells and macrophages.
  • Mannose 6-phosphate glycoproteins can be used to target fibroblasts.
  • Pulmonary surfactants, such as Protein A can be used to target epithelial cells (see e.g., Ross et al. (1995) Human Gene Therapy 6:31-40).
  • Anti-secretory component antibodies can also be used to target the polymeric immunoglobulin receptor on lung and liver epithelial cells (see e.g., Perales et al. (1994) Ewr. J. Biochem.
  • Transfe ⁇ in can be used to target smooth muscle cells (see e.g., Wagner et al. (1990) PNAS 87:3410-3414 and U.S. Patent No. 5, 354,844 (Beug et al.)).
  • Intrinsic factor- vitamin B 12 and bile acids can be used to target enterocytes.
  • Insulin can be used to target fat cells and muscle cells (see e.g., Rosenkranz et al. (1992) Experimental Cell Research 199:323-329 and Huckett et al. (1990) Chemical Pharmacology 40(2):253-263).
  • Apolipoprotein E can be used to target nerve cells.
  • the bifunetional carrier molecule comprises polyethylene glycol (PEG) as one of its constituent components.
  • the bifunetional carrier molecule can be modified using any reagent known in the art to chemically link or create positively charged groups (e.g., amine groups) on the surface of the carrier in a manner which allows for interaction with negatively charged DNA.
  • protein ligands can be reacted with agents such as lysine methyl ester or N-acylurea.
  • Standard cross-linking reagents which can be used for this purpose are well known in the art (e.g., l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), as described by McKee et al (1994) Bioconjugate Chem. 5: 306- 311 or Jung. G. et al.
  • linkages which can be used to produce modified ligands are disulfide bonds which can be formed using cross-linking reagents, such as N-Succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N- hydroxysuccinimidyl ester of chlorambucil, N-Succinimidyl-(4- Iodoacetyl)aminobenzoate) (SIAB), Sulfo-SIAB, and Sulfo-succinimidyl-4- maleimidophenyl-butyrate (Sulfo-SMPB). Strong noncovalent linkages, such as avidin- biotin interactions, can also be used to link cationic moieties to a variety of cell binding agents to form suitable carrier molecules.
  • SPDP N-Succinimidyl 3-(2-pyridyldithio)propionate
  • SIAB N-Succinimidyl-(4- Iodoace
  • the linkage reaction can be optimized for the particular cationic moiety and cell binding agent used to form the carrier.
  • the optimal ratio (w:w) of cationic moiety to cell binding agent can be determined empirically. This ratio will vary with the size of the cationic moiety (e.g., polycation) being used in the carrier, and with the size of the polynucleotide to be complexed. However, this ratio generally ranges from about 0.2- 5.0 (cationic moiety : ligand). Uncoupled components and aggregates can be separated from the carrier by molecular sieve or ion exchange chromatography (e.g., AquaporeTM cation exchange, Rainin).
  • Novel polynucleotide complexes of the present invention can also include additional components to further enhance transfection efficiency and/or expression.
  • the complex comprises an endosomolytic agent capable of releasing the complex from an endosome once internalized into a cell.
  • endosomolytic agent refers to any agent (e.g., protein, peptide, chemical etc.) which, upon internalization into an endosome, is capable of lysing the endosome so that the agent (and any linked components) are released intracellularly.
  • Suitable endosomolytic agents include, viral and bacterial components (e.g., fusogenic peptides and bacterial cytolysins).
  • lysteriolysin O can be incorporated into the complex as described in U.S. Serial No. 08/484,009, the contents of which are incorporated by reference herein).
  • a fusogenic peptide from the vesicular stomatitis virus (VSV) having the following amino acid sequence Lys-Phe-Thr-Ile-Val-Phe-Pro-His-Asn-Gln-Lys-Gly-Asn-Trp-Lys-Asn-Val-Pro-Ser- Asn-Tyr-His-Tyr-Cys-Pro (SEQ ID NO: 1).
  • the polynucleotide complex also contains a nuclear targeting agent which directs the polynucleotide of the complex to the nucleus where the polynucleotide can be expressed.
