EP2391389A1 - Vecteur de transfection non viral - Google Patents

Vecteur de transfection non viral

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Publication number
EP2391389A1
EP2391389A1 EP10709985A EP10709985A EP2391389A1 EP 2391389 A1 EP2391389 A1 EP 2391389A1 EP 10709985 A EP10709985 A EP 10709985A EP 10709985 A EP10709985 A EP 10709985A EP 2391389 A1 EP2391389 A1 EP 2391389A1
Authority
EP
European Patent Office
Prior art keywords
polymer
nucleic acid
transfection agent
viral transfection
agent according
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP10709985A
Other languages
German (de)
English (en)
Inventor
Joachim H. Wendorff
Achim Aigner
Roland Dersch
Sabrina HÖBEL
Markus Rudisile
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Philipps Universitaet Marburg
Original Assignee
Philipps Universitaet Marburg
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Filing date
Publication date
Application filed by Philipps Universitaet Marburg filed Critical Philipps Universitaet Marburg
Publication of EP2391389A1 publication Critical patent/EP2391389A1/fr
Withdrawn legal-status Critical Current

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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
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the invention relates to a non-viral transfection agent according to claim 1, a method for its preparation according to claim 17, the use according to claims 20 and 21, and a medicament containing a non-viral transfection agent and its use according to claims 22 and 23.
  • Transfection agents are well known and, in particular with regard to the use of nucleic acid molecules in the field of gene therapy of increasing importance. Their task is to transport nucleic acid molecules based on DNA (for example expression plasmids), increasingly but also short-chain nucleic acid molecules based on RNA (for example siRNAs) into the cells of a culture or a target tissue to be treated.
  • DNA for example expression plasmids
  • RNA for example siRNAs
  • virus-based systems For stable but also transient transfection of eukaryotic cells with DNA plasmids usually physical or chemical methods or virus-based systems are used, for example. Modified adeno- or retroviruses. While most physical or chemical methods - especially in mammalian cells - are often less effective and difficult to apply to the entire organism, the virus-based systems usually have quite a high efficiency. However, these also have crucial disadvantages. On the one hand, they are very time-consuming to prepare and highly sensitive to handling. On the other hand, they pose not insignificant risks, especially with regard to imminent defense reactions of the immune system or oncogenic diseases.
  • WO 96/02655 A1 describes polycation / DNA complexes for the in vivo transfer of DNA. However, these show in comparison to viral transfection a drastically reduced transfection efficiency. This is usually associated with nonspecific interactions of the polycation / DNA complexes - for example with blood components - and aggregate formations.
  • the polycation / DNA complexes have been further developed in WO 98/59064 A1 by modifying the cationic polymers, especially polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • Another problem is the storage of the complexes. Although the complexes can be stored in the solution for a limited period of time. However, there is once again the risk of aggregation and, as a result, an enormous loss of transfection efficiency. In addition, storage in lyophilized or frozen form is possible in a few cases. However, this is associated with further not inconsiderable preparation effort. In addition, it is not absolutely certain that the preparation has a comparable bio-activity when reconstituted or thawed as the freshly prepared preparation.
  • RNA in particular of ribozymes
  • PEI polycation / RNA complexes
  • it should make it possible to protect the nucleic acids against degradation and their efficient introduction into tissue and cells, as well as improved loco-regional and, above all, temporal control of the release of the nucleic acids.
  • the transfection should be storable in a simple way and with the simplest possible means and inexpensive to produce.
  • the invention provides a non-viral transfection agent consisting of polymer / nucleic acid complexes and nanofibers, wherein the polymer / nucleic acid complexes consist of at least one nucleic acid and at least one cationic polymer.
  • the invention provides a method for producing a non-viral transfection agent comprising the steps
  • a significant advantage of the non-viral transfection agent of the invention is that it contains nanofibers which support the polymer / nucleic acid complexes.
  • the transfection agent can be stored without problems.
  • the dry nanofibers with the polymer / nucleic acid complexes can be stored at 4 ° C for several weeks without any loss of bioactivity.
  • Another advantage of the invention is that the complexes are released from the nanofibers in a protracted manner. This significantly improves the loco-regional and temporal control of the uptake of the complexes into the target cells.
