KR20020007382A - Membrane disruptive peptides covalently oligomerized - Google Patents

Abstract

The present invention relates to modified membrane disrupting peptides. The invention also relates to a delivery complex consisting of said modified membrane disrupting peptide and to the use of said delivery complex. The modified membrane disrupting peptides are modified to form covalently attached multimers.

Description

Membrane disruptive peptides covalently oligomerized

In the field of gene therapy, there is a need for simple non-viral gene delivery agents that can be used to efficiently transfect cells. Cationic peptides have been used to condense DNA by electrostatic interactions and to prepare simple and reproducible peptide: DNA preparations. Methods for their preparation and purification are well documented. Cationic peptides are also used as chemical anchors for the introduction of another peptidic or non-peptidic entities that aid in the passage of genes packaged into the nucleus of the target cell required at an extracellular location. Has been used.

When using cationic peptides as in vitro delivery agents, the presence of exogenous agents believed to increase the ability of the gene to transport away from intracellular vesicles that encapsulate during or after cell uptake. Transfection has proven to be optimal only under Examples of such exogenous agents are chlorokines or membrane disrupting peptides. Destabilization of biological membranes is central to the role of these agents. Structural breakdown of any membrane that acts as a barrier to the passage of the delivery complex is believed to increase the ability to access the nucleus. However, in many in vivo applications, the use of exogenous agents, such as small organic molecules chlorokines, apart from complicating formulations, and by the in vivo clearance mechanisms and diffusion rates found between small and large molecules, It is reasonable to assume that it is disturbed. Fusogenic and membrane disrupting peptides, such as those described by Wagner (Wagner et al., PNAS, 89, 7934-8, 1992) or Smith (international patent application WO 97/35070), can be condensed prior to complex formation. It may be immobilized on structure or preformed peptide: DNA, but this will also complicate preparation of the preparation. Moreover, these peptides appear to be less efficient than adenovirus particles when enhancing gene transfer (Gottshalk et al., Gene Therapy, 3, 448-457, 1996; Wagner et al., PNAS, 89, 7934-8, 1992). It has emerged, and so the search for more effective peptides continues.

Peptides that can condense DNA and destroy biological membranes will simplify formulation and may lead to more efficient transfection.

Melittin is a well known membrane disrupting peptide; However, despite being one of the most widely studied peptide sequences, only two examples of its use in gene transfer have been described. First, it was associated with melittin (Legendre et al., Bioconjugate Chem., 8, 57-63, 1997) dioleoylphosphatidylethanolamine-N- [3- (2-pyridyldithio) propionate] ( DOPE derivatives) and, secondly, in US Pat. No. UA-A-5,547,932. In Legendre et al., The conjugation of lipids is expensive and complicated to step, and problems have been reported for the use of DOPE in vivo. US Patent US-A-5,547,932 describes the formulation of endosomolytic agents attached to nucleic acid binders. The formulations mentioned form relatively inefficient transfection formulations which are possibly toxic to the transfected cells. In fact, the toxicity of melittin may explain why it is not used as a sole peptide in successful non-viral formulations.

The present invention relates to modified membrane disrupting peptides. The invention also relates to delivery complexes and to the use of delivery complexes containing modified membrane disrupting peptides.

1 shows erythrocyte lysis activity of CP36 dimer (CP36D), CP36 monomer (CP36M) and melittin (CP1).

2 shows the erythrocyte lytic activity of CP36 and CP48.

3A and 3B show CP1 2, 18, 36, 39, 41, 42, 43, 44, complexed with various charge ratios in the presence or absence of DTT and in the presence of 10% fetal calf serum. When transfected with 45 and 48, transfection of HepG2 cells is shown.

4A and 4B show trans with CP1 2, 18, 36, 39, 41, 42, 43, 44, 45 and 48 complexed at various charge ratios in the presence or absence of DTT and in the presence of 10% fetal calf serum. When done, the protein levels of HepG2 cells are shown. Reduced protein levels indicate a toxic effect on the cells.

5A and 5B show trans of HepG2 with pCMVβ complexed with CP36 / 1 (first batch), CP36 / 1.2 (second batch) with or without DTT and with or without 10% fetal calf serum Represents a specification. Red means reduced form, i.e. in the absence of 5 mM DTT; And NR means unreduced form, ie no DTT is added.

FIG. 6 shows data from experiments comparing luciferase expression from HUVECs transfected with CP36 complexed-DNA from different batches of four charge ratio CP36 peptides. The batches of peptides meant different synthesis and were called CP36 / 1, / 3, / 4, / 5, / 6.

FIG. 7 shows a number of experiments (number of experiments are indicated in parentheses) comparing mean luciferase expression from HUVECs transfected with CP36 complexed-DNA and PEI complexed-DNA at various charge or N: P ratios. Compilation was shown and the PEI used was 22 kDa linear PEI (Exgen-500).

FIG. 8 shows a number of experiments comparing average% cells expressing GFP from HUVECs transfected with CP36 complexed-DNA and PEI complexed-DNA at various charge or N: P ratios (numbers of experiments are shown in parentheses). ), And the PEI used was 22 kDa linear PEI (Exgen-500).

9 shows luciferase expression in vivo after dosing with 75 μg DNA using various peptides in many tissues.

10 shows luciferase average expression from HUVEC transfected with NBC28: DNA particles coated with CP36 dimer.

The present invention provides modified membrane disrupting peptides, which are modified to form covalently linked multimers.

Preferably, the modified membrane disrupting peptide is further modified to form a substantially continuous α helix.

By modifying the membrane disrupting peptides to form multimers and preferably to form substantially continuous α helices, the peptide's toxicity is reduced and the peptides are increased when used to deliver nucleic acids to cells. It is found to give a specification level.

