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WO2002010197A1 - Peptides cationiques amphipathiques et leur application dans les vecteurs de transfert genique - Google Patents

Peptides cationiques amphipathiques et leur application dans les vecteurs de transfert genique Download PDF

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Publication number
WO2002010197A1
WO2002010197A1 PCT/ES2001/000274 ES0100274W WO0210197A1 WO 2002010197 A1 WO2002010197 A1 WO 2002010197A1 ES 0100274 W ES0100274 W ES 0100274W WO 0210197 A1 WO0210197 A1 WO 0210197A1
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Prior art keywords
peptide
polynucleotide
dna
cell
vector
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English (en)
Spanish (es)
Inventor
Jesús FOMINAYA GUTIERREZ
Antonio Bernad Miana
María Angustias GASSET VEGA
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MEDPLANT GENETICS SL
Consejo Superior de Investigaciones Cientificas CSIC
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MEDPLANT GENETICS SL
Consejo Superior de Investigaciones Cientificas CSIC
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Priority to AU2001269146A priority Critical patent/AU2001269146A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • 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 is included within the area of Biotechnology and Biomedicine, and relates, in general, to the transport of nucleic acids into the cellular interior (gene transfer) and their applications both in the experimental area (transfections ) as therapeutic (gene therapy).
  • the invention relates to new amphipathic cationic peptides useful as gene transfer systems and non-viral vectors comprising said peptides.
  • Viral DNA transfer vectors have attracted the attention of gene therapy specialists and are the basis of the majority of clinical trials currently underway [Anderson F. Nature 1998; 392: 25-30] although efficiency levels can be very variable and there may be a risk of severe immune response to viral vector elements.
  • non-viral vectors seem to be more successful as gene transfer agents because they are easier to handle because they have a greater range of Applications.
  • the exponential development in this field reveals a promising future for non-viral vectors as a logical alternative to viral vectors for gene therapy, providing similar advantages and eliminating the inherent drawbacks of viruses.
  • the term "non-viral” is a concept that encompasses a wide variety of unrelated structures, such as polylysine conjugates, linear and branched polycations, complete or fractured dendrimers, nanospheres, polysaccharides, cationic liposomes, modular fusion proteins and peptides.
  • peptides as gene transfer vectors is in its initial research and development period.
  • the peptides offer simplicity, ease of production and correct control of their composition compared to most of the other non-viral vectors, which are based on complex, and often poorly defined structures of a polymeric nature.
  • the peptides were initially used as enhancers of other systems, contributing their fusogenic capacity [Plank C, Oberhauser B, Mechtler K, Koch C, Wagner. J. Bi ol. Chem 1994; 269: 12918-12924].
  • peptides offer other functions subsequently J exploited, including cell recognition and DNA binding, which make them true vectors of gene transfer, either alone or in association with other structures [Hart SL, Collins L, Gustafsson K, Fabre J. Gene Ther. 1997; 4: 1225-1230; Niidome T, Ohmori N, Ichinose A, ada A, Mihara H, Hirayama T, et al. J. Biol. Chem 1997; 272: 15307-15312].
  • advantages of peptides can be considered 1) their biological nature, 2) their biodegradability, 3) the possibility of making modifications, which in turn allow the incorporation of additional structures to confer new characteristics (solubility, stability, specificity, etc. ), and 4) its small molecular size.
  • a common denominator of the mentioned activities is the requirement of positive charges, which (i) allow to establish ionic interactions with nucleotide phosphates and induce their compaction, (ii) allow to recognize acidic components present in the membranes (proteoglycans, acidic lipids, etc.). ), facilitating membrane permeabilization phenomena, and even (iii) favor nuclear tropism.
  • the KALA peptide [Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr. Biochemistry 1997; 36: 3008-3017], useful as a gene transfer system, simultaneously combines the ability to bind DNA and alter membranes in the same peptide molecule.
  • said peptide has a condensation capacity of relatively poor genetic material, a relatively large cytotoxicity and a transfer efficiency of relatively small genetic material, which limits its potential application.
  • the invention generally faces the problem of obtaining new non-viral vectors for the transfer of genetic material, and, in particular, the problem of searching for new non-viral vectors of transfer of genes based on cationic peptides that simultaneously combine the ability to bind polynucleotides and alter biological membranes, with an improved condensation capacity of genetic material, and / or with reduced cytotoxicity and / or with improved transfer efficiency of genetic material.
