WO2003038103A1 - Methode de transfection genetique faisant appel a des combinaisons synergiques de lipides cationiques et de polymeres cationiques - Google Patents

Methode de transfection genetique faisant appel a des combinaisons synergiques de lipides cationiques et de polymeres cationiques Download PDF

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Publication number
WO2003038103A1
WO2003038103A1 PCT/FI2001/000943 FI0100943W WO03038103A1 WO 2003038103 A1 WO2003038103 A1 WO 2003038103A1 FI 0100943 W FI0100943 W FI 0100943W WO 03038103 A1 WO03038103 A1 WO 03038103A1
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composition
dna
transfection
dosper
pei
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PCT/FI2001/000943
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English (en)
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Atso Raasmaja
Pasi Lampela
Pekka T. MÄNNISTÖ
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Atso Raasmaja
Pasi Lampela
Maennistoe Pekka T
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Priority to PCT/FI2001/000943 priority Critical patent/WO2003038103A1/fr
Publication of WO2003038103A1 publication Critical patent/WO2003038103A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
    • C08L79/02—Polyamines
    • 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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • 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
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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

Definitions

  • the present invention relates to a synergistic method of gene transfection, including gene therapy of human diseases.
  • the invention provides the use and preparation of specific combinations of cationic lipids (e.g., Dosper) and cationic polymers (e.g., PEIs or poly- ethyl enimines) for the transfection of DNA or RNA or synthetic nucleic acids (including plain nucleic acids, genomic or nongenomic DNA, nonviral expression plasmids and viral vectors) into host cells in vitro and in vivo.
  • This invention includes also the use of described combinations in any systems for gene transfection, expression, repair, activation, inhibition and regulation, when a specific combination of cationic lipids and cationic polymers is used for the procedure.
  • the present invention includes any delivery of other molecules or compounds into the cells, especially negatively charged, when any combinations of cationic liposomes and cationic polymers are used for the procedure, especially in therapeutic aims.
  • Gene therapy is a potential technique for the treatment of genetic and acquired diseases.
  • the aim of this therapy is to correct a pathophysiological dysfunction by an introduction of therapeutic genes into a patient's target cells 1 .
  • Gene therapy may have a better specificity and selectivity, and a longer duration when compared to a traditional drug treatment. It could also be possible to treat by gene therapy rather the cause than the symptoms of the diseases.
  • Nonviral vectors have been less efficient than viral vectors.
  • the use of viral vectors e.g., adenoviral or retroviral vectors
  • modified plasmids could be used instead of viral vectors.
  • these plasmids are often large and negatively charged which properties reduce their ability to enter the target cells. Indeed, the use of nonviral gene transfection has been limited by the low transfection efficiency.
  • Cationic lipids have been tested in the gene transfection.
  • Dosper Liposomal Transfection Reagent l,3-di-oleoyloxy-2-(6-carboxy-spermyl)- propylamid, Boehringer Mannheim, Germany
  • the cationic liposomes form electrostatic complexes with negatively charged DNA, and these complexes can enter the cell via an endocytotic pathway 7 .
  • the liposome-DNA complex destabilizes the endosomal membrane, and induces a flip-flop of anionic lipids from the cytoplasm-facing monolayer.
  • anionic lipids diffuse laterally into the liposome-DNA complex and form a charged neutral ion pair with the liposomes (cationic lipids). In this way, the liposome-DNA complex is broken and the DNA is released into the cytoplasm 8 .
  • PEIs are cationic polymers, and especially their branched forms are efficient vectors for delivering plasmids into cells 4 . Every third atom of each PEI molecule is an amino nitrogen that can be protonated. PEI has the ability to condense DNA 9 . In addition, PEI retains a substantial buffering capacity at virtually any pH 10 , protects DNA from endosomal de- gration 4 and targets DNA into nucleus 11 . When considering transfection efficiency, the ratio of PEI nitrogens to DNA phosphates (N/P ratio) is important, and maximal transfection efficiencies have been obtained with N P ratios of 5-13.5 12 . Transfection efficiency of PEI increases with the increase of molecular weight.
