EP0743984A1 - Nucleic acid-containing composition, its preparation and use - Google Patents

Nucleic acid-containing composition, its preparation and use

Info

Publication number
EP0743984A1
EP0743984A1 EP95907715A EP95907715A EP0743984A1 EP 0743984 A1 EP0743984 A1 EP 0743984A1 EP 95907715 A EP95907715 A EP 95907715A EP 95907715 A EP95907715 A EP 95907715A EP 0743984 A1 EP0743984 A1 EP 0743984A1
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
composition according
oligopeptide
transfer
nucleic acids
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Withdrawn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
EP95907715A
Other languages
German (de)
French (fr)
Inventor
Didier Bazile
Carole Emile
Claude Helene
Gilles Spenlehauer
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.)
Aventis Pharma SA
Original Assignee
Rhone Poulenc Rorer SA
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 Rhone Poulenc Rorer SA filed Critical Rhone Poulenc Rorer SA
Publication of EP0743984A1 publication Critical patent/EP0743984A1/en
Withdrawn legal-status Critical Current

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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • 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
    • 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
    • A61K47/6905—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 colloid or an emulsion
    • A61K47/6917—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 colloid or an emulsion the form being a lipoprotein vesicle, e.g. HDL or LDL proteins
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Definitions

  • the present invention relates to compositions based on nucleic acids, their preparation and their use. More particularly, it relates to compositions comprising nucleic acids and oligopeptides and their use in gene therapy, in particular for the transfer of nucleic acids.
  • Gene therapy consists of correcting a deficiency or an abnormality (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ.
  • This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue.
  • Various techniques have been described for the transfer of this genetic information, among which various transfection techniques involving DNA and DEAE-dextran complexes (Pagano et al., J. Virol.
  • viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques.
  • retroviruses RSV, HMS, MMS, etc.
  • the HSV virus adeno-associated viruses
  • adenoviruses adenoviruses
  • nucleic acids prevent their passage through cell membranes. If it has been shown that naked nucleic acids are capable of crossing the plasma membrane ex vivo (see in particular application No. WO90 / 11092), the transfection efficiency remains quite low. In addition, naked nucleic acids have a short plasma half-life, due to their degradation by enzymes and their elimination through the urinary tract. Furthermore, if the recombinant viruses make it possible to improve the efficiency of transfer of nucleic acids, their use presents certain risks such as pathogenicity, transmission, replication, recombination, transformation, etc.
  • the present invention provides an advantageous solution to these various problems.
  • the Applicant has indeed shown that it is possible to form ion pairs between particular cationic oligopeptides and the phosphate groups of the nucleic acids, and that the complexes thus formed are stable, and are capable of penetrating cells or be encapsulated in transfer vectors such as liposomes, nanoparticles or low density lipoproteins (LDL) with high yields.
  • transfer vectors such as liposomes, nanoparticles or low density lipoproteins (LDL) with high yields.
  • a first object of the invention therefore resides in a composition comprising a nucleic acid and a cationic oligopeptide capable of forming secondary structures.
  • the term secondary structure designates peptides capable of adopting a particular spatial conformation under physiological conditions, in contrast to peptides which do not exhibit any particular organization of their primary structure.
  • the secondary structure can appear either in certain solvents, or in aqueous solution, or after complexation with the nucleic acid.
  • oligopeptides used in the context of the invention are capable of forming ⁇ helices or ⁇ sheets.
  • complexation of a nucleic acid with a polylysine has already been described in the prior art.
  • the complexation rate and the stability of the complex formed with polylysine are relatively low, and these complexes cannot be encapsulated in transfer vectors in a satisfactory manner (see examples).
  • the complexes according to the invention which involve cationic oligopeptides capable of forming secondary structures ( ⁇ helices, ⁇ sheets) exhibit high stability, can be obtained with yields close to 100%, and are capable of being encapsulated with high yields in transfer vectors.
  • the compositions of the invention can be used on cells extracted from the body (ex vivo) for their re-administration, or directly in vivo.
  • the oligopeptides used in the context of the present invention correspond to the formula (A 0 AyAyAo) n or (AoAyA ⁇ AyJn in which A Q is a hydrophobic amino acid, Ay is a hydrophilic amino acid, and n is a number integer greater than or equal to 4.
  • a Q is a hydrophobic amino acid
  • Ay is a hydrophilic amino acid
  • n is a number integer greater than or equal to 4.
  • the hydrophobic amino acid is chosen from leucine, valine, isoleucine and phenylalanine; and the hydrophilic amino acid is chosen from lysine, arginine and histidine.
  • the capacity of the oligopeptides to form secondary structures can be verified by circular dichroism or by NMR, as indicated in the examples.
  • the oligopeptide is chosen from oligopeptides of formula (LKKL) n, (LKLK) n, (LRRL) n, in which n is defined as above.
  • n can vary between 4 and 100, preferably between 10 and 50.
  • the value of n is adapted by a person skilled in the art depending on the length and the nature nucleic acid, oligopeptide composition, intended use, etc.
  • the respective proportions of the oligopeptide and of the nucleic acid are preferably determined so that the ratio of positive charges of the oligopeptide / negative charges of nucleic acid is equal or greater than 1 (this ratio is designated R in the examples).
  • R this ratio
  • the longer the nucleic acid the higher the number of positive charges provided by the oligopeptide to obtain a maximum effect. This can result either by the use of oligopeptides in which the value of n is higher, or by the use of higher quantities of oligopeptides, or even by both.
  • the oligopeptides used in the context of the present invention can be prepared by any technique known to a person skilled in the art. Preferably, they are synthesized chemically, using a peptide synthesizer, using any type of chemistry known to those skilled in the art (F-moc, T-boc, etc.). When the values of n are high, it is also possible to synthesize the oligopeptides into several fragments which are then assembled. Furthermore, according to the synthesis technique used (for example in the homogeneous phase), the oligopeptide obtained may not be a defined compound, but a mixture of oligopeptides having different lengths centered around an average. In this case, the value of n of the formula of the invention represents the average of the values of the n of the various constituents of the mixture. Appropriate synthetic methods are given in general molecular biology techniques and in the examples.
  • nucleic acid includes both deoxyribonucleic acids and ribonucleic acids. They may be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic v . Semi-synthetic sequences. These nucleic acids ⁇ ⁇ can ⁇ be of human, animal, vegetable, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks. They can also be incorporated into vectors, such as plasmid vectors.
  • deoxyribonucleic acids With regard more particularly to deoxyribonucleic acids, they can be single or double stranded. These deoxyribonucleic acids can carry therapeutic genes, transcriptional regulatory sequences, antisense sequences, regions of binding to other cellular components, etc.
  • therapeutic gene is understood in particular any gene coding for one or more proteins (or peptide or polypeptide) having pharmacological activity. These can be enzymes, hormones, growth factors, lymphokines, apolipoproteins, etc.
  • antisense sequence is meant any sequence capable, directly or indirectly (after transcription into RNA) of reducing the expression levels of a desired protein, or even of suppressing them (EP 140 308).
  • Antisense also includes sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321,201).
  • RNA ribonucleic acids
  • they may be antisense RNAs capable of at least partially blocking the translation of target mRNAs (cellular, viral, bacterial, etc.); or also ribozymes or nucleic acids capable of binding to another nucleic acid by the formation of a triple helix.
  • compositions according to the invention can be used in vitro, ex vivo or in vivo.
  • in vitro they can make it possible to transfer to cell lines desired nucleic acid sequences, for example for the purpose of expressing a recombinant protein or an antisense activity, or for the purpose of inhibiting a protein, by fixation of said protein. protein on nucleic acid.
  • ex vivo they can be used for the therapeutic transfer of a nucleic acid into a cell originating from an organism, with a view to imparting to said cell new or enhanced properties, before its re-administration to an organism.
  • they can be used for direct administration of nucleic acid.
  • Another object of the invention therefore lies in the use of a cationic oligopeptide capable of forming secondary structures for the transfer of nucleic acids into cells. As indicated above, this transfer can be carried out in vitro, ex vivo or in vivo.
  • compositions according to the invention can make it possible to transfer nucleic acids into various types of cells.
  • these are animal cells, preferably human. They may in particular be hematopoietic, endothelial, myoblastic cells, etc. Furthermore, these can be both healthy cells and cells affected by dysfunctions (tumor, viral infection, etc.).
  • the invention also relates to a method for transferring a nucleic acid into a cell, characterized in that said cell is cultured in the presence of the nucleic acid and a cationic oligopeptide capable of forming secondary structures.
  • nucleic acid- oligopepti.de complexes of the present invention also allow the encapsulation of nucleic acids in transfer vectors with considerably improved yields.
  • nucleic acids can in fact be combined with appropriate carriers or drug vectors.
  • the encapsulation of nucleic acids in such transfer vectors makes it possible to protect them from serum nucleases, to facilitate their penetration into the cells where their pharmacological target is found, and to slow down their elimination.
  • the major difficulty limiting the use of these vectors lies in the low yields of nucleic acid encapsulation.
  • nucleic acid-oligopeptide complexes of the invention can be encapsulated in transfer vectors with high yields. More particularly, the encapsulation yields of the complexes of the invention in nanoparticles are greater than 50%, while they are less than 1% with naked nucleic acids, or with other oligopeptides which do not form secondary structures. (See examples).
  • Another object of the invention therefore lies in the use of a cationic oligopeptide capable of forming secondary structures for the encapsulation of nucleic acids in a transfer vector.
  • a subject of the invention is also the nucleic acid transfer vectors comprising a composition as defined above.
  • vectors which are biocompatible, biodegradable, hydrophobic and of protein or polymeric nature.
  • preferred vectors according to the invention are liposomes, nanoparticles or low density lipoproteins (LDL).
  • Liposomes are phospholipid vesicles with an internal aqueous phase in which nucleic acids can be encapsulated.
  • the synthesis of liposomes and their use for the transfer of nucleic acids is known in the prior art (WO91 / 06309, WO92 / 19752, WO92 / 19730).
  • the use of complexes according to the invention makes it possible to improve the efficiency of encapsulation of nucleic acids in liposomes.
  • Nanoparticles are small particles, generally less than 500 nm, capable of transporting or vectorizing an active ingredient (such as a nucleic acid) in cells or in the bloodstream.
  • the present invention also makes it possible to considerably improve the yields of encapsulation of nucleic acids in nanoparticles.
  • the nanoparticles according to the invention consist of polymers comprising a majority of degradable units such as polylactic acid, optionally copolymerized with polyethylene glycol.
  • Other polymers usable in the nanoparticles have been described in the prior art (see for example EP 275 796; EP 520 889).
  • the invention therefore also relates to a method of encapsulating nucleic acids in transfer vectors according to which the transfer vector or the constituent components, the nucleic acid and a cationic oligopeptide capable of forming secondary structures are brought into contact. , or optionally a nucleic acid - oligopeptide complex already formed, under conditions allowing the encapsulation of the nucleic acid in said transfer vector, then the transfer vector formed is recovered.
  • the method according to the invention is preferably applied for the preparation of liposomes, nanoparticles or low density lipoproteins.
  • the invention also relates to pharmaceutical compositions comprising a therapeutic nucleic acid and a cationic oligopeptide capable of forming secondary structures, optionally encapsulated in a transfer vector.
  • This oligopeptide was synthesized as a salt of trifluoroacetic acid using an Applied Biosystem 431A peptide synthesizer, on an HMP resin (Applied Biosystem) and according to an F-MOC strategy. After synthesis, the peptide was released from the resin by treatment for 90 minutes in the presence of a TFA / water solution 95/5 (v / v), precipitated by addition of tert-butyl methyl ether, then purified by reverse phase HPLC on a column. Cl 8 100 A (Biorad RSL). The purity of the peptide obtained is greater than 95% and its solubility in water by 50 mg ml.
  • This oligopeptide was synthesized in the form of a trifluoroacetic acid salt by following the protocol described above.
  • the purity of the peptide obtained is greater than 90% and its solubility in water of 100 mg / ml. It has been shown that the polytetrapeptide LKLK, not structured in water, adopts a conformation in ⁇ sheets in saline solution or in the presence of nucleic acids, due to the interaction between phosphates and amino groups. Along a ⁇ sheet, the distance separating 2 positive charges is 6.9 A, which is compatible with the 6.2 A separating 2 phosphate groups from a single-stranded nucleic acid. This conformation should therefore improve the stability of the complex.
  • This peptide (supplied by A. Brack, Center for Molecular Biophysics, La) is not structured in saline solution and was used as a control.
  • the polylysine used is of commercial origin. This peptide is not structured in saline solution and was used as a control.
  • Anti ras anti nucleic acids were prepared. These nucleic acids are oligonucleotides of 12 and 13 residues, synthesized by the company Eurogentec (Belgium). These oligonucleotides are directed against a sequence of the mutated ras mRNA at the twelfth codon.
  • the nucleic acids used are the antisense (AS-Val), its inverse (IN V- Val: the sequence is identical but oriented in the opposite direction), as well as the antisense of normal ras mRNA (AS-Gly) and that a control nucleic acid comprising 2 unpaired nucleotides in the middle of the sequence (AS-mut2).
  • the sequence of these nucleic acids is as follows:
  • the nucleic acid (d (Tp) 15T) is composed of 16 thymidines. It is of commercial origin (Pharmacia).
  • This example illustrates the formation of complexes between the antisense nucleic acids described in Example 2 and the oligopeptides described in Example 1, under different ionic strength and concentration conditions (all the nucleic acid concentrations are expressed as phosphate). H shows that very high complexation yields can be obtained, thus testifying to the stability of the complexes.
  • the A / AO value is determined, making it possible to evaluate the fraction of free nucleic acid and, by difference, the complexed fraction.
  • a ratio R 1, the concentration of oligopeptide expressed as lysine is therefore 10 " 4 M.
  • the nucleic acid (10 ⁇ 4 M) and the oligopeptide (variable concentration) were brought into contact in a 50 mM Tris-HCl buffer pH 7.4. The solution was then centrifuged, and the absorbance at 256 nm determined in the supernatant. For each value of the R ratio tested, the A / AO value is determined as above.
  • Example 4 Study of the encapsulation in a transfer vector of the nanoparticles.
  • This example illustrates the very advantageous properties of the complexes of the invention, allowing the encapsulation of nucleic acid in transfer vectors with very high yields.
  • Nanoparticles used are diblock copolymers consisting of a poly (D, L lactic acid) linked by an ester bond to a poly (ethylene glycol): PLAp (M) -PEG (N) where M and N are the average molecular weights (in kD) of PLA and PEG, respectively, and p, the percentage of L-lactic acid.
  • the 2 types of nanoparticles used are PLA50 (30) -PEG (2) and PLA50 (30) -PEG (5). These copolymers can be synthesized by any technique known to a person skilled in the art (see for example EP 520 889).
  • the nucleic acids were treated with phage T4 polynucleotide kinase (Biolabs) in the presence of ATP. ⁇ 32p (Amersham) in kinase buffer. After 30 min of incubation at 37 ° C., the nucleic acid was separated from the unreacted ATP by deposition on a Sephadex G25 column (Quick Spin) and centrifugation.
  • This example describes the encapsulation of the INV-Vall3 nucleic acid in a PLA5Q (30) -PEG (2) nanoparticle in the presence or absence of an oligopeptide (LKKL) 10 .
  • the polymer used (PLA50 (30) -PEG (2)) was dissolved in 1 ml of acetone at a concentration of 10 g 1.
  • the organic solution obtained was then poured dropwise into 5 ml of an aqueous solution (50 mM Tris-HCl buffer, pH 7.4), under The polymer insoluble in the water / acetone mixture precipitates in the form of nanoparticles, trapping the nucleic acid-oligopeptide complex, then the acetone is removed by evaporation under partial vacuum, to a final volume of 2.5 ml.
  • the nanoparticulate suspension was filtered through a Sartorius filter with a porosity of 1.2 ⁇ m, which acts as a screen for dispersion and injectability.
  • the encapsulation yield corresponds to the percentage of nucleic acid encapsulated in the nanoparticles, relative to the total amount of nucleic acid present at the start.
  • - LKKLn Propeller ⁇ synthesized in homogeneous phase in the form of a mixture of peptides of average molecular weight of 34900.
  • - LKLKn Sheet ⁇ synthesized in homogeneous phase in the form of a mixture of peptides of average molecular weight of 7400.
  • sphingosine a positively charged membrane lipid
  • the nucleic acid (d (Tp) 15T) was mixed with the oligopeptide in solution in water.
  • sphingosine it is dissolved in a water / ethanol mixture 50% v / v.
  • concentrations used are indicated in the table below. 100 ⁇ l of a polylactic acid solution (PLA50) dissolved in l acetone at a concentration of 20 g / 1 were added to the mixture, as well as 300 ⁇ l of pure acetone (final concentration in PLA50: 5 g / 1).
  • the mixture was poured drop by drop into a 2.5% (w / v) aqueous solution of pluronic F68 in order to precipitate the polymer in the form of a turbid nanoparticulate colloidal suspension.
  • the turbidity was appreciated by the eye.
  • the diameter of the nanoparticles (175 +/- 40 nm) was measured by quasi elastic scattering " of light on a BI 90 device (Brookhaven Instrument Corporation).
  • the acetone was then evaporated in vacuo for one hour.
  • the suspension was then filtered through an AP20 millipore filter (pore diameter: 1.5 ⁇ m), previously wetted with the 2.5% pluronic F68 solution.
  • the encapsulation yield has been determined and is reported in the table below.
  • This example illustrates the capacity of the compositions of the invention to transfer nucleic acids into cells.
  • Cells used The nucleic acid transfer tests described in this example were carried out on a cell line originating from a human bladder carcinoma, designated T24 / EJ accessible to ATCC. The cells were cultured in MEM-EAGLE medium ("minimum essential medium", Biological Industries) supplemented with L-glutamine ((5 mM), streptomycin (50 u / ml), penicillin (50 u / ml), in the presence 7% fetal calf serum decomplemented 30 min at 60 ° C.
  • MEM-EAGLE medium minimum essential medium
  • L-glutamine ((5 mM)
  • streptomycin 50 u / ml
  • penicillin 50 u / ml
  • the cells (1500 / well) were seeded in 96-well microplates, in the absence or presence of nucleic acid, whether or not encapsulated in nanoparticles, in a total volume of 100 ⁇ l. Each point was made in triplicate.
  • the nucleic acid concentration used was adjusted by dilution in water or by concentration by centrifugation for 1 hour at 12,000 rpm. In this case, the suspension is centrifuged, the pellet weighed and then the volume adjusted.
  • the efficiency of the transfer was determined by measuring the inhibition of cell proliferation induced by the antisense nucleic acid, after 72 or 96 hours of culture at 37 ° C. in the presence of 5% CO 2. For this, 2 methods were used:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • General Engineering & Computer Science (AREA)
  • Nanotechnology (AREA)
  • Epidemiology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Medical Informatics (AREA)
  • Optics & Photonics (AREA)
  • Dispersion Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

