US20110053829A1 - Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids - Google Patents

Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids Download PDF

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US20110053829A1
US20110053829A1 US12/553,559 US55355909A US2011053829A1 US 20110053829 A1 US20110053829 A1 US 20110053829A1 US 55355909 A US55355909 A US 55355909A US 2011053829 A1 US2011053829 A1 US 2011053829A1
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Patrick Baumhof
Thomas Schlake
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CureVac AG
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K48/00Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A61K47/6455Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids

Abstract

The present invention is directed to an inventive polymeric carrier molecule according to generic formula (I) and variations thereof, which allows for efficient transfection of nucleic acids into cells in vivo and in vitro, a polymeric carrier cargo complex formed by a nucleic acid and the inventive polymeric carrier molecule, but also methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier complex. The present invention also provides methods of application and use of this inventive polymeric carrier and the inventive polymeric carrier cargo complex as a medicament, for the treatment of various diseases, and in the preparation of a pharmaceutical composition for the treatment such diseases.

Description

  • The present invention is directed to an inventive polymeric carrier molecule according to generic formula (I) and variations thereof, which allows for efficient transfection of nucleic acids into cells in vivo and in vitro, a polymeric carrier cargo complex formed by a nucleic acid and the inventive polymeric carrier molecule, but also methods of preparation of this inventive polymeric carrier molecule and of the inventive polymeric carrier complex. The present invention also provides methods of application and use of this inventive polymeric carrier and the inventive polymeric carrier cargo complex as a medicament, for the treatment of various diseases, and in the preparation of a pharmaceutical composition for the treatment such diseases.
  • Various diseases today require a treatment which involves administration of peptide-, protein-, and nucleic acid-based drugs, particularly the transfection of nucleic acids into cells or tissues. The full therapeutic potential of peptide-, protein-, and nucleic acid-based drugs is frequently compromised by their limited ability to cross the plasma membrane of mammalian cells, resulting in poor cellular access and inadequate therapeutic efficacy. Today this hurdle represents a major challenge for the biomedical development and commercial success of many biopharmaceuticals (see e.g. Foerg and Merkle, Journal of Pharmaceutical Sciences, published online at www.interscience.wiley.com, 2008, 97(1): 144-62).
  • For some diseases or disorders, gene therapeutic approaches have been developed as a specific form of such treatments. These treatments in general utilize transfection of nucleic acids or genes into cells or tissues, whereas gene therapeutic approaches additionally involve the insertion of one or more of these nucleic acids or genes into an individual's cells and tissues to treat a disease, e.g. hereditary diseases, in which a defective mutant allele is replaced with a functional one.
  • Transfer or insertion of one or more of these nucleic acids or genes into an individual's cells, however, still represents a major challenge today and is absolutely necessary for ensuring a good therapeutical effect of a nucleic acid based medicament, particularly in the field of gene therapy.
  • To achieve successful transfer of nucleic acids or genes into an individual's cells, a number of different hurdles have to be passed. The transport of nucleic acids typically occurs via association of the nucleic acid with the cell membrane and subsequent uptake by the endosomes. In the endosomes, the introduced nucleic acids are separated from the cytosol. As expression occurs in the cytosol, these nucleic acids have to depart the cytosol. If the nucleic acids do not manage departing the cytosol, either the endosome fuses with the lysosome leading to a degradation of its content, or the endosome fuses with the cell membrane leading to a return of its content into the extracellular medium. For efficient transfer of nucleic acids, the endosomal escape thus appears to be one of the most important steps additional to the efficiency of transfection itself. Until now, there are different approaches addressing these issues. However, no approach was at least successful in all aspects.
  • Transfection agents used in the art today typically comprise peptides, different polymers, lipids as well as nano- and microparticles (see e.g. Gao, X., K. S. Kim, et al. (2007), Aaps 9(1): E92-104). These transfection agents typically have been used successfully only in in vitro reactions. When transfecting nucleic acids in vivo into cells of a living animal, further requirements have to be fulfilled. As an example, the complex has to be stable in physiological salt solutions with respect to agglomerisation. Furthermore, it does not interact with parts of the complement system of the host. Additionally, the complex shall protect the nucleic acid from early extracellular degradation by ubiquitously occurring nucleases. For therapeutical applications it is furthermore of utmost importance, that the complex is not recognized by the immune system and does not stimulate secretion of cytokines (see Gao, Kim et al., (2007, supra); Martin, M. E. and K. G. Rice (2007), Aaps J 9(1): E18-29; and Foerg and Merkle, (2008, supra)).
  • Foerg and Merkle (2008, supra), discuss therapeutic potential of peptide-, protein and nucleic acid-based drugs. According to their analysis, the full therapeutic potential of these drugs is frequently compromised by their limited ability to cross the plasma membrane of mammalian cells, resulting in poor cellular access and inadequate therapeutic efficacy. Today this hurdle represents a major challenge for the biomedical development and commercial success of many biopharmaceuticals.
  • In this context, Gao et al. (Gao et al. The AAPS Journal 2007; 9(1) Article 9) see the primary challenge for gene therapy in the development of a method that delivers a therapeutic gene to selected cells where proper gene expression can be achieved. Gene delivery and particularly successful transfection of nucleic acids into cells or tissue is, however, not simple and typically dependent on many factors. For successful delivery, e.g., delivery of nucleic acids or genes into cells or tissue, many barriers must be overcome. According to Gao et al. (2007) an ideal gene delivery method needs to meet 3 major criteria: (1) it should protect the transgene against degradation by nucleases in intercellular matrices, (2) it should bring the transgene across the plasma membrane and (3) it should have no detrimental effects.
  • These goals may be achieved by using a combination of different compounds or vectors. Notably, there are some compounds or vectors, which overcome at least some of these barriers.
  • Most usually, transfection, e.g. of nucleic acids, is carried out using viral or non-viral vectors. For successful delivery, these viral or non-viral vectors must be able to overcome the above mentioned barriers. The most successful gene therapy strategies available today rely on the use of viral vectors, such as adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses. Viral vectors are able to mediate gene transfer with high efficiency and the possibility of long-term gene expression, and satisfy 2 out of 3 criteria. However, the acute immune response, immunogenicity, and insertion mutagenesis uncovered in gene therapy clinical trials have raised serious safety concerns about some commonly used viral vectors.
  • A solution to this problem may be found in the use of non-viral vectors. Although non-viral vectors are not as efficient as viral vectors, many non-viral vectors have been developed to provide a safer alternative in gene therapy. Methods of nonviral gene delivery have been explored using physical (carrier-free gene delivery) and chemical approaches (synthetic vector-based gene delivery). Physical approaches usually include needle injection, electroporation, gene gun, ultrasound, and hydrodynamic delivery, employ a physical force that permeates the cell membrane and facilitates intracellular gene transfer. The chemical approaches typically use synthetic or naturally occurring compounds (cationic lipids, cationic polymers, lipid-polymer hybrid systems) as carriers to deliver the transgene into cells. Although significant progress has been made in the basic science and applications of various nonviral gene delivery systems, the majority of nonviral approaches is still much less efficient than viral vectors, especially for in vivo gene delivery (see e.g. Gao et al. The AAPS Journal 2007; 9(1) Article 9).
  • Over the past decade, attractive prospects for a substantial improvement in the cellular delivery of nucleic acids have been announced that were supposed to result from their physical assembly or chemical ligation to so-called cell penetrating peptides (CPPs) also denoted as protein-transduction domains (PTDs) (see Foerg and Merkle, (2008, supra)), CPPs represent short peptide sequences of 10 to about 30 amino acids which can cross the plasma membrane of mammalian cells and may thus offer unprecedented opportunities for cellular drug delivery. Nearly all of these peptides comprise a series of cationic amino acids in combination with a sequence, which forms an α-helix at low pH. As the pH is continuously lowered in vivo by proton pumps, a conformational change of the peptide is usually initiated rapidly. This helix motif mediates an insertion into the membrane of the endosome leading to a release of its content into the cytoplasma (see Foerg and Merkle, (2008, supra); and Vives, E., P. Brodin, et al. (1997). “A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.” J Biol Chem 272(25): 16010-7). Despite these advantages, a major obstacle to CPP mediated drug delivery is thought to consist in the often rapid metabolic clearance of the peptides when in contact or passing the enzymatic barriers of epithelia and endothelia. In conclusion, metabolic stability of CPPs represents an important biopharmaceutical factor for their cellular bioavailability. However, there are no CPPs available in the art, which are on the one hand side stable enough to carry their cargo to the target before they are metabolically cleaved, and which on the other hand side can be cleared from the tissue before they can accumulate and reach toxic levels.
  • One further approach in the art for delivering cargo molecules into cells, e.g. for gene therapy, comprises peptide ligands (see Martin and Rice (see Martin and Rice, The AAPS Journal 2007; 9 (1) Article 3)). Peptide ligands can be short sequences taken from larger proteins that represent the essential amino acids needed for receptor recognition, such as EGF peptide used to target cancer cells. Other peptide ligands have been identified including the ligands used to target the lectin-like oxidized LDL receptor (LOX-1). Up-regulation of LOX-1 in endothelial cells is associated with dysfunctional states such as hypertension and atherosclerosis. Such peptide ligands, however, are not suitable for many gene therapeutic approaches, as they cannot be linked to their cargo molecules by complexation or adhesion but require covalent bonds, e.g. crosslinkers, which typically exhibit cytotoxic effects in the cell.
  • Synthetic vectors may also be used in the art for delivering cargo molecules into cells, e.g., for the purpose of gene therapy. However, one main disadvantage of many synthetic vectors is their poor transfection efficiency compared to viral vectors and significant improvements are required to enable further clinical development. Several barriers that limit nucleic acid transfer both in vitro and in vivo have been identified, and include poor intracellular delivery, toxicity and instability of vectors in physiological conditions (see. e.g. Read, M. L., K. H. Bremner, et al. (2003). “Vectors based on reducible polycations facilitate intracellular release of nucleic acids.” J Gene Med 5(3): 232-45).
  • One specific approach in gene therapy uses cationic lipids. However, although many cationic lipids show excellent transfection activity in cell culture, most do not perform well in the presence of serum, and only a few are active in vivo. A dramatic change in size, surface charge, and lipid composition occurs when lipoplexes are exposed to the overwhelming amount of negatively charged and often amphipatic proteins and polysaccharides that are present in blood, mucus epithelial lining fluid, or tissue matrix. Once administered in vivo, lipoplexes tend to interact with negatively charged blood components and form large aggregates that could be absorbed onto the surface of circulating red blood cells, trapped in a thick mucus layer or embolized in microvasculatures, preventing them from reaching the intended target cells in the distal location. Furthermore, toxicity related to gene transfer by lipoplexes has been observed. Symptomes include inter alia induction of inflammatory cyokines. In humans, various degrees of adverse inflammatory reactions, including flulike symptoms were noted among subjects who received lipoplexes. Accordingly, it appears questionable, as to whether lipoplexes can be safely used in humans at all.
  • One further, more promising approach in gene therapy utilizes cationic polymers. Cationic polymers turned out to be efficient in transfection of nucleic acids, as they can tightly complex and condense a negatively charged nucleic acid. Thus, a number of cationic polymers have been explored as carriers for in vitro and in vivo gene delivery. These include polyethylenimine (PEI), polyamidoamine and polypropylamine dendrimers, polyallylamine, cationic dextran, chitosan, cationic proteins and cationic peptides. Although most cationic polymers share the function of condensing DNA into small particles and facilitating cellular uptake via endocytosis through charge-charge interaction with anionic sites on cell surfaces, their transfection activity and toxicity differ dramatically. Interestingly, cationic polymers exhibit a better transfection efficiency with rising molecular weight. However, a rising molecular weight also leads to a rising toxicity of the cationic polymer. PEI is perhaps the most active and most studied polymer for gene delivery, but its main drawback as a transfection reagent relates to its non-biodegradable nature and toxicity.
  • For this reason, Read et al. (see Read, M. L., K. H. Bremner, et al. (2003). “Vectors based on reducible polycations facilitate intracellular release of nucleic acids.” J Gene Med 5(3): 232-45; and Read, M. L., S. Singh, et al. (2005), “A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids.” Nucleic Acids Res 33(9): e86) developed a new type of synthetic vector based on a linear reducible polycation (RPC) prepared by oxidative polycondensation of the peptide Cys-Lys10-Cys that can be cleaved by the intracellular environment to facilitate release of nucleic acids. They could show that polyplexes formed by RPC are destabilised by reducing conditions enabling efficient release of DNA and mRNA. Cleavage of the RPC also reduced toxicity of the polycation to levels comparable with low molecular weight peptides. The disadvantage of this approach of Read et al. (2003, supra) was that chloroquine was required to facilitate endosomal release of the complexed nucleic acids. Read et al. (2003, supra) therefore included histidine residues in the RPCs which have a known endosomal buffering capacity. They could show that histidine-rich RPCs can be cleaved by the intracellular reducing environment enabling efficient cytoplasmic delivery of a broad range of nucleic acids, including plasmid DNA, mRNA and siRNA molecules without the requirement for the endosomolytic agent chloroquine. Although polyplexes formed with higher molecular weight polymers have improved stability under physiological conditions in Read et al. (2003, supra), data have indicated that high molecular weight polymers can hinder vector unpacking. An important consideration in developing vectors based on high molecular weight histidine-rich polycations was therefore to include cysteine residues to enable them to be triggered by reduction to facilitate release of nucleic acids.
  • Advantageously, low molecular polycationic polymers are less toxic than high molecular polycationic polymers. Hence, the negligible toxicity observed with histidine-rich RPCs may reflect its capacity to be cleaved by reduction into shorter peptides with a lower toxicity profile. The size of the polycation can also influence gene expression by determining the rate of unpacking of DNA from polyplexes. For example, poly (L-lysine) (PLL) of 19 and 36 residues was shown to dissociate from DNA more rapidly than PLL of 180 residues resulting in significantly enhanced short-term gene expression. A minimum length of six to eight cationic amino acids is required to compact DNA into structures active in receptor-mediated gene delivery. However, polyplexes formed with short polycations are unstable under physiological conditions and typically aggregate rapidly in physiological salt solutions.
  • For this reason Read et al. (2003, supra) developed a vector which combines the advantage of short polycations and high-molecular polycations by cationic peptides which are linked together by disulfide bonds. Unfortunately; Read et al. (2003, supra) did not assess whether histidine-rich RPCs can be directly used for in vivo applications. In their study, transfections were performed in the absence of serum to avoid masking the ability of histidine residues to enhance gene transfer that may have arisen from binding of serum proteins to polyplexes restricting cellular uptake. Preliminary experiments indicate that the transfection properties of histidine-rich RPC polyplexes can be affected by the presence of serum proteins with a 50% decrease in GFP-positive cells observed in 10% FCS. For in vivo application they propose modifications with the hydrophilic polymer poly-[N-(2hydroxy-propyl)methacrylamide]. Unfortunately, Read et al. (2003, supra) did not prevent aggregation of polyplexes and binding of polycationic proteins to serum proteins. Furthermore, due to the large excess of polymer, which is characterized by the high N/P ratio, strong cationic complexes are formed when complexing the nucleic acid, which are only of limited use in vivo due to their strong tendency of salt induced agglomeration and interactions with serum contents (opsonization). Additionally, these complexes may excite an immunostimulatory response, when used for purposes of gene therapy. Read et al. (2003, supra) did also not provide in vivo data for the RPC based complexes shown in the publication. It has also turned out that these RPC based complexes are completely inactive subsequent to local administration into the dermis.
  • Summarizing the above, the present prior art as exemplified above suffers from various disadvantages. One particular disadvantage of the reagents described by Read et al. (2003, supra) concerns the high positive charge on the surface of the particles formed. Due to the high positive charge the particles exhibit a high instability towards agglomeration when subjecting these particles in vivo to raised salt concentrations. Such salt concentrations, however, typically occur in vivo in cells or extracellular media. Furthermore, high positively charged complexes show a strong tendency of opsonization. This leads to an enhanced uptake by macrophages and furthermore to a fast inactivation of the complex due to degradation. Particularly the uptake of these complexes by cells of the immune system in general leads to a downstream stimulation of different cytokines. The activation of the immune system, however, represents a severe disadvantage of these systems and should be avoided, particularly for the purpose of several aspects of gene therapy, where an immune response is strictly to be avoided. Additionally, in biological systems positively charged complexes can easily be bound or immobilized by negatively charged components of the extracellular matrix or the serum. Also, the nucleic acids in the complex may be released too early, leading to reduced efficiency of the transfer and half life of the complexes in vivo. Finally, methods which require a surface coating of the complexes e.g. with hydrophilic polymers such as PEI, are to be voided as these are technically expensive and laborious and the products are difficult to control and validate.
  • In consequence, no feasible method or carrier has been presented until today, which allows both compacting and stabilizing a nucleic acid for the purposes of gene therapy, which show a good transfection activity and low or even no toxicity. Accordingly, there is still an intensive need in the art to provide carriers for the purpose of gene transfer, which are on the one hand side stable enough to carry their cargo to the target before they are metabolically cleaved, and which on the other hand side can be cleared from the tissue before they can accumulate and reach toxic levels.
  • The object underlying the present invention is therefore to provide a carrier or a complexing agent, particularly for the transfection of nucleic acids for the purposes of gene therapy, which is capable to compact nucleic acids, preferably coding DNA or coding RNA, such as mRNA, and which allows efficient transfection of the nucleic acid into different cell lines in vitro but also transfection in vivo. As uptake by cell occurs via the endosomal route, such a carrier or a complexing agent shall also allow or provide for efficient release of the nucleic acid, e.g. mRNA, from the endosomes (endosomal release or endosomal escape). Additionally, it is preferred, that the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and such carrier or a complexing agent shows improved resistance to agglomeration. It should preferably also confer enhanced stability to the nucleic acid cargo with respect to serum containing media. Most importantly, in vivo activity shall be obtained, thereby suppressing at least in part an immune reaction of the transfected nucleic acid and the cytokine stimulation.
  • This object is solved by the subject matter of the present invention, preferably by the subject matter of the attached claims. Particularly, according to a first aspect of the present invention the above object is solved by a polymeric carrier molecule according to generic formula (I):

  • L-P1—S—[S—P2—S]n—S—P2-L
  • wherein,
    • P1 and P3 represent a linear or branched hydrophilic polymer chain, each P1 and P3 exhibiting at least one —SH-moiety, capable to form a disulfide linkage upon condensation with component P2, or alternatively with further components, the linear or branched hydrophilic polymer chain preferably selected independent from each other from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkyl glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 100 kDa, preferably of about 5 kDa to about 25 kDa;
    • P2 is a cationic or polycationic peptide or protein, and preferably having a length of about 3 to 100 amino acids, more preferably having a length of about 3 to 50 amino acids, even more preferably having a length of about 3 to 25 amino acids, e.g. having a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to 25 amino acids, or
      • is a cationic or polycationic polymer, typically having a molecular weight of about 0.5 kDa to about 100 kDa, including a molecular weight of about 10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30 kDa, each P2 exhibiting at least two —SH-moieties, capable to form a disulfide linkage upon condensation with component(s) P1 and/or P3 or alternatively with further components;
    • —S—S— is a (reversible) disulfide bond (the brackets are omitted for better readability), wherein S preferably represents sulphur or a —SH carrying moiety, which has formed (reversible) disulfide bond. The (reversible) disulfide bond is preferably formed by condensation of —SH-moieties of either components P1 and P2, P2 and P2, or P2 and P3, or optionally of further components as defined herein (e.g. L, (AA)x, [(AA)x]z, etc.); The —SH-moiety may be part of the structure of these components or added by a modification as defined below;
    • L is an optional ligand, which may be present or not, and may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, (e.g. TAT), etc.;
    • n is an integer, typically selected from a range of about 1 to 50, preferably from a range of about 1, 2 or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10.
  • The inventive polymeric carrier molecule according to generic formula (I) is prepared by a new synthesis strategy and advantageously allows to combine desired properties of different (short) polymers in one polymer, e.g. to efficiently compact nucleic acids for the purpose of efficient transfection of nucleic acids for the purposes of gene therapy without loss of activity, particularly efficient transfection of a nucleic acid into different cell lines in vitro but also transfection in vivo. The inventive polymeric carrier molecule is furthermore not toxic to cells and provides for efficient release of its nucleic acid cargo. Finally, it shows improved resistance to agglomeration due to addition of PEG-monomers in the inventive polymeric carrier molecule according to generic formula (I), which additionally confer enhanced stability of the nucleic acid cargo with respect to serum containing media.
  • Even more advantageously, the inventive polymeric carrier molecule according to generic formula (I) allows to considerably vary its peptide or polymeric content and thus to modulate its biophysical/biochemical properties, particularly the cationic properties of component [S—P2—S]n, quite easily and fast, e.g. by incorporating as components P2 the same or different cationic peptide(s) or polymer(s) and optionally adding other components, e.g. repetitive amino acid component(s) (AA)x, into repetitive component [S—P2—S] to form a modified repetitive component such as {[S—P2—S]1-y/[S-(AA)n-S]y}n as a core motif of the inventive polymeric carrier (see below). Even though consisting of quite small non-toxic monomer units the inventive polymeric carrier molecule forms a long cationic binding sequence providing a strong condensation of the nucleic acid cargo and complex stability. Under the reducing conditions of the cytosole (e.g. cytosolic GSH), the complex is rapidly degraded into its monomers, which are further degraded (e.g. oligopeptides) or secreted (e.g. PEG). This supports deliberation of the nucleic acid cargo in the cytosol. Due to degradation into small oligopeptides in the cytosole, no toxicity is observed as known for high-molecular oligopeptides, e.g. from high-molecular oligoarginine. The PEG-“coating” also allows to somehow “coat” the polymeric carrier with a hydrophilic coating at its terminal ends, which prevents salt-mediated agglomeration and undesired interactions with serum contents. In the cytosole, this “coating” is easily removed under the reducing conditions of the cell. Also, this effect promotes deliberation of the nucleic acid cargo in the cytosol. When using a difunctional PEG derivative, even more components, such as ligands (L), may be used, e.g. for direction of the inventive carrier polymer and its complexed nucleic acid into specific cells.
  • In the context of formula (I) of the present invention components P1 and P3 represent a linear or branched hydrophilic polymer chain, containing at least one —SH-moiety, each P1 and P3 independently selected from each other, e.g. from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkyl L-glutamine). Preferably, each of hydrophilic polymers P1 and P3 exhibits a molecular weight of about 1 kDa to about 100 kDa, preferably of about 1 kDa to about 75 kDa, more preferably of about 5 kDa to about 50 kDa, even more preferably of about 5 kDa to about 25 kDa. Additionally, each of hydrophilic polymers P1 and P3 typically exhibits at least one —SH-moiety, wherein the at least one —SH-moiety is capable to form a disulfide linkage upon condensation with component P2 as defined below and optionally with a further component, e.g. L and/or (AA)x, and/or [(AA)x]z, e.g. if two or more —SH-moieties are contained. The following subformulas “P1—S—S—P2” and “P2—S—S—P3” of generic formula (I) above (the brackets are omitted for better readability), wherein any of S, P1 and P3 are as defined herein, typically represent a situation, wherein one-SH-moiety of hydrophilic polymers P1 and P3 was condensed with one —SH-moiety of component P2 of generic formula (I) above, wherein both sulphurs of these —SH-moieties form a disulfide bond —S—S— as defined herein in formula (I). These —SH-moieties are typically provided by each of the hydrophilic polymers P1 and P3, e.g. via an internal cysteine or any further (modified) amino acid, which carries a —SH moiety. Accordingly, the subformulas “P1—S—S—P2” and “P2—S—S—P3” may also be written as “P1-Cys-Cys-P2” and “P2-Cys-Cys-P3”, wherein the term Cys-Cys represents two cysteines coupled via a disulfide bond, not via a peptide bond. In this case, the term “—S—S—” in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their —SH-moieties to form a disulfide bond. Accordingly, the term “-Cys-Cys-” may also be understood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates the sulfur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a —SH containing moiety and a cysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”. Alternatively, the hydrophilic polymers P1 and P3 may be modified with a —SH moiety, preferably via a chemical reaction with a compound carrying a —SH moiety, such that each of the hydrophilic polymers P1 and P3 carries at least one such —SH moiety. Such a compound carrying a —SH moiety may be e.g. an (additional) cysteine or any further (modified) amino acid, which carries a —SH moiety. Such a compound may also be any non-amino compound or moiety, which contains or allows to introduce a —SH moiety into hydrophilic polymers P1 and P3 as defined herein. Such non-amino compounds may be attached to the hydrophilic polymers P1 and P3 of formula (I) according to the present invention via chemical reactions or binding of compounds, e.g. by binding of a 3-thio propionic acid or thioimolane, by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc.), by Michael addition (e.g maleinimide moieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow Sn-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components. In each case, the SH-moiety, e.g. of a cysteine or of any further (modified) amino acid or compound, may be present at the terminal ends or internally at any position of hydrophilic polymers P1 and P3. As defined herein, each of hydrophilic polymers P1 and P3 typically exhibits at least one —SH-moiety, but may also contain two or even more —SH-moieties, which may be used to additionally attach further components as defined herein, e.g. a ligand, a repetitive amino acid component (AA)x, antibodies, cell penetrating peptides (e.g. TAT), etc.
  • According to a further preferred embodiment, each of hydrophilic polymers P1 and P3 may also contain at least one further functional moiety, which allows attaching further components as defined herein, e.g. a ligand, a repetitive amino acid component (AA)x, etc. Such functional moieties may be selected from functionalities which allow the attachment of further components, e.g. functionalities as defined herein, e.g. by amide formation (e.g. carboxylic acids, sulphonic acids, amines, etc.), by Michael addition (e.g maleinimide moieties, α,β unsatured carbonyls, etc.), by click chemistry (e.g. azides or alkines), by alkene/alkine methatesis (e.g. alkenes or alkines), imine or hydrozone formation (aldehydes or ketons, hydrazins, hydroxylamins, amines), complexation reactions (avidin, biotin, protein G) or components which allow Sn-type substitution reactions (e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) or other chemical moieties which can be utilized in the attachment of further components.
  • Component P2 in the context of formula (I) of the present invention preferably represents a cationic or polycationic peptide or protein or alternatively a cationic or polycationic polymer. Each component P2 typically exhibits at least two —SH-moieties, capable to form a disulfide linkage upon condensation with component(s) P1 and/or P3 or alternatively with further components. Component P2 typically occurs within repetitive component [—S—P2—S—]n of formula (I) of the present invention. The term “cationic or polycationic” typically refers to a charged molecule, which is positively charged (cation) at a pH value of about 1 to 9, preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4. Accordingly, a cationic or polycationic peptide or protein as component P2 or alternatively a cationic or polycationic polymer as component P2 according to the present invention is positively charged under physiological conditions, particularly under physiological salt conditions of the cell in vivo.
  • In the specific case that component P2 of formula (I) of the present invention is a cationic or polycationic peptide or protein the cationic properties of repetitive component [S—P2—S]n may be determined upon its content of cationic amino acids in the entire repetitive component [S—P2—S]n. Preferably, the content of cationic amino acids in repetitive component [S—P2—S]n is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 15% to 75%, even more preferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a range formed by any two of the afore mentioned values, provided, that the content of all amino acids, e.g. cationic, lipophilic, hydrophilic, aromatic and further amino acids, in the entire repetitive component [S—P2—S]n is 100%.
  • In the specific case that component P2 of formula (I) of the present invention is a cationic or polycationic polymer the cationic properties of repetitive component [S—P2—S]n may be determined upon its content of cationic charges in the entire repetitive component [S—P2—S]n when compared to the overall charges of repetitive component [S—P2—S]n. Preferably, the content of cationic charges in repetitive component [S—P2—S]n at a (physiological) pH as defined herein is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 15% to 75%, even preferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a range formed by any two of the afore mentioned values, provided, that the content of alt charges, e.g. positive and negative charges at a (physiological) pH as defined herein, in the entire repetitive component [S—P2—S]n is 100%.
  • The cationic or polycationic peptide or protein as component P2, or the cationic or polycationic polymer as component P2, is preferably a linear molecule, however, branched cationic or polycationic peptides or proteins as component P2 or branched cationic or polycationic polymers as component P2 may also be used.
  • Typically, component P2, e.g. the cationic or polycationic peptide or protein or the cationic or polycationic polymer as defined herein, is linked to its neighboring components, e.g. components P1 and P2, and/or as a part of repetitive component [S—P2—S]n to further components P2, via disulfide bonds (—S—S—). In this context, the sulfurs adjacent to component P2 in repetitive component [S—P2—S]n and as defined in generic formula (I) L-P1—S—[S—P2—S]n—S—P3-L, necessary for providing a disulfide bond, may be provided by component P2 itself by a —SH moiety as defined herein or may be provided by modifying component P2 accordingly to exhibit a —SH moiety within the above definition of repetitive component [S—P2—S]n. The —SH moieties for component P2 are preferably as defined herein for components P1 and P3. If such —SH-moieties, necessary to form a disulfide bond (—S—S—) within the above meaning, are provided by component P2 itself this may occur e.g. by at least two cysteines or any further (modified) amino acids or chemical compounds, which carry a —SH moiety, already occurring within the amino acid sequence of component P2 at whatever position of the amino acid sequence of component P2. Alternatively, component P2 may be modified accordingly with a chemical compound, e.g. a cysteine or any further (modified) amino acid or chemical compound, which carries a (free)-SH moiety. Thereby, component P2 preferably carries at least two —SH-moieties, which sulphurs atoms are capable to form a disulfide bond upon condensation with a-SH-moiety of components P1 or P3 as defined herein, or between a first component P2 and a further component P2, etc. Such —SH-moieties are preferably as defined herein.
  • According to one specific embodiment, component P2 within repetitive component [S—P2—S]n of generic formula (I) above may comprise a cysteine as a —SH moiety. In this context, repetitive component [S—P2—S]n may thus be written as follows:

  • [Cys-P2-Cys]n
  • wherein n and P2 are as defined herein and each Cys provide for the —SH-moiety for the disulfide bond. Cys is the amino acid cysteine in its three letter code. (For illustrative purposes, in the present description the disulfide bond —S—S— generally may also be written as -(Cys-S)—(S-Cys)-, wherein Cys-S represents a Cysteine with an naturally occurring —SH moiety, wherein this —SH moiety forms a disulfide bond with a —SH moiety of a second cysteine. Accordingly, repetitive component [Cys-P2-Cys]n may also be written as [(S-Cys)-P2-(Cys-S)]n, which indicates that the —SH-moiety is provided by a cysteine and the Cysteine itself provides for the sulfur of the disulfide bond.)
  • In the context of the entire formula (I) above, this specific embodiment thus may be defined as follows:

  • L-P1—S-[Cys-P2-Cys]n-S—P3-L
  • wherein L, P1, P2, P3 and N are as defined herein, S is sulphur and each Cys provides for one —SH-moiety for the disulfide bond.
  • In each case, the SH-moiety, e.g. of a cysteine or any further (modified) amino acid or further compound used for modification of component P2, may be present in the cationic or polycationic peptide or protein as component P2, internally or at one or both of its terminal ends, e.g. if a cationic or polycationic peptide or protein is used as component P2 at the N-terminal end or at the C-terminal end, at both these terminal ends, and/or internally at any position of the cationic or polycationic peptide or protein as component P2. Preferably, the —SH moiety may be present component P2 at least at one terminal end, e.g. at the N-terminal end and/or at the C-terminal end, more preferably at both the N-terminal and the C-terminal end of a cationic or polycationic peptide or protein as component P2.
  • Due to its repetitive character component [S—P2—S]n may represent a situation, wherein one of the at least two —SH-moieties of component P2 was condensed with a —SH-moiety of a further component P2 of generic formula (I) above, wherein both sulphurs of these —SH-moieties form a disulfide bond (—S—S—) between a first component P2 and at least one further component P2.
  • In this context, the number of repetitions of component P2 in formula (I) according to the present invention is defined by integer n. n is an integer, typically selected from a range of about 1 to 50, preferably from a range of about 1, 2 or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10. If, for example, in repetitive component [S—P2—S]n integer n is 5, repetitive component [S—P2—S]n preferably reads as follows:

  • [S—P2—S—S—P2—S—S—P2—S—S—P2—S—S—P2—S]
  • In the above example component P2 occurs 5 times (preferably in a linear order), wherein each component P2 is linked to its neighbor component by a disulfide bond within the above definition of repetitive component [S—P2—S]n. Any of components P2 may be the same or different from each other.
  • According to one particular embodiment, component P2 represents a cationic or polycationic peptide or protein having a length of about 3 to 100 amino acids, preferably having a length of about 3 to 50 amino acids, more preferably having a length of about 3 to 25 amino acids, e.g. having a length of about 3 to 10, 5 to 15, 10 to 20 or 15 to 25 amino acids.
  • The cationic or polycationic peptide or protein as component P2 may be any protein or peptide suitable for this purpose, particular any cationic or polycationic peptide or protein capable to complex a nucleic acid as defined according to the present invention, and thereby preferably condensing the nucleic acid.
  • Particularly preferred, cationic or polycationic peptides or proteins as component P2 may be selected from protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, *1, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-rich peptides, Loligomere, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc.
  • According to one particular preferred embodiment, cationic or polycationic peptides or proteins as component P2 are selected from following cationic peptides having the following total sum formula (II):