  • the solution presented by this invention is based on the fact that the inventors have observed that the combination of two or more characteristics selected from the use of some basic amino acid residues (arginine) instead of others (lysine), the modification of the angle of said residues of amino acids with respect to the axis of the helix, the absence of acidic amino acid residues, the placement of basic amino acid residues at the two ends of the peptide and the placement of, preferably, a residue of an aromatic amino acid at position 3 of the peptide sequence, a cationic, amphipathic, small peptide can be obtained, which simultaneously has the ability to bind a polynucleotide and alter biological membranes, useful for the construction of a non-viral transfer vector, with a capacity to condensation of improved genetic material, and / or with a reduced cytotoxicity and / or with an improved transfer efficiency of genetic material.
  • the efficiency in the transfer of genetic material of said vector is very high and favorably comparable with that of other well established and / or commercial gene transfer systems.
  • the absence of inhibition by serum components and the low toxicity of this new vector place it in an advantageous situation for its application in vivo.
  • the ability of a peptide developed in this invention to bind and condense genetic material is shown in Examples 2 and 3, while in Example 4 its ability to interact with biological membranes is illustrated.
  • Examples 5 and 6 show the efficacy of the peptide developed in this invention to transfer genetic material and show that said efficiency is equivalent to or greater than that obtained by other known systems (using the luciferase gene as indicator gene values have been obtained of activity greater than 10 10 URL / mg of protein).
  • Example 7 illustrates a possible in vivo application of a non-viral vector provided by this invention and the use of a permeabilization enhancing compound (peptide permeabilizing capacity) of membranes, such as a fusogenic peptide.
  • a permeabilization enhancing compound peptide permeabilizing capacity of membranes, such as a fusogenic peptide.
  • an object of this invention is a cationic, amphipathic peptide, which simultaneously has the ability to bind polynucleotides and alter biological membranes.
  • a synthetic or recombinant process for obtaining said peptide constitutes an additional object of this invention.
  • Another additional object of this invention is a non-viral gene transfer vector comprising said peptide and a polynucleotide coupled (not covalently) to said peptide.
  • Another additional object of this invention is a cell transfected with said non-viral gene transfer vector.
  • Another additional object of this invention is a composition comprising said non-viral gene transfer vector.
  • Another additional object of this invention is a pharmaceutical composition comprising said non-viral gene transfer vector.
  • Another additional object of this invention is the use of said peptide in the preparation of a non-viral gene transfer vector.
  • Another additional object of this invention is the use of said peptide in the preparation of a pharmaceutical composition, suitable for gene therapy, which comprises a non-viral gene transfer vector containing said peptide suitable for gene therapy.
  • Figure 1A illustrates the binding of the RAWA peptide to DNA analyzed by electrophoresis in agarose gels (Example 2).
  • Figure IB is a graph showing the binding of RAWA and KALA peptides to DNA, studied by means of extinction measurements of peptide tryptophan fluorescence (Qobs) as a function of the load ratio (Example 3).
  • Figure 1C is a graph showing the extent of peptide induced DNA condensation.
  • RAWA and KALA determined as a percentage of the displacement of the intercalating agent ethidium bromide (Example 3).
  • the values for the RAWA peptide have been represented by white dots, while the values for the KALA peptide have been represented by black dots.
  • Figure 2 illustrates the ability to interact with membranes of the RAWA and KALA peptides.
  • Figure 2A is the representation of the variation in the percentage of lipid mixture induced by RAWA (white dots) and by KALA (black dots) as a function of the peptide / phospholipid molar ratio.
  • Figure 2B is a graph that shows the membrane permeabilizing capacity, measured as a percentage of intravesicular content release, induced by RAWA (white dots) and by KALA (black dots) as a function of the peptide / phospholipid molar ratio.
  • Figure 2C is a graph showing the decrease in membrane permeabilizing activity of RAWA and DNA peptide mixtures, with respect to that of free RAWA, as a function of loading ratio, to total peptide to lipid molar ratios. of 0.003 (white dots) and 0.005 (black dots).
  • Figure 4 illustrates the characteristics of RAWA-mediated gene transfer.
  • Figure 4A is a bar chart showing the functional transport capacity •, in relation to the value obtained in a chloroquine treatment, obtained with the gene transfer vectors based on the RAWA peptide as a function of the ratio of loading them.