  • the low molecular weight PEIs (600, 1200, 1800) have been virtually ineffective 13 .
  • Large and small PEIs have also been used together to improve transfection efficiency 16 .
  • cationic liposomes and polyethylenimines have been successfully used in vitro in non- viral gene transfer. They have different mechanisms of action. Polycationic liposomes form complexes with DNA which then enter the cell via an endocytotic pathway. PEIs condense DNA and protect them from endosomal degradation thereby improving transfection efficiency. In earlier reports, only large PEI molecules (MW>20 000) have been effective in the gene transfection.
  • the transfection method described here uses the advantage of the synergism between cationic lipids and cationic polymers to potentiate significantly the transfection efficiency. This synergism may result from the different mechanisms of described compounds, therefore allowing a higher transfection efficiency of gene expression plasmids.
  • the synergistic effect of PEI and cationic liposomes was a novel unexpected finding in the gene transfection.
  • a general object of the present invention is to provide a method to be used in in vitro and in vivo gene transfection.
  • a specific object of the present invention is the use of a combination of cationic lipids and cationic polymers in the gene transfection, especially Dosper and polyethylenimines.
  • a further object of the invention is a composition for transfecting a cell, which comprises one or more nucleic acid molecules, polycations or cationic polymers, and cationic liposomes or lipids.
  • Another specific object of the present invention is the use of small polyethylenimines (e.g. PEI 700 and PEI 2000) in the combination of cationic lipids and cationic polymers for the gene transfection.
  • Another specific object is the use of such amounts of reagents which are alone ineffective in the gene transfection. These amounts are varying from low and ineffective to high and effective.
  • Another specific object is the expression of a therapeutic gene in the cells of a subject.
  • the expression is achieved by transfecting the cells with the help of the present method.
  • Another specific object of the present invention is the transfection and expression of any plasmids or vectors containing a therapeutic gene, to be used in the gene therapy of human diseases.
  • the polyethylenimines are used at equivalencies from 1 to 150 N P, and other polycations are used at equivalent amounts and principle.
  • transfection reagents of the invention i.e. polycations or cationic polymers and cationic liposomes or lipids are used in very low amounts, which are inefficient alone.
  • Fig. 1 shows the effect of addition of Dosper to the PEI-DNA complexes on the ⁇ - galactosidase activity.
  • the cells were transfected with TkBPNlacZ plasmid (1 ⁇ g) com- plexed with three different PEIs (average MW 700, 2000 and 25 000) with different ⁇ /P ratios (1-50). The transfections were carried out without (A) or with (B) addition of Dosper (Dosper/D ⁇ A ratio of 1) to the PEI-D ⁇ A complexes.
  • the cells were incubated in the transfection solution for 6 h and then in the growth medium for 42 h.
  • the ⁇ -galactosidase activity was measured by O ⁇ PG assay.
  • Fig. 2 shows the effect of different Dosper/DNA ratios on the ⁇ -galactosidase activity.
  • the cells were transfected with 1 ⁇ g TkBPNlacZ plasmid at a Dosper/D ⁇ A ratios 0-7.5 (A) or D ⁇ A was condensed with PEIs at ⁇ /P ratios 1 (B) and 30 (C) before the addition of Dosper.
  • the cells were incubated in the transfection solution for 6 hours and then in the growth medium for 42 h.
  • the ⁇ -galactosidase activity was measured by O ⁇ PG assay.
  • Fig. 3 shows the analysis of transfection efficiency by X-gal staining.
  • the cells were transfected with 1 ⁇ g TkBPNlacZ plasmid complexed with PEI700 and PEI2K at the ⁇ /P ratio of 30 and/or Dosper at a Dosper/D ⁇ A ratios of 0, 2.5 and 5.
  • the cells were incubated in the transfection solution for 6 h and then incubated in the growth medium for 42 h. After the incubation, the cells were washed with PBS and fixed with 4 % paraformaldehyde for 15 min. Then, the cells were stained with X-gal (1 mg/ml) for 3 h at 37°C and washed with PBS.
  • Fig. 4 shows the analysis of complex formation by agarose gel electrophoresis.
  • TkBPNlacZ plasmids were complexed with PEIs (MW 700, 2000 and 25 000) at the ⁇ /P ratios 1, 2.5 and 5. With ⁇ /P ratios above 1, all the D ⁇ A was complexed and remained in the well.
  • TkBPNlacZ plasmid (2 ⁇ g) was diluted with 150 mM ⁇ aCl to total volume of 30 ⁇ l. Different amounts of PEI were also diluted with ⁇ aCl to the total volume of 30 ⁇ l. After a 10 min incubation, the solutions were mixed and the PEI-D ⁇ A complexes were allowed to form during another 10 min incubation.
  • DMEM Dulbecco's Modified Eagle Medium