Without being bound by any theory, it is suggested that the membrane disrupting peptides should be in the form of monomers in order to be able to insert into the membrane and aggregate to form pores. The formation of pores is thought to be toxic to cells because it enhances passive ion permeability. Multimerization of the peptide prevents the formation of monomers, impedes the formation of pores, and reduces cytotoxicity. Multimers still retain some membrane destructive properties because of the widespread non-polar amphiphilic α helix. By modifying the peptide to obtain a substantially continuous α helix, the membrane disruptive properties of the peptide are increased because the substantially continuous α helix can interact with the cell membrane. Multimerization of this peptide also has the advantage that the peptide is less separated from all bound nucleic acids.

The term "membrane destructive peptide" means a peptide capable of enhancing membrane destabilization and lowering the energy required for molecules to cross the membrane. Assays such as erythrocyte lysis assays can be used to determine if a peptide is a membrane disrupting peptide; However, different membrane disrupting peptides have different cell specificities and may dissolve different cell types into red blood cells. Therefore, other cell types should be used to determine if the peptide is a membrane disrupting peptide in a lysis assay. The membrane disrupting peptide is preferably a toxic membrane disrupting peptide. It is also preferred that the membrane disrupting peptide is toxic by insertion into the membrane in the form of monomers. Membrane destructive peptides are toxic α helical membrane destructive agents such as melittin, cecropin A, cecropin P1, cecropin D, magainin 2, bombolittins or pardaxin. Also preferred are peptides (Saberwal et al., Biochemica et Biophysica Acta, 1197, 109-131, 1994). The membrane disrupting peptide preferably forms an amphiphilic α helix having one side rich in cationic residues that enable condensation of DNA and a hydrophobic side that can interact with membrane lipids. It is also preferred that the membrane disrupting peptide consists of an α-helix region and a base region at the C-terminus of the peptide to condense the nucleic acid. Preferably, the membrane disrupting peptide is not a human bactericidal / permeable-increasing protein (BPI) (Gray et al., J. Biol. Chem., 264, 9505, 1989 and US Patent US-A-5,856,302). Preferably, the membrane disrupting peptide is melittin.

As used herein, the term "peptide" refers to a polymer of amino acids having a chain length of 10 to 150 amino acids. This term does not refer to or exclude modifications of peptides such as, for example, glycosylation, acetylation, phosphorylation. Within the definition, peptides include one or more amino acid analogs (eg, artificial amino acids), peptides with substituted bonds, as well as other modifications known in the art that occur naturally or are synthesized.

The term "modified to form a multimer" means that the membrane disrupting peptide is covalently modified to bind one or more other membrane disruptive peptides. Preferably the multimer is a dimer, trimer or tetramer, more preferably a dimer or tetramer, most preferably a dimer. The membrane disrupting peptides that form multimers may be the same or different.

Membrane disrupting peptides can be bound to each other at any point along the length of the peptide; However, it is preferred that the peptides bind to each other at the N-terminus and / or C-terminus of each peptide, and most preferably, the peptides bind to each other at the N-terminus of each peptide.

Preferably, the membrane disrupting peptide is obtained by substituting an amino acid of a membrane disrupting peptide having an amino acid which forms a covalent bond either directly through a disulfide bond or through a linking group with an amino acid of another modified membrane disrupting peptide. Is modified to form a dimer. Alternatively, amino acids that form covalent bonds either directly through disulfide bonds or through linkage groups can be added to the membrane disrupting peptide. Preferably, the amino acid added or substituting the amino acid of the peptide is a cysteine amino acid, which can form a disulfide bond with the cysteine residue of another peptide. The membrane disrupting peptides forming the dimers do not have to be identical, but it is preferred that the peptides forming the dimers are identical.

Preferably, the membrane disrupting peptide replaces two or more amino acids with two or more amino acids that form a covalent bond either directly or via a linking group via disulfide bonds with amino acids of another membrane disrupting peptide modified to form a trimer or tetramer. To form a trimer or tetramer. Alternatively, two or more amino acids that form disulfide bonds, either directly or through a linking group, can be added to the membrane disrupting peptide. The membrane disrupting peptides that form the trimers or tetramers do not have to be identical, but the peptides that formed the trimers or tetramers are preferably the same.

The membrane disrupting peptides can be linked to each other via linkers, such as commercially available linkers, including bismaleimide or bisvinylsulphone linkers.

The term “substantially continuous α helix” refers to a region of a membrane disrupting peptide that forms α helix and does not contain one or more amino acids that disrupt the formation of the α helix. Preferably the substantially continuous helix is at least 10 amino acids in length. Preferably the substantially continuous helix is at least 5% of the length of the modified membrane disrupting peptide, more preferably 40 to 90% of the length of the membrane disrupting peptide and most preferably about 80% of the length of the membrane disrupting peptide to be. The presence of α helices in the peptide can be readily determined using standard cyclic dichroism assays.

Amino acids that can disrupt α helix formation include proline, glycine, tyrosine, threonine and serine. However, as will be appreciated by those skilled in the art, the ability of an amino acid to disrupt α helix formation depends on other factors such as the overall peptide sequence and the pH of the solution in which the peptide is folded.

Preferably the amino acid capable of disrupting α helix formation is proline.

Preferably, the membrane disrupting peptide is modified to form a substantially continuous α helix by replacing an amino acid that disrupts α helix formation with an amino acid that does not disrupt α helix formation.

In a preferred embodiment, the modified membrane disrupting peptides of the invention have the amino acid sequence of CIGAVLKVLTTGLAALISWIKRKRQQ. It is also preferred that the modified membrane disrupting peptide forms a direct disulfide bond between the N-terminal cysteine amino acids.