  • Figure 4B is a bar chart showing the functional transport capacity, in relation to the value obtained in a chloroquine treatment, of the gene transfer vectors based on the RAWA peptide as a function of the load ratio ; optionally in the presence of chloroquine or free peptide
  • peptiplexes peptide-DNA complexes
  • cells treated with peptiplex CR (+/-) 2 were incubated together either with 50 ⁇ M chloroquine (2 + Ch) or with an additional amount of free peptide equivalent to that used for the preparation of peptiplex CR (+/-) 20] .
  • Figure 5 illustrates the effect of the dose of DNA on the efficiency of DNA transfer mediated by the RAWA peptide.
  • Figure 5A is a bar chart illustrating the effect of the dose of DNA on the functional transport of the RAWA-DNA vector formed at a load ratio 4 (+/-).
  • Figure 5B is a bar chart showing the effect of the load ratio of RAWA-based transfer vectors on the degree of functional transport using 0.5 ⁇ g of DNA.
  • Figure 6 illustrates the potential of the RAWA peptide as an effective gene transfer system.
  • Figure 6A is a bar chart showing the results of a comparative study of the RAWA peptide with the best reagents of known transfer using the COS-7 cellular system as a reference.
  • the abbreviations used are: CaPh, optimized protocol of calcium phosphate [Jordan M, Schallhorn A, Wurm FM. Nucleic Acids Res 1996; 24: 596-601], PEI, polyethyleneimine [Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. Proc. Na you. Acad. Sci.
  • FIG. 6B is a bar chart showing the effect of cellular context on RAWA-mediated DNA transfer activity; for this, the various mammalian cell lines were treated under conventional conditions with transfer vectors consisting of 2 ⁇ g of pSV2LUC and RAWA at a loading ratio of 4 (+/-), using as equal control the same amount of DNA transported by Lipofectamine Plus (dot bars).
  • the invention provides a cationic, amphipathic peptide, which simultaneously has the ability to bind a polynucleotide and biological membranes, hereinafter, peptide of the invention, comprising an amino acid sequence of general formula (I) aal-aa2-aa3-aa4- aa5-aa6-aa7-aa8-aa9-aal0-aall-aal2-aal3-aal4- aal5-aal6-aal7-aal8-aal9-aa20-aa21-aa22-aa23
  • the peptide of the invention may be formed by repeated units of the sequence of general formula (I), consecutive and / or spaced by flexible or proline-rich sequences.
  • the general formula sequences are not limited to:
  • the peptide of the invention comprises two or more sequences of general formula (I) linked each other, independently, consecutively or spaced by flexible or proline-rich sequences.
  • Another preferred class of peptides of formula (I) is constituted by those peptides (I) in which aa3 is a residue of an aromatic amino acid, preferably tryptophan.
  • the peptide of the invention has the amino acid sequence shown in the SEC. ID. N °: 1. This peptide has been called RAWA (for its first four amino acid residues of the amino terminus) and will serve as a reference to illustrate the nature of this invention.
  • the RAWA peptide is a cationic, amphipathic peptide, capable of forming an amphipathic helix whose polar face is the amino acid residues of a basic (cationic) character. Its amphipathic character contributes to generating a destabilizing or disruptive activity of biological membranes. Unlike other gene transfer vectors, the RAWA peptide is small in molecular size and resembles natural DNA condensation agents (protamines, HMGs). The RAWA peptide is capable of binding and compacting polynucleotides as well as altering membrane permeability.
  • the RAWA peptide interacts spontaneously with polynucleotides and forms stable complexes that bind to cell membranes and enable the effective introduction of genetic material.
  • This peptide is soluble in aqueous solutions and has an approximately 40% helical structure determined by circular dichroism [Example 1].
  • the peptide of the invention includes a number of features that They increase the binding and condensation capacity of polynucleotides and reduce cellular toxicity, maintaining the amphipathic capacity necessary for biological membrane binding. Among these characteristics are (i) the absence of acidic amino acid residues and the modification of the ends of the peptide in order to maintain positive charges, thereby avoiding the presence of additional structures outside the interaction edges that could alter the potential condensation capacity of polynucleotides, and (ii) the introduction of an aromatic amino acid at position 3 (aa3) that seems to exert an effect on the anchoring of the peptide to the biological membrane.
  • the RAWA peptide has the following characteristics in relation to the KALA peptide:
  • the combination of all or some of the previously mentioned characteristics has led, surprisingly, to the development of the peptide of the invention, that is, a cationic, amphipathic, small peptide, which simultaneously has the ability to bind a polynucleotide and alter biological membranes, useful for the construction of a non-viral gene transfer vector, with an improved condensation capacity of genetic material, and / or with a reduced cytotoxicity and / or with an improved transfer efficiency of genetic material.