Compositions including nucleic acids and cationic oligopeptides capable of forming secondary structures, and their use in therapeutics and gene therapy, in particular for transferring nucleic acids into cells, are disclosed.

Description

COMPOSITION CONTENANT DES ACIDES NUCLEIQUES- PREPARATION ET UTILISATIONS COMPOSITION CONTAINING NUCLEIC ACIDS - PREPARATION AND USES
La présente invention concerne des compositions à base d'acides nucléiques, leur préparation et leur utilisation. Plus particulièrement, elle concerne des compositions comprenant des acides nucléiques et des oligopeptides et leur utilisation en thérapie génique, notamment pour le transfert d'acides nucléiques.The present invention relates to compositions based on nucleic acids, their preparation and their use. More particularly, it relates to compositions comprising nucleic acids and oligopeptides and their use in gene therapy, in particular for the transfer of nucleic acids.
La thérapie génique consiste à corriger une déficience ou une anormalité (mutation, expression aberrante, etc) par introduction d'une information génétique dans la cellule ou l'organe affecté. Cette information génétique peut être introduite soit in vitro dans une cellule extraite de l'organe, la cellule modifiée étant alors réintroduite dans l'organisme, soit directement in vivo dans le tissu approprié. Différentes techniques ont été décrites pour le transfert de cette information génétique, parmi lesquelles des techniques diverses de transfection impliquant des complexes d'ADN et de DEAE-dextran (Pagano et al., J.Virol. 1 (1967) 891), d'ADN et de protéines nucléaires (Kaneda et al., Science 243 (1989) 375), d'ADN et de lipides (Felgner et al., PNAS 84 (1987) 7413), l'emploi de liposomes (Fraley et al., J.Biol.Chem. 255 (1980) 10431), etc. Plus récemment, l'emploi de virus comme vecteurs pour le transfert de gènes est apparu comme une alternative prometteuse à ces techniques physiques de transfection. A cet égard, différents virus ont été testés pour leur capacité à infecter certaines populations cellulaires. En particulier, les rétrovirus (RSV, HMS, MMS, etc), le virus HSV, les virus adéno-associés, et les adénovirus.Gene therapy consists of correcting a deficiency or an abnormality (mutation, aberrant expression, etc.) by introducing genetic information into the affected cell or organ. This genetic information can be introduced either in vitro into a cell extracted from the organ, the modified cell then being reintroduced into the organism, or directly in vivo into the appropriate tissue. Various techniques have been described for the transfer of this genetic information, among which various transfection techniques involving DNA and DEAE-dextran complexes (Pagano et al., J. Virol. 1 (1967) 891), DNA and nuclear proteins (Kaneda et al., Science 243 (1989) 375), DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), etc. More recently, the use of viruses as vectors for gene transfer has emerged as a promising alternative to these physical transfection techniques. In this regard, different viruses have been tested for their ability to infect certain cell populations. In particular, retroviruses (RSV, HMS, MMS, etc.), the HSV virus, adeno-associated viruses, and adenoviruses.
Toutefois, les techniques développées jusqu'à présent ne permettent pas de résoudre de manière satisfaisante les difficultés liées au transfert de gènes dans les cellules et/ou l'organisme. En particulier, les problèmes liés à la pénétration de l'acide nucléique dans les cellules ne sont pas entièrement résolus. En effet, la nature polyanionique des acides nucléiques prévient leur passage à travers les membranes cellulaires. S'il a été montré que les acides nucléiques nus sont capables de traverser la membrane plasmique ex vivo (voir notamment la demande n° WO90/11092), l'efficacité de transfection reste assez faible. De plus, les acides nucléique nus ont une demi-vie plasmatique courte, en raison de leur dégradation par les enzymes et de leur élimination par les voies urinaires. Par ailleurs, si les virus recombinants permettent d'améliorer l'efficacité de transfert des acides nucléiques, leur emploi présente certains risques tels que la pathogénicité, la transmission, la réplication, la recombinaison, la transformation, etc.However, the techniques developed so far do not allow a satisfactory resolution of the difficulties linked to the transfer of genes into cells and / or the organism. In particular, the problems linked to the penetration of nucleic acid into cells have not been entirely resolved. Indeed, the polyanionic nature of nucleic acids prevents their passage through cell membranes. If it has been shown that naked nucleic acids are capable of crossing the plasma membrane ex vivo (see in particular application No. WO90 / 11092), the transfection efficiency remains quite low. In addition, naked nucleic acids have a short plasma half-life, due to their degradation by enzymes and their elimination through the urinary tract. Furthermore, if the recombinant viruses make it possible to improve the efficiency of transfer of nucleic acids, their use presents certain risks such as pathogenicity, transmission, replication, recombination, transformation, etc.
La présente invention apporte une solution avantageuse à ces différents problèmes. La demanderesse a en effet montré qu'il est possible de former des paires d'ions entre des oligopeptides cationiques particuliers et les groupements phosphates des acides nucléiques, et que les complexes ainsi formés sont stables, et sont capables de pénétrer les cellules ou d'être encapsulés dans des vecteurs de transfert tels que des liposomes, des nanoparticules ou des lipoprotéines de faible densité (LDL) avec des rendements élevés.The present invention provides an advantageous solution to these various problems. The Applicant has indeed shown that it is possible to form ion pairs between particular cationic oligopeptides and the phosphate groups of the nucleic acids, and that the complexes thus formed are stable, and are capable of penetrating cells or be encapsulated in transfer vectors such as liposomes, nanoparticles or low density lipoproteins (LDL) with high yields.
Un premier objet de l'invention réside donc dans une composition comprenant un acide nucléique et un oligopeptide cationique capable de former des structures secondaires. Le terme structure secondaire désigne les peptides capables d'adopter une conformation spaciale particulière dans des conditions physiologiques, par opposition aux peptides ne présentant pas d'organisation particulière de leur structure primaire. La structure secondaire peut apparaître soit dans certains solvants, soit en solution aqueuse, soit après complexation avec l'acide nucléique.A first object of the invention therefore resides in a composition comprising a nucleic acid and a cationic oligopeptide capable of forming secondary structures. The term secondary structure designates peptides capable of adopting a particular spatial conformation under physiological conditions, in contrast to peptides which do not exhibit any particular organization of their primary structure. The secondary structure can appear either in certain solvents, or in aqueous solution, or after complexation with the nucleic acid.
Plus particulièrement, les oligopeptides utilisés dans le cadre de l'invention sont capables de former des hélices α ou des feuillets β.More particularly, the oligopeptides used in the context of the invention are capable of forming α helices or β sheets.
La complexation d'un acide nucléique avec une polylysine a déjà été décrite dans l'art antérieur. Cependant, le taux de complexation et la stabilité du complexe formé avec la polylysine sont relativement faibles, et ces complexes ne peuvent être encapsulés dans des vecteurs de transfert de façon satisfaisante (voir exemples). Au contraire, les complexes selon l'invention, qui impliquent des oligopeptides cationiques capables de former des structures secondaires (hélices α, feuillets β) présentent une stabilité élevée, peuvent être obtenus avec des rendements proches de 100%, et sont capables d'être encapsulés avec des rendements élevés dans des vecteurs de transfert.Complexation of a nucleic acid with a polylysine has already been described in the prior art. However, the complexation rate and the stability of the complex formed with polylysine are relatively low, and these complexes cannot be encapsulated in transfer vectors in a satisfactory manner (see examples). On the contrary, the complexes according to the invention, which involve cationic oligopeptides capable of forming secondary structures (α helices, β sheets) exhibit high stability, can be obtained with yields close to 100%, and are capable of being encapsulated with high yields in transfer vectors.
Ces complexes constituent donc des outils particulièrement avantageux pour le transfert d'acides nucléiques dans les cellules. Par ailleurs, selon la nature du vecteur de transfert utilisé, les compositions de l'invention peuvent être utilisées sur des cellules extraites de l'organisme (ex vivo) en vue de leur réadministration, ou directement in vivo. Plus particulièrement, les oligopeptides utilisés dans le cadre de la présente invention répondent à la formule (A0AyAyAo)n ou (AoAyAφAyJn dans laquelle AQ est un acide aminé hydrophobe, Ay est un acide aminé hydrophile, et n est un nombre entier supérieur ou égal à 4. La demanderesse a en effet montré que de tels oligopeptides sont capables, une fois complexés aux acides nucléiques, de former des structures secondaires qui stabilisent fortement lesdits complexes.These complexes therefore constitute particularly advantageous tools for the transfer of nucleic acids into cells. Furthermore, depending on the nature of the transfer vector used, the compositions of the invention can be used on cells extracted from the body (ex vivo) for their re-administration, or directly in vivo. More particularly, the oligopeptides used in the context of the present invention correspond to the formula (A 0 AyAyAo) n or (AoAyA φ AyJn in which A Q is a hydrophobic amino acid, Ay is a hydrophilic amino acid, and n is a number integer greater than or equal to 4. The Applicant has indeed shown that such oligopeptides are capable, once complexed with nucleic acids, of forming secondary structures which strongly stabilize said complexes.
Plus préférentiellement, l'acide aminé hydrophobe est choisi parmi la leucine, la valine, l'isoleucine et la phénylalanine; et l'acide aminé hydrophile est choisi parmi la lysine, l'arginine et l'histidine.More preferably, the hydrophobic amino acid is chosen from leucine, valine, isoleucine and phenylalanine; and the hydrophilic amino acid is chosen from lysine, arginine and histidine.
La capacité des oligopeptides à former des structures secondaires peut être vérifiée par dichroïsme circulaire ou en RMN, comme indiqué dans les exemples.The capacity of the oligopeptides to form secondary structures can be verified by circular dichroism or by NMR, as indicated in the examples.
Encore plus -préférentiel-ement, l'oligopeptide est choisi parmi les oligopeptides de formule (LKKL)n, (LKLK)n, (LRRL)n, dans lesquelles n est défini comme précédemment.Even more preferably, the oligopeptide is chosen from oligopeptides of formula (LKKL) n, (LKLK) n, (LRRL) n, in which n is defined as above.
D'une manière générale, dans les oligopeptides de l'invention, n peut varier entre 4 et 100, de préférence entre 10 et 50. La valeur de n est adaptée par l'homme du métier en fonction de la longueur et de la nature de l'acide nucléique, de la composition de l'oligopeptide, de l'utilisation recherchée, etc.In general, in the oligopeptides of the invention, n can vary between 4 and 100, preferably between 10 and 50. The value of n is adapted by a person skilled in the art depending on the length and the nature nucleic acid, oligopeptide composition, intended use, etc.
Pour obtenir un effet optimum des compositions de l'invention, les proportions respectives de l'oligopeptide et de l'acide nucléique sont de préférence déterminées de sorte que le rapport charges positives de l'oligopeptide / charges négatives de l'acide nucléique soit égal ou supérieur à 1 (ce rapport est désigné R dans les exemples). Ainsi, plus l'acide nucléique est long, plus le nombre de charges positives apporté par l'oligopeptide doit être élevé pour obtenir un effet maximum. Ceci peut se traduire soit par l'utilisation d'oligopeptides dans lesquels la valeur de n est plus élevée, soit par l'utilisation de quantités plus élevées d'oligopeptides, soit encore par les deux.To obtain an optimum effect of the compositions of the invention, the respective proportions of the oligopeptide and of the nucleic acid are preferably determined so that the ratio of positive charges of the oligopeptide / negative charges of nucleic acid is equal or greater than 1 (this ratio is designated R in the examples). Thus, the longer the nucleic acid, the higher the number of positive charges provided by the oligopeptide to obtain a maximum effect. This can result either by the use of oligopeptides in which the value of n is higher, or by the use of higher quantities of oligopeptides, or even by both.
Les oligopeptides utilisés dans le cadre de la présente invention peuvent être préparés par toute technique connue de l'homme du métier. Préférentiellement, ils sont synthétisés par voie chimique, au moyen d'un synthétiseur de peptides, en utilisant tout type de chimie connue de l'homme du métier (F-moc, T-boc, etc). Lorsque les valeurs de n sont élevées, il est par ailleurs possible de synthétiser les oligopeptides en plusieurs fragments qui sont ensuite assemblés. Par ailleurs, selon la technique de synthèse utilisée (par exemple en phase homogène), l'oligopeptide obtenu peut être non pas un composé défini, mais un mélange d'oligopeptides ayant des longueurs différentes centrées autour d'une moyenne. Dans ce cas, la valeur de n de la formule de l'invention représente la moyenne des valeurs des n des différents constituants du mélange. Des méthodes de synthèse appropriées sont données dans les techniques générales de biologie moléculaire et dans les exemples.The oligopeptides used in the context of the present invention can be prepared by any technique known to a person skilled in the art. Preferably, they are synthesized chemically, using a peptide synthesizer, using any type of chemistry known to those skilled in the art (F-moc, T-boc, etc.). When the values of n are high, it is also possible to synthesize the oligopeptides into several fragments which are then assembled. Furthermore, according to the synthesis technique used (for example in the homogeneous phase), the oligopeptide obtained may not be a defined compound, but a mixture of oligopeptides having different lengths centered around an average. In this case, the value of n of the formula of the invention represents the average of the values of the n of the various constituents of the mixture. Appropriate synthetic methods are given in general molecular biology techniques and in the examples.