  • {(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x},
  • wherein l+m+n+o+x=8−15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the oligopeptide. Any of amino acids Arg, Lys, His, Orn and Xaa may be positioned at any place of the peptide. Particularly preferred peptides of this formula are oligoarginines such as e.g. Arg7, Arg8, Arg9, Arg7, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc.
  • According to a particular preferred embodiment, cationic or polycationic peptides or proteins as component P2, having the empirical formula (II) as shown above, may be, without being restricted thereto, selected from the following subgroup of formulae:

  • Arg8, Arg9, Arg10, Arg11, Arg12, Arg13, Arg14, Arg15;

  • Lys8, Lys9, Lys10, Lys11, Lys12, Lys13, Lys14, Lys15;

  • His8, His9, His10, His11, His12, His13, His14, His15;

  • Orn8, Orn9, Orn10, Orn11, Orn12, Orn13, Orn14, Orn15;
  • According to a further particularly preferred embodiment, cationic or polycationic peptides or proteins as component P2, having the empirical formula (II) as shown above, may be, without being restricted thereto, selected from the following subgroup of formulae, wherein the following formulae (as with empirical formula (II)) do not specify any amino acid order, but are intended to reflect empirical formulae by exclusively specifying the (number of) amino acids as components of the respective peptide. Accordingly, as an example, empirical formula Arg(7-14)Lys1 is intended to mean that peptides falling under this formula contain 7 to 14 Arg residues and 1 Lys residue of whatsoever order. If the peptides contain 7 Arg residues and 1 Lys residue, all variants having 7 Arg residues and 1 Lys residue are encompassed. The Lys residue may therefore be positioned anywhere in the e.g. 8 amino acid long sequence composed of 7 Arg and 1 Lys residues. The subgroup preferably comprises:
  • Arg(7-14)Lys1, Arg(7-14)His1, Arg(7-14)Orn1, Lys(7-14)His1, Lys(7-14)Orn1, His(7-14)Orn1,;
    Arg(6-13)Lys2, Arg(6-13)His2, Arg(6-13)Orn2, Lys(6-13)His2, Lys(6-13)Orn2, His(6-13)Orn2,;
    Arg(5-12)Lys3, Arg(5-12)His3, Arg(5-12)Orn3, Lys(5-12)His3, Lys(5-12)Orn3, His(5-12)Orn3,;
    Arg(4-11)Lys4, Arg(4-11)His4, Arg(4-11)Orn4, Lys(4-11)His4, Lys(4-11)Orn4, His(4-11)Orn4,;
    Arg(3-10)Lys5, Arg(3-10)His5, Arg(3-10)Orn5, Lys(3-10)His5, Lys(3-10)Orn5, His(3-10)Orn5,;
    Arg(2-9)Lys6, Arg(2-9)His6, Arg(2-9)Orn6, Lys(2-9)His6, Lys(2-9)Orn6, His(2-9)Orn6,;
    Arg(1-8)Lys7, Arg(1-8)His7, Arg(1-8)Orn7, Lys(1-8)His7, Lys(1-8)Orn7, His(1-8)Orn7,;
    Arg(6-13)Lys1His1, Arg(6-13)Lys1Orn1, Arg(6-13)His1Orn1, Arg1Lys(6-13)His1, Arg1Lys(6-13)Orn1,
    Lys(6-13)His1Orn1, Arg1Lys1His(6-13), Arg1His(6-13)Orn1, Lys1His(6-13)Orn1;
    Arg(5-12)Lys2His1, Arg(5-12)Lys1His2, Arg(5-12)Lys2Orn1, Arg(5-12)Lys1Orn2, Arg(5-12)His2Orn1,
    Arg(5-12)His1Orn2, Arg2Lys(5-12)His1, Arg1Lys(5-12)His2, Arg2Lys(5-12)Orn1, Arg1Lys(5-12)Orn2,
    Lys(5-12)His2Orn1, Lys(5-12)His1Orn2, Arg2Lys1His(5-12), Arg1Lys2His(5-12), Arg2His(5-12)Orn1,
    Arg1His(5-12)Orn2, Lys2His(5-12)Orn1, Lys1His(5-12)Orn2;
    Arg(4-11)Lys3His1, Arg(4-11)Lys2His2, Arg(4-11)Lys1His3, Arg(4-11)Lys3Orn1, Arg(4-11)Lys2Orn2,
    Arg(4-11)Lys1Orn3, Arg(4-11)His3Orn1, Arg(4-11)His2Orn2, Arg(4-11)His1Orn3, Arg3Lys(4-11)His1,
    Arg2Lys(4-11)His2, Arg1Lys(4-11)His3, Arg3Lys(4-11)Orn1, Arg2Lys(4-11)Orn2, Arg1Lys(4-11)Orn3,
    Lys(4-11)His3Orn1, Lys(4-11)His2Orn2, Lys(4-11)His1Orn3, Arg3Lys1His(4-11), Arg2Lys2His(4-11),
    Arg1Lys3His(4-11), Arg3His(4-11)Orn1, Arg2His(4-11)Orn2, Arg1His(4-11)Orn3, Lys3His(4-11)Orn1,
    Lys2His(4-11)Orn2, Lys1His(4-11)Orn3;
    Arg(3-10)Lys4His1, Arg(3-10)Lys3His2, Arg(3-10)Lys2His3, Arg(3-10)Lys1His4, Arg(3-10)Lys4Orn1,
    Arg(3-10)Lys3Orn2, Arg(3-10)Lys2Orn3, Arg(3-10)Lys1Orn4, Arg(3-10)His4Orn1, Arg(3-10)His3Orn2,
    Arg(3-10)His2Orn3, Arg(3-10)His1Orn4, Arg4Lys(3-10)His1, Arg3Lys(3-10)His2, Arg2Lys(3-10)His3,
    Arg1Lys(3-10)His4, Arg4Lys(3-10)Orn1, Arg3Lys(3-10)Orn2, Arg2Lys(3-10)Orn3, Arg1Lys(3-10)Orn4,
    Lys(3-10)His4Orn1, Lys(3-10)His3Orn2, Lys(3-10)His2Orn3, Lys(3-10)His1Orn4, Arg4Lys1His(3-10),
    Arg3Lys2His(3-10), Arg2Lys3His(3-10), Arg1Lys4His(3-10), Arg4His(3-10)Orn1, Arg3His(3-10)Orn2,
    Arg2His(3-10)Orn3, Arg1His(3-10)Orn4, Lys4His(3-10)Orn1, Lys3His(3-10)Orn2, Lys2His(3-10)Orn3,
    Lys1His(3-10)Orn4;
    Arg(2-9)Lys5His1, Arg(2-9)Lys4His2, Arg(2-9)Lys3His3, Arg(2-9)Lys2His4, Arg(2-9)Lys1His5,
    Arg(2-9)Lys5Orn1, Arg(2-9)Lys4Orn2, Arg(2-9)Lys3Orn3, Arg(2-9)Lys2Orn4, Arg(2-9)Lys1Orn5,
    Arg(2-9)His5Orn1, Arg(2-9)His4Orn2, Arg(2-9)His3Orn3, Arg(2-9)His2Orn4, Arg(2-9)His1Orn5,
    Arg5Lys(2-9)His1, Arg4Lys(2-9)His2, Arg3Lys(2-9)His3, Arg2Lys(2-9)His4, Arg1Lys(2-9)His5,
    Arg5Lys(2-9)Orn1, Arg4Lys(2-9)Orn2, Arg3Lys(2-9)Orn3, Arg2Lys(2-9)Orn4, Arg1Lys(2-9)Orn5,
    Lys(2-9)His5Orn1, Lys(2-9)His4Orn2, Lys(2-9)His3Orn3, Lys(2-9)His2Orn4, Lys(2-9)His1Orn5,
    Arg5Lys1His(2-9), Arg4Lys2His(2-9), Arg3Lys3His(2-9), Arg2Lys4His(2-9), Arg1Lys5His(2-9),
    Arg5His(2-9)Orn1, Arg4His(2-9)Orn2, Arg3His(2-9)Orn3, Arg2His(2-9)Orn4, Arg1His(2-9)Orn5,
    Lys5His(2-9)Orn1, Lys4His(2-9)Orn2, Lys3His(2-9)Orn3, Lys2His(2-9)Orn4, Lys1His(2-9)Orn5;
    Arg(1-8)Lys6His1, Arg(1-8)Lys5His2, Arg(1-8)Lys4His3, Arg(1-8)Lys3His4, Arg(1-8)Lys2His5,
    Arg(1-8)Lys1His6, Arg(1-8)Lys6Orn1, Arg(1-8)Lys5Orn2, Arg(1-8)Lys4Orn3, Arg(1-8)Lys3Orn4,
    Arg(1-8)Lys2Orn5, Arg(1-8)Lys1Orn6, Arg(1-8)His6Orn1, Arg(1-8)His5Orn2, Arg(1-8)His4Orn3,
    Arg(1-8)His3Orn4, Arg(1-8)His2Orn5, Arg(1-8)His1Orn6, Arg6Lys(1-8)His1, Arg5Lys(1-8)His2,
    Arg4Lys(1-8)His3, Arg3Lys(1-8)His4, Arg2Lys(1-8)His5, Arg1Lys(1-8)His6, Arg6Lys(1-8)Orn1,
    Arg5Lys(1-8)Orn2, Arg4Lys(1-8)Orn3, Arg3Lys(1-8)Orn4, Arg2Lys(1-8)Orn5, Arg1Lys(1-8)Orn6,
    Lys(1-8)His6Orn1, Lys(1-8)His5Orn2, Lys(1-8)His4Orn3, Lys(1-8)His3Orn4, Lys(1-8)His2Orn5,
    Lys(1-8)His1Orn6, Arg6Lys1His(1-8), Arg5Lys2His(1-8), Arg4Lys3His(1-8), Arg3Lys4His(1-8),
    Arg2Lys5His(1-8), Arg1Lys6His(1-8), Arg6His(1-8)Orn1, Arg5His(1-8)Orn2, Arg4His(1-8)Orn3,
    Arg3His(1-8)Orn4, Arg2His(1-8)Orn5, Arg1His(1-8)Orn6, Lys6His(1-8)Orn1, Lys5His(1-8)Orn2,
    Lys4His(1-8)Orn3, Lys3His(1-8)Orn4, Lys2His(1-8)Orn5, Lys1His(1-8)Orn6;
    Arg(5-12)Lys1His1Orn1, Arg1Lys(5-12)His1Orn1, Arg1Lys1His(5-12)Orn1, Arg1Lys1His1Orn(5-12);
    Arg(4-11)Lys2His1Orn1, Arg(4-11)Lys1His2Orn1, Arg(4-11)Lys1His1Orn2, Arg2Lys(4-11)His1Orn1,
    Arg1Lys(4-11)His2Orn1, Arg1Lys(4-11)His1Orn2, Arg2Lys1His(4-11)Orn1, Arg1Lys2His(4-11)Orn1,
    Arg1Lys1His(4-11)Orn2, Arg2Lys1His1Orn(4-11), Arg1Lys2His1Orn(4-11), Arg1Lys1His2Orn(4-11);
    Arg(3-10)Lys3His1Orn1, Arg(3-10)Lys2His2Orn1, Arg(3-10)Lys2His1Orn2, Arg(3-10)Lys1His2Orn2,
    Arg(3-10)Lys1His1Orn3, Arg3Lys(3-10)His1Orn1, Arg2Lys(3-10)His2Orn1, Arg2Lys(3-10)His1Orn2,
    Arg1Lys(3-10)His2Orn2, Arg1Lys(3-10)His1Orn3, Arg3Lys1His(3-10)Orn1, Arg2Lys2His(3-10)Orn1,
    Arg2Lys1His(3-10)Orn2, Arg1Lys2His(3-10)Orn2, Arg1Lys1His(3-10)Orn3, Arg3Lys1His1Orn(3-10),
    Arg2Lys2His1Orn(3-10), Arg2Lys1His2Orn(3-10), Arg1Lys2His2Orn(3-10), Arg1Lys1His3Orn(3-10);
    Arg(2-9)Lys4His1Orn1, Arg(2-9)Lys1His4Orn1, Arg(2-9)Lys1His1Orn4, Arg(2-9)Lys3His2Orn1,
    Arg(2-9)Lys3His1Orn2, Arg(2-9)Lys2His3Orn1, Arg(2-9)Lys2His1Orn3, Arg(2-9)Lys1His2Orn3,
    Arg(2-9)Lys1His3Orn2, Arg(2-9)Lys2His2Orn2, Arg4Lys(2-9)His1Orn1, Arg1Lys(2-9)His4Orn1,
    Arg1Lys(2-9)His1Orn4, Arg3Lys(2-9)His2Orn1, Arg3Lys(2-9)His1Orn2, Arg2Lys(2-9)His3Orn1,
    Arg2Lys(2-9)His1Orn3, Arg1Lys(2-9)His2Orn3, Arg1Lys(2-9)His3Orn2, Arg2Lys(2-9)His2Orn2,
    Arg4Lys1His(2-9)Orn1, Arg1Lys4His(2-9)Orn1, Arg1Lys1His(2-9)Orn4, Arg3Lys2His(2-9)Orn1,
    Arg3Lys1His(2-9)Orn2, Arg2Lys3His(2-9)Orn1, Arg2Lys1His(2-9)Orn3, Arg1Lys2His(2-9)Orn3,
    Arg1Lys3His(2-9)Orn2, Arg2Lys2His(2-9)Orn2, Arg4Lys1His1Orn(2-9), Arg1Lys4His1Orn(2-9),
    Arg1Lys1His4Orn(2-9), Arg3Lys2His1Orn(2-9), Arg3Lys1His2Orn(2-9), Arg2Lys3His1Orn(2-9),
    Arg2Lys1His3Orn(2-9), Arg1Lys2His3Orn(2-9), Arg1Lys3His2Orn(2-9), Arg2Lys2His2Orn(2-9);
    Arg(1-8)Lys5His1Orn1, Arg(1-8)Lys1His5Orn1, Arg(1-8)Lys1His1Orn5, Arg(1-8)Lys4His2Orn1,
    Arg(1-8)Lys2His4Orn1, Arg(1-8)Lys2His1Orn4, Arg(1-8)Lys1His2Orn4, Arg(1-8)Lys1His4Orn2,
    Arg(1-8)Lys4His1Orn2, Arg(1-8)Lys3His3Orn1, Arg(1-8)Lys3His1Orn3, Arg(1-8)Lys1His3Orn3,
    Arg5Lys(1-8)His1Orn1, Arg1Lys(1-8)His5Orn1, Arg1Lys(1-8)His1Orn5, Arg4Lys(1-8)His2Orn1,
    Arg2Lys(1-8)His4Orn1, Arg2Lys(1-8)His1Orn4, Arg1Lys(1-8)His2Orn4, Arg1Lys(1-8)His4Orn2,
    Arg4Lys(1-8)His1Orn2, Arg3Lys(1-8)His3Orn1, Arg3Lys(1-8)His1Orn3, Arg1Lys(1-8)His3Orn3,
    Arg5Lys1His(1-8)Orn1, Arg1Lys5His(1-8)Orn1, Arg1Lys1His(1-8)Orn5, Arg4Lys2His(1-8)Orn1,
    Arg2Lys4His(1-8)Orn1, Arg2Lys1His(1-8)Orn4, Arg1Lys2His(1-8)Orn4, Arg1Lys4His(1-8)Orn2,
    Arg4Lys1His(1-8)Orn2, Arg3Lys3His(1-8)Orn1, Arg3Lys1His(1-8)Orn3, Arg1Lys3His(1-8)Orn3,
    Arg5Lys1His1Orn(1-8), Arg1Lys5His1Orn(1-8), Arg1Lys1His5Orn(1-8), Arg4Lys2His1Orn(1-8),
    Arg2Lys4His1Orn(1-8), Arg2Lys1His4Orn(1-8), Arg1Lys2His4Orn(1-8), Arg1Lys4His2Orn(1-8),
    Arg4Lys1His2Orn(1-8), Arg3Lys3His1Orn(1-8), Arg3Lys1His3Orn(1-8), Arg1Lys3His3Orn(1-8);
  • According to another particular preferred embodiment, cationic or polycationic peptides or proteins as component P2, having the empirical formula (II) as shown above, may be, without being restricted thereto, selected from following formulae: Arg8, Arg9, Arg10, Arg11, Arg12, Arg13, Arg14, Arg15; Lys8, Lys9, Lys10, Lys11, Lys12, Lys13, Lys14, Lys15; His8, His9, His10, His11, His12, His13, His14, His15; Orn8, Orn9, Orn10, Orn11, Orn12, Orn13, Orn14, Orn15.
  • According to a further particular preferred embodiment, cationic or polycationic peptides or proteins as component P2, having the empirical formula (II) as shown above, may be, without being restricted thereto, selected from the subgroup consisting of generic formulas Arg9 (also termed R9), Arg9His3 (also termed R9H3), His3Arg9His3 (also termed H3R9H3), TyrSerSerArg9SerSerTyr (also termed YSSR9SSY), His3Arg9SerSerTyr (also termed H3R9SSY), (ArgLysHis)4 (also termed (RKH)4), Tyr(ArgLysHis)2Arg (also termed Y(RKH)2R). Even more preferably, these generic formulas are defined as follows:
  • According to a one further particular preferred embodiment, the cationic or polycationic peptide or protein as component P2, when defined according to formula {(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (II)) as shown above, may be, without being restricted thereto, selected from formula (IIa):

  • (Arg)l;(Lys)m;(His)n;(Orn)o;(Cys)x  (formula (IIa))
  • wherein (Arg)l;(Lys)m;(His)n;(Orn)o; and x are as defined herein. Xaa of formula (II) has been replaced with Cys.
  • According to another particular preferred embodiment, the cationic or polycationic peptide or protein as component P2, when defined according to formula {(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (II)) as shown above, may be, without being restricted thereto, selected from formula (IIb):

  • Cys{(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x}Cys  (formula (IIb))
  • wherein empirical formula {(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x} (formula (II)) is as defined herein and forms a core of an amino acid according to (semiempirical) formula (II) or (IIa). Exemplary examples may comprise any of the above sequences flanked by two Cys and following sequences:
  • (SEQ ID NOs: 1 to 32)
    Cys(Arg8)Cys, Cys(Arg9)Cys, Cys(Arg10)Cys,
    Cys(Arg11)Cys, Cys(Arg12)Cys, Cys(Arg13)Cys,
    Cys(Arg14)Cys, Cys(Arg15)Cys; Cys(Lys8)Cys,
    Cys(Lys9)Cys, Cys(Lys10)Cys, Cys(Lys11)Cys,
    Cys(Lys12)Cys, Cys(Lys13)Cys, Cys(Lys14)Cys,
    Cys(Lys15)Cys; Cys(His8)Cys, Cys(His9)Cys,
    Cys(His10)Cys, Cys(His11)Cys, Cys(His12)Cys,
    Cys(His13)Cys, Cys(His14)Cys, Cys(His15)Cys;
    Cys(Orn8)Cys, Cys(Orn9)Cys, Cys(Orn10)Cys,
    Cys(Orn11)Cys, Cys(Orn12)Cys, Cys(Orn13)Cys,
    Cys(Orn14)Cys, Cys(Orn15)Cys,

    more preferably following exemplary sequences (SEQ ID NOs: 33 to 41):
  • CysArg9Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Cys-Cys
    CysArg9His3Cys: Cys-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-His-Cys
    CysHis3Arg9His3Cys: Cys-His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-
    His-Cys
    CysTyrSerSerArg9SerSerTyrCys: Cys-Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-
    Ser-Ser-Tyr-Cys
    CysHis3Arg9SerSerTyrCys: Cys-His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Ser-
    Ser-Tyr-Cys
    Cys (ArgLysHis)4Cys: Cys-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Cys
    CysTyr(ArgLysHis)2ArgCys: Cys-Tyr-Arg-Lys-His-Arg-Lys-His-Arg-Cys
    CysHis3Arg9His3Cys: Cys-His-His-His-Arg-Arg-Arg-Arg-His-His-His-Cys
    CysHis6Arg9His6Cys: Cys-His-His-His-His-His-His-Arg-Arg-Arg-Arg-His-His-His-His-His-
    His-Cys
  • This embodiment may apply to situations, wherein the polycationic peptide or protein as component P2, e.g. when defined according to empirical formula (Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x (formula (II)) as shown above, has been modified with at least two cysteines as —SH moieties in the above meaning such that component P2 carries at least two (terminal) cysteines, which are capable to form a disulfide bond with other components of formula (I).
  • According to another embodiment, component P2 represents a cationic or polycationic polymer, selected from e.g. any cationic polymer suitable in this context, provided that this cationic polymer exhibits at least two —SH-moieties, which provide for a disulfide bond linking component P2 with component P1 or P3, or with further component(s) P2. Thus, likewise as defined herein, component P2 may occur as a repetitive component as defined herein as represented by subformula [S—P2—S]n, wherein the same or different cationic or polycationic polymers P2 may be used in said repetitive component.
  • Preferably, component P2 represents a cationic or polycationic polymer, typically exhibiting a molecular weight of about 0.5 kDa to about 100 kDa, preferably of about 1 kDa to about 75 kDa, more preferably of about 5 kDa to about 50 kDa, even more preferably of about 5 kDa to about 30 kDa, or a molecular weight of about 10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30 kDa. Additionally, the cationic or polycationic polymer as component P2 typically exhibits at least two —SH-moieties, which are capable to form a disulfide linkage upon condensation with either components P1 or P3 or with other components P2 as defined herein.
  • When component P2 represents a cationic or polycationic polymer, such a polymer may be selected from acrylates, modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), chitosanes, aziridines or 2-ethyl-2-oxazoline (forming oligo ethylenimines or modified oligoethylenimines), polymers obtained by reaction of bisacrylates with amines forming oligo beta aminoesters or poly amino amines, or other polymers like polyesters, polycarbonates, etc. Each molecule of these cationic or polycationic polymers typically exhibits at least two —SH-moieties, wherein these at least two —SH-moieties may be introduced into the cationic or polycationic polymer by chemical modifications, e.g. using imonothiolan, 3-thio propionic acid or introduction of —SH-moieties containing amino acids, such as cystein, methionine or any further (modified) amino acid. Such —SH-moieties are preferably as already defined above for components P1, P2 or P3.
  • Component P2 of formula (I) of the present invention preferably occurs as repetitive component [—S—P2—S—]n. Such a repetitive component [S—P2—S]n may be prepared using at least one or even more of the same or different of the above defined components P2 and polymerizing same in a polymerization condensation reaction via their —SH-moieties.
  • According to one specific embodiment, such a repetitive component [S—P2—S]n may be prepared using at least one or even more of the same or different of the above defined cationic or polycationic peptides, and polymerizing same in a polymerization condensation reaction via their —SH-moieties. Accordingly, such a repetitive component [S—P2—S]n contains a number of at least one or even more of the same or different of the above defined cationic or polycationic proteins or peptides determined by integer n.
  • According to another specific embodiment, such a repetitive component [S—P2—S]n may be prepared using at least one or even more of the same or different of the above defined cationic or polycationic polymers, and polymerizing same in a polymerization condensation reaction via their —SH-moieties. Accordingly, such a repetitive component [S—P2—S]n contains a number of at least one or even more of the same or different of the above defined cationic or polycationic polymers determined by integer n.
  • According to a further specific embodiment, such a repetitive component [S—P2—S]n may be prepared using at least one or even more of the same or different of the above defined cationic or polycationic polymers and at least one or even more of the same or different of the above defined cationic or polycationic proteins or peptides, and polymerizing same in a polymerization condensation reaction via their —SH-moieties. Accordingly, such a repetitive component [S—P2—S]n contains a number of at least one or even more of the same or different of the above defined cationic or polycationic polymers and at least one or even more of the same or different of the above defined cationic or polycationic proteins or peptides, both together determined by integer n.
  • According to a further embodiment, the inventive polymeric carrier according to formula (I) above, may comprise at least one repetitive amino acid component (AA)x, wherein AA is preferably an amino acid as defined in the following, which, when occurring as repetitive amino acid component (AA)x, allows to (substantially) modify the biophysical/biochemical properties of the inventive polymeric carrier according to formula (I) as defined herein. According to the present invention, the repetitions of such a repetitive amino acid component (AA)x, are defined by x. In the above context, x is preferably an integer and may be selected from a range of about 1 to 30, preferably from a range of about 1 to 15, more preferably selected from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from a range formed by any two of the afore mentioned values.
  • The repetitive amino acid component (AA)x may contain or may be flanked (e.g. terminally) by a —SH containing moiety, which allows introducing this component (AA)x via a disulfide bond into the polymeric carrier according to formula (I) as defined herein. In this context, the repetitive amino acid component (AA)x may also be read as a component —S-(AA)x-S—, wherein S represents a —SH containing moiety (or, of course, one sulfur of a disulfide bond), e.g. a cysteine residue. In the specific case that the —SH containing moiety represents a cysteine, the repetitive amino acid component (AA)x may also be read as -Cys-(AA)x-Cys- wherein Cys represents Cysteine and provides for the necessary —SH-moiety for a disulfide bond. (Accordingly, -Cys-(AA)x-Cys- may also be written as —(S-Cys)-(AA)x-(Cys-S)—.) The —SH containing moiety may be also introduced into repetitive amino acid component (AA)x using any of modifications or reactions as shown above for components P1, P2 or P3.
  • The repetitive amino acid component (AA)x may also occur as a mixed repetitive amino acid component [(AA)x]z, wherein the number of repetitions of repetitive amino acid component (AA)x are further defined by z. In this context, z is an integer and may be selected from a range of about 1 to 30, preferably from a range of about 1 to 15, more preferably selected from a number comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, or may be selected from a range formed by any two of the afore mentioned values. Such a mixed repetitive amino acid component [(AA)x]n may be used to combine different repetitive amino acid components (AA)x as defined herein. Preferably, in the mixed repetitive amino acid component [(AA)x]z the repetitive amino acid component (AA)x may contain or may be flanked (e.g. terminally) by a —SH containing moiety as already defined above, which allows coupling the different repetitive amino acid component (AA)x using a disulfide bond via a condensation polymerization. Likewise as above, the mixed repetitive amino acid component [(AA)x]z may also be read as [S-(AA)x-S]z, wherein S represents a —SH containing moiety, e.g. a cysteine residue. In the specific case that the —SH containing moiety represents a cysteine, the mixed repetitive amino acid component [(AA)x]z may also be read as [Cys-(AA)x-Cys]z, wherein Cys represents Cysteine and provides for the necessary —SH-moiety for a disulfide bond. The —SH containing moiety may be also introduced into repetitive amino acid component (AA)x using any of modifications or reactions as shown above for components P1, P2 or P3.
  • The repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be provided with one —SH-moiety, e.g. in a form represented by formula (AA)x-SH. Then, the repetitive component (AA)x according to formula (AA)x-SH or the mixed repetitive amino acid component [(AA)x]z according to formula [(AA)x]z-SH, may be bound to any of components P1, P2 and/or P3 via a disulfide bond. If bound to component P1 and/or component P3, components P1 and/or P3 preferably exhibit at least two —SH-moieties to allow further binding of components P1 and/or P3 to a component P2 via a —SH-moiety forming a disulfide bond (see above). The repetitive amino acid component (AA)x in a form represented by formula (AA)x-SH or the mixed repetitive amino acid component [(AA)x]z according to formula [(AA)x]z-SH may be also used to terminate a condensation reaction due to its single —SH moiety. In this case, repetitive amino acid component (AA)x in a form represented by formula (AA)x-SH is preferably coupled terminally to components P1 and/or P3. The repetitive amino acid component (AA)x in a form represented by formula (AA)x-SH or the mixed repetitive amino acid component [(AA)x]z according to formula [(AA)x]z-SH may be also used to bind internally to any of components P1, P2 and/or P3 via a further internal —SH-moiety of any of components P1, P2 and/or P3.
  • Furthermore, the repetitive amino acid component (AA)x may be provided with two —SH-moieties (or even more), e.g. in a form represented by formula HS-(AA)x-SH. Additionally, the mixed repetitive amino acid component [(AA)x]z may be provided with two —SH-moieties (or even more), e.g. in a form represented by formula HS-[(AA)x]z-SH, to allow binding to two functionalities via disulfide bonds, e.g. if the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z is used as a linker between two further components (e.g. as a linker between components L and P1, between components P1 and P2, in or as a part of repetitive component [S—P2—S]n, between components P2 and P3 and/or between components P3 and L). In this case, one —SH moiety is preferably protected in a first step using a protecting group as known in the art, leading to a repetitive amino acid component (AA)x of formula HS-(AA)x-S-protecting group or to a mixed repetitive amino acid component [(AA)x]z of formula HS-[(AA)x]z-S-protecting group. Then, the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be bound to a component L, P1, P2 and/or P3, to form a first disulfide bond via the non-protected —SH moiety. The protected-SH-moiety is then typically deprotected and bound to a further free —SH-moiety of a further component L, P1, P2 and/or P3 to form a second disulfide bond.
  • Alternatively, the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be provided with other fuctionalities as already described above for components P1 and P2 and/or P3, which allow binding of the repetitive amino acid component (AA)x or binding of the mixed repetitive amino acid component [(AA)x]z to any of components P1, P2 and/or P3 and optionally to component L.
  • Thus, according to the present invention, the repetitive amino acid component [(AA)x]z and/or the mixed repetitive amino acid component [(AA)x]z may be bound to P1, P2, P3 and/or L with or without using a disulfide linkage. Binding without using a disulfide linkage may be accomplished by any of the reactions described above, preferably by binding the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z to P1, P2, P3 and/or L using an amid-chemistry as defined herein. If desired or necessary, the other terminus of the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z, e.g. the N- or C-terminus, may be used to couple another component, e.g. a ligand L. For this purpose, the other terminus of the repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z preferably comprises or is modified to comprise a further functionality, e.g. an alkyn-species (see above), which may be used to add the other component via e.g. click-chemistry. Such a construct, e.g. L-(AA)x-P1—S-or L-[(AA)x]z-P1—S—, may be used to terminate the polymerization condensation reaction of repetitive component [S—P2—S]n. If the ligand is bound via an acid-labile bond, the bond is preferably cleaved off in the endosome and the inventive polymeric carrier presents repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z at its surface.
  • The repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may occur as a further component of generic formula (I) above, e.g. as a linker between components P1 or P3 and P2, as a linker between components L and P1 or P2 or as an additional component of repetitive component [S—P2—S]n.
  • According to a first alternative, such a repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be present as a linker between components P1 or P3 and component P2. This is preferably represented in the context of the entire inventive polymeric carrier according to formula (I) by following formulae:

  • L-P1—S—S-(AA)x-S—[S—P2—S]n—S-(AA)x-S—S—P3-L, or

  • L-P1—S—[S-(AA)x-S]z—[S—P2—S]n—[S-(AA)x-S]z—S—P3-L,
  • wherein n, x, z, S, L, AA, Cys, P1, P2 and P3 are preferably as defined herein. In the above formulae, the term “—S—S—” represents a disulfide bond, wherein this at least one sulfur of the disulfide bond may also be provided by a cysteine. In this case, the term “—S—S—” in these formulae may also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”. In this context, the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their —SH-moieties to form a disulfide bond. Accordingly, the term “-Cys-Cys-” may also be understood generally as “-(Cys-S)—(S-Cys)-”, wherein in this specific case S indicates the sulfur of the —SH-moiety of cysteine. Likewise, the terms “—S-Cys” and “-Cys-S” indicate a disulfide bond between a —SH containing moiety and a cysteine, which may also be written as “—S—(S-Cys)” and “-(Cys-S)—S”.
  • According to a second alternative, such a repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be present as a linker between components P1 or P3 and component L. This is preferably represented in the context of the entire inventive polymeric carrier according to formula (I) by following formulae:

  • L-(AA)x-P1—S—[S—P2—S]nS—P3-(AA)x-L, or

  • L-[(AA)x]z-P1—S—[S—P2—S]n—S—P3-[(AA)x]z-L,

  • or alternatively

  • L-(AA)x-S—S—P1—S—[S—P2—S]nS—P3—S—S-(AA)x-S—S-L, or

  • L-S—S-(AA)x-S—S—P1—S—[S—P2—S]n—S—P3—S—S-(AA)x-S—S-L, or

  • L-S—[S-(AA)x-S]z—S—P1—S—[S—P2—S]n—S—P3—S—[S-(AA)x-S]2—S-L, etc.
  • wherein n, x, z, S, L, AA, Cys, P1, P2 and P3 are preferably as defined herein. In the above formulae, the term “—S—S—” represents a disulfide bond, as already defined above.
  • According to a third alternative, such a repetitive amino acid component (AA)x or the mixed repetitive amino acid component [(AA)x]z may be present as a part of components P1 and/or P3, wherein the repetitive amino acid component (AA)x may be directly bound to (e.g. the terminus of) component P1 and/or P3 without a further ligand L. This is preferably represented in the context of the entire inventive polymeric carrier according to formula (I) by following formulae:

  • (AA)x-P1—S—[S—P2—S]n—S—P3-(AA)x, or

  • [(AA)x]z-P1—S—[S—P2—S]n—S—P3—[(AA)x]z, or

  • or alternatively

  • (AA)x-S—S—P1—S—[S—P2—S]n—S—P3—S—S-(AA)x, or

  • H—[S-(AA)x-S]2—S—P1—S—[S—P2—S]n—S—S—P3—S—[S-(AA)x-S]z—H,
  • wherein n, x, z, S, L, AA, Cys, P1, P2 and P3 are preferably as defined herein. In the above formulae, the term “—S—S—” represents a disulfide bond, as already defined above. The free —SH moiety at the terminal ends in the last formula may also be terminated using a monothiol compound as defined herein.
  • According to a fourth and particularly preferred alternative, the repetitive amino acid component (AA)x, preferably written as S-(AA)x-S or [S-(AA)x-S] may be used to modify component P2, particularly the content of component S—P2—S in repetitive component [S—P2—S]n of formula (I) above. This may be represented in the context of the entire polymeric carrier according to formula (I) e.g. by following formula (Ia):