  • the peptide of the invention can be obtained either by chemical synthesis using conventional solid phase synthesis methodology, for example, with standard Fmoc chemistry [Gausepohl H, Boulin C, Kraft M, Frank RW. Pept Res. 1992; 5: 315-320; King DS, Fields CG, Fields GB. Int. J. Pept. Protein Res. 1990; 36: 255-266], or by recombinant DNA techniques, for example, by expression in a suitable expression system of a DNA sequence encoding the peptide of the invention or fusion proteins or not containing it. Accordingly, the invention further provides a nucleic acid sequence encoding the peptide of the invention, as well as a method for obtaining a peptide of the invention comprising the expression of said nucleic acid sequence in a system of appropriate expression
  • the peptide of the invention can be used in the construction of a non-viral gene transfer vector due to its binding and condensation properties • of polynucleotides, binding to biological membranes and transfer of polynucleotides into the cell interior, as will be described in greater detail below.
  • the polynucleotide that can be used in the vector of the invention comprises any nucleic acid and mixtures thereof, for example, DNA, RNA, or hybrid molecules thereof [both between said molecules (DNA-RNA hybrids) and between said molecules and PNA (for example, PNA-DNA hybrids)]; with linear or circular structures; single-stranded, double-stranded or multi-stranded, for example three-stranded or four-stranded.
  • the polynucleotide is part of an artificial chromosome.
  • said polynucleotide is a nucleic acid with therapeutic value, for example, an antisense polynucleotide, or a polynucleotide encoding a product.
  • polynucleotide may have all or part of its nucleotide bonds stabilized by non-phosphodiester type linkages, for example, phosphorothioate, etc. [US Patent 5,990,089, the description of which is included as reference].
  • the binding capacity of a peptide of the invention, in particular of the RAWA peptide, to a polynucleotide, as well as the ability to condense and transfer genetic material thereof, has been tested using as indicator gene the Luciferase gene.
  • the vector of the invention has all or most of the advantages of the different gene transfer systems known to date, related to the ability of polynucleotide condensation, biological membrane binding, transport through them and size of the conveyor
  • the vector of the invention has a good ability to bind and condense polynucleotides, which is a critical feature to increase the stability of the genetic material and reduce the size of the peptide-polynucleotide complexes (peptiplexes), and larger than other systems.
  • the polynucleotide condensation capacity of the RAWA peptide is superior to that of the system based on the use of the KALA peptide (Example 3); in that case, it seems that the presence of arginine basic amino acid residues as cationic moieties (which provide guanidinium groups) for interaction with the phosphate anions present in the polynucleotide allows a correct equivalent for the peptide-polynucleotide interaction when compared with a amino group, based on the characteristic parallel binding "hydrogen z itterionic" with the phosphate group and with the potential to form hydrogen bonds with the nitrogen bases [Vigneron JP, Oudrhiri N, Fauquet M, Vergley L, Bradley JC, Basseville M, Lehn P, Lehn JM Proc.
  • the peptide of the invention presents. an optimized helical structure, which together with the placement of basic amino acid residues, which provide cationic groups, at both ends of the peptide, increases the binding capacity thereof to polynucleotides.
  • the peptide of the invention shows an important helical fraction and an improvement in the levels of polynucleotide condensation compared to other peptides that do not have the innovative characteristics present in the peptide of the invention.
  • the levels of condensation induced by the peptide of the invention are within the defined range for protamines, which may be related to an adequate dissociation of the polynucleotide in the nucleus.
  • maximum condensation is achieved at a CR (+/-) loading ratio of 4.0, and is associated with the formation of oligomers of the peptide bound to the polynucleotide.
  • the degree of membrane alteration depends on several factors including the hydrophilic / hydrophobic balance, the length of the propeller and the presence of residues in certain positions, such as tryptophan on the first turn of the propeller, which imposes restrictions on orientation [Braun P, and von Heijne G. Biochemistry. 1999; 38: 9778-9782]. Taking into account all these factors in the design of the peptide of the invention, in particular, of the RAWA peptide, it has been possible to reduce the damage to the membrane and the cytotoxicity of the vector of the invention, which are characteristics that limit the application of the systems of gene transfer.