  • Foetal Bovine Serum and Penicillin- Streptomycin were purchased from Gibco BRL (U.K.)
  • the pTKBPNlacZ plasmid was synthesized by prof. Mart Us- tav at University of Tartu, Estonia.
  • the pTKBPNlacZ plasmid was produced inE. coli (DH5 ⁇ ) and purified using the commercial kits (Qiagen, Germany). All other chemicals were of cell culture and molecular biological quantity.
  • the pTKBPNlacZ expression plasmids (1 ⁇ g/well) were transfected in the subconfluent CN1-P cell cultures (a monkey fibroblastoma cell line). The cells were cultured in 24-well plates in the atmosphere containing 5 % CO 2 at 37 °C. The bacterial lacZ gene encoding the ⁇ -galactosidase enzyme was used as a reporter gene. Preparation of transfection mixtures were made separately for P ⁇ I, Dosper and P ⁇ I/Dosper combinations. First, 10 ⁇ g of pTKBPNlacZ plasmid was diluted to a final volume of 150 ⁇ l of 150 mM ⁇ aCl.
  • P ⁇ I transfection mixture 10 mM P ⁇ I was diluted to a final volume of 150 mM ⁇ aCl, incubated for 10 min, added to plasmid D ⁇ A dilution and incubated for another 10 min be- fore the gene transfection.
  • Dosper transfection mixture Dosper was diluted at the ratio of 1/5 in 150 mM ⁇ aCl and incubated for 15 min before the use for gene transfection. In the case of P ⁇ I/Dosper combination, Dosper solution was added to the D ⁇ A-P ⁇ I mixture which was then incubated for an additional 15 min.
  • the transfection mixtures were pipetted dropwise to cell cultures with 1 ml of freshly added DM ⁇ M without serum and antibiotics. After 6 h exposure, the transfection mixture was replaced with 1 ml of fresh DM ⁇ M (with 9% Foetal Bovine Serum and 90 U Penicillin- Streptomycin). The cells were incubated further up to 42 h before analyses of the ⁇ - galactosidase activity.
  • the cells were washed with PBS, lysed with 150 ⁇ l lysis reagent (25 mM glycylglycine, 15 mM MgSO 4 , 4 mM ⁇ GTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF), and centrifuged at 13 000 rpm for 5 min ( ⁇ ppendorf Centrifuge 5415C, ⁇ p- pendorf- ⁇ etheler-Hinz, Germany).
  • 150 lysis reagent 25 mM glycylglycine, 15 mM MgSO 4 , 4 mM ⁇ GTA, 1% Triton X-100, 1 mM DTT, 1 mM PMSF
  • the activity of ⁇ -galactosidase was measured with O ⁇ PG assay from the supernatant: 20 ⁇ l of the supernatant, 80 ⁇ l H 2 O and 100 ⁇ l of 2x ⁇ - gal solution (2 mM MgCl 2 , 1 mM ⁇ -mercaptoethanol, 1.33 mg/ml O ⁇ PG in sodium phosphate buffer (0.2M)) were put in a 96-well plate and incubated up to 1 h at RT.
  • the detection is based on the cleavage of ⁇ -bond from O ⁇ PG by the ⁇ - galactosidase enzyme resulting in the yellow o-nitrophenol molecule
  • the reaction was stopped with 1M Na 2 CO 3 , when o-nitrophenol anionizes and absorbs lightwaves at 405 nm wavelength
  • Samples were analysed by measuring absorbance at 405 nm wavelength with the Bio-Tek Elx-800 microplate reader (Bio-Tek Instruments, USA) and KC-3 PC- program
  • Protein concentrations were measured with Bio-Rad Protein Assay (Coomassie Brilliant Blue, Bio-Rad Laboratories, USA) 15 ⁇ l of supernatant was diluted to 800 ⁇ l of H 2 O, and 200 ⁇ l of Protein Assay Dye Reagent Concentrate was added Absorbance was read at 595 nm wavelength using Hitachi U-2000 spectrophotometer
  • the X-gal staining was used for histochemical analysis of ⁇ -galactosidase enzyme There- fore, the cells were washed with PBS, fixed with 4 % paraformaldehyde (15 min, RT) and washed again twice with PBS Then, the cells were incubated in the X-gal staining solution (X-gal 1 mg/ml, MgCl 2 2 mM, 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN 6 )x3H 2 O, 0 01 % sodi- umdeoxycholate, 0 02 % Nonidet P-40) for 3 h at +37 °C
  • the activity of ⁇ -galactosidase was detected as a blue color of 3,5'-dichromo-4,4'-dichloroindigo molecule resulting from the cleavage of X-gal substrate by the ⁇ -galactosidase in the transfected cells After X-
  • the role ofPEI/DNA ratios on the transfection efficiency of the PEI/Dosper combination In the first experiments, the effect of different N/P ratios of PEI/DNA complexes was studied on the transfection efficiency of the PEI/Dosper combination. Here, the transfection efficiency was measured as a total ⁇ -galactosidase activity in the cell extracts using the colorimetric ONPG assay.
  • the effect of PEI/Dosper combination on the number of transfected cells was measured using the histochemical X-gal staining.
  • the number of blue-colored cells (indicating successful lacZ gene transfections and expressions) was increased in comparison to PEI or Dosper alone ( Figure 3).
  • the Dosper-mediated gene transfection at the Dosper/DNA ratio of 5 there was less than 1 % of the cells were stained.
  • the number of stained cells increased significantly, when PEI 700 or 2K at the N/P ratio of 30, was used to condense the plasmid DNA prior addition of Dosper, showing the averages of with 4.8 ⁇ 0.8 % for PEI 700 and 4.3 + 1 % for PEI 2K (data not shown).

