Patent Application
20110212028
The present invention relates to a system for intracellular delivery of a cargo comprising at least one component A chosen from aliphatic linear or branched moieties with at least 4 carbon and/or cyclic ring systems comprising 2-4 rings which may contain several hetero atoms chosen from N, S, O and P, wherein component(s) A is (are) attached to a cell penetrating peptide B and/or a non-peptide analogue thereof.
It also relates to the use of the system in diagnosis of deceases, as research tool and as a targeting system, a composition comprising the system and especially a pharmaceutical composition a material covered with the system and a material having the delivery systems into the material. Finally it relates to novel peptides.
The hydrophobic plasma membrane constitutes an essential barrier for cells in living animals, allowing the constitutive and regulated influx of essential molecules while preventing access to the interior of cells of other macromolecules. Although being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development.
During the past 40 years, several oligonucleotide (ON)-based methods have been developed with the purpose of manipulating gene expression. The basic method involves the use of bacterial plasmids for expression of genes of interest. In addition, to evaluate functional aspects of different genes, this is a highly appealing strategy to utilize in clinical settings, i.e. gene therapy. Gene therapy was originally thought to serve as corrective treatment for inherited genetic diseases. However, over the past 15 years, experimental gene therapy for cancer has become a frequent application although other acquired diseases have also been investigated [1].
Other versatile approaches utilizing shorter ON-sequences to interfere with gene expression have emerged. Antisense approaches based on short interfering RNAs (siRNAs) that are utilized to confer gene silencing and splice correcting ONs (SCOs), applied for the manipulation of splicing patterns, have recently been rigorously exploited [2,3]. Although being efficient compounds for regulating gene expression, their hydrophilic nature prohibits cellular internalization.
Despite the great potential gene therapy holds for future treatment of various disorders, it suffers from some severe drawbacks. First, plasmids are large, usually exceeding one MDa in size, making them impermeable over cellular membranes. Secondly, viruses have been used to confer cellular internalization of therapeutic genes in clinical trials. Albeit providing an effective means of delivering genes, they might cause severe immunological responses. Thus, in order to progress current gene therapy, safer delivery systems are required, preferably not reliant on the use of viruses.
The search for efficient non-viral delivery vectors has therefore intensified. In the field today, the vectors based on cationic liposomes or polycationic polymers have been employed and these are highly efficient for transfection of commonly used cell lines. However, a great number of these vectors are either sensitive to serum proteins, are unable to transfect the entire cell populations, are inefficient in “hard to transfect” cells, or are simply too toxic. For the vectors on the market today it seems to be a direct correlation between high efficacy and high cytotoxicity. Therefore, there is an urgent need to find delivery vehicles that can overcome the above mentioned problems.
Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus the last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion, as reviewed in [4]. The peptides are usually less than 30 amino acids (aa) in length with a cationic and/or amphipathic nature and have been extensively applied for delivery of various ONs both in vitro and in vivo [5]. Even though the peptides are non-toxic in general, there are some problems associated with their use [6]. One shortcoming with the CPP technology, in terms of ON-delivery, is that peptides usually need a covalent attachment to ONs, which is a cumbersome procedure and high concentrations of peptide conjugates are generally needed to obtain a significant biological response [7,8]. A few studies have convincingly shown that a non-covalent co-incubation strategy of simply mixing CPPs with ONs works efficiently and in a non-toxic fashion. When using the co-incubation strategy with unmodified CPPs, it seems that the complexes remain trapped inside endosomes and are therefore unable to exert a biological response [9]. Ideally, CPPs would be designed to more efficiently escape endosomal compartments following endocytosis thereby allowing them to be non-covalently complexed with oligonucleotides or plasmids. Attempts have been made to combine the use of CPPs with known transfection reagents to reduce the amount of transfection regent needed to obtain biological responses or CPPs have been co-added with known fusogenic peptides. Another strategy has been to co-add the lysosomotrophic agent chloroquine at high concentrations to increase the efficacy of the CPP/ON complexes, which significantly increases transfections but is limited to in vitro use and furthermore, the high concentrations of chloroquine needed raises toxicity concerns.
A related patent, US 2007/0059353 discloses a liposome having cellular and nuclear entry ability. The provided liposome has on its surface a peptide comprising multiple consecutive arginine residues, and specifically a liposome is provided wherein the peptide is modified with a hydrophobic group or hydrophobic compound and the hydrophobic group or hydrophobic compound is inserted into a lipid bilayer so that the peptide is exposed on the surface of the bilayer. The problem with this delivery system, apart from the difficulty of constructing such complex vectors, is that they are based on liposomes. Several groups have reported on alterations in gene expression profiles after transfections with liposome-based delivery systems which greatly hamper their use. In addition, oligoarginines are prone to remain bound to endosomal compartments and are therefore not optimal for delivery. An improved strategy would be to chemically modify newly designed or existing CPPs with one or more chemical entities that could promote endosomal escape.
The drug of choice today for endosomal escape is Chloroquine (CQ) and its analogues. It is a, as it is also called, lysosomotropic agent, inhibiting endosome acidification, leading, at higher concentrations, to endosomal swelling and rupture.
There are several U.S. patents disclosing chloroquine for use against a variety of diseases either alone or in combination with other drugs. For instance, U.S. Pat. No. 4,181,725 and A. M. Krieg, et al, U.S. Patent Applic. 20040009949 disclose the use of chloroquine for treating various autoimmune diseases in combination with inhibitory nucleic acids.
The ability of chloroquine to act as “lysosomotropic”agent to enable release of substances from cellular endosomes/lysosomes is well-documented. [Marches, 2004; A. Cuatraro 1990 etc]. Nevertheless, in vivo use of chloroquine was claimed to be prohibited by its toxicity, as high concentration of free chloroquine needs to be administrated to reach endosomes. (Citing J. M. Benns, et al, 1.sup.st paragraph, Bioconj. Chem. 11, 637-645, (2000): “Although chloroquine has proven to aid in the release of the plasmid DNA into the cytoplasm, it has been found to be toxic and thus cannot be used in vivo.”)
Recent USPTO Application #: 20070166281 entitled “Chloroquine coupled antibodies and other proteins with methods for their synthesis” discloses coupling of chloroquine and thereof derived structures to different carrier compositions that contain biocleavable linkages allowing release of chloroquines under controlled conditions. Application #: 20070166281 is aimed to provide controlled release of the chloroquines from protein or peptide active agent or antibody after the carrier has reached its site of action.