  • L-P1—S—{[S—P2—S]a[S-(AA)x-S]b}—S—P3-L,
  • wherein x, z, S, L, AA, P1, P2 and P3 are preferably as defined herein. In formula (Ia) above, any of the single components [S—P2—S] and [S-(AA)x-S] may occur in any order in the subformula {[S—P2—S]a[S-(AA)x-S]b}. The numbers of single components [S—P2—S] and [S-(AA)x-S] in the subformula {[S—P2—S]a[S-(AA)x-S]b} are determined by integers a and b, wherein a+b=n. n is an integer and is defined as above for formula (I).
  • a is an integer, typically selected independent from integer b from a range of about 1 to 50, preferably from a range of about 1, 2 or 3 to 30, more preferably from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1, 2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10.
  • b is an integer, typically selected independent from integer a from a range of about 0 to 50, preferably from a range of about 0, 1, 2 or 3 to 30, more preferably from a range of about 0, 1, 2, 3, 4, or 5 to 25, or a range of about 0, 1, 2, 3, 4, or 5 to 20, or a range of about 0, 1, 2, 3, 4, or 5 to 15, or a range of about 0, 1, 2, 3, 4, or 5 to 10, including e.g. a range of about 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of about 6 to 11 or 7 to 10.
  • In the above formula, the term “—S—S—” (the brackets are omitted for better readability) represents a disulfide bond as already defined above.
  • The modification of component P2, particularly of component S—P2—S of repetitive component [S—P2—S]n, by “diluting” same with repetitive amino acid component (AA)x may be also realized in the context of any of the afore mentioned alternatives of the entire polymeric carrier according to formula (I),