  • the cell binding of the peptiplexes requires a positive net charge and presumably takes place through the interaction with anionic cell surface proteoglycans as described for some viruses and other non-viral cationic vectors [Summerford C, Samulski RJ. J. Virol. 1998; 72: 1438-1445; Mounkes LC, Zhong W, Cypress- Palacin G, Heath TD, Debs RJ. J. Biol. Chem 1998; 273: 26164-26170].
  • the peptide-polynuleotide complex exhibits cell binding properties, the ability to alter the membrane is inherent in the peptide of the invention in the free state.
  • the peptiplexes follow the endocytosis pathway and the final expression of the gene is dependent on the addition of endosomolytic agents such as chloroquine.
  • endosomolytic agents such as chloroquine.
  • the peptide of the invention alters the membrane permeability, probably plasma and facilitates the entry of peptiplex regardless of the presence of chloroquine.
  • the size of the polynucleotide transporter determines the pharmacokinetics of peptiplex and is, therefore, another important factor to consider.
  • Most commercially available vectors are based on large structures.
  • the use of small and well-defined covalent structures, such as peptides, can facilitate the process of producing gene transfer vectors and the formation and characterization of peptiplexes.
  • the unfriendly peptides exhibit self-aggregation properties that basically depend on the concentration of the peptide and environmental conditions such as pH, ionic strength and temperature, which can hinder the molecular definition of the system or at least require it. definition conditions away from those used in cell cultures.
  • the results obtained in the present invention with the RAWA peptide demonstrate the existence of oligomeric forms of said peptide in the peptiplexes.
  • the supposed The presence of these forms under the experimental conditions for gene transfer does not constitute a limitation, since high levels of efficiency in gene transfer are observed as there is no significant inhibition by serum components.
  • serum inhibition is usually related to the formation of large aggregates as a result of neutralization of complexes with serum components [Yang P, Huang L. Gene ' her. 1997; 4: 950-960].
  • the presence of oligomers of the RAWA peptide in the peptiplex allows consideration of the possibility of including a fusogenic peptide and taking advantage of its properties to increase the overall effectiveness of the process of functional gene transfer.
  • the JTS-1 fusogenic peptide [Gottschalk S, Sparro JT, Hauer J, Mims MP, Leland FE, Woo SL, et al. Gene Ther. nineteen ninety six; 3: 448-457], an amphipathic peptide, with a helical structure, was incorporated into the RAWA-polynucleotide complexes (Example 7) to obtain a ternary complex with the intrinsic ability to alter the membrane, which opens up the possibility of adapting this system for in vivo gene therapy.
  • the JTS-1 peptide was incorporated into the outer surface of the peptiplex through interaction with the RAWA peptide that gives it an efficient polynuleotide transfer capacity under stoichiometry conditions that still preserve the overall load positive necessary for cell binding.
  • the vector of the invention has numerous applications related to the transport of polynucleotides into the cellular interior, both in basic or applied research (transfections) and in Biomedicine (gene therapy).
  • the invention provides an improved gene transport system, in particular, a non-viral gene transfer vector, based on a peptide cationic, amphipathic, with an improved capacity for polynucleotide condensation and reduced cytotoxicity, but which maintains the binding and disruption activities of biological membranes.
  • the efficiency observed with the peptide of the invention, in particular with the RAWA peptide is very high and favorably comparable with other well recognized gene transfer systems. In fact, it seems that it is the first time that a gene transfer vector based on a peptide reaches that level. The absence of serum inhibition and the low toxicity place the vector of the invention in an advantageous situation for its application in vivo.
  • the invention provides a transformed cell comprising a vector of the invention.
  • said cell is a eukaryotic cell, preferably mammalian.
  • composition of the invention comprising a vector of the invention, together with, optionally, one or more agents selected from polynucleotide masking agents, cell recognition agents, permeabilization agents. biological membranes, subcellular localization agents, and mixtures thereof, even when part of these activities are already contemplated in the vector.
  • the agents that mask the polynucleotide are chemical compounds, for example, polyethylene glycol, lipids, etc., which mask all or part of a polynucleotide in order to increase its half-life in the bloodstream by inhibiting the attack of degrading products such as nucleases present in the circulatory system and / or interfering with its uptake by the reticulum endoplasmic
  • the cell recognition agents are molecules, for example, antibodies against cell surface antigens, ligands for cell surface receptors, peptide hormones, etc., capable of recognizing a component present on the surface of a target cell.