Au sens de la présente invention, le terme acide nucléique comprend aussi bien les acides désoxyribonucléiques que les acides ribonucléiques. Il peut s'agir de séquences d'origine naturelle ou artificielle, et notamment d'ADN génomique, d'ADNc, d'ARNm, d'ARNt, d'ARNr, de séquences hybrides ou de séquences synthétiques vOU .semiηsynthétiques. Ces acides nucléiques Λ^peuvent^être d'origine humaine, animale, végétale, bactérienne, virale, etc. Ils peuvent être obtenus par toute technique connue de l'homme du métier, et notamment par criblage de banques, par synthèse chimique, ou encore par des méthodes mixtes incluant la modification chimique ou enzymatiqe de séquences obtenues par criblage de banques. Ils peuvent par ailleurs être incorporés dans des vecteurs, tels que des vecteurs plasmidiques.Within the meaning of the present invention, the term nucleic acid includes both deoxyribonucleic acids and ribonucleic acids. They may be sequences of natural or artificial origin, and in particular genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic v . Semi-synthetic sequences. These nucleic acids Λ ^ can ^ be of human, animal, vegetable, bacterial, viral, etc. origin. They can be obtained by any technique known to those skilled in the art, and in particular by screening of banks, by chemical synthesis, or also by mixed methods including chemical or enzymatic modification of sequences obtained by screening of banks. They can also be incorporated into vectors, such as plasmid vectors.
Concernant plus particulièrement les acides désoxyribonucléiques, ils peuvent être simple ou double brin. Ces acides désoxyribonucléiques peuvent porter des gènes thérapeutiques, des séquences régulatrices de la transcription, des séquences antisens, des régions de liaison à d'autres composants cellulaires, etc.With regard more particularly to deoxyribonucleic acids, they can be single or double stranded. These deoxyribonucleic acids can carry therapeutic genes, transcriptional regulatory sequences, antisense sequences, regions of binding to other cellular components, etc.
Au sens de l'invention, on entend par gène thérapeutique notamment tout gène codant pour une ou des protéines (ou peptide ou polypeptide) ayant une activité pharmacologique. Il peut s'agir d'enzymes, d'hormones, de facteurs de croissance, de lymphokines, d'apolipoprotéines, etc. Par séquence antisens, on entend toute séquence capable, directement ou indirectement (après transcription en ARN) de réduire les niveaux d'expression d'une protéine désirée, voire de les supprimer (EP 140 308). Les antisens comprennent également les séquences codant pour des ribozymes, qui sont capables de détruire sélectivement des ARN cibles (EP 321 201).Within the meaning of the invention, the term therapeutic gene is understood in particular any gene coding for one or more proteins (or peptide or polypeptide) having pharmacological activity. These can be enzymes, hormones, growth factors, lymphokines, apolipoproteins, etc. By antisense sequence is meant any sequence capable, directly or indirectly (after transcription into RNA) of reducing the expression levels of a desired protein, or even of suppressing them (EP 140 308). Antisense also includes sequences encoding ribozymes, which are capable of selectively destroying target RNAs (EP 321,201).
Concernant plus particulièrement les acides ribonucléiques (ARN), il peut s'agir d'ARN antisens, capables de bloquer au moins partiellement la traduction d'ARNm cibles (cellulaires, viraux, bactériens, etc); ou également de ribozymes ou d'acides nucléiques capables de se lier à un autre acide nucléique par formation d'une triple hélice.With regard more particularly to ribonucleic acids (RNA), they may be antisense RNAs capable of at least partially blocking the translation of target mRNAs (cellular, viral, bacterial, etc.); or also ribozymes or nucleic acids capable of binding to another nucleic acid by the formation of a triple helix.
Les compositions selon l'invention peuvent être utilisés in vitro, ex vivo ou in vivo. In vitro, elles peuvent permettre de transférer à des lignées cellulaires des séquences d'acides nucléiques désirées, par exemple dans le but d'exprimer une protéine recombinante ou une activité antisens, ou dans le but d'inhiber une protéine, par fixation de ladite protéine sur l'acide nucléique. Ex vivo, elles peuvent être utilisés pour le transfert thérapeutique d'un acide nucléique dans une cellule issue d'un organisme, en vue de conférer à ladite cellule des propriétés nouvelles eu renforcées, avant sa réadministration à un organisme. In vivo, elles peuvent être utilisés pour l'administration directe d'acide nucléique.The compositions according to the invention can be used in vitro, ex vivo or in vivo. In vitro, they can make it possible to transfer to cell lines desired nucleic acid sequences, for example for the purpose of expressing a recombinant protein or an antisense activity, or for the purpose of inhibiting a protein, by fixation of said protein. protein on nucleic acid. Ex vivo, they can be used for the therapeutic transfer of a nucleic acid into a cell originating from an organism, with a view to imparting to said cell new or enhanced properties, before its re-administration to an organism. In vivo, they can be used for direct administration of nucleic acid.
Un autre objet de l'invention réside donc dans l'utilisation d'un oligopeptide cationique capable de former des -rtructures secondaires pour le transfert d'acides nucléiques dans les cellules. Comme indiqué plus haut, ce transfert peut être effectué in vitro, ex vivo ou in vivo.Another object of the invention therefore lies in the use of a cationic oligopeptide capable of forming secondary structures for the transfer of nucleic acids into cells. As indicated above, this transfer can be carried out in vitro, ex vivo or in vivo.
Les compositions selon l'invention peuvent permettre de transférer des acides nucléiques dans des types variés de cellules. Préférentiellement, il s'agit de cellules animales, de préférence humaine. Il peut s'agir notamment de cellules hématopoiétiques, endothéliales, myoblastiques, etc. Par ailleurs, il peut s'agir aussi bien de cellules saines que de cellules affectées par des dysfonctionnements (tumeur, infection virale, etc).The compositions according to the invention can make it possible to transfer nucleic acids into various types of cells. Preferably, these are animal cells, preferably human. They may in particular be hematopoietic, endothelial, myoblastic cells, etc. Furthermore, these can be both healthy cells and cells affected by dysfunctions (tumor, viral infection, etc.).
L'invention concerne également un procédé pour le transfert d'un acide nucléique dans une cellule caractérisé en ce que l'on cultive ladite cellule en présence de l'acide nucléique et d'un oligopeptide cationique capable de former des structures secondaires.The invention also relates to a method for transferring a nucleic acid into a cell, characterized in that said cell is cultured in the presence of the nucleic acid and a cationic oligopeptide capable of forming secondary structures.
Par ailleurs, les complexes acide nucleique-oligopepti.de de la présente invention permettent également l'encapsulation des acides nucléiques dans des vecteurs de transfert avec des rendements considérablement améliorés. Pour diminuer les problèmes de stabilité et de pénétration dans les cellules, les acides nucléiques peuvent en effet été associés à des transporteurs ou à des vecteurs de médicament appropriés. L'encapsulation des acides nucléiques dans de tels vecteurs de transfert permet de les protéger des nucléases sériques, de faciliter leur pénétration dans les cellules où se trouve leur cible pharmacologique, et de ralentir leur élimination. Toutefois, la difficulté majeure limitant l'utilisation de ces vecteurs réside dans les faibles rendements d'encapsulation des acides nucléiques. La demanderesse a maintenant montré que les complexes acide nucléique-oligopeptides de l'invention peuvent être encapsulés dans des vecteurs de transfert avec des rendements élevés. Plus particulièrement, les rendements d'encapsulation des complexes de l'invention dans des nanoparticules sont supérieurs à 50%, alors qu'ils sont inférieurs à 1% avec des acides nucléiques nus, ou avec d'autres oligopeptides ne formant pas de structure secondaires (Cf exemples).Furthermore, the nucleic acid- oligopepti.de complexes of the present invention also allow the encapsulation of nucleic acids in transfer vectors with considerably improved yields. To reduce the problems of stability and penetration into cells, nucleic acids can in fact be combined with appropriate carriers or drug vectors. The encapsulation of nucleic acids in such transfer vectors makes it possible to protect them from serum nucleases, to facilitate their penetration into the cells where their pharmacological target is found, and to slow down their elimination. However, the major difficulty limiting the use of these vectors lies in the low yields of nucleic acid encapsulation. The Applicant has now shown that the nucleic acid-oligopeptide complexes of the invention can be encapsulated in transfer vectors with high yields. More particularly, the encapsulation yields of the complexes of the invention in nanoparticles are greater than 50%, while they are less than 1% with naked nucleic acids, or with other oligopeptides which do not form secondary structures. (See examples).
Un autre objet de l'invention réside donc dans l'utilisation d'un oligopeptide cationique capable de former des structures secondaires pour l'encapsulation d'acides nucléiques dans un vecteur de transfert.Another object of the invention therefore lies in the use of a cationic oligopeptide capable of forming secondary structures for the encapsulation of nucleic acids in a transfer vector.
L'invention a également pour objet les vecteurs de transfert d'acides nucléique comprenant une composition telle que définie ci-avantA subject of the invention is also the nucleic acid transfer vectors comprising a composition as defined above.
Parmi les différents vecteurs de transfert, on préfère utiliser dans le cadre de la présente invention des vecteurs biocompatibles, biodégradables, hydrophobes et de nature protéique ou polymérique. En particulier, les vecteurs préférés selon l'invention sont les liposomes, les nanoparticules ou les lipoprotéines de faible densité (LDL).Among the various transfer vectors, it is preferred to use, within the framework of the present invention, vectors which are biocompatible, biodegradable, hydrophobic and of protein or polymeric nature. In particular, the preferred vectors according to the invention are liposomes, nanoparticles or low density lipoproteins (LDL).
Les liposomes sont des vésicules phospholipidiques comportant une phase aqueuse interne dans laquelle les acides nucléiques peuvent être encapsulés. La synthèse de liposomes et leur utilisation pour le transfert d'acides nucléiques est connue dans l'art antérieur (WO91/06309, WO92/19752, WO92/19730). L'utilisation de complexes selon l'invention permet d'améliorer l'efficacité d'encapsulation des acides nucléiques dans les liposomes.Liposomes are phospholipid vesicles with an internal aqueous phase in which nucleic acids can be encapsulated. The synthesis of liposomes and their use for the transfer of nucleic acids is known in the prior art (WO91 / 06309, WO92 / 19752, WO92 / 19730). The use of complexes according to the invention makes it possible to improve the efficiency of encapsulation of nucleic acids in liposomes.
Les nanoparticules sont des particules de petite dimension, généralement inférieure à 500 nm, capables de transporter ou de vectoriser un principe actif (tel qu'un acide nucléique) dans les cellules ou dans la circulation sanguine. La présente invention permet également d'améliorer considérablement les rendements d'encapsulation d'acides nucléiques dans des nanoparticules. Préférentiellement, les nanoparticules selon l'invention sont constituées par des polymères comportant une majorité de motifs dégradables tels que l'acide polylactique, éventuellement copolymérisé à du polyéthylène glycol. D'autres polymères utilisables dans la réalisation de nanoparticules ont été décrits dans l'art antérieur (voir par exemple EP 275 796; EP 520 889).Nanoparticles are small particles, generally less than 500 nm, capable of transporting or vectorizing an active ingredient (such as a nucleic acid) in cells or in the bloodstream. The present invention also makes it possible to considerably improve the yields of encapsulation of nucleic acids in nanoparticles. Preferably, the nanoparticles according to the invention consist of polymers comprising a majority of degradable units such as polylactic acid, optionally copolymerized with polyethylene glycol. Other polymers usable in the nanoparticles have been described in the prior art (see for example EP 275 796; EP 520 889).
L'invention concerne donc également un procédé d'encapsulation d'acides nucléiques dans des vecteurs de transfert selon lequel on met en contact le vecteur de transfert ou les composants le constituant, l'acide nucléique et un oligopeptide cationique capable de former des structures secondaires, ou éventuellement un complexe acide nucléique - oligopeptide déjà formé, dans des conditions permettant l'encapsulation de l'acide nucléique dans ledit vecteur de transfert, puis on récupère le vecteur de transfert formé. Comme indiqué plus haut, le procédé selon l'invention est préférentiellement appliqué pour la préparation de liposomes, de nanoparticules ou de lipoprotéines de faible densité.The invention therefore also relates to a method of encapsulating nucleic acids in transfer vectors according to which the transfer vector or the constituent components, the nucleic acid and a cationic oligopeptide capable of forming secondary structures are brought into contact. , or optionally a nucleic acid - oligopeptide complex already formed, under conditions allowing the encapsulation of the nucleic acid in said transfer vector, then the transfer vector formed is recovered. As indicated above, the method according to the invention is preferably applied for the preparation of liposomes, nanoparticles or low density lipoproteins.