Modified membrane disrupting peptides of the invention are further modified by the addition of other functional groups such as lipids, peptides such as antibodies or antibody fragments, targeting groups, antigenic peptides, sugars and neutral hydrophilic polymers such as PEG and PVP. Can be. Suitable functional groups that can be added to the modified membrane disrupting peptides of the present invention are described in International Patent Application WO 96/41606, along with methods of attaching such functional groups to the peptide.

Preferably, the modified membrane disrupting peptides of the present invention are further modified by the addition of amino acids to be substantially continuous α helix regions of the peptide, wherein the added amino acids extend the length of the substantially continuous α helix region. . Preferably, the substantially continuous α helix region extends so that the α helix region is at least 10, more preferably at least 20 amino acids in length.

Preferably, the modified membrane disrupting peptides of the invention are also modified by the addition of basic amino acids to the C-terminal region of the peptide.

The C-terminal region of the modified membrane disrupting peptide of the present invention is a region extending from the C-terminal amino acid to a region that forms a substantially continuous α-helix region.

Basic amino acids are well known to those skilled in the art and include lysine, arginine and histidine. Preferably, the C-terminal region is modified by the addition of between 1 and 50 amino acids, more preferably between 5 and 15 amino acids.

The invention also provides functional homologues of the modified membrane disrupting peptides of the invention.

Preferred functional homologues of the modified membrane disrupting peptides of the present invention still have activity, preferably 80%, more preferably 90%, and most preferably 95% homologous to the peptides of the present invention. Such functional homologs, preferably comprising fragments of the peptides of the invention, differ only by 1 to 10 amino acids. It is also preferred that the amino change is conservative. Conservative changes replace one amino acid with one of the amino acid families involved in the side chain. For example, it is expected that the substitution of isosin or valine, aspartate to glutamate, threonine to serine, or similar conservative substitution of amino acids to structurally related amino acids would not have a major effect on the biological activity of the peptide. It is reasonable.

However, it is sometimes desirable to alter amino acids to alter the biological activity of the peptide. For example, mutations that abolish or enhance one or more functions of a peptide may be particularly useful. Such mutations can generally be made by changing any conserved sequence of the peptide. Such homologues preferably have a homology of at least 80%, more preferably 90% and most preferably 95% with the peptides of the invention or fragments thereof. Preferably such altered protein or fragment thereof differs by only 1 to 10 amino acids.

The invention also provides the use of the modified membrane disrupting peptides of the invention in a delivery complex that delivers nucleic acids to cells.

The modified membrane disrupting peptides of the present invention can be used to deliver negatively charged polymers, preferably nucleic acids, to any cell type. Preferred cell types include mammalian cells, including prokaryotic cell types, such as E. coli and in vitro primary cells, such as HUVEC and DC cells, and eukaryotic cell types, such as mammalian cell lines, including HeLa, HepG2, CHO, and myeloma cell lines, and yeast. The same lower prokaryotic cell types are included. Preferably the modified membrane disrupting peptides of the invention are used to deliver nucleic acids to mammalian cells.

The invention also provides a delivery complex consisting of the modified membrane disrupting peptides and nucleic acids of the invention.

Preferably, the delivery complex consists of the nucleic acid to be delivered and the modified membrane disrupting peptide of the invention.

The delivery complex of the invention may be any delivery complex consisting of the nucleic acid to be delivered and the modified membrane disrupting peptide of the invention.

Many delivery complexes for delivering nucleic acids to cells are well known to those skilled in the art. Peptides derived from the amino acid sequence of viral envelope proteins have been used in gene transfer when co-administered with polylysine DNA complexes (Plant et al., 1994, J. Biol. Chem., 269: 12918-47; Trubetskoy et al, 1992, Bioconjugate Chem., 3: 323-327; Mark et al, 1994, Am. J. Med. Sci., 307: 138-143) .Co-condensation of polylysine conjugates with cationic lipids It suggests that progress can be made in the efficiency of gene transfer. WO 95/02698 discloses the use of viral components to increase the efficiency of cationic lipid gene delivery.

In a preferred embodiment, the delivery complex of the invention comprises modified membrane disrupting peptides and nucleic acids encapsulated within nano- or micro-particles such as polylactide glycolide particles and liposomes and the like.

In another preferred embodiment, the delivery complexes of the invention consist of nucleic acids, nucleic acid condensation peptides and modified membrane disrupting peptides of the invention.

Nucleic acid condensation peptides can be any peptide that condenses nucleic acids, including peptides derived from polylysine and histones. Preferred nucleic acid condensation peptides are described in international patent applications WO 96/41606 and WO 98/35984.

Preferably, the delivery complex is formed by condensing the nucleic acid with a nucleic acid condensation peptide to form a condensed nucleic acid complex. The condensed nucleic acid complex is then coated with the modified membrane disrupting peptides of the invention.

The present invention provides a method for forming a delivery complex according to the present invention consisting of:

1. condensing the nucleic acid with a nucleic acid condensation peptide to form a condensed nucleic acid complex; And

2. coating the condensed nucleic acid complexes with the modified membrane disrupting peptides according to the invention.

The presence of serum during transfection has been found to increase the level of transfection. Preferably the serum is fetal calf serum. Other suitable serums that can be used are well known to those skilled in the art and include normal human serum and normal mouse serum.

The modified membrane disrupting peptides of the invention can be used to deliver therapeutic nucleic acids to cells in vivo, in vitro and ex vivo treatments. Therapeutic uses of nucleic acids in various diseases are well known to those skilled in the art.

The therapeutic nucleic acid delivered to the cell can be in the form of any DNA or RNA vector, including plasmids, linear nucleic acid molecules, ribozymes, deoxyribozymes, and episomal vectors. Expression of heterologous genes is characterized by muscle (Wolff JA et al., 1990, Science, 247: 1465-1468; Carson DA et al., US Pat. No. 5,580,859), thyroid gland (Sykes et al., 1994, Human Gene Ther., 5: 837-844), melanoma cells (Vile et al., 1993, Cancer Res., 53: 962-967), skin (Hengge et al., 1995., Nature Genet., 10: 161-166) and After injection of plasmid DNA into the liver (Hickman et al., 1994, Human Gene Therapy, 5: 1477-1483) and after exposure of airway epithelial tissue (Meyer et al., 1995, Gene Therapy, 2: 450-460 ) Has been observed.