  • L-P1—S—S-(AA)x-S-{[S—P2—S]a[S-(AA)x-S]b}—S-(AA)x-S—S—P3-L, or

  • L-P1—S—[S-(AA)x-S]z—{[S—P2—S]a[S-(AA)x-S]b}-[S-(AA)x-S]z—S—P3-L, or

  • L-(AA)x-P1—S-{[S—P2—S]a[S-(AA)x-S]b}S—P3-(AA)x-L, or

  • L-[(AA)x]zP1—S-{[S—P2—S]a[S-(AA))x—S]b}—S—P3-[(AA)x]z-L, or

  • L-(AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x-S—S-L, or

  • L-S—S-(AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x-S—S-L, or

  • L-S—[S-(AA)x-S]z-S—P1—S-{[S—P2—S]a[S-(AA)-S]b}—S—P3—S—[S-(AA)x-S]z—S-L, or

  • (AA)x-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P8-(AA)x, or

  • [(AA)x]z-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—[(AA)x]z, or

  • (AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x, or

  • H—[S-(AA)x-S]z—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—[S-(AA)x-S]z—H,
  • wherein n, x, z, S, L, AA, P1, P2 and P3 are preferably as defined herein. Likewise, the term “—S—S—” represents a disulfide bond and is preferably as defined herein.
  • In the above alternatives, wherein the component [S—P2—S] is preferably “diluted” with repetitive amino acid component [S-(AA)x-S], the ratio is determined by integers a and b, wherein a+b=n. Preferably, integers a and b are selected such that the cationic binding properties of component [S—P2—S] are not lost but remain to a minimum extent in subformula/component {[S—P2—S]a[S-(AA)x-S]b}. This allows to weaken (“dilute”) the cationic binding strength of component [S—P2—S] in repetitive component [S—P2—S]n of inventive polymeric carrier of formula (I) to a desired extent.
  • In this specific context the (desired) cationic binding strength of subformula/component {[S—P2—S]a[S-(AA)x-S]b} may be determined using different methods.
  • According to a first alternative, component P2 of formula (I) of the present invention is particularly preferable a cationic or polycationic peptide as defined herein. Furthermore, the repetitive amino acid component (AA)x, preferably written as [S-(AA)x-S], typically resembles a peptide sequence. In this specific case, the cationic properties of subformula/component {[S—P2—S]a[S-(AA)x-S]b} may be determined upon their content of cationic amino acids in the entire subformula/component. Preferably, the content of cationic amino acids in subformula/component {[S—P2—S]a[S-(AA)x-S]b} is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 15% to 75%, even preferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a range formed by any two of the afore mentioned values, provided, that the content of all amino acids, e.g. cationic, lipophilic, hydrophilic, aromatic and further amino acids, in the entire subformula/component {[S—P2—S]a[S-(AA)x-S]b} is 100%.
  • According to a second alternative, component P2 of formula (I) of the present invention is particularly preferable a cationic or polycationic polymer as defined herein. The repetitive amino acid component (AA)x, preferably written as [S-(AA)x-S], typically resembles a peptide sequence. In this specific case, the cationic properties of subformula/component {[S—P2—S]a[S -(AA)x-S]b} may be determined upon their content of cationic charges in the entire subformula/component. Preferably, the content of cationic charges in subformula/component {[S—P2—S]a[S-(AA)x-S]b} at a (physiological) pH as defined herein is at least 10%, 20%, or 30%, preferably at least 40%, more preferably at least 50%, 60% or 70%, but also preferably at least 80%, 90%, or even 95%, 96%, 97%, 98%, 99% or 100%, most preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, or may be in the range of about 10% to 90%, more preferably in the range of about 15% to 75%, even preferably in the range of about 20% to 50%, e.g. 20, 30, 40 or 50%, or in a range formed by any two of the afore mentioned values, provided, that the content of all charges, e.g. positive and negative charges at a (physiological) pH as defined herein, in the entire subformula/component {[S—P2—S]a[S-(AA)x-S]b} is 100%.
  • In the context of the present invention, the repetitive amino acid component (AA)x may be selected from the following alternatives.
  • According to a first alternative, the repetitive amino acid component (AA)x may be a repetitive aromatic amino acid component (AA)x. The incorporation of aromatic amino acids or sequences as repetitive amino aromatic acid component (AA)x into the inventive polymeric carrier according to formula (I) of the present invention enables a different (second) binding of the inventive polymeric carrier to the nucleic acid due to interactions of the aromatic amino acids with the bases of the nucleic acid cargo in contrast to the binding thereof by cationic charged sequences of the polymeric carrier molecule to the phosphate backbone. This interaction may occur e.g. by intercalations or by minor or major groove binding. This kind of interaction is not prone to decompaction by anionic complexing partners (e.g. Heparin, Hyaluronic acids) which are found mainly in the extracellular matrix in vivo and is also less susceptible to salt effects.
  • For this purpose, the amino acid AA in the repetitive aromatic amino acid component (AA)x may be selected from either the same or different aromatic amino acids e.g. selected from Trp, Tyr, or Phe. Alternatively, the amino acid AA (or the entire repetitive aromatic amino acid component (AA)x) may be selected from following peptide combinations Trp-Tyr, Tyr-Trp, Trp-Trp, Tyr-Tyr, Trp-Tyr-Trp, Tyr-Trp-Tyr, Trp-Tip-Trp, Tyr-Tyr-Tyr, Trp-Tyr-Trp-Tyr, Tyr-Trp-Tyr-Trp, Trp-Trp-Trp-Trp, Phe-Tyr, Tyr-Phe, Phe-Phe, Phe-Tyr-Phe, Tyr-Phe-Tyr, Phe-Phe-Phe, Phe-Tyr-Phe-Tyr, Tyr-Phe-Tyr-Phe, Phe-Phe-Phe-Phe, Phe-Trp, Trp-Phe, Phe-Phe, Trp-Trp, Phe-Trp-Phe, Trp-Phe-Trp, Phe-Phe-Phe, Trp-Trp-Trp, Phe-Trp-Phe-Trp, Trp-Phe-Trp-Phe, Phe-Phe-Phe-Phe, or Tyr-Tyr-Tyr-Tyr, etc.
  • Additionally, the repetitive aromatic amino acid component (AA)x may contain or may be flanked by a—SH containing moiety, which allows introducing this component via a disulfide bond as a further part of generic formula (I) above, e.g. as a linker. Such a—SH containing moiety may be any moiety as defined herein suitable to couple one component as defined herein to a further component as defined herein. As an example, such a —SH containing moiety may be a cysteine. Then, e.g. the repetitive aromatic amino acid component (AA)x may be selected from e.g. peptide combinations Cys-Tyr-Cys, Cys-Trp-Cys, Cys-Trp-Tyr-Cys, Cys-Tyr-Trp-Cys, Cys-Trp-Trp-Cys, Cys-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Cys, Cys-Tyr-Trp-Tyr-Cys, Cys-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Cys, Cys-Trp-Tyr-Trp-Tyr-Cys, Cys-Tyr-Trp-Tyr-Trp-Cys, Cys-Trp-Trp-Trp-Trp-Cys, Cys-Tyr-Tyr-Tyr-Tyr-Cys, Cys-Phe-Cys, Cys-Phe-Tyr-Cys, Cys-Tyr-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Cys, Cys-Tyr-Phe-Tyr-Cys, Cys-Phe-Phe-Phe-Cys, Cys-Tyr-Tyr-Tyr-Cys, Cys-Phe-Tyr-Phe-Tyr-Cys, Cys-Tyr-Phe-Tyr-Phe-Cys, or Cys-Phe-Phe-Phe-Phe-Cys, Cys-Phe-Tip-Cys, Cys-Trp-Phe-Cys, Cys-Phe-Phe-Cys, Cys-Trp-Trp-Cys, Cys-Phe-Trp-Phe-Cys, Cys-Trp-Phe-Trp-Cys, Cys-Phe-Phe-Phe-Cys, Cys-Phe-Trp-Phe-Trp-Cys, Cys-Trp-Phe-Trp-Phe-Cys, etc. Each Cys above may also be replaced by any modified peptide or chemical compound carrying a free —SH-moiety as defined herein.
  • Additionally, the repetitive aromatic amino acid component (AA)x may contain at least one proline, which may serve as a structure breaker of longer repetitive sequences of Trp, Tyr and Phe in the aromatic repetitive amino acid component (AA)x, preferably two, three or more prolines.
  • According to a second alternative, the repetitive amino acid component (AA)x may be a repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x. The incorporation of hydrophilic (and preferably non charged polar) amino acids or sequences as repetitive amino hydrophilic (and preferably non charged polar) acid component (AA)x into the inventive polymeric carrier according to formula (I) of the present invention enables a more flexible binding to the nucleic acid cargo. This leads to a more effective compaction of the nucleic acid cargo and hence to a better protection against nucleases and unwanted decompaction. It also allows provision of a (long) inventive polymeric carrier according to formula (I) which exhibits a reduced cationic charge over the entire carrier or preferably within repetitive component [S—P2—S]n and in this context to better adjusted binding properties, if desired or necessary.
  • For this purpose, the amino acid AA in the repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x may be selected from either the same or different hydrophilic (and preferably non charged polar) amino acids e.g. selected from Thr, Ser, Asn or Gln. Alternatively, the amino acid AA (or the entire repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x) may be selected from following peptide combinations Ser-Thr, Thr-Ser, Ser-Ser, Thr-Thr, Ser-Thr-Ser, Thr-Ser-Thr, Ser-Ser-Ser, Thr-Thr-Thr, Ser-Thr-Ser-Thr, Thr-Ser-Thr-Ser, Ser-Ser-Ser-Ser, Thr-Thr-Thr-Thr, Gln-Asn, Asn-Gln, Gln-Gln, Asn-Asn, Gln-Asn-Gln, Asn-Gln-Asn, Gln-Gln-Gln, Asn-Asn-Asn, Gln-Asn-Gln-Asn, Asn-Gln-Asn-Gln, Gln-Gln-Gln-Gln, Asn-Asn-Asn-Asn, Ser-Asn, Asn-Ser, Ser-Ser, Asn-Asn, Ser-Asn-Ser, Asn-Ser-Asn, Ser-Ser-Ser, Asn-Asn-Asn, Ser-Asn-Ser-Asn, Asn-Ser-Asn-Ser, Ser-Ser-Ser-Ser, or Asn-Asn-Asn-Asn, etc.
  • Additionally, the repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x may contain or may be flanked by a —SH containing moiety, which allows introducing this component via a disulfide bond as a further part of generic formula (I) above, e.g. as a linker. Such a —SH containing moiety may be any moiety as defined herein suitable to couple one component as defined herein to a further component as defined herein. As an example, such a —SH containing moiety may be a cysteine. Then, e.g. the repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x may be selected from e.g. peptide combinations Cys-Thr-Cys, Cys-Ser-Cys, Cys-Ser-Thr-Cys, Cys-Thr-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Cys, Cys-Thr-Ser-Thr-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Cys, Cys-Ser-Thr-Ser-Thr-Cys, Cys-Thr-Ser-Thr-Ser-Cys, Cys-Ser-Ser-Ser-Ser-Cys, Cys-Thr-Thr-Thr-Thr-Cys, Cys-Asn-Cys, Cys-Gln-Cys, Cys-Gln-Asn-Cys, Cys-Asn-Gln-Cys, Cys-Gln-Gln-Cys, Cys-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Cys, Cys-Asn-Gln-Asn-Cys, Cys-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Gln-Asn-Gln-Asn-Cys, Cys-Asn-Gln-Asn-Gln-Cys, Cys-Gln-Gln-Gln-Gln-Cys, Cys-Asn-Asn-Asn-Asn-Cys, Cys-Asn-Cys, Cys-Ser-Cys, Cys-Ser-Asn-Cys, Cys-Asn-Ser-Cys, Cys-Ser-Ser-Cys, Cys-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Cys, Cys-Asn-Ser-Asn-Cys, Cys-Ser-Ser-Ser-Cys, Cys-Asn-Asn-Asn-Cys, Cys-Ser-Asn-Ser-Asn-Cys, Cys-Asn-Ser-Asn-Ser-Cys, Cys-Ser-Ser-Ser-Ser-Cys, or Cys-Asn-Asn-Asn-Asn-Cys, etc. Each Cys above may also be replaced by any modified peptide or chemical compound carrying a free —SH-moiety as defined herein.
  • Additionally, the repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x may contain at least one proline, which may serve as a structure breaker of longer repetitive sequences of Ser, Thr and Asn in the repetitive hydrophilic (and preferably non charged polar) amino acid component (AA)x, preferably two, three or more prolines.
  • According to a third alternative, the repetitive amino acid component (AA)x may be a repetitive lipohilic amino acid component (AA)x. The incorporation of lipohilic amino acids or sequences as repetitive amino lipohilic acid component (AA)x into the inventive polymeric carrier according to formula (I) of the present invention enables a stronger compaction of the nucleic acid cargo and/or the polymeric carrier according to formula (I) and its nucleic acid cargo when forming a complex. This is particularly due to interactions of one or more polymer strands of the inventive polymeric carrier, particularly of lipophilic sections of lipohilic amino acid component (AA)x, preferably in the context of subformula/component {[S—P2—S]a[S-(AA)x-S]b}, and the nucleic acid cargo. This interaction will preferably add an additional stability to the complex between the polymeric carrier according to formula (I) and its nucleic acid cargo. This stabilization may somehow be compared to a sort of non covalent crosslinking between different polymerstrands. Especially in aqueous environment this interaction is typically strong and provides a significant effect.
  • For this purpose, the amino acid AA in the repetitive lipophilic amino acid component (AA)x may be selected from either the same or different lipophilic amino acids e.g. selected from Leu, Val, Ile, Ala, Met. Alternatively, the amino acid AA (or the entire repetitive lipophilic amino acid component (AA)x) may be selected from following peptide combinations Leu-Val, Val-Leu, Leu-Leu, Val-Val, Leu-Val-Leu, Val-Leu-Val, Leu-Leu-Leu, Val-Val-Val, Leu-Val-Leu-Val, Val-Leu-Val-Leu, Leu-Leu-Leu-Leu, Val-Val-Val-Val, Ile-Ala, Ala-Ile, Ile-Ile, Ala-Ala, Ile-Ala-Ile, Ala-Ile-Ala, Ile-Ile-Ile, Ala-Ala-Ala, Ile-Ala-Ile-Ala, Ala-Ile-Ala-Ile, Ile-Ile-Ile-Ile, Ala-Ala-Ala-Ala, Met-Ala, Ala-Met, Met-Met, Ala-Ala, Met-Ala-Met, Ala-Met-Ala, Met-Met-Met, Ala-Ala-Ala, Met-Ala-Met-Ala, Ala-Met-Ala-Met, or Met-Met-Met-Met etc.
  • Additionally, the repetitive lipophilic amino acid component (AA)x may contain or may be flanked by a —SH containing moiety, which allows introducing this component via a disulfide bond as a further part of generic formula (I) above, e.g. as a linker. Such a —SH containing moiety may be any moiety as defined herein suitable to couple one component as defined herein to a further component as defined herein. As an example, such a —SH containing moiety may be a cysteine. Then, e.g. the repetitive lipophilic amino acid component (AA)x may be selected from e.g. peptide combinations Cys-Val-Cys, Cys-Leu-Cys, Cys-Leu-Val-Cys, Cys-Val-Leu-Cys, Cys-Leu-Leu-Cys, Cys-Val-Val-Cys, Cys-Leu-Val-Leu-Cys, Cys-Val-Leu-Val-Cys, Cys-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Cys, Cys-Leu-Val-Leu-Val-Cys, Cys-Val-Leu-Val-Leu-Cys, Cys-Leu-Leu-Leu-Leu-Cys, Cys-Val-Val-Val-Val-Cys, Cys-Ala-Cys, Cys-Ile-Cys, Cys-Ile-Ala-Cys, Cys-Ala-Ile-Cys, Cys-Ile-Ile-Cys, Cys-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Cys, Cys-Ala-Ile-Ala-Cys, Cys-Ile-Ile-Ile-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Ile-Ala-Ile-Ala-Cys, Cys-Ala-Ile-Ala-Ile-Cys, Cys-Ile-Ile-Ile-Ile-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, Cys-Met-Cys, Cys-Met-Ala-Cys, Cys-Ala-Met-Cys, Cys-Met-Met-Cys, Cys-Ala-Ala-Cys, Cys-Met-Ala-Met-Cys, Cys-Ala-Met-Ala-Cys, Cys-Met-Met-Met-Cys, Cys-Ala-Ala-Ala-Cys, Cys-Met-Ala-Met-Ala-Cys, Cys-Ala-Met-Ala-Met-Cys, Cys-Met-Met-Met-Met-Cys, or Cys-Ala-Ala-Ala-Ala-Cys, etc. Each Cys above may also be replaced by any modified peptide or chemical compound carrying a free —SH-moiety as defined herein.
  • Additionally, the repetitive lipophilic amino acid component (AA)x may contain at least one praline, which may serve as a structure breaker of longer repetitive sequences of Leu, Val, Ile, Ala and Met in the repetitive lipophilic amino acid component (AA)x, preferably two, three or more prolines.
  • Finally, according to a fourth alternative, the repetitive amino acid component (AA)x may be a repetitive weak basic amino acid component (AA)x. The incorporation of weak basic amino acids or sequences as repetitive weak basic amino acid component (AA)x into the inventive polymeric carrier according to formula (I) of the present invention may serve as a proton sponge and facilitates endosomal escape (also called endosomal release) (proton sponge effect). Incorporation of such a repetitive weak basic amino acid component (AA)x preferably enhances transfection efficiency.
  • For this purpose, the amino acid AA in the repetitive weak basic amino acid component (AA)x may be selected from either the same or different weak amino acids e.g. selected from histidine or aspartate (aspartic acid). Alternatively, the weak basic amino acid AA (or the entire repetitive weak basic amino acid component (AA)x) may be selected from following peptide combinations Asp-His, His-Asp, Asp-Asp, His-His, Asp-His-Asp, His-Asp-His, Asp-Asp-Asp, His-His-His, Asp-His-Asp-His, His-Asp-His-Asp, Asp-Asp-Asp-Asp, or His-His-His-His, etc.
  • Additionally, the repetitive weak basic amino acid component (AA)x may contain or may be flanked by a —SH containing moiety, which allows introducing this component via a disulfide bond as a further part of generic formula (I) above, e.g. as a linker. Such a —SH containing moiety may be any moiety as defined herein suitable to couple one component as defined herein to a further component as defined herein. As an example, such a —SH containing moiety may be a cysteine. Then, e.g. the repetitive weak basic amino acid component (AA)x may be selected from e.g. peptide combinations Cys-His-Cys, Cys-Asp-Cys, Cys-Asp-His-Cys, Cys-His-Asp-Cys, Cys-Asp-Asp-Cys, Cys-His-His-Cys, Cys-Asp-His-Asp-Cys, Cys-His-Asp-His-Cys, Cys-Asp-Asp-Asp-Cys, Cys-His-His-His-Cys, Cys-Asp-His-Asp-His-Cys, Cys-His-Asp-His-Asp-Cys, Cys-Asp-Asp-Asp-Asp-Cys, or Cys-His-His-His-His-Cys, etc. Each Cys above may also be replaced by any modified peptide or chemical compound carrying a free —SH-moiety as defined herein.
  • Additionally, the repetitive weak basic amino acid component (AA)x may contain at least one proline, which may serve as a structure breaker of longer repetitive sequences of histidine or aspartate (aspartic acid) in the repetitive weak basic amino acid component (AA)x, preferably two, three or more prolines.
  • Additionally, the inventive polymeric carrier according to formula (I) above (or according to any of its subformulas herein), may comprise as an additional component a signal peptide, a signal peptide coding sequence, a localization signal or sequence or a nuclear localization signal or sequence (NLS), which allows a translocalization of the inventive polymeric carrier according to formula (I) above to a specific target, e.g. into the cell, into the nucleus, into the endosomal compartiment, sequences for the mitochondrial matrix, localisation sequences for the plasma membrane, localisation sequences for the Golgi apparatus, the nucleus, the cytoplasm and the cytosceleton, etc. Such signal peptide, a localization signal or sequence or a nuclear localization signal may be used for the transport of any of the herein defined nucleic acids, preferably an RNA or a DNA, more preferably an shRNA or a pDNA, e.g. into the nucleus. Without being limited thereto, such a signal peptide, a localization signal or sequence or a nuclear localization signal may comprise, e.g., localisation sequences for the endoplasmic reticulum. Particular localization signals or sequences or a nuclear localization signals may include e.g. KDEL (SEQ ID NO: 42), DDEL (SEQ ID NO: 43), DEEL (SEQ ID NO: 44), QEDL (SEQ ID NO: 45), RDEL (SEQ ID NO: 46), and GQNLSTSN (SEQ ID NO: 47), nuclear localisation sequences, including PKKKRKV (SEQ ID NO: 48), PQKKIKS (SEQ ID NO: 49), QPKKP (SEQ ID NO: 50), RKKR (SEQ ID NO: 51), RKKRRQRRRAHQ (SEQ ID NO: 52), RQARRNRRRRWRERQR (SEQ ID NO: 53), MPLTRRRPAASQALAPPTP (SEQ ID NO: 54), GAALTILV (SEQ ID NO: 55), and GAALTLLG (SEQ ID NO: 56), localisation sequences for the endosomal compartiment, including MDDQRDLISNNEQLP (SEQ ID NO: 57), localisation sequences for the mitochondrial matrix, including MLFNLRXXLNNAAFRHGHNFMVRNFRCGQPLX (SEQ ID NO: 58), localisation sequences for the plasma membrane: GCVCSSNP (SEQ ID NO: 59), GQTVTTPL (SEQ ID NO: 60), GQELSQHE (SEQ ID NO: 61), GNSPSYNP (SEQ ID NO: 62), GVSGSKGQ (SEQ ID NO: 63), GQTITTPL (SEQ ID NO: 64), GQTLTPL (SEQ ID NO: 65), GQIFSRSA (SEQ ID NO: 66), GQIHGLSP (SEQ ID NO: 67), GARASVLS (SEQ ID NO: 68), and GCTLSAEE (SEQ ID NO: 69), localisation sequences for the endoplasmic reticulum and the nucleus, including GAQVSSQK (SEQ ID NO: 70), and GAQLSRNT (SEQ ID NO: 71), localisation sequences for the Golgi apparatus, the nucleus, the cytoplasm and the cytosceleton, including GNAAAAKK (SEQ ID NO: 72), localisation sequences for the cytoplasm and cytosceleton, including GNEASYPL (SEQ ID NO: 73), localisation sequences for the plasma membrane and cytosceleton, including GSSKSKPK (SEQ ID NO: 74), etc. A signal peptide coding sequence as defined herein preferably encodes a signal peptide which allows for the transport of the protein or peptide as encoded by the nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, into a defined cellular compartiment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Examples of secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201), signal sequences of cytokines or immunoglobulines as defined herein, signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Particularly preferably, signal sequences of MHC class I molecule HLA-A*0201 may be used according to the present invention. Such an additional component may also occur as component L as defined herein. Alternatively, such an additional component may also be bound e.g. to a component P1, P2, P3 as defined herein, e.g. to a side chain of any of components P1, P2, or P3, preferably via a side chain of component P2, or optionally as a linker between components L and P1 or P3 and L. The binding to any of components P1, P2, or P3 may also be accomplished using an acid-labile bond, preferably via a side chain of any of components P1, P2, P3, which allows to detach or release the additional component at lower pH-values, e.g. at physiological pH-values as defined herein.
  • Additionally, the inventive polymeric carrier according to formula (I) above (or according to any of its subformulas herein), may comprise further functional peptides or proteins, which may modulate the functionality of the polymer accordingly. According to one alternative, such further functional peptides or proteins may comprise so called cell penetrating peptides (CPPs) or cationic peptides for transportation. Particularly preferred are CPPs, which induce a pH-mediated conformational change in the endosome and lead to an improved release of the inventive polymeric carrier (in complex with a nucleic acid) from the endosome by insertion into the lipid layer of the liposome. Such called cell penetrating peptides (CPPs) or cationic peptides for transportation, may include, without being limited thereto protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-rich peptides, Loligomers, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, Calcitonin peptide(s), etc. Likewise, such an additional component may also occur as component L as defined herein. Alternatively, such an additional component may also be bound to a component P1, P2, P3 as defined herein, e.g. to a side chain of any of components P1, P2, or P3, preferably via a side chain of component P2, or optionally as a linker between components L and P1 or P3 and L. The binding to any of components P2, or P3 may also be accomplished using an acid-labile bond, preferably via a side chain of any of components P1, P2, P3, which allows to detach or release the additional component at lower pH-values, e.g. at physiological pH-values as defined herein.
  • The object underlying the present invention is furthermore solved according to a second aspect of the present invention by the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) L-P1—S—[S—P2—S]n—S—P3-L as defined herein (or according to any of its subformulas herein). This complex may also be termed “complexed nucleic acid” for the purposes of the present application.
  • In the inventive polymeric carrier cargo complex, the polymeric carrier molecule according to generic formula (I) L-P1—S—[S—P2—S]n—S—P2-L as defined herein (or according to any of its subformulas herein) and the nucleic acid cargo are typically provided in a molar ratio of about 5 to 10000, preferably in a molar ratio of about 10 to 5000, more preferably in a molar ratio of about 20 to 2500, even more preferably in a molar ratio of about 25 to 2000, and most preferably in a molar ratio of about 50 to 1000 of inventive polymeric carrier molecule: nucleic acid.
  • Furthermore, in the inventive polymeric carrier cargo complex, the polymeric carrier molecule according to generic formula (I) L-P1—S—[S—P2—S]n—S—P3-L as defined herein (or according to any of its subformulas herein) and the nucleic acid cargo are preferably provided in an N/P-ratio of about 0.1 to 20, preferably in an N/P-ratio of about 0.2 to 12, and even more preferably in an N/P-ratio of about 0.4 to 10. In this context, an N/P-ratio is defined as the nitrogen/phosphate ratio (N/P-ratio) of the entire inventive polymeric carrier cargo complex. This is typically illustrative for the content/amount of peptides, if peptides are used, in the inventive polymeric carrier and characteristic for the content/amount of nucleic acids bound or complexed in the inventive polymeric carrier cargo complex. It may be calculated on the basis that, for example, 1 μg RNA typically contains about 3 nmol phosphate residues, provided the RNA exhibits a statistical distribution of bases. Additionally, 1 μg peptide typically contains about x nmol nitrogen residues, dependent on the molecular weight and the number of its (cationic) amino acids.
  • In the context of the present invention such a nucleic acid cargo of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may be any suitable nucleic acid, selected e.g. from any DNA, preferably, without being limited thereto, e.g. genomic DNA, single-stranded DNA molecules, double-stranded DNA molecules, coding DNA, DNA primers, DNA probes, a pDNA or may be selected e.g. from any PNA (peptide nucleic acid) or may be selected e.g. from any RNA, preferably, without being limited thereto, a coding RNA, a messenger RNA (mRNA), an siRNA, an shRNA, an antisense RNA, or riboswitches, ribozymes or aptamers; etc. The nucleic acid may also be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA). Preferably, the nucleic acid is RNA, more preferably a coding RNA. Even more preferably, the nucleic acid may be a (linear) single-stranded RNA, even more preferably an mRNA. In the context of the present invention, an mRNA is typically an RNA, which is composed of several structural elements, e.g. an optional 5′-UTR region, an upstream positioned ribosomal binding site followed by a coding region, an optional 3′-UTR region, which may be followed by a poly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
  • Furthermore, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may be a single- or a double-stranded nucleic acid (molecule) (which may also be regarded as a nucleic acid (molecule) due to non-covalent association of two single-stranded nucleic acid(s) (molecules)) or a partially double-stranded or partially single stranded nucleic acid, which are at least partially self complementary (both of these partially double-stranded or partially single stranded nucleic acid molecules are typically formed by a longer and a shorter single-stranded nucleic acid molecule or by two single stranded nucleic acid molecules, which are about equal in length, wherein one single-stranded nucleic acid molecule is in part complementary to the other single-stranded nucleic acid molecule and both thus form a double-stranded nucleic acid molecule in this region, i.e. a partially double-stranded or partially single stranded nucleic acid (molecule). Preferably, the nucleic acid (molecule) may be a single-stranded nucleic acid molecule. Furthermore, the nucleic acid (molecule) may be a circular or linear nucleic acid molecule, preferably a linear nucleic acid molecule.
  • According to one alternative, the nucleic acid of the inventive polymeric carrier cargo complex may be a coding nucleic acid, e.g. a DNA or RNA. Such a coding DNA or RNA may be any DNA or RNA as defined herein. Preferably, such a coding DNA or RNA may be a single-or a double-stranded DNA or RNA, more preferably a single-stranded DNA or RNA, and/or a circular or linear DNA or RNA, more preferably a linear DNA or RNA. Even more preferably, the coding DNA or RNA may be a (linear) single-stranded DNA or RNA. Most preferably, the nucleic acid according to the present invention may be a ((linear) single-stranded) messenger RNA (mRNA). Such an mRNA may occur as a mono-, di-, or even multicistronic RNA, i.e. an RNA which carries the coding sequences of one, two or more proteins or peptides. Such coding sequences in di-, or even multicistronic mRNA may be separated by at least one IRES sequence, e.g. as defined herein.
  • The nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may encode a protein or a peptide, which may be selected, without being restricted thereto, e.g. from therapeutically active proteins or peptides, from antigens, e.g. tumor antigens, pathogenic antigens (e.g. selected from pathogenic proteins as defined herein or from animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens), autoimmune antigens, or further antigens, from allergens, from antibodies, from immunostimulatory proteins or peptides, from antigen-specific T-cell receptors, or from any other protein or peptide suitable for a specific (therapeutic) application, wherein the coding DNA or RNA may be transported into a cell, a tissue or an organism and the protein may be expressed subsequently in this cell, tissue or organism.
  • a) Therapeutically Active Proteins
  • In this context, therapeutically active proteins may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex. These may be selected from any naturally occurring recombinant or isolated protein known to a skilled person from the prior art. Without being restricted thereto therapeutically active proteins may comprise proteins, capable of stimulating or inhibiting the signal transduction in the cell, e.g. cytokines, antibodies, etc. Therapeutically active proteins may thus comprise cytokines of class I of the family of cytokines, having 4 positionally conserved cysteine residues (CCCC) and comprising a conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS), wherein X is a non-conserved amino acid. Cytokines of class I of the family of cytokines comprise the GM-CSF subfamily, e.g. IL-3, IL-5, GM-CSF, the IL-6-subfamily, e.g. IL-6, IL-11, IL-12, or the IL-2-subfamily, e.g. IL-2, IL-4, IL-7, IL-9, IL-15, etc., or the cytokines IL-1alpha, IL-1beta, IL-10 etc. Therapeutically active proteins may also comprise cytokines of class II of the family of cytokines, which also comprise 4 positionally conserved cystein residues (CCCC), but no conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS). Cytokines of class II of the family of cytokines comprise e.g. IFN-alpha, IFN-beta, IFN-gamma, etc. Therapeutically active proteins may additionally comprise cytokines of the family of tumor necrose factors, e.g. TNF-alpha, TNF-beta, etc., or cytokines of the family of chemokines, which comprise 7 transmembrane helices and interact with G-protein, e.g. IL-8, MIP-1, RANTES, CCR5, CXH4, etc., or cytokine specific receptors, such as TNF-I, TNF-RII, CD40, OX40 (CD134), Fas, etc.
  • Therapeutically active proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex may also be selected from any of the proteins given in the following: 0ATL3, 0FC3, 0PA3, 0PD2, 4-1BBL, 5T4, 6Ckine, 707-AP, 9D7, A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABM, ABCB11, ABCB2, ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABL1, ABO, ABR ACAA1, ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE, ACHE, ACHM3, ACHM1, ACLS, ACPI, ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA, ADAMTS13, ADAMTS2, ADFN, ADHIB, ADH1C, ADLDH3A2, ADRB2, ADRB3, ADSL, AEZ, AFA, AFD1, AFP, AGA, AGL, AGMX2, AGPS, AGS1, AGT, AGTR1, AGXT, AH02, AHCY, AHDS, AHHR, AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE, AK1, ALAD, ALAS2, ALB, HPG1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDHIA1, ALDOA, ALDOB, ALMS1, ALPL, ALPP, ALS2, ALX4, AMACR, AMBP, AMCD, AMCD1, AMCN, AMELX, AMELY, AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR, ANK1, ANOP1, AOM, AP0A4, AP0C2, AP0C3, AP3B1, APC, aPKC, AP0A2, AP0A1, APOB, AP0C3, AP0C2, APOE, APOH, APP, APRT, APS1, AQP2, AR, ARAF1, ARG1, ARHGEF12, ARMET, ARSA, ARSB, ARSC2, ARSE, ART-4, ARTC1/m, ARTS, ARVD1, ARX, AS, ASAH, ASAT, ASD1, ASL, ASMD, ASMT, ASNS, ASPA, ASS, ASSP2, ASSP5, ASSP6, AT3, ATD, ATHS, ATM, ATP2A1, ATP2A2, ATP2C1, ATP6B1, ATP7A, ATP7B, ATP8B1, ATPSK2, ATRX, ATXN1, ATXN2, ATXN3, AUTS1, AVMD, AVP, AVPR2, AVSD1, AXIN1, AXIN2, AZF2, B2M, B4GALT7, B7H4, BAGE, BAGE-1, BAX, BBS2, BBS3, BBS4, BCA225, BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10, BCL2, BCD, BCL5, BCL6, BCPM, BCR, BCR/ABL, BDC, SDE, BDMF, BDMR, BEST1, beta-Catenin/m, BF, BFHD, BFIC, BFLS, BFSP2, BGLAP, BGN, BHD, BHR1, BING-4, BIRC5, BJS, BLM, BLMH, BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1/m, BRCA2, BRCA2/m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, STK, BURL BWS, BZX, C0L2A1, C0L6A1, C1NH, C1QA, C1QB, C1QG, C1S, C2, C3, C4A, C4B, C5, C6, C7, C7orf2, CSA, CSB, C9, CA125, CA15-3/CA 27-29, CA195, CA19-9, CA72-4, CA2, CA242, CA50, CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F, CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA, CALCR, CALM, CALR, CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5/m, CASP-8, CASP-8/m, CASR, CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1, CCAL2, CCAL1, CCAT, CCL-1, CCL-11, CCL-12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19, CCL-2, CCL-20, CCL-21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-27, CCL-3, CCL-4, CCL-5, CCL-7, CCL-8, CCM1, CCNB1, CCND1, CCO, CCR2, CCR5, CCT, CCV, CCZS, CD1, CD19, CD20, CD22, CD25, CD27, CD27L, cD3, CD30, CD30, CD30L, CD33, CD36, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD44v, CD44v6, CD52, CD55, CD56, CD59, CD80, CD86, CDAN1, CDAN2, CDAN3, CDC27, CDC27/m, CDC2L1, CDH1, CDK4, CDK4/m, CDKN1C, CDKN2A, CDKN2A/m, CDKN1A, CDKN1C, CDL1, CDPD1, CDR1, CEA, CEACAM1, CEACAM5, CECR, CECR9, CEPA, CETP, CFNS, CFTR, CGF1, CHAC, CHED2, CHED1, CHEK2, CHM, CHML, CHR39c, CHRNA4, CHRNA1, CHRNB1, CHRNE, CHS, CHS1, CHST6, CHX10, CIAS1, CIDX, CKN1, CLA2, CLA3, CLA1, CLCA2, CLCN1, CLCN5, CLCNKB, CLDN16, CLP, CLN2, CLN3, CLN4, CLN5, CLN6, CLN8, C1QA, C1QB, C1QG, C1R, CLS, CMCWTD, CMDJ, CMD1A, CMDIB, CMH2, MH3, CMH6, CMKBR2, CMKBR5, CML28, CML66, CMM, CMT2B, CMT2D, CMT4A, CMT1A, CMTX2, CMTX3, C-MYC, CNA1, CND, CNGA3, CNGA1, CNGB3, CNSN, CNTF, COA-1/m, COCH, COD2, COD1, COH1, COL10A, COL2A2, COL11A2, COL17A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL4A4, COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COLSA2, COL9A2, COL9A3, COL11A1, COL1A2, COL23A1, COL1A1, COLQ, COMP, COMT, CORD5, CORD1, COX10, COX-2, CP, CPB2, CPO, CPP, CPS1, CPT2, CPT1A, CPX, CRAT, CRB1, CRBM, CREBBP, CRH, CRHBP, CRS, CRV, CRX, CRYAB, CRYBA1, CRYBB2, CRYGA, CRYGC, CRYGD, CSA, CSE, CSF1R, CSF2RA, CSF2RB, CSF3R, CSF1R, CST3, CSTB, C1, CT7, CT-9/BRD6, CTAA1, CTACK, CTEN, CTH, CTHM, CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB, CTSC, CTSK, CTSL, CTS1, CUBN, CVD1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CYB5, CYBA, CYBB, CYBB5, CYFRA 21-1, CYLD, CYLD1, CYMD, CYP11B1, CYP11B2, CYP17, CYP17A1, CYP19, CYP19A1, CYP1A2, CYP1B1, CYP21A2, CYP27A1, CYP27B1, CYP2A6, CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6, CYP2D7P1, CYP3A4, CYP7B1, CYPB1, CYP11B1, CYP1A1, CYP1B1, CYRAA, D40, DAD1, DAM, DAM-10/MAGE-B1, DAM-6/MAGE-B2, DAX1, DAZ, DBA, DBH, D131, DBT, DCC, DC-CK1, DCK, DCR, DCX, DDB 1, DDB2, DDIT3, DDU, DECR1, DEK-CAN, DEM, DES, DF, DFN2, DFN4, DFN6, DFNA4, DFNA5, DENS5, DGCR, DHCR7, DHFR, DHOF, DHS, DIA1, DIAPH2, DIAPH1, DIH1, D101, DISC1, DKC1, DLAT, DLD, DLL3, DLX3, DMBT1, DMD, DMI, DMPK, DMWD, DNAI1, DNASE1, DNMT3B, DPEP1, DPYD, DPYS, DRD2, DRD4, DRPLA, DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR, DURS1, DWS, DYS, DYSF, DYT2, DYT3, DYT4, DYT2, DYT1, DYXI, EBAF, ESM, EBNA, ESP, EBR3, ESS1, ECA1, ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA, EDAR, ECA1, EDN3, EDNRB, EEC1, EEF1A1L14, EEGV1, EFEMP1, EFTUD2/m, EGFR, EGFR/Her1, EGI, EGR2, EIF2AK3, eIF4G, EKV, El IS, ELA2, ELF2, ELF2M, ELK1, ELN, ELONG, EMD, EML1, EMMPRIN, EMX2, ENA-78, ENAM, END3, ENG, ENO1, ENPP1, ENUR2, ENUR1, EOS, EP300, EPB41, EPB42, EPCAM, EPD, EphA1, EphA2, EphA3, EphrinA2, EphrinA3, EPHX1, EPM2A, EPO, EPOR, EPX, ERBB2, ERCC2 ERCC3, ERCC4, ERCC5, ERCC6, ERVR, ESR1, ETFA, ETFB, ETFDH, ETM1, ETV6-AML1, ETV1, EVC, EVR2, EVR1, EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2, EYCL3, EYCL1, EZH2, F10, F11, F12, F13A1, F13B, F2, F5, F5F8D, F7, F8, F8C, F9, FABP2, FACIA FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL, FBN2, FBN1, FBP1, FCG3RA, FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1, FDPSL5, FECH, FEO, FEOM1, FES, FGA, FGB, FGD1, FGF2, FGF23, FGF5, FGFR2, FGFR3, FGFR1, FGG, FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FMO3, FMO4, FMR2, FMR1, FN, FN1/m, FOXC1, FOXEI, FOXL2, FOXO1A, FPDMM, FPF, Fra-1, FRAXF, FRDA, FSHB, FSHMDIA, FSHR, FTH1, FTHL17, FTL, FT2F1, FUCA1, FUT2, FUT6, FUT1, FY, G250, G250/CAIX, G6PC, G6PD, G6PT1, G6PT2, GAA, GABRA3, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GALC, GALE, GALK1, GALNS, GALT, GAMT, GAN, GAST, GASTRIN17, GATA3, GATA, GSA, GBE, GC, GCDH, GCGR, GCH1, GCK, GCP-2, GCS1, G-CSF, GCSH, GCSL, GCY, GDEP, GDF5, GDI1, GDNF, GDXY, GFAP, GFND, GGCX, GGT1, GH2, GH1, GHR, GNRHR, GHS, GIF, GINGF, GIP, GJA3, GJA8, GJB2, GJB3, GJB6, GJB1, GK, GLA, GLB, GLB1, GLC3B, GLC1B, GLC1C, GLDC, GLI3, GLP1, GLRA1, GLUD1, GM1 (fuc-GM1), GM2A, GM-CSF, GMPR, GNAI2, GNAS, GNAT1, GNB3, GNE, GNPTA, GNRH, GNRH1, GNRHR, GNS, GnT-V, gp100, GP1BA, GP1BB, GP9, GPC3, GPD2, GPDS1, GP1, GP1BA, GPN1LW, GPNMB/m, GPSC, GPX1, GRHPR, GRK1, GROα, GROβ, GROγ, GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS, GUCA1A, GUCY2D, GULOP, GUSB, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, H0KPP2, H0MG2, HADHA, HADHB, HAGE, HAGH, HAL, HAST-2, HB 1, HBA2, HBA1, HBB, HBBP1, HBD, HBE1, HBG2, HBG1, HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1, HCC-4, HCF2, HCG, HCL2, HCL1, HCR, HCVS, HD, HPN, HER2, HER2/NEU, HER3, HERV-K-MEL, HESX1, HEXA, HEXB, HF1, HF1, HGD, HHC2, HHC3, HHG, HK1 HLA-A, HLA-A*0201-R170I, HLA-A11/m, HLA-A2/m, HLA-DPB1 HLA-DRA, HLCS, HLXB9, HMBS, HMGA2, HMGCL, HMI, HMN2, HMOX1, HMS1 HMW-MAA, FIND, HNE, HNF4A, HOAC, HOMEOBOX NKX 3.1, HOM-TES-14/SCP-1, HOM-TES-85, HOXA1 HOXD13, HP, HPC1, HPD, HPE2, HPE1, HPFH, HPFH2, HPRT1, HPS1, HPT, HPV-E6, HPV-E7, HR, HRAS, HRD, HAG, HRPT2, HRPT1, HRX, HSD11B2, HSD17B3, HSD17B4, HSD3B2, HSD3B3, HSN1, HSP70-2M, HSPG2, HST-2, HTC2, HTC1, hTERT, HTN3, HTR2c, HVBS6, HVBS1, HVEC, HV1S, HYAL1, HYR, 1-309, IAB, IBGC1, IBM2, ICAM1, ICAM3, iCE, ICHQ, ICR5, ICR1, ICS 1, IDDM2, IDDM1, IDS, IDUA, IF, □IFNa/b, □IFNGR1, IGAD1, IGER, IGF-1R, IGF2R, IGF1, IGH, IGHC, IGHG2, IGHG1, IGHM, IGHR, IGKC, 1HG1, 1HH, IKBKG, ILL IL-1 RA, IL10, IL-11, IL12, IL12RB1, IL13, IL-13Rα2, IL-15, IL-16, IL-17, IL18, IL-1a, IL-1α, IL-1b, IL-1β, IL1RAPL1, IL2, I24, IL-2R, IL2RA, IL2RG, 13, IL3RA, IL4, IL4R, IL4R, IL-5, IL6, IL-7, IL7R, IL-8, IL-9, Immature laminin receptor, IMMP2L, INDX, INEGR1, INFGR2, INFα, IFN□□□□INFγ, INS, INSR, INVS, IP-10, IP2, IPF1, IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3, ITGB4, IT1H1; ITM2B, IV, IVD, JAG1, JAK3, J13S, JBTS1, JMS, JPD, KAL1, KAL2, KALI, KLK2, KLK4, KCNA1, KCNE2, KCNE1, KCNH2, KCNJ1, KCNJ2, KCNJ1, KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA, KFM, KFS, KFSD, KHK, ki-67, KIAA0020, KIAA0205, KIAA0205/m, KIF1B, KIT, KK-LC-1, KLK3, KLKB1, KM-HN-1, KMS, KNG, KNO, K-RAS/m, KRAS2, KREV1, KRT1, KRT10, KRT12, KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3, KRT16, KRT16L1, KRT16L2, KRT17, KRT18, KRT2A, KRT3, KRT4, KRT5, KRT6A, KRT6B, KRT9, KRTHB1, KRTHB6, KRT1, KSA, KSS, KWE, KYNU, LOH19CR1, L1CAM, LAGE, LAGE-1, LALL, LAMA2, LAMA3, LAMB3, LAMB1, LAMC2, LAMP2, LAP, LCA5, LCAT, LCCS, LCCS1, LCFS2, LCS1, LCT, LDHA, LDHB, LDHC, LDLR, LDLR/FUT, LEP, LEWISY, LGCR, LGGF-PBP, LGI1, LGMD2H, LGMD1A, LGMDIB, LHB, LHCGR, LHON, LHRH, LHX3, LIF, LIG1, LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN, L1CAM, LMAN1, LMNA, LMX1B, LOLR, LOR, LOX, LPA, LPL, LPP, LQT4, LRP5, LRS1, LSFC, LT-β, LTBP2, LTC4S, LYL1, XCLI, LYZ, M344, MA50, MAA, MADH4, MAFD2, MAFD1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGEB1, MAGE-B10, MAGE-B16, MAGE-817, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, MGBI, MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA, MAOB, MAPK81P1, MAPT, MART-1, MART-2, MART2/m, MAT1A, MBL2, MBP, MBS1, MC1R, MC2R, MC4R, MCC, MCCC2, MCCC1, MCDR1, MCF2, MCKD, MCL1, MC1R, MCOLN1, MCOP, MCOR, MCP-1, MCP-2, MCP-3, MCP-4, MCPH2, MCPH1, MCS, M-CSF, MDS, MDCR, MDM2, MDRV, MDS 1, ME1, ME1/m, ME2, ME20, ME3, MEAX, MEB, MEC CCL-28, MECP2, MEFV, MELANA, MELAS, MEN1 MSLN, MET, MF4, MG50, MG50/PXDN, MGAT2, MGAT5, MGC1 MGCR, MGCT, MGI, MGP, MHC2TA, MHS2, MHS4, MIC2, MICS, MIDI, MIF, MIP, MIP-5/HCC-2, MITF, MJD, MKI67, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3, MLLT7, MLLT1, MLS, MLYCD, MMA1a, MMP 11, MMVP1, MN/CA IX-Antigen, MNG1, MN1, MOC31, MOCS2, MOCS1, MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPI, MPIF-1, MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD, MRX14, MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3, MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTCO3, MTCO1, MTCYB, MTHFR, MTM1, MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2, MTRNR1, MTRR, MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP, MTTS1, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MUM-1/m, MUM-2, MUM-2/m, MUM-3, MUM-3/m, MUT, mutant p21 ras, MUTYH, MVK, MX2, MXI1, MY05A, MYB, MYBPC3, MYC, MYCL2, MYH6, MYL2, MYL3, MYMY, MYO15A, MYO1G, MYO5A, MYO7A, MYOC, Myosin/m, MYP2, MYP1, NA88-A, N-acetylglucosaminyltransferase-V, NAGA, NAGLU, NAMSD, NAPS, NAT2, NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP, NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEB, NEFH, NEM1, Neo-PAP, neo-PAP/m, NEU1, NEUROD1, NF2, NF1, NFYC/m, NGEP, NHS, NKSI, NKX2E, NM, NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1, NPHL2, NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2, NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-CO1, NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1, NYX, OA3, OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRLI, OCT, ODDD, ODT1, 0FC1, OFD1, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1, OPEM, OPG, OPN, OPNILW, OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1, ORP1, OS-9, 0S-9/m, OSM LIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15, P190 MINOR BCR-ABL, P2RY12, P3, P16, P40, P4HB, P-501, P53, P53/m, P97, PABPN1, PAFAH1B1, PAFAH1P1, PAGE-4, PAGE-S, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1, PATE, PAX2, PAX3, PAX6, PAX7, PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1, PBXP1, PC, PCBD, PCCA, PCCB, PCK2, PCK1, PCLD, PCOS1, PCSK1, PDB1, PDCN, PDE6A, PDE6B, PDEF, PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1, PEE1, PEO1, PEPD, PEXIO, PEX12, PEX13, PEX3, PEXS, PEX6, PEX7, PEX1, PF4, PFBI, PFC, PFKFB1, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS, PHA2A, PHS, PHEX, PHGDH, PHKA2, PHKA1, PHKB, PHKG2, PHP, PHYH, P1, PI3, PIGA, PIM1-KINASE, PIN1, PIP5KIB, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS, PKHD1, PKLR, PKP1, PKU1, PLA2G2A, PLA2G7, PLAT, PLEC1, PLG, PLI, PLOD, PLP1, PMEL17, PML, PML/RARα, PMM2, PMP22, PMS2, PMS1, PNKD, PNLIP, POF1, POLA, POLH, POMC, PON2, PON1, PORC, POTE, POU1F1, POU3F4, POU4F3, POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1, PPKB, PPMX, PPDX, PPP1R3A, PPP2R2B, PPT1, PRAME, PRS, PRB3, PRCA1, PRCC, PRD, PRDX5/m, PRF1, PRG4, PRKAR1A, PRKCA, PRKDC, PRKWNK4, PRNP, PROC, PRODH, PROM1, PROP1, PROS1, PRST, PRP8, PRPF31, PRPF8, PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1, PRTN3, PAX, PSA, PSAP, PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA, PSORS1, PTC, PTCH, PTCH1, PTCH2, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1, PTPN12, PTPNI 1, PTPRK, PTPRK/m, PTS, PUJO, PVR, PVRL1, PWCR, PXE, PXMP3, PXR1, PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE, RAGE-1, RAG1, RAP1, RARA, RASA1, RBAF600/m, RB1, RBP4, RBP4, RBS, RCA1, RCAS1, RCCP2, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4, REG1A, REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFXAP, RGA, RHAG, RHAMM/CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP, ROM1, ROR2, RP, RP1, RP14, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR, RPGRIP1, RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1, RSN, RSS, RU1, RU2, RUNX2, RUNX1, RWS, RYR1, S-100, SAA1, SACS, SAG, SAGE, SALL1, SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4, SCA5, SCAT, SCA8, SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A, SCN5A, SCNN1A, SCNN1B, SCNN1G, SCO2, SCP1, SCZD2, SCZD3, SCZD4, SCZD6, SCZD1, SDF-1□/□□□SDHA, SDHD, SDYS, SEDL, SERPENA7, SERPINA3, SERPINA6, SERPINA1, SERPINC1, SERPIND1, SERPINE1, SERPINF2, SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC, SFTPD, SGCA, SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2, SHFM3, SHFM1, SHH, SHOX, SI, SIAL, SIALYL I EWISX SIASD, S11, SIM1, SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5, SLC19A2, SLC22A1L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4, SLC25A5, SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2, SLC2A4, SLC3A1, SLC4A1, SLC4A4, SLC5A1, SLC5A5, SLC6A2, SLC6A3, SLC6A4, SLC7A7, SLC7A9, SLC11A1, SLOS, SMA, SMAD1, SMAL, SMARCB1, SMAX2, SMCR, SMCY, SM1, SMN2, SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3, SOD1, SOS1, SOST, SOX9, SOX10, Sp17, SPANXC, SPG23, SPG3A, SPG4, SPG5A, SPG5B, SPG6, SPG7, SPINK1, SPINK5, SPPK, SPPM, SPSMA, SPTA1, SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS, SRY, βhCG, SSTR2, SSX1, SSX2 (HOM-MEL-40/SSX2), SSX4, ST8, STAMP-1, STAR, STARP1, STATH, STAMP, STK2, STK11, STn/KLH, STO, STOM, STS, SUOX, SURF1, SURVIVIN-2B, SYCP1, SYM1, SYN1, SYNS1, SYP, SYT/SSX, SYT-SSX-1, SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG72, TAF7L, TAF1, TAGE, TAG-72, TALI, TAM, TAP2, TAP1, TAPVR1, TARC, TARP, TAT, TAZ, TBP, TBX22, TBX3, TBX5, TBXA2R, TBXAS1, TCAP, TCF2, TCF1, TCIRG1, TCL2, TCL4, TCL1A, TCN2, TCOF1, TCR, TCRA, TDD, TDFA, TDRD1, TECK, TECTA, TEK, TEL/AML1, TELAB1, TEX15, TF, TFAP2B, TFE3, TFR2, TG, TGFα, TGF-□□β, TGFB1, TGFB1, TGFBR2, TGEBRE, TGFβ, TGRβII, TGIF, TGM-4, TGM1, TH, THAS, THBD, THC, THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3, TIMP1, TITF1, TKCR, TKT, TLP, TLR1, TLR10, TLR2, TLR3, TLR4, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLX1, TM4SF1, TM4SF2, TMC1, TMD, TMIP, TNDM, TNF, TNFRSF11A, TNFRSF1A, TNFRSF6, TNFSF5, TNFSF6, TNFα, TNFβ, TNNI3, TNNT2, TOC, TOP2A, TOP1, TP53, TP63, TPA, TPBG, TPI, TPI/m, TPI1, TPM3, TPM1, TPMT, TPO, TPS, TPTA, TRA, TRAG3, TRAPPC2, TRC8, TREH, TRG, TRH, TRIM32, TRIM37, TRP1, TRP2, TRP-2/6b, TRP-2/1NT2, Trp-p8, TRPS1, TS, TSC2, TSC3, TSC1, TSG101, TSHB, TSHR, TSP-180, TST, TTGA2B, TTPA, TTR, TU M2-PK, TULP1, TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1, TYS, UBE2A, UBE3A, UBE1, UCHL1, UFS, UGT1A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1, URO5, UROD, UPKIB, UROS, USH2A, USH3A, USH1A, USH1C, USP9Y, UV24, VBCH, VCF, VDI, VDR, VEGF, VEGFR-2, VEGFR-1, VEGFR-2/FLK-1, VHL, VIM, VMD2, VMD1, VMGLOM, VNEZ, VNF, VP, VRNI, VWF, VWS, WAS, WBS2, WFS2, WFS1, WHCR, WHN, W1SP3, WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1, WTS, WWS, XAGE, XDH, XIC, XIST, XK, XM, XPA, XPC, XRCC9, XS, ZAP70, ZFHX1B, ZFX, ZFY, ZIC2, ZIC3, ZNF145, ZNF261, ZNF35, ZNF41, ZNF6, ZNF198, ZWS1.
  • Therapeutically active proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex may further be selected from apoptotic factors or apoptosis related proteins including AIF, Apaf e.g. Apaf-1, Apaf-2, Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2, Bcl-xL, Bd-xS, bik, CAD, Calpain, Caspase e.g. Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, ced-3, ced-9, c-Jun, c-Myc, crm A, cytochrom C, CdR1, DcR1, DD, DED, DISC, DNA-PKCS, DR3, DR4, DR5, FADD/MORT-1, FAK, Fas (Fas-ligand CD95/fas (receptor)), FLICE/MACH, FLIP, fodrin, fos, G-Actin, Gas-2, gelsolin, granzyme NB, ICAD, ICE, JNK, lamin NB, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD, NF-kappaB, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKCdelta, pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidinkinase from herpes simplex, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3, transglutaminase, etc.
  • A therapeutically active protein, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, can also be an adjuvant protein. In this context, an adjuvant protein is preferably to be understood as any protein, which is capable to elicit an innate immune response as defined herein. Preferably, such an innate immune response comprises activation of a pattern recognition receptor, such as e.g. a receptor selected from the Toll-like receptor (TLR) family, including e.g. a Toll like receptor selected from human TLR1 to TLR10 or from murine Toll like receptors TLR1 to TLR13. Preferably, an innate immune response is elicited in a mammal as defined herein. More preferably, the adjuvant protein is selected from human adjuvant proteins or from pathogenic adjuvant proteins, in particular from bacterial adjuvant proteins. In addition, mRNA encoding human proteins involved in adjuvant effects may be used as well.