US2006/0040879 Kosak and colleagues patent discloses compositions and methods for preparing chloroquine-coupled nucleic acid compositions. The prior art has shown that chloroquine given as free drug in high enough concentration, enhances the release of various agents from cellular endosomes into the cytoplasm. The purpose of these compositions is to provide a controlled amount of chloroquine at the same site where the nucleic acid needs to be released, thereby reducing the overall dosage needed. This patent is aimed at achieving controlled release of chloroquine conjugated to nucleic acid compositions, this is not the subject of the present invention, but rather to enhance and simplify delivery in gene therapy in vitro and in vivo.
The present invention provides a system for intracellular cargo delivery comprising a new series of molecules that overcome the described drawbacks for non-covalent gene-delivery, ie low and heterogeneous delivery as well as toxicity. The present invention is in no need of biocleavable linkers to cleave chloroquine analogues. The system according to the present invention comprises irreversibly chloroquine-coupled compounds.
The system comprises compounds which are improved CPPs with fatty acid modifications which can be utilized for efficient delivery of a wide variety of ONs, without the toxicity of the delivery agents on the market today. The next generation of further derivatised CPPs can both efficiently deliver the drug load into all of the cells in a population as well as releasing the ONs from their entrapment in endosomes. The claims of the invention describe the modified and derivatised delivery peptides and their tested applications; enhanced transfection, splice correction as well as siRNA delivery.
The present invention relates to a system designed for intracellular cargo delivery comprising at least one component A chosen from aliphatic linear or branched moieties with at least 4 carbon and/or cyclic ring systems comprising 2-4 rings which may contain several hetero atoms chosen from N, S, O and P, wherein component(s) A is (are) attached to a cell penetrating peptide B and/or a non-peptide analogue thereof, and in which said delivery system is capable of delivering a cargo by covalent or non-covalent attachment. The delivery system is called PepFect (see examples table 1 and
According to one embodiment the delivery system, further comprises at least one component C which is a targeting moiety capable of reaching specific cells or tissue of interest. The targeting moiety may be an aptamer or a targeting peptide such as a homing peptide or a receptor ligand.
According to another embodiment the delivery system further comprises a cargo, which may be delivered into cells, tissue or across a cell layer.
One or more components A, one or more components C and one or more cargos can be coupled covalently either to an amino acid side chain and/or to the N- and/or C-terminal of the peptide (B). In some PepFect compounds, a branched tree-like structured spacer has been applied. The targeting moiety C, may be added non-covalently or through covalent conjugation.
The cell delivery system may comprise more than one peptide B which may be bound to each other through peptide bonds.
Moreover, one or more of the components A, C and the cargo may be attached to one ore more peptides B via a spacer arm.
According to the invention, the delivery system may comprise one ore more components A, one or more peptides B, one or more targeting components C coupled to each other in any order without any cargo. One ore more peptides B may be coupled to one or more components A in any order and without any targeting components C and without any cargoes. These may be delivered for further coupling of cargoes at a later stage. The invention relates to a method of delivering cargoes into a target cell in vivo or in vitro by using such a delivery system.
One or more peptides B and one or more cargoes may be coupled to one or more components A in any order without any targeting components C. One ore more peptides B and one or more cargoes may be coupled to one or more components A and one or more targeting components C in any order.
The invention also relates to novel cell-penetrating peptides as well as the method how to produce the PepFect constructs.
Component A
Component A can be one or several aliphatic linear or branched moieties with at least 4 carbon and/or ring systems comprising 2-4 rings which may contain several hetero atoms chosen from N, S, O and P, wherein component(s) A is(are) attached to a cell penetrating peptide B and/or a non-peptide analogue thereof.
A may also be any Acyl deriving from any organic compound, preferentially a fatty acid, a stearyl, bile acid or its derivatives, cholesteryl, cholic acid, deoxycholate, lithocholate or palmitate.
The aliphatic component A may be 4-30 carbon atoms and may be a fatty acid. Such an aliphatic acid may comprise 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 carbon atoms or any interval created by these figures. It may also be a derivative thereof. The functional group(s) could instead of a carboxylic acid be any of but not limited to —OH, —SH, —NH2, —CHO, COXR wherein X is either O or S, R is any aliphatic or aromatic moiety, or a counter ion in a salt formation like Na+, K+ etc or a halogen. According to one embodiment the fatty acid may comprise 10-30 carbon atoms and can be chosen from stearic acid or a C18 derivate thereof or lauric-, myristic-, palmitic-, arachidic-, and behenic acid, attached to a side chain residue, C- or N-terminally on the cell-penetrating peptide. Moreover the chain may contain saturated/non-saturated bonds.
In addition, the component A, may also be one or more copies of a two-four fused cyclic system of 2 to 4 rings of 3- to 8 membered rings, saturated or non-saturated, possibly comprising one to several hetero atoms in the ring systems chosen from N, S, O, B or P. These may be but not limited to biphenyl, diphenyl ether, amine, sulphide or peri-and/or 'ortho-fused and be chosen from but not limited to quinoline, isoquinoline, quinoxaline, pentalene, naphthalene, heptalene, octalene, norbonane, adamantane, indole, indoline, azulene, benzazepine, acridine, anthracene, biphenylene, triphenylene and benzanthracene and analogues thereof. Such analogues may comprise one or more carboxylic groups and/or one or more additional functional groups such as but not limited to one or more amines, one or more thiols, one or more hydroxyls, one or more esters and one or more aldehydes.
According to one embodiment, four copies of component A may be conjugated on a side chain residue via a lysine branched spacer.
These ring systems may also be substituted e. g with other groups with pH buffering capacity to destabilize endosomes or a as a condensing moiety for nucleotide interactions. Examples of substituents but not limited to, could be one to several primary, secondary and/or tertiary amines, substituted or included in as any aliphatic or aromatic moiety or combinations thereof, also spaced by zero to several atoms in a linear, branched or cyclic fashion or a combination thereof.
Examples are N′-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine(chloroquine) or derivatives thereof, di- to tetra ring systems (naphthalene and/or biphenyl connected), 4- to 8 membered rings, one to several hetero atoms in ring systems attached anywhere to the construct described in 1 (
Introduction of a quinoline analogue is accomplished by coupling to activated succinylated side-chains of multiple lysine residues, providing multiple copies of the quinoline analogue covalently bound to the carrier. The preferred conditions are described in Example 12.