Useful therapeutic nucleic acid sequences include those encoding receptors, enzymes, ligands, regulatory factors and structural proteins. Therapeutic nucleic acid sequences also encode nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmaal-related proteins, serum proteins, viral antigens, bacterial antigens, protozoan and parasitic antigens. Sequence. Useful therapeutic nucleic acid sequences according to the invention also include sequences encoding proteins, fatty proteins, glycoproteins, phosphorus-proteins and nucleic acids (eg, RNAs such as ribozymes or antisense nucleic acids). Proteins or polypeptides encoded by nucleic acids include hormones, growth factors, neurotransmitters, enzymes, coagulation factors, apolipoproteins, receptors, drugs, tumor genes, tumor antigens, tumor suppressors, structural proteins. , Viral antigens, parasitic animal antigens and bacterial antigens. Specific examples of these compounds include proinsulin, growth hormone, dystrophin, androgen receptor, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding protein, epidermal growth factor TFG-α, TGF-β , PDGF, angiogenesis factor (acidic fibroblast growth factor, basic fibroblast growth factor and angiogenin), mattress protein (type IV collagen, type VII collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxy Silases, tumor genes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transformation sequences, p53 proteins, Rb gene products, cytokines (e.g. IL-1, IL-6, IL- 8) or its receptors, viral capsid proteins, proteins derived from viruses, bacteria and parasitic organisms that can be used to induce immunological responses, and other proteins of significant utility to the human body. . Compounds that can be incorporated are only limited by the availability of the nucleic acid sequence to the protein or polypeptide to be incorporated. Those skilled in the art will readily recognize that as more proteins and polypeptides are recognized, they can be incorporated into selective delivery complexes that are transfected and expressed using the modified membrane disrupting peptides of the invention.

Nucleic acids delivered using the modified membrane disrupting peptides of the present invention include those designed to eliminate translation of unwanted proteins as well as those that are deficient or clinically effective at higher than normal concentrations. Nucleic acids used for the removal of harmful proteins are not only antisense RNAs and ribozymes, but also DNA expression constructs encoding them.

Hammerhead ribozymes are the smallest known ribozymes and are used for in vitro synthesis and delivery to cells (Sullivan, 1994, J. Invest. Dermatol., 103: 85S-98S; Usman et al., 1996 , Summarized by Curr. Opin.Struct. Biol., 6: 527-533).

The invention also provides a modified membrane disrupting peptide or a delivery complex of the invention for use in therapy.

The invention also provides the use of a membrane disrupting peptide or a delivery complex of the invention in the preparation of a composition for the treatment of a genetic disorder.

Genetic disorders are defined as those that can be treated at the genetic level, i.e., by delivering nucleic acids to patients in need of such treatment. Genetic disorders include, but are not limited to, enzyme deficiencies (eg, enzyme deficiencies in the liver, digestive system, skin and nervous system), endocrine gland deficiencies (eg, growth hormone, regenerative hormone, vascular action, and hydrostatic). Hormone deficiency), exocrine gland deficiency (such as deficiency of pancreatic hormone secretion), neurodegenerative disorders (Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Tay-Sachs disease, etc.), cancer, muscular atrophy and pigment deficiency.

The invention also provides a method of treating a genetic disorder, comprising administering to a patient in need thereof an effective amount of a delivery complex containing a therapeutic nucleic acid and a modified membrane disrupting peptide of the invention.

The invention is illustrated in the accompanying embodiments with reference to the following figures.

Materials and methods

Peptide Synthesis and Purification

Method 1: Multiple Peptide Synthesiser (MPS)

Device software operating version 1.7 with P.E. Peptides were synthesized in a Biosystems Pioneer peptide synthesizer. The device was equipped with a single MPS unit attached to column position 2 and controlled by a workstation with operating software version 1.3.

Each synthesis was performed on a scale of 0.05 mmol using Fmoc-PAL-PEG-PS resin (P.E. Biosystems). For synthesis, extended and slow activation and device-supplied coupling cycles were used. Deprotection was carried out using a 20% pyreridine solution in DMF. The following amino acid derivatives were used as appropriate for the peptides; Fmoc-L-Ala-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-L-Cys (Trt) -OH, Fmoc-L-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-L -Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc) -OH, Fmoc-Nle-OH, Fmoc-L-Pro-OH, Fmoc-L-Ser (tBu) -OH, Fmoc -L-Thr (tBu) -OH, Fmoc-L-Trp (Boc) -OH, Fmoc-L-Val-OH and Fmoc-L-Lys (Fmoc) -OH. Activation of amino acids was achieved using TBTU and DIPEA. At the end of the synthesis, the Fmoc group was removed, followed by exchange of the solvent with dichloromethane and drying of the resin under a dry nitrogen stream. Then, the column containing the resin was taken and dried under vacuum for 2-3 hours at room temperature.

