EP1363675A2 - Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique - Google Patents

Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique

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Publication number
EP1363675A2
EP1363675A2 EP02716783A EP02716783A EP1363675A2 EP 1363675 A2 EP1363675 A2 EP 1363675A2 EP 02716783 A EP02716783 A EP 02716783A EP 02716783 A EP02716783 A EP 02716783A EP 1363675 A2 EP1363675 A2 EP 1363675A2
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EP
European Patent Office
Prior art keywords
leu
ala
peptide
lys
cells
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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.)
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EP02716783A
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German (de)
English (en)
Inventor
Karola Rittner
Eric Jacobs
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Transgene SA
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Transgene SA
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Priority to EP02716783A priority Critical patent/EP1363675A2/fr
Publication of EP1363675A2 publication Critical patent/EP1363675A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the use of non complexing peptides for the preparation of compositions useful for improving transfer of substances of interest into cells.
  • Such compositions are specially useful in gene therapy, vaccination, and any therapeutic or prophylactic situation in which a substance of interest, particularly a nucleic acid is administered to cells in vivo.
  • Gene therapy can be defined as the transfer of genetic material into a cell or an organism.
  • the possibility of treating human, disorders by gene therapy has changed in a few years from the stage of theoretical considerations to that of clinical applications.
  • the first protocol applied to man was initiated in ' the USA in September 1990 on a patient suffering from adenine deaminase (ADA) deficiency.
  • ADA adenine deaminase
  • Therapeutic genes can be transferred into cells using a wide variety of vectors resulting in either transient expression or permanent transformation of the host genome.
  • vectors resulting in either transient expression or permanent transformation of the host genome.
  • a large number of viral, as well as non-viral, vectors has been developed for gene transfer (see for example Robbins et al., 1998, Tibtech 16, 35-40 and Rolland, 1998, Therapeutic Drug Carrier Systems 15, 143-198 for reviews).
  • retroviral vectors cannot accommodate large-sized nucleotide sequences (e.g.
  • the retroviral genome is integrated into host cell DNA and may thus cause genetic changes in the recipient cell and infectious viral particles can disseminate within the organism or into the environment; adenoviral vectors can induce a strong immune response in treated patients (Mc Coy et al, 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996, Immunity, 1, 433-442).
  • non-viral systems presenting special advantages with respect to large-scale production, safety, low immunogenicity, and capacity to deliver large fragments of DNA have been proposed.
  • alternative non-viral vectors have been proposed (see Rolland, 1998, Therapeutic Drug Carrier Systems, 15, 143-
  • cationic compounds are capable of forming complexes with anionic molecules, thus tending to neutralize their negative charges and allowing to compact them in complexed form which favors their introduction into the cell.
  • non-viral delivery systems are, for example, based on receptor-mediated mechanisms (Perales et al., 1994, Eur. J. Biochem.
  • Endocytosis is the natural process by which eukaryotic cells ingest segments of the plasma membrane in the form of small endocytosis vesicles, i.e. endosomes, entrapping extracellular fluid and molecular material, e.g. nucleic acid molecules.
  • endosomes fuse with lysosomes which are specialized sites of intracellular degradation.
  • the lysosomes are acidic and contain a wide variety of degradative enzymes to digest the molecular contents of the endosomal vesicles.
  • the internalized material After endocytosis the internalized material is thus still separated from the cytoplasm by a membrane and therefore is not available for performing its desired function.
  • said desired function i.e. the desired therapeutic effect, depends in most of the nucleic acids transfer approaches on their delivery at least into the cytoplasm (e.g. for RNA) or rather into the nucleus of the cell (e.g. for DNA encoding a polypeptide or antisense oligonucleotides) where their functional effect can occur. Consequently, the internalized nucleic acid accumulation into endosomal vesicles strongly reduces the efficiency of nucleic acid functional transfer to the cell, and therefore the efficiency of gene therapy (Zabner et al.,1995, J. Biol. Chem., 270, 18997-19007).
  • the efficient delivery to and expression of genetic information within the cells of a living organism depend both on the capability of the delivery system to transfer the nucleic acid molecule into the cell and on its capability to promote nucleic acid escape from endosomal retention and degradation.
  • the general strategy is to promote endosomolysis, e.g. by using fusogenic or membranolytic/endosomolytic peptides (see Mahato et al., 1999,Current Opinion in Mol. Therapeutics, 1, 226-243).
  • microorganisms e.g. viruses
  • Some microorganisms are naturally internalized via receptor-mediated endocytosis and have developed systems for escaping from the above-mentioned endosomal degradation.
  • gene transfer systems have been proposed including the endosome-destabilizing activity of replication- defective adenovirus particles or rhinovirus particles which were either added to the transfection medium (Gotten et al., 1992, Proc. Natl. Acad. Sci. USA, 89, 6094-6098) or directly linked to the delivery complex (Wu et al., 1994, J. Biol. Chem., 269, 11542-11546 ; US 5,928,944).
  • the synthetic peptide JTS-1 developped by Gottschalk et al.(1996, Gene Therapy, 3, 448-457) starts with the INF sequence GLFEA followed by an optimized peptide sequence.
  • This JTS-1 peptide was shown to be capable of lysing calcein containing phosphatidylcholine liposomes at pH 5, more efficiently than at a pH 7.
  • the intracellular delivery of nucleic acids requires that said peptides combine their fusogenic activity with a nucleic acid complexing activity to form delivery complexes capable of transferring said nucleic acid into cells.
  • the technical problem underlying the present invention is the provision of improved methods and means for the delivery into cells of substances of interest, preferably of nucleic acid molecules, which are useful for therapy, preferably for gene therapy. This problem is solved by the provision of the embodiments as characterized in the claims.
  • the present invention relates to the use of a peptide for the preparation of a composition for transferring at least one substance of mterest into a cell, and more specifically for the preparation of a composition for improving the transfer of at least one substance of interest into a cell, wherein said peptide is selected from the group consisting of : (i) a peptide comprising or consisting of the amino acid sequence Gly Leu Phe Xaa Ala Leu Leu
  • Xaa Leu Leu Xaa Ser Leu Trp Xaa Leu Leu Leu Xaa Ala (SEQ ID NO:l), wherein Xaa is selected independently of one another from the group consisting of Alanine (Ala or A), Isoleucine (He or I), Leucine (Leu or L), Phenylalanine (Phe or F), Proline (Pro or P), Tryptophane (Trp or W), Valine (Val or V), Asparagine (Asn or N), Cysteine (Cys or C), Glutamine (Glu or Q), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T) and
  • Tyrosine (Tyr or Y) ;
  • cationic peptides which are capable to cause cell membrane disruption, to bind an anionic substance, in particular a nucleic acid molecule, in order to form complexes and thereby to enhance transfer of said complexed anionic substance into a cell. More specifically, these cationic peptides do not comprise acidic amino acids, and more particularly do not comprise glutamic amino acid (Glu or E).
  • cationic peptides are peptides which comprise or consist of the amino acid sequence Gly Leu Phe Xaa Ala Leu Leu Xaa Leu Leu Xaa Ser Leu Trp Xaa Leu Leu Leu Xaa Ala (SEQ ID NO: 1) wherein Xaa is selected independently of one another from the group consisting of lysine (Lys or K), histidine (His or H) and arginine (Arg or R) amino acid.
  • the results provided in the present application demonstrate that it is possible to use non- charged peptides, which do not allow complex formation with nucleic acids, for the preparation of a pharmaceutical composition for an improved transfer of a nucleic acid into a cell.
  • the term "substance of interest” designates preferably a charged molecule without limitation of the number of charges.
  • said molecule is an anionic substance of interest, and more preferably it is selected from the group consisting of proteins and nucleic acid molecules.
  • said anionic substance of interest is a nucleic acid.
  • nucleic acid or “nucleic acid molecule” as used in the scope of the present invention means a DNA or RNA or a fragment or combination thereof, which is single- or double-stranded, linear or circular, natural or synthetic, modified or not (see US 5525711, US 4711955, US 5792608 or EP 302 175 for modification examples) without size limitation. It may, inter alia, be a genomic DNA, a cDNA, an mRNA, an antisense RNA, a ribozyme, or a DNA encoding such RNAs.
  • nucleic acid may be in the form of a linear or circular polynucleotide, and preferably in the form of a plasmid.