The invention also relates to method for synthesizing a quinoline analogue-coupled peptide, or a non-peptide analogue thereof, comprising (a) the steps for activating the lysine on peptide and (b) covalent coupling the quinoline analogue. Activation of the lysine pendant groups of peptide, in order to enable coupling of chloroquine-amine derivative (further disclosed in the Example 12), is achieved by suitable modification of epsilon-amino groups of lysine residues using succinic anhydride. Thus obtained multiple carboxyl groups of the peptide are further activated in situ, i.e. simultaneously with the coupling of quinoline analogue. This procedure is superior to the procedure described in literature up to date, where semi-stable active ester, like NHS, are formed and then coupling hydroxychloroquine, which was derivatised by amine. The method here described is novel and gives better control over the reaction and higher yield. This quinoline analogue is novel, there is no aminoalkylation of chloro-trifluoromethyl-quinoline by a primary diamine derivative in the literature, to our knowledge.
By introducing for example an extra amine at the end of the alkyl chain, covalent attachment is facilitated. The function of the alkyl chain is to provide space and buffer capacity for the aromatic ring system to interact. The chloroquine analogue should consist of but not limited to, quinoline system substituted with a trifluormethylgroup, an alkyl chain with two amines separated with a number of atoms and a functional group at the other end of the alkyl chain separated from the second amine by several atoms for further attachment.
Preferable four copies of component A are conjugated on a side chain via an lysine branched spacer.
The invention further envisages coupling of multiple copies of the quinoline derivative to the peptide B containing appropriate number of poly(L-lysine) pendant groups, all modified by succinic anhydride or any other suitable derivatization reagent known to the skilled in the art.
Peptide Component B
The peptide component B may be selected from one or several copies of the following sequences:
The peptides may be synthesized with a synthesizing device e.g. on Applied Biosystems stepwise synthesizer model 433A. Amino acids may be assembled by t-Boc chemistry using a 4-methylbenzhydrylamine-polysterene resin (MBHA) to generate amidated C-terminus or by F-moc chemistry on a Rink resin.
Moreover, the peptide B may selected from a peptide that contains a sequence of the form Ny1-Bx1-Ny2-Bx2-Ny3, where B is a basic amino acid (such as arg, lys, orn, or his) and N is a neutral aminoacid (such as leu, ile, ala, val, phe, trp, ser, thr, gly, cys, gin, met, pro, tyr) and x and y are integers between 2 and 8.
According to one embodiment the peptide B is selected from LLOOLAAAALOOLL [SEQ ID No 6] and especially AGYLLGKLLOOLAAAALOOLL [SEQ ID No 2] or INLKALAALAKKIL[SEQ ID No28 ] and especially AGYLLGKINLKALAALAKKIL [SEQ ID No 1] and deletions, additions insertions and substitutions of amino acids. The invention also relates to peptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99,5% homology with these sequences. The invention also relates to sub-fragments of the above mentioned peptides having the same properties.
The peptide B may also be non-peptide analogue of a CPP or a scrambled component of the CPP or of the non-peptide analogue thereof.
The term non-peptide analogue is in the present context employed to describe any amino acid sequence comprising at least one non-coded amino acid and/or having a backbone modification resulting in an amino acid sequence without a peptide linkage, i.e. a CO—NH bond formed between the carboxyl group of one amino acid and the amino group of another amino acid. Examples of non-coded amino acids are D-form amino acids, diamino acids, diphenylalanine, Gly, Pro and Pyr derivates.
Furthermore, the present amino acid sequences may either be amidated or occur as free acids.
A scrambled component B means a peptide with the exact amino acid composition but with completely randomized order. Furthermore, a partly inverted sequence is when two or more of the amino acids of the original sequence have been added in reversed order.
Amino acids can be added, inserted, substituted or deleted from the sequences, also non-natural amino acids, without changing their over all cell-penetrating abilities.
The C-terminus of the cell penetrating peptide B and/or the non-peptide analoge thereof may be modified and be chosen from cysteamine or a thiol containing compound, a linear or branched, cyclic or non-cyclic amine containing compound with preferably one additional functional group such as but not limited to COXR wherein X is O or S, R is any aliphatic or aromatic moiety or, a counter ion in a salt formation like Na, K etc, halogen, —OH, —NHR wherein R is a protective group or any aliphatic or aromatic moiety, —SSR wherein R is a protective/activating group or any aliphatic or aromatic moiety.
The cysteamide group on the C-terminal of entity B, is responsible for the unique property, being activated in serum thereby forming a dimer. This dimerasation reaction is catalyzed by oxidative enzymes present in serum. According to the invention a Cysteamide group of one peptide molecule may interact with the Cysteamide group of another peptide molecule in an oxidation reaction. One such sequence may be AGYLLGKINLKALAALAKKIL-Cysteamide. Such a reaction may lead to the formation of a peptide dimer, by creation of a disulfide bridge between thiol-groups located on two different cysteamide-modified peptides. Hence, the sulfhydryl-groups (—SH) of the cysteamide-modified peptides forms disulfide bonds (S—S-bond, disulfide bridge, C—S—S—C) when exposed to oxidative environment.
The delivery system may comprise at least one peptide B, which may be different or the same peptide. Thus, it may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 peptides B. They may be dimers and multiples of CPP and may be cyclic and/or branched.
Component C
The C component is a targeting moiety, such as a ligand for a known or unknown receptor. The substrate may be an aptamer and/or targeting peptide.
An aptamer is a double stranded DNA or single stranded RNA molecule that bind to a specific molecular targets, such as a protein or metabolite.
A targeting peptide is a peptide that binds to specific molecular targets, such as a protein or metabolite, for example a homing peptide. A homing peptide is a peptide sequence which have been selected to bind a certain tissue or cell type, usually by phage display.
In addition, the component C may be another molecule that directs the delivery system to a certain cell type or tissue, well known examples are over-expression of growth factors as tumour targets.
The targeting moiety C may also be non-covalently complexed with the component A and B of the delivery system, as a part of a composition.
Generally, a cell-selective CPP will be useful in the targeted transport of any kind of drug or pharmaceutical substance to a variety of specific eukaryotic and/or prokaryotic cellular targets. A cell-selective transport of such cargo is e.g. envisioned for an improved treatment or prevention of infectious diseases, such as diseases caused by a viral, bacterial or parasital infection.