Peptides without cysteine residues were cleaved from the resin using 95: 2.5: 2.5 trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS), and those containing cysteine residues were 92.5: 2.5: 2.5: The cleavage was carried out using 2.5 trifluuroacetic acid (TFA) / water / triisopropylsilane (TIS) / ethanedithiol (EDT). An empty 5 ml syringe was attached to one end of each column and a 2.5 ml syringe containing the cleavage mixture was attached to the other end. The cleavage mixture (1 mL) was injected into each column, left for 30 minutes, then 1 mL more, and after the last 0.5 mL. After leaving for 30 minutes, the empty 2.5 ml syringe was removed and the remaining cleavage mixture was drawn out with a 5 ml syringe. The column was removed from the 5 ml syringe, turned over and exchanged, and then a new 2.5 ml syringe containing the cleavage mixture was attached to the other end and the cleavage procedure was repeated. The contents of the 5 ml syringe were then pushed into a 50 ml centrifuge tube containing diethyl ether (45 ml). The resulting precipitate was fixed at room temperature for 1-2 hours and the supernatant was poured out. The remaining diethyl ether was blown under a stream of dry nitrogen gas and the pellet further dried under vacuum for 2-3 hours.

Method 2: Continuous Flow Synthesis of Peptides

P.E with device software operating version 1.7, controlled by the workstation with operating software version 1.3. Peptides were synthesized in a Biosystems Pioneer peptide synthesizer.

Each synthesis was performed on a scale of 0.2 mmol using Fmoc-PAL-PEG-PS resin (P.E. Biosystems). For the coupling of all amino acids except cysteine, an extended coupling cycle supplied with the device was used. A special extended (1 hour coupling time) solvent exchange cycle for kerning of cysteine was used to allow coupling to occur in 1: 1 DMF / DCM. Deprotection was carried out using a 20% pyreridine solution in DMF. The following amino acid derivatives were used as appropriate for the peptides; Fmoc-L-Ala-OH, Fmoc-L-Arg (Pbf) -OH, Fmoc-L-Cys (Trt) -OH, Fmoc-L-Gln (Trt) -OH, Fmoc-Gly-OH, Fmoc-L -His (Trt) -OH, Fmoc-L-Ile-OH, Fmoc-L-Leu-OH, Fmoc-L-Lys (Boc) -OH, Fmoc-L-Pro-OH, Fmoc-L-Ser (tBu ) -OH, Fmoc-L-Thr (tBu) -OH, Fmoc-L-Trp (Boc) -OH, Fmoc-L-Val-OH and Fmoc-L-Lys (Fmoc) -OH. Activation of amino acids except cysteine was achieved using TBTU and DIPEA in DMF. Activation of cysteine was achieved using Sym-Collidine in TBTU and DCM in DMF. At the end of the synthesis, the Fmoc group was removed, followed by exchange of solvent with dichloromethane and drying of the resin under a dry nitrogen stream. The resin was washed into a filter funnel that was allowed to air dry outside the column for several minutes, transferred to a pre-weighed flask, and further dried under vacuum until constant weight was achieved.

Peptides without cysteine residues were cleaved from the resin using 95: 2.5: 2.5 (20 mL) trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) for 1.5 hours at room temperature, and the cysteine residues were Inclusions were cleaved using 92.5: 2.5: 2.5: 2.5 (20 mL) trifluuroacetic acid (TFA) / water / triisopropylsilane (T IS) / ethanedithiol (EDT) for 1.5 hours at room temperature. It was. The resin was then filtered in each case, washed with unburned TFA (3 × 5 mL) and the mixed filtrate and washes evaporated to a volume of about 3 mL. The residue was transferred to a 50 ml centrifuge tube containing diethyl ether (45 ml). The resulting precipitate was fixed at room temperature for 1-2 hours and the suspension was poured out. The remaining diethyl ether was blown under a stream of dry nitrogen gas and the pellet further dried under vacuum for 2-3 hours.

Purification of Peptides

Peptides were purified by reverse phase h.p.l.c. Shandon Hypersil SAS columns that typically use a gradient of water (0.1% TFA) acetonitrile (0.1% TFA) with 10-65% acetonitrile in water at a flow rate of 24 ml / min over 20 minutes. 120 μs, 10 μ, 150 × 21.2 mm), or a gradient of water (0.1% TFA) acetonitrile (0.1% TFA), typically with 30-100% acetonitrile in water, over 6 ml / More polar peptides were purified on a Phenomenex Jupiter C4 column (120 μs, 5μ, 250 × 10 mm) using a flow rate of minutes.

Fractions comprising the peptide of interest were pooled and lyophilized, as measured by mattress assisted laser desorption / ionization time (MALDI-TOF MS) and HPLC analysis on a flight mass spectrometer.

Peptide Multimer Formation

For complete dimerization or oligomerization via disulfide or cystine bonds, pure peptides were dissolved in 20 mM fresh ammonium bicarbonate and left at room temperature for 16 hours. Dimerization was confirmed by analytical gel filtration chromatography using a Pharmacia Superdex Peptide HR 10/30 column operating at a flow rate between 0.8 and 1.0 ml / min in water containing 0.1% TFA with 20% acetonitrile. Reduction of the elution time of the dimerized peptide was confirmed by monitoring the absorption of the elution at 214 nm.

For the synthesis of bisvinylsulfone linked to conjugate (CP48), 2.0 mg Biolink 6 (Molecular Biosciences, Colerodo) was dissolved in 100 μl of acetonitrile and a volume of 1.0 ml was made using 25 mM HEPES, pH7.2. 200 μl of this solution was then added to 15 mg of CP36 monomer (5.2 μmol) dissolved in 800 μl buffer. The reaction was monitored by MALDI and RP-HPLC and judged complete after 1 hour at 24 ° C. Phenomenex Jupiter C ₄ semiprepared using a gradient of 30% to 100% increased acetonitrile in water containing 0.1% TFA. The final product was separated by a semipreparative column. The weight of this final product was confirmed by MALDI-TOF MS.