  • the nucleic acid can also be an oligonucleotide which is to be delivered to the cell, e.g., for antisense or ribozyme functions.
  • the nucleic acid is preferably a naked polynucleotide (Wolff et al., Science 247 (1990), 1465-1468) or is formulated with at least one compound such as a polypeptide, preferably a viral polypeptide, or a cationic lipid, or a cationic polymer, or combination thereof, which can participate in the uptake of the polynucleotide into the cells (see Ledley, Human Gene Therapy 6 (1995), 1129-1144 for a review) or a protic polar compound (examples are provided below in the present application or in EP 890362).
  • a naked polynucleotide preferably a naked polynucleotide (Wolff et al., Science 247 (1990), 1465-1468) or is formulated with at least one compound such as a polypeptide, preferably a viral polypeptide, or a cationic lipid, or a cationic polymer, or combination thereof, which can
  • nucleic acid further designate a viral vector (adenoviral vector, retroviral vector, poxviral vector, etc.).
  • the term « viral vector » as used in the present invention encompasses the vector genome, the viral particles (i.e. the viral capsid including the viral genome) as well as empty viral capsids.
  • “Plasmid” refers to an extrachromosomic circular DNA. The choice of the plasmids is very large.
  • Plasmids can be purchased from a variety of manufacturers. Suitable plasmids include but are not limited to those derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene), pCI (Promega) and p Poly (Lathe et al., Gene 57 (1987), 193-201). It is also possible to engineer such a plasmid by molecular biology techniques (Sambrook et al., Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), NY). A plasmid may also comprise a selection gene in order to select or identify the transfected cells (e.g. by complementation of a cell auxotrophy, antibiotic resistance), stabilizing elements (e.g. cer sequence; Summers and Sherrat, Cell 36 (1984), 1097-1103) or integrative elements (e.g. LTR viral sequences).
  • pBR322 Gibco
  • said nucleic acid molecule includes at least one encoding gene sequence of interest (i.e. a transcriptional unit) that can be transcribed and translated to generate a polypeptide of interest and the elements enablin its expression (i.e. an expression cassette). If the nucleic acid contains this proper genetic information when it is placed in an environment suitable for gene expression, its transcriptional unit will thus express the encoded gene product. The level and cell specificity of expression will depend to a significant extent on the strength and origin of the associated promoter and the presence and activation of an associated enhancer element.
  • the transcriptional control element includes the promoter/enhancer sequences such as the CMV promoter/enhancer.
  • promoter and/or enhancer sequences are known which may be obtained from any viral, prokaryotic, e.g. bacterial, or eukaryotic organism, which are constitutive or regulable, which are suitable for expression in eukaryotic cells, and particularly in target cells or tissues. More precisely, this genetic information necessary for expression by a target cell or tissue comprises all the elements required for transcription of said gene sequence (if this gene sequence is DNA) into RNA, preferably into mRNA, and, if necessary, for translation of the mRNA into a polypeptide. Promoters suitable for use in various vertebrate systems are widely described in literature.
  • Suitable promoters include but are not limited to the adenoviral Ela, MLP, PGK (Phospho Glycero Kinase ; Adra et al. Gene 60 (1987) 65-74 ; Hitzman et al. Science 219 (1983) 620-625), RSV, MPSV, SV40, CMV or 7.5k, the vaccinia promoter, inducible promoters, MT (metallothioneine; Mc Ivor et al., Mol. Cell Biol. 7 (1987), 838-848), alpha-1 antitrypsin, CFTR, immunoglobulin, alpha-actin (Tabin et al., Mol. Cell Biol.
  • promoters can be used which are active in tumor cells. Suitable examples include but are not limited to the promoters isolated from the gene encoding a protein selected from the group consisting of MUC-1 (overexpressed in breast and prostate cancers ; Chen et al, J. Clin. Invest. 96 (1995), 2775- 2782), CEA (Carcinoma Embryonic Antigen ; overexpressed in colon cancers ; Schrewe et al., Mol. Cell. Biol.
  • the nucleic acid can also include intron sequences, targeting sequences, transport sequences, sequences involved in replication or integration.
  • nucleic acid can also be modified in order to be stabilized with specific components, for example spermine. It can also be substituted, for example by chemical modification, in order to facilitate its binding with specific polypeptides such as, for example the peptides of the present invention.
  • nucleic acid can be homologous or heterologous to the target cells into which it is introduced.
  • the nucleic acid contains at least one gene sequence of interest encoding a gene product which is a therapeutic molecule (i.e. a therapeutic gene).
  • a therapeutic molecule is one which has a pharmacological or protective activity when administered, or expressed, appropriately to a patient, especially patient suffering from a disease or illness condition or who should be protected against this disease or condition.
  • a pharmacological or protective activity is one which is expected to be related to a beneficial effect on the course or a symptom of said disease or said condition.
  • the sequence of interest can be homologous or heterologous to the target cells into which it is introduced.
  • said sequence of interest encodes all or part of a polypeptide, especially a therapeutic or prophylactic polypeptide giving a therapeutic or prophylactic effect.
  • a polypeptide is understood to be any translational product of a polynucleotide regardless of size, and whether glycosylated or not, and includes peptides and proteins.
  • Therapeutic polypeptides include as a primary example those polypeptides that can compensate for defective or deficient proteins in an animal or human organism, or those that act through toxic effects to limit or remove harmful cells from the body. They can also be immunity conferring polypeptides which act as an endogenous antigen to provoke a humoral or cellular response, or both.
  • The- following encoding gene sequences are of particular interest.
  • genes coding for a cytokine V,3 or (-interferon, interleukine (IL), in particular IL-2, IL-6, IL-10 or IL-12, a tumor necrosis factor (TNF), a colony stimulating factor (such as GM-CSF, C-CSF, M-CSF), an immunostimulatory polypeptide (such as B7.1, B7.2, CD40, CD4, CD8, ICAM and the like), a cell or nuclear receptor, a receptor ligand (such as fas ligand), a coagulation factor (such as FVIII, FIX), a growth factor (such as Transforming Growth Factor TGF, Fibroblast Growth Factor FGF and the like), an enzyme (such as urease, renin, thrombin, metalloproteinase, nitric oxide synthase NOS, SOD, catalase), an enzyme inhibitor (such as Vl-antitrypsine, antithrombine III, viral protease inhibitor,
  • a functional allele of a defective gene for example a gene encoding factor VIII or IX in the context of haemophilia A or B, dystrophin (or minidystrophm) in the- context -of myopathies, insulin in the context of diabetes, CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) in the context of cystic fibrosis.
  • a functional allele of a defective gene for example a gene encoding factor VIII or IX in the context of haemophilia A or B, dystrophin (or minidystrophm) in the- context -of myopathies, insulin in the context of diabetes, CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) in the context of cystic fibrosis.
  • Suitable anti-tumor genes include but are not limited to those encodmg an antisense RNA, a ribozyme, a cytotoxic product such as thymidine kinase of herpes- 1 simplex virus (TK-HSV-1), ricin, a bacterial toxin, the expression product of yeast genes FCY1 and/or FUR1 having UPRTase (Uracile Phosphoribosyltransferase) and CDase (Cytosine Deaminase) activity respectively, an antibody, a polypeptide inhibiting cellular division or transduction signals, a tumor suppressor gene (p53, Rb, p73), a polypeptide activating host immune system, a tumor-associated antigen (MUC-1, BRCA-1, an HPV early or late antigen (E6, E7, LI, L2), optionally in combination with a cytokine gene.
  • TK-HSV-1 herpes- 1 simplex virus
  • the polynucleotide can also encode an antibody.
  • antibody encompasses whole immunoglobulins of any class, chimeric antibodies and hybrid antibodies with dual or multiple antigen or epitope specificities, and fragments, such as F(ab)'2, Fab', Fab including hybrid fragments and anti-idiotypes
  • nucleic acid encodes all or part of a polypeptide which is an immunity conferring polypeptide and acts as endogenous immunogen to provoke a humoral or cellular response, or both, against infectious agents, mcluding intracellular viruses, or against tumor cells.
  • An "immunity-conferring polypeptide” means that said polypeptide when it is produced in the transfected cells will participate in an immune response in the treated patient. More specifically, said polypeptide produced in or taken up by macropinocyte cells such as APCs will be processed and the resulting fragments will be presented on the surface of these cells by MHC class I and/or II molecules in order to elicit a specific immune response.