  • Biological membrane permeabilization agents are compounds that help the polynucleotide pass a biological membrane and include compounds that neutralize the polynucleotide load and allow the polynucleotide to pass through the hydrophobic interior of the membrane, as well as antipathic compounds, peptides, glycolipids, phospholipids, bile salts and detergents.
  • the vector of the invention has a good ability to transport the polynucleotide into the cell through the membrane, which may make the addition of additional membrane permeabilization agents unnecessary, sometimes, if desired, the composition of the invention may contain other permeabilization agents that contribute to the peptide of the invention in membrane disturbance.
  • Subcellular localization agents are compounds, generally, small peptide sequences, capable of recognizing a subcellular component, for example, nucleus, ribosomes, lysosomes, mitochondria, etc., in a target cell.
  • the invention also provides a pharmaceutical composition, hereinafter pharmaceutical composition of the invention, comprising a vector of the invention, or a composition of the invention, and one or more pharmaceutically acceptable excipients.
  • the vector or composition of the invention, for therapeutic applications can be formulated in multiple ways, in a form of administration suitable for administration by any appropriate route, including, orally, parenterally or topically.
  • a review of the different drug dosage forms and of the excipients for their preparation may be found in Tratado de Farmacia Galénica, C. Faul ⁇ i Trillo of, Luzan 5, SA de Ediations, I edition, 1993.
  • the vector of the invention, or a composition of the invention or a pharmaceutical composition of the invention can be employed for the introduction of a polynucleotide into a cell.
  • the cell preferably, will be a eukaryotic cell, more preferably, a mammalian cell, including cells of humans.
  • the introduction of the polynucleotide into the cell can be performed in vivo, ex vivo or in vitro. Therefore, the invention provides a method for in vitro introduction of a polynucleotide into a cell comprising contacting said cell with a vector of the invention, or a composition of the invention or a pharmaceutical composition of the invention.
  • the RAWA peptide is soluble in an aqueous solution and has a helical structure of approximately 40%, according to its circular dichroism (CD) spectrum at 25 ° C [Padmanabhan S, Zhang W, Capp MW, Anderson, CF, Record MT Jr. Biochemi stry 1997, 36: 5193-5206].
  • CD circular dichroism
  • EXAMPLE 3 Determination of DNA binding and condensation by fluorescence
  • the binding of RAWA and KALA peptides to DNA has been determined, as well as the condensation of DNA by said peptides by fluorescence assays based on the extinction of tryptophan fluorescence from peptides and ethidium bromide displacement, previously intercalated in the nucleic acid.
  • Figures IB and 1C show the DNA binding profiles of the RAWA and KALA peptides obtained in the tryptophan fluorescence extinction and ethidium bromide displacement assays, respectively.
  • Fluorescence measurements were performed on an SLM-Aminco 8100 spectrofluorimeter (Urbana, IL) at 37 ° C, using a magical angle polarizer configuration.
  • Peptide binding to DNA pSV2LUC
  • Peptide solutions 3-5 ⁇ M
  • Peptide solutions were titrated by successive additions of a DNA plasmid (pSV2LUC).
  • the observed fluorescence extinction (Qobs) was calculated as (FF 0 ) / F 0 , where F is the intensity of the fluorescence observed in the presence of DNA and F 0 is the fluorescence intensity of the free peptide.
  • EtBr ethidium bromide
  • the displacement of ethidium bromide (EtBr) was determined by measuring the reduction in fluorescence emission at 595 nm (excitation at 510 nm) of a plasmid DNA solution (30 ⁇ M phosphate-DNA) labeled with the intercalating agent (molar ratio DNA: EtBr of 29: 1) by adding increasing amounts of the RAWA and KALA peptides [Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr. Bi ochemistry 1991; 36: 3008-3017].
  • the ethidium bromide displacement was calculated as (FF £ ) / (F b -F f ) where F is the intensity of the fluorescence observed in the presence of DNA and F f and F b are the fluorescence intensities of the EtBr in the absence and presence of DNA, respectively.
  • the binding of the RAWA peptide to the DNA is biphasic. The extent of "shutdown" (extinction) increases sharply to a maximum at a CR (- / +) of 0.25, decreasing to a plateau at a CR (- / +) of 1.0.
  • lipid vesicles (POPC: POPG, 70:30) of 100 nm in diameter were prepared by hydration-extrusion in 25 mM Hepes buffer pH 7.0, 100 mM NaCl and 1 mM EDTA.