Erythrocyte Lysis Assay

To 9.0 ml of blood 1.0 ml of 110 mM citrate, pH5, was added to stop coagulation. Blood was spun at 2000 rpm for 5 minutes. The plasma supernatant was aspirated and the pellet was washed at least 6 times with HBS by continuous mixing, centrifugation and aspiration. The clear supernatant was aspirated and the pellet washed twice with appropriate buffer: HBS (pH 7.4) or 15 mM sodium acetate, 150 mM NaCl, pH 5. Appropriate buffer was added to the blood pellet to a total volume of 6 ml, from which 1 ml was taken and diluted 15 times with buffer to the final working solution. With three 96-well plates, peptides were tested by serial dilution and mixing by adding 75 ml of blood solution in appropriate buffer to 100 ml of peptide solution in corresponding buffer. Blood was incubated with peptides at 37 ° C. for 1 hour. In this step, 1% Triton X-100 was added to the blood solution without peptide to serve as a control for 100% dissolution. The cells were spun at 2500 rpm for 5 minutes and 80 ml of supernatant was harvested for spectrometric analysis at 450 nm.

Preparation of the complex

40 μg / ml of plasmid DNA in HBS (HEPES buffered saline), pH 7.4, was quickly mixed with a transfection formulation of the same concentration in HBS and incubated for 1 hour at room temperature. Concentration of the transfection agent was determined by PEI (Exgen-500, Euromedex, France) with respect to the peptide by the final required charge ratio or by the final ratio of nitrogen (derived from PEI): phosphate (derived from DNA). ). The charge ratio was calculated according to the definition by Felgner et al (1997) (Nomenclature for synthetic gene delivery systems. Gene Therapy, (1997) 8: 511-512).

HepG2 Transfection Analysis

1.β-Gal Transfection Protocol

One day prior to transfection with 5 × 10 ⁴ cells / well, HepG2 was plated in 96-well plates with DMEM + 10% FCS (with antibiotics) and incubated at 37 ° C. The next day the cells were washed with 100 μl / well PBS. Tran containing a plasmid pCMVβ reporter plasmid (plasmid encoding for β-galactosidase) prepared by buffering 90 μl HEPES with RPMI containing 10% FCS and adding antibiotics to the cells and prepared as described above. 10 μl of the specification complex was then added. Complexes were formulated with or without 5 mM DDT to reduce all peptide disulfide bonds. Using the appropriate controls, it was confirmed that the presence of DDT in the formulation did not have a noticeable effect on transfection. Cells were transfected in triplicate with each complex. Cells were centrifuged at 1100 rpm and incubated under damp conditions in a non-gas treated incubator at 37 ° C. for 5 hours. After 5 hours, transfection medium was removed and cells were washed with 100 μl / well PBS before incubation in DMEM / 10% FCS (with antibiotics) medium for 19-20 hours in CO ₂ -gassed incubator. . Finally, cells were washed twice with PBS, lysed with 30 ml 0.1% Triton, 250 mM Tris, pH 8 and frozen at −20 ° C. prior to β-gal analysis.

2. β-Gal Analysis

Frozen lysed cells were thawed at room temperature and 10 μl from each well was removed for protein analysis. The remaining cell lysates were analyzed for β-gal reporters using Galacton-Sstar β-gal assay (TROPLX) and luminescence was measured using a 96 well TopCount scintillation counter operating in SPC mode. DC Protein Assay Kit (BioRad) was used to analyze lysates for total protein amount. Transfection counts were reported as pg β-gal / ng total protein using a β-gal standard curve made in cell lysates from untransfected cells on the same 96-well plate.

HUVEC Transfection Analysis

Luciferase Transfection Assay

Primary HUVEC (Promocell, Germany) was taken in endothelial growth medium (EGMS) (Promocell, Germany) with a supplement in a 96-well plate coated with 0.1% gelatin one day before transfection with 1 x 10 ⁴ cells / well. Plates were incubated at 37 ° C. in a CO ₂ -gassed incubator. The next day the cells were washed with PBS. 10 μl of M199 (Sigma) per well medium containing 10% FCS and antibiotics was added to the cells and the transfection complex prepared with the pCMVLuc reporter plasmid (plasmid encoding firefly luciferase) at 10 μl per well. Added. Cells were transfected in triplicate for each complex. Cells were centrifuged at 1100 rpm for 5 minutes and incubated in a CO ₂ -gassed incubator at 37 ° C. for 1 hour. After 1 hour, the transfection medium was removed and cells were washed with 100 μl / well PBS before incubation in 100 μl / well EGMS at 37 ° C. for 20-24 hours in a CO ₂ -gassed incubator.

Luciferase Analysis

Luciferase expression was determined using LucScreen Assay (TROPIX) with luminescence measured using a 96 well TopCount scintillation counter (Packard) operating in SPC mode after 20-24 hours.

GFP Transfection Protocol

In 6-well plates, one day prior to transfection in 2 ml EGMS, primary HUVECs were inoculated at 1.5 × 10 ⁵ cells / well. The following day, 100 μl transfection complex prepared twice with pCMVEGEP reporter plasmid (plasmid encoding green fluorescent protein) mixed with 2 mL PBS per well and mixed with 900 μl M119 containing 10% FCS and antibiotics. A transfection solution consisting of was added at 1 ml per well. Cells were transfected in duplicate for each complex. The cells were incubated for 2 hours at 37 ° C. in a CO ₂ -gassed incubator and then the medium was changed to EGMS at 2 ml per well. Cells were again incubated for 24 hours at 37 ° C. in a CO ₂ -gassed incubator.

Gfp Analytics

After 24 hours, cells were washed twice with 2 ml PBS per well, trypsinized and resuspended in M119 medium + 10% FCS. The percentage of transfected cells was determined by FACS analysis.