  • the nucleic acid may comprise one or more gene(s) of interest.
  • a suicide gene product e.g. , 3 or ⁇ interferons, interleukins, preferably selected among IL-2, IL-4, IL-6, IL-10 or IL-12, TNF factors, GM-CSF, C-CSF, M-CSF and the like
  • an immunostimulatory gene e.g. B7.1, B7.2, ICAM
  • a chi iokine gene e.g. MIP , RANTES, MCP 1
  • the different gene expression may be controlled by a unique promoter (polycistronic cassette) or by independent promoters. Moreover, they may be inserted in a unique site or in various sites along the nucleic acid either in the same or opposite directions.
  • the encoding gene sequence of interest may be isolated from any organism or cell by conventional techniques of molecular biology (PCR, cloning with appropriate probes, chemical synthesis) and if needed its sequence may be modified by mutagenesis, PCR or any other protocol.
  • the "substance of interest" is a peptide (polypeptide, protein and peptide are synonyms) including variant or modified peptides, peptide-like molecules, antibodies or fragments thereof, chimeric antibody or peptide, ....
  • the introduction or transfer process of an substance of interest into a cell is by itself well known.
  • “Introduction or transfer” means that the substance is transferred into the cell and is located, at the end of the process, inside said cell or within or on its membrane. If the substance is a nucleic acid, “introduction or transfer” is also referred to as “transfection”. Transfection can be verified by any appropriate method, for example by measuring the expression of a gene encoded by said nucleic acid or by measuring the concentration of the expressed protein or its mRNA, or by measuring its biological effect.
  • improved transfer in the scope of the present invention means, in this regard, a more efficient transfer of a substance of interest by cells when the peptide according to the invention is present compared to an introduction performed in absence of said peptide. This can be determined by comparing the amount of the substance taken up without the use of the peptide as disclosed in the present invention and comparing this amount with the amount taken up by the cells when using said peptide under the same experimental conditions.
  • the improved transfer can be determined by a higher amount of expression of the gene present in the nucleic acid transferred into the cells when using compositions comprising the peptide of the present invention, and/or compositions having pH over 6, and preferably having pH 8 in comparison to a situation where no peptide is used.
  • the peptide of the mvention is capable of causing membrane disruption.
  • the term "peptide capable of causing membrane disruption" as used herein refers to a peptide which is capable of interacting with a membrane, particularly with a cellular membrane, and more particularly with an endosomal and/or lysosomal membrane, in such a manner that said interaction results in destabilizing and/or leaking of the membrane, and particularly in freeing the contents of the endosomes.
  • said interaction results in freeing the endosome and/or lysosome contents into the cytoplasm of the cell.
  • membrane disrupting property of the peptide can be easily measured for example by the method described in the appended Examples or in Olson et al.( 1979, Biochim. Biophys. Acta, 557, 19-23).
  • the term "membrane” as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Generally, it designates a mono or bi-layer consisting mainly of lipids, and eventually contains proteins. Included are natural (e.g. membrane of the cells) and synthetic (e.g. liposomal) membranes. Preferred membranes are natural membranes such as for example cellular membranes, endosomal or lysosomal membranes, trans-Golgi network membranes, virus membranes, nuclear membranes. Said property can be evaluated as done in the Experimental section.
  • peptide refers to a polymer of amino acids residues that is less than 50 residues in length, more preferably less than 30 residues in length and most preferably less than 20 residues in length.
  • the peptide implemented in the present invention has a molecular weight of less than 5 lcD and most preferably of less than 3 kD.
  • Peptides of the invention may be produced de novo by synthetic methods or by expression of the appropriate DNA fragment by recombinant DNA techniques in eukaryotic or prokaryotic cells.
  • said peptide contains one or more non-hydrolyzable chemical moieties in place of those which exist in naturally occurring peptides, such as carboxyl moieties.
  • the naturally hydrolyzable moities are replaced by non-hydrolizable ones such as for example methylene moities.
  • the present invention also encompasses analogs of the above described peptide, wherein at least one amino acid is replaced by another amino acid having similar properties, including retro or inverso peptides (W095/24916).
  • the ligand moiety in use in the invention may include modifications of its original structure by way of substitution or addition of chemical moieties (e.g.
  • the present invention also contemplates modifications that render the peptides of the invention detectable.
  • the peptides of the invention can be modified with a detectable moiety (i.e. a scintigraphic, radioactive, a fluorescent moiety, an enzyme, a dye label and the like).
  • Suitable radioactive labels include but are not limited to Tc 99m , I 123 and In 111 . Such labels can be attached to the peptide of the invention in a known manner, for example via a cysteine residue. Other techniques are described elsewhere.
  • the labeled peptides of the invention may be used for diagnostic purposes (e.g. imaging of tumoral cells, of transformed cells, and the like).
  • the peptide of the invention is modified by addition of at least one cysteine residue at its N- and/or C-terminal extremities.
  • This modification allows for example the formation of di-, tri- or multimeric association of peptides of the present mvention. Said association of modified peptides can be linear or cyclized.
  • the peptide of the invention is further modified with a ligand capable of cell-specific targeting or with a ligand capable of nuclear targeting.
  • ligand capable of cell- specific targeting refers to a ligand moiety which binds to a surface receptor of a cellular membrane (i.e. anti- ligand).
  • Said cell membrane surface receptor is a molecule or structure which can bind said ligand with high affinity and preferably with high specificity.
  • Said cell membrane surface receptor is preferably specific for a particular cell, i.e. it is found predominantly in one type of cells rather than in another type of cells (e.g. galactosyl residues to target the asialoglycoprotein receptor on the surface of hepatocytes).
  • the cell membrane surface receptor facilitates cell targeting and internalization into the target cell of the ligand (i.e. the peptide involved in cell-specific targeting) and attached molecules (i.e. the peptide of the invention).
  • ligand moieties /anti-ligands that may be used in the context of the present invention are widely described in the literature.
  • a ligand moiety is capable of conferring to the peptide of the invention, the ability to bind to a given anti-ligand molecule or a class of anti-ligand molecules localized at the surface of at least one target cell.
  • Suitable anti-ligand molecules include without limitation polypeptides selected from the group consisting of cell-specific markers, tissue-specific markers, cellular receptors, viral antigens, antigenic epitopes and tumor-associated markers.
  • Anti-ligand molecules may moreover consist of or comprise one or more sugar, lipid, glycolipid or antibody molecules.
  • a ligand moiety may be for example a lipid, a glycolipid, a hormone, a sugar, a polymer (e.g. PEG, polylysine, PE1), an oligonucleotide, a vitamin, an antigen, all or part of a lectin, all or part of a polypeptide such as for example JTS1 (WO 94/40958), an antibody or a fragment thereof, or a combination thereof.
  • the ligand moiety used in the present invention is a peptide or polypeptide having a minimal length of 7 amino acids. It is either a native polypeptide or a polypeptide derived from a native polypeptide. "Derived” means containing (a) one or more modifications with respect to the native sequence (e.g. addition, deletion and/or substitution of one or more residues), (b) amino acid analogs, including not naturally occurring amino acids or (c) substituted linkages or (d) other modifications known in the art.
  • polypeptides serving as ligand moiety encompass variant and chimeric polypeptides obtained by fusing sequences of various origins, such as for example a humanized antibody which combines the variable region of a mouse antibody and the constant region of a human immunoglobulin.
  • polypeptides may have a linear or cyclized structure (e.g. by flanking at both extremities a polypeptide ligand by cysteine residues).
  • the polypeptide in use as ligand moiety may include modifications of its original structure by way of substitution or addition of chemjcal moieties (e.g. glycosylation, alkylation, acetylation, amidation, phosphorylation, addition of sulfhydryl groups and the like).
  • the invention further contemplates modifications that render the ligand moiety detectable.
  • modifications with a detectable moiety can be envisaged (i.e. a scintigraphic, radioactive, or fluorescent moiety, or a dye label and the like).
  • Suitable radioactive labels include but are not limited to Tc 99m , I 123 and In 111 .
  • detectable labels may be attached to the ligand moiety by any conventional techniques and may be used for diagnostic purposes (e.g. imaging of tumoral cells).
  • the anti-ligand molecule is an antigen (e.g.