In yet a further embodiment of the present invention, a cell-penetrating peptide and/or a non-peptide analogue thereof is provided that will enter selectively into a certain cell type/tissue/organ, or that transports a cargo that will only be activated in a certain cell type, tissue, or organ type.
We have shown that adding a moiety for targeting the delivery system to a specific cell type or tissue, does not abolish the delivery properties. As seen by Pepfect 7 (ortogonally SA and with CREKA N-term) can deliver both SCO and plasmid in HeLa and CHO cells (data not shown).
Spacer
Spacers may be used for the attachment of component A, C and the cargoe to the component B.
According to one embodiment the spacer comprises one or more amino acids e.g. lysine units, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. e.g. lysine units, which may be straight or branched and modified with functional groups or extra carbon atoms for further attachment.
The spacer may be a linear or branched moiety with one to several substituents facilitating attachment of component A or a tree-like structure comprised of preferably but not limited to Lysine or Ornithine residues arranged in a dendrimeric fashion comprising of 1, 2, 3, 4 or unlimited number of Lys or Orn residues as depicted in an example in
A spacer is preferable used in conjugating four copies of the ring system (component A) via a side chain in component B. The spacer may be a dendrimer.
Dendrimers are repeatedly branched molecules, in this case preferable with a peptide backbone.
Examples of the Pepfect system for intracellular delivery are here presented without cargo, as shown below:
According to the present invention a new series of CPPs are provided, especially with stearyl modifications. For example (PepFect 1-4 and 14) can be utilized for efficient delivery of SCOs using a non-covalent approach. It is shown that the PepFect peptides are equally, or even more efficient than the commercial transfection reagent Lipofectamine 2000 in conveying SCOs inside cells, still being less toxic. In addition, the low toxicity of Pepfect 1-4 renders them suitable for transfection in sensitive cell systems where Lipofectamine 2000 is not functional. Furthermore, the PepFect peptides are more potent than conventionally used CPPs and far more potent than the pre-clinically used CPP-conjugate (RXR)4-PMO. In addition, the peptides can be effectively exploited for plasmid transfections, also this difficult task in hard to transfect primary glial cells. Moreover, of great importance, only very low amounts of delivery agent and ONs are needed to gain a biological response, which decrease both labours and costs.
The PepFect5-13 analogs may be further chemically modified. Instead of, or in addition to, being modified with a stearic acid entity, these may also be conjugated to a lysine tree bearing e.g. one or more such as four QN analogs that facilitates release of the ONs from vesicular compartments. These are not only active for transportation of ON compounds acting in the nucleus of cells but can additionally be efficiently utilized for the delivery of cytoplasmically active ONs such as anti-miRs and siRNAs. In fact, both PepFect5 and PepFect6, and in particular the latter peptide is significantly more active than Lipofectamine 2000 for the delivery of siRNAs in various cell types. While Lipofectamine/siRNA complexes rarely generates more than 80% down-regulation of gene expression at any given siRNA concentration, both PepFects complexed with siRNA confers almost complete RNAi at low siRNA concentrations. Furthermore, they transfect entire cell populations and not only dividing cells. Finally, PepFect6 is highly active even in serum containing media and is able to transfect very “difficult to transfect” cells including SHSY-5Y, N2a, Jurkat suspension cells, embryonal fibroblasts and primary glia cells. The above described properties, in combination with the lower toxicity compared to Lipofectamine 2000 or Oligofectamine, makes this particular vector highly unique.
The Cargo
The cargo may be chosen from gene modulating compounds, such as oligonucleotides or plasmids. They may be attached to the delivery system by covalent attachment or complex formation.
The family of oligonucleotides includes antisense oligonucleotides for mRNA silencing, splice correcting oligonucleotides for manipulation of pre-mRNA splicing patterns, and short interfering RNAs for gene silencing.
The cargo may be selected from the group consisting of oligonucleotides and modified versions thereof, single strand oligonucleotides (DNA, RNA, PNA, LNA and all synthetic oligonucleotides), double-strand oligonucleotides (siRNA, shRNA, decoy dsDNA etc.), plasmids and other varieties thereof, synthetic nucleotide analogs for the purpose of inhibition of viral replication or antiviral ONs.
The delivery system makes it possible to release ONs (as cargoes) at the correct intracellular location without addition of extra chloroquine. Because the attachment of four copies of the ring system A increases the local effect of the chloroquine analogues. This is a valuable property for in vivo applications. Also, by conjugating chloroquine to the peptide, the effective concentration is resuced by more than a log, most likely explaining the lack of toxicity otherwise seen with chloroquine at 100 μM concentrations.
It has been estimated that 20-30% of all disease-causing mutations affects pre-mRNA splicing. Several genetic disorders and other diseases, including β-thalassemia, cystic fibrosis, muscular dystrophies, cancers, and several neurological disorders, are associated with alterations in alternative splicing, reviewed in. The majority of mutations that disrupt splicing is single nucleotide substitutions within the intronic or exonic segments of the classical splice sites. These mutations result in either exon skipping, use of a nearby pseudo 3′- or 5′splice site, or retention of the mutated intron. Mutations can also introduce new splice sites within an exon or intron.
One of the first splicing mutations described was found in β-thalassemia patients, where mutations in intron 2 of β-globin pre-mRNA create an aberrant 5′splice site, concomitantly activating a cryptic 3′splice site. This in turn leads to an intron inclusion and non-functional protein. Same type of mutations has been identified in the cystic fibrosis transmembrane conductance regulatorgene, resulting in aberrant splicing and development of cystic fibrosis. Duchenne muscular dystrophy (DMD), characterized by progressive degenerative myopathy, and its milder allelic disorder, Becker muscular dystrophy (BMD), are both caused by mutations in the dystrophin gene. Most nonsense mutations within this gene result in premature termination of protein synthesis and to the severe DMD, whereas a nonsense mutation within a regulatory sequence generates partial in-frame skipping of an exon and is associated with the milder BMD. Also, several types of cancers are known to emenate from mutations affecting alternative splicing. Thus, by using oligonucleotides that sequence specifically bind to these intronic/exonic mutations, these mutations are masked and splicing restored.