L'invention concerne également des compositions pharmaceutiques comprenant un acide nucléique thérapeutique et un oligopeptide cationique capable de former des structures secondaires, éventuellement encapsulés dans un vecteur de transfert.The invention also relates to pharmaceutical compositions comprising a therapeutic nucleic acid and a cationic oligopeptide capable of forming secondary structures, optionally encapsulated in a transfer vector.
La présente invention sera plus complètement décrite à l'aide des exemples qui suivent, qui doivent être considérés comme illustratifs et non limitatifs.The present invention will be more fully described with the aid of the following examples, which should be considered as illustrative and not limiting.
Exemple 1 : Préparation des oligopeptidesExample 1: Preparation of the oligopeptides
Les trois oligopeptides suivants ont été synthétisés :The following three oligopeptides have been synthesized:
1.1. -(H)-(Leucine-Lysine-Lysine-Leucine)iQ-(OH) ou (LKKL) L.1.1. - (H) - (Leucine-Lysine-Lysine-Leucine) iQ- (OH) or (LKKL) L.
Cet oligopeptide a été synthétisé sous forme de sel d'acide trifluoroacétique au moyen d'un synthétiseur de peptides Applied Biosystem 431A, sur une résine HMP (Applied Biosystem) et selon une stratégie F-MOC. Après la synthèse, le peptide a été libéré de la résine par traitement 90 minutes en présence d'une solution TFA/eau 95/5 (v/v), précipité par addition d'ether tertiobutylméthylique, puis purifié par HPLC phase inverse sur colonne Cl 8 100 A (Biorad RSL). La pureté du peptide obtenu est supérieure à 95 % et sa solubilité dans l'eau de 50 mg ml. Il a été montré que le polytétrapeptide LKKL, non structuré dans l'eau, adopte une conformation en hélice α en solution saline. Cette conformation devrait améliorer la stabilité du complexe par formation de paires d'ions entre les charges positives des lysines de l'oligopeptide et les phosphates ionisés à pH physiologique de l'acide nucléique. 1.2. -(H)-(Leucine-Lysine-Leucine-Lysine)|Q-(OH) ou (LKLK)|Q^ This oligopeptide was synthesized as a salt of trifluoroacetic acid using an Applied Biosystem 431A peptide synthesizer, on an HMP resin (Applied Biosystem) and according to an F-MOC strategy. After synthesis, the peptide was released from the resin by treatment for 90 minutes in the presence of a TFA / water solution 95/5 (v / v), precipitated by addition of tert-butyl methyl ether, then purified by reverse phase HPLC on a column. Cl 8 100 A (Biorad RSL). The purity of the peptide obtained is greater than 95% and its solubility in water by 50 mg ml. It has been shown that the polytetrapeptide LKKL, not structured in water, adopts an α-helical conformation in saline solution. This conformation should improve the stability of the complex by the formation of ion pairs between the positive charges of the lysines of the oligopeptide and the phosphates ionized at physiological pH of the nucleic acid. 1.2. - (H) - (Leucine-Lysine-Leucine-Lysine) | Q - (OH) or (LKLK) | Q ^
Cet oligopeptide a été synthétisé sous forme de sel d'acide trifluoroacétique en suivant le protocole décrit ci-dessus. La pureté du peptide obtenu est supérieure à 90% et sa solubilité dans l'eau de 100 mg/ml. Il a été montré que le polytétrapeptide LKLK, non structuré dans l'eau, adopte une conformation en feuillets β en solution saline ou en présence d'acides nucléiques, en raison de l'interaction entre phosphates et groupements aminés. Le long d'un feuillet β, la distance séparant 2 charges positives est de 6,9 A, ce qui est compatible avec les 6,2 A séparant 2 groupements phosphate d'un acide nucléique simple brin. Cette conformation devrait donc améliorer la stabilité du complexe.This oligopeptide was synthesized in the form of a trifluoroacetic acid salt by following the protocol described above. The purity of the peptide obtained is greater than 90% and its solubility in water of 100 mg / ml. It has been shown that the polytetrapeptide LKLK, not structured in water, adopts a conformation in β sheets in saline solution or in the presence of nucleic acids, due to the interaction between phosphates and amino groups. Along a β sheet, the distance separating 2 positive charges is 6.9 A, which is compatible with the 6.2 A separating 2 phosphate groups from a single-stranded nucleic acid. This conformation should therefore improve the stability of the complex.
Ce peptide (fourni par A. Brack, Centre de Biophysique Moléculaire, Orléans) n'est pas structuré en solution saline et a été utilisé comme contrôle.This peptide (supplied by A. Brack, Center for Molecular Biophysics, Orleans) is not structured in saline solution and was used as a control.
1.4. Polylysine.1.4. Polylysine.
La polylysine utilisée est d'origine commerciale. Ce peptide n'est pas structuré en solution saline et a été utilisé comme contrôle.The polylysine used is of commercial origin. This peptide is not structured in saline solution and was used as a control.
Exemple 2 : Acides nucléiques utilisésEXAMPLE 2 Nucleic Acids Used
2.1. Acides nucléiques antisens anti-ras Vall22.1. Vall2 anti-ras anti-sense nucleic acids
Des acides nucléiques antisens anti ras ont été préparés. Ces acides nucléiques sont des oligonucléotides de 12 et 13 résidus, synthétisés par la société Eurogentec (Belgique). Ces oligonucléotides sont dirigés contre une séquence de l'ARNm de ras muté au niveau du douzième codon. Les acides nucléiques utilisés sont l'antisens (AS- Val), son inverse (IN V- Val : la séquence est identique mais orientée dans le sens inverse), ainsi que l'antisens de l'ARNm ras normal (AS-Gly) et qu'un acide nucléique témoin comportant 2 nucléotides non appariés au milieu de la séquence (AS-mut2). La séquences de ces acides nucléiques est la suivante :Anti ras anti nucleic acids were prepared. These nucleic acids are oligonucleotides of 12 and 13 residues, synthesized by the company Eurogentec (Belgium). These oligonucleotides are directed against a sequence of the mutated ras mRNA at the twelfth codon. The nucleic acids used are the antisense (AS-Val), its inverse (IN V- Val: the sequence is identical but oriented in the opposite direction), as well as the antisense of normal ras mRNA (AS-Gly) and that a control nucleic acid comprising 2 unpaired nucleotides in the middle of the sequence (AS-mut2). The sequence of these nucleic acids is as follows:
3'-CGCGGCAGCCAC-5' (AS-Val12) 3'- GCGGCAGCCACAC-5' (AS-Val13) 3 ' -CACCGACGGCGC-5 ' (INV-Val12) 3 ' -CACACCGACGGCG- 5 ' ( INV-Val1 3 )3'-CGCGGCAGCCAC-5 '(AS-Val12) 3'- GCGGCAGCCACAC-5' (AS-Val13) 3 '-CACCGACGGCGC-5' (INV-Val12) 3 '-CACACCGACGGCG- 5' (INV-Val1 3)
3 ' -CGCGGCCGCCAC- 5 ' ' (AS-Gly1 2 )3 '-CGCGGCCGCCAC- 5'' (AS-Gly1 2)
3 ' -CGCCGGAGCCAC- 5 ' AS-mut21 2 )3 '-CGCCGGAGCCAC- 5' AS-mut21 2)
2.2. Acide nucléique (d(Tp)15T)2.2. Nucleic acid (d (Tp) 15T)
L'acide nucléique (d(Tp)15T) est composé de 16 thymidines. Il est d'origine commerciale (Pharmacia).The nucleic acid (d (Tp) 15T) is composed of 16 thymidines. It is of commercial origin (Pharmacia).
Exemple 3 : Etude de complexationExample 3: Complexation study
Cet exemple illustre la formation de complexes entre les acides nucléiques antisens décrits dans l'exemple 2 et les oligopeptides décrits dans l'exemple 1, dans différentes conditions de force ionique et de concentration (toutes les concentrations en acides nucléiques sont exprimées en phosphate). H montre que des rendements de complexation très élevés peuvent être obtenus, témoignant ainsi de la stabilité des complexes.This example illustrates the formation of complexes between the antisense nucleic acids described in Example 2 and the oligopeptides described in Example 1, under different ionic strength and concentration conditions (all the nucleic acid concentrations are expressed as phosphate). H shows that very high complexation yields can be obtained, thus testifying to the stability of the complexes.
3.3. Complexation en tampon phosphate3.3. Phosphate buffer complexation
L'acide nucléique (10~4 M exprimé en phosphate : Cp^osphate = 12 X Cacicie nucléique^ et l'oligopeptide (concentration variant entre 2.10"3, 10"4 et 2.10"^ exprimées en charges positives) ont été mis en contact dans un tampon phosphate 50 mM pH 7,4. La solution a ensuite été centrifugée, et rabsorbance (A) à 256 nm (maximum d'absorption des acides nucléiques) déterminée dans le surnageant. Pour chaque valeur du rapport R (Nombre de charges positives de l'oligopeptide / Nombre de charges négatives de l'acide nucléique) testée, la valeur A/AO est déterminée, permettant d'évaluer la fraction d'acide nucléique libre et, par différence, la fraction complexée. Pour un rapport R = 1, la concentration en oligopeptide exprimée en lysine est donc de 10"4 M.The nucleic acid (10 -4 M phosphate expressed as: Cp = 12 ^ osphate XC ac nucleic here e ^ and oligopeptide (concentration ranging from 2.10 "3, 10" 4 and 2.10 "^ expressed in positive charges) were brought into contact in a 50 mM phosphate buffer pH 7.4. The solution was then centrifuged, and absorbance (A) at 256 nm (maximum absorption of nucleic acids) determined in the supernatant. For each value of the ratio R ( Number of positive charges of the oligopeptide / Number of negative charges of nucleic acid tested), the A / AO value is determined, making it possible to evaluate the fraction of free nucleic acid and, by difference, the complexed fraction. a ratio R = 1, the concentration of oligopeptide expressed as lysine is therefore 10 " 4 M.
Les résultats obtenus montrent que la fraction de complexe précipitant AS- Vall3/LKKLιo ou INV-Vall3/LKKL!0 est de 85 % pour un rapport R = 2. Pour un rapport R = 1, la fraction de complexe précipitant AS-Vall3/LKLK10 et INV- Vall3/LKLK10 est inférieure : 3Q %. Ces résultats peuvent s'expliquer par une compétition entre les phosphates du tampon et les phosphates de l'acide nucléique pour la formation du complexe. Ces résultats sont cependant supérieurs à ceux obtenus avec l'oligopeptide contrôle : 15 % de complexe INV-Vall3/PKKL10 précipitant seulement.The results obtained show that the fraction of AS-Vall3 / LKKLιo or INV-Vall3 / LKKL! 0 precipitating complex is 85% for a ratio R = 2. For a R = 1 ratio, the fraction of AS-Vall3 / precipitating complex LKLK10 and INV-Vall3 / LKLK10 is lower: 3Q%. These results can be explained by a competition between the phosphates in the buffer and the phosphates in the nucleic acid for the formation of the complex. These results are however superior to those obtained with the control oligopeptide: 15% of INV-Vall3 / PKKL10 complex only precipitating.
3.2. Complexation en tampon Tris-HCl3.2. Complexation in Tris-HCl buffer
L'acide nucléique (10~4 M) et l'oligopeptide (concentration varriable) ont été mis en contact dans un tampon Tris-HCl 50 mM pH 7,4. La solution a ensuite été centrifugée, et l'absorbance à 256 nm déterminée dans le surnageant Pour chaque valeur du rapport R testée, la valeur A/AO est déterminée comme précédemmentThe nucleic acid (10 ~ 4 M) and the oligopeptide (variable concentration) were brought into contact in a 50 mM Tris-HCl buffer pH 7.4. The solution was then centrifuged, and the absorbance at 256 nm determined in the supernatant. For each value of the R ratio tested, the A / AO value is determined as above.
Les résultats obtenus montrent que la fraction de complexe précipitant AS-The results obtained show that the fraction of AS- precipitating complex
Vall3/LKKL10 et AS-Vall3/LKLK10 est de 100 % pour un rapport R = 1. Pour un rapport R = 2, la fraction de complexe précipitant AS-Vall3/LKL 10 est également de 100 %. Par ailleurs. 100 % de complexe précipitant AS-Vall3/LKKL10 ont également été obtenus avec une concentration d'acide nucléique de 2.10~ M et un rapport R = 1.Vall3 / LKKL10 and AS-Vall3 / LKLK10 is 100% for an R = 1 ratio. For an R = 2 ratio, the fraction of AS-Vall3 / LKL 10 precipitating complex is also 100%. Otherwise. 100% of AS-Vall3 / LKKL10 precipitating complex were also obtained with a nucleic acid concentration of 2.10 ~ M and an R = 1 ratio.
En revanche, en ce qui concerne le complexe AS-Vall3/PKKL10, seulement 70 % des acides nucléiques impliqués dans le complexe précipite ce qui démontre que l'affinité des oligopeptides selon l'invention est supérieure.On the other hand, with regard to the AS-Vall3 / PKKL10 complex, only 70% of the nucleic acids involved in the complex precipitates, which demonstrates that the affinity of the oligopeptides according to the invention is higher.
3.3. Complexation dans l'eau3.3. Complexation in water
Le protocole ci-dessus a été répété en remplaçant le tampon Tris-HCl par de l'eau. Les mêmes résultats ont été obtenus (100 % de complexation pour R = 1), démontrant l'affinité élevée des oligopeptides de l'invention pour les acides nucléiques.The above protocol was repeated, replacing the Tris-HCl buffer with water. The same results were obtained (100% complexation for R = 1), demonstrating the high affinity of the oligopeptides of the invention for nucleic acids.
3.4. Conclusions3.4. Conclusions
L'ensemble de ces résultats démontre le rendement élevé de complexation des oligopeptides de l'invention aux acides nucléiques, et le fait que ce rendement est indépendant de la séquence et de la concentration de l'acide nucléique utilisé. Par ailleurs, comme le montre le tableau ci-après, l'étude des spectres dichroïques a permis de confirmer les structures secondaires adoptées par les oligopeptides cationiques utilisés, en solution ou après complexation. Peptide Tampon Phosphate -Tampon Phosphate Eau Eau / acide 50mM 50mM / NaCl 0.2M nucléiqueAll of these results demonstrate the high yield of complexing of the oligopeptides of the invention with nucleic acids, and the fact that this yield is independent of the sequence and of the concentration of the nucleic acid used. Furthermore, as shown in the table below, the study of the dichroic spectra made it possible to confirm the secondary structures adopted by the cationic oligopeptides used, in solution or after complexation. Peptide Phosphate Buffer - Phosphate Buffer Water Water / acid 50mM 50mM / NaCl 0.2M nucleic acid
LKKL hélice α hélice α non structuré hélice αLKKL α propeller unstructured α propeller α propeller
LKLK feuillet β feuillet β non structuré feuillet βLKLK β-sheet unstructured β-sheet β-sheet