  • the ligand moiety is an antibody, a fragment or a minimal recognition unit thereof (i.e. a fragment still presenting an antigenic specificity) such as those described in detail in immunology manuals (see for example Immunology, third edition 1993, Roitt, Brostoff and Male, ed Gambli, Mosby).
  • the ligand moiety may be a monoclonal antibody. Monoclonal antibodies which will bind to many of these antigens are already known but in any case, with today's techniques in relation to monoclonal antibody technology, antibodies may be prepared to most antigens.
  • the ligand moiety may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example, ScFv).
  • Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in “Monoclonal Antibodies: A manual of techniques", H. Zola (CRC Press, 1988) and in “Monoclonal Hybridoma Antibodies: Techniques and Applications", J. G. R. Hurrell (CRC Press, 1982).
  • Suitably prepared non-human antibodies may be “humanized” in known ways, for example by inserting the CDR regions of mouse antibodies into the framework of human antibodies.
  • variable heavy (VH) and variable light (VL) domains of the antibody are involved in antigen recognition
  • variable domains of rodent origin may be fused to constant domains of human origin such that the resultant antibody retains the antigenic specificity of the rodent parental antibody (Morrison et al (1984) Proc. Natl. Acad. Sci. USA 81, 6851-6855).
  • variable domains including Fab-like molecules (Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al (1988) Science 240, 1038); ScFv molecules where the VH and VL partner domains are linked via a flexible oligopeptide (Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl. Acad. Sci. USA 85, 5879) and dAbs comprising isolated V domains (Ward et al (1989) Nature 341, 544).
  • the ligand moiety is selected among antibody fragments, rather than whole antibodies. Effective functions of whole antibodies, such as complement binding, are removed. ScFv and dAb antibody fragments may be expressed as a fusion with one or more other polypeptides. Minimal recognition units may be derived from the sequence of one or more of the complementary-determining regions (CDR) of the Fv fragment.
  • Whole antibodies, and F(ab')2 fragments are "bivalent". By “bivalent” we mean that said antibodies and F(ab') 2 fragments have two antigen binding sites.
  • Fab, Fv, ScFv, dAb fragments and minimal recognition units are monovalent, having only one antigen binding sites.
  • the ligand moiety is at least part of a specific moiety implicated in natural cell- surface receptor binding.
  • said natural receptors e.g. hormone receptors
  • said natural receptors may also be target cell- specific antigens and may be recognized by ligand moieties which have the property of a monoclonal antibody, a ScFv, a dAb or a minimal recognition unit.
  • the ligand moiety allows to target a vitally infected cell and is capable of recognizing and binding to a viral component (e.g. envelope glycoprotein) or capable of interfering with the virus biology (e.g. entry or replication).
  • a viral component e.g. envelope glycoprotein
  • virus biology e.g. entry or replication
  • the targeting of an HIV (Human Immunodeficiency Virus) infected cell can be performed with a ligand moiety specific for an epitope of the HTV envelope, such as a ligand moiety derived from the 2F5 antibody (Buchacher et al., 1992, Vaccines 92, 191-195) recognizing a highly conserved epitope of the transmembrane glycoprotein gp41 or with a ligand moiety interfering with HIV attachment to its cellular receptor CD4 (e.g. the extracellular domain of the CD4 molecule).
  • a ligand moiety specific for an epitope of the HTV envelope such as a ligand moiety derived from the 2F5 antibody (Buchacher et al., 1992, Vaccines 92, 191-195) recognizing a highly conserved epitope of the transmembrane glycoprotein gp41 or with a ligand moiety interfering with HIV attachment to its cellular receptor CD4 (e.g
  • the ligand moiety allows to target a tumor cell and is capable of recognizing and binding to a molecule related to the tumor status, such as a tumor-specific antigen, a cellular protein differentially or over-expressed in tumor cells or a gene product of a cancer-associated virus.
  • tumor-specific antigens include but are not limited to MUC-1 related to breast cancer (Hareuveni et al., 1990, Eur. J. Biochem 189, 475-486), the products encoded by the mutated BRCA ⁇ and BRCA2 genes related to breast and ovarian cancers (Miki et al., 1994, Science 226, 66-71 ; Futreal et al., 1994, Science 226, 120-122 ; Wooster et al., 1995, Nature 378, 789-792), APC related to colon cancer (Polakis, 1995, Curr. Opin. Genet. Dev.
  • PSA prostate specific antigen
  • CEA carcinoma embryonic antigen
  • tyrosinase related to melanomas
  • MSH melanocyte-stimulating hormone
  • a special ligand moiety in use in the present invention is a fragment of an antibody capable of recognizing and binding to the MUC-1 antigen and thus targeting the MUC-1 positive tumor cells.
  • a more preferred ligand moiety is the scFv fragment of the SM3 monoclonal antibody which recognizes the tandem repeat region of the MUC-1 antigen (Burshell et al., 1987, Cancer Res. 47, 5476-5482 ; Girling et al., 1989, Int J.
  • cellular proteins differentially or overexpressed in tumor cells include but are not limited to the receptor for interleukin 2 (IL-2) overexpressed in some lymphoid tumors, GRP (Gastrin Release Peptide) overexpressed in lung carcinoma cells, pancreas, prostate and stomach tumors (Michael et al., 1995, Gene Therapy 2, 660-668), TNF (Tumor Necrosis Factor) receptor, epidermal growth factor receptors, Fas receptor, CD40 receptor, CD30 receptor, CD27 receptor, OX-40, Vv integrins (Brooks et al., 1994, Science 264, 569) and receptors for certain angiogenic growth factors (Hanahan, 1997, Science 277, 48). Based on these indications, it is within the scope of those skilled in the art to define an appropriate ligand moiety capable of recognizing and binding to
  • Suitable gene products of cancer-associated viruses include but are not limited to human papilloma virus (HPV) E6 and E7 early polypeptides as well as LI and L2 late polypeptides (EP 0 462 187, US 5,744,133 and WO98/04705) that are expressed in cervical cancer and EBNA-1 antigen of Epstein-Barr virus (EBV) associated with Burkitt's lymphomas (Evans et al., 1997, Gene Therapy 4, 264-267).
  • HPV human papilloma virus
  • E6 and E7 early polypeptides as well as LI and L2 late polypeptides
  • LI and L2 late polypeptides EP 0 462 187, US 5,744,133 and WO98/04705
  • EBV Epstein-Barr virus
  • the ligand moiety allows to target tissue-specific molecules.
  • ligand moieties suitable for targeting liver cells include but are not limited to those derived from ApoB (apolipoprotein) capable of binding to the LDL receptor, alpha-2-macroglobulin capable of binding to the LPR receptor, alpha- 1 acid glycoprotein capable of binding to the asialoglycoprotein receptor and transferrin capable of binding to the transferrin receptor.
  • a ligand moiety for targeting activated endothelial cells may be derived from the sialyl-Lewis-X antigen (capable of binding to ELAM-1), from VLA-4 (capable of binding to the VCAM-1 receptor) or from LFA-1 (capable of binding to the ICAM-1 receptor).
  • a ligand moiety derived from CD34 is useful to target hematopo ⁇ etic progenitor cells through binding to the CD34 receptor.
  • a ligand moiety derived from ICAM-1 is more intended to target lymphocytes through binding to the LFA-1 receptor.
  • the targeting of T-helper cells may use a ligand moiety derived from HTV gp-120 or a class II MHC antigen capable of binding to the CD4 receptor.
  • target cells we refer to the cells that the peptide of the invention can selectively target or the type of cell where transfer of the substance of interest is desirable.
  • target cells may designate a unique type of cell or a group of different types of cells having as a common feature on their surface an anti-ligand molecule(s) recognized by ligand moiety(s) present in the complex of the invention.
  • a target cell is any mammalian cell
  • to target refers to addressing a certain type of cells or a group of types of cells for gene transfer in favour of the remaining part of the totality of cells being contacted with the composition of the present invention.
  • the target cell may be a primary cell, a transformed cell or a tumor cell.
  • Suitable target cells include but are not limited to hematopo ⁇ etic cells (totipotent, stem cells, leukocytes, lymphocytes, monocytes, macrophages, APC, dendritic cells , non-human cells and the like), muscle cells (satellite, myocytes, myoblasts, skeletal or smooth muscle cells, heart cells), pulmonary cells , tracheal cells, hepatic cells, epithelial cells, endothelial cells or fibroblasts.