Further, the invention relates to a method of delivering cargoes into a target cell in vivo or in vitro. Formation of the complex between PepFect and the ONs described here (siRNA, plasmid, SCO (splice correcting Ons)) may be carried out in a small volume of sterile water 30 minutes in RT, and then added, in most experiments, in full serum containing media. An example of the complex formation with cargo: Phosphorothioate 2′O methyl RNA (SCO) or anti-miR21 2′OMe RNA may be mixed with CPPs at different molar ratios (1:0-1:20) in MQ water in 1/10th of the final treatment volume (i.e 50 μl). Complexes can be formed for about 30 min in RT. After 30 min, complexes were added to cells grown in 450 μl of fresh serum free media.
The cargo may also be selected from a fluorescent marker, a cell- or a linker comprising a cleavable site coupled to an inactivating peptide, peptide ligands, cytotoxic peptides, bioactive peptides, diagnostic agents, proteins, pharmaceuticals e.g. anticancer drugs, antibiotica, chemotherapeutics.
The cargo may be attached to any of the components A, B and/or C by covalent or non-covalent bonds. According to one embodiment the cargo may be attached to the peptide component B. In one embodiment of the invention, the cell-penetrating peptide may be coupled by a S—S bridge to said cargo. Naturally, there are a broad variety of methods for coupling a cargo to a CPP, selected individually depending on the nature of CPP, cargo and intended use. A mode for coupling can be selected from the group consisting of covalent and non-covalent binding, as biotin-avidin binding, ester linkage, amide bond, antibody bindings, etc.
The anticancer drugs may be an alkylating agent, an antimetabolite and a cytotoxic antibiotic. The alkylating agent may include 4-[4-Bis(2-chloroethyl)amino)phenyl]butyric acid (chlorambucil) or 3-[4-(Bis(2-chloroethyl)amino)phenyl]-L-alanine (Melphalan), the antimetabolite is N-[4-(N-(2,4-Diamino-6-pteridinylmethyl)methylamino)-benzoyl]-glutamic acid (Methotrexate) and the cytotoxic antibiotic is (8S,10S)-10-[(3-Amino-2,3,6-trideoxy-α-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione (Doxorubicin).
The delivery system may further comprise at least one imaging agent and/or labelling molecule and/or chemotherapeutics. The delivery system of the invention may then be used as chemotherapeutics and/or imaging agents. Such composition may possibly also comprise targeting sequences. The chemotherapeutics and/or imaging agent may be used for delivery of antiviral oligonucleotides.
The labelling molecules may be molecular beacons, including quenched fluorescence based beacons and FRET technology based beacons, for labelling or quantification of intracellular mRNA.
Molecular beacons are molecules, e.g. single-stranded oligonucleotides, with internal fluorophore and a corresponding quenching moiety organized in a hair-pin structure so that the two moieties are in close proximity. Upon binding a target nucleic acid sequence or exposure to other structural modification, the fluorophore is set apart from the quenching moiety resulting in possibility to detect the fluorophore. The most commonly used molecular beacons are oligonucleotide hybridisation probes used for detection of specific DNA or RNA motifs. Similarly, FRET probes are a pair of fluorescent probes placed in close proximity. Fluorophores are so chosen that the emission spectrum of one overlaps significantly with the excitation spectrum of the other. The energy transferred from the donor fluorophore to the acceptor fluorophore is distance-dependent and therefore FRET-technology based beacons can be used for investigating a variety of biological phenomena that produce changes in molecular proximity of the two fluorophores.
The delivery system may also be conjugated to, or complexed with circulation clearance modifiers, like PEG. Such systems may be used for retarded delivery of cargoes. Circular clearance modifiers are molecules that prolong the half-life of drugs in the body, examples are pegyl, albumin binding or sequence capping.
The delivery system may be used in diagnosis of diseases, as research tool and as a targeting system.
The invention also relates to a composition comprising one or more delivery system as defined herein. In such a composition the delivery systems may comprise different components A, and/or different peptides B, and/or different targeting components C and/or different cargoes. These delivery systems may comprise different combinations of A, B, C and cargo as mentioned above.
The invention also relates to a pharmaceutical composition comprising the delivery system according and/or a composition as defined above.
It also relates to the use of one or more delivery systems for the production of a pharmaceutical composition.
Especially the composition may comprise at least two different delivery systems that may act additative or synergistic. These may be present in the composition in different ratios. For example, the compositions may comprise any combination of the Pepfects disclosed in table 1 e.g. Pepfect 5 and 6.
Such a composition may also comprise a mixture of at least two peptides in the same or in different delivery systems which peptides each bring a different property to the complex, such as targetting and transfection.
Such a pharmaceutical composition may be in the form of a oral dosage unit; an injectable fluid; a suppository; a gel; and a cream and may comprise excipients, lubricants, binders, disintegrating agents, solubilizers, suspending agents, isotonizing agents, buffers, soothing agents, preservatives, antioxidants, colorants, sweeteners.
For example, the delivery agent may also be used as an antimicrobial composition, as cell-penetrating peptides resembling those of lytic peptides.
The invention also relates to a material covered with one or more of the delivery systems according to the invention.
Further, it relates to a material having one or more of the delivery systems according to any of claims 1-16 incorporated into the material. The delivery system according to the invention may be incorporated into the dendrimers, liposomes etc. Liposomes are composite structures made of phospholipids and may contain small amounts of other molecules
The invention also relates to a novel peptide that contains a sequence of the form Ny1-Bx1-Ny2-Bx2-Ny3, where B is a basic amino acid (such as arg,lys,orn, or his) and N is a neutral aminoacid (such as leu, ile, ala, val, phe, trp, ser, thr, gly, cys, gln, met, pro, tyr) and x and y are integers between 2 and 8.
The invention especially relates to peptides wherein the entity B is selected from LLOOLAAAALOOLL [SEQ ID No 6] and especially AGYLLGKLLOOLAAAALOOLL[SEQ ID No2] or INLKALAALAKKIL [SEQ ID No28] and especially AGYLLGKINLKALAALAKKIL [SEQ ID No 1] and the sequences with deletions, additions insertions and substitutions of amino acids. The invention also relates to peptides having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% homology with these sequences. The invention also relates to sub-fragments of the above mentioned peptides having the same properties.
As described previously, a CPP can be coupled to a cargo to function as a carrier of said cargo into cells, various cellular compartments, tissue or organs. The cargo may be selected from the group consisting of any pharmacologically interesting substance, such as a peptide, polypeptide, protein, small molecular substance, drug, mononucleotide, oligonucleotide, polynucleotide, antisense molecule, double stranded as well as single stranded DNA, RNA and/or any artificial or partly artificial nucleic acid, e.g. PNA, a low molecular weight molecule, saccharid, plasmid, antibiotic substance, cytotoxic and/or antiviral agent. Furthermore, the transport of cargo can be useful as a research tool for delivering e.g. tags and markers as well as for changing membrane potentials and/or properties, the cargo may e.g. be a marker molecule, such as biotin.