  • the transfection agent according to the invention is distinguished by a very simple handling, thanks to the support of the polymer / nucleic acid complexes by the nanofibers. It is advantageous if the non-viral transfection agent is produced by electrospinning. In this case, the polymer / nucleic acid complexes can be spun together with a carrier polymer. The resulting nanofibers are collected, for example, on a glass slide and can be removed, for example, with a pair of tweezers as a braid and further processed. Thus, the mesh in a cell culture dish or directly in or on a tissue - for example, in the area of a diseased organ - are inserted. It is also conceivable z. B.
  • the further processing into oral, anal, subcutaneous, intramuscular, intraperitoneal or inhalation administrable formulations is particularly advantageous that the polymer / nucleic acid complexes can be released directly from nanofibers on the spot, which reduces the risk of the unwanted (and harmful) co-treatment of unaffected organs and body parts compared to an application with the help of a simple solution significantly reduced.
  • Another advantage is that the concentration of the polymer / nucleic acid complexes in the nanofibers can be easily adjusted by appropriate mixing of the spinning solution. In this way, a very exact dosage of the polymer / nucleic acid complexes is possible. It is particularly advantageous that the nanofibers, which support the complexes, are in solid form and therefore, for example, are readily weighable. It is also advantageous that the integrity of the complexes and the release of the nucleic acid from the complexes are surprisingly not affected by prior electrospinning.
  • the cationic polymer is a polyimine, preferably polyethyleneimine (PEI) or polypropyleneimine (PPI), particularly preferably polyethyleneimine (PEI).
  • PEI polyethyleneimine
  • PPI polypropyleneimine
  • the polyethyleneimine (PEI) may be a linear or branched polyethylenimine (PEI) and / or a modified polyethylenimine (PEI).
  • the cationic polymer may in particular also be modified with one or more hydrophilic polymers coupled thereto.
  • hydrophilic polymers are, for example but not exhaustively, selected from the group of polyethylene glycols (PEG), polyvinylpyrollidones, polyacrylamides, polyvinyl alcohols or copolymers thereof.
  • PEG polyethylene glycols
  • polyvinylpyrollidones polyvinylpyrollidones
  • polyacrylamides polyacrylamides
  • polyvinyl alcohols or copolymers thereof e.g., polyacrylamides, polyvinyl alcohols or copolymers thereof.
  • the hydrophilic polymer is PEG.
  • the cationic polymer is coupled with one or more carbohydrates.
  • the cationic polymer is coupled to a receptor-specific ligand, preferably with transferrin or EGF.
  • ligands bind to receptors that are specifically expressed by the targeted cells, such as the EGF receptor.
  • EGF receptor transferrin
  • other ligands that bind to other receptors or antibodies that recognize surface structures of cells and bind to them are also conceivable.
  • the cationic polymer can be attached to the Polymer / nucleic acid complexes coupled ligands - depending on the nature of the ligand - then perform different functions, such as binding to target cell structures on the outside of the cell membrane, increasing the uptake efficiency of nanoscale particles into the cells and / or the increased release of the Polymer / nucleic acid complexes from intracellular organelles.
  • the respective optimum for the size and type of the polymer is dependent on the nucleic acid to be transported. However, it is altogether advantageous if the molecular weight of the cationic polymer is between 0.6 kD and 2000 kD, preferably between 0.6 kD and 800 kD, particularly preferably between 4 kD and 25 kD. In this area, complexes of nucleic acid and polymer are obtained which are optimal in terms of complexing of the nucleic acid, biological activity / transfection efficiency and toxicity. The optimal complexation of the nucleic acid is particularly important if the polymer / nucleic acid complexes are to be processed by electrospinning.
  • the complexation Since the complexation is based on electrostatic interactions, it must have a certain stability in order not to be destroyed in the electrical field of the spinning apparatus or to prematurely disintegrate in the aqueous medium outside the target cells. At the same time, however, it must not be too stable, otherwise it would be difficult to resolve it again in the target cells. As a result, the nucleic acids could not be effective.
  • the strength of the complex binding affects the size of the polymer / nucleic acid complex. In this case, the larger the polymer, the smaller the complex, because then the DNA assumes more compact structures.