  • nuclear targeting agent refers to any agent (e.g., protein, peptide etc.) which promotes nuclear localization of the polynucleotide when linked to the polynucleotide.
  • Suitable nuclear targeting agents include but are not limited to peptides known in the art to be involved in cellular trafficking of proteins to the nucleus.
  • a peptide derived from the Simian Virus-40 having the following sequence is used: Cys-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Glu-Tyr-Gly (SEQ ID NO:2).
  • SV40 Simian Virus-40
  • Such peptides can be linked to the complex using any standard cross-linking agent known in the art, including those previously described herein.
  • an N-terminal cysteine can be added to permit coupling to the complex.
  • nuclear targeting proteins include, for example, DNA binding proteins which localize to and interact with the nuclear matrix, such as SATB-1 (Dickinson et al. (1992) Cell 70, 631-645), ARBP (Buhrmester et al. (1995) Biochemistry 34, 4108-4117; von Kries et al. (1991) Cell 64, 123-135), Lamin B
  • the polynucleotide is linked to the carrier so that (a) the polynucleotide is sufficiently stable (either in vivo, ex vivo, or in vitro) to prevent significant uncoupling of the gene extracellularly prior to internalization by the target cell, (b) the polynucleotide is released in functional form under appropriate conditions within the cell, (c) the polynucleotide is not damaged and (d) the carrier retains its capacity to bind to cells.
  • the linkage between the carrier and the gene is noncovalent.
  • Noncovalent bonds include, for example, electrostatic bonds, hydrogen bonds, hydrophobic bonds, anti-polynucleotide antibody binding, linkages mediated by intercalating agents, and streptavidin or avidin binding to polynucleotide-containing biotinylated nucleotides.
  • the carrier can also be directly (e.g., covalently) linked to the gene using, for example, chemical cross- linking agents (e.g., as described in WO-A-91/04753 (Cetus Corp.), entitled "Conjugates of Antisense Oligonucleotides and Therapeutic Uses Thereof).
  • Polynucleotides delivered to cells in the form of a molecular complex of the invention can be DNAs or RNAs. Typically, they are in a form suitable for expression, meaning that they are operably linked (e.g., within an expression vector) to genetic regulatory elements necessary for expression in a cell. Such regulatory elements include, for example, promoter sequences which drive transcription of the gene, enhancers and other expression control elements. Such regulatory sequences are known and discussed in Goeddel, Gene expression Technology: Methods in Enzvmology, p. 185, Academic Press, San Diego, CA (1990).
  • the polynucleotide also may be operably linked to appropriate signal sequences which provide for trafficking of the encoded protein to intracellular destinations and/or extracellular secretion.
  • the signal sequence may be a natural sequence of the protein or an exogenous sequence.
  • the term "operably linked” means linked in trans so that the regulatory element exerts its function on the polynucleotide to be expressed.
  • the soluble molecular complex of the invention can be used to deliver polynucleotides, e.g., genes encoding therapeutic proteins either in vitro, ex vivo or in vivo to selected cells, and to obtain expression of the polynucleotides in the cells.
  • polynucleotides e.g., genes encoding therapeutic proteins either in vitro, ex vivo or in vivo to selected cells, and to obtain expression of the polynucleotides in the cells.
  • the complex is contacted (e.g., incubated) with a target cell in culture in an appropriate medium under conditions conducive to endocytotic uptake by the cells.