With respect to the intended transport of a cargo across the blood-brain barrier, both intracellular and extracellular substances are equally preferred cargo.
All specific details relate mutatis mutandis to all embodiments described herein. When for example specific chemical components are described in relation to the components A, B, and C of the delivery system, these also applies when the delivery system is incorporated into the dendrimers or liposomes, a material covered with the delivery system as well as when a delivery system it is covered with circulation clearance modifiers.
The invention will now be further illustrated by the following description of embodiments, including short description of the drawings, materials and methods, examples including figures and figure legends as well as sequence listing, but it should be understood that the scope of the invention is not limited to any specifically mentioned embodiments or details.
The invention is below described by examples and comparisons with different PepFect systems and also with Lipofectamine 2000, which is market leading for transfections in vitro today. First the improvements and remodeling of the delivery system are described. Then the testing of various methods to illustrate how versatile PepFects are for delivery of splice correcting ONs, plasmid as well as miRNA and siRNA is being described. In addition, the overall improvement of PepFect compared to Lipofectamine 2000, which have been applied according to the manufacturers instruction, is shown by lower toxicity, homogeneous transfections and high yield transfections in “hard to transfect cells”. All the experiments have been performed in more than one cell type and in most cases with serum present.
Synthesis of Peptides and Oligonucleotides
The peptides in PepFect 1, PepFect 2, PepFect 3, PepFect 4, penetratin [10], TP10 [11], M918 [12], Arg9 [13], and stearyl-Arg9 were synthesized on Applied Biosystems stepwise synthesizer model 433A. Peptides were assembled by t-Boc chemistry using a 4-methylbenzhydrylamine-polysterene resin (MBNA) to generate amidated C-terminus. Solid phase peptide synthesis (SPPS) can also be synthesised using standard Fmoc SPPS conditions well-known to those skilled in the art. Peptides used in the present invention are obtained using standard protocols on a SYRO multiple peptide synthesizer (MultiSynTech GmbH). The method involves repeated coupling of Fmoc-protected amino acids from the carboxy terminal end to the N-terminal end of the peptide, assisted by HBTU activating reagent and DIEA as a base component. The polystyrene resin solid support employed is Rink amide resin (preferable substitution level 0.4 to 0.6 milliequivalents per gram of resin). Amino acids were purchased from Neosystem, France and coupled as hydroxybenzotriazole (HOBt) esters. After cleavage of peptides from the resin using HF, synthesis products were purified by reverse phase HPLC Iomega C18 column and analyzed using Perkin Elmer prOTOF™ 2000 MALDI O-TOF Mass Spectrometer. Masses of peptides correlated well with theoretical values. The sequences of the peptides are presented in Table 1.
For the branched spacer: Resin-bound fully protected peptide sequence Fmoc-AGYLLGK(ε-Mtt)INLKALAALAKKIL-Rink (Rink=Rink amide resin, all amino acids are protected by standard protecting groups if not stated otherwise) is used as starting material. The general manual procedure (Step 1 to Step 7) is to be followed to obtain branched structure of four free carboxyl groups (designated as point of attachment for four copies of novel QN analogues). After each step qualitative ninhydrine colour test (Kaiser test) is performed to monitor the completeness of reaction.
1. The peptide resin is treated with 35% piperidine for 40 minutes to achieve deprotection of amino group.
2. Stearic acid is coupled (the preferred method is use of DCM as solvent and BOP/DIEA for activation and coupling for 1 hour).
3. For Mtt removal, repeated washes by 1% TFA, 3-4% TIS, DCM are employed (1-1.5 hours of total treatment). In order to insure the completeness of removal 1% TFA in DCM without addition of TIS is added to monitor the yellow color of leaving trityl group.
4. 3-5 equivalents of Fmoc-Lys(Fmoc)-OH is coupled for 45 minutes. Preferred coupling reagents are BOP (even more preferably, its non-cancerogenic analog PyBOP) or DIPCDI/HOAt.
5. Fmoc removal according to Step 1.
6. Repeat steps 4 and 5.
7. Coupling of 1.5 equivalents of succinic anhydride in DMF in the presence of 3 equivalents of DIEA for 10 minutes.
Phosphorothioate 2′-O-methyl RNA oligonucleotides were synthesized on an ÄKTA™ oligopilot™ plus 10 with Oligosynt™ 15, pre-packed synthesis columns. Phosphoroamidites (ChemGenes Corporation, Boston, Mass.) at 0.1 M concentration were added in 5 equivalents excess and the recycle time of coupling reagents was 5 minutes. Thiolation was performed with 0.2 M bis-phenylacetyl disulfide (ISIS Pharmaceuticals, Carlsbad, Calif.) in 3-picoline/acetonitrile (1:1) during 3 minutes using 3 ml per synthesis cycle. Oligonucleotides were cleaved from the solid support and deprotected overnight in 25% aqueous ammonium hydroxide (Merck, Darmstadt, Germany) at 55° C. Purification was made with a Tricorn™ column packed with Source™ 15Q anion exchange media utilizing an ÄKTAexplorer™ 100 system and basic NaCl buffers. HiTrap™ desalting columns were used for subsequent work-up of purified oligonucleotides followed by HPLC analysis (Agilent 1100, Santa Clara, Calif.) utilizing a DNAPac™-100 analysis column (Dionex, Sunnyvale, Calif.) confirming >97% full length purity. Correct product was confirmed by mass analysis on a Finnigan™ LCQ™ Deca XP plus mass spectrometer (ThermoFischer Scientific, Waltham, Mass.).
Cell Culture
HeLa pLuc 705 cells, kindly provided by R. Kole and B. Leblue, and hek 293 cells were grown in Dulbecco's Modified Eagle's Media (DMEM) with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin and 200 μg/ml hygromycin. CHO cells were grown in DMEM-F12 media with glutamax supplemented with 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin. BHK 21 cells were grown in GMEM +2 mM Glutamine +5% Tryptose Phosphate Broth +5-10% Fetal Bovine Serum. Cells were grown at 37° C. in 5% CO2 atmosphere. All media and chemicals were purchased from Invitrogen (Sweden).