  • the vesicles were labeled with 1% NBD-PE and 0.6% Rh-PE, and diluted in a 1: 9 ratio with unlabeled vesicles.
  • hydration was performed in 25 mM Hepes buffer pH 7.0, 20 mM NaCl, 12.5 mM ANTS, DPX 45 mM and 1 mM EDTA.
  • the permeabilization percentage was calculated as the relative increase in fluorescence intensity at 530 nm (excitation at 386 nm), with F 0 and Fioo being intensity values in the absence of peptides and in the presence of 0.5% Triton X-100 (v / v), respectively.
  • the KALA peptide has a disturbing membrane action in such a way that it makes it competent for lipid mixing (fusogenic capacity) at levels of peptide / lipid ratios greater than those observed in the lithic activity test (Figure 2A).
  • the RAWA peptide lacks a fusogenic potential and maximum lytic activity requires a higher peptide / lipid ratio than the KALA peptide. Both results indicate that the RAWA peptide acts as a transmembrane pore-forming sequence and with interference of species added in aqueous solution [Brasseur RJ Biol. Chem 1991; 266: 16120-16127; Rapaport D, Peled R, Nir S, Shai Y. Biophys. J. 1996; 70: 2503-2512].
  • the pSV2LUC expression plasmid includes the luciferase gene under the control of the SV40 early promoter.
  • Peptide / DNA complexes were prepared by incubating 2 ⁇ g of plasmid DNA (p ⁇ V2LUC) with amounts variables of each peptide in 50 mM Hepes buffer pH 7.5, 50 mM KC1, 5 mM MgCl 2 , at room temperature for 15 minutes.
  • plasmid DNA p ⁇ V2LUC
  • cells were seeded in 12-well plates at a density of 5 x 10 4 cells / well, grown overnight at 37 ° C. The growth medium was changed with 1 ml / well of fresh medium 30 minutes before adding the peptide-DNA complexes (100 ⁇ l / well). The cells were incubated for 16 h at 37 ° C with the peptiplexes and, after a medium change, an additional 24 h, before collection for analysis. All trials were performed in duplicate.
  • Luciferase activity was determined using the conventional methodology [Fominaya J, Wels W. J. Biol. Chem. 1996; 271: 10560-10568], using 150 ⁇ l of Used and 5 ⁇ l of test sample. Enzymatic activity was followed for 10 seconds on a luminometer (LUMAT LB 9501,
  • Luciferic activity was calculated as relative units of light per mg of total cellular protein (URL / mg).
  • Figure 3A shows the levels of cytotoxicity, expressed as a percentage of viability of COS-7 cells transfected with said complexes, at different CRs (as a reference value of 100% viability cells treated with a placebo were used).
  • CR (+/-) chloroquine is essential for a successful gene transfer.
  • DNA-RAWA complexes act independently of additive and offer efficiencies that increase in parallel with the total amount of peptide. This demonstrates that the peptide / DNA complexes are effective in terms of cell binding and initial transport of DNA into the cell interior but complexes at low stoichiometry (CR (+/-)) are unable to reach the cytosol and are retained in endosomes. . This behavior suggests the existence of at least two peptide forms, each responsible for a different activity.
  • peptiplex peptide can mediate the binding of complexes to the cell membrane, while the free peptide could modify the permeability of the membrane. This hypothesis was confirmed by analyzing the effect of the addition of the free peptide to cells incubated with peptiplexes in a CR (+/-) ratio of 2 in the absence (negative control) and in the presence (positive control) of chloroquine compared to prepared peptiplexes at elevated CR (+/-) levels of 20.
  • Figure 5A describes the effect of increasing amounts of plasmid DNA on a gene transfer system mediated by the RAWA peptide.
  • CR molar loading ratio
  • the RAWA peptide offers similar results to those obtained with most of the best commercially available gene transfer agents, whether used at CR (+/-) levels of 4.0 in the presence of chloroquine ( Figure 6A) and in CR levels (+/-) of 16.0 in the absence of additives (data not shown).
  • the efficiency of a vector based on the RAWA peptide in COS-7 cells is similar to that obtained with Lipofectamine Plus® (Gibco-Life Technologies), Superfect® (Quiagen), GenePorter® (Gene Delivery Systems) or FuGene® (Boehringer Mannheim , Ind) ( Figure 6A).
  • Figure 6B shows a comparative study to evaluate the efficiency of the RAWA peptide on different cell lines, using Lipofectamine Plus® as internal control.