  • the complex is administered in vivo to a subject in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any physiologically acceptable carrier for stabilizing polynucleotide-carrier complexes of the present invention for administration in vivo, including, for example, saline and aqueous buffer solutions, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • the pharmaceutical composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action or microorganisms such as bacteria and fungi. Protection of the polynucleotide-carrier complexes from degradative enzymes (e.g., nucleases) can be achieved by including in the composition a protective coating or nuclease inhibitor.
  • degradative enzymes e.g., nucleases
  • Molecular complexes of the invention may be administered in vivo by any suitable route of administration.
  • the complex can be administered by injection (e.g., intravenous, intraperitoneal, intramuscular, intravascular, or subcutaneous injection).
  • Other suitable routes of administration include slow-release implants, topical and oral administration.
  • the appropriate dosage of complex may vary according to the selected route of administration.
  • the complexes are preferably injected intravenously in solution containing a pharmaceutically acceptable carrier, as defined herein.
  • Sterile injectable solutions can be prepared by incorporating the polynucleotide-carrier complexes in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the complex is contained in 0.15 M NaCl or in glycine.
  • mice can be administered dosages of up to 1.0 mg of polynucleotide per 20 g of mouse, or about 1.0 mL of complex per 1.4 mL of mouse blood.
  • This invention is illustrated further by the following examples which should not be construed as further limiting the subject invention. The contents of all references and published patent applications cited throughout this application are hereby incorporated by reference.
  • L-Lysine methyl ester 50 mg (Sigma Chemical Co., St. Louis, MO) was dissolved in 1 ml deionized water and mixed with ASOR. The pH of the solution was adjusted to 6.5 by addition of NaOH. l-(3- dimethylaminopropyl)-3-ethylcarbodiimide 4 mg was dissolved in 1 ml H2O, and directly added to the ASOR and lysine methyl ester mixture. The solution was stirred at 37°C for 24 hrs., and then dialyzed through membranes with 12-14 Kd exclusion limits (Spectrapore) against deionized water at 4°C for 24 Hrs.
  • the samples were lyophilized and then redissolved in 0.5 ml 0.15 M NaCl, filtered through 0.22 ⁇ membranes (Millipore) and applied on a Waters HPLC system using a Rainin LW-804 column eluted with 0.15 M NaCl at a flow rate of 0.12 ml/min.
  • An acid urea gel revealed a single band that migrated faster than ASOR indicating an increase in overall positive charge.
  • the molar ratio of methyl lysine to ASOR was calculated to be 24:1 based on amino acid analysis of the purified material.
  • VSV Vesicular Stomatitis virus
  • the G-protein on its spikes possess the property of membrane fusion that is induced by a decrease in the pH of its environment. After internalization within endosomes, the decrease in pH triggers a conformational change in the G-protein which results in insertion into the endosomal membrane and release of the virus from the endosome.
  • the region of the protein responsible has been identified and a sequence has been shown to have pH dependent hemolytic properties (4). Based on this information, we prepared the following 25 amino acid peptide:
  • the cysteine residue could be used to link this molecule to the ASOR- methyllysine carrier.
  • the N-terminal cysteine was added to permit coupling to carriers, and the Tyr residue used for radiolabeling.
  • samples were dialyzed through 12-14 kD exclusion limit membranes against 50 L de-ionized water 4°C for 24 hrs.
  • Samples were lyophilized and then purified by HPLC using a Shodex KW-804 column eluted with 0.15 M NaCl, at a flow rate of 0.12 ml/min.
  • a 12-15% gradient SDS polyacrylamide gel, and an acid urea gel showed a purified bank that had no free VSV or NP. The band was more slowly migrating on SDS gel and released both VSV and NP on reduction in acid area gels.
  • the amount of conjugate needed for complete complexation to DNA was determined by (a) complete retardation of DNA on 1% agarose gel (TBE running buffer) (6) and (b) ethidium bromide exclusion from DNA as measured by fluorescence with 260 nm excitation 591 nm emission on a fluorimeter (7).