In vivo Delivery of pCMVLuc Using Peptides

PCMVLuc at 500 μg / ml in HBS was mixed with the same volume (typically 500 μl) of peptide or polylysine (127 mer) (Sigma) at the appropriate concentration to give the required charge ratio. The charge ratio was as defined by Felgner et al (Hum. Gene Ther., 8 (5), 511-2,1997). Peptide or polylysine was added to the DNA over 3-4 seconds while mixing in a vortex mixer at 800 rpm. The complex was incubated for 1 hour at room temperature. 300 μl of the complex was injected into the tail vein of CD-1 mice. After 20 hours, mice were sacrificed and each organ removed 80-200 mg, simply wiped to remove excess fluid, and frozen in liquid nitrogen and stored at -80 ° C. The frozen tissue is weighed and then in lysis buffer (containing 1 mM EDTA, 1% Triton X-100, 15% glycerol, 8 mM MgCl ₂ , 0.5 mM PMSF and 1 mM DTT with 10 mM sodium phosphate). And homogenized using Mini bead mixer-8 (Stratech Ltd) and 1 mm glass beads. The homogenate was removed and the glass beads were washed with lysis buffer and the wash was mixed with the homogenate. Particles were removed at 13000 rpm for 5 minutes by centrifugation, and 80 μl was analyzed for luciferase activity on Berthold LB593 luminometer using 0.1 mM luciferin, 0.44 mM ATP and 4 sec acquisition time. Results were expressed in RLU modified with mg weight of each tissue.

The following peptides were used where possible as monomers or as dimers or other multimers using the methods described above.

CP1 is melittin and is the main toxic component of bee venom.

CP2 is CP1 (G1 to C). This peptide was designed to form an N-terminal cysteine linked CP1 dimer.

CP18 is CP1 (A in P14). This peptide was modified to form a substantially continuous helix.

CP36 is CP1 (G1 to C; A at P14). This peptide was modified to form a dimer and have a substantially continuous helix.

CP39 is CP36 (Norleucine at C1). Modifications were introduced to increase hydrophobicity at the N-terminus.

Peptides CP41 to CP45 were designed to study the effect of conserved amino acid changes on CP36 activity.

CP37 is CP18 (L at I20) and all lysines are substituted with glutamate.

CP41 is CP36 (L in I20).

CP42 is CP36 (L at I17; L at I20).

CP43 is CP36 (A14 to W; I20 to L).

CP44 is CP36 (A15 to W; I20 to L).

CP45 is CP36 (W in T11; L in I20).

CP46 is a functional homologue of melittin, including conserved amino acid changes, DeGrado et al., (1981), J. Am. Chem. Based on the sequence described by Soc., 103, 679-681.

CP47 is CP46 with N-terminal cysteine added for dimer formation.

CP49 is CP36 with C-terminal cysteine added for multimer formation.

CP50 is CP18 with C-terminal cysteine added for multimer formation.

CP51 is CP41 with all lysine residues substituted by glutamate.

CP52 is a dimer of CP36 formed by the bis-amine modification of an additional C-terminal lysine.

CP53 is a dimer of CP36 with an added C-terminal lysine and a cysteine residue linked to CP36.

CP54 is CP36 with LAALISW inserted at position 20 to increase the length of the α helix.

CP55 is a heterodimer of CP36 and CP51.

CP56 is a tetramer consisting of CP52 and two CP36 peptides.

CP57 is CP1 (K23 to C).

CP58 is a heterodimer of CP36 and CP50.

CP59 is a tetramer consisting of a dimer of CP52 linked to two CP37 peptides.

CP60 is a tetramer consisting of a dimer of CP53.

CP61 is a dimer consisting of CP36 and CP51, wherein disulfide bonds are formed between N-terminal cysteines.

Although melittin (CP1) can be effectively bound to DNA, it was observed that melittin: DNA complex delivered little or no transfection of HepG2 cells, as determined by plasmid delay on agarose gels. And melittin was observed to be toxic to cells. After incubation of the cells with this complex, the total protein amount per cell decreased with increasing melittin ratio to DNA (FIG. 4). This meant a toxic effect caused by melittin's ability to form numerous structures that disturb biological membranes. The literature on the mechanism of melittin states that even if the peptide is present as a tetramer in solution at high concentrations and / or high ionic strength, it must be able to separate in the form of monomers for insertion into the membrane, where it aggregates into pore-forming tetramers. It can be done. The pore forming tetramer of melittin is thought to enhance passive ion permeability and is considered toxic to cells. Dimerization or tetramerization of melittin has been suggested to be less toxic to cells, and dimerization or tetramerization will result in dimerization or tetramerization, constructs that effectively prevent the onset of pore-forming tetramer formation. Melitin dimers or tetramers nevertheless retain membrane destructive properties because of the dominant non-polar amphiphilic helix. At position 14 of the melittin, it was determined to optimize the interaction between the amphipathic helix of the melittin and the membrane surface by substituting the pro moiety for the defect causing the defect in the helix structure.

As mentioned above, dimers were constructed and their membrane breaking activity was measured by erythrocyte lysis assay. Surprisingly, even though the lytic activity of dimers was greater than melittin at pH 7 and 5 (see FIGS. 1 and 2), dimers appeared less toxic to mammalian cell lines (see FIG. 4). Thus, it is proposed that pore formation is toxic but other membrane destruction / stabilization mechanisms are not.

Dimerization of melittin with spiral elongation, as described, resulted in a structure capable of binding DNA; The resulting DNA complexes delivered significantly less harmful to HepG2 cells and efficient transfection of various cell types in the absence of exogenous agents, as determined by protein content determination (see FIG. 4). See). It was also found that the presence of fetal calf serum during transfection confers enhanced transfection levels (see FIG. 5).

9 shows that peptides CP36 and CP61 of the present invention are effective in increasing the transfection of DNA into numerous tissues in vivo.

Delivery Complex Coated with CP36 Dimer

The delivery complex was prepared as follows.