  • ligand capable of nuclear targeting refers to a particular ligand which is capable of binding to a nuclear receptor (nuclear anti-ligand).
  • Said nuclear receptor is a molecule or structure localized in or/and on the nuclear membrane which can bind to said ligand, thereby facilitating intracellular transport of the peptide of the present invention towards the nucleus and its internalization into the nucleus.
  • a ligand involved in nuclear targeting are the nuclear signal sequences derived from the T-antigen of the SV40 virus (Lanford and Butel, 1984, Cell 37, 801-813) and from the EBNA-1 protein of the Epstein Barr virus (Ambinder et al., 1991, J. Virol. 65, 1466-1478).
  • the invention also encompasses a composition, preferably for transferring a substance of interest into a cell, wherein said composition comprises at least one peptide as defined herein above and at least one substance of interest.
  • said composition has a pH value preferably higher than 6, more preferably higher than 6.5, still preferably higher than 7, even more preferably higher than 7.5 and most preferably of about 8.
  • said substance of interest is nucleic acids and said composition is particularly useful for the delivery of nucleic acids to cells or tissues of a subject in connection with gene therapy methods but are not limited to such uses.
  • the term "gene therapy method” is preferably understood as a method for the introduction of a nucleic acids into cells either in vivo or by introduction into cells in vitro followed by re- implantation into a subject. "Gene therapy” in particular concerns the case where the gene product is expressed in a tissue as well as the case where the gene product is excreted, especially into the blood stream.
  • the amount of peptide in the compositions prepared according to the use of the present invention ranges between about 0.05 micrograms to about 100 micrograms, preferably between about 0.1 micrograms to about 50 micrograms, and more preferably between about 0.5 micrograms and about 15 micrograms. These concentration and pH conditions may also be adapted by those skilled in the art.
  • the concentration of the nucleic acid in the composition is from about 0.01 mM to about 1 M, and in a preferred embodiment is from about 0.1 mM to 10 mM.
  • composition can be formulated in various forms, e.g. in solid, liquid, powder, aqueous, lyophilized form.
  • this composition further comprises a pharmaceutically acceptable carrier, allowing its use in a method for the therapeutic treatment of humans or animals.
  • the carrier is preferably a pharmaceutically suitable injectable carrier or diluent (for examples, see Remington's
  • Such a carrier or diluent is pharmaceutically acceptable, i.e. is non-toxic to a recipient at the dosage and concentration employed. It is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as provided by a sucrose solution.
  • aqueous or partly aqueous liquid carriers comprising sterile, pyrogen-free water, dispersion media, coatings, and equivalents, or diluents (e.g. Tris-HCl, acetate, phosphate), emulsif ⁇ ers, solubilizers or adjuvants.
  • diluents e.g. Tris-HCl, acetate, phosphate
  • emulsif ⁇ ers emulsif ⁇ ers
  • solubilizers or adjuvants e.g. Tris-HCl, acetate, phosphate
  • the pH of the pharmaceutical preparation is suitably adjusted and buffered in order to be useful in in vivo applications.
  • the pH of the composition is adjusted and buffered in order to be over pH 6, and preferably to be pH 8. It may be prepared either as a liquid solution or in a solid form (e.g.
  • lyophilized which can be suspended in a solution prior to administration.
  • carriers or diluents for an injectable composition include water, isotonic saline solutions which are buffered at desirable pH (such as phosphate buffered saline or Tris buffered saline), mannitol, dextrose, glycerol and ethanol. as well as polypeptides or proteins such as human serum albumin.
  • such composition comprise 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris pH 7.2 and 150 mM NaCl.
  • the invention more particularly relates to a composition as described above which further comprises at least one adjuvant capable of improving the transfection capacity of said substane of interest.
  • Adjuvants may be selected from the group consisting of a chloroquine, protic polar compounds, such as propylene glycol, polyethylene glycol, glycerol, EtOH, 1 -methyl L -2-pyrrolidone or their derivatives, or aprotic polar compounds such as dimethylsulfoxide (DMSO), diethylsulfoxide, di-n-propylsulfoxide, dimethylsulfone, sulfolane, dimethylforrnamide, dimethylacetamide, tetramethylurea, acetonitrile or their derivatives. These compounds are added in conditions respecting pH limitations mentioned above.
  • composition of the present invention can be administered into a vertebrate tissue.
  • This administration may be carried out by an intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, by means of a syringe or other devices.
  • Transdermal administration is also contemplated, such as inhalation, aerosol routes, instillation or topical application. Intratumoral administration is preferred.
  • composition prepared according to the invention is for the transfer into muscle cells, more preferably, by intramuscular injection routes or intravascular route
  • the administration method can be advantageously improved by combining injection in a afferent and/or efferent vessel with increases of permeability of said vessel.
  • said increases is obtained by increasing hydrostatic pressure (i.e. by obstructing outflow and/or inflow), osmotic pressure (with hypertonic solution) and/or introducing a biologically-active molecule (e.g. histamine into administered composition) (see WO 98/58542).
  • Vertebrate as used herein is intended to have the same meaning as commonly understood by one of ordinary skill in the art. Particularly, “vertebrate” encompasses mammals, and more particularly humans.
  • this invention allows repeated administration to the patient without any risk of the administered preparation to induce a significant immune reaction.
  • Administration may be by single or repeated dose, once or several times after a certain period of time. Repeated administration allows a reduction of the dose of active substance, in particular DNA, administered at a single time.
  • the route of administration and the appropriate dose varies depending on several parameters, for example the individual patient, the disease being treated, or the nucleic acid being transfen-ed.
  • the peptide of the present invention can be administered independently from a second administration consisting in administration of a composition containing at least one nucleic acid into the same target tissue.
  • the first administration can be done prior to, concurrently with or subsequent to the second administration, and vice-versa.
  • the composition administration and second administration can be performed by different or identical delivery routes (systemic delivery and targeted delivery, or targeted deliveries for example).
  • each should be done into the same target tissue and most preferably by injection.
  • the present invention also relates to a process for transferring a substance of interest into cells wherein said process comprises contacting said cells with at least one composition according to the invention.
  • This process may be applied by direct administration of said composition to cells of the animal in vivo, or by in vitro treatment of cells which were recovered from the animal and then re-introduced into the animal body (ex vivo process).
  • cells cultivated on an appropriate medium are placed in contact with a suspension containing a composition of the invention. After an incubation time, the cells are washed and recovered. Introduction of the active substance can be verified (eventually after lysis of the cells) by any appropriate method.
  • the patient in order to improve the transfection rate, the patient may undergo a macrophage depletion treatment prior to administration of the composition as described above.
  • a macrophage depletion treatment prior to administration of the composition as described above.
  • the invention further concerns a process for transferring a substance of interest into cells wherein said process comprises contacting said cells with a composition comprising at least one peptide, as defined herein above, before, simultaneously or subsequently to contacting it with the nucleic acid and wherein said . peptide is at a pH preferably higher than 6, more preferably higher than 6.5, still preferably higher than 7, even more preferably higher than 7.5 and most preferably about 8, in said composition, when a peptide is used which comprises or consists of the amino acid sequence shown in SEQ ID N0:7.
  • the invention provides the use of a peptide as defined herein above for improving the transfer of a substance of interest into a cell, wherein said peptide is at a pH preferably higher than 6, more preferably higher than 6.5, still preferably higher than 7, even more preferably higher than 7.5 and most preferably about 8, in said composition, when a peptide is used which comprises or consists of the amino acid sequence shown in SEQ ID N0:7.
  • said substance of interest is nucleic acid.
  • Said improvement of transfer of substances of interest into cells can be implemented either in vitro (or ex vivo, see above) or in vivo.
  • Treatment refers to prophylaxis and therapy. It concerns both the treatment of humans and animals.
  • a “therapeutally effective amount of a peptide or a composition” is a dose sufficient for the alleviation of one or more symptoms normally associated with the disease desired to be treated.
  • a method according to the invention is preferentially intended for the treatment of the diseases listed above.
  • the invention further concerns the use of a peptide as defined above for the preparation of a composition for curative, preventive or vaccine treatment of man or animals, preferably mammals, and more specifically for gene therapy use.
  • the present invention also relates to a process for transferring a substance of interest into cells wherein said process comprises contacting said cells with a composition prepared according to the use of the invention before, simultaneously or after contacting them with the substance.