  • the complex was incubated at room temperature for 30 minutes followed by filtration through Millipore GV 0.22 ⁇ 10 mm diameter membranes when the volume of complex was 1 ml or less, or through Gelman Acridisc HT tuffryn membrane filter, 0.2 ⁇ , 25 mm diameter when volumes of complex were greater than 1 ml.
  • UV absorbances of filtered complexes were determined to calculate recovery of complexes. An aliquot of the final complex was applied onto 1% agarose gel to ensure completeness of DNA binding in the complex after filtration. If filtered complex was more concentrated than 20 ⁇ g/ml, the complex was diluted with sterile saline to make a final concentration of 20 ⁇ g/ml.
  • Linear CMVHCVLuciferase DNA was used as the template for T7 RNA polymerase for in vitro transcription assays.
  • a 1 % agarose gel in TBE was used to determine the amount of conjugate necessary to retard linear T7 luciferase DNA.
  • the calculate titration ratios based on retardation points are shown below in Table 1.
  • DNA template 0.375 ⁇ g, alone or complexed to either ASOR-PL, ASOR- methyllysine, ASOR-methyllysine-VSV or ASOR-methyllysine-VSV-NP conjugate were incubated with 50 Units of T7 RNA polymerase, 5 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 40 Units RNasin, 2.5 mg BSA, IX transcription buffer (50 mM Tris HCI pH 8.0, 8 mM MgCl 2 , 2 mM spermidine and 50 mM NaCl) and 2 ⁇ l of [ 32 P]- UTP 3000 Ci/mmol ⁇ Amersham ⁇ in a 20 ⁇ l total volume.
  • T7 RNA polymerase 5 mM DTT, 0.5 mM ATP, 0.5 mM GTP, 0.5 mM CTP, 40 Units RNasin, 2.5 mg BSA, IX transcription buffer
  • luciferase lysis buffer Promega, Luciferase Assay kit #E1501
  • Livers were homogenized for 10 strokes in a glass Dounce homogenizer with tight pestle B.
  • One ml of homogenized liver was removed, and centrifuged at 10,000 rpm for 5 minutes at room temperature. Aliquots of liver supernatant (20 ⁇ 1 - 100 ⁇ 1) were used for to measure luciferase activity by the method described by Promega using a Monolight Luminometer.
  • a standard curve for luciferase using firefly luciferase was performed along with the test samples. Results were expressed as pg luciferase /mg of cell protein or per gram liver.
  • samples were incubated for 30 min in the presence of 100 U/20 ⁇ l heparin, 4 M urea, whole mouse serum, or DMEM medium 10% fetal bovine serum. Samples of serum or medium were dissociated with 4 M urea immediately prior to analysis on agarose gels.
  • liver cell nuclei were isolated from mice 24 hours after injection of the plasmid carrying the luciferase gene complexed by the various conjugates.
  • a semiquantitative competitive PCR assay was performed on the episomal DNA samples extracted from the nuclei.
  • Liver tissue was homogenized in 4 ml buffer 1 (Sucrose 0.32 M, CaCl2 3 mM, Mg acetate 2 mM, Na2EDTA 0.1 mM, Tris HC1 pH 8.0 10 mM, DTT 1 mM, NP40 0.5%)) using a Dounce Tissue Grinder (Kontes) and applying 5 strokes with a loose and tight fitting pestel, respectively.
  • buffer 1 Sacrose 0.32 M, CaCl2 3 mM, Mg acetate 2 mM, Na2EDTA 0.1 mM, Tris HC1 pH 8.0 10 mM, DTT 1 mM, NP40 0.5%)
  • the homogenate was loaded on top of a sucrose gradient consisting of four layers with 1.0, 1.4, 1.6 and 1.8 M sucrose, respectively, in buffer 2 (Mg acetate 5 mM, Na 2 EDTA 0.1 mM, Tris HC1 pH 8.0 mM, DTT 1 mM and centrifuged for 40 min, 34,000xg at 4°C (Beckmann SW28 rotor).
  • the nuclei containing pellet was resuspended in 1 ml buffer 1 (without NP40) and pelleted again by low speed centrifugation (750xg, 2 min).
  • nuclei were resuspended in 1 ml TE50/10 (Tris HC1 pH 7.5, 50 mM, Na 2 EDTA 10 mM) and lysed with 1.5 ml lysis buffer (SDS 1%, NaOH 0.2 N).
  • modified Hirt extraction was performed by adding 1.25ml of 3M-5M acetate. After incubation for 15 min on ice the precipitate was pelleted and the supernatant was extracted with phenol/chloroform. After precipitation with ethanol, samples were resuspended in 100 ⁇ l of TE (Tris HC1 pH 7.5, 10 mM, Na2EDTA 1 mM) and subjected to PCR analysis.