1.Preparation of NBC28: DNA at DNA concentrations of charge ratio ± 4 and 25 μg:

Solutions of 92.6 μg / ml NBC28 and 50 μg / ml pCMVluc DNA were prepared in 10 mM HEPES at pH 7.4. The peptide solution was added to the DNA in a ratio of 1: 1 (volume / volume) and left at room temperature for 1 hour. NBC28 is a nucleic acid condensation peptide having the amino acid sequence: TKKKKKKKKKKKKKKKKKKYCG.

2. Coating of the composite:

The coated peptide was added to the complex and made up to 97% of the final volume required with some 10 mM HEPES buffer. The resulting complex was vortexed for 10 seconds and stored at 4 ° C. overnight.

3. Salt spikes:

The next morning (after about 18 hours) 5M salt (3% of final volume to become 150 mM salt) was added to the complex, then vortexed for 10 seconds and left for several hours at room temperature.

4. Reconstitution of the Complex:

The complex in 1.5 mL Eppendorf was spun at 1300 rpm for 30 minutes in a MSE Microcentaur centrifuge. Supernatant (75%) was removed and an equal amount of fresh HBS was added while gently vortexing for 30 seconds and sucking up and down with a Gilson pipette.

5. Ultrasonic Treatment of Complexes

The complex in a 1.5 ml Eppendorf tube was sonicated for 30 seconds in an ultrasonic bath prior to transfection.

The complex was then transfected into HUVEC cells according to the method described above.

10 shows that the complex coated with CP36 confers good transfection of HUVEC cells. In particular, the degree of transfection is greater than that obtained with CP36 or NBC28 alone.

Claims (28)

A modified membrane disrupting peptide, wherein the membrane disrupting peptide is modified to form a covalently linked multimer. The modified membrane disrupting peptide of claim 1, further modified to form a substantially continuous α helix. 3. A modified membrane disrupting peptide according to claim 1 or 2, wherein the membrane disrupting peptide is an α helix membrane disrupting peptide and is toxic to cells. The modified membrane disrupting peptide of any one of the preceding claims, wherein the membrane disrupting peptide is melittin. The method of claim 2, wherein the membrane disrupting peptide is modified to form a substantially continuous α helix by replacing an amino acid that disrupts α helix formation with an amino acid that does not disrupt α helix formation. Modified membrane disrupting peptides. 6. The modified membrane disrupting peptide according to claim 5, wherein the proline at position 14 of the melittin peptide is substituted with alanine or tryptophan. The modified membrane disrupting peptide according to any one of claims 2 to 4, wherein the membrane disrupting peptide is modified to form a substantially continuous α helix by removing an amino acid that stops α helix formation. . The modified membrane disrupting peptide according to any one of the preceding claims, which is modified to form a dimer. 9. The membrane-breaking peptide according to claim 8, wherein the membrane-breaking peptide is modified by replacing the amino acid of the membrane-breaking peptide with an amino acid that forms a covalent bond directly via a disulfide bond or a linking group with an amino acid of another modified membrane-breaking peptide. Modified membrane disrupting peptides. 10. The modified method of claim 8, wherein the membrane disrupting peptide is modified by adding an amino acid capable of forming a covalent bond directly via a disulfide bond or linking group together with an amino acid of another modified membrane disrupted peptide. Membrane destructive peptides. 8. The modified membrane disrupting peptide according to any one of claims 1 to 7, characterized in that it is modified to form trimers. 8. The modified membrane disrupting peptide according to any one of claims 1 to 7, characterized in that it has been modified to form tetramers. 13. The method of claim 11 or 12, wherein the membrane disrupting peptide comprises two or more amino acids having two or more amino acids that form disulfide bonds, either directly or through a linking group, to form a trimer or tetramer. Modified membrane disrupting peptides, characterized by modification by substitution with amino acids. 13. The method of claim 11 or 12, wherein the membrane disrupting peptide is combined with amino acids of other modified membrane disrupting peptides to form two or more amino acids which form a covalent bond directly via a disulfide bond or linker to form a trimer or tetramer. Modified membrane disrupting peptides, characterized by modification by addition. Modified membrane disrupting peptides having the following amino acid sequences: CIGAVLKVLTTGLAALISWIKRKRQQ. The modified membrane disrupting peptide of any one of the preceding claims, comprising a lipid. The modified membrane disrupting peptide according to any one of the preceding claims, having an extended α helix. The modified membrane disrupting peptide according to any one of the preceding claims, having basic amino acid residues added to the C-terminus of the membrane disrupting peptide. Functional homolog of the modified membrane disrupting peptide according to any one of the preceding claims. Use of said modified membrane disrupting peptide according to any one of the preceding claims in a delivery complex for delivering a negatively charged polymer to a cell. 21. The use of claim 20, wherein said negatively charged polymer is a nucleic acid. 20. A delivery complex for delivering nucleic acid to a cell consisting of a negatively charged polymer and said modified membrane disrupting peptide according to any one of claims 1 to 19. 23. The delivery complex of claim 22, wherein said negatively charged polymer is a nucleic acid. A delivery complex consisting of a nucleic acid, a nucleic acid condensation peptide and said modified membrane disrupting peptide according to any one of claims 1 to 19. A method for forming a delivery complex according to claim 24: 1. condensing the nucleic acid with a nucleic acid condensation peptide to form a condensed nucleic acid complex; And 2. A method comprising coating the condensed nucleic acid complex with the modified membrane disrupting peptide according to any one of items 1 to 19. 20. A modified membrane disrupting peptide according to any one of claims 1 to 19 for therapeutic use. Use of said modified membrane disrupting peptide according to any one of claims 1 to 19 in the preparation of a composition for the treatment of a genetic disorder. 20. A method for treating a genetic disorder comprising administering to a patient in need thereof an effective amount of a therapeutic nucleic acid and a delivery complex consisting of said modified membrane disrupting peptide according to any one of claims 1 to 19. Way.