PKKL non structuré non structuré non structuré non structuréPKKL unstructured unstructured unstructured unstructured
Exemple 4 : Etude de l'encapsulation dans un vecteur de transfert les nanoparticules.Example 4: Study of the encapsulation in a transfer vector of the nanoparticles.
Cet exemple illustre les propriétés très avantageuses des complexes de l'invention, permettant l'encapsulation d'acide nucléique dans des vecteurs de transfert avec des rendements très élevés.This example illustrates the very advantageous properties of the complexes of the invention, allowing the encapsulation of nucleic acid in transfer vectors with very high yields.
4.1. Nanoparticules utilisées : Les nanoparticules utilisées dans cet exemple sont des copolymères dibloc constituées d'un poly (D,L acide lactique) lié par une liaison ester à un poly (éthylène glycol) : PLAp(M)-PEG(N) où M et N sont respectivement les masses moléculaires moyennes (en kD) du PLA et du PEG, et p, le pourcentage d'acide L-lactique. Les 2 types de nanoparticules utilisés sont du PLA50(30)-PEG(2) et du PLA50(30)-PEG(5). Ces copolymères peuvent être synthétisés par toute technique connue de l'homme du métier (voir par exemple EP 520 889).4.1. Nanoparticles used: The nanoparticles used in this example are diblock copolymers consisting of a poly (D, L lactic acid) linked by an ester bond to a poly (ethylene glycol): PLAp (M) -PEG (N) where M and N are the average molecular weights (in kD) of PLA and PEG, respectively, and p, the percentage of L-lactic acid. The 2 types of nanoparticles used are PLA50 (30) -PEG (2) and PLA50 (30) -PEG (5). These copolymers can be synthesized by any technique known to a person skilled in the art (see for example EP 520 889).
4.2. Marquage radioactif des acides nucléiques :4.2. Radioactive labeling of nucleic acids:
Les acides nucléiques ont été traités avec de la polynucléotide kinase du phage T4 (Biolabs) en présence d'ATP.γ32p (Amersham) dans un tampon kinase. Après 30 min d'incubation à 37°C, l'acide nucléique a été séparé de l'ATP n'ayant pas réagi par dépôt sur colonne Séphadex G25 (Quick Spin) et centrifugation.The nucleic acids were treated with phage T4 polynucleotide kinase (Biolabs) in the presence of ATP.γ32p (Amersham) in kinase buffer. After 30 min of incubation at 37 ° C., the nucleic acid was separated from the unreacted ATP by deposition on a Sephadex G25 column (Quick Spin) and centrifugation.
4.3. Encapsulation de l'acide nucléique INV-Vall34.3. INV-Vall3 nucleic acid encapsulation
Cet exemple décrit l'encapsulation de l'acide nucléique INV-Vall3 dans une nanoparticule de PLA5Q(30)-PEG(2) en présence ou en l'absence d'oligopeptide (LKKL)10. Le polymère utilisé (PLA50(30)-PEG(2)) a été solubilisé dans 1 ml d'acétone à une concentration de 10 g 1. La dilution isotopique de l'acide nucléique (concentration finale 10" * M) a été ajoutée, puis l'oligopeptide (à une concentration telle que R = 1). La solution organique obtenue a ensuite été versée goutte à goutte dans 5 ml d'une solution aqueuse (tampon Tris-HCl 50 mM, pH 7,4), sous agitation. Le polymère insoluble dans le mélange eau/acétone précipite sous forme de nanoparticules, emprisonnant le complexe acide nucléique-oligopeptide. L'acétone a ensuite été éliminée par évaporation sous vide partiel, jusqu'à un volume final de 2,5 ml. Enfin, la suspension nanoparticulaire a été filtrée à travers un filtre Sartorius de porosité 1,2 μm, qui tient lieu de crible de dispersion et d'injectabilité.This example describes the encapsulation of the INV-Vall3 nucleic acid in a PLA5Q (30) -PEG (2) nanoparticle in the presence or absence of an oligopeptide (LKKL) 10 . The polymer used (PLA50 (30) -PEG (2)) was dissolved in 1 ml of acetone at a concentration of 10 g 1. The isotopic dilution of the nucleic acid (final concentration 10 "* M) was added , then the oligopeptide (at a concentration such that R = 1) The organic solution obtained was then poured dropwise into 5 ml of an aqueous solution (50 mM Tris-HCl buffer, pH 7.4), under The polymer insoluble in the water / acetone mixture precipitates in the form of nanoparticles, trapping the nucleic acid-oligopeptide complex, then the acetone is removed by evaporation under partial vacuum, to a final volume of 2.5 ml. Finally, the nanoparticulate suspension was filtered through a Sartorius filter with a porosity of 1.2 μm, which acts as a screen for dispersion and injectability.
Une expérience contrôle a été effectuée dans les mêmes conditions, mais en l'absence de l'oligopeptide (LKKL)ιo-A control experiment was carried out under the same conditions, but in the absence of the oligopeptide (LKKL) ιo-
Les résultats obtenus montrent que le rendement d'encapsulation de INV-Vall3 dans la nanoparticule est de 56 % en présence de l'oligopeptide, et de 5 % seulement sans l'oligopeptide.The results obtained show that the encapsulation yield of INV-Vall3 in the nanoparticle is 56% in the presence of the oligopeptide, and only 5% without the oligopeptide.
Le rendement d'encapsulation correspond au pourcentage d'acide nucléique encapsulé dans les nanoparticules, par rapport à la quantité totale d'acide nucléique présente au départ.The encapsulation yield corresponds to the percentage of nucleic acid encapsulated in the nanoparticles, relative to the total amount of nucleic acid present at the start.
4.4. Encapsulation de l'acide nucléique (d(Tp)15T)4.4. Encapsulation of nucleic acid (d (Tp) 15T)
Le rendement d'encapsulation de l'acide nucléique (d(Tp)15T) (exemple 2.2.) dans une nanoparticule de PLA50 a été étudié, en présence des oligopeptides cationiques suivants :The encapsulation yield of nucleic acid (d (Tp) 15T) (example 2.2.) In a nanoparticle of PLA50 was studied, in the presence of the following cationic oligopeptides:
- LKKLn Hélice α : synthétisé en phase homogène sous forme d'un mélange de peptides de poids moléculaire moyen de 34900. - LKLKn Feuillet β : synthétisé en phase homogène sous forme d'un mélange de peptides de poids moléculaire moyen de 7400.- LKKLn Propeller α: synthesized in homogeneous phase in the form of a mixture of peptides of average molecular weight of 34900. - LKLKn Sheet β: synthesized in homogeneous phase in the form of a mixture of peptides of average molecular weight of 7400.
- PKKLn Pas de structure secondaire- PKKLn No secondary structure
- Kn Pas de structure secondaire- Kn No secondary structure
ou de sphingosine (lipide membranaire chargé positivement). Pour cela, l'acide nucléique (d(Tp)15T) a été mélangé à l'oligopeptide en solution dans l'eau. En ce qui concerne la "sphingosine, elle est solubilisée dans un mélange eau/éthanol 50 % v/v. Les concentrations utilisées sont indiquées dans le tableau qui suit. 100 μl d'une solution d'acide polylactique (PLA50) solubilisée dans l'acétone à une concentration de 20 g/1 ont été ajoutés au mélange, ainsi que 300 μl d'acétone pure (concentration finale en PLA50 : 5 g/1). Après homogénéisation au vortex, le mélange a été versé goutte à goutte dans une solution aqueuse de pluronic F68 à 2,5 % (p/v) afin de précipiter le polymère sous forme d'une suspension colloïdale nanoparticulaire turbide. La turbidité a été appréciée à l'oeil. Le diamètre des nanoparticules (175 +/- 40 nm) a été mesuré par diffusion quasi élastique" de la lumière sur un appareil BI 90 (Brookhaven Instrument Corporation). L'acétone- a ensuite été évaporée sous vide pendant une heure. La suspension a ensuite été filtrée sur filtre millipore AP20 (diamètre des pores : 1,5 μm), préalablement mouillé avec la solution de pluronic F68 à 2,5 %. Le rendement d'encapsulation a été déterminé et est reporté dans le tableau qui suit.or sphingosine (a positively charged membrane lipid). For this, the nucleic acid (d (Tp) 15T) was mixed with the oligopeptide in solution in water. As regards " sphingosine, it is dissolved in a water / ethanol mixture 50% v / v. The concentrations used are indicated in the table below. 100 μl of a polylactic acid solution (PLA50) dissolved in l acetone at a concentration of 20 g / 1 were added to the mixture, as well as 300 μl of pure acetone (final concentration in PLA50: 5 g / 1). After homogenization with a vortex, the mixture was poured drop by drop into a 2.5% (w / v) aqueous solution of pluronic F68 in order to precipitate the polymer in the form of a turbid nanoparticulate colloidal suspension. The turbidity was appreciated by the eye. The diameter of the nanoparticles (175 +/- 40 nm) was measured by quasi elastic scattering " of light on a BI 90 device (Brookhaven Instrument Corporation). The acetone was then evaporated in vacuo for one hour. The suspension was then filtered through an AP20 millipore filter (pore diameter: 1.5 μm), previously wetted with the 2.5% pluronic F68 solution. The encapsulation yield has been determined and is reported in the table below.
Ces résultats montrent clairement que les oligopeptides de l'invention permettent d'encapsuler les acides nucléiques avecdes rendements nettement supérieurs à ceux obtenus avec des oligopeptides de l'art antérieur (polylysine) ou ne formant pas de structures secondaires (PKKL). Exemple 5 : Transfert d'acide nucléique dans les cellulesThese results clearly show that the oligopeptides of the invention make it possible to encapsulate the nucleic acids with yields clearly higher than those obtained with oligopeptides of the prior art (polylysine) or which do not form secondary structures (PKKL). Example 5 Transfer of Nucleic Acid into Cells
Cet exemple illustre la capacité des composition de l'invention à transférer des acides nucléiques dans les cellules.This example illustrates the capacity of the compositions of the invention to transfer nucleic acids into cells.
5.1. Cellules utilisées : Les essais de transfert d'acide nucléique décrits dans cet exemple ont été effectués sur une lignée de cellules issue d'un carcinome vésical humain , désignée T24/EJ accessible à l'ATCC. Les cellules ont été cultivées dans un milieu MEM-EAGLE ("minimum essential médium", Biological Industries) supplémenté en L-glutamine ((5 mM), streptomycine (50 u/ml), pénicilline (50 u/ml), en présence de 7 % de sérum de veau foetal décomplémenté 30 min à 60°C.5.1. Cells used: The nucleic acid transfer tests described in this example were carried out on a cell line originating from a human bladder carcinoma, designated T24 / EJ accessible to ATCC. The cells were cultured in MEM-EAGLE medium ("minimum essential medium", Biological Industries) supplemented with L-glutamine ((5 mM), streptomycin (50 u / ml), penicillin (50 u / ml), in the presence 7% fetal calf serum decomplemented 30 min at 60 ° C.
5.2. Transfert des acides nucléiques : Les cellules (1500 / puits) ont été ensemencées dans des microplaques de 96 puits, en absence ou en présence d'acide nucléique, encapsulé ou non dans des nanoparticules, dans un volume total de 100 μl. Chaque point a été effectué en triple. La concentration en acide nucléique utilisée a été ajustée par dilution dans de l'eau ou par concentration par centrifugation 1 h à 12000 rpm. Dans ce cas, la suspension est centrifugée, le culot pesé puis le volume ajusté.5.2. Transfer of nucleic acids: The cells (1500 / well) were seeded in 96-well microplates, in the absence or presence of nucleic acid, whether or not encapsulated in nanoparticles, in a total volume of 100 μl. Each point was made in triplicate. The nucleic acid concentration used was adjusted by dilution in water or by concentration by centrifugation for 1 hour at 12,000 rpm. In this case, the suspension is centrifuged, the pellet weighed and then the volume adjusted.
L'efficacité du transfert a été déterminée par mesure de rinhibition de la prolifération cellulaire induite par l'acide nucléique antisens, après 72 ou 96 heures de culture à 37°C en présence de 5 % de CO2. Pour cela, 2 méthodes ont été utilisées :The efficiency of the transfer was determined by measuring the inhibition of cell proliferation induced by the antisense nucleic acid, after 72 or 96 hours of culture at 37 ° C. in the presence of 5% CO 2. For this, 2 methods were used:
- Tout d'abord, rincorporation de thymidine tritiée, qui a été déterminée après addition de 2 μCi de 3H thymidine par puits. Après 6 heures d'incubation à l'étuve, les plaques ont été lavées au Skatron (Lier, Norvège), l'ADN recueilli sur un filtre, et chaque rondelle du filtre (correspondant à chaque puits de la plaque) placée dans du liquide à scintillation puis comptée au compteur (LKB Wallac 1211 Minibeta). Les résultats correspondent à la moyenne des valeurs obtenues dans chacun des 3 puits. - Ensuite, la prolifération cellulaire a été évaluée par comptage des cellules en cellule de Malassez. Pour cela, le surnageant a été éliminé, les cellules trypsinisées (10 min à 37°C), puis additionnées de milieu de culture avant d'être comptées.- First, the incorporation of tritiated thymidine, which was determined after addition of 2 μCi of 3H thymidine per well. After 6 hours of incubation in the oven, the plates were washed with Skatron (Lier, Norway), the DNA collected on a filter, and each washer of the filter (corresponding to each well of the plate) placed in liquid scintillation then counted on the counter (LKB Wallac 1211 Minibeta). The results correspond to the average of the values obtained in each of the 3 wells. - Next, cell proliferation was assessed by counting cells in the Malassez cell. For this, the supernatant was removed, the cells trypsinized (10 min at 37 ° C), then added with culture medium before being counted.
Les résultats obtenus sont les suivants :The results obtained are as follows:
- AS-Vall2, nu, 30 μM 42 % d'inhibition - AS- Vall2/LKKL/nanoparticule, 100 nM (exprimé en antisens) .50 % d'inhibition- AS-Vall2, naked, 30 μM 42% inhibition - AS- Vall2 / LKKL / nanoparticle, 100 nM (expressed in antisense). 50% inhibition
- nanoparticule blanche, même quantité de polymère < 5 % d'inhibition - AS-Vall2/LKKL/nanoparticule, 500 nM 95 % d'inhibition- white nanoparticle, same amount of polymer <5% inhibition - AS-Vall2 / LKKL / nanoparticle, 500 nM 95% inhibition
Ces résultats démontrent clairement l'efficacité des compositions de l'invention pour le transfert d'acides nucléiques. De plus, ils montrent que l'acide nucléique transféré conserve ses propriétés fonctionnelles, dans le cas présent, son activité d'antisens. These results clearly demonstrate the effectiveness of the compositions of the invention for the transfer of nucleic acids. In addition, they show that the transferred nucleic acid retains its functional properties, in this case, its antisense activity.