  • Human adjuvant proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, typically comprise any human protein, which is capable of eliciting an innate immune response (in a mammal), e.g. as a reaction of the binding of an exogenous TLR ligand to a TLR. More preferably, human adjuvant proteins encoded by the nucleic acid according to the present invention may be selected from the group consisting of, without being limited thereto, cytokines which induce or enhance an innate immune response, including IL-2, IL-12, IL-15, IL-18, IL-21CCL21, GM-CSF and TNF-alpha; cytokines which are released from macrophages, including IL-1, IL-6, IL-8, IL-12 and TNF-alpha; from components of the complement system including C1q, MBL, C1r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, Cab, C5a, C3a, C4a, C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4 bp, MCP, DAF, H, I, P and CD59; from proteins which are components of the signalling networks of the pattern recognition receptors including TLR and IL-1R1, whereas the components are ligands of the pattern recognition receptors including IL-1alpha, IL-1 beta, Beta-defensin, heat shock proteins, such as HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIII repeat extra domain A of fibronectin; the receptors, including IL-1R1, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; the signal transducers including components of the Small-GTPases signalling (RhoA, Ras, Rac1, Cdc42 etc.), components of the PIP signalling (PI3K, Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88, IRAK1, IRAK2, etc.), components of the MyD88-independent signalling (TICAM1, TICAM2 etc.); activated transcription factors including e.g. NF-κB, c-Fos, c-Jun, c-Myc; and induced target genes including e.g. IL-1 alpha, IL-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFN beta; from costimulatory molecules, including CD28 or CD40-ligand or PD1; protein domains, including LAMP; cell surface proteins; or human adjuvant proteins including CD80, CD81, CD86, trif, flt-3 ligand, thymopentin, Gp96 or fibronectin, etc., or any species homolog of any of the above human adjuvant proteins.
  • Pathogenic adjuvant proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, typically comprise any pathogenic (adjuvant) protein, which is capable of eliciting an innate immune response (in a mammal), more preferably selected from pathogenic (adjuvant) proteins derived from bacteria, protozoa, viruses, or fungi, animals, etc., and even more preferably from pathogenic adjuvant proteins selected from the group consisting of, without being limited thereto, bacterial proteins, protozoan proteins (e.g. profilin—like protein of Toxoplasma gondii), viral proteins, or fungal proteins, animal proteins, etc.
  • In this context, bacterial (adjuvant) proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may comprise any bacterial protein, which is capable of eliciting an innate immune response (preferably in a mammal) or shows an adjuvant character. More preferably, such bacterial (adjuvant) proteins are selected from the group consisting of bacterial heat shock proteins or chaperons, including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein) from gram-negative bacteria; bacterial porins, including OmpF; bacterial toxins, including pertussis toxin (PT) from Bordetella pertussis, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylate cyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin, cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from cholera toxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin (LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coli heat-labile enterotoxin mutants with reduced toxicity, including LTK63, LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP) from Helicobacter pylori; Surfactant protein D; Outer surface protein A lipoprotein from Borrelia burgdorferi, Ag38 (38 kDa antigen) from Mycobacterium tuberculosis; proteins from bacterial fimbriae; Enterotoxin CT of Vibrio cholerae, Pilin from pili from gram negative bacteria, and Surfactant protein A; etc., or any species homolog of any of the above bacterial (adjuvant) proteins.
  • Bacterial (adjuvant) proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may also be selected from bacterial adjuvant proteins, even more preferably selected from the group consisting of, without being limited thereto, bacterial flagellins, including flagellins from organisms including Agrobacterium, Aquifex, Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia, Campylobacter, Caulobacte, Clostridium, Escherichia, Helicobacter, Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas, Rhizobium, Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia, Shigella, Treponema, Vibrio, Wolinella, Yersinia, more preferably flagellins from the species, without being limited thereto, Agrobacterium tumefaciens, Aquifex pyrophilus, Azospirillum brasilense, Bacillus subtilis, Bacillus thuringiensis, Bartonella bacilliformis, Bordetella bronchiseptica, Borrelia burgdorferi, Burkholderia cepacia, Campylobacter jejuni, Caulobacter crescentus, Clostridium botulinum strain Bennett clone 1, Escherichia coli, Helicobacter pylori, Lachnospiraceae bacterium, Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis, Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti, Rhodobacter sphaeroides, Roseburia cecicola, Roseburis hominis, Salmonella typhimurium, Salmonella bongori, Salmonella typhi, Salmonella enteritidis, Serpulina hyodysenteriae, Serratia marcescens, Shigella boydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio cholerae, Vibrio parahaemolyticus, Wolinella succinogenes and Yersinia enterocolitica.
  • Bacterial flagellins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, even more preferably comprise a sequence selected from the group comprising any of the following sequences as referred to their accession numbers:
  • accession
    organism species gene name No GI No
    Agrobacterium Agrobacterium FlaD (flaD) U95165 GI: 14278870
    tumefaciens FlhB (flhB)
    FliG (fliG)
    FliN (fliN)
    FliM (fliM)
    MotA (motA)
    FlgF (flgF)
    Flil (flil)
    FlgB (flgB)
    FlgC (flgC)
    FliE (fliE)
    FlgG (flgG)
    FlgA (flgA)
    FlgI (flgI)
    FlgH (flgH)
    FliL (fliL)
    FliP (fliP)
    FlaA (flaA)
    FlaB (flaB)
    FlaC (flaC)
    Aquifex Aquifex U17575 GI: 596244
    pyrophilus
    Azospirillum Azospirillum Laf1 U26679 GI: 1173509
    brasilense
    Bacillus Bacillus subtilis hag AB033501 GI: 14278870
    Bacillus Bacillus flab X67138 GI: 46019718
    thuringiensis
    Bartonella Bartonella L20677 GI: 304184
    bacilliformis
    Bordetella Bordetella flaA L13034 GI: 289453
    bronchiseptica
    Borrelia Borrelia X16833 GI: 39356
    burgdorferi
    Burkholderia Burkholderia fliC AF011370 GI: 2935154
    cepacia
    Campylobacter Campylobacter flaA J05635 GI: 144197
    jejuni flaB
    Caulobacter Caulobacter J01556 GI: 144239
    crescentus
    Clostridium Clostridium FlaA DQ845000 GI: 114054886
    botulinum strain
    Bennett clone 1
    Escherichia Escherichia coli hag M14358 GI: 146311
    AJ884569
    (EMBL-SVA)
    Helicobacter Helicobacter flaA X60746 GI: 43631
    pylori
    Lachnospiraceae Lachnospiraceae DQ789131 GI: 113911615
    bacterium
    Legionella Legionella flaA X83232 GI: 602877
    pneumophila
    Listeria Listeria flaA X65624 GI: 44097
    monocytogenes
    Proteus Proteus mirabilis FlaD (flaD) AF221596 GI: 6959881
    FlaA (flaA)
    FlaB (flaB)
    FliA (fliA)
    FliZ (fliZ)
    Pseudomonas Pseudomonas flaA M57501 GI: 151225
    aeroguinosa
    Pseudomonas Pseudomonas fliC EF544882 GI: 146335619
    syringae
    Rhizobium Rhizobium flaA M24526 GI: 152220
    meliloti flaB
    Rhodobacter Rhodobacter fliC AF274346 GI: 10716972
    sphaeroides
    Roseburia Roseburia M20983 GI: 152535
    cecicola
    Roseburia Roseburis Fla2 DQ789141 GI: 113911632
    hominis
    Salmonella Salmonella D13689 GI: 217062
    typhimurium (NCBI ID)
    Salmonella Salmonella fliC AY603412 GI: 51342390
    bongori
    Salmonella Salmonella typhi flag L21912 GI: 397810
    Salmonella Salmonella fliC M84980 GI: 154015
    enteritidis
    Serpulina Serpulina flaB2 X63513 GI: 450669
    hyodysenteriae
    Serratia Serratia hag M27219 GI: 152826
    marcescens
    Shigella Shigella boydii fliC-SB D26165 GI: 442485
    Treponema Treponema flaB2 M94015 GI: 155060
    phagedenis
    Vibrio Vibrio flaA EF125175 GI: 119434395
    alginolyticus
    Vibrio s Vibrio AF069392 G1: 7327274
    parahaemolyticus
    Wolinella Wolinella flag M82917 GI: 155337
    succinogenes
    Yersinia Yersinia L33467 GI: 496295
    enterocolitica
  • Protozoan proteins, which may also be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may be selected from any protozoan protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Tc52 from Trypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shock proteins, LeIF from Leishmania spp., profilin-like protein from Toxoplasma gondii, etc.
  • Viral proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may be selected from any viral protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Respiratory Syncytial Virus fusion glycoprotein (F-protein), envelope protein from MMT virus, mouse leukemia virus protein, Hemagglutinin protein of wild-type measles virus, etc.
  • Fungal proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may be selected from any fungal protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, fungal immunomodulatory protein (FIP; LZ-8), etc.
  • Finally, pathogenic adjuvant proteins, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may finally be selected from any further pathogenic protein showing adjuvant character, more preferably, from the group consisting of, without being limited thereto, Keyhole limpet hemocyanin (KLH), OspA, etc.
  • b) Antigens
  • The nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may alternatively encode an antigen. According to the present invention, the term “antigen” refers to a substance which is recognized by the immune system and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies as part of an adaptive immune response. In this context, the first step of an adaptive immune response is the activation of naïve antigen-specific T cells by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naïve T cells are constantly passing. The three cell types that can serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Tissue dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by infection to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents to express MHC class II molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may be important to induce T cells. By presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8 cytotoxic T cells and the activation of macrophages by TH1 cells which together make up cell-mediated immunity, and the activation of B cells by both TH2 and TH1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which does not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogens' protein antigens, which are bound to MHC molecules on the surfaces of other cells.
  • In the context of the present invention, antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex typically comprise any antigen, falling under the above definition, more preferably protein and peptide antigens, e.g. tumor antigens, allergy antigens, auto-immune self-antigens, pathogens, etc. In accordance with the invention, antigens as encoded by the nucleic acid according to the present invention may be antigens generated outside the cell, more typically antigens not derived from the host organism (e.g. a human) itself (i.e. non-self antigens) but rather derived from host cells outside the host organism, e.g. viral antigens, bacterial antigens, fungal antigens, protozoological antigens, animal antigens (preferably selected from animals or organisms as disclosed herein), allergy antigens, etc. Allergy antigens are typically antigens, which cause an allergy in a human and may be derived from either a human or other sources. Antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex may be furthermore antigens generated inside the cell, the tissue or the body, e.g. by secretion of proteins, their degradation, metabolism, etc. Such antigens include antigens derived from the host organism (e.g. a human) itself, e.g. tumor antigens, self-antigens or auto-antigens, such as auto-immune self-antigens, etc., but also (non-self) antigens as defined herein, which have been originally been derived from host cells outside the host organism, but which are fragmented or degraded inside the body, tissue or cell, e.g. by (protease) degradation, metabolism, etc.
  • Antigens as encoded by nucleic acid of the inventive polymeric carrier cargo complex may furthermore comprise fragments of such antigens as mentioned herein, particularly of protein or peptide antigens. Fragments of such antigens in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their naïve form.
  • Fragments of antigens as defined herein, encoded by the nucleic of the inventive polymeric carrier cargo complex, may also comprise epitopes of those antigens. Epitopes (also called “antigen determinants”) are typically fragments located on the outer surface of (naïve) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their naïve form.
  • One class of antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex comprises tumor antigens. “Tumor antigens” are preferably located on the surface of the (tumor) cell. Tumor antigens may also be selected from proteins, which are overexpressed in tumor cells compared to a normal cell. Furthermore, tumor antigens also includes antigens expressed in cells which are (were) not themselves (or originally not themselves) degenerate but are associated with the supposed tumor. Antigens which are connected with tumor-supplying vessels or (re)formation thereof, in particular those antigens which are associated with neovascularization, e.g. growth factors, such as VEGF, bFGF etc., are also included herein. Antigens connected with a tumor furthermore include antigens from cells or tissues, typically embedding the tumor. Further, some substances (usually proteins or peptides) are expressed in patients suffering (knowingly or not-knowingly) from a cancer disease and they occur in increased concentrations in the body fluids of said patients. These substances are also referred to as “tumor antigens”, however they are not antigens in the stringent meaning of an immune response inducing substance. The class of tumor antigens can be divided further into tumor-specific antigens (TSAs) and tumor-associated-antigens (TAAs). TSAs can only be presented by tumor cells and never by normal “healthy” cells. They typically result from a tumor specific mutation. TAAs, which are more common, are usually presented by both tumor and healthy cells. These antigens are recognized and the antigen-presenting cell can be destroyed by cytotoxic T cells. Additionally, tumor antigens can also occur on the surface of the tumor in the form of, e.g., a mutated receptor. In this case, they can be recognized by antibodies.
  • Examples of tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex are shown in Tables 1 and 2 below. These tables illustrate specific (protein) antigens (i.e. “tumor antigens”) with respect to the cancer disease, they are associated with. According to the invention, the terms “cancer diseases” and “tumor diseases” are used synonymously herein.
  • TABLE 1
    Antigens expressed in cancer diseases
    Cancers or cancer diseases
    Tumor antigen Name of tumor antigen related thereto
    5T4 colorectal cancer, gastric cancer,
    ovarian cancer
    707-AP 707 alanine praline Melanoma
    9D7 renal cell carcinoma
    AFP alpha-fetoprotein hepatocellular carcinoma, gallbladder
    cancer, testicular cancer, ovarian
    cancer, bladder cancer
    AlbZIP HPG1 prostate cancer
    alpha5beta1-
    Integrin
    alpha5beta6- colon cancer
    Integrin
    alpha-methylacyl- prostate cancer
    coenzyme A
    racemase
    ART-4 adenocarcinoma antigen lung cancer, head and neck cancer,
    recognized by T cells 4 leukemia, esophageal cancer, gastric
    cancer, cervical cancer, ovarian
    cancer, breast cancer, squamous cell
    carcinoma
    B7H4 ovarian cancer
    BAGE-1 B antigen bladder cancer, head and neck
    cancer, lung cancer, melanoma,
    squamous cell carcinoma
    BCL-2 leukemia
    BING-4 melanoma
    CA 15-3/CA 27-29 breast cancer, ovary cancer, lung
    cancer, prostate cancer
    CA 19-9 gastric cancer, pancreatic cancer, liver
    cancer, breast cancer, gallbladder
    cancer, colon cancer, ovary cancer,
    lung cancer
    CA 72-4 ovarian cancer
    CA125 ovarian cancer, colorectal cancer,
    gastric cancer, liver cancer, pancreatic
    cancer, uterus cancer, cervix
    carcinoma, colon cancer, breast
    cancer, lung cancer
    calreticulin bladder cancer
    CAMEL CTL-recognized antigen on melanoma
    melanoma
    CASP-8 caspase-8 head and neck cancer
    cathepsin B breast cancer
    cathepsin L breast cancer
    CD19 B-cell malignancies
    CD20
    CD22
    CD25
    CD30
    CD33
    CD4
    CD52
    CD55
    CD56
    CD80
    CEA carcinoembryonic antigen gut carcinoma, colorectal cancer,
    colon cancer, hepatocellular cancer,
    lung cancer, breast cancer, thyroid
    cancer, pancreatic cancer, liver
    cancer cervix cancer, bladder cancer,
    melanoma
    CLCA2 calcium-activated chloride lung cancer
    channel-2
    CML28 leukemia
    Coactosin-like pancreatic cancer
    protein
    Collagen XXIII prostate cancer
    COX-2 ovarian cancer, breast cancer,
    colorectal cancer
    CT-9/BRD6 bromodomain testis-specific
    protein
    Cten C-terminal tensin-like protein prostate cancer
    cyclin B1
    cyclin D1 ovarian cancer
    cyp-B cyclophilin B bladder cancer, lung cancer, T-cell
    leukemia, squamous cell carcinoma,
    CYPB1 cytochrom P450 1B1 leukemia
    DAM-10/MAGE- differentiation antigen melanoma melanoma, skin tumors, ovarian
    B1 10 cancer, lung cancer
    DAM-6/MAGE- differentiation antigen melanoma 6 melanoma, skin tumors, ovarian
    B2 cancer, lung cancer
    EGFR/Her1 lung cancer, ovarian cancer, head
    and neck cancer, colon cancer,
    pancreatic cancer, breast cancer
    EMMPRIN tumor cell-associated extracellular lung cancer, breast cancer, bladder
    matrix metalloproteinase inducer/ cancer, ovarian cancer, brain cancer,
    lymphoma
    EpCam epithelial cell adhesion molecule ovarian cancer, breast cancer, colon
    cancer, lung cancer
    EphA2 ephrin type-A receptor 2 glioma
    EphA3 ephrin type-A receptor 2 melanoma, sarcoma, lung cancer
    ErbB3 breast cancer
    EZH2 (enhancer of Zeste homolog 2) endometrium cancer, melanoma,
    prostate cancer, breast cancer
    FGF-5 fibroblast growth factor-5 renal cell carcinoma, breast cancer,
    prostate cancer
    FN fibronectin melanoma
    Fra-1 Fos-related antigen-1 breast cancer, esophageal cancer,
    renal cell carcinoma, thyroid cancer
    G250/CAIX glycoprotein 250 leukemia, renal cell carcinoma, head
    and neck cancer, colon cancer,
    ovarian cancer, cervical cancer
    GAGE-1 G antigen 1 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-2 G antigen 2 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-3 G antigen 3 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-4 G antigen 4 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-5 G antigen 5 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-6 G antigen 6 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-7b G antigen 7b bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GAGE-8 G antigen 8 bladder cancer, lung cancer,
    sarcoma, melanoma, head and neck
    cancer
    GDEP gene differentially expressed in prostate cancer
    prostate
    GnT-V N-acetylglucosaminyltransferase V glioma, melanoma
    gp100 glycoprotein 100 kDa melanoma
    GPC3 glypican 3 hepatocellular carcinoma, melanoma
    HAGE helicase antigen bladder cancer
    HAST-2 human signet ring tumor-2
    hepsin prostate
    Her2/neu/ErbB2 human epidermal receptor- breast cancer, bladder cancer,
    2/neurological melanoma, ovarian cancer, pancreas
    cancer, gastric cancer
    HERV-K-MEL melanoma
    HNE human neutrophil elastase leukemia
    homeobox NKX prostate cancer
    3.1
    HOM-TES- ovarian cancer
    14/SCP-1
    HOM-TES-85
    HPV-E6 cervical cancer
    HPV-E7 cervical cancer
    HST-2 gastric cancer
    hTERT human telomerase reverse breast cancer, melanoma, lung
    transcriptase cancer, ovarian cancer, sarcoma,
    Non-Hodgkin-lymphoma, acute
    leukemia
    iCE intestinal carboxyl esterase renal cell carcinoma
    IGF-1R colorectal cancer
    IL-13Ra2 interleukin 13 receptor alpha 2 glioblastoma
    chain
    IL-2R colorectal cancer
    IL-5
    immature laminin renal cell carcinoma
    receptor
    kallikrein 2 prostate cancer
    kallikrein 4 prostate cancer
    Ki67 prostate cancer, breast cancer, Non-
    Hodgkin-lymphoma, melanoma
    KIAA0205 bladder cancer
    KK-LC-1 Kita-kyushu lung cancer antigen 1 lung cancer
    KM-HN-1 tongue cancer, hepatocellular
    carcinomas, melanoma, gastric
    cancer, esophageal, colon cancer,
    pancreatic cancer
    LAGE-1 L antigen bladder cancer, head and neck
    cancer, melanoma
    livin bladder cancer, melanoma
    MAGE-A1 melanoma antigen-A1 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A10 melanoma antigen-A10 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A12 melanoma antigen-A12 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia,
    prostate cancer, myeloma, brain
    tumors
    MAGE-A2 melanoma antigen-A2 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A3 melanoma antigen-A3 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A4 melanoma antigen-A4 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A6 melanoma antigen-A6 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-A9 melanoma-antigen-A9 bladder cancer, head and neck
    cancer, melanoma, colon cancer,
    lung cancer, sarcoma, leukemia
    MAGE-B1 melanoma-antigen-B1 melanoma
    MAGE-B10 melanoma-antigen-B10 melanoma
    MAGE-B16 melanoma-antigen-B16 melanoma
    MAGE-B17 melanoma-antigen-B17 melanoma
    MAGE-B2 melanoma-antigen-B2 melanoma
    MAGE-B3 melanoma-antigen-B3 melanoma
    MAGE-B4 melanoma-antigen-B4 melanoma
    MAGE-B5 melanoma-antigen-B5 melanoma
    MAGE-B6 melanoma-antigen-B6 melanoma
    MAGE-C1 melanoma-antigen-C1 bladder cancer, melanoma
    MAGE-C2 melanoma-antigen-C2 melanoma
    MAGE-C3 melanoma-antigen-C3 melanoma
    MAGE-D1 melanoma-antigen-D1 melanoma
    MAGE-D2 melanoma-antigen-D2 melanoma
    MAGE-D4 melanoma-antigen-D4 melanoma
    MAGE-E1 melanoma-antigen-E1 bladder cancer, melanoma
    MAGE-E2 melanoma-antigen-E2 melanoma
    MAGE-F1 melanoma-antigen-F1 melanoma
    MAGE-H1 melanoma-antigen-H1 melanoma
    MAGEL2 MAGE-like 2 melanoma
    mammaglobin A breast cancer
    MART-1/Melan-A melanoma antigen recognized by melanoma
    T cells-1/melanoma antigen A
    MART-2 melanoma antigen recognized by melanoma
    T cells-2
    matrix protein 22 bladder cancer
    MC1R melanocortin 1 receptor melanoma
    M-CSF macrophage colony-stimulating ovarian cancer
    factor gene
    mesothelin ovarian cancer
    MG50/PXDN breast cancer, glioblastoma,
    melanoma
    MMP 11 M-phase phosphoprotein 11 leukemia
    MN/CA IX- renal cell carcinoma
    antigen
    MRP-3 multidrug resistance-associated lung cancer
    protein 3
    MUC1 mucin 1 breast cancer
    MUC2 mucin 2 breast cancer, ovarian cancer,
    pancreatic cancer
    NA88-A NA cDNA clone of patient M88 melanoma
    N-acetylglucos-
    aminyltransferase-V
    Neo-PAP Neo-poly(A) polymerase
    NGEP prostate cancer
    NMP22 bladder cancer
    NPM/ALK nucleophosmin/anaplastic
    lymphoma kinase fusion protein
    NSE neuron-specific enolase small cell cancer of lung,
    neuroblastoma, Wilm' tumor,
    melanoma, thyroid cancer, kidney
    cancer, testicle cancer, pancreas
    cancer
    NY-ESO-1 New York esophageous 1 bladder cancer, head and neck
    cancer, melanoma, sarcoma, B-
    lymphoma, hepatoma, pancreatic
    cancer, ovarian cancer, breast cancer
    NY-ESO-B
    OA1 ocular albinism type 1 protein melanoma
    OFA-iLRP oncofetal antigen-immature leukemia
    laminin receptor
    OGT O-linked N-acetylglucosamine
    transferase gene
    OS-9
    osteocalcin prostate cancer
    osteopontin prostate cancer, breast cancer,
    ovarian cancer
    p15 protein 15
    p15 melanoma
    p190 minor bcr-
    abl
    p53
    PAGE-4 prostate GAGE-like protein-4 prostate cancer
    PAI-1 plasminogen acitvator inhibitor 1 breast cancer
    PAI-2 plasminogen acitvator inhibitor 2 breast cancer
    PAP prostate acic phosphatase prostate cancer
    PART-1 prostate cancer
    PATE prostate cancer
    PDEF prostate cancer
    Pim-1-Kinase
    Pin1 Propyl isomerase prostate cancer
    POTE prostate cancer
    PRAME preferentially expressed antigen of melanoma, lung cancer, leukemia,
    melanoma head and neck cancer, renal cell
    carcinoma, sarcoma
    prostein prostate cancer
    proteinase-3
    PSA prostate-specific antigen prostate cancer
    PSCA prostate cancer
    PSGR prostate cancer
    PSM
    PSMA prostate-specific membrane prostate cancer
    antigen
    RAGE-1 renal antigen bladder cancer, renal cancer,
    sarcoma, colon cancer
    RHAMM/CD168 receptor for hyaluronic acid leukemia
    mediated motility
    RU1 renal ubiquitous 1 bladder cancer, melanoma, renal
    cancer
    RU2 renal ubiquitous 1 bladder cancer, melanoma, sarcoma,
    brain tumor, esophagel cancer, renal
    cancer, colon cancer, breast cancer
    S-100 melanoma
    SAGE sarcoma antigen
    SART-1 squamous antigen rejecting tumor 1 esophageal cancer, head and neck
    cancer, lung cancer, uterine cancer
    SART-2 squamous antigen rejecting tumor 1 head and neck cancer, lung cancer,
    renal cell carcinoma, melanoma,
    brain tumor
    SART-3 squamous antigen rejecting tumor 1 head and neck cancer, lung cancer,
    leukemia, melanoma, esophageal
    cancer
    SCC squamous cell carcinoma antigen lung cancer
    Sp17 sperm protein 17 multiple myeloma
    SSX-1 synovial sarcoma X breakpoint 1 hepatocellular cell carcinom, breast
    cancer
    SSX-2/HOM- synovial sarcoma X breakpoint 2 breast cancer
    MEL-40
    SSX-4 synovial sarcoma X breakpoint 4 bladder cancer, hepatocellular cell
    carcinoma, breast cancer
    STAMP-1 prostate cancer
    STEAP six transmembrane epithelial prostate cancer
    antigen prostate
    survivin bladder cancer
    survivin-2B intron 2-retaining survivin bladder cancer
    TA-90 melanoma
    TAG-72 prostate carcinoma
    TARP prostate cancer
    TGFb TGFbeta
    TGFbRII TGFbeta receptor II
    TGM-4 prostate-specific transglutaminase prostate cancer
    TRAG-3 taxol resistant associated protein 3 breast cancer, leukemia, and
    melanoma
    TRG testin-related gene
    TRP-1 tyrosine related protein 1 melanoma
    TRP-2/6b TRP-2/novel exon 6b melanoma, glioblastoma
    TRP-2/INT2 TRP-2/intron 2 melanoma, glioblastoma
    Trp-p8 prostate cancer
    Tyrosinase melanoma
    UPA urokinase-type plasminogen breast cancer
    activator
    VEGF vascular endothelial growth factor
    VEGFR-2/FLK-1 vascular endothelial growth factor
    receptor-2
    WT1 Wilm' tumor gene gastric cancer, colon cancer, lung
    cancer, breast cancer, ovarian cancer,
    leukemia
  • TABLE 2
    Mutant antigens expressed in cancer diseases
    Cancers or cancer diseases related
    Mutant antigen Name of mutant antigen thereto
    alpha-actinin-4/m lung carcinoma
    ARTC1/m melanoma
    bcr/abl breakpoint cluster region-Abelson CML
    fusion protein
    beta-Catenin/m beta-Catenin melanoma
    BRCA1/m breast cancer
    BRCA2/m breast cancer
    CASP-5/m colorectal cancer, gastric cancer,
    endometrial carcinoma
    CASP-8/m head and neck cancer, squamous cell
    carcinoma
    CDC27/m cell-division-cycle 27
    CDK4/m cyclin-dependent kinase 4 melanoma
    CDKN2A/m melanoma
    CML66 CML
    COA-1/m colorectal cancer
    DEK-CAN fusion protein AML
    EFTUD2/m melanoma
    ELF2/m Elongation factor 2 lung squamous cell carcinoma
    ETV6-AML1 Ets variant gene6/acute myeloid ALL
    leukemia 1 gene fusion protein
    FN1/m fibronectin 1 melanoma
    GPNMB/m melanoma
    HLA-A*0201-R170I arginine to isoleucine exchange at renal cell carcinoma
    residue 170 of the alpha-helix of the
    alpha2-domain in the HLA-A2 gene
    HLA-A11/m melanoma
    HLA-A2/m renal cell carcinoma
    HSP70-2M heat shock protein 70-2 mutated renal cell carcinoma, melanoma,
    neuroblastoma
    KIAA0205/m bladder tumor
    K-Ras/m pancreatic carcinoma, colorectal
    carcinoma
    LDLR-FUT LDR-Fucosyltransferase fusion melanoma
    protein
    MART2/m melanoma
    ME1/m non-small cell lung carcinoma
    MUM-1/m melanoma ubiquitous mutated 1 melanoma
    MUM-2/m melanoma ubiquitous mutated 2 melanoma
    MUM-3/m melanoma ubiquitous mutated 3 melanoma
    Myosin class I/m melanoma
    neo-PAP/m melanoma
    NFYC/m lung squamous cell carcinoma
    N-Ras/m melanoma
    OGT/m colorectal carcinoma
    OS-9/m melanoma
    p53/m
    Pml/RARa promyelocytic leukemia/retinoic acid APL, PML
    receptor alpha
    PRDX5/m melanoma
    PTPRK/m receptor-type protein-tyrosine melanoma
    phosphatase kappa
    RBAF600/m melanoma
    SIRT2/m melanoma
    SYT-SSX-1 synaptotagmin I/synovial sarcoma X sarcoma
    fusion protein
    SYT-SSX-2 synaptotagmin I/synovial sarcoma X sarcoma
    fusion protein
    TEL-AML1 translocation Ets-family AML
    leukemia/acute myeloid leukemia 1
    fusion protein
    TGFbRII TGFbeta receptor II colorectal carcinoma
    TPI/m triosephosphate isomerase Melanoma
  • In a preferred embodiment according to the present invention, the tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) are selected from the group consisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinin-4/m, alpha-methylacyl-coenzyme A racemase, ART-4, ARTC1/m, B7H4, SAGE-1, BCL-2, bcr/abl, beta-catenin/m, BING-4, BRCA1/m, BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL, CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2, CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2, CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, F7H2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R17I, HLA-A11/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67, KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A, MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m, mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2, MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A, N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP, NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, 0A1, OFA-iLRP, OGT, OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-Kinase, Pin-1, Pml/PARalpha, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Spl7, SSX-1, SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFbeta, TGFbetaRII, TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8, tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1.
  • In a particularly preferred embodiment, the tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex are selected from the group consisting of MAGE-A1 (e.g. MAGE-A1 according to accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (e.g. MAGE-A6 according to accession number NM005363), MAGE-C1, MAGE-C2, melan-A (e.g. melan-A according to accession number NM005511), GP100 (e.g. GP100 according to accession number M77348), tyrosinase (e.g. tyrosinase according to accession number NM000372), surviving (e.g. survivin according to accession number AF077350), CEA (e.g. CEA according to accession number NM004363), Her-2/neu (e.g. Her-2/neu according to accession number M11730), WT1 (e.g. WT1 according to accession number NM000378), PRAME (e.g. PRAME according to accession number NM006115), EGFRI (epidermal growth factor receptor 1) (e.g. EGFR1 (epidermal growth factor receptor 1) according to accession number AF288738), MUC1, mucin-1 (e.g. mucin-1 according to accession number NM002456), SEC61G (e.g. SEC61G according to accession number NM014302), hTERT (e.g. hTERT accession number NM198253), 5T4 (e.g. 5T4 according to accession number NM006670), NY-Eso-1 (e.g. NY-Eso1 according to accession number NM001327), TRP-2 (e.g. TRP-2 according to accession number NM001922), STEAP, PCA, PSA, PSMA, etc.
  • According to a further particularly preferred embodiment, the tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex may form a cocktail of antigens, e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of prostate cancer (PCa), preferably of neoadjuvant and/or hormone-refractory prostate cancers, and diseases or disorders related thereto. For this purpose, the nucleic acid of the inventive polymeric carrier cargo complex is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three or even four (preferably different) antigens of the following group of antigens:
      • PSA (Prostate-Specific Antigen)=KLK3 (Kallikrein-3),
      • PSMA (Prostate-Specific Membrane Antigen),
      • PSCA (Prostate Stem Cell Antigen),
      • STEAP (Six Transmembrane Epithelial Antigen of the Prostate).
  • According to another particularly preferred embodiment, the tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may form a cocktail of antigens, e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of non-small cell lung cancers (NSCLC), preferably selected from the three main sub-types squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma, or of disorders related thereto. For this purpose, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) is preferably at least one RNA, more preferably at least one mRNA, which may encode at least one, preferably two, three, four, five, six, seven, eight, nine, ten eleven or twelve (preferably different) antigens of the following group of antigens:
      • hTERT,
      • WT1,
      • MAGE-A2,
      • 5T4,
      • MAGE-A3,
      • MUC1,
      • Her-2/neu,
      • NY-ESO-1,
      • CEA,
      • Survivin,
      • MAGE-C1, and/or
      • MAGE-C2,
        wherein any combination of these antigens is possible.
  • According to a further particularly preferred embodiment, the tumor antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex may form a cocktail of antigens, e.g. in an active (immunostimulatory) composition or a kit of parts (wherein preferably each antigen is contained in one part of the kit), preferably for eliciting an (adaptive) immune response for the treatment of non-small cell lung cancers (NSCLC), preferably selected from the three main sub-types squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma, or of disorders related thereto. For this purpose, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) is preferably at least one RNA, more preferably at least one mRNA, which may encode at least two (preferably different) antigens,
    • a) wherein at least one, preferably at least two, three, four, five or even six, of these at least two antigens is (are) selected from:
      • 5T4
      • NY-ESO-1,
      • MAGE-A2,
      • MAGE-A3,
      • MAGE-C1, and/or
      • MAGE-C2, and
    • b) wherein the further antigen(s) is (are) selected from at least one antigen as defined herein, preferably in any of the herein mentioned combinations, groups or subgroups of antigens, e.g. the further antigen(s) is (are) selected from, e.g.:
      • hTERT,
      • WT1,
      • MAGE-A2,
      • 5T4,
      • MAGE-A3,
      • MUC1,
      • Her-2/neu,
      • NY-ESO-1,
      • CEA,
      • Survivin,
      • MAGE-C1, and/or
      • MAGE-C2.
  • In the above embodiments, each of the above defined proteins, e.g. therapeutically active proteins, antibodies, antigens, etc., as defined herein may be encoded by one (monocistronic) RNA, preferably one (monocistronic) mRNA. In other words, the nucleic acid of the inventive polymeric carrier cargo complex may comprise at least two (monocistronic) RNAs, preferably mRNAs, wherein each of these at least two (monocistronic) RNAs, preferably mRNAs, may encode, e.g. just one (preferably different) protein, e.g. an antigen, preferably selected from one of the above mentioned antigen combinations.
  • According to another particularly preferred embodiment, the nucleic acid of the inventive polymeric carrier cargo complex may comprise (at least) one bi-or even multicistronic RNA, preferably mRNA, i.e. (at least) one RNA which carries, e.g. two or even more of the coding sequences of at least two (preferably different) proteins, e.g. antigens, preferably selected from one of the above mentioned antigen combinations. Such coding sequences, e.g. of the at least two (preferably different) proteins, e.g. antigens, of the (at least) one bi-or even multicistronic RNA may be separated by at least one IRES (internal ribosomal entry site) sequence, as defined below. Thus, the term “encoding at least two (preferably different) proteins” may mean, without being limited thereto, that the (at least) one (bi-or even multicistronic) RNA, preferably an mRNA, may encode e.g. at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve or more (preferably different) proteins, e.g. antigens of the above mentioned group(s) of antigens, or their fragments or variants, therapeutically active proteins, antibodies, adjuvant proteins, etc. More preferably, without being limited thereto, the (at least) one (bi-or even multicistronic) RNA, preferably mRNA, may encode e.g. at least two, three, four, five or six or more (preferably different) proteins, e.g. antigens of the above mentioned subgroup(s) of antigens, or their fragments or variants within the above definitions. In this context, a so-called IRES (internal ribosomal entry site) sequence as defined herein can function as a sole ribosome binding site, but it can also serve to provide a bi-or even multicistronic RNA as defined herein which codes for several proteins, which are to be translated by the ribosomes independently of one another. Examples of IRES sequences which can be used according to the invention are those from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV).
  • According to a further particularly preferred embodiment, the nucleic acid of the inventive polymeric carrier cargo complex may comprise a mixture of at least one monocistronic RNA, preferably mRNA, as defined herein, and at least one bi-or even multicistronic RNA, preferably mRNA, as defined herein. The at least one monocistronic RNA and/or the at least one bi-or even multicistronic RNA preferably encode different proteins, e.g. antigens, or their fragments or variants, the antigens preferably being selected from one of the above mentioned groups or subgroups of antigens, more preferably in one of the above mentioned combinations. However, the at least one monocistronic RNA and the at least one bi-or even multicistronic RNA may preferably also encode (in part) identical proteins as defined herein, e.g. antigens selected from one of the above mentioned groups or subgroups of antigens, preferably in one of the above mentioned combinations, provided that the nucleic acid of the present invention as a whole provides at least two (preferably different) proteins, e.g. antigens, as defined herein. Such an embodiment may be advantageous e.g. for a staggered, e.g. time dependent, administration of one or several complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein), e.g. as a pharmaceutical composition, to a patient in need thereof. The components of such a pharmaceutical composition of the present invention, particularly the different complexed RNAs encoding the at least two (preferably different) proteins, may be e.g. contained in (different parts of) a kit of parts composition or may be e.g. administered separately as components of different pharmaceutical compositions according to the present invention.
  • According to another embodiment, one further class of antigens as encoded by the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) comprises allergy antigens. Such allergy antigens may be selected from antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergy antigens typically belong to different classes of compounds, such as nucleic acids and their fragments, proteins or peptides and their fragments, carbohydrates, polysaccharides, sugars, lipids, phospholipids, etc. Of particular interest in the context of the present invention are antigens, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, i.e. protein or peptide antigens and their fragments or epitopes, or nucleic acids and their fragments, particularly nucleic acids and their fragments, encoding such protein or peptide antigens and their fragments or epitopes.
  • Particularly preferred, antigens derived from animals, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may include antigens derived from, without being limited thereto, insects, such as mite (e.g. house dust mites), mosquito, bee (e.g. honey bee, bumble bee), cockroache, tick, moth (e.g. silk moth), midge, bug, flea, wasp, caterpillar, fruit fly, migratory locust, grasshopper, ant aphide, from crustaceans, such as shrimps, crab, krill, lobster, prawn, crawfish, scampi, from birds, such as duck, goose, seagull, turkey, ostrich, chicken, from fishes, such as eel, herring, carp, seabream, codfish, halibut, catfish, beluga, salmon, flounder, mackerel, cuttlefish, perch, form molluscs, such as scallop, octopus, abalone, snail, whelk, squid, clam, mussel, from spiders, from mammals, such as cow, rabbit, sheep, lion, jaguar, leopard, rat, pig, buffalo, dog, loris, hamster, guinea pig, fallow deer, horse, cat, mouse, ocelot, serval, from arthropod, such as spider, or silverfish, from worms, such as nematodes, from trichinella species, or roundworm, from amphibians, such as frogs, or from sea squirt, etc.
  • Antigens derived from plants, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex may include antigens derived from, without being limited thereto, fruits, such as kiwi, pineapple, jackfruit, papaya, lemon, orange, mandarin, melon, sharon fruit, strawberry, lychee, apple, cherry paradise apple, mango, passion fruit, plum, apricot, nectarine, pear, passion fruit, raspberry, grape, from vegetables, such as garlic, onion, leek, soya bean, celery, cauliflower, turnip, paprika, chickpea, fennel, zucchini, cucumber, carrot, yam, bean, pea, olive, tomato, potato, lentil, lettuce, avocado, parsley, horseradish, chirimoya, beet, pumkin, spinach, from spices, such as mustard, coriander, saffron, pepper, aniseed, from crop, such as oat, buckwheat, barley, rice, wheat, maize, rapeseed, sesame, from nuts, such as cashew, walnut, butternut, pistachio, almond, hazelnut, peanut, brazil nut, pecan, chestnut, from trees, such as alder, hornbeam, cedar, birch, hazel, beech, ash, privet, oak, plane tree, cypress, palm, from flowers, such as ragweed, carnation, forsythia, sunflower, lupine, chamomile, lilac, passion flower, from grasses, such as quack grass, common bent, brome grass, Bermuda grass, sweet vernal grass, rye grass, or from other plants, such as opium poppy, pellitory, ribwort, tobacco, asparagus, mugwort, cress, etc.
  • Antigens derived from fungi, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may include antigens derived from, without being limited thereto, e.g. Altemia sp., Aspergillus sp., Beauveria sp., Candida sp., Cladosporium sp., Endothia sp., Curcularia sp., Embellisia sp., Epicoccum sp., Fusarium sp., Malassezia sp., Penicillum sp., Pleospora sp., Saccharomyces sp., etc.
  • Antigens derived from bacteria, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may include antigens derived from, without being limited thereto, e.g. Bacillus tetani, Staphylococcus aureus, Streptomyces griseus, etc.
  • c) Antibodies
  • According to a further alternative, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may encode an antibody. According to the present invention, such an antibody may be selected from any antibody, e.g. any recombinantly produced or naturally occurring antibodies, known in the art, in particular antibodies suitable for therapeutic, diagnostic or scientific purposes, or antibodies which have been identified in relation to specific cancer diseases. Herein, the term “antibody” is used in its broadest sense and specifically covers monoclonal and polyclonal antibodies (including agonist, antagonist, and blocking or neutralizing antibodies) and antibody species with polyepitopic specificity. According to the invention, “antibody” typically comprises any antibody known in the art (e.g. IgM, IgD, IgG, IgA and IgE antibodies), such as naturally occurring antibodies, antibodies generated by immunization in a host organism, antibodies which were isolated and identified from naturally occurring antibodies or antibodies generated by immunization in a host organism and recombinantly produced by biomolecular methods known in the art, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e. antibodies expressed in cells and optionally localized in specific cell compartments, and fragments and variants of the aforementioned antibodies. In general, an antibody consists of a light chain and a heavy chain both having variable and constant domains. The light chain consists of an N-terminal variable domain, VL, and a C-terminal constant domain, CL. In contrast, the heavy chain of the IgG antibody, for example, is comprised of an N-terminal variable domain, VH, and three constant domains, CH1, CH2 and CH3. Single chain antibodies may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex as well.
  • According to a first alternative, the nucleic acid of the inventive polymeric carrier cargo complex may encode a polyclonal antibody. In this context, the term, “polyclonal antibody” typically means mixtures of antibodies directed to specific antigens or immunogens or epitopes of a protein which were generated by immunization of a host organism, such as a mammal, e.g. including goat, cattle, swine, dog, cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit. Polyclonal antibodies are generally not identical, and thus usually recognize different epitopes or regions from the same antigen. Thus, in such a case, typically a mixture (a composition) of different nucleic acids of the inventive polymeric carrier cargo complex will be used, each nucleic acid encoding a specific (monoclonal) antibody being directed to specific antigens or immunogens or epitopes of a protein.
  • According to a further alternative, the nucleic acid of the inventive polymeric carrier cargo complex may encode a monoclonal antibody. The term “monoclonal antibody” herein typically refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed to a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. For example, monoclonal antibodies as defined herein may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods, e.g. as described in U.S. Pat. No. 4,816,567. “Monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990), for example. According to Kohler and Milstein, an immunogen (antigen) of interest is injected into a host such as a mouse and B-cell lymphocytes produced in response to the immunogen are harvested after a period of time. The B-cells are combined with myeloma cells obtained from mouse and introduced into a medium which permits the B-cells to fuse with the myeloma cells, producing hybridomas. These fused cells (hybridomas) are then placed into separate wells of microliter plates and grown to produce monoclonal antibodies. The monoclonal antibodies are tested to determine which of them are suitable for detecting the antigen of interest. After being selected, the monoclonal antibodies can be grown in cell cultures or by injecting the hybridomas into mice. However, for the purposes of the present invention, the peptide sequences of these monoclonal antibodies have to be sequenced and the nucleic acid sequences encoding these antibodies can be present as the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein).
  • For therapeutical purposes in humans, non-human monoclonal or polyclonal antibodies, such as murine antibodies may also be encoded by the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein). However, such antibodies are typically only of limited use, since they generally induce an immune response by production of human antibodies directed to the said non-human antibodies, in the human body. Therefore, a particular non-human antibody can only be administered once to the human. To solve this problem, chimeric, humanized non-human and human antibodies are also envisaged encoded by the nucleic acid of the inventive polymeric carrier cargo complex. “Chimeric” antibodies, which may be encoded by the nucleic acid according to the present invention, are preferably antibodies in which the constant domains of an antibody described above are replaced by sequences of antibodies from other organisms, preferably human sequences. “Humanized” (non-human) antibodies, which may be also encoded by nucleic acid according to the present invention, are antibodies in which the constant and variable domains (except for the hypervariable domains) described above of an antibody are replaced by human sequences. According to another alternative, the nucleic acid according to the present invention may encode human antibodies, i.e. antibodies having only human sequences. Such human antibodies can be isolated from human tissues or from immunized non-human host organisms which are transgene for the human IgG gene locus, and nucleic acid sequences may be prepared according to procedures well known in the art. Additionally, human antibodies can be provided by the use of a phage display.
  • In addition, the nucleic acid of the inventive polymeric carrier cargo complex may encode bispecific antibodies. “Bispecific” antibodies in context of the invention are preferably antibodies which act as an adaptor between an effector and a respective target by two different Fa/b-domains, e.g. for the purposes of recruiting effector molecules such as toxins, drugs, cytokines etc., targeting effector cells such as CTL, NK cells, makrophages, granulocytes, etc. (see for review: Kontermann R. E., Acta Pharmacol. Sin, 2005, 26(1): 1-9). Bispecific antibodies as defined herein are, in general, configured to recognize by two different Fa/b-domains, e.g. two different antigens, immunogens, epitopes, drugs, cells (or receptors on cells), or other molecules (or structures) as defined herein. Bispecificity means herewith that the antigen-binding regions of the antibodies are specific for two different epitopes. Thus, different antigens, immunogens or epitopes, etc. can be brought close together, what, optionally, allows a direct interaction of the two components. For example, different cells such as effector cells and target cells can be connected via a bispecific antibody. Encompassed, but not limited, by the present invention are antibodies or fragments thereof which bind, on the one hand, a soluble antigen as defined herein, and, on the other hand, an antigen or receptor on the surface of a tumor cell.