  • This process may be applied by direct administration of said composition to cells of the animal in vivo.
  • targeted "cells” and "in vivo administration route” are defined as above described.
  • “Targeted cells” are those where polynucleotide uptake and expression occur ; they are not necessarily located into the injected tissue (site of administration).
  • administration is done into vessel and polynucleotide transfection or infection occurs at a proximal or distal site, for example in organ or tissue, such as lung, muscle, liver, kidney, heart,....
  • tumoral tissues are used as sites for the delivery and expression of a substance of interest, especially nucleic acid.
  • compositions and uses of the invention can be applied in the treatment of all kinds of diseases the treatment and/or diagnostic of which is related to or dependent on the transfer of nucleic acids in cells.
  • the compositions, and uses of the present invention may be desirably employed in humans, although animal treatment is also encompassed by the uses described herein.
  • the invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced different from what is specifically described herein.
  • Figure 1 Liposome leakage assay. Increasing amounts of the peptides JTS1, ppTGl, JTS1-K13, KALA and ppTG20 are indicated.
  • Figure 1A shows the results of the liposome leakage assay carried out at pH5.
  • Figure IB summarizes the results obtained at pH7.
  • FIG. 2 Transfection study in vitro - comparison of ppTGl, PE1 and Lipofectin.293-EBNA cells were transfected with 0.5 ⁇ g, 0.1 ⁇ g, 0.05 ⁇ g or 0.01 ⁇ g of the plasmid pTGl 1056 formulated with Lipofectin, PEI or ppTGl at the indicated charge ratio (+/-). Mock represents transfection with buffer.
  • Figure 3 Transfection study in vitro - comparison of ppTGl and Lipofectin; effect of charge ratios. 7 x 10 4 HeLa cells were plated on 24 well plates. The next day cells were transfected with 0.5 ⁇ g-or 0.05 ⁇ g pTGl 1236 formulated with Lipofectin or ppTGl at the indicated charge ratio.
  • FIG. 4 Transfection study in vitro - comparison of ppTGl and JTS1-K13. 5 x 10 4 HeLa cells were transfected with 0.5 ⁇ g or 0.05 ⁇ g pTGl 1236 formulated with ppTGl or JTS1-K13 at the indicated charge ratio. The calculations for JTS1-K13 are based on a net positive charge of +5 per molecule.
  • FIG. 6 Transfection study in vitro -comparison of ppTGl with and without pcTG90/DOPE.
  • HeLa or CHO cells were transfected with 500ng and 50ng of pTG11236, complexed with ppTGl or KALA.
  • the charge ratio +/- varied from 1, 2, 3, 4, 7 to 10.
  • Figure 9 Liposome leakage assay. Increasing amounts of the indicated peptides were incubated with POPC/Cholesterol (3:2 mol/mol) liposomes for 30 min at RT. Emitted fluorescence was plotted against peptide concentration.
  • D Comparison of ppTGl, ppTG20-D, ppTG22, ppTG23 and ppTG24 at pH7.
  • Figure 11 In vivo experiments. Numbers with an asterisk indicate the number of dead mice per group of five.
  • B) Five B6SJL mice per group were intravenously injected with 60 ⁇ g or 50 ⁇ g of pTGl 1236 complexed with ppTGl, JTS-1- K13 and ppTG20.
  • mice were sacrificed day 1 after injection and luciferase activities in the lungs were analyzed.
  • FIG. 12 Luciferase activity in RENCA tumors one day after intratumoral administration of 10 micrograms of pTGl 1236 plasmid (noted p) in the presence or absence of HPC or ppTG21 at pH8.
  • EXAMPLES :
  • a new low molecular weight cationic peptide has been synthesized, ppTGl (SEQ ID NO:2). This peptide does not contain glutamic acid residues and is capable of binding and compacting DNA, and further of causing membrane disruption.
  • HeLa cells ATCC
  • 293-EBNA cells Invitrogen were cultivated in DMEM medium supplemented with 10% fetal calf serum, 1% gentamycine, 1% glutamine and 3 g/1 glucose, in an incubator at 37°C and 5% C02.
  • WiDr (ATCC CCL-218), MDA-MB-435S (ATCC HTB-129), SW480 (ATCC CCL-228) and LoVo cell (CCL-229) were cultivated in the appropriate medium with 10% fetal calf serum, 1% gentamycine, 1% glutamine and 3 g/1 glucose, in an incubator at 37°C and 5% C0 2 .
  • Plasmids The plasmid pTGl 1056 (13787 bp; Langle-Rouault et al., 1998, J. Virol., 72, 6181-6185) is employed which carries besides the EBV oriP sequences a luciferase gene under the control of the CMV promoter, intron 1 of the HMG gene and the SV40 polyA signal. Additionally, the plasmid pTG11236 (5738 bp) with a luciferase expression cassette comprising the CMV promoter, the short SV40 16S/19S intron and the SV40 polyA signal, is also used in the experiments.
  • the plasmid pTG 11022 (7998 bp) represents a plasmid with an "empty" CMV IE promoter-driven expression cassette, containing the intron 1 of the HMG gene and the SV40 polyA signal.
  • Polypeptides The chemical synthesis of the following peptides was carried out ' by Neosystem (France). ppTG 1 (20-mer, MW 2297) (SEQ ID N0:2)
  • JTS-1 (20-mer, MW 2301) (SEQ ID N0:3)
  • JTS-1-K13 (40-mer, MW 4826) (SEQ ID N0:4)
  • the peptides were received as lyophilized powder at a purity of 80 - 97% with acetate as counter-ion in case of the cationic peptides.
  • the peptide JTS-1-K13 was synthesized in two steps. First: synthesis of JTS-1- Cystein and Cystein-K13, then formation of disulfide bridge. All peptides are diluted in milliQ water to a final concentration of at least l ⁇ g/ ⁇ l.
  • the N-termini of the peptides ppTGl and ppTG20 were covalently linked to polyethyleneglycol (PEG) MW 2000 resulting in PEG-ppTGl and PEG-ppTG20. These products were not HPLC-purified. All peptides were dissolved in milliQ water if not indicated differently at a concentration between 3.3 ⁇ g/ ⁇ l and 0.5 ⁇ g/ ⁇ l.
  • the lipid pcTG90 is as disclosed in EP 901463 the content of which is incorporated herein in its entirety (see also the formula provided above).
  • Liposomes' diameter was determined by dynamic laser light scattering using a Coulter N4 Plus submicron particle sizer (Coultronics France S.A, France). Measurements were performed within a 3nm -10 OOOnm size window with a fixed 90° scattered light angle.
  • the liposome leakage assay was carried out as described in Planck et al. (1994, J. Biol. Chem. 269, 12918-12924).
  • the liposome stock solution was diluted to a lipid concentration of 45 ⁇ M in 1.8 x assay buffer (360mM NaCl, 36 mM sodium citrate, pH 5 and pH 7).
  • a 1 :5 serial dilution of the tested peptide was carried out in a 96-well microtiter plate by transferring 20 ⁇ l of the peptide solution from one well to the next well and diluting with 80 ⁇ l H 2 0.
  • Cells were plated on 24 well plates at a density of 4 x 10 4 (293-EBNA ) or 7 xlO 4 (HeLa, Renca) cells / well in DMEM supplemented with 10% FCS. The next day, the medium was replaced by 200 ⁇ l serum-free DMEM and plasmid (pTGl 1056 or pTGl 1236) / peptide complexes or plasmid / peptide /lipid complexes were prepared in 30 ⁇ l 0.9 % NaCl or 5% glucose. After 20 min at room temperature these complexes were added to the cells which were then, incubated for 2-3 h at 37°C and 5% C0 2 before 1 ml serum-containing DMEM was added.
  • Indicated amounts of the luciferase expression plasmid pTG11236 were mixed with ppTGl and /or pcTG90/DOPE.
  • the resulting fonnulations were injected intramuscularly(30 ⁇ l), intravenously(250 ⁇ l) into mice or intratumorally (Renca tumors, lOO ⁇ l). Muscles, tumors or organs were recovered at the indicated points of time, macerated and tested for luciferase activity with the luciferase assay kit from Promega.