Claims

REVENDICATIONS
1. Composition comprenant un acide nucléique et un oligopeptide cationique capable de former des structures secondaires.1. Composition comprising a nucleic acid and a cationic oligopeptide capable of forming secondary structures.
2. Composition selon la revendication 1 caractérisée en ce que l'oligopeptide est capable de former des hélices ou des feuillets β.2. Composition according to claim 1 characterized in that the oligopeptide is capable of forming helices or β sheets.
3. Composition selon la revendication 2 caractérisée en ce que l'oligopeptide répond à la formule (A0AyAyA0)n ou (AoAyAQAy)n dans laquelle AQ est un acide aminé hydrophobe, Ay est un acide aminé hydrophile, et n est un nombre entier supérieur ou égal à 4.3. Composition according to claim 2 characterized in that the oligopeptide corresponds to the formula (A 0 AyAyA 0 ) n or (AoAyA Q Ay) n in which A Q is a hydrophobic amino acid, Ay is a hydrophilic amino acid, and n is an integer greater than or equal to 4.
4. Composition selon la revendication 3 caractérisée en ce que l'acide aminé hydrophobe est choisi parmi la leucine, la valine, l'isoleucine et la phénylalanine.4. Composition according to claim 3 characterized in that the hydrophobic amino acid is chosen from leucine, valine, isoleucine and phenylalanine.
5. Composition selon la revendication 3 caractérisée en ce que l'acide aminé hydrophile est choisi parmi la lysine, l'arginine et lliistidine.5. Composition according to claim 3 characterized in that the hydrophilic amino acid is chosen from lysine, arginine and lliistidine.
6. Composition selon la revendication 3 caractérisée en ce que l'oligopeptide est choisi parmi (LKKL)n, (LKLK)n, (LRRL)n.6. Composition according to claim 3 characterized in that the oligopeptide is chosen from (LKKL) n, (LKLK) n, (LRRL) n.
7. Composition selon la revendication 6 caractérisée en ce que n est compris entre 4 et 100, de préférence entre 10 et 50.7. Composition according to claim 6 characterized in that n is between 4 and 100, preferably between 10 and 50.
8. Composition selon la revendication 1 caractérisée en ce que l'acide nucléique est un acide désoxyribonucléique.8. Composition according to claim 1 characterized in that the nucleic acid is a deoxyribonucleic acid.
9. Composition selon la revendication 1 caractérisée en ce que l'acide nucléique est un acide ribonucléique.9. Composition according to claim 1 characterized in that the nucleic acid is a ribonucleic acid.
10. Composition selon la revendication 8 ou 9 caractérisée en ce que l'acide nucléique est modifié chimiquement.10. Composition according to claim 8 or 9 characterized in that the nucleic acid is chemically modified.
11. Composition selon la revendication 8 ou 9 caractérisée en ce que l'acide nucléique est un antisens.11. Composition according to claim 8 or 9 characterized in that the nucleic acid is an antisense.
12. Composition selon la revendication 8 caractérisée en ce que l'acide nucléique comporte un gène thérapeutique. 12. Composition according to claim 8 characterized in that the nucleic acid comprises a therapeutic gene.
13. Composition selon l'une des revendications 8 à 12 caractérisée en ce que le rapport charges positives de Poligopeptide / charges négatives de l'acide nucléique est égal ou supérieur à 1.13. Composition according to one of claims 8 to 12 characterized in that the ratio positive charges of Poligopeptide / negative charges of the nucleic acid is equal to or greater than 1.
14. Vecteur de transfert d'acides nucléique comprenant une composition selon l'une des revendications précédentes.14. A nucleic acid transfer vector comprising a composition according to one of the preceding claims.
15. Vecteur de transfert selon la revendication 14 caractérisé en ce qu'il s'agit d'une nanoparticule, d'un liposome ou d'une lipoprotéine de faible densité.15. Transfer vector according to claim 14 characterized in that it is a nanoparticle, a liposome or a low density lipoprotein.
16. Procédé d'encapsulation d'acides nucléiques dans des vecteurs de transfert caractérisé en ce que l'on met en contact le vecteur de transfert ou ces composants, l'acide nucléique et un oligopeptide cationique capable de former des structures secondaires, ou éventuellement un complexe acide nucléique-oligopeptide déjà formé, dans des conditions permettant -'encapsulation de l'acide nucléique dans ledit vecteur de transfert, puis on récupère le vecteur de transfert formé.16. Method for encapsulating nucleic acids in transfer vectors, characterized in that the transfer vector or these components, the nucleic acid and a cationic oligopeptide capable of forming secondary structures, or optionally, are brought into contact a nucleic acid-oligopeptide complex already formed, under conditions allowing the nucleic acid to be encapsulated in said transfer vector, then the transfer vector formed is recovered.
17. Procédé pour le transfert d'un acide nucléique dans une cellule caractérisé en ce que l'on cultive ladite cellule en présence de l'acide nucléique et d'un oligopeptide cationique capable de former des structures secondaires.17. Method for the transfer of a nucleic acid into a cell characterized in that said cell is cultured in the presence of the nucleic acid and a cationic oligopeptide capable of forming secondary structures.
18. Utilisation d'un oligopeptide cationique capable de former des structures secondaires pour le transfert d'acides nucléiques dans les cellules.18. Use of a cationic oligopeptide capable of forming secondary structures for the transfer of nucleic acids into cells.
19. Utilisation selon la revendication 18 pour le transfert in vitro, ex vivo ou in vivo.19. Use according to claim 18 for transfer in vitro, ex vivo or in vivo.
20. Composition pharmaceutique comprenant un acide nucléique thérapeutique et un oligopeptide cationique capable de former des structures secondaires, éventuellement encapsulés dans un vecteur de transfert ' 20. Pharmaceutical composition comprising a therapeutic nucleic acid and a cationic oligopeptide capable of forming secondary structures, optionally encapsulated in a transfer vector.
EP95907715A 1994-02-08 1995-01-27 Nucleic acid-containing composition, its preparation and use Withdrawn EP0743984A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9401381A FR2715847B1 (en) 1994-02-08 1994-02-08 Composition containing nucleic acids, preparation and uses.
FR9401381 1994-02-08
PCT/FR1995/000098 WO1995021931A1 (en) 1994-02-08 1995-01-27 Nucleic acid-containing composition, its preparation and use