Complex Formation
Formation of ON/CPP complexes: Phosphorothioate 2′O methyl RNA (SCO) or anti-miR21 2′OMe RNA (with 33% LNA substitution) was mixed with CPPs at different molar ratios (1:0-1:20) in MQ water in 1/10th of the final treatment volume (i.e 50 μl). Complexes were formed for 30 min in RT and meanwhile media was replaced in 24-well plates to fresh serum free DMEM (450 μl). Thereafter, complexes were added to each well. The final concentration of SCO was kept constant at 200 nM and peptide concentration was varied or complexes were formed at a given molar ratio using 400 nM SCO and then serial diluted in water. Complexes were prepared according to manufacturers protocol when using the commercial transfection reagent Lipofectamine 2000 (Promega, USA). PepFect/siRNA complexes were essentially formed in a similar manner but using 100 nM siRNA as starting concentrations and molar ratios ranging from 20-40. Lower concentrations were generated by doing serial dilutions.
Formation of plasmid/CPP complexes:0.5 μg of pGL3 luciferase expressing plasmid or pEGFP-C1 plasmid was mixed with CPPs at different charge ratios (1:1-1:4) in MQ water in 1/10th of the final treatment volume (50 μl). After 30 min, complexes were added to cells grown in 450 μl of fresh serum free media. When using Lipofectamine 2000, complexes were prepared according to manufacturers protocol (Promega, USA).
Gel Shift Assay
Peptides were mixed with SCO as previously described. 0.5 μg SCO was mixed with increasing concentrations of peptides giving rise to peptide/SCO molar ratios ranging from 5 to 20. Complexes were analyzed by electrophoresis on a 20% polyacrylamidic gel at 150V for 1 h in TBE buffer, containing ethidium bromide (Sigma, Sweden). Pictures were taken in Fujifilm LAS-1000 Intelligent Dark box II using IR LAS-1000 Lite v1.2 software.
Quantitative Uptake 100 000 HeLa pLuc 705 cells, seeded 24 h prior experiment, were treated with 200 nM Cy5 labeled SCO complexed with peptides at molar ratios for 1 h. After treatment, cells were washed twice in HKR before trypsination. Cells were centrifuged at 1000 g for 5 min at 4° C. and cell pellets were lysed with 250 μl 0.1 M NaOH for 30 min after which 200 μl lysate was transferred to a black 96-well plate. Fluorescence was measured at 643/670 nm on a Spectra Max Gemeni XS fluorometer (Molecular devices, USA) and recalculated to amount of internalized compound by using the linearity of fluorescein and normalizing to amount of protein (Lowry BioRad, USA).
Luciferase Assay
Splice correction experiments: 100 000 HeLa pLuc 705 cells were seeded 24 h prior experiments in 24-well plates in all experiments. Cells were treated for 4 h with complexes in serum free media followed by replacement to serum medium for additionally 20 h. Thereafter, the cells were washed twice with Hepes Krebs Ringer (HKR) buffer and lysed using 100 μl 0.1% triton X100 in HKR for 15 min at room temperature. Luciferase activity was measured on Flexstation II (Molecular devices, USA) using Promega luciferase assay system. RLU values were normalized to protein content and results are displayed as RLU/mg or as fold-increase in splicing over untreated cells. In experiments with the agent promoting endosomal escape, chloroquine, complexes were co-incubated for 2 h with 75 μM chloroquine in serum free DMEM and subsequently the cells were grown for 20 h in complete DMEM.
Plasmid transfections: For plasmid transfection, 80 000 CHO or Jurkat cells were seeded 24 h before treatment. Cells were treated for 4 h with pGL3/CPP complexes in serum free DMEMF12 after which media was replaced for full growth media for additionally 20 h. Cells were lysed and analyzed in accordance with the splice correction assay.
siRNA transfections: Different luciferase-stable cell lines including HeLa, BHK21, and U2OS cells were seeded as in the other experiments. Cells were treated for 4 h in serum-free or full growth media after which 1 ml of full growth media was added to the wells. Cells were lysed and measured for luminescence 20 h later as described previously. The luminescence was normalized to protein content in each well and the RLU/mg value from untreated cells were considered as 100%. Different treatments are then presented as % of untreated cells.
MicroRNA-21 assay: A plasmid, psi-CHECK2, carrying one internal firefly luciferase gene and a second renilla luciferase gene carrying a microRNA-21 target site in the 3′UTR, was transfected into HeLa cells grown in a 6 cm dish. One day after transfection, cells were detached by trypsination and seeded at a density of 70 000 cells/well in a 24-well plate. After another 24 h, cells were treated with antimiR-21 complexes as previously described for SCOs. Cells were lysed and then assessed for fire fly luciferase expression, that act as an internal standard for transfection, and then for renilla expression. If an ON reaches the cytoplasm, miR21 is sequestered and an increase in renilla expression is expected, generating a positive read-out similar to that of the splice correction assay. The firefly/renilla luciferase ratio of untreated cells was set to 1 and increases in renilla after treatment are presented as fold-increases compared to untreated cells. HeLa cells were used since they are known to express high levels of miR21.
Toxicity Measurements
Membrane integrity was measured using the Promega Cytox-ONE™ assay. In brief, 104 cells were seeded in 96-well plates two days before treatment with peptides in serum free DMEM. After 30 min, media was transferred to a black fluorescence plate and incubated for 10 min with Cytolox-ONE™ reagent followed by stop solution. Fluorescence was measured at 560/590 nm. Untreated cells were defined as zero and LDH released by lysating in 0.18% triton in HKR as 100% leakage.
Wst-1 Assay
Long-term toxic effects of peptides were evaluated using the Wst-1 proliferation assay. HeLa pLuc 705 cells were seeded onto 96-well plates, 104 cells/well, two days before treatment. Cells were treated with complexes in 100 μl serum or serum free DMEM for 24 h. Cells were then exposed to Wst-1 according to manufacturers protocol (Sigma, Sweden). Absorbance (450-690 nm) was measured on absorbance reader Digiscan (Labvision, Sweden). Untreated cells are defined as 100% viable.