  • a quantitative PCR assay was performed as described by Stieger et al. (1991) J
  • the new Kpn I site was created by PCR site-directed mutagenesis, using S2 (5 '-3') CTGGATCTACTGGGGTACCTAAGGGTGTG (SEQ ID NO:5) and AS2 CACACCCTTAGGTACCCCAGTAGATCCAG (SEQ ID NO:6) as primers and pCMVluc, carrying the luciferase gene as published (Wet et al. (1987) Mol Cell Biol. 7:725-737) as a template. After Kpn I digestion PCR products generated from target sequences (1538bp) or internal standard (596/942bp), respectively, could be discriminated.
  • DNA amplification experiments were carried out with 2.5 U Taq ploymerase (Life Technologies) per reaction and 1.5 mM MgCl2.
  • a hot start protocol was used with annealing at 60°C (45 s), elongation at 72°C (3 min) and denaturation at 94°C (45 s) for 35 cycles, followed by a final elongation at for 72°C 10 min.
  • VSV VSV 25 amino acid peptide
  • NP SV-40 nuclear localizing peptide
  • ASOR-PL ASOR-methyllysine
  • ASOR-methyllysine-VSV ASOR-methyllysine-VSV-NP
  • NP nuclear localizing peptide
  • ASOR-PL and ASOR-methyllysine as well as NP complexes could not be dissociated.
  • coupling of either VSV alone or in combination with NP resulted in increased dissociability of DNA complexes.
  • ASOR-PL and NP complexes were dissociable with 100 U/20 ⁇ l heparin, while the ASOR-methyllysine conjugates were not. This suggests that ASOR-methyllysine derivatives bind DNA differently compared to polylysine conjugates.
  • a plasmid containing a luciferase gene driven by a CMV promoter was complexed with ASOR-methyllysine-VSV-NT. Controls consisting of ASOR- methyllysine alone, ASOR-methyllysine-VSV, and ASOR-polylysine were also prepared. All complexes were filtered through 0.2 ⁇ membranes. Complexes containing 10 ⁇ of DNA in 0.5 ml were injected intravenously into mice over a minimum of 10 seconds. After 24 hours, the animals were sacrificed and liver luciferase activity determined by luminometry.
  • ASOR-methyllysine-VSV-NP The means of the multi-component conjugates, ASOR-methyllysine-VSV-NP, were approximately two orders of magnitude greater than expression by ASOR-PL complexes of the same amount of DNA. ASOR-methyllysine-DNA alone produced only background luciferase activity. ASOR-VSV conjugates resulted in a 10-fold enhancement. Expression from tri-component complexes was substantially blocked by competition by a 100-fold molar excess of ASOR, but essentially unaffected by co- administration of 100-fold molar excess of OR.
  • the amount of luciferase DNA was increased by approximately a factor 10 after injection of AsOR-lysine-VSV complexed DNA and by a factor 20 after injection of AsOR-lysine-VSV/NP complexed DNA compared to injection of the same amount of DNA in the form of AsOR-lysine complexes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne de nouveaux complexes polynucléotidiques multiconstituants destinés à la transfection efficace de cellules. Les complexes comprennent un constituant support bifonctionnel lié à un polynucléotide. Le support se fixe au polynucléotide et comprend un ou plusieurs constituants augmentant l'efficacité de la transfection et/ou l'expression du polynucléotide une fois internalisé dans une cellule. Dans un mode de réalisation, le support est cationique de manière qu'il forme un complexe avec le polynucléotide. Dans un mode de réalisation préféré, le support comprend un ligand cellulaire. Dans d'autres modes de réalisation, le support comprend également un agent endosomolytique et/ou un agent de ciblage nucléaire. Les complexes multiconstituants peuvent être utilisés pour transfecter une variété de cellules soit in vitro, ex vivo, soit in vivo d'une manière hautement efficace et spécifique.
PCT/US1998/020154 1997-09-26 1998-09-25 Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite WO1999015687A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU96677/98A AU9667798A (en) 1997-09-26 1998-09-25 Multicomponent polynucleotide complex for high efficiency cell transfection