Publications (1)

Publication Number Publication Date
EP0743984A1 true EP0743984A1 (en) 1996-11-27

Family

ID=9459869

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95907715A Withdrawn EP0743984A1 (en) 1994-02-08 1995-01-27 Nucleic acid-containing composition, its preparation and use

Country Status (7)

Country Link
US (1) US5856435A (en)
EP (1) EP0743984A1 (en)
JP (1) JPH09508530A (en)
AU (1) AU1581895A (en)
CA (1) CA2182118A1 (en)
FR (1) FR2715847B1 (en)
WO (1) WO1995021931A1 (en)

Families Citing this family (131)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1116424A (en) * 1993-09-20 1996-02-07 钟渊化学工业株式会社 Process for producing 3-amino-2-hydroxy-1-propanol derivative
JP2002514892A (en) * 1995-06-08 2002-05-21 コブラ セラピューティクス リミテッド Use of synthetic virus-like particles in gene therapy
WO1997017063A1 (en) † 1995-11-09 1997-05-15 Microbiological Research Authority Microencapsulated dna for vaccination and gene therapy
US6344436B1 (en) 1996-01-08 2002-02-05 Baylor College Of Medicine Lipophilic peptides for macromolecule delivery
WO1997045069A1 (en) * 1996-05-29 1997-12-04 Cell Genesys, Inc. Cationic polymer/lipid nucleic acid delivery vehicles
US7008776B1 (en) * 1996-12-06 2006-03-07 Aventis Pharmaceuticals Inc. Compositions and methods for effecting the levels of high density lipoprotein (HDL) cholesterol and apolipoprotein AI very low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol
AP1313A (en) 1996-12-06 2004-10-04 Aventis Pharma Inc Polypeptides encoded by a human lipase-like gene, composition and method.
EP0860167A1 (en) * 1997-01-30 1998-08-26 Robert Gurny RNA-and DNA- based active agents in nanoparticles
BR9808697A (en) 1997-04-28 2000-07-11 Rhone Poulenc Rorer Sa Process to inhibit the growth of a tumor, defective adenovirus vector, virus vector, use of it, and pharmaceutical composition
US6875606B1 (en) 1997-10-23 2005-04-05 The United States Of America As Represented By The Department Of Veterans Affairs Human α-7 nicotinic receptor promoter
US6254890B1 (en) * 1997-12-12 2001-07-03 Massachusetts Institute Of Technology Sub-100nm biodegradable polymer spheres capable of transporting and releasing nucleic acids
US6958148B1 (en) * 1998-01-20 2005-10-25 Pericor Science, Inc. Linkage of agents to body tissue using microparticles and transglutaminase
KR100314721B1 (en) * 1998-01-22 2001-11-23 김일웅 Biologically active peptides
US6225456B1 (en) 1998-05-07 2001-05-01 University Technololy Corporation Ras suppressor SUR-5
US6506889B1 (en) 1998-05-19 2003-01-14 University Technology Corporation Ras suppressor SUR-8 and related compositions and methods
US6441156B1 (en) 1998-12-30 2002-08-27 The United States Of America As Represented By The Department Of Health And Human Services Calcium channel compositions and methods of use thereof
GB9918670D0 (en) 1999-08-06 1999-10-13 Celltech Therapeutics Ltd Biological product
US7070807B2 (en) 1999-12-29 2006-07-04 Mixson A James Branched histidine copolymers and methods for using same
US7163695B2 (en) 1999-12-29 2007-01-16 Mixson A James Histidine copolymer and methods for using same
JP4987205B2 (en) * 2000-03-03 2012-07-25 ジェネトロニクス， インコーポレイテッド Nucleic acid preparations for gene delivery and methods of use
EP1276500B1 (en) 2000-03-13 2011-06-08 Cornell Research Foundation, Inc. Blocking leukocyte emigration and inflammation by interfering with cd99/hec2
US20040033600A1 (en) 2001-03-21 2004-02-19 Palli Subba Reddy Ecdysone receptor-based inducible gene expression system
GB0018307D0 (en) 2000-07-26 2000-09-13 Aventis Pharm Prod Inc Polypeptides
JP2003531149A (en) 2000-04-13 2003-10-21 ザ・ロツクフエラー・ユニバーシテイ Enhancement of antibody-derived immune response
US8105825B2 (en) 2000-10-03 2012-01-31 Intrexon Corporation Multiple inducible gene regulation system
US7060442B2 (en) 2000-10-30 2006-06-13 Regents Of The University Of Michigan Modulators on Nod2 signaling
EP1348034B1 (en) * 2000-11-15 2016-07-20 Minerva Biotechnologies Corporation Oligonucleotide identifiers
EP1355918B9 (en) 2000-12-28 2012-01-25 Wyeth LLC Recombinant protective protein from $i(streptococcus pneumoniae)
FR2818981A1 (en) * 2001-01-03 2002-07-05 Synt Em AMPHIPATHIC PEPTIDES AND THEIR USE FOR THE TRANSFER OF SUBSTANCES OF INTEREST TO CELLS
MXPA03007493A (en) 2001-02-20 2004-10-15 Rheogene Holdings Inc Novel ecdysone receptor/invertebrate retinoid x receptor-based inducible gene expression system.
ES2392508T3 (en) 2001-02-20 2012-12-11 Intrexon Corporation Chimeric X retinoid receptors and their use in an inducible gene expression system based on novel ecdysone receptors
WO2002066615A2 (en) 2001-02-20 2002-08-29 Rheogene, Inc. Novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system
US8715959B2 (en) 2001-02-20 2014-05-06 Intrexon Corporation Substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system
US20030039660A1 (en) 2001-03-02 2003-02-27 The Rockefeller University Recombinant hybrid allergen constructs with reduced allergenicity that retain immunogenicity of the natural allergen
FR2825364A1 (en) * 2001-05-29 2002-12-06 Genethon Iii PEPTIIDIC VECTOR FOR THE TRANSFER OF NUCLEIC ACIDS IN EUKARYOTIC CELLS
US7919269B2 (en) 2001-09-26 2011-04-05 Intrexon Corporation Whitefly ecdysone receptor nucleic acids, polypeptides, and uses thereof
MXPA04002810A (en) 2001-09-26 2005-06-06 Rheogene Holdings Inc Leafhopper ecdysone receptor nucleic acids, polypeptides, and uses thereof.
US20040023910A1 (en) * 2001-09-28 2004-02-05 Zhiming Zhang Use of cyr61 in the treatment and diagnosis of human uterine leiomyomas
MX339524B (en) 2001-10-11 2016-05-30 Wyeth Corp Novel immunogenic compositions for the prevention and treatment of meningococcal disease.
US7375093B2 (en) 2002-07-05 2008-05-20 Intrexon Corporation Ketone ligands for modulating the expression of exogenous genes via an ecdysone receptor complex
US7785608B2 (en) 2002-08-30 2010-08-31 Wyeth Holdings Corporation Immunogenic compositions for the prevention and treatment of meningococcal disease
US7465708B2 (en) 2002-11-25 2008-12-16 Mixson A James Branched cationic copolymers and methods for antimicrobial use
US7304161B2 (en) 2003-02-10 2007-12-04 Intrexon Corporation Diaclhydrazine ligands for modulating the expression of exogenous genes in mammalian systems via an ecdysone receptor complex
US7091185B2 (en) * 2003-02-24 2006-08-15 Dow Global Technologies Inc. Periodic antimicrobial peptides
US7456315B2 (en) 2003-02-28 2008-11-25 Intrexon Corporation Bioavailable diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex
US7432057B2 (en) 2004-01-30 2008-10-07 Michigan State University Genetic test for PSE-susceptible turkeys
CA2605574A1 (en) 2004-04-20 2005-11-03 Galapagos N.V. Methods, compositions and compound assays for inhibiting amyloid-beta protein production
JP5128273B2 (en) 2004-04-27 2013-01-23 ガラパゴス・ナムローゼ・フェンノートシャップ Methods, agents, and compound screening assays for inducing differentiation of undifferentiated mammalian cells into osteoblasts
US7935510B2 (en) 2004-04-30 2011-05-03 Intrexon Corporation Mutant receptors and their use in a nuclear receptor-based inducible gene expression system
ATE534736T1 (en) 2004-06-14 2011-12-15 Galapagos Nv IDENTIFICATION METHODS AND COMPOUNDS FOR THE TREATMENT OF DEGENERATIVE AND INFLAMMATORY DISEASES
JP5022217B2 (en) 2004-06-18 2012-09-12 デューク ユニバーシティー Odorant receptor modulators
US20070004658A1 (en) 2004-06-21 2007-01-04 Nick Vandeghinste Method and means for treatment of osteoarthritis
US7604798B2 (en) 2004-07-15 2009-10-20 Northwestern University Methods and compositions for importing nucleic acids into cell nuclei
ATE527281T1 (en) 2004-07-16 2011-10-15 Us Gov Health & Human Serv VACCINES AGAINST AIDS COMPRISING CMV/R NUCLEIC ACID CONSTRUCTS
US7485468B2 (en) 2004-10-15 2009-02-03 Galapagos Bv Molecular targets and compounds, and methods to identify the same, useful in the treatment of joint degenerative and inflammatory diseases
US9034650B2 (en) 2005-02-02 2015-05-19 Intrexon Corporation Site-specific serine recombinases and methods of their use
CN101437539B (en) 2005-07-05 2013-10-02 康奈尔研究基金会（有限公司） Blocking leukocyte emigration and inflammation by interfering with CD99l2
WO2007028147A2 (en) 2005-09-01 2007-03-08 Philadelphia Health & Education Corporation D.B.A. Drexel University College Of Medicin Identification of a prostatic intraepithelial neoplasia (pin)-specific gene and protein (pin-1) useful as a diagnostic treatment for prostate cancer
JP5336853B2 (en) 2005-11-02 2013-11-06 プロチバ バイオセラピューティクス インコーポレイティッド Modified siRNA molecules and methods of use thereof
MY151438A (en) 2006-07-28 2014-05-30 Sanofi Aventis Compositions and method for treatment of tumors
US8124598B2 (en) 2006-09-14 2012-02-28 Sharon Sageman 7-keto DHEA for psychiatric use
AR064642A1 (en) 2006-12-22 2009-04-15 Wyeth Corp POLINUCLEOTIDE VECTOR THAT INCLUDES IT RECOMBINATING CELL THAT UNDERSTANDS THE VECTOR POLYPEPTIDE, ANTIBODY, COMPOSITION THAT UNDERSTANDS THE POLINUCLEOTIDE, VECTOR, RECOMBINATING CELL POLYPEPTIDE OR ANTIBODY, USE OF THE COMPOSITION AND A COMPOSITION AND A METHOD
WO2008137993A1 (en) 2007-05-08 2008-11-13 Duke University Compositions and methods for characterizing and regulating olfactory sensation
CA2689137C (en) 2007-05-29 2017-05-02 Intrexon Corporation Chiral diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex
CA2690685A1 (en) 2007-06-20 2008-12-24 Galapagos N.V. Molecular targets and compounds, and methods to identify the same, useful in the treatment of bone and joint degenerative diseases
US20090069266A1 (en) 2007-06-27 2009-03-12 Northwestern University Methods and compositions for nucleic acid transfer into cells
JP2010536373A (en) 2007-08-23 2010-12-02 イントレキソン コーポレーション Methods and compositions for diagnosing disease
US20100160274A1 (en) * 2007-09-07 2010-06-24 Sharon Sageman 7-KETO DHEA for Psychiatric Use
BRPI0817009A2 (en) * 2007-09-17 2014-10-07 Rohm & Haas Gene Expression System for Controllable Expression of Ethylene Response in a Plant Cell, Composition, and Methods for Producing a Transgenic Plant, and for Modulating Ethylene Sensitivity in a Plant.
EP2042193A1 (en) 2007-09-28 2009-04-01 Biomay AG RNA Vaccines
CA2715080C (en) 2007-09-28 2021-09-28 Intrexon Corporation Therapeutic gene-switch constructs and bioreactors for the expression of biotherapeutic molecules, and uses thereof
CA2702058A1 (en) 2007-10-08 2009-04-16 Intrexon Corporation Engineered dendritic cells and uses for the treatment of cancer
CA2710713C (en) 2007-12-27 2017-09-19 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
EP2281041B1 (en) 2008-04-15 2014-07-02 Protiva Biotherapeutics Inc. Silencing of csn5 gene expression using interfering rna
WO2010029118A1 (en) 2008-09-11 2010-03-18 Biofocus Dpi B.V. Method for identifying compounds useful for increasing the functional activity and cell surface expression of cf-associated mutant cystic fibrosis transmembrane conductance regulator
CN102203122A (en) 2008-11-05 2011-09-28 惠氏有限责任公司 Multicomponent immunogenic composition for the prevention of beta-hemolytic streptococcal (bhs) disease
WO2010096561A1 (en) 2009-02-18 2010-08-26 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Synthetic hiv/siv gag proteins and uses thereof
EP2398481A2 (en) 2009-02-19 2011-12-28 Galapagos N.V. Methods for identifying and compounds useful for the diagnosis and treatment of diseases involving inflammation
WO2010094734A2 (en) 2009-02-19 2010-08-26 Biofocus Dpi B.V. Methods for identifying and compounds useful for the diagnosis and treatment of diseases involving inflammation
WO2010094732A1 (en) 2009-02-19 2010-08-26 Biofocus Dpi B.V. Methods for identifying and compounds useful for the diagnosis and treatment of diseases involving inflammation
EP2414830A2 (en) 2009-03-31 2012-02-08 Robert Zimmermann Modulation of adipose triglyceride lipase for prevention and treatment of cachexia, loss of weight and muscle atrophy and methods of screening therefor
WO2010112569A1 (en) 2009-03-31 2010-10-07 Robert Zimmermann Modulation of adipose triglyceride lipase for prevention and treatment of cachexia, loss of weight and muscle atrophy and methods of screening therefor
WO2010115841A1 (en) 2009-04-01 2010-10-14 Galapagos Nv Methods and means for treatment of osteoarthritis
CA2758542A1 (en) 2009-04-17 2010-10-21 New York University Peptides targeting tnf family receptors and antagonizing tnf action, compositions, methods and uses thereof
WO2011015572A1 (en) 2009-08-03 2011-02-10 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of neurodegenerative diseases
WO2011015573A1 (en) 2009-08-03 2011-02-10 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of neurodegenerative diseases
US9186370B2 (en) 2010-03-19 2015-11-17 University Of South Alabama Methods and compositions for the treatment of cancer
WO2011119773A1 (en) 2010-03-23 2011-09-29 Roeth Jeremiah F Vectors conditionally expressing therapeutic proteins, host cells comprising the vectors, and uses thereof
US20130164380A1 (en) 2010-06-17 2013-06-27 Actogenix Nv Compositions and methods for treating inflammatory conditions
SI3246044T2 (en) 2010-08-23 2024-06-28 Wyeth Llc Stable formulations of neisseria meningitidis rlp2086 antigens
ES2585328T5 (en) 2010-09-10 2022-12-14 Wyeth Llc Non-lipidated variants of Neisseria meningitidis ORF2086 antigens
CA2819552C (en) 2010-12-09 2023-09-26 Institut Pasteur Mgmt-based method for obtaining high yield of recombinant protein expression
EP2492279A1 (en) 2011-02-25 2012-08-29 Laboratorios Del. Dr. Esteve, S.A. Rapid immunogen selection method using lentiviral display
CN103534355A (en) 2011-03-04 2014-01-22 英特瑞克斯顿股份有限公司 Vectors conditionally expressing protein
US9458456B2 (en) 2011-04-01 2016-10-04 University Of South Alabama Methods and compositions for the diagnosis, classification, and treatment of cancer
EP2546358A1 (en) 2011-07-15 2013-01-16 Laboratorios Del. Dr. Esteve, S.A. Methods and reagents for efficient control of HIV progression
BR112014006535A2 (en) 2011-09-20 2017-03-28 Univ North Carolina Chapel Hill regulation of sodium channels by plunc proteins
WO2013053765A1 (en) 2011-10-11 2013-04-18 Proyecto De Biomedicina Cima, S.L. A non-human animal model of mucosa-associated lymphoid tissue (malt) lymphoma
JP2014533953A (en) 2011-11-17 2014-12-18 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ Therapeutic RNA switch compositions and methods of use
NO2788478T3 (en) 2011-12-09 2018-01-20
US9035039B2 (en) 2011-12-22 2015-05-19 Protiva Biotherapeutics, Inc. Compositions and methods for silencing SMAD4
US9464291B2 (en) 2012-01-06 2016-10-11 University Of South Alabama Methods and compositions for the treatment of cancer
SA115360586B1 (en) 2012-03-09 2017-04-12 فايزر انك Neisseria meningitidis compositions and methods thereof
RU2665841C2 (en) 2012-03-09 2018-09-04 Пфайзер Инк. Neisseria meningitidis compositions and methods of use thereof
EP2698377A1 (en) 2012-08-17 2014-02-19 Laboratorios Del. Dr. Esteve, S.A. Enhanced rapid immunogen selection method for HIV gp120 variants
JP6446377B2 (en) 2013-03-08 2018-12-26 ファイザー・インク Immunogenic fusion polypeptide
WO2014139884A2 (en) 2013-03-14 2014-09-18 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of fibrosis
JP2016522675A (en) 2013-03-14 2016-08-04 ガラパゴス・ナムローゼ・フェンノートシャップＧａｌａｐａｇｏｓ Ｎ．Ｖ． Molecular targets useful in the treatment of diseases associated with epithelial-mesenchymal transition and inhibitors of said targets
EP2972322B1 (en) 2013-03-14 2019-03-06 Galapagos NV Molecular targets and compounds, and methods to identify the same, useful in the treatment of fibrotic diseases
AU2014227571B2 (en) 2013-03-15 2017-02-02 Intrexon Corporation Boron-containing diacylhydrazines
US20160130585A1 (en) 2013-05-28 2016-05-12 The Johns Hopkins University Aptamers for the treatment of sickle cell disease
EP3041502A2 (en) 2013-09-08 2016-07-13 Pfizer Inc. Neisseria meningitidis compositions and methods thereof
KR101605421B1 (en) 2014-03-05 2016-03-23 국립암센터 A monoclonal antibody which specifically recognizes B cell lymphoma and use thereof
EP3194359B1 (en) 2014-09-17 2020-04-08 Intrexon Corporation Boron-containing diacylhydrazine compounds
KR20170075742A (en) 2014-10-02 2017-07-03 프로티바 바이오쎄라퓨틱스, 인코포레이티드 Compositions and methods for silencing hepatitis b virus gene expression
KR102554850B1 (en) 2015-02-06 2023-07-13 더 유니버시티 오브 노쓰 캐롤라이나 엣 채플 힐 Optimized human coagulation factor VIII gene expression cassette and use thereof
RU2723045C2 (en) 2015-02-19 2020-06-08 Пфайзер Инк. Compositions of neisseria meningitidis and methods for preparation thereof
US20180245074A1 (en) 2015-06-04 2018-08-30 Protiva Biotherapeutics, Inc. Treating hepatitis b virus infection using crispr
TW201710503A (en) 2015-06-12 2017-03-16 長庚醫療財團法人林口長庚紀念醫院 Novel polynucleotides, vactor, pharmaceutical compositions and use thereof
WO2017019891A2 (en) 2015-07-29 2017-02-02 Protiva Biotherapeutics, Inc. Compositions and methods for silencing hepatitis b virus gene expression
EP3347373A1 (en) 2015-10-10 2018-07-18 Intrexon Corporation Improved therapeutic control of proteolytically sensitive, destabilized forms of interleukin-12
WO2017147128A1 (en) 2016-02-22 2017-08-31 The University Of North Carolina At Chapel Hill Peptide inhibitors of calcium channels
WO2018089527A1 (en) 2016-11-09 2018-05-17 Intrexon Corporation Frataxin expression constructs
IL267733B2 (en) 2017-01-31 2023-10-01 Pfizer Neisseria meningitidis compositions and methods thereof
US20200393446A1 (en) 2017-12-08 2020-12-17 Centre National De La Recherche Scientifique (Cnrs) Bioluminescent screening test for detecting cell cytolysis
EP3539975A1 (en) 2018-03-15 2019-09-18 Fundació Privada Institut d'Investigació Oncològica de Vall-Hebron Micropeptides and uses thereof
MX2021001672A (en) 2018-08-10 2021-07-15 Eutilex Co Ltd Chimeric antigen receptor binding to hla-dr, and car-t cell.
SG11202109225WA (en) 2019-03-08 2021-09-29 Obsidian Therapeutics Inc Cd40l compositions and methods for tunable regulation
KR20220139319A (en) 2020-01-08 2022-10-14 옵시디안 테라퓨틱스, 인크. Compositions and methods for modulation of tunable transcription
EP3885440A1 (en) 2020-03-26 2021-09-29 Splicebio, S.L. Split inteins and their uses
WO2023242436A1 (en) 2022-06-17 2023-12-21 Danmarks Tekniske Universitet Allergy vaccines based on consensus allergens