  • the size of the complex is, inter alia, important for transfection efficiency.
  • the polymer / nucleic acid complexes have a size between 20 nm and about 800 nm, more preferably between 50 nm and 500 nm.
  • the nucleic acid in the non-viral transfection agent, may be a DNA or an RNA or a DNA or RNA derivative. It is advantageous if the nucleic acid is a therapeutically active nucleic acid.
  • the nucleic acid is a DNA plasmid.
  • This may for example comprise 4,000 to 10,000 bp and one to be expressed Include gene sequence which is to be introduced by means of the transfection system in cell cultures.
  • DNAs which comprise 500 to 25,000 bp, for example, smaller helper or marker expressing plasmids or particularly large plasmids for the stable introduction of a particular gene including control or exon / intron structures in the target cells, for example in the generation of transgenic organisms or as part of a gene therapy.
  • antisense DNA or decoy DNA can also be used as nucleic acids in the polymer / nucleic acid complex. These usually have less than 500 bp, preferably less than 100 bp.
  • siRNAs longer double-stranded RNA molecules, preferably 27-29mers, siLNAs, sisLNAs and siRNA molecules with other chemical modifications for increasing the stability, the selectivity and / or come in particular (but by no means exhaustive) as therapeutically effective nucleic acids efficiency in question.
  • Ribozymes are also conceivable, preferably hamsterhead ribozymes, but also ribozymes with a hairpin motif or HDV ribozymes.
  • messenger RNAs (mRNA) as well as transfer RNAs (tRNA) can be contained as nucleic acids in the transfection agent and thus be brought into the desired cells.
  • the nanofibers consist of biodegradable, biocompatible polymers, preferably selected from the group polylactides, polyvinyl alcohols, polyethylene glycols, polycaprylactone, polyhydroxybutyrates, polyester urethanes, cellulose, cellulose acetate, chitosan, alginates, collagen or copolymers and blends thereof.
  • the composition of the polymers determine the solubility properties of the nanofibers. It is advantageous if the fibers are water-soluble.
  • the solubility properties of the nanofibers are of importance in particular with regard to a protracted release of the polymer / nucleic acid complexes. Depending on how fast the nanofibers dissolve, the complexes are also released quickly or slowly and thus dosed. Thus, the release kinetics of the polymer / nucleic acid complexes can be controlled by the choice of nanofiber polymers.
  • This release kinetics can additionally be influenced if the entire transfection agent, ie the nanofibers including the polymer / nucleic acid complexes are provided with an additional coating of a biodegradable polymer, preferably with a polymer selected from the group of polyethylene glycols (PEG), polyethyleneimines (PEI), poly (diallyldimethylammonium chloride) (PDDA), polylactides (PLA), poly-p-xylylene (PPX), copolymers and blends thereof or lipids.
  • This coating can have a thickness of, for example, 10 nm up to many micrometers.
  • the size of the nanofibers also plays a role in the amount of total released polymer / nucleic acid complexes.
  • These may for example have a diameter in the range of 1 nm to 100 microns, preferably between 10 nm and 10 microns, more preferably between 50 nm and 3 microns.
  • the length of the fibers may be in the range of between about 2 ⁇ m and 100 ⁇ m, other dimensions e.g. up to the centimeter range and beyond are application-dependent conceivable.
  • a particular advantage of the method for producing the non-viral transfection agent according to the invention is that the polymer / nucleic acid complexes are electrospun together with the carrier polymers to give the nanofibers. Consequently, only the provision of the polymer / nucleic acid complexes and the provision of a spinning solution for preparation are necessary.
  • the provision of the polymer / nucleic acid complex comprises the steps of preparing a nucleic acid solution
  • Example 1 shows an exemplary protocol for the preparation of PEI-F25LMW polymer / nucleic acid complexes.
  • the provision of the spinning solution is not particularly complicated. It comprises the steps according to the invention:
  • the first step namely the provision of the solution containing polymer / nucleic acid complexes
  • the first step namely the provision of the solution containing polymer / nucleic acid complexes
  • the second step namely the provision of the solution containing polymer / nucleic acid complexes
  • a suitable medium eg. In 'EPES buffer' (10 mM to 1 M HEPES, 150 mM NaCl, pH 7.4) takes place.