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US5993797P 1997-09-26 1997-09-26
US60/059,937 1997-09-26

Publications (1)

Publication Number Publication Date
WO1999015687A1 true WO1999015687A1 (fr) 1999-04-01

Family

ID=22026264

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/020154 WO1999015687A1 (fr) 1997-09-26 1998-09-25 Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite

Country Status (2)

Country Link
AU (1) AU9667798A (fr)
WO (1) WO1999015687A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025608A1 (fr) * 1993-04-27 1994-11-10 Baylor College Of Medicine Proteines de fixations d'adn naturel ou recombine utilisees comme vecteurs dans le transfert de genes ou la therapie genique
WO1996000792A1 (fr) * 1994-06-29 1996-01-11 University Of Connecticut Utilisation d'un composant bacterien pour ameliorer l'apport cible de polynucleotides a des cellules
WO1996040958A1 (fr) * 1995-06-07 1996-12-19 Baylor College Of Medicine Transporteurs d'acide nucleique servant a introduire des acides nucleiques dans une cellule
WO1997025070A2 (fr) * 1996-01-08 1997-07-17 Baylor College Of Medicine Peptides lipophiles destines a l'apport de macromolecules

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994025608A1 (fr) * 1993-04-27 1994-11-10 Baylor College Of Medicine Proteines de fixations d'adn naturel ou recombine utilisees comme vecteurs dans le transfert de genes ou la therapie genique
WO1996000792A1 (fr) * 1994-06-29 1996-01-11 University Of Connecticut Utilisation d'un composant bacterien pour ameliorer l'apport cible de polynucleotides a des cellules
WO1996040958A1 (fr) * 1995-06-07 1996-12-19 Baylor College Of Medicine Transporteurs d'acide nucleique servant a introduire des acides nucleiques dans une cellule
WO1997025070A2 (fr) * 1996-01-08 1997-07-17 Baylor College Of Medicine Peptides lipophiles destines a l'apport de macromolecules

Also Published As

Publication number Publication date
AU9667798A (en) 1999-04-12

Similar Documents

Publication Publication Date Title
US6069133A (en) Targeted delivery of genes encoding interferon
US5994316A (en) Method of preparing polynucleotide-carrier complexes for delivery to cells
US6794189B2 (en) Polyampholytes for delivering polyions to a cell
Guy et al. Delivery of DNA into mammalian cells by receptor-mediated endocytosis and gene therapy
US8211468B2 (en) Endosomolytic polymers
US5728399A (en) Use of a bacterial component to enhance targeted delivery of polynucleotides to cells
US7098030B2 (en) Polyampholytes for delivering polyions to a cell
US8217015B2 (en) Endosomolytic polymers
US7682626B2 (en) Polyvinylethers for delivery of polynucleotides to mammalian cells
US5843770A (en) Antisense constructs directed against viral post-transcriptional regulatory sequences
EP1272222A2 (fr) Composition pour administration de medicament
Hellgren et al. Factors controlling the efficiency of Tat-mediated plasmid DNA transfer
US5744326A (en) Use of viral CIS-acting post-transcriptional regulatory sequences to increase expression of intronless genes containing near-consensus splice sites
WO1999015687A1 (fr) Complexe polynucleotidique multiconstituant pour transfection cellulaire a haute efficacite
Carmichael et al. Targeted delivery of anti-hepatitis B antisense oligonucleotides
EP1248838A1 (fr) Polyampholytes destines a introduire des polyions dans une cellule
Liang et al. Targeted gene delivery: the role of peptide nucleic acid
Kren et al. 1081. Targeted Nanocapsules for Liver Cell-Type Delivery of Plasmids In Vivo
Huang Cytotoxicity of reducible polycations and subcellular trafficking of their DNA nanoparticles
Zhang Enhancement of targeted gene expression by incorporation of Listeriolysin O into protein-DNA complexes
Rettig Strategies to test nuclear localization of non-viral gene delivery vectors in vitro and in vivo
OGRIS et al. DELIVERY TECHNIQUES AND THERAPEUTIC

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载