  • According to the invention, the nucleic acid of the inventive polymeric carrier cargo complex may also encode intrabodies, wherein these intrabodies may be antibodies as defined herein. Since these antibodies are intracellular expressed antibodies, i.e. antibodies which may be encoded by nucleic acids localized in specific areas of the cell and also expressed there, such antibodies may be termed intrabodies.
  • Antibodies as encoded by the nucleic acid of the inventive polymeric carrier cargo complex may preferably comprise full-length antibodies, i.e. antibodies composed of the full heavy and full light chains, as defined herein. However, derivatives of antibodies such as antibody fragments, variants or adducts may also be encoded by the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I).
  • The nucleic acid of the inventive polymeric carrier cargo complex may also encode antibody fragments selected from Fab, Fab′, F(ab′)2, Fc, Facb, pFc′, Fd and Fv fragments of the aforementioned (full-length) antibodies. In general, antibody fragments are known in the art. For example, a Fab (“fragment, antigen binding”) fragment is composed of one constant and one variable domain of each of the heavy and the light chain. The two variable domains bind the epitope on specific antigens. The two chains are connected via a disulfide linkage. A scFv (“single chain variable fragment”) fragment, for example, typically consists of the variable domains of the light and heavy chains. The domains are linked by an artificial linkage, in general a polypeptide linkage such as a peptide composed of 15-25 glycine, proline and/or serine residues.
  • According to a further alternative, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may be in the form of dsRNA, preferably siRNA. A dsRNA, or a siRNA, is of interest particularly in connection with the phenomenon of RNA interference. The in vitro technique of RNA interference (RNAi) is based on double-stranded RNA molecules (dsRNA), which trigger the sequence-specific suppression of gene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). In the transfection of mammalian cells with long dsRNA, the activation of protein kinase R and RnaseL brings about unspecific effects, such as, for example, an interferon response (Stark et al. (1998) Annu. Rev. Biochem. 67: 227-264; He and Katze (2002) Viral Immunol. 15: 95-119). These unspecific effects are avoided when shorter, for example 21-to 23-mer, so-called siRNA (small interfering RNA), is used, because unspecific effects are not triggered by siRNA that is shorter than 30 bp (Elbashir et al. (2001) Nature 411: 494-498).
  • The nucleic acid of the inventive polymeric carrier cargo complex may thus be a double-stranded RNA (dsRNA) having a length of from 17 to 29, preferably from 19 to 25, and preferably being at least 90%, more preferably 95% and especially 100% (of the nucleotides of a dsRNA) complementary to a section of the nucleic acid sequence of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore, either a coding or a non-coding section, preferably a coding section. 90% complementary means that with a length of a dsRNA described herein of, for example, 20 nucleotides, this contains not more than 2 nucleotides without corresponding complementarity with the corresponding section of the mRNA. The sequence of the double-stranded RNA used according to the invention as the nucleic acid of the inventive polymeric carrier cargo complex is, however, preferably wholly complementary in its general structure with a section of the nucleic acid of a therapeutically relevant protein or antigen described hereinbefore. In this context the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) may be a dsRNA having the general structure 5-(N17-29)-3′, preferably having the general structure 5-(N19-25)-3′, more preferably having the general structure 5′-(N19-24)-3′, or yet more preferably having the general structure 5′-(N21-23)-3′, wherein for each general structure each N is a (preferably different) nucleotide of a section of the mRNA of a therapeutically relevant protein or antigen described hereinbefore, preferably being selected from a continuous number of 17 to 29 nucleotides of the mRNA of a therapeutically relevant protein or antigen and being present in the general structure 5′-(N17-29)-3′ in their natural order. In principle, all the sections having a length of from 17 to 29, preferably from 19 to 25, base pairs that occur in the coding region of the mRNA can serve as target sequence for a dsRNA herein. Equally, dsRNAs used as nucleic acid of the inventive polymeric carrier cargo complex can also be directed against nucleotide sequences of a (therapeutically relevant) protein or antigen described (as active ingredient) hereinbefore that do not lie in the coding region, in particular in the 5′ non-coding region of the mRNA, for example, therefore, against non-coding regions of the mRNA having a regulatory function. The target sequence of the dsRNA used as nucleic acid of the inventive polymeric carrier cargo complex can therefore lie in the translated and untranslated region of the mRNA and/or in the region of the control elements of a protein or antigen described hereinbefore. The target sequence of a dsRNA used as nucleic acid of the inventive polymeric carrier cargo complex can also lie in the overlapping region of untranslated and translated sequence; in particular, the target sequence can comprise at least one nucleotide upstream of the start triplet of the coding region of the mRNA.
  • According to another alternative, the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) (or according to any of its subformulas herein) may be in the form of a CpG nucleic acid, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA used according to the invention can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid of the inventive polymeric carrier cargo complex is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). Also preferably, such CpG nucleic acids have a length as defined herein.
  • The nucleic acid of the inventive polymeric carrier cargo complex as defined herein may also be in the form of a modified nucleic acid, wherein any modification, as defined herein, may be introduced into the nucleic acid. Modifications as defined herein preferably lead to a further stabilized nucleic acid of the present invention.
  • According to a first embodiment, the nucleic acid of the inventive polymeric carrier cargo complex as defined herein may thus be provided as a “stabilized nucleic acid”, preferably as a stabilized mRNA, more preferably as an mRNA that is essentially resistant to in vivo degradation (e.g. by an exo-or endo-nuclease). Such stabilization can be effected, for example, by a modified phosphate backbone of the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I). A backbone modification in connection with the present invention is a modification in which phosphates of the backbone of the nucleotides contained in the nucleic acid are chemically modified. Nucleotides that may be preferably used in this connection contain e.g. a phosphorothioate-modified phosphate backbone, preferably at least one of the phosphate oxygens contained in the phosphate backbone being replaced by a sulfur atom. Stabilized nucleic acids may further include, for example: non-ionic phosphate analogues, such as, for example, alkyl and aryl phosphonates, in which the charged phosphonate oxygen is replaced by an alkyl or aryl group, or phosphodiesters and alkylphosphotriesters, in which the charged oxygen residue is present in alkylated form. Such backbone modifications typically include, without implying any limitation, modifications from the group consisting of methylphosphonates, phosphoramidates and phosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).
  • The nucleic acid of the inventive polymeric carrier cargo complex may additionally or alternatively also contain sugar modifications. A sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid and typically includes, without implying any limitation, sugar modifications selected from the group consisting of 2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate), 2′-deoxy-2′-deamine oligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate, 2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyl oligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide (2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5-triphosphate), 2′-C-alkyl oligoribonucleotide, and isomers thereof (2′-aracytidine-5′-triphosphate, 2′-arauridine-5-triphosphate), or azidotriphosphate (2′-azido-2′-deoxycytidine-5-triphosphate, 2′-azido-2′-deoxyuridine-5′-triphosphate).
  • The nucleic acid of the inventive polymeric carrier cargo complex may additionally or alternatively also contain at least one base modification, which is preferably suitable for increasing the expression of the protein coded for by the nucleic acid as compared with the unaltered, i.e. natural (=native), nucleic acid sequence. Significant in this case means an increase in the expression of the protein compared with the expression of the native nucleic acid sequence by at least 20%, preferably at least 30%, 40%, 50% or 60%, more preferably by at least 70%, 80%, 90% or even 100% and most preferably by at least 150%, 200% or even 300% or more. In connection with the present invention, a nucleotide having such a base modification is preferably selected from the group of the base-modified nucleotides consisting of 2-amino-6-chloropurineriboside-5′-triphosphate, 2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate, 2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate, 5-aminoallylcytidine-5′-triphosphate, 5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5-triphosphate, 5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate, 5-methyluridine-5-triphosphate, 6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate, 6-chloropurineriboside-5-triphosphate, 7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate, benzimidazole-riboside-5′-triphosphate, N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate, N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate, pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate, xanthosine-5′-triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate, 5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.
  • According to another embodiment, the nucleic acid of the inventive polymeric carrier cargo complex can likewise be modified (and preferably stabilized) by introducing further modified nucleotides containing modifications of their ribose or base moieties. Generally, the nucleic acid of the inventive polymeric carrier cargo complex may contain any native (=naturally occurring) nucleotide, e.g. guanosine, uracil, adenosine, and/or cytosine or an analogue thereof. In this connection, nucleotide analogues are defined as non-natively occurring variants of naturally occurring nucleotides. Accordingly, analogues are chemically derivatized nucleotides with non-natively occurring functional groups, which are preferably added to or deleted from the naturally occurring nucleotide or which substitute the naturally occurring functional groups of a nucleotide. Accordingly, each component of the naturally occurring nucleotide may be modified, namely the base component, the sugar (ribose) component and/or the phosphate component forming the backbone (see above) of the nuecleic acid sequence. Exemplary analogues of guanosine, uracil, adenosine, and cytosine include, without implying any limitation, any naturally occurring or non-naturally occurring guanosine, uracil, adenosine, thymidine or cytosine that has been altered chemically, for example by acetylation, methylation, hydroxylation, etc., including 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6-diaminopurine, 2′-Amino-2′-deoxyadenosine, 2′-Amino-2′-deoxycytidine, 2′-Amino-2′-deoxyguanosine, 2′-Amino-2′-deoxyuridine, 2-Amino-6-chloropurineriboside, 2-Aminopurine-riboside, 2′-Araadenosine, 2′-Aracytidine, 2′-Arauridine, 2′-Azido-2′-deoxyadenosine, 2′-Azido-2′-deoxycytidine, 2′-Azido-2′-deoxyguanosine, 2′-Azido-2′-deoxyuridine, 2-Chloroadenosine, 2′-Fluoro-2′-deoxyadenosine, 2′-Fluoro-2′-deoxycytidine, 2′-Fluoro-2′-deoxyguanosine, 2′-Fluoro-2′-deoxyuridine, 2′-Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-Methyl-2-aminoadenosine, 2′-O-Methyl-2′-deoxyadenosine, 2′-O-Methyl-2′-deoxycytidine, 2′-O-Methyl-2′-deoxyguanosine, 2′-O-Methyl-2′-deoxyuridine, 2′-O-Methyl-5-methyluridine, 2′-O-Methylinosine, 2′-O -Methylpseudouridine, 2-Thiocytidine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 4-Thiouridine, 5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine, 5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine, 5-carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine, 5-Fluoro-uridine, 5-Iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-Azauridine, 6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside, 6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside, 7-Deaza-2′-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine, 8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo-guanosine, 8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside, Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine, N1-Methyladenosine, N6-([6-Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acid methyl ester, Puromycin, Queosine, Uracil-5-oxyacetic acid, Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, and Xylo-adenosine. The preparation of such analogues is known to a person skilled in the art, for example from U.S. Pat. Nos. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642. In the case of an analogue as defined herein, particular preference may be given according to the invention to those analogues that do not interfere with a further modification of the nucleic acid that has been introduced.
  • The nucleic acid of the inventive polymeric carrier cargo complex may likewise be stabilized in order to prevent degradation of the nucleic acid in vivo by various approaches, particularly, when RNA or mRNA is used as a nucleic acid. It is known in the art that instability and (fast) degradation of mRNA or of RNA in vivo in general may represent a serious problem in the application of RNA based compositions. This instability of RNA is typically due to RNA-degrading enzymes, “RNAases” (ribonucleases), wherein contamination with such ribonucleases may sometimes completely degrade RNA in solution. Accordingly, the natural degradation of mRNA in the cytoplasm of cells is very finely regulated and RNase contaminations may be generally removed by special treatment prior to use of said compositions, in particular with diethyl pyrocarbonate (DSPC). A number of mechanisms of natural degradation are known in this connection in the prior art, which may be utilized as well. E.g., the terminal structure is typically of critical importance for an mRNA in vivo. As an example, at the 5′ end of naturally occurring mRNAs there is usually a so-called “cap structure” (a modified guanosine nucleotide), and at the 3′ end is typically a sequence of up to 200 adenosine nucleotides (the so-called poly-A tail).
  • The nucleic acid of the inventive polymeric carrier cargo complex, particularly if provided as an mRNA, can therefore be stabilized against degradation by RNases by the addition of a so-called “5′ cap” structure. Particular preference is given in this connection to an m7G(5′)ppp (5′(A,G(5′)ppp(5′)A or G(5′)ppp(5′)G as the 5′ cap″ structure. However, such a modification is introduced only if a modification, for example a lipid modification, has not already been introduced at the 5′ end of the nucleic acid of the present invention if provided as an mRNA or if the modification does not interfere with the immunogenic properties of the (unmodified or chemically modified) nucleic acid of the present invention.
  • According to a further preferred embodiment, the nucleic acid of the inventive polymeric carrier cargo complex may contain, especially if the nucleic acid is in the form of an mRNA, a poly-A tail on the 3′ terminus of typically about 10 to 200 adenosine nucleotides, preferably about 10 to 100 adenosine nucleotides, more preferably about 20 to 100 adenosine nucleotides or even more preferably about 40 to 80 adenosine nucleotides.
  • According to a further preferred embodiment, the nucleic acid of the inventive polymeric carrier cargo complex may contain, especially if the nucleic acid is in the form of an mRNA, a poly-C tail on the 3′ terminus of typically about 10 to 200 cytosine nucleotides, preferably about 10 to 100 cytosine nucleotides, more preferably about 20 to 70 cytosine nucleotides or even more preferably about 20 to 60 or even 10 to 40 cytosine nucleotides.
  • According to another embodiment, the nucleic acid of the inventive polymeric carrier cargo complex may be modified, and thus stabilized, especially if the nucleic acid is in the form of an mRNA, by modifying the G/C content of the nucleic acid, particularly an mRNA, preferably of the coding region thereof.
  • In a particularly preferred embodiment of the present invention, the G/C content of the coding region of the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is modified, particularly increased, compared to the G/C content of the coding region of its particular wild-type mRNA, i.e. the unmodified mRNA. The encoded amino acid sequence of the at least one mRNA is preferably not modified compared to the coded amino acid sequence of the particular wild-type mRNA.
  • This modification of the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA. Thus, the composition and the sequence of various nucleotides is important. In particular, sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content. According to the invention, the codons of the mRNA are therefore varied compared to its wild-type mRNA, while retaining the translated amino acid sequence, such that they include an increased amount of G/C nucleotides. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage).
  • Depending on the amino acid to be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, there are various possibilities for modification of the at least one mRNA sequence, compared to its wild-type sequence. In the case of amino acids which are encoded by codons which contain exclusively G or C nucleotides, no modification of the codon is necessary. Thus, the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U is present.
  • In contrast, codons which contain A and/or U nucleotides can be modified by substitution of other codons which code for the same amino acids but contain no A and/or U. Examples of these are:
  • the codons for Pro can be modified from CCU or CCA to CCC or CCG;
  • the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGC or CGG;
  • the codons for Ala can be modified from GCU or GCA to GCC or GCG;
  • the codons for Gly can be modified from GGU or GGA to GGC or GGG.
  • In other cases, although A or U nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and U content by using codons which contain a lower content of A and/or U nucleotides. Examples of these are:
  • the codons for Phe can be modified from UUU to UUC;
  • the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC or CUG;
  • the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG or AGC;
  • the codon for Tyr can be modified from UAU to UAC;
  • the codon for Cys can be modified from UGU to UGC;
  • the codon for His can be modified from CAU to CAC;
  • the codon for Gln can be modified from CAA to CAG;
  • the codons for Ile can be modified from AUU or AUA to AUC;
  • the codons for Thr can be modified from ACU or ACA to ACC or ACG;
  • the codon for Asn can be modified from MU to AAC;
  • the codon for Lys can be modified from MA to MG;
  • the codons for Val can be modified from GUU or GUA to GUC or GUG;
  • the codon for Asp can be modified from GAU to GAC;
  • the codon for Glu can be modified from GM to GAG;
  • the stop codon UAA can be modified to UAG or UGA.
  • In the case of the codons for Met (AUG) and Trp (UGG), on the other hand, there is no possibility of sequence modification.
  • The substitutions listed above can be used either individually or in all possible combinations to increase the G/C content of the nucleic acid of the present invention, especially if the nucleic acid of the inventive polymeric carrier cargo complex is in the form of an mRNA, compared to its particular wild-type mRNA (i.e. the original sequence). Thus, for example, all codons for Thr occurring in the wild-type sequence can be modified to ACC (or ACG).
  • Preferably, however, for example, combinations of the above substitution possibilities are used:
  • substitution of all codons coding for Thr in the original sequence (wild-type mRNA) to ACC (or ACG) and
  • substitution of all codons originally coding for Ser to UCC (or UCG or AGC);
  • substitution of all codons coding for Ile in the original sequence to AUC and
  • substitution of all codons originally coding for Lys to MG and
  • substitution of all codons originally coding for Tyr to UAC;
  • substitution of all codons coding for Val in the original sequence to GUC (or GUG) and
  • substitution of all codons originally coding for Glu to GAG and
  • substitution of all codons originally coding for Ala to GCC (or GCG) and
  • substitution of all codons originally coding for Arg to CGC (or CGG);
  • substitution of all codons coding for Val in the original sequence to GUC (or GUG) and
  • substitution of all codons originally coding for Glu to GAG and
  • substitution of all codons originally coding for Ala to GCC (or GCG) and
  • substitution of all codons originally coding for Gly to GGC (or GGG) and
  • substitution of all codons originally coding for Asn to AAC;
  • substitution of all codons coding for Val in the original sequence to GUC (or GUG) and
  • substitution of all codons originally coding for Phe to UUC and
  • substitution of all codons originally coding for Cys to UGC and
  • substitution of all codons originally coding for Leu to CUG (or CUC) and
  • substitution of all codons originally coding for Gln to CAG and
  • substitution of all codons originally coding for Pro to CCC (or CCG); etc.
  • Preferably, the G/C content of the coding region of nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is increased by at least 7%, more preferably by at least 15%, particularly preferably by at least 20%, compared to the G/C content of the coded region of the wild-type mRNA which codes for an antigen, antigenic protein or antigenic peptide as deinined herein or its fragment or variant thereof. According to a specific embodiment at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%, 95% or even 100% of the substitutable codons in the region coding for a protein or peptide as defined herein or its fragment or variant thereof or the whole sequence of the wild type mRNA sequence are substituted, thereby increasing the GC/content of said sequence.
  • In this context, it is particularly preferable to increase the G/C content of the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, to the maximum (i.e. 100% of the substitutable codons), in particular in the region coding for a protein, compared to the wild-type sequence.
  • According to the invention, a further preferred modification of the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, if so-called “rare codons” are present in the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I), especially if the nucleic acid is in the form of an mRNA, to an increased extent, the corresponding modified nucleic acid sequence is translated to a significantly poorer degree than in the case where codons coding for relatively “frequent” tRNAs are present.
  • According to the invention, in the modified nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, the region which codes for the adjuvant protein is modified compared to the corresponding region of the wild-type mRNA such that at least one codon of the wild-type sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and carries the same amino acid as the relatively rare tRNA. By this modification, the sequences of the nucleic acid, especially if the nucleic acid is in the form of an mRNA, is modified such that codons for which frequently occurring tRNAs are available are inserted. In other words, according to the invention, by this modification all codons of the wild-type sequence which code for a tRNA which is relatively rare in the cell can in each case be exchanged for a codon which codes for a tRNA which is relatively frequent in the cell and which, in each case, carries the same amino acid as the relatively rare tRNA.
  • Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. The codons which use for the particular amino acid the tRNA which occurs the most frequently, e.g. the Gly codon, which uses the tRNA which occurs the most frequently in the (human) cell, are particularly preferred.
  • According to the invention, it is particularly preferable to link the sequential G/C content which is increased, in particular maximized, in the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, with the “frequent” codons without modifying the amino acid sequence of the protein encoded by the coding region of the nucleic acid. This preferred embodiment allows provision of a particularly efficiently translated and stabilized (modified) nucleic acid for the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA.
  • The determination of a modified nucleic acid of the inventive polymeric carrier cargo complex as defined herein (increased G/C content; exchange of tRNAs) can be carried out using the computer program explained in WO 02/098443—the disclosure content of which is included in its full scope in the present invention. Using this computer program, the nucleotide sequence of any desired nucleic acid or mRNA can be modified with the aid of the genetic code or the degenerative nature thereof such that a maximum G/C content results, in combination with the use of codons which code for tRNAs occurring as frequently as possible in the cell, and the amino acid sequence coded by the modified nucleic acid of the present invention preferably not being modified compared to the non-modified sequence. Alternatively, it is also possible to modify only the G/C content or only the codon usage compared to the original sequence. The source code in Visual Basic 6.0 (development environment used: Microsoft Visual Studio Enterprise 6.0 with Servicepack 3) is also described in WO 02/098443.
  • In a further preferred embodiment of the present invention, the A/U content in the environment of the ribosome binding site of the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is increased compared to the A/U content in the environment of the ribosome binding site of its particular wild-type mRNA. This modification (an increased A/U content around the ribosome binding site) increases the efficiency of ribosome binding to the nucleic acid. An effective binding of the ribosomes to the ribosome binding site (Kozak sequence: GCCGCCACCAUGG (SEQ ID NO: 75), the AUG forms the start codon) in turn has the effect of an efficient translation of the nucleic acid.
  • According to a further embodiment of the present invention the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, may be modified with respect to potentially destabilizing sequence elements. Particularly, the coding region and/or the 5′ and/or 3′ untranslated region of this nucleic acid may be modified compared to the particular wild-type nucleic acid such that is contains no destabilizing sequence elements, the coded amino acid sequence of the modified nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, preferably not being modified compared to its particular wild-type nucleic acid. It is known that, for example, in sequences of eukaryotic RNAs destabilizing sequence elements (DSE) occur, to which signal proteins bind and regulate enzymatic degradation of RNA in vivo. For further stabilization of the modified nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, optionally in the region which encodes for a protein or a peptide as defined herein, one or more such modifications compared to the corresponding region of the wild-type nucleic acid can therefore be carried out, so that no or substantially no destabilizing sequence elements are contained there. According to the invention, DSE present in the untranslated regions (3′- and/or 5′-UTR) can also be eliminated from the nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, by such modifications.
  • Such destabilizing sequences are e.g. AU-rich sequences (AURES), which occur in 3′-UTR sections of numerous unstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, is therefore preferably modified compared to the wild-type nucleic acid such that the modified nucleic acid contains no such destabilizing sequences. This also applies to those sequence motifs which are recognized by possible endonucleases, e.g. the sequence GAACAAG, which is contained in the 3′-UTR segment of the gene which codes for the transferrin receptor (Binder et al., EMBO J. 1994, 13: 1969 to 1980). These sequence motifs are also preferably removed in the nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA.
  • Also preferably according to the invention, the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, has, in a modified form, at least one IRES as defined herein and/or at least one 5′ and/or 3′ stabilizing sequence, in a modified form, e.g. to enhance ribosome binding or to allow expression of different encoded antigens located on an at least one (bi-or even multicistronic) RNA of the inventive polymeric carrier cargo complex.
  • According to the invention, the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, furthermore preferably has at least one 5′ and/or 3′ stabilizing sequence. These stabilizing sequences in the 5′ and/or 3′ untranslated regions have the effect of increasing the half-life of the nucleic acid in the cytosol. These stabilizing sequences can have 100% sequence identity or identity to naturally occurring sequences which occur in viruses, bacteria and eukaryotes, but can also be partly or completely synthetic. The untranslated sequences (UTR) of the βglobin gene, e.g. from Homo sapiens or Xenopus laevis may be mentioned as an example of stabilizing sequences which can be used in the present invention for a stabilized nucleic acid. Another example of a stabilizing sequence has the generic formula (C/U)CCANxCCC(U/A)PyxUC(C/U)CC (SEQ ID NO: 76), which is contained in the 3′UTR of the very stable RNA which codes for α-globin, α(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Such stabilizing sequences can of course be used individually or in combination with one another and also in combination with other stabilizing sequences known to a person skilled in the art. The nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I), especially if the nucleic acid is in the form of an mRNA, is therefore preferably present as globin UTR (untranslated regions)-stabilized RNA, in particular as βglobin UTR-stabilized RNA.
  • Nevertheless, substitutions, additions or eliminations of bases are preferably carried out with the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, using a DNA matrix for preparation of the nucleic acid by techniques of the well known site directed mutagenesis or with an oligonucleotide ligation strategy (see e.g. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, N.Y., 2001). In such a process, for preparation of the nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, a corresponding DNA molecule may be transcribed in vitro. This DNA matrix preferably comprises a suitable promoter, e.g. a T7 or SP6 promoter, for in vitro transcription, which is followed by the desired nucleotide sequence for the nucleic acid, e.g. mRNA, to be prepared and a termination signal for in vitro transcription. The DNA molecule, which forms the matrix of an at least one RNA of interest, may be prepared by fermentative proliferation and subsequent isolation as part of a plasmid which can be replicated in bacteria. Plasmids which may be mentioned as suitable for the present invention are e.g. the plasmids pT7Ts (GenBank accession number U26404; Lai et al., Development 1995, 121: 2349 to 2360), pGEM® series, e.g. pGEM®-1 (GenBank accession number X65300; from Promega) and pSP64 (GenBank accession number X65327); cf. also Mezei and Storts, Purification of PCR Products, in: Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.
  • According to another particularly preferred embodiment, the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, may additionally or alternatively encode a secretory signal peptide. Such signal peptides are sequences, which typically exhibit a length of about 15 to 30 amino acids and are preferably located at the N-terminus of the encoded peptide, without being limited thereto. Signal peptides as defined herein preferably allow the transport of the protein or peptide as encoded by the nucleic acid of the present invention, especially if the nucleic acid is in the form of an mRNA, into a defined cellular compartiment, preferably the cell surface, the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Examples of secretory signal peptide sequences as defined herein include, without being limited thereto, signal sequences of classical or non-classical MHC-molecules (e.g. signal sequences of MHC I and II molecules, e.g. of the MHC class I molecule HLA-A*0201), signal sequences of cytokines or immunoglobulines as defined herein, signal sequences of the invariant chain of immunoglobulines or antibodies as defined herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin, Calnexin, and further membrane associated proteins or of proteins associated with the endoplasmic reticulum (ER) or the endosomal-lysosomal compartiment. Particularly preferably, signal sequences of MHC class I molecule HLA-A*0201 may be used according to the present invention.
  • Any of the above modifications may be applied to the nucleic acid of the inventive polymeric carrier cargo complex, especially if the nucleic acid is in the form of an mRNA, and further to any nucleic acid as used in the context of the present invention and may be, if suitable or necessary, be combined with each other in any combination, provided, these combinations of modifications do not interfere with each other in the respective nucleic acid. A person skilled in the art will be able to take his choice accordingly.
  • Proteins or peptides as encoded by the nucleic acid of the inventive polymeric carrier cargo complex as defined herein, may comprise fragments or variants of those sequences. Additionally, the nucleic acid of the inventive polymeric carrier cargo complex may comprise fragments or variants of those coding sequences. Such fragments or variants may typically comprise a sequence having a sequence identity with one of the above mentioned proteins or peptides or sequences of their encoding nucleic acid sequences of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, equally more preferably at least 85%, even more preferably at least 90% and most preferably at least 95% or even 97%, to the entire wild-type sequence, either on nucleic acid level or on amino acid level.
  • “Fragments” of proteins or peptides in the context of the present invention may comprise a sequence of an protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid sequence), N-terminally, C-terminally and/or intrasequentially truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid sequence). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid sequence of such a protein or peptide. The same applies accordingly to nucleic acids.
  • Fragments of proteins or peptides in the context of the present invention may furthermore comprise a sequence of a protein or peptide as defined herein, which has a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form.
  • Fragments of proteins or peptides as defined herein may also comprise epitopes of those proteins or peptides. Epitopes (also called “antigen determinants”) in the context of the present invention are typically fragments located on the outer surface of (native) proteins or peptides as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies or B-cell receptors, i.e. in their native form. Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • “Variants” of proteins or peptides as defined herein may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, wherein nucleotides of the nucleic acid, encoding the protein or peptide as defined herein, are exchanged. Thereby, a protein or peptide may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property.
  • The nucleic acid of the inventive polymeric carrier cargo complex may also encode a protein or peptide as defined herein, wherein the encoded amino acid sequence comprises conservative amino acid substitution(s) compared to its physiological sequence. Those encoded amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids which originate from the same class are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).
  • Furthermore, variants of proteins or peptides as defined herein, which may be encoded by the nucleic acid of the inventive polymeric carrier cargo complex, may also comprise those sequences, wherein nucleotides of the nucleic acid sequence are exchanged according to the degeneration of the genetic code, without leading to an alteration of respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.
  • In order to determine the percentage to which two sequences (nucleic acid sequences, e.g. nucleic acid sequences of the inventive polymeric carrier cargo complex, or their encoded amino acid sequences, e.g. the amino acid sequences of the proteins or peptides as defined herein) are identical, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. gaps can be inserted into the sequence of the first sequence and the component at the corresponding position of the second sequence can be compared. If a position in the first sequence is occupied by the same component as is the case at a position in the second sequence, the two sequences are identical at this position. The percentage to which two sequences are identical is a function of the number of identical positions divided by the total number of positions. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.
  • The present invention also provides a method of preparing the inventive polymeric carrier according to formula (I) L-P1—S—[S—P2—S]n—S—P3-L as defined herein (or of any subformula thereof as defined herein, e.g. (Ia)) and the product prepared by such a method, the method comprising following steps:
      • a) providing at least one cationic or polycationic protein or peptide as component P2 as defined herein and/or at least one cationic or polycationic polymer as component P2 as defined herein, preferably in the ratios indicated above by formula (I), mixing these components, preferably in a basic milieu as defined herein, preferably in the presence of oxygen or a further starter as defined herein, preferably at a pH, at a temperature and at time as defined herein, and thereby condensing and thus polymerizing these components with each other via disulfide bonds (in a polymerization condensation or polycondensation) to obtain a repetitive component H—[S—P2—S]n—H;
      • b) providing a hydrophilic polymer P1 and/or P3 as defined herein, optionally modified with a ligand L and/or a repetitive amino acid component (AA)x as defined herein;
      • c) mixing the hydrophilic polymer P1 and/or P2 provided according to step b) with the repetitive component H—[S—P2—S]n—H obtained according to step a), typically in a ratio of about 2:1, (and thereby typically terminating the polymerization condensation or polycondensation reaction) and obtaining the inventive polmeric carrier according to formula (I);
      • d) optionally purifying the inventive polymeric carrier obtained according to step c), preferably using a method as defined herein;
      • e) optionally adding a nucleic acid as defined herein to the inventive polymeric carrier obtained according to step c) or d), preferably in the above mentioned ratios, and complexing the nucleic acid with the polymeric carrier obtained according to step c) or d) to obtain an inventive polymeric carrier cargo complex as defined herein.
  • The inventive method of preparing the inventive polymeric carrier according to formula (I) as defined herein represents a multi-step condensation polymerization or polycondensation reaction via —SH moieties of the educts, e.g. component(s) P2 as defined herein, further components P1 and/or P3 and optionally further components (AA)x. The condensation polymerization or polycondensation reaction preferably leads to the inventive polymeric carrier as a condensation polymer, wherein the single components are linked by disulfide bonds. This condensation polymerization leads to the inventive polymeric carrier according to formula (I) preparing in a first step a) of the condensation reaction the inventive repetitive component H—[S—P2—S]n—H or a variant thereof as a sort of a “core” or “central motif” of the inventive polymeric carrier. In a second step b) components P1 and/or P3 are provided, which allow to terminate or to somehow “coat” the inventive repetitive component H—[S—P2—S]n—H or a variant thereof in a third step c) by adding components P1 and/or P3 as defined herein (optionally modified with a ligand L and/or a repetitive amino acid component (AA)x as defined herein) to the condensation product obtained according to step a). In subsequent step d), this product may be purified and further used to complex a nucleic acid cargo as defined herein to obtain an inventive complex.
  • It is important to understand that the inventive method is based on an equibrility reaction in steps a), (b)) and c), which, upon balancing the equilibirity state, allows to obtain the inventive polymeric carrier according to formula (I) above or according to any of its subformulas comprising the selected components in the desired molar ratios. For this purpose, long reaction times are envisaged to achieve an equibrility state in steps a), (b)) and c). If for example a condensation polymerization is to be carried out using a molar ratio of 5 components P2 in step a), the equilibrium is surprisingly settled at a polymer length of about 5 after sufficient time, preferably e.g. >12 hours.
  • As defined herein in a step a) of the inventive method of preparing the inventive polymeric carrier according to formula (I) at least one cationic or polycationic protein or peptide as component P2 as defined herein and/or at least one cationic or polycationic polymer as component P2 as defined herein are provided, preferably in the ratios indicated above by formula (I). These components are mixed, preferably in a basic milieu as defined herein, preferably in the presence of oxygen or a further starter as defined herein, preferably at a pH, and at a temperature and at a time as defined herein, and thereby condensing and thus polymerizing these components with each other via disulfide bonds (in a polymerization condensation or polycondensation) to obtain a repetitive component H—[S—P2—S]n—H.
  • According to an alternative, in step a) of the inventive method of preparing the inventive polymeric carrier at least one cationic or polycationic protein or peptide and/or at least one cationic or polycationic polymer are provided and used as component(s) P2 as defined herein, and additionally at least one repetitive amino acid component (AA)x is provided as defined herein, and components P2 and (AA)x, are used for a polymerization condensation or polycondensation according to step a). Preferably, the components are all provided in the ratios indicated above by formula (Ia), mixed, preferably in a basic milieu as defined herein, preferably in the presence of oxygen or a further starter as defined herein, preferably at a pH, at a temperature and at time as defined herein. Upon mixing and starting the reaction, the components are condensed and thus polymerized with each other via disulfide bonds (in a polymerization condensation or polycondensation) to obtain a repetitive component H—{[S—P2—S]a[S-(AA)x-S]b}-H.
  • In both of the above alternatives, different component(s) P2, particularly different peptides and/or different polymers as component P2, may be selected in the condensation polymerization as indicated above. In this context, the selection of different component(s) P2 is typically dependent upon the desired properties of the final inventive polymeric carrier and the desired cationic strength of the final inventive polymeric carrier or its central core motif. Accordingly, the repetitive component [S—P2—S]n, may furthermore be “diluted” or modified in the above alternative of step a) e.g. by introducing a repetitive amino acid component (AA)x as defined herein, preferably in the above defined ratios. Thereby, a modified central core motif {[S—P2—S]a[S-(AA)x-S]b} may be obtained, wherein the cationic character of (unmodified) repetitive component [S—P2—S]n typically remains in the limitations as defined herein. The properties of the final inventive polymeric carrier may thus be adjusted as desired with properties of components (AA)x by inserting repetitive amino acid component (AA)x as defined herein in steps a), b) and/or c).
  • In all cases, step a) is based on an equibrility reaction which, upon balancing the equilibirity state, allows to obtain either inventive repetitive component H—[S—P2—S]n—H or inventive repetitive component H-{[S—P2—S]a[S-(AA)x-S]b}—H in the desired molar ratios. For this purpose, long reaction times are envisaged to achieve an equibrility state in step a), most preferably e.g. >12 hours. Accordingly, step a) of the inventive method of preparing a polymeric carrier typically requires at least about 5 hours, even more preferably at least about 7.5 hours or even 10 hours, most preferably at least about 12 hours, e.g. a reaction time of about 12 to 60 hours, a reaction time of about 12 to 48 hours, a reaction time of about 12 to 36 hours, or a reaction time of about 12 to 24 hours, etc, wherein the lower border of 12 hours of the latter ranges may also be adjusted to 10, 7.5, or even 5 hours.
  • In step a), the at least one cationic or polycationic protein or peptide as component P2 as defined herein and/or at least one cationic or polycationic polymer as component P2 as defined herein, and optionally at least one repetitive amino acid component (AA)x as defined herein, are preferably contained in a basic milieu in the step a) of the inventive method of preparing the inventive polymeric carrier according to formula (I) (or any of its subformulas, e.g. (Ia)). Such a basic milieu typically exhibits a pH range of about 6 to about 12, preferably a pH range of about 7 to about 10, more preferably a pH range of about 8 to about 10, e.g. about 8, 8.5, 9, 9.5, or 10 or any range selected from any two of these or the aforementioned values.
  • Furthermore, the temperature of the solution in step a) is preferably in a range of about 5° C. to about 60° C., more preferably in a range of about 15° C. to about 40° C., even more preferably in a range of about 20° C. to about 30° C., and most preferably in a range of about 20° C. to about 25° C., e.g. about 25° C.
  • In step a) of the inventive method of preparing the inventive polymeric carrier according to formula (I) (or any of its subformulas, e.g. (Ia)) as defined herein buffers may be used as suitable. Preferred buffers may comprise, but are not limited to carbonate buffers, borate buffers, Bicine buffer, CHES buffer, CAPS buffer, Ethanolamine containing buffers, HEPES, MOPS buffer, Phosphate buffer, PIPES buffer, Tris buffer, Tricine buffer, TAPS buffer, and/or TES buffer as buffering agents. Particularly preferred is a carbonate buffer.
  • Upon mixing the components, preferably in the presence of oxygen, preferably in the presence of a basic mileu as defined herein, the condensation polymerization or polycondensation reaction is started. For this purpose, the mixture in step a) is preferably exposed to oxygen or may be started using a further starter, e.g. a catalytical amount of an oxidizing agent, e.g. DMSO, etc. Upon start of the condensation polymerization or polycondensation reaction the at least one cationic or polycationic protein or peptide and/or at least one cationic or polycationic polymer as component P2 and optionally at least one repetitive amino acid component (AA)x as defined herein, are condensed and thus polymerized with each other via disulfide bonds (polymerization condensation or polycondensation). In this reaction step a) preferably linear polymers are created using monomers with at least two reactive —SH moieties, i.e. at least one cationic or polycationic protein or peptide and/or at least one cationic or polycationic polymer as component P2 as defined herein, each component P2 exhibiting at least two free —SH-moieties as defined herein, e.g. at their terminal ends. However, components P2 with more than two free moieties may be used, which may lead to branched polymers.
  • According to one other specific embodiment, the condensation product obtained according to step a) may be modified (e.g. in a step a1)) by adding a repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z as defined herein to the terminal ends of the condensation product of step a). This may occur via any functionality as defined herein, e.g a —SH moiety or any further functionality described herein, preferably a —SH moiety. For this purpose, repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z may be provided with two (or even more) —SH-moieties, e.g. in a form represented by formulae “H(S-AA-S)x—H” or “H[S-(AA)x-S]zH”. Then, a polycondensation reaction may be carried out with the products of step a), i.e. inventive repetitive component H—[S—P2—S]n—H or inventive repetitive component H-{[S—P2—S]a[S-(AA)x-S]b}—H, leading to intermediate components