  • the electrospinning takes place.
  • the non-viral transfection agent is not only easy to store, but can also be produced inexpensively by simple means and in proportion.
  • the nucleic acids are further protected against degradation.
  • the loco-regional and temporal control of the release of the nucleic acids is made possible in particular by the support by the nanofibers.
  • the simple handling of the solid-like transfection agent is also advantageous.
  • the non-viral transfection agent according to the invention can be used for the transfection of eukaryotic cells. Both applications in the cell culture laboratory and directly in the organi- mus conceivable. In view of the latter, the non-viral transfection agent can be used for the preparation of a medicine.
  • a medicament containing a non-viral transfection agent according to the invention is advantageously distinguished by the simple handling and easily controllable loco-regional delivery of therapeutic nucleic acids. Even a protracted release is good and easy to control.
  • the use of such a medicament for gene therapy treatment is appropriate.
  • the non-viral transfection agent according to the invention makes a variety of formulations and administration forms problem-free, the drug can be specifically tailored to the corresponding individual treatment.
  • FIG. 3a knockdown efficiency (PEO solution), corresponding to example 6a
  • FIG. 3a knockdown efficiency (PEO solution) corresponding to example 6a
  • FIG. 3b Knockdown efficiency (PEO / dichloromethane solution), corresponding to Example 6b, FIG.
  • PEI F25-LMW 22 ⁇ l of PEI F25-LMW (0.6 ⁇ g / ⁇ l) are dissolved in 80 ⁇ l of HEPES buffer (1 mM to 1 M HEPES, 15 mM NaCl, pH 7.4 Solution pipetted.
  • the complexation is carried out by incubation for up to 1 hour at room temperature.
  • the complexes can then be aliquoted and frozen.
  • the complexes then only have to be thawed.
  • the complexes are mixed by brief vortexing before use and incubated again for 30-60 minutes.
  • nucleic acid to be complexed all amounts and volume information as well as the mixing ratios of the polymer solution and the nucleic acid solution can be adapted to the respective requirements.
  • the total volume of the complex solution prepared is to be understood only as an example and can of course also in Midi or Maxitial. Industrial scale to be produced.
  • PEI F25-LMW / DNA complexes For the production of a polymer / nucleic acid complex for lyophilization, mention may be made, by way of example, of the known preparation of PEI F25-LMW / DNA complexes. For this purpose, 260 ⁇ g of DNA are dissolved in 1040 ⁇ l of 5% glucose in water and incubated for 10 minutes. 213 ⁇ l of PEI F25-LMW (6.1 ⁇ g / ⁇ l) are dissolved in 1040 ⁇ l of 5% glucose in water and pipetted after 10 minutes to the DNA solution. After a short vortex, incubate for 1 h and vortex briefly again.
  • the spinning solution between 0.2 and 20% by weight of the polymer to be spun are dissolved in a solvent.
  • the solvents used are preferably water, but also organic solvents, e.g. Dichloromethane or hexafluoroisopropanol in question. The selection depends on the carrier polymer chosen.
  • 165 ⁇ g of the PEI / siRNA complexes (with a luciferase-specific or with a nonspecific siRNA) dissolved in 100 ⁇ l HEPES buffer (10 mM HEPES, 150 mM NaCl) were thawed and added to 200 ⁇ l of a 6 Wt .-% PEO / water solution given. After mixing in a circular The spinning solution was spun (2500 rpm) at a voltage of about 17 kV and a distance of 14 cm on glass slides as carrier matrix. The flow rate was 0.2 mL / h.
  • Carrier plate removed or rinsed with liquid and added to the medium.
  • the glass slides with the applied nanofibers can also be placed inverted in the well on the medium.
  • the measurement of luciferase activity is typically between 48 h and 96 h after introduction of the
  • Nanofibers via chemiluminescence in the luminometer Nanofibers via chemiluminescence in the luminometer.
  • a further advantage of the transfection agent according to the invention is the improved shelf life. This makes it conceivable, for example, to process the polymer / nucleic acid complex-containing nanofibers in a form of administration such as, for example, tablets, which makes it possible to administer the transfection agent as a therapeutic agent to a patient as simply as possible.