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5187260A (en) * 1988-09-06 1993-02-16 Sharifa Karali Process for the preparation of a high purity protamine-DNA complex and process for use of same
US5354844A (en) * 1989-03-16 1994-10-11 Boehringer Ingelheim International Gmbh Protein-polycation conjugates
US5286634A (en) * 1989-09-28 1994-02-15 Stadler Joan K Synergistic method for host cell transformation
DE59106279D1 (en) * 1990-05-18 1995-09-21 Genentech Inc NEW PROTEIN POLYCATION CONJUGATES.
WO1992022651A1 (en) * 1991-06-14 1992-12-23 Isis Pharmaceuticals, Inc. Antisense oligonucleotide inhibition of the ras gene
WO1993007892A1 (en) * 1991-10-16 1993-04-29 The Children's Hospital Of Philadelphia Composition and treatment with biologically active peptides and antibiotic
US5585479A (en) * 1992-07-24 1996-12-17 The United States Of America As Represented By The Secretary Of The Navy Antisense oligonucleotides directed against human ELAM-I RNA
US5670347A (en) * 1994-05-11 1997-09-23 Amba Biosciences Llc Peptide-mediated gene transfer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9521931A1 *

Also Published As

Publication number Publication date
US5856435A (en) 1999-01-05
AU1581895A (en) 1995-08-29
CA2182118A1 (en) 1995-08-17
FR2715847B1 (en) 1996-04-12
WO1995021931A1 (en) 1995-08-17
JPH09508530A (en) 1997-09-02
FR2715847A1 (en) 1995-08-11

Similar Documents

Publication Publication Date Title
EP0743984A1 (en) Nucleic acid-containing composition, its preparation and use
CA2194797C (en) Nucleic acid containing composition, preparation and uses of same
Wang et al. Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies
JP4796063B2 (en) Methods and articles for delivering therapeutic agents
EP1513506B1 (en) Targeting system comprising uniformly-sized nanoparticles with at least one polymer and at least one positively-charged polysaccharide and preparation method thereof
US20100285111A1 (en) Self-assembling micelle-like nanoparticles for systemic gene delivery
FR2730637A1 (en) PHARMACEUTICAL COMPOSITION CONTAINING NUCLEIC ACIDS, AND USES THEREOF
Cheng et al. The effect of guanidinylation of PEGylated poly (2-aminoethyl methacrylate) on the systemic delivery of siRNA
FR2766195A1 (en) CATIONIC POLYMERS, COMPLEXES ASSOCIATING THE SAID CATIONIC POLYMERS AND THERAPEUTICALLY ACTIVE SUBSTANCES INCLUDING AT LEAST ONE NEGATIVE CHARGES, ESPECIALLY NUCLEIC ACIDS, AND THEIR USE IN GENE THERAPY
Chiarantini et al. Comparison of novel delivery systems for antisense peptide nucleic acids
Li et al. Recent developments of polymeric delivery systems in gene therapeutics
WO1998034648A1 (en) Formulation of stabilised cationic transfection agent(s)/nucleic acid particles
Vlasov Starlike branched and hyperbranched biodegradable polymer systems as DNA carriers
CN114642734A (en) Drug and siRNA co-delivery nano-composite as well as preparation method and application thereof
FR2724935A1 (en) Compsns. contg. ras oncogene anti-sense oligo:nucleotide(s)
Haidar NanoDentistry: Perspectives on the role of NanoBiotechnology in biomaterials, pharmaceutics and BioDental tissue engineering
Delas Formulation and stabilization of colloidal polyelectrolyte complexes of chitosan and siRNA
WO1998017693A1 (en) Composition containing chitosan
KR102013908B1 (en) Composition for delivery of nucleic acid comprising fatty acid modified phospholipid and cationic polipeptides conjugate
EP0951560A1 (en) Conjugates of a particle vector and oligonucleotides, process for their preparation, and pharmaceutical compositions containing them
CN111704705A (en) Block polymer, preparation method thereof, medicine-carrying vesicle and targeted medicine-carrying vesicle
WO2002096928A2 (en) Peptide vector for transferring molecules of interest to eurocaryotic cells
MOFFATT et al. ENHANCED TARGETING OF POLYETHYLENIMINE (PEI)-BASED TUMOR-SPECIFIC ANTISENSE NANO-OLIGONUCLEOTIDES (TANOL) IN CANCER THERAPY

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19960801

AK Designated contracting states

Kind code of ref document: A1

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

17Q First examination report despatched

Effective date: 20000113

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20041023