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Abstract

L'invention se rapporte à une méthode synergique de transfection génétique, comprenant la thérapie génique appliquée aux maladies humaines. L'invention concerne l'utilisation et la préparation de combinaisons spécifiques de lipides cationiques et de polymères cationiques pour la transfection d'acides nucléiques dans des cellules hôtes. L'invention concerne également l'utilisation desdites combinaisons dans des systèmes spécifiques destinés à la transfection, l'expression, la réparation, l'activation, l'inhibition et la régulation génétiques.
PCT/FI2001/000943 2001-10-29 2001-10-29 Methode de transfection genetique faisant appel a des combinaisons synergiques de lipides cationiques et de polymeres cationiques WO2003038103A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2415375A (en) * 2004-05-25 2005-12-28 Coletica Hydrated lamellar phases or liposomes containing a fatty monoamine or cationic polymer for intracellular penetration
CN105295055A (zh) * 2015-11-24 2016-02-03 南开大学 一种基于超分子自组装的磁转染试剂

Citations (6)

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Publication number Priority date Publication date Assignee Title
EP0424688A2 (fr) * 1989-09-28 1991-05-02 Iowa State University Research Foundation, Inc. Méthode synergique pour la transformation de cellules hôtes
US5627159A (en) * 1994-10-27 1997-05-06 Life Technologies, Inc. Enhancement of lipid cationic transfections in the presence of serum
EP0905254A2 (fr) * 1997-09-30 1999-03-31 Hoechst Marion Roussel Deutschland GmbH Vecteur d'ADN associés avec des polyéthylèneimines à bas poids moléculaire, biocompatibles
WO1999058694A1 (fr) * 1998-05-12 1999-11-18 The Regents Of The University Of California Procedes de formation de microparticules lipidiques liees a des proteines et leurs compositions
WO2001015755A2 (fr) * 1999-09-01 2001-03-08 Genecure Pte Ltd Procedes et compositions d'administration d'agents pharmaceutiques
US6210708B1 (en) * 1996-05-08 2001-04-03 Nika Health Products Limited Cationic virosomes as transfer system for genetic material

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0424688A2 (fr) * 1989-09-28 1991-05-02 Iowa State University Research Foundation, Inc. Méthode synergique pour la transformation de cellules hôtes
US5627159A (en) * 1994-10-27 1997-05-06 Life Technologies, Inc. Enhancement of lipid cationic transfections in the presence of serum
US6210708B1 (en) * 1996-05-08 2001-04-03 Nika Health Products Limited Cationic virosomes as transfer system for genetic material
EP0905254A2 (fr) * 1997-09-30 1999-03-31 Hoechst Marion Roussel Deutschland GmbH Vecteur d'ADN associés avec des polyéthylèneimines à bas poids moléculaire, biocompatibles
WO1999058694A1 (fr) * 1998-05-12 1999-11-18 The Regents Of The University Of California Procedes de formation de microparticules lipidiques liees a des proteines et leurs compositions
WO2001015755A2 (fr) * 1999-09-01 2001-03-08 Genecure Pte Ltd Procedes et compositions d'administration d'agents pharmaceutiques

Non-Patent Citations (2)

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Title
GOOMER R.S. ET AL.: "High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects", OSTEOARTHRITIS AND CARTILAGE, vol. 9, 2001, pages 248 - 256 *
XIANG GAO ET AL.: "Potentiation of cationic liposome-mediated gene delivery by polycations", BIOCHEMISTRY, vol. 35, 1996, pages 1027 - 1036 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2415375A (en) * 2004-05-25 2005-12-28 Coletica Hydrated lamellar phases or liposomes containing a fatty monoamine or cationic polymer for intracellular penetration
GB2415375B (en) * 2004-05-25 2007-01-31 Coletica Liposomes containing a fatty monoamine or cationic polymer which promote intracellular penetration and a method of screening such substances
US9655822B2 (en) 2004-05-25 2017-05-23 Basf Beauty Care Solutions France S.A.S. Hydrated lamellar phases or liposomes which contain a fatty monoamine or a cationic polymer which promotes intracellular penetration, and a cosmetic or pharmaceutical composition containing same, as well as a method of screening such a substance
CN105295055A (zh) * 2015-11-24 2016-02-03 南开大学 一种基于超分子自组装的磁转染试剂

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