  • the nano-fibers with spun nanoplexes are stored dry at 4 ° C. or at RT for a relatively long period of time, typically several weeks. The measurement of the transfection efficiency then takes place as in Example 4.
  • the polymer / nucleic acid complexes spun into nanofibers according to the invention can only detect a decrease of slightly more than one tens of thousands ,
  • Examples 6a to 6d the results of which are shown in FIGS. 3a to 3d, moreover show that with the aid of the transfection agent according to the invention a targeted knockdown of genes by the introduction of siRNA is possible.
  • different spinning solutions are suitable (examples 6a to 6d, 3a to 3d)
  • the obtained nanofibers can additionally be provided with a polymer coating (compare example 6d, 3d).
  • PEI / siRNA complexes are prepared as described in Example 1. These are then spun according to Example 3. The transfection agent thus obtained is used for the following experiment:
  • luciferase-expressing SKOV-3 / Luc ovarian carcinoma cells / well are seeded in a 24-well plate and cultured for 24 h in IMDM / 10% FCS medium. Subsequently, e.g. between 1 .mu.g and 10 .mu.g nanofibers with supported PEI / luciferase siRNA complexes with tweezers removed from the glass support plate or rinsed with liquid and added to the medium.
  • These may be, for example, PEO nanofibers with 0.63 ⁇ g of PEI-complexed luciferase siRNA or a nonspecific control siRNA.
  • the luciferase activity is typically measured between 48 h and 96 h after introduction of the nanofibers via chemiluminescence in the lumino-meter.
  • siRNA complexes treated cells versus the luciferase activity of nanofiber-supported PEI / unspecific. siRNA complexes treated cells.
  • FIG. 3 a shows the chemiluminescence of the cells treated with specific siRNA decreases markedly in comparison with the cells treated with unspecific siRNA.
  • column 9 shows the luciferase expression-induced chemiluminescence of the cultured cells which were treated with the control siRNA.
  • Column 10 shows the chemiluminescence diminished by treatment with specific luciferase siRNA. Treatment with the control siRNA excludes nonspecific, eg cytotoxic, effects as the cause of gene knockdown.
  • Example 6b Knockdown efficiency of PEI / siRNA complexes electrospun in PEO / dichloromethane solution
  • PEI / siRNA complexes are prepared as described in Example 1.
  • the knockdown efficiency was determined according to Example 6a.
  • Example 6a or Fig. 3a it can be seen here that the specific Luciferase siRNA, in contrast to the non-specific control RNA causes a knockdown of luciferase expression.
  • column 1 1 shows the luminescence of the cultured cells caused by the luciferase expression, which were treated with the control siRNA.
  • Column 12 shows luminescence diminished by treatment with specific luciferase siRNA.
  • PEI / siRNA complexes are prepared as described in Example 1. These are further processed by electrospinning as follows.
  • 165 ⁇ g of the PEI / siRNA complexes (of both the luciferase-specific and the non-specific siRNA) dissolved in 100 ⁇ L of 'HEPES buffer' (10 mM HEPES and 150 mM NaCl) were thawed and added to 200 ⁇ L of a 12% by weight PVA / water solution. After mixing in a rotary shaker (2500 rpm), the spinning solution at a voltage of about 21 kV and Spaced a distance of 14 cm on glass slides as a carrier matrix. The flow rate was 0.15 mL / h.
  • the knockdown efficiency was determined according to Example 6a.
  • Example 6d Knockdown efficiency after four and seven days of PEI / siRNA complexes electrospun in PEO / water solution, coating the fibers with PPX
  • PEI / siRNA complexes are prepared as described in Example 1. These are as described in Example 3 and electrospun.
  • the coating agent Labcoater 1 PDS 2010 from the company SPECIALY COATING SYSTEMS was used for chemical vapor deposition (CVD).
  • the fiber mats were fixed to a bent metal bar which was attached to the bottom of the coating apparatus.
  • the knockdown efficiency was determined according to Example 6a.
  • column 15 shows the luminescence of the cultured cells caused by the luciferase expression, which is treated with the control siRNA, four days after the transfection.