  • H(S-AA-S)x-[S—P2—S]n—(S-AA-S)xH, or

  • H[S-(AA)x-S]z—[S—P2—S]n—[S-(AA)x-S]zH, or

  • H(S-AA-S)x-{[S—P2—S]a[S-(AA)x-S]b}-(S-AA-S)xH, or

  • H[S-(AA)x-S]z—{[S—P2—S]a[S-(AA)x-S]b}[S-(AA)x-S]zH.
  • Any single or all of these intermediate components or the inventive repetitive component

  • H—[S—P2—S]n—H
  • or the inventive repetitive component

  • H-{[S—P2—S]a[S-(AA)x-S]b}-H
  • obtained according to step a), may be used to be coupled to the polymers provided in step b) of the inventive method.
  • According to a second step b) of the inventive method of preparing the inventive polymeric carrier according to formula (I) as defined herein (or according to any of its subformulas), a hydrophilic polymer P1 and/or P3 as defined herein is added to the condensation product obtained according to step a). In this context, the hydrophilic polymers P1 and/or P3 as defined herein, preferably exhibit at least one —SH-moiety, more preferably only one —SH-moiety per hydrophilic polymers P1 and/or P3 as defined herein, thereby terminally stopping the polymerization condensation or polycondensation according to step a) in step c). Hydrophilic polymers P1 and/or P3 as defined herein may be the same or different, wherein these polymers may be selected according to the desired properties. Typically, hydrophilic polymers P1 and/or P3 as a whole may be added to the condensation product obtained according to step a) in a ratio of about 2:1 hydrophilic polymer P1 and/or P3:condensation product obtained according to step a).
  • According to one alternative, the hydrophilic polymer(s) P1 and/or P3 additionally may be modified with either a component L (ligand) as defined herein or a component (AA)x or [(AA)x]z as defined herein or both a component L (ligand) as defined herein and a component (AA)x or [(AA)x]z as defined herein.
  • According to a first example, a ligand is attached to component(s) P1 and/or P3 as component L prior to providing component(s) P1 and/or P3 in step b) via any functionality as defined herein, e.g a —SH moiety. This ligand is preferably attached to the hydrophilic polymer(s) Pr and/or P3 at one terminus of these polymers. If the attachment is carried out via —SH bonds, the hydrophilic polymer(s) P1 and/or P3 are preferably provided with two (or even more) —SH-moieties., e.g. in a form represented by formulae HS—P1—SH or HS—P3—SH. Ligand L preferably carries only one —SH moiety. In this case, one —SH moiety of hydrophilic polymer(s) P1 and/or P3 is preferably protected in a first step using a protecting group as known in the art. Then, the hydrophilic polymer(s) P1 and/or P3 may be bound to a component L to form a first disulfide bond via the non-protected —SH moiety. The protected —SH-moiety of hydrophilic polymer(s) P1 and/or P3 is then typically deprotected for further reactions. This preferably leads to following intermediate components

  • L-S—S—P1—SH, or

  • HS—P3—S—S-L.
  • Alternatively, the above intermediate components may be provided similarly without the necessity of blocking the free —SH-moieties. These intermediate components may be used in step c) to be coupled with the condensation products obtained according to step a) above, e.g. to form a second disulfide bond with inventive repetitive component H—[S—P2—S]n—H or inventive mixed repetitive component H-{[S—P2—S]a[S-(AA)x-S]b}-H obtained according to step a) or any of its modifications, e.g. according to step a1). If the attachment is carried out via other moieties, any of the reactions as defined herein may be used accordingly.
  • According to a further example, a repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z as defined herein may be attached to component(s) P1 and/or P3 prior to providing component(s) P1 and/or P3 via any functionality as defined herein, e.g a —SH moiety. The repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z may be attached to the hydrophilic polymer(s) P1 and/or P3 at any position within these polymersor at one or both termini of these polymers. In one specific case, the repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z may be provided as a linker between component(s) P1 and/or P3 and the condensation product obtained according to step a) above or as a linker between component(s) P1 and/or P3 and a further component, e.g. a linker L, or according to another alternative, as a terminating component at one terminus of component(s) P1 and/or P3. In any of these cases, the attachment preferably may carried out via —SH bonds, wherein the hydrophilic polymer(s) P1 and/or P3 are preferably provided with two (or even more) —SH-moieties., e.g. in a form represented by formula “HS—P1—SH” or “HS—P3—SH”, wherein preferably one of these to —SH moieties is protected, e.g. in a form represented by formula “HS—P1—S-protecting group” or “protecting group-S—P3—SH”. Furthermore, repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z are also preferably provided with two (or even more) —SH-moieties, e.g. in a form represented by formulae “H(S-AA-S)x—H” or “H[S-(AA)x-S]zH”, wherein preferably one of these to —SH moieties is protected, e.g. in a form represented by formulae “protecting group-(S-AA-S)x—SH” or “H[S-(AA)x-S]z-protecting group”. Then, a polycondensation reaction may be carried out with polymers “HS—P1—S-protecting group” or “protecting group-S—P3—SH” leading to intermediate components
      • “protecting group-S—P1—S—(S-AA-S)x—S-protecting group”,
      • “protecting group-(S-AA-S)x—S—S—P3—S-protecting group”,
      • “protecting group-S—P1—S—[S-(AA)x-S]z-protecting group”, or
      • “protecting group-[S-(AA)x-S]z—P3—S-protecting group”.
  • Any single or all of these intermediate components may then be used in step c) of the inventive method to be coupled to the condensation product according to step a).
  • For this purpose, at least one or both protecting groups (selected upon the desired direction of the component in the final carrier) of each intermediate compound may be deprotected prior to providing them in step b), leading to following intermediate components

  • “HS—P1—S—(S-AA-S)x—SH”,

  • “H(S-AA-S)x—S—S—P3—SH”,

  • “HS—P1—S—[S-(AA)x-S]zH”, or

  • “H[S-(AA)x-S]2—S—P3—SH”,
  • Alternatively, the above intermediate components may be provided similarly without the necessity of blocking the free —SH-moieties. Any single or all of these intermediate components may then be provided in step b) of the inventive method to be coupled to the condensation product according to step a).
  • If any of the afore mentioned intermediate components is provided in step b), this condensation reaction may be terminated in a step c) by adding a linker component as defined herein with one —SH-moiety (e.g. L-SH) or any further component with a single —SH moiety, e.g. as defined herein. In one further specific case, the repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z may be used as a terminal component at one terminus of component(s) P1 and/or P3 without adding a further component to the repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z.
  • According to a further example, a repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z as defined herein may be attached to component(s) P1 and/or P3 prior to step c), wherein component(s) P1 and/or P3 have been already modified with a linker. For this purpose, component(s) P1 and/or P3 preferably carry (at least) two —SH moieties as defined herein, wherein a polycondensation is carried out with a linker, carrying e.g. one —SH moiety. This reaction may be carried out by using protecting groups as defined herein, or, preferably, without protecting groups. Alternatively, any further functionality as defined herein except —SH moieties may be used for coupling. Then, the second —SH moiety of component(s) P1 and/or P3 may be used to couple a repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z as defined herein via —SH-moieties, e.g. in a form represented by formulae “H(S-AA-S)x—H” or “H[S-(AA)x-S]zH”. The reaction preferably leads to following intermediate compounds

  • “L-S—S—P1—S—(S-AA-S)x—SH”,

  • “L-S—S—P1—S—[S-(AA)x-S]zH”, or

  • “HS—(S-AA-S)x(S—S—P3—S—S-L”, or

  • “HS—[S-(AA)x-S], —S—P3—S—S-L”;
  • or, if component L has been linked without a dislufide bond to following intermediate products

  • “L-P1—S—(S-AA-S), —SH”,

  • “L-P1—S—[S-(AA)x-S]zH”, or

  • “HS—(S-AA-S)x—S—S—P3-L”, or

  • “HS—[S-(AA)x-S]z—S—P3-L”;
  • In step c) the hydrophilic polymers P1 and/or P3 (or any of the intermediate components provided according to step b)) as defined herein, are provided and mixed with the repetitive component H—[S—P2—S]n—H, with the mixed repetitive component H-{[S—P2—S]a[S-(AA)x-S]b}—H, or any of the intermediate components obtained according to step a), typically in a ratio of about 2:1. The reaction is typically started and carried out under conditions already described above for step a) (pH, temperature, reaction time, buffers, etc.). Step c) allows to terminate the polymerization condensation or polycondensation reaction and to obtain the inventive polmeric carrier according to formula (I) or (Ia) or according to any of subformulas thereof as defined herein, preferably the inventive polmeric carrier according to formula (I)

  • L-P1—S[S—P2S]n—S—P3-L
  • or according to formula (Ia)