  • mice fibroblasts NIH-3T3; human kidney embryonic cells transformed with E1A, 293; human kidney embryonic cells transformed with E1A and SV 40, 293T; MCF-7 human breast adenocarcinoma cells; HT-1080 human fibrosarcoma cells; HeLa human adenocarcinoma cells; canine osteosarcoma D17; human umbilical vein endothelial cells (HU ⁇ / EC); human umbilical cord cells ÉCV304; and B16 mouse melanoma cells.
  • Peptiplexes and luciferase assays were prepared as described in Example 5.
  • the RAWA peptide meets all the criteria required for a gene vector in transfection assays. However, with the exception of pharmacokinetic doubts, its in vivo applications may be limited because it requires a free peptide fraction to be able to exert a disruptive membrane activity. To overcome this limitation, a fusogenic peptide has been incorporated into the peptiplex based on the RAWA peptide ( Figure 7). As an example fusogenic entity, JTS-1, an amphipathic acid peptide optimized from the amino-terminal sequence of influenza hemagglutinin [Gottschalk S, Sparrow JT, Hauer J, Mims MP, Leland, has been used FE, Woo SL, et al. Gene Ther.
  • JTS-1 binds to peptiplex by interaction with the RAWA peptide.
  • Preincubation of both peptides before DNA addition eliminates the fusogenic effect obtained when JTS-1 is added to the peptiplex comprising the RAWA peptide.
  • the activity of said fusogenic peptide is much lower when it is added trans to cells treated with RAWA-DNA complexes.
  • the stoichiometry used in this experiment is the result of the JTS-1 and different CR (+/-) titrations of the RAWA-DNA complexes, in a range of 3 to 8.

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Abstract

L'invention concerne des peptides comprenant des restes d'acides aminés de base aliphatiques et aromatiques, des restes d'acides aminés de base dans les deux extrémités du peptide et, de préférence, un reste d'un acide aminé aromatique dans la position 3 de la séquence du peptide. Ces peptides présentent simultanément la capacité d'unir un polynucléotide et d'altérer des membranes biologiques, et servent à construire des vecteurs non viraux de transfert génique, présentant une capacité de condensation de matière génétique améliorée, et/ou une cytotoxicité réduite et/ou une efficacité de transfert de matière génétique améliorée. L'invention trouve son application dans le domaine du transfert génique à des fins expérimentales et thérapeutiques.
PCT/ES2001/000274 2000-07-20 2001-07-06 Peptides cationiques amphipathiques et leur application dans les vecteurs de transfert genique Ceased WO2002010197A1 (fr)

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US20150314011A1 (en) * 2012-12-07 2015-11-05 The Queen's University Of Belfast Amphipathic peptide

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WO1999001379A1 (fr) * 1997-07-05 1999-01-14 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Procede de preparation de composes de titanate zirconate de plomb

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CA2303908A1 (fr) * 1997-09-18 1999-03-25 Gene Therapy Systems, Inc. Modification chimique d'adn a l'aide de conjugues d'acide nucleique peptidique

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Publication number Priority date Publication date Assignee Title
WO1999001379A1 (fr) * 1997-07-05 1999-01-14 The Secretary Of State For Defence In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland Procede de preparation de composes de titanate zirconate de plomb

Non-Patent Citations (2)

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Title
UHEREK C. ET AL.: "Modular fusion proteins for receptor-mediated gene delivery", NATO ASI SER. H., vol. 105, 1998, pages 167 - 170, XP002909658 *
WYMAN T.B. ET AL.: "Design, synthesis and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers", BIOCHEMISTRY, vol. 36, 1997, pages 3008 - 3017, XP002088731 *

Cited By (5)

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Publication number Priority date Publication date Assignee Title
US20150314011A1 (en) * 2012-12-07 2015-11-05 The Queen's University Of Belfast Amphipathic peptide
US9744244B2 (en) * 2012-12-07 2017-08-29 The Queen's University Of Belfast Amphipathic peptide
US10188744B2 (en) 2012-12-07 2019-01-29 The Queen's University Of Belfast Amphipathic peptide
US20190091344A1 (en) * 2012-12-07 2019-03-28 The Queen's University Of Belfast Amphipathic peptide
US10500287B2 (en) * 2012-12-07 2019-12-10 The Queen's University Of Belfast Amphipathic peptide

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