  • Column 16 shows luminescence diminished by treatment with specific luciferase siRNA over the same time period.
  • the invention is not limited to one of the above-described embodiments, but can be modified in many ways. For example, it is conceivable to couple ligands to the nanofibers after spinning.
  • the solubility of the polymer / nucleic acid complexes can be influenced by the additional application of an outer shell, for example of lipids.
  • a non-viral transfection agent advantageously consists of polymer / nucleic acid complexes and nanofibers, wherein the polymer / nucleic acid complexes consist of at least one nucleic acid and at least one cationic polymer.
  • the nanofibers carry the polymer / nucleic acid complexes, the non-viral transfection agent being conveniently made by electrospinning.
  • the cationic polymer is desirably a polyimine, preferably polyethylenimine, and may be modified with one or more hydrophilic polymers coupled thereto. It may also be expedient to couple the cationic polymer with one or more carbohydrates and / or with a receptor-specific ligand.
  • the nucleic acid is a DNA or an RNA or a DNA or RNA Dehvat, advantageously a therapeutically active nucleic acid.
  • the nanofibers are made of biodegradable, biocompatible polymers. They or the entire transfection agent can be provided with a polymer coating.
  • a method for producing a non-viral transfection agent comprises the following steps: provision of the polymer / nucleic acid complex, preparation of a spinning solution containing the polymer / nucleic acid complexes, electrospinning.
  • the non-viral transfection agent can be used to transfect eukaryotic cells and to produce a drug.
  • the medicine can be used to advantage for gene therapy treatments.
  • the present invention therefore relates to a non-viral transfection agent consisting of polymer / nucleic acid complexes and nanofibers, wherein the polymer / nucleic acid complexes consist of at least one nucleic acid and at least one cationic polymer.
  • the nanofibers carry the polymer / nucleic acid complexes, the non-viral transfection agent being conveniently made by electrospinning.
  • the cationic polymer is conveniently a polyimine or polyethylenimine and may be modified with one or more hydrophilic polymers coupled thereto.
  • the cationic polymer may also be expedient to couple the cationic polymer with one or more carbohydrates and / or with a receptor-specific ligand.
  • the nucleic acid is a DNA or an RNA or a DNA or RNA Dehvat, advantageously a therapeutically active nucleic acid.
  • the nanofibers are made of biodegradable, biocompatible polymers. They or the entire transfection agent can be provided with a polymer coating.
  • a method for producing a non-viral transfection agent comprises the following steps: provision of the polymer / Nucleic acid complex, preparation of a spinning solution containing the polymer / nucleic acid complexes, electrospinning.
  • non-viral transfection agent can be used to transfect eukaryotic cells and to produce a drug.
  • the medicine can be used to advantage for gene therapy treatments.

Abstract

L'invention concerne un vecteur de transfection non viral constitué de complexes polymère(s)/acide(s) nucléique(s) et de nanofibres, les complexes comprenant au moins un acide nucléique et au moins un polymère cationique. Les nanofibres portent les complexes polymère(s)/acide(s) nucléique(s), le vecteur de transfection non viral étant produit de façon avantageuse par électrofilage couplés à cette dernière. Le polymère cationique est de préférence une polyimine ou une polyéthylènimine et peut être modifié à l'aide d'un ou de plusieurs polymères hydrophyles couplés à cette dernière. Avantageusement, le polymère cationique peut également se coupler à un ou à plusieurs glucides et/ou à un ligand spécifique à un récepteur. L'acide nucléique est un ADN ou un ARN ou un dérivé d'ADN ou d'ARN, avantageusement un acide nucléique thérapeutiquement efficace. Les nanofibres sont constituées de polymères biocompatibles et biodégradables. Les nanofibres ou le vecteur de transfection total peuvent présenter un enrobage polymère. Un procédé de production d'un vecteur de transfection non viral selon l'invention comprend les étapes consistant à fournir des complexes polymère(s)/acide(s) nucléique(s), à produire une solution de filage contenant lesdits complexes, et à procéder à l'électrofilage.