  • Example 1 DNA-binding activity The ability of ppTGl peptide to complex DNA was analyzed by gel retardation assays. 3 ⁇ g pTGl 1236 were mixed with increasing amounts of ppTGl (O.Ol ⁇ g to 27 ⁇ g) adding 0.9% NaCl to a final volume of 30 ⁇ l. After 20 minutes incubation at room temperature, loading buffer was added and 10 ⁇ l of the resulting solution were analyzed on a 1% agarose gel. Similar studies were carried out with JTS-1, JTS-1-K13 and KALA peptides.
  • the peptide ppTGl was tested for its capacity to cause membrane disruption in a liposome leakage assay with POPC liposomes containing the fluorescent product calcein.
  • the liposome leakage assay was carried out as described in Planck et al. (1994- supra).- Release of calcein by Triton X-100 treatment was taken as positive control reaction, incubation with water served as negative control.
  • the membranolytic activity of peptide ppTGl (20 ⁇ g as starting point) was compared with those of equal quantities (20 ⁇ g) of JTS-1, JTS-1- K13 and KALA.
  • Figures 1A 1B show that ppTGl is capable of causing membrane disruption both at pH5 (Figure lA)and pH7 (Figure IB), at least as efficiently as JTS-1 and JTS-1-K13 did.
  • Example 3 Transfection efficiency in vitro - comparison of ppTGl, Lipofectin and PEI.
  • 4xl0 4 293-EBNA cells plated on 24 well plates the day before, were transfected with 0.5 ⁇ g, O.l ⁇ g, 0.05 ⁇ g and 0.01 ⁇ g of the plasmid pTG11056.
  • the plasmid was either mixed with completely or uncompletely DNA-complexing amounts of ppTGl in 30 ⁇ l 5% glucose and after 20 min at room temperature the mixture was added to the cells which were incubated in 200 ⁇ l serum-free medium.
  • pTGl 1056 was formulated with the established transfection reagents Lipofectin and PEI. Lipofectin (Gibco BRL) and PEI were used as recommended by the manufacturer.
  • Lipofectin was added to plasmid DNA in a fourfold weight excess in 200 ⁇ l serum fi-ee medium, which was then (after 20 min at room temperature) added to the cells.
  • PEI was diluted to a lOmM solution, e.g. 75 ⁇ l were added to 0.5 ⁇ g DNA in 30 ⁇ l 5% glucose, after 30 min at room temperature transfer on cells incubated in 200 ⁇ l serum-free medium.
  • 1 ml of serum-containing DMEM was added to the cells. After twenty hours, cells were washed and luciferase activity was determined in 1/5 of the lysed cells. The luciferase activities are shown in Figure 2.
  • Figure 2 shows that transfection of 293-EBNA1 cells with 0.5 ⁇ g pTG11056 complexed with 0.65 ⁇ g ppTGl (charge ratio around 1) led to luciferase activities higher than those observed with complexes formed with Lipofectin or PEI.
  • Transfection with 0.05 ⁇ g pTG11056 mixed with 0.065 ⁇ g ppTGl (same charge ratio) still showed high transfection efficiency, while PEI and even Lipofectin formulations were at least 10-times less efficient.
  • the plasmid / peptide complexes were prepared in 30 ⁇ l of 5% glucose or 0.9% NaCl and added to the cells (200 ⁇ l serum-free medium) after 20 min at room temperature. Serum-containing medium was added after 3 h, the cells were harvested the next day.
  • the luciferase activities in the total protein lysate are presented in Figure 3.
  • Figure 3 shows that the formulation of 0.5 ⁇ g or 0.05 ⁇ g of pTGU236 with l.IJ ⁇ g or 0.117 ⁇ g of ppTGl (totally DNA-complexing amount of peptide; charge ratio 1.8) resulted in comparable luciferase activities as observed for DNA formulated with Lipofectin. Comparing transfection efficiencies of 0.05 ⁇ g ⁇ TG11236, it appeared that plasmid DNA mixed with ppTGl in 0.9% NaCl resulted in higher luciferase activities than observed for Lipofectin formulations.
  • Figure 2 and figure 3 demonstrate, that the peptide ppTGl alone is sufficient to efficiently transfer plasmid DNA into cells. At low DNA dose this efficiency is superior to established transfection reagents such as for example Lipofectin or PEI reagents.
  • Example 4 Transfection efficiency : comparison of ppTGl and JTS-1 -K13 in Hela cells
  • ppTGl and KALA were carried out in HeLa and CHO cells. 5x10 4 cells were seeded on 24 well plates. The next day, cells were transfected with 500ng and 50ng of pTG11236, complexed with ppTGl or KALA in 30 ⁇ l 0.9% NaCl. The charge ratio +/- varied from 1, 2, 3, 4, 7 to 10. Cells were harvested 20h after transfection and lysis was obtained in 100 ⁇ l Promega lysis buffer. Luciferase activity and total protein concentrations in 20 ⁇ l were determined.
  • Figures 5 a/b show that transfection with complexes comprising ppTGl was more efficient than with those formed with the peptide KALA.
  • the best charge ratio condition in HeLa cells was either 1 or 2.
  • KALA showed optimal gene transfer at a charge ratio of 7 which was 200-fold (50 ng pTGl 1236) to 4-fold (500ng) lower than for ppTGl.
  • the complex comprising ppTGl was 3000-fold more efficient than the complex comprising KALA.
  • Example 6 Transfection efficiency in vitro of complexes comprising peptide ppTGl and pcTG90 / DOPE
  • a mixture of pcTG90 and DOPE (1:1) was prepared in 350 ⁇ l of chloroform. The solution was evaporated using the vortex evaporator. The lipid film obtained was resuspended in 1ml of 5% glucose to a concentration of about 0.5mg/nil.
  • ppTGl was dissolved to 3mg/ml in water and added to the lipide suspension. The mixture was then added to the DNA (pTGl 1236) diluted in 5% glucose, and vortexed.
  • HeLa cells were seeded at a density of 6 x 10 4 cells on 24 well plates. The next day, cells were incubated with 200 ⁇ l serum-free medium, 30 ⁇ l of plasmid (50ng) /peptide / lipid mixtures were added and incubated for 3h at 37°C in 5% C0 2 . 1ml of DMEM+10% FCS was then added. The next day, the medium was removed and cells were washed with 500 ⁇ l of PBS and subsequently treated with lOO ⁇ l of Promega lysis buffer. Plates were stored at -80°C until luciferase activity was measured of 20 ⁇ l of the cell lysate. The protein assay was performed using the Pierce BCA kit. Figure 6 presents the results of this experiment.
  • Figure 6 shows that at 50ng of plasmid and a final charge ratio of 3, 5 and 10, the addition of small amounts of ppTGl (contributing 1/3, 1/5 and 1/10, respectively, of the positive charge of the complex) improved by at least 1 log the transfection efficiency of pcTG90/DOPE. This improvement was even better (2 log) when the quantity of ppTGl was increased (contributing 2/3 and 7/10, respectively, of the positive charge of the complex) at a final charge ratio of 5 and 10. At a final charge ratio of 3, ppTGl alone gave better results than pcTG90/DOPE alone.
  • 60 ⁇ g or 30 ⁇ g of the plasmid pTG11236 were mixed with peptide ppTGl and or with a pcTG90 / DOPE 1:2 mixture in 250 ⁇ l 5% glucose. After 20 min incubation at room temperature, the complexes were intravenously injected into B6SJL mice. Mice were sacrificed at day 1. The lungs were recovered, total protein was extracted and luciferase activity was analysed.
  • Figure 7 shows that gene expression into the lung can be achieved with complexes comprising pTG'11236 and ppTGl (in 5 out of 5 mice).
  • the presence of ppTGl in complexes further comprising cationic lipids improved gene expression by a factor of 10.
  • JTS1 was dissolved to lmg/ml in ImM NaOH and mixed with DNA diluted in 5% glucose. ppTGl was then added to this solution.
  • the pcTG90/DOPE ppTGl mixtures were prepared as described in Example 6. Transfection assays were performed on HeLa cells with 50ng of plasmid as described in Example 6. The results are shown on Figure 8.
  • Figure 8 shows that pTG11236 complexed with 0.9 ⁇ g of ppTGl and O.l ⁇ g of JTS1 (final charge ratio 5) increased luciferase activity by a factor of about 10 in comparison with the best formulation of ppTGl/pcTG90/DOPE (final charge ratio 5, ppTGl contributing a charge ratio of 2 (+/-), and by a factor of about 1000 in comparison with pcTG90/DOPE alone at a final charge ratio of 5.