FACS
A 24-well plate with EGFP-stable CHO cells were seeded one day prior transfection. On the day of transfection, transfect the cells with PF6-siRNA complexes, molar ratios between siRNA:PF6 a) for serum free media incubations: 1:20 and 1:40 with siRNA concentrations 50 nM, 25 nM and 12.5 nM; nd 1:80 with siRNA concentrations 20 nM, 10 nM and 5 nM. b) for full media incubations: 1:20 and 1:40 with siRNA concentrations 100 nM, 50 nM and 25 nM. For control experiments the cells were transfected with EGFP siRNA using Lipofectamine (100 nM siRNA and 2.8 ul of Lipofectamine), or mock transfected without complexes or only with siRNA (to get native EGFP levels). The incubation vol. during transfections was 500 ul. Incubate the cells with the complexes in serum free media or in full media, as required, for 4 hours. Then add 1 ml of full media into each well and let the cells grow for 24 (or, alternatively, for 48 hours).
On the day of measurment, wash the cells with PBS, and detach from the wells by trypsination. Use 125 ul trypsin solution. After the cells have detached, add 500 ul full media and transfer the cells into eppendorf tubes. Centrifuge at 500×g for 10 min, remove supernatant and resuspend in 500 ul of PBS supplemented with 2% FBS (this FBS is quite important, keeps the cells in better shape when the tubes are waiting to be analyzed). Transfer the cell suspensions info FACS tubes and put them on ice. Analyze as soon as possible. Measure the cell suspensions in FACS. Gate the living cell population on FSC-SSC plot. On the FSC/FITC-A or SSC/FITC-A plot find the gates for moctransfected cells (gate/cloud for cells with native EGFP levels). Run the samples using the same gates. All the cells falling out from the cloud with native EGFP-levels and having lower EGFP levels will be counted as cells where the siRNA transfection has taken place. Present the results as % of cells transfected with siRNA. Alternatively, choose the gates in a similar way based on mock-transfected cells using FITC-A histogram graph.
In order to evaluate the potency of the different unmodified CPPs to convey oligonucleotides into cells, we used a so-called splice correction assay [14]. This assay is based on a stably transfected HeLa cell line, HeLa pLuc 705 cells. The cells have a stable transfection of a plasmid carrying the luciferase coding sequence, interrupted by an insertion of intron 2 from β-globin pre-mRNA carrying a cryptic splice site (
In the first set of experiments we wanted to evaluate the ability of several established CPPs to deliver splice correcting phosphorothioate 2′O-methyl RNA (i.e SCO) into cells using the co-incubation strategy [15]. As seen in
Next, modifications were introduced in the CPPs in an attempt to increase their efficiency. A fatty acid moiety (i.e stearic acid) was introduced to existing CPPs and also to a newly designed sequence (see
An increasing number of diseases, such as β-thalassemia, cystic fibrosis, muscular dystrophies, cancer etc, are caused by mutations leading to aberrant splicing which now may be restored using different SCOs [3,17,18]. However, high doses of SCOs are needed in order to attain significant biological effects in vivo. Therefore, these results are extremely promising for future treatment of various diseases emanating from defective alternative splicing.
To evaluate and characterise the position of the fatty acid modification PepFect 3 (Stearyl modification at N-terminal) and PepFect4 (side chain stearylated on Lys7) where compared in efficiency to deliver SCO.
In the next set of experiments, the ability of the above mentioned PepFect peptides to promote uptake and expression of a 4.7 kbp luciferase expressing pGL3 plasmid was evaluated. Surprisingly, all PepFect peptides were able to significantly increase gene delivery (
Importantly when working with living tissue, is to keep the toxic effects as low as possible. As seen in
MicroRNAs (miRNAs) represent a new class of noncoding RNAs encoded in the genomes of plants, invertebrates, and vertebrates. MicroRNAs regulate translation and stability of target mRNAs based on (partial) sequence complementarity. miRNA alterations are involved in the initiation and progression of human cancer. It has been shown that miR-21 functions as an oncogene and modulates tumorigenesis through regulation of genes such as bcl-2 and thus, it may serve as a novel therapeutic target [19].
Here we show miR-21 delivery by PepFect 5 as compared to Lipofectamine (
In this example, the CPPs are instead of, or in addition to, being modified with a stearic acid entity, also conjugated to a lysine tree bearing four chloroquine analogues (
A user friendly reagent should tolerate minor variations in for instance molar ratio in complex formation. PF 6 works almost equally well at different molar ratio (
In
Several different cell penetrating peptides have been tested in the PepFect delivery system and here is an example of a novel sequence with attachment of stearyl called Pepfect 14. PepFect 14 is able to effectively deliver both siRNA and SCO also in the presence of serum as
In addition, the PepFect delivery system can be added together as shown in
A mixture of 3.8 g (16.3 mmol) 4-chloro-7-(trifluoromethyl)quinoline and 12 times molar excess of N-methyl-2,2′-diaminodiethylamine (25 ml) in a 50 mL round-bottom flask equipped with a magnetic stirrer is heated using PEG 400 bath from room temperature to 80° C. over 2.5 h with stirring, then temperature is raised to 130° C. over the period of 3 h, and finally heated 2.5 h at 140° C. The reaction mixture is cooled down to room temperature, and cold DCM is added, causing immediate precipitation, which is filtered off. The organic layer is washed twice with 5% aqueous NaHCO3, then washed twice by water. The organic phase is dried over anhydrous MgSO4, and solvent is removed under reduced pressure (rotavapor) and the residue is left in freeze-drier. Weight 4.5 g (MW 312.3) 14.4 mmol, 83% yield of crude product which is used for conjugation to peptides without further purification.
Coupling of N-(2-aminoethyl)-N-methyl-N′-[7-(trifluoromethyl)-quinolin-4-yl]ethane-1,2-diamine to the succininic acid modified side-chains of multiple lysine residues, providing multiple copies of QN analogue covalently bound to molecule of carrier. Activation of solid supported free carboxyl groups is achieved with 3 equivalents of TBTU/HOBt and 6 equivalents of DIEA. An excess (2-5 equivalents) of novel derivative of chloroquine to be attached, N-(2-aminoethyl)-N-methyl-N′-[7-(trifluoromethyl)-quinolin-4-yl]ethane-1,2-diamine, is dissolved in DMF and added to the peptide-resin simultaneously with activating reagent, to couple QN analogue via its free amino group group to the activated resin. To assure complete coupling, this reaction is allowed to run for prolonged period of time (typically overnight) as it can not be monitored by Kaiser test.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/SE2009/051032 | 9/16/2009 | WO | 00 | 5/18/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61136574 | Sep 2008 | US |