EP10709985A 2009-01-29 2010-01-29 Vecteur de transfection non viral Withdrawn EP2391389A1 (fr)

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* Cited by examiner, † Cited by third party
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US10034737B2 (en) 2011-02-22 2018-07-31 Institut National De La Sante Et De La Recherche Medicale (Inserm) Nano-reservoirs technology for use in bone and/or cartilage regeneration
GB201117538D0 (en) 2011-10-11 2011-11-23 Royal Veterinary College The Methods
US9533068B2 (en) 2012-05-04 2017-01-03 The Johns Hopkins University Drug loaded microfiber sutures for ophthalmic application
US9758606B2 (en) 2012-07-31 2017-09-12 The Trustees Of Columbia University In The City Of New York Cyclopropenium polymers and methods for making the same
CN103884695B (zh) * 2012-12-21 2016-07-13 武汉纺织大学 一种具有快速检测细菌功能的纳米纤维膜传感器及其制备方法
WO2014124006A1 (fr) 2013-02-05 2014-08-14 The Johns Hopkins University Nanoparticules pour le suivi de l'imagerie par résonance magnétique et procédés de fabrication et d'utilisation associés
US10335500B2 (en) * 2014-05-12 2019-07-02 The Johns Hopkins University Highly stable biodegradable gene vector platforms for overcoming biological barriers
JP6846351B2 (ja) 2015-01-27 2021-03-24 ザ・ジョンズ・ホプキンス・ユニバーシティー 粘膜表面における活性薬剤の増強された輸送のための低張ヒドロゲル製剤
JP2018515479A (ja) * 2015-05-05 2018-06-14 イッサム リサーチ ディベロップメント カンパニー オブ ザ ヘブライ ユニバーシティー オブ エルサレム リミテッドＹｉｓｓｕｍ Ｒｅｓｅａｒｃｈ Ｄｅｖｅｌｏｐｍｅｎｔ Ｃｏｍｐａｎｙ Ｏｆ Ｔｈｅ Ｈｅｂｒｅｗ Ｕｎｉｖｅｒｓｔｙ Ｏｆ Ｊｅｒｕｓａｌｅｍ Ｌｔｄ． 核酸−カチオン性ポリマー組成物、並びにその作製方法及び使用方法
KR101980819B1 (ko) * 2016-12-23 2019-05-21 주식회사 제놉시 자성 나노 입자가 탑재된 전도성 고분자 및 양이온성 고분자를 포함하는 세포-유리(cell-free) DNA 검출 및 회수용 자성 나노 구조체
CN110438162B (zh) * 2019-08-22 2021-06-25 广州晶欣生物科技有限公司 一种改良型非脂质体转染试剂盒及其应用方法
CN113622084B (zh) * 2021-08-12 2022-11-11 闽江学院 一种阳离子纳米纤维膜的制法及所得纳米纤维膜和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002100628A1 (fr) * 2001-06-12 2002-12-19 University Of Akron, The Conservation de materiaux biologiques par des techniques de formation de fibres

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2722506B1 (fr) 1994-07-13 1996-08-14 Rhone Poulenc Rorer Sa Composition contenant des acides nucleiques, preparation et utilisations
DE19726186A1 (de) 1997-06-20 1998-12-24 Boehringer Ingelheim Int Komplexe für den Transport von Nukleinsäure in höhere eukaryotische Zellen
DE19960206A1 (de) 1999-12-14 2001-07-19 Frank Czubayko Komplexierung von RNS mit Polyethyleniminen zur deren Stabilisierung und zellulären Einschleusung
WO2006110813A2 (fr) * 2005-04-12 2006-10-19 Intradigm Corporation Composition d'agents therapeutiques a arn interferent (arni) et procedes pour traiter le cancer et d'autres maladies de neovascularisation
WO2008048272A2 (fr) * 2006-10-21 2008-04-24 University Of South Florida Procédé de délivrance de médicament par des nanotube de carbone-nanocomplexes de chitosane
US20080312174A1 (en) * 2007-06-05 2008-12-18 Nitto Denko Corporation Water soluble crosslinked polymers

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002100628A1 (fr) * 2001-06-12 2002-12-19 University Of Akron, The Conservation de materiaux biologiques par des techniques de formation de fibres

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