  • ppTGl/ plasmid DNA complexes with a final charge ratio of 1-2 mediate efficient transfection of a variety of cell lines. The efficiency is comparable to, or superior to, transfection levels observed with complexes comprising Lipofectin, PEI, or multicomponent peptide complexes according to Gottschalk et al.,1996.
  • ppTG28 and ppTG29 were comparable to ppTGl and ppTG20 in liposome leakage and gene transfer assays.
  • Replacement of Leu by He or Val diminished liposome leakage activity (ppTG32 and ppTG33), and diminished gene transfer efficiency in vitro (ppTG30, ppTG31, ppTG32 and ppTG33).
  • ppTGl derivatives with wild type or mutated basic nuclear localisation signal (SV40 large T antigen) at the C-terminus retained liposome leakage and gene transfer efficiency.
  • the addition of Cys residues N- and/or C-tenninal of ppTGl did not abolish liposome leakage activity, gene transfer efficiencies were reduced.
  • ppTGl and ppTG20 derivatives with polyethylenglycol (PEG)2000 covalently linked to the N-terminus of the peptides retained the liposome leakage activity, gene transfer efficiencies, however, were strongly reduced. Further tested was a ppTG20 derivative with all amino acids in D-configuration (ppTG20-D). Liposome leakage and gene transfer activities with this peptide were retained.
  • PEG polyethylenglycol
  • Example 9 DNA binding activity ppTGl derivatives as they are indicated in Materials and Methods, were tested for their capacity to bind plasmid DNA by gel retardation assays.
  • Liposome leakage activities were analyzed on liposomes consisting of POPC and cholesterol at a molar ratio of 3:2 (mol/mol). Cholesterol is an important ubiquitous component of natural membranes determining their fluidity. Tests with such liposomes are thus closer to in vivo conditions than tests with pure POPC liposomes.
  • the peptides ppTGl, JTS-1-K13, KALA and JTS-1 were compared at pH5 and pH7.
  • FIG. 9A clearly shows that KALA could not liberate • calcein from cholesterol(chol)- containing liposomes.
  • JTS-1-K13 was also impeded in calcein release atpH7, while low level release occurred at pH5.
  • the pH-sensitive peptide JTS-1 showed high lysis activity at pH5, at pH7 this activity was reduced. ppTGl could efficiently liberate calcein from POPC/chol liposomes at pH5 and pH7.
  • Figure 9B clearly shows that at an excess of peptide over plasmid DNA (P/N 5 or 10), liposome leakage was comparable to free ppTGl.
  • P/N charge ratios
  • FIGS. 9C, 9D and 9G clearly show that the replacement of Lys in ppTGl by Arg or His, ppTG20 in D-configuration or the addition of PEG2000 N-terminally to ppTGl or ppTG20 did not reduce the membrane destructive activities of the resulting peptides.
  • Figure 9F demonstrates that the addition of two basic amino acids (ppTG28 and ppTG29), or the replacement of Leu by He (ppTG31) slightly reduced liposome leakage activity.
  • ppTGl / plasmid complexes can efficiently transfect human tumor cell lines, especially SW480 cells.
  • Figure 10B clearly shows that the replacement of Lys by Arg residues did not change the transfection efficiency, while replacing Lys by His resulted in significant reduction.
  • Figure 10C demonstrates that the C- terminal addition of SV40 large T antigen-derived NLS peptide did not influence the efficiency of gene transfer.
  • Figure 10D shows that the addition of two basic amino acid residues (ppTG28 and ppTG29) did not change transfection efficiencies. The replacement of Leu by He (ppTG30 and ppTG31), however, reduced the efficiency of gene transfer, and the replacement of Leu by Val reduced this activity even further.
  • Bafilomycin A is a specific inhibitor of the vacuolar proton pump. Treatment with Bafilomycin inhibits the acidification of late endosomes. HeLa cells were treated with Bafilomycin A (175 nM) 30 min before and throughout the transfection (1 h incubation with transfection complexes in the absence of serum). 6x10 4 cells were transfected with 150 ng pTG11236 using PEI or ppTGl. The luciferase assay was perfonned 1 day after transfection.
  • Figure 11A clearly demonstrates that gene transfer with ppTGl complexes (at charge ratios [+/-] between 2 and 3) led to luciferase activities in the lung which were comparable to those obtained with the lipoplexes.
  • Gene transfer with ppTG20 showed a general tendency to be more efficient and less toxic than ppTGl, while complexes with the peptide ppTG32 did not lead to detectable reporter gene expression. This implies that membrane-destructive activity is necessary for successful gene delivery with ppTGl -derived peptides.
  • Example 13 In vivo studies : ppTG21 as non-condensing transfection enhancer after intratumoral injection
  • RENCA cells urine kidney tumor cells, ATCC
  • RENCA cells urine kidney tumor cells, ATCC
  • the peptide ppTG21 was tested for its capacity to enhance plasmid transfer after intratumoral injection in RENCA tumor-bearing mice.
  • Ten ⁇ g of the luciferase expression plasmid pTG11236 were mixed with increasing amounts (i.e.. 0.1, 0.6, 3 or 15 micrograms) of ppTG21 at pH 8 (10 mM Tris pH8). Under these conditions ppTG21 is not capable to bind to plasmid DNA (see above).
  • experiments have been conducted in identical conditions by administering the same plasmid DNA in the presence of increasing amounts (i.e. 0.1%, 0.2%, 0.4% and 0.6 %) of HPC (Hexadecyl-phospho-choline).
  • Plasmid DNA alone, or plasmid DNA in the presence of HPC or ppTG21 were intratumorally injected in similar conditions. Next day, luciferase activities in the tumors was determined (RLU/g tumour). The results are shown in Figure 12.
  • Figure 12 clearly indicates that co-injection of 10 ⁇ g of pTG11236 with 0.6 ⁇ g ppTG21 leads to an increase of apparent gene expression. This improvement is comparable to results obtained with the non- condensing lipid HPC.
  • Trp Glu Leu Leu Leu Glu Ala 15 20
  • Trp Glu Leu Leu Leu Glu Ala Cys Cys Tyr Lys Ala Lys 15 20 25
  • Trp Glu Ala Lys Leu Ala Lys Ala Leu Ala Lys Ala Leu Ala Lys Ala Leu 1 5 10

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Abstract

L'invention concerne l'utilisation de peptides non complexants pour préparer des compositions permettant d'améliorer le transfert de substances d'intérêt dans des cellules. De telles compositions sont particulièrement utiles en thérapie génique, en vaccination et en toute situation thérapeutique ou prophylactique dans laquelle une substance d'intérêt, notamment un acide nucléique, est administrée à des cellules in vivo.
EP02716783A 2001-02-27 2002-02-15 Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique Withdrawn EP1363675A2 (fr)

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EP02716783A EP1363675A2 (fr) 2001-02-27 2002-02-15 Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique

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EP01440049 2001-02-27
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EP01440133 2001-05-15
US29318701P 2001-05-25 2001-05-25
US293187P 2001-05-25
PCT/EP2002/001646 WO2002074794A2 (fr) 2001-02-27 2002-02-15 Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique
EP02716783A EP1363675A2 (fr) 2001-02-27 2002-02-15 Utilisation de peptides non complexants pour la preparation d'une composition destinee a la transfection d'un polynucleotide dans une cellule et compositions utiles en therapie genique

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EP1969000A2 (fr) * 2005-12-06 2008-09-17 Centre National de la Recherche Scientifique Peptides penetrant dans les cellules a delivrance intracellulaire des molecules
EP1795539B1 (fr) * 2005-12-06 2010-12-01 Centre National de la Recherche Scientifique Peptides pénétrant dans les cellules à délivrance intracellulaire des molecules
CA2805403A1 (fr) * 2009-07-10 2011-01-13 Trustees Of Tufts College Systeme d'administration d'acide nucleique a base de proteine de soie biologiquement modifiee

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EP0832269A1 (fr) * 1995-06-07 1998-04-01 Baylor College Of Medicine Transporteurs d'acide nucleique servant a introduire des acides nucleiques dans une cellule
EP1161957A1 (fr) * 2000-05-26 2001-12-12 Transgene S.A. Complexe pour le transfert d'une substance anionique d' intérêt dans une cellule

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