  • L-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3-L.
  • According to a further step d) of the inventive method of preparing the inventive polymeric carrier according to formula (I) or (Ia) as defined herein, or according to any of subformulas thereof as defined herein, the inventive polymeric carrier obtained according to step c) is optionally purified. Purification may occur by using chromatographic methods, such as HPLC, FPLC, GPS, dialysis, etc.
  • According to a final step e) of the inventive method of preparing the inventive polymeric carrier according to formula (I) or (Ia) as defined herein, or according to any of subformulas thereof as defined herein, a nucleic acid as defined herein is optionally added to the inventive polymeric carrier obtained according to step c) or d), preferably in the above mentioned ratios. Typically, in the inventive complex, the polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, as defined herein and the nucleic acid are provided in a molar ratio of about 10 to 10000, preferably in a molar ratio of about 10 to 5000, more preferably in a molar ratio of about 25 to 2500, even more preferably in a molar ratio of about 50 to 1000 polymeric carrier:nucleic acid. The N/P ratios are preferably as indicated above.
  • The inventive method of preparing the inventive polymeric carrier according to formula (I) or (Ia) or according to any of subformulas thereof as defined herein is particularly suitable to adapt the chemical properties of the desired inventive polymeric carrier due to specific selection of its components P2, (AA)x, or [(AA)x]z, thereby avoiding agglomeration and toxicity in vivo.
  • Furthermore, a skilled person would not have expected to obtain an inventive polymeric carrier using the above inventive method as the skilled person would always have expected that the polymer obtained according to the inventive method due to general rules of equilibrity reactions leads to a monomeric content of component P2, flanked by monomeric components P1 and/or P3, wherein the linkages are formed by disulfide bonds. In contrast, the present inventors were surprisingly able to show that when using a specific ratio of polymers and method steps as defined herein, the polymerization condensation can be directed to specifically obtain a desired distribution of polymers and a desired average length and the desired inventive polymeric carrier according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein without the necessity of blocking the free —SH-moieties. This was not expected by a skilled person.
  • According to a further embodiment, the present invention also provides a pharmaceutical composition, comprising the inventive polymeric carrier cargo complex formed by a nucleic acid cargo as defined herein and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein and optionally a pharmaceutically acceptable carrier and/or vehicle.
  • As a first ingredient, the inventive pharmaceutical composition comprises the polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein.
  • Furthermore, the inventive pharmaceutical composition may comprise a pharmaceutically acceptable carrier and/or vehicle. In the context of the present invention, a pharmaceutically acceptable carrier typically includes the liquid or non-liquid basis of the inventive pharmaceutical composition. If the inventive pharmaceutical composition is provided in liquid form, the carrier will typically be pyrogen-free water; isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions. Particularly for injection of the inventive pharmaceutical composition, water or preferably a buffer, more preferably an aqueous buffer, may be used, containing a sodium salt, preferably at least 50 mM of a sodium salt, a calcium salt, preferably at least 0.01 mM of a calcium salt, and optionally a potassium salt, preferably at least 3 mM of a potassium salt. According to a preferred embodiment, the sodium, calcium and, optionally, potassium salts may occur in the form of their halogenides, e.g. chlorides, iodides, or bromides, in the form of their hydroxides, carbonates, hydrogen carbonates, or sulfates, etc. Without being limited thereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na2CO3, NaHCO3, Na2SO4, examples of the optional potassium salts include e.g. KCl, KI, KBr, K2CO3, KHCO3, K2SO4, and examples of calcium salts include e.g. CaCl2, Cal2, CaBr2, CaCO3, CaSO4, Ca(OH)2. Furthermore, organic anions of the aforementioned cations may be contained in the buffer. According to a more preferred embodiment, the buffer suitable for injection purposes as defined herein, may contain salts selected from sodium chloride (NaCl), calcium chloride (CaCl2) and optionally potassium chloride (KCl), wherein further anions may be present additional to the chlorides. CaCl2 can also be replaced by another salt like KCl. Typically, the salts in the injection buffer are present in a concentration of at least 50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl2). The injection buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e. the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of cells due to osmosis or other concentration effects. Reference media are e.g. liquids occurring in “in vivo” methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro” methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.
  • However, one or more compatible solid or liquid fillers or diluents or encapsulating compounds may be used as well for the inventive pharmaceutical composition, which are suitable for administration to a patient to be treated. The term “compatible” as used here means that these constituents of the inventive pharmaceutical composition are capable of being mixed with the complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions. Pharmaceutically acceptable carriers, fillers and diluents must, of course, have sufficiently high purity and sufficiently low toxicity to make them suitable for administration to a person to be treated. Some examples of compounds which can be used as pharmaceutically acceptable carriers, fillers or constituents thereof are sugars, such as, for example, lactose, glucose and sucrose; starches, such as, for example, corn starch or potato starch; cellulose and its derivatives, such as, for example, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid glidants, such as, for example, stearic acid, magnesium stearate; calcium sulfate; vegetable oils, such as, for example, groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from theobroma; polyols, such as, for example, polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.
  • According to another embodiment, the inventive pharmaceutical composition may comprise an adjuvant. In this context, an adjuvant may be understood as any compound, which is suitable to initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response. With other words, when administered, the inventive pharmaceutical composition typically elicits an innate immune response due to the adjuvant, optionally contained therein. Such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an innate immune response in a mammal. Preferably, the adjuvant may be selected from the group consisting of, without being limited thereto, including chitosan, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™ (propanediamine); BAY R1005™ ((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amide hydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calcium phosphate gel; CAPTM (calcium phosphate nanoparticles); cholera holotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein, sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205); cytokine-containing liposomes; DDA (dimethyldioctadecylammonium bromide); DHEA (dehydroepiandrosterone); DMPC (dimyristoylphosphatidylcholine); DMPG (dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acid sodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant; gamma inulin; Gerbu adjuvant (mixture of: i) N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP), ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline salt complex (ZnPro-8); GM-CSF); GMDP (N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine); imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine); ImmTher™ (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glycerol dipalmitate); DRVs (immunoliposomes prepared from dehydration-rehydration vesicles); interferon-gamma; interleukin-1beta; interleukin-2; interleukin-7; interleukin-12; ISCOMS™; 1SCOPREP 7.0.3.™; liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E. coli labile enterotoxin-protoxin); microspheres and microparticles of any composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™ (purified incomplete Freund's adjuvant); MONTANIDE ISA 720™ (metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipid A); MTP-PE and MTP-PE liposomes ((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide, monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™ and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl); NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles of any composition; NISVs (non-ionic surfactant vesicles); PLEURAN™ (β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid and glycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA (polymethyl methacrylate); PODDS™ (proteinoid microspheres); polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylic acid-polyuridylic acid complex); polysorbate 80 (Tween 80); protein cochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™ (QS-21); Quil-A (Quil-A saponin); S-28463 (4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo [4,5-c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendai proteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitan trioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85); squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane); stearyltyrosine (octadecyltyrosine hydrochloride); Theramie (N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide); Theronyl-MDP (Termurtide™ or [thr 1]-MDP; N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs or virus-like particles); Walter-Reed liposomes (liposomes containing lipid A adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys, in particular aluminium salts, such as Adju-phos, Alhydrogel, Rehydragel; emulsions, including CPA, SAP, IFA, MF59, Provax, TiterMax, Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121, Poloaxmer4010), etc.; liposomes, including Stealth, cochleates, including BIORAL; plant derived adjuvants, including QS21, Quil A, Iscomatrix, ISCOM; adjuvants suitable for costimulation including Tomatine, biopolymers, including PLG, PMM, Inulin, microbe derived adjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleic acid sequences, CpG7909, ligands of human TLR 1-10, ligands of murine TLR 1-13, 155-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine, IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPI anchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusion protein, cdiGMP; and adjuvants suitable as antagonists including CGRP neuropeptide. Suitable adjuvants may furthermore be selected from lipid modified nucleic acids.
  • The inventive pharmaceutical composition may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
  • Preferably, the inventive pharmaceutical composition may be administered by parenteral injection, more preferably by subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or via infusion techniques. Sterile injectable forms of the inventive pharmaceutical compositions may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation of the inventive pharmaceutical composition.
  • The inventive pharmaceutical composition as defined herein may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive immunostimulatory composition, particularly its components as defined herein, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • The inventive pharmaceutical composition typically comprises a “safe and effective amount” of the components of the inventive pharmaceutical composition, particularly of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein or the nucleic acid as such. As used herein, a “safe and effective amount” means an amount of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule or the nucleic acid cargo as such that is sufficient to significantly induce a positive modification of a disease or disorder as defined herein. At the same time, however, a “safe and effective amount” is small enough to avoid serious side-effects, that is to say to permit a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment. A “safe and effective amount” of the components of the inventive pharmaceutical composition, particularly of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, will furthermore vary in connection with the particular condition to be treated and also with the age and physical condition of the patient to be treated, the body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the activity of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule as herein employed or the nucleic acid as such, the severity of the condition, the duration of the treatment, the nature of the accompanying therapy, of the particular pharmaceutically acceptable carrier used, and similar factors, within the knowledge and experience of the accompanying doctor. The inventive pharmaceutical composition may be used for human and also for veterinary medical purposes, preferably for human medical purposes, as a pharmaceutical composition in general or as a vaccine.
  • According to a specific embodiment, the inventive pharmaceutical composition may be provided as a vaccine. Such an inventive vaccine is typically composed like the inventive pharmaceutical composition and preferably supports or elicits an immune response of the immune system of a patient to be treated, e.g. an innate immune response, if an RNA or mRNA is used as the nucleic acid molecule of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein. Furthermore or alternatively, the inventive vaccine may elicit an adaptive immune response, preferably, if the nucleic acid of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein encodes any of the above mentioned antigens or proteins, which elicit an adaptive immune response.
  • The inventive vaccine may also comprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicle as defined herein for the inventive pharmaceutical composition. In the specific context of the inventive vaccine, the choice of a pharmaceutically acceptable carrier is determined in principle by the manner in which the inventive vaccine is administered. The inventive vaccine can be administered, for example, systemically or locally. Routes for systemic administration in general include, for example, transdermal, oral, parenteral routes, including subcutaneous, intravenous, intramuscular, intraarterial, intradermal and intraperitoneal injections and/or intranasal administration routes. Routes for local administration in general include, for example, topical administration routes but also intradermal, transdermal, subcutaneous, or intramuscular injections or intralesional, intracranial, intrapulmonal, intracardial, and sublingual injections. More preferably, vaccines may be administered by an intradermal, subcutaneous, or intramuscular route. Inventive vaccines are therefore preferably formulated in liquid (or sometimes in solid) form. The suitable amount of the inventive vaccine to be administered can be determined by routine experiments with animal models. Such models include, without implying any limitation, rabbit, sheep, mouse, rat, dog and non-human primate models. Preferred unit dose forms for injection include sterile solutions of water, physiological saline or mixtures thereof. The pH of such solutions should be adjusted to about 7.4. Suitable carriers for injection include hydrogels, devices for controlled or delayed release, polylactic acid and collagen matrices. Suitable pharmaceutically acceptable carriers for topical application include those which are suitable for use in lotions, creams, gels and the like. If the inventive vaccine is to be administered orally, tablets, capsules and the like are the preferred unit dose form. The pharmaceutically acceptable carriers for the preparation of unit dose forms which can be used for oral administration are well known in the prior art. The choice thereof will depend on secondary considerations such as taste, costs and storability, which are not critical for the purposes of the present invention, and can be made without difficulty by a person skilled in the art.
  • The inventive vaccine can additionally contain one or more auxiliary substances in order to increase its immunogenicity, if desired. A synergistic action of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and a polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein and of an auxiliary substance, which may be optionally contained in the inventive vaccine as defined herein, is preferably achieved thereby. Depending on the various types of auxiliary substances, various mechanisms can come into consideration in this respect. For example, compounds that permit the maturation of dendritic cells (DCs), for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a first class of suitable auxiliary substances. In general, it is possible to use as auxiliary substance any agent that influences the immune system in the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow an immune response to be enhanced and/or influenced in a targeted manner. Particularly preferred auxiliary substances are cytokines, such as monokines, lymphokines, interleukins or chemokines, that further promote the innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, INF-alpha, IFN-beta, INF-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.
  • Further additives which may be included in the inventive vaccine are emulsifiers, such as, for example, Tween®; wetting agents, such as, for example, sodium lauryl sulfate; colouring agents; taste-imparting agents, pharmaceutical carriers; tablet-forming agents; stabilizers; antioxidants; preservatives.
  • The inventive vaccine can also additionally or alternatively contain any further compound, which is known to be immune-stimulating due to its binding affinity (as ligands) to human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its binding affinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
  • Another class of compounds, which may be added to an inventive vaccine in this context, may be CpG nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA, more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). The CpG nucleic acid preferably contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to a first preferred alternative, at least one CpG motif contained in these sequences, that is to say the C (cytosine) and the G (guanine) of the CpG motif, is unmethylated. All further cytosines or guanines optionally contained in these sequences can be either methylated or unmethylated. According to a further preferred alternative, however, the C (cytosine) and the G (guanine) of the CpG motif can also be present in methylated form.
  • The inventive vaccine can also additionally or alternatively contain an immunostimulatory RNA, i.e. an RNA derived from an immunostimulatory RNA, which triggers or increases an (innate) immune response. Preferably, such an RNA may be in general be as defined herein for RNAs. In this context, those classes of RNA molecules, which can induce an innate immune response, may be selected e.g. from ligands of Toll-like receptors (TLRs), particularly from RNA sequences representing and/or encoding ligands of TLRs, preferably selected from human family members TLR1-TLR10 or murine family members TLR1TLR13, more preferably from TLR7 and TLR8, ligands for intracellular receptors for RNA (such as RIG-I or MDA-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mal. Cell 22, 561-569), or any other immunostimulatory RNA sequence. Such an immunostimulatory RNA may comprise a length of 1000 to 5000, of 500 to 5000, of 5 to 5000, or of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides.
  • The present invention furthermore provides several applications and uses of the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule, a pharmaceutical composition comprising same or of kits comprising same.
  • According to one embodiment, the present invention is directed to the first medical use of the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule, or of kits comprising same, as a medicament, preferably for gene therapy. The medicament may be in the form of a pharmaceutical composition or in the form of a vaccine as a specific form of pharmaceutical compositions. A pharmaceutical composition in the context of the present invention typically comprises the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein or the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule, optionally further ingredients, e.g. as defined herein for the inventive nucleic acid, and optionally a pharmaceutically acceptable carrier and/or vehicle, preferably as defined herein.
  • According to one further embodiment, the present invention is directed to the use of the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, or of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule for the prophylaxis, treatment and/or amelioration of diseases as defined herein, preferably selected from cancer or tumor diseases, infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases, neuronal diseases.
  • According to another embodiment, the present invention is directed to the second medical use of the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, or of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule for the treatment of diseases as defined herein, preferably to the use of the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, or of the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule, of a pharmaceutical composition comprising same or of kits comprising same for the preparation of a medicament for the prophylaxis, treatment and/or amelioration of various diseases as defined herein.
  • According to a further embodiment, the present invention is directed to the treatment of diseases as defined herein, particularly prophylaxis, treatment and/or amelioration of various diseases as defined herein, preferably using or administering to a patient in need thereof the inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, or the inventive pharmaceutical composition as defined herein.
  • According to one specific embodiment, diseases as defined herein comprise cancer or tumor diseases, preferably selected from melanomas, malignant melanomas, colon carcinomas, lymphomas, sarcomas, blastomas, renal carcinomas, gastrointestinal tumors, gliomas, prostate tumors, bladder cancer, rectal tumors, stomach cancer, oesophageal cancer, pancreatic cancer, liver cancer, mammary carcinomas (=breast cancer), uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chronic lymphocytic leukaemia (CLL), hepatomas, various virus-induced tumors such as, for example, papilloma virus-induced carcinomas (e.g. cervical carcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors (e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma), heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1- and HTLV-2-induced lymphomas, acoustic neuroma, lung carcinomas (=lung cancer=bronchial carcinoma), small-cell lung carcinomas, pharyngeal cancer, anal carcinoma, glioblastoma, rectal carcinoma, astrocytoma, brain tumors, retinoblastoma, basalioma, brain metastases, medulloblastomas, vaginal cancer, pancreatic cancer, testicular cancer, Hodgkin's syndrome, meningiomas, Schneeberger disease, hypophysis tumor, Mycosis fungoides, carcinoids, neurinoma, spinalioma, Burkitt's lymphoma, laryngeal cancer, renal cancer, thymoma, corpus carcinoma, bone cancer, non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/neck tumors, oligodendroglioma, vulval cancer, intestinal cancer, colon carcinoma, oesophageal carcinoma (=Oesophageal cancer), wart involvement, tumors of the small intestine, craniopharyngeomas, ovarian carcinoma, genital tumors, ovarian cancer (=Ovarian carcinoma), pancreatic carcinoma (=pancreatic cancer), endometrial carcinoma, liver metastases, penile cancer, tongue cancer, gall bladder cancer, leukaemia, plasmocytoma, lid tumor, prostate cancer (=prostate tumors), etc.
  • According to one further specific embodiment, diseases as defined herein comprise infectious diseases, preferably (viral, bacterial or protozoological) infectious diseases. Such infectious diseases, preferably to (viral, bacterial or protozoological) infectious diseases, are typically selected from influenza, malaria, SANS, yellow fever, AIDS, Lyme borreliosis, Leishmaniasis, anthrax, meningitis, viral infectious diseases such as AIDS, Condyloma acuminata, hollow warts, Dengue fever, three-day fever, Ebola virus, cold, early summer meningoencephalitis (FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplex type II, Herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburg virus, measles, foot-and-mouth disease, mononucleosis, mumps, Norwalk virus infection, Pfeiffer's glandular fever, smallpox, polio (childhood lameness), pseudo-croup, fifth disease, rabies, warts, West Nile fever, chickenpox, cytomegalic virus (CMV), bacterial infectious diseases such as miscarriage (prostate inflammation), anthrax, appendicitis, borreliosis, botulism, Camphylobacter, Chlamydia trachomatis (inflammation of the urethra, conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhus fever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori, whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease, leprosy, listeriosis, pneumonia, meningitis, bacterial meningitis, anthrax, otitis media, Mycoplasma hominis, neonatal sepsis (Chorioamnionitis), noma, paratyphus, plague, Reiter's syndrome, Rocky Mountain spotted fever, Salmonella paratyphus, Salmonella typhus, scarlet fever, syphilis, tetanus, tripper, tsutsugamushi disease, tuberculosis, typhus, vaginitis (colpitis), soft chancre, and infectious diseases caused by parasites, protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease, Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm, athlete's foot, canine tapeworm, candidosis, yeast fungus spots, scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice, malaria, microscopy, onchocercosis (river blindness), fungal diseases, bovine tapeworm, schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness), visceral Leishmaniosis, nappy/diaper dermatitis or miniature tapeworm.
  • According to another specific embodiment, diseases as defined herein comprise autoimmune diseases as defined in the following. Autoimmune diseases can be broadly divided into systemic and organ-specific or localised autoimmune disorders, depending on the principal clinico-pathologic features of each disease. Autoimmune diseases may be divided into the categories of systemic syndromes, including systemic lupus erythematosus (SLE), Sjögren's syndrome, Scleroderma, Rheumatoid Arthritis and polymyositis or local syndromes which may be endocrinologic (type I diabetes (Diabetes mellitus Type 1), Hashimoto's thyroiditis, Addison's disease etc.), dermatologic (pemphigus vulgaris), haematologic (autoimmune haemolytic anaemia), neural (multiple sclerosis) or can involve virtually any circumscribed mass of body tissue. The autoimmune diseases to be treated may be selected from the group consisting of type I autoimmune diseases or type II autoimmune diseases or type III autoimmune diseases or type IV autoimmune diseases, such as, for example, multiple sclerosis (MS), rheumatoid arthritis, diabetes, type I diabetes (Diabetes mellitus Type 1), chronic polyarthritis, Basedow's disease, autoimmune forms of chronic hepatitis, colitis ulcerosa, type I allergy diseases, type II allergy diseases, type III allergy diseases, type IV allergy diseases, fibromyalgia, hair loss, Bechterew's disease, Crohn's disease, Myasthenia gravis, neurodermitis, Polymyalgia rheumatica, progressive systemic sclerosis (PSS), Reiter's syndrome, rheumatic arthritis, psoriasis, vasculitis, etc, or type II diabetes. While the exact mode as to why the immune system induces an immune reaction against autoantigens has not been elucidated so far, there are several findings with regard to the etiology. Accordingly, the autoreaction may be due to a T-Cell bypass. A normal immune system requires the activation of B-cells by T-cells before the former can produce antibodies in large quantities. This requirement of a T-cell can be by-passed in rare instances, such as infection by organisms producing super-antigens, which are capable of initiating polyclonal activation of B-cells, or even of T-cells, by directly binding to the B-subunit of T-cell receptors in a non-specific fashion. Another explanation deduces autoimmune diseases from a Molecular Mimicry. An exogenous antigen may share structural similarities with certain host antigens; thus, any antibody produced against this antigen (which mimics the self-antigens) can also, in theory, bind to the host antigens and amplify the immune response. The most striking form of molecular mimicry is observed in Group A beta-haemolytic streptococci, which shares antigens with human myocardium, and is responsible for the cardiac manifestations of rheumatic fever.
  • Additionally, according to one further specific embodiment, diseases as defined herein comprise allergies or allergic diseases, i.e. diseases related to allergies. Allergy is a condition that typically involves an abnormal, acquired immunological hypersensitivity to certain foreign antigens or allergens, such as the allergy antigens as defined herein. Such allergy antigens or allergens may be selected from allergy antigens as defined herein antigens derived from different sources, e.g. from animals, plants, fungi, bacteria, etc. Allergens in this context include e.g. grasses, pollens, molds, drugs, or numerous environmental triggers, etc. Allergies normally result in a local or systemic inflammatory response to these antigens or allergens and lead to immunity in the body against these allergens. Without being bound to theory, several different disease mechanisms are supposed to be involved in the development of allergies. According to a classification scheme by P. Gell and R. Coombs the word “allergy” was restricted to type I hypersensitivities, which are caused by the classical IgE mechanism. Type I hypersensitivity is characterised by excessive activation of mast cells and basophils by IgE, resulting in a systemic inflammatory response that can result in symptoms as benign as a runny nose, to life-threatening anaphylactic shock and death. Well known types of allergies include, without being limited thereto, allergic asthma (leading to swelling of the nasal mucosa), allergic conjunctivitis (leading to redness and itching of the conjunctiva), allergic rhinitis (“hay fever”), anaphylaxis, angiodema, atopic dermatitis (eczema), urticaria (hives), eosinophilia, respiratory, allergies to insect stings, skin allergies (leading to and including various rashes, such as eczema, hives (urticaria) and (contact) dermatitis), food allergies, allergies to medicine, etc. Treatment of such allergic disorders or diseases may occur preferably by desensitizing the immune reaction which triggers a specific immune response. Such a desensitizing may be carried out by administering an effective amount of the allergen or allergic antigen encoded by the nucleic acid as defined herein, preferably, when formulated as a pharmaceutical composition, to induce a slight immune reaction. The amount of the allergen or allergic antigen may then be raised step by step in subsequent administrations until the immune system of the patient to be treated tolerates a specific amount of allergen or allergic antigen.
  • Additionally, diseases to be treated in the context of the present invention likewise include (hereditary) diseases, or genetic diseases in general monogenetic diseases, i.e. (hereditary) diseases, or genetic diseases in general. Such (mono-)genetic diseases, (hereditary) diseases, or genetic diseases in general are typically caused by genetic defects, e.g. due to gene mutations resulting in loss of protein activity or regulatory mutations which do not allow transcription or translation of the protein. Frequently, these diseases lead to metabolic disorders or other symptoms, e.g. muscle dystrophy. The present invention allows treating the following (hereditary) diseases or genetic diseases: 3-beta-hydroxysteroid dehydrogenase deficiency (type II); 3-ketothiolase deficiency; 6-mercaptopurine sensitivity; Aarskog-Scott syndrome; Abetalipoproteinernia; Acatalasemia; Achondrogenesis; Achondrogenesis-hypochondrogenesis; Achondroplasia; Achromatopsia; Acromesomelic dysplasia (Hunter-Thompson type); ACTH deficiency; Acyl-CoA dehydrogenase deficiency (short-chain, medium chain, long chain); Adenomatous polyposis coli; Adenosin-deaminase deficiency; Adenylosuccinase deficiency; Adhalinopathy; Adrenal hyperplasia, congenital (due to 11-beta-hydroxylase deficiency; due to 17-alpha-hydroxylase deficiency; due to 21-hydroxylase deficiency); Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism; Adrenogenital syndrom; Adrenoleukodystrophy; Adrenomyeloneuropathy; Afibrinogenemia; Agammaglobulinemia; Alagille syndrome; Albinism (brown, ocular, oculocutaneous, rufous); Alcohol intolerance, acute; Aldolase A deficiency; Aldosteronism, glucocorticoid-remediable; Alexander disease; Alkaptonuria; Alopecia universalis; Alpha-1-antichymotrypsin deficiency; Alpha-methylacyl-CoA racemase deficiency; Alpha-thalassemia/mental retardation syndrome; Alport syndrome; Alzheimer disease-1 (APP-related); Alzheimer disease-3; Alzheimer disease-4; Amelogenesis imperfecta; Amyloid neuropathy (familial, several allelic types); Amyloidosis (Dutch type; Finnish type; hereditary renal; renal; senile systemic); Amytrophic lateral sclerosis; Analbuminemia; Androgen insensitivity; Anemia (Diamond-Blackfan); Anemia (hemolytic, due to PK deficiency); Anemia (hemolytic, Rh-null, suppressor type); Anemia (neonatal hemolytic, fatal and nearfatal); Anemia (sideroblastic, with ataxia); Anemia (sideroblastic/hypochromic); Anemia due to G6PD deficiency; Aneurysm (familial arterial); Angelman syndrome; Angioedema; Aniridia; Anterior segment anomalies and cataract; Anterior segment mesenchymal dysgenesis; Anterior segment mesenchymal dysgenesis and cataract; Antithrombin III deficiency; Anxiety-related personality traits; Apert syndrome; Apnea (postanesthetic); ApoA-1 and apoC-III deficiency (combined); Apolipoprotein A-II deficiency; Apolipoprotein B-100 (ligand-defective); Apparent mineralocorticoid excess (hypertension due to); Argininemia; Argininosuccinicaciduria; Arthropathy (progressive pseudorheumatoid, of childhood); Aspartylglucosaminuria; Ataxia (episodic); Ataxia with isolated vitamin E deficiency; Ataxia-telangiectasia; Atelosteogenesis II; ATP-dependent DNA ligase I deficiency; Atrial septal defect with atrioventricular conduction defects; Atrichia with papular lesions; Autism (succinylpurinemic); Autoimmune polyglandular disease, type I; Autonomic nervous system dysfunction; Axenfeld anomaly; Azoospermia; Bamforth-Lazarus syndrome; Bannayan-Zonana syndrome; Barthsyndrome; Bartter syndrome (type 2 or type 3); Basal cell carcinoma; Basal cell nevus syndrome; BCG infection; Beare-Stevenson cutis gyrata syndrome; Becker muscular dystrophy; Beckwith-Wiedemann syndrome; Bernard-Soulier syndrome (type B; type C); Bethlem myopathy; Bile acid malabsorption, primary; Biotinidase deficiency; Bladder cancer; Bleeding disorder due to defective thromboxane A2 receptor; Bloom syndrome; Brachydactyl)-(type B1 or type C); Branchiootic syndrome; Branchiootorenal syndrome; Breast cancer (invasive intraductal; lobular; male, with Reifenstein syndrome; sporadic); Breast cancer-1 (early onset); Breast cancer-2 (early onset); Brody myopathy; Brugada syndrome; Brunner syndrome; Burkitt lymphoma; Butterfly dystrophy (retinal); C1q deficiency (type A; type B; type C); Cir/C is deficiency; C is deficiency, isolated; C2 deficiency; C3 deficiency; C3b inactivator deficiency; C4 deficiency; CS deficiency, type II; C9 deficiency; Campomelic dysplasia with autosomal sex reversal; Camptodactylyl-arthropathy-coxa varapericarditis syndrome; Canavan disease; Carbamoylphosphate synthetase I deficiency; Carbohydrate-deficient glycoprotein syndrome (type I; type Ib; type II); Carcinoid tumor of lung; Cardioencephalomyopathy (fatal infantile, due to cytochrome c oxidase deficiency); Cardiomyopathy (dilated; X-linked dilated; familial hypertrophic; hypertrophic); Carnitine deficiency (systemic primary); Carnitine-acylcarnitine translocase deficiency; Carpal tunnel syndrome (familial); Cataract (cerulean; congenital; crystalline aculeifarm; juvenile-onset; polymorphic and lamellar; punctate; zonular pulverulent); Cataract, Coppock-like; CD59 deficiency; Central core disease; Cerebellar ataxia; Cerebral amyloid angiopathy; Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy; Cerebral cavernous malformations-1; Cerebrooculofacioskeletal syndrome; Cerebrotendinous xanthomatosis; Cerebrovascular disease; Ceroid lipofuscinosis (neuronal, variant juvenile type, with granular osmiophilic deposits); Ceroid lipofuscinosis (neuronal-1, infantile); Ceroid-lipofuscinosis (neuronal-3, juvenile); Char syndrome; Charcot-Marie-Tooth disease; Charcot-Marie-Tooth neuropathy; Charlevoix-Saguenay type; Chediak-Higashi syndrome; Chloride diarrhea (Finnish type); Cholestasis (benign recurrent intrahepatic); Cholestasis (familial intrahepatic); Cholestasis (progressive familial intrahepatic); Cholesteryl ester storage disease; Chondrodysplasia punctata (brachytelephalangic; rhizomelic; X-linked dominant; X-linked recessive; Grebe type); Chondrosarcoma; Choroideremia; Chronic granulomatous disease (autosomal, due to deficiency of CYBA); Chronic granulomatous disease (X-linked); Chronic granulomatous disease due to deficiency of NCF-1; Chronic granulomatous disease due to deficiency of NCF-2; Chylomicronemia syndrome, familial; Citrullinemia; classical Cockayne syndrome-1; Cleft lip, cleft jaw, cleft palate; Cleft lip/palate ectodermal dysplasia syndrome; Cleidocranial dysplasia; CMO II deficiency; Coats disease; Cockayne syndrome-2, type B; Coffin-Lowry syndrome; Colchicine resistance; Colon adenocarcinoma; Colon cancer; Colorblindness (deutan; protan; tritan); Colorectal cancer; Combined factor V and VIII deficiency; Combined hyperlipemia (familial); Combined immunodeficiency (X-linked, moderate); Complex I deficiency; Complex neurologic disorder; Cone dystrophy-3; Cone-rod dystrophy 3; Cone-rod dystrophy 6; Cone-rod retinal dystrophy-2; Congenital bilateral absence of vas deferens; Conjunctivitis, ligneous; Contractural arachnodactylyl-; Coproporphyria; Cornea plana congenita; Corneal clouding; Corneal dystrophy (Avellino type; gelatinous drop-like; Groenouw type I; lattice type I; Reis-Bucklers type); Cortisol resistance; Coumarin resistance; Cowden disease; CPT deficiency, hepatic (type I; type II); Cramps (familial, potassium-aggravated); Craniofacial-deafness-hand syndrome; Craniosynostosis (type 2); Cretinism; Creutzfeldt-Jakob disease; Crigler-Najjar syndrome; Crouzon syndrome; Currarino syndrome; Cutis laxa; Cyclic hematopoiesis; Cyclic ichthyosis; Cylindromatosis; Cystic fibrosis; Cystinosis (nephropathic); Cystinuria (type II; type III); Daltonism; Darier disease; D-bifunctional protein deficiency; Deafness, autosomal dominant 1; Deafness, autosomal dominant 11; Deafness, autosomal dominant 12; Deafness, autosomal dominant 15; Deafness, autosomal dominant 2; Deafness, autosomal dominant 3; Deafness, autosomal dominant 5; Deafness, autosomal dominant 8; Deafness, autosomal dominant 9; Deafness, autosomal recessive 1; Deafness, autosomal recessive 2; Deafness, autosomal recessive 21; Deafness, autosomal recessive 3; Deafness, autosomal recessive 4; Deafness, autosomal recessive 9; Deafness, nonsyndromic sensorineural 13; Deafness, X-linked 1; Deafness, X-linked 3; Debrisoquine sensitivity; Dejerine-Sottas disease; Dementia (familial Danish); Dementia (frontotemporal, with parkinsonism); Dent disease; Dental anomalies; Dentatorubro-pallidoluysian atrophy; Denys-Drash syndrome; Dermatofibrosarcoma protuberans; Desmoid disease; Diabetes insipidus (nephrogenic); Diabetes insipidus (neurohypophyseal); Diabetes mellitus (insulin-resistant); Diabetes mellitus (rare form); Diabetes mellitus (type II); Diastrophic dysplasia; Dihydropyrimidinuria; Dosage-sensitive sex reversal; Doyne honeycomb degeneration of retina; Dubin-Johnson syndrome; Duchenne muscular dystrophy; Dyserythropoietic anemia with thrombocytopenia; Dysfibrinogenemia (alpha type; beta type; gamma type); Dyskeratosis congenita-1; Dysprothrombinemia; Dystonia (DOPAresponsive); Dystonia (myoclonic); Dystonia-1 (torsion); Ectodermal dysplasia; Ectopia lentis; Ectopia pupillae; Ectrodactylyl-(ectodermal dysplasia, and cleft lip/palate syndrome 3); Ehlers-Danlos syndrome (progeroid form); Ehlers-Danlos syndrome (type I; type II; type III; type IV; type VI; type VII); Elastin Supravalvar aortic stenosis; Elliptocytosis-1; Elliptocytosis-2; Elliptocytosis-3; Ellis-van Creveld syndrome; Emery-Dreifuss muscular dystrophy; Emphysema; Encephalopathy; Endocardial fibroelastosis-2; Endometrial carcinoma; Endplate acetylcholinesterase deficiency; Enhanced S-cone syndrome; Enlarged vestibular aqueduct; Epidermolysis bullosa; Epidermolysis bullosa dystrophica (dominant or recessive); Epidermolysis bullosa simplex; Epidermolytic hyperkeratosis; Epidermolytic palmoplantar keratoderma; Epilepsy (generalize; juvenile; myoclonic; nocturnal frontal lobe; progressive myoclonic); Epilepsy, benign, neonatal (type1 or type2); Epiphyseal dysplasia (multiple); Episodic ataxia (type 2); Episodic ataxia/myokymia syndrome; Erythremias (alpha-; dysplasia); Erythrocytosis; Erythrokeratoderma; Estrogen resistance; Exertional myoglobinuria due to deficiency of LDH-A; Exostoses, multiple (type 1; type 2); Exudative vitreoretinopathy, X-linked; Fabry disease; Factor H deficiency; Factor VII deficiency; Factor X deficiency; Factor XI deficiency; Factor XII deficiency; Factor XIIIA deficiency; Factor XIIIB deficiency; Familial Mediterranean fever; Fanconi anemia; Fanconi-Bickel syndrome; Farber lipogranulomatosis; Fatty liver (acute); Favism; Fish-eye disease; Foveal hypoplasia; Fragile X syndrome; Frasier syndrome; Friedreich ataxia; fructose-bisphosphatase Fructose intolerance; Fucosidosis; Fumarase deficiency; Fundus albipunctatus; Fundus flavimaculatus; G6PD deficiency; GABA-transaminase deficiency; Galactokinase deficiency with cataracts; Galactose epimerase deficiency; Galactosemia; Galactosialidosis; GAMT deficiency; Gardner syndrome; Gastric cancer; Gaucher disease; Generalized epilepsy with febrile seizures plus; Germ cell tumors; Gerstmann-Straussler disease; Giant cell hepatitis (neonatal); Giant platelet disorder; Giant-cell fibroblastoma; Gitelman syndrome; Glanzmann thrombasthenia (type A; type B); Glaucoma 1A; Glaucoma 3A; Glioblastoma multiforme; Glomerulosclerosis (focal segmental); Glucose transport defect (blood-brain barrier); Glucose/galactose malabsorption; Glucosidase I deficiency; Glutaricaciduria (type I; type JIB; type IIC); Gluthation synthetase deficiency; Glycerol kinase deficiency; Glycine receptor (alpha-1 polypeptide); Glycogen storage disease I; Glycogen storage disease II; Glycogen storage disease III; Glycogen storage disease IV; Glycogen storage disease VI; Glycogen storage disease VII; Glycogenosis (hepatic, autosomal); Glycogenosis (X-linked hepatic); GM1-gangliosidosis; GM2-gangliosidosis; Goiter (adolescent multinodular); Goiter (congenital); Goiter (nonendemic, simple); Gonadal dysgenesis (XY type); Granulomatosis, septic; Graves disease; Greig cephalopolysyndactylyl-syndrome; Griscelli syndrome; Growth hormone deficient dwarfism; Growth retardation with deafness and mental retardation; Gynecomastia (familial, due to increased aromatase activity); Gyrate atrophy of choroid and retina with ornithinemia (136 responsive or unresponsive); Hailey-Hailey disease; Haim-Munk syndrome; Hand-foot-uterus syndrome; Harderoporphyrinuria; HDL deficiency (familial); Heart block (nonprogressive or progressive); Heinz body anemia; HELLP syndrome; Hematuria (familial benign); Heme oxygenase-1 deficiency; Hemiplegic migraine; Hemochromotosis; Hemoglobin H disease; Hemolytic anemia due to ADA excess; Hemolytic anemia due to adenylate kinase deficiency; Hemolytic anemia due to band 3 defect; Hemolytic anemia due to glucosephosphate isomerase deficiency; Hemolytic anemia due to glutathione synthetase deficiency; Hemolytic anemia due to hexokinase deficiency; Hemolytic anemia due to PGK deficiency; Hemolytic-uremic syndrome; Hemophagocytic lymphohistiocytosis; Hemophilia A; Hemophilia B; Hemorrhagic diathesis due to factor V deficiency; Hemosiderosis (systemic, due to aceruloplasminemia); Hepatic lipase deficiency; Hepatoblastoma; Hepatocellular carcinoma; Hereditary hemorrhagic telangiectasia-1; Hereditary hemorrhagic telangiectasia-2; Hermansky-Pudlak syndrome; Heterotaxy (X-linked visceral); Heterotopia (periventricular); Hippel-Lindau syndrom; Hirschsprung disease; Histidine-rich glycoprotein Thrombophilia due to HAG deficiency; HMG-CoA lyase deficiency; Holoprosencephaly-2; Holoprosencephaly-3; Holoprosencephaly-4; Holoprosencephaly-5; Holt-Oram syndrome; Homocystinuria; Hoyeraal-Hreidarsson; HPFH (deletion type or nondeletion type); HPRT-related gout; Huntington disease; Hydrocephalus due to aqueductal stenosis; Hydrops fetalis; Hyperbetalipoproteinemia; Hypercholesterolemia, familial; Hyperferritinemia-cataract syndrome; Hyperglycerolemia; Hyperglycinemia; Hyperimmunoglobulinemia D and periodic fever syndrome; Hyperinsulinism; Hyperinsulinism-hyperammonemia syndrome; Hyperkalemic periodic paralysis; Hyperlipoproteinemia; Hyperlysinemia; Hypermethioninemia (persistent, autosomal, dominant, due to methionine, adenosyltransferase I/III deficiency); Hyperomithinemia-hyperammonemiahomociirullinernia syndrome; Hyperoxaluria; Hyperparathyroidism; Hyperphenylalaninemia due to pterin-4acarbinolamine dehydratase deficiency; Hyperproinsulinemia; Hyperprolinemia; Hypertension; Hyperthroidism (congenital); Hypertriglyceridemia; Hypoalphalipoproteinemia; Hypobetalipoproteinemia; Hypocalcemia; Hypochondroplasia; Hypochromic microcytic anemia; Hypodontia; Hypofibrinogenemia; Hypoglobulinemia and absent B cells; Hypogonadism (hypergonadotropic); Hypogonadotropic (hypogonadism); Hypokalemic periodic paralysis; Hypomagnesemia; Hypomyelination (congenital); Hypoparathyroidism; Hypophosphatasia (adult; childhood; infantile; hereditary); Hypoprothrombinemia; Hypothyroidism (congenital; hereditary congenital; nongoitrous); Ichthyosiform erythroderma; Ichthyosis; Ichthyosis bullosa of Siemens; IgG2 deficiency; Immotile cilia syndrome-1; Immunodeficiency (T-cell receptor/CD3 complex); Immunodeficiency (X-linked, with hyper-IgM); Immunodeficiency due to defect in CD3-gamma; Immunodeficiency-centromeric instabilityfacial anomalies syndrome; Incontinentia pigmenti; Insensitivity to pain (congenital, with anhidrosis); Insomnia (fatal familial); Interleukin-2 receptor deficiency (alpha chain); Intervertebral disc disease; Iridogoniodysgenesis; Isolated growth hormone deficiency (Illig type with absent GH and Kowarski type with bioinactive GH); Isovalericacidemia; Jackson-Weiss sydnrome; Jensen syndrome; Jervell and Lange-Nielsen syndrome; Joubert syndrom; Juberg-Marsidi syndrome; Kallmann syndrome; Kanzaki disease; Keratitis; Keratoderma (palmoplantar); Keratosis palmoplantaris striata I; Keratosis palmoplantaris striata II; Ketoacidosis due to SCOT deficiency; Keutel syndrome; Klippel-Trenaumay syndrom; Kniest dysplasia; Kostmann neutropenia; Krabbe disease; Kurzripp-Polydaktylie syndrom; Lacticacidemia due to PDX1 deficiency; Langer mesomelic dysplasia; Laron dwarfism; Laurence-Moon-Biedl-Bardet syndrom; LCHAD deficiency; Leber congenital amaurosis; Left-right axis malformation; Leigh syndrome; Leiomyomatosis (diffuse, with Alport syndrome); Leprechaunism; Leri-Weill dyschondrosteosis; Lesch-Nyhan syndrome; Leukemia (acute myeloid; acute promyelocytic; acute T-cell lymphoblastic; chronic myeloid; juvenile myelomonocytic; Leukemia-1 (T-cell acute lymphocytic); Leukocyte adhesion deficiency; Leydig cell adenoma; Lhermitte-Duclos syndrome; Liddle syndrome; L1-Fraumeni syndrome; Lipoamide dehydrogenase deficiency; Lipodystrophy; Lipoid adrenal hyperplasia; Lipoprotein lipase deficiency; Lissencephaly (X-linked); Lissencephaly-1; liver Glycogen storage disease (type 0); Long QT syndrome-1; Long QT syndrome-2; Long QT syndrome-3; Long QT syndrome-5; Long QT syndrome-6; Lowe syndrome; Lung cancer; Lung cancer (nonsmall cell); Lung cancer (small cell); Lymphedema; Lymphoma (B-cell non-Hodgkin); Lymphoma (diffuse large cell); Lymphoma (follicular); Lymphoma (MALT); Lymphoma (mantel cell); Lymphoproliferative syndrome (X-linked); Lysinuric protein intolerance; Machado-Joseph disease; Macrocytic anemia refractory (of 5q syndrome); Macular dystrophy; Malignant mesothelioma; Malonyl-CoA decarboxylase deficiency; Mannosidosis, (alpha-or beta-); Maple syrup urine disease (type Ia; type Ib; type II); Marfan syndrome; Maroteaux-Lamy syndrome; Marshall syndrome; MASA syndrome; Mast cell leukemia; Mastocytosis with associated hematologic disorder; McArdle disease; McCune Albright polyostotic fibrous dysplasia; McKusick-Kaufman syndrome; McLeod phenotype; Medullary thyroid carcinoma; Medulloblastoma; Meesmann corneal dystrophy; Megaloblastic anemia-1; Melanoma; Membroproliferative glomerulonephritis; Meniere disease; Meningioma (NF2-related; SIS-related); Menkes disease; Mental retardation (X-linked); Mephenyloin poor metabolizer; Mesothelioma; Metachromatic leukodystrophy; Metaphyseal chondrodysplasia (Murk Jansen type; Schmid type); Methemoglobinemia; Methionine adenosyltransferase deficiency (autosomal recessive); Methylcobalamin deficiency (cbl G type); Methylmalonicaciduria (mutase deficiency type); Mevalonicaciduria; MHC class II deficiency; Microphthalmia (cataracts, and iris abnormalities); Miyoshi myopathy; MODY; Mohr-Tranebjaerg syndrome; Molybdenum cofactor deficiency (type A or type. B); Monilethrix; Morbus Fabry; Morbus Gaucher; Mucopolysaccharidosis; Mucoviscidosis; Muencke syndrome; Muir-Tone syndrome; Mulibrey nanism; Multiple carboxylase deficiency (biotinresponsive); Multiple endocrine neoplasia; Muscle glycogenosis; Muscular dystrophy (congenital merosindeficient); Muscular dystrophy (Fukuyama congenital); Muscular dystrophy (limb-girdle); Muscular dystrophy) Duchenne-like); Muscular dystrophy with epidermolysis bullosa simplex; Myasthenic syndrome (slow-channel congenital); Mycobacterial infection (atypical, familial disseminated); Myelodysplastic syndrome; Myelogenous leukemia; Myeloid malignancy; Myeloperoxidase deficiency; Myoadenylate deaminase deficiency; Myoglobinuria/hemolysis due to PGK deficiency; Myoneurogastrointestinal encephalomyopathy syndrome; Myopathy (actin; congenital; desmin-related; cardioskeletal; distal; nemaline); Myopathy due to CPT II deficiency; Myopathy due to phosphoglycerate mutase deficiency; Myotonia congenita; Myotonia levior; Myotonic dystrophy; Myxoid liposarcoma; NAGA deficiency; Nailpatella syndrome; Nemaline myopathy 1 (autosomal dominant); Nemaline myopathy 2 (autosomal recessive); Neonatal hyperparathyroidism; Nephrolithiasis; Nephronophthisis (juvenile); Nephropathy (chronic hypocomplementemic); Nephrosis-1; Nephrotic syndrome; Netherton syndrome; Neuroblastoma; Neurofibromatosis (type 1 or type 2); Neurolemmomatosis; neuronal-5 Ceroid-lipofuscinosis; Neuropathy; Neutropenia (alloimmune neonatal); Niemann-Pick disease (type A; type B; type C1; type D); Night blindness (congenital stationary); Nijmegen breakage syndrome; Noncompaction of left ventricular myocardium; Nonepidermolytic palmoplantar keratoderma; Norrie disease; Norum disease; Nucleoside phosphorylase deficiency; Obesity; Occipital homsyndrome; Ocular albinism (Nettleship-Falls type); Oculopharyngeal muscular dystorphy; Oguchi disease; Oligodontia; Omenn syndrome; Opitz G syndrome; Optic nerve coloboma with renal disease; Ornithine transcarbamylase deficiency; Oroticaciduria; Orthostatic intolerance; OSMED syndrome; Ossification of posterior longitudinal ligament of spine; Osteoarthrosis; Osteogenesis imperfecta; Osteolysis; Osteopetrosis (recessive or idiopathic); Osteosarcoma; Ovarian carcinoma; Ovarian dysgenesis; Pachyonychia congenita (Jackson-Lawler type or Jadassohn-Lewandowsky type); Paget disease of bone; Pallister-Hall syndrome; Pancreatic agenesis; Pancreatic cancer; Pancreatitis; Papillon-Lefevre syndrome; Paragangliomas; Paramyotonia congenita; Parietal foramina; Parkinson disease (familial or juvenile); Paroxysmal nocturnal hemoglobinuria; Pelizaeus-Merzbacher disease; Pendred syndrome; Perineal hypospadias; Periodic fever; Peroxisomal biogenesis disorder; Persistent hyperinsulinemic hypoglycemia of infancy; Persistent Mullerian duct syndrome (type II); Peters anomaly; Peutz-Jeghers syndrome; Pfeiffer syndrome; Phenylketonuria; Phosphoribosyl pyrophosphate synthetaserelated gout; Phosphorylase kinase deficiency of liver and muscle; Piebaldism; Pilomatricoma; Pinealoma with bilateral retinoblastoma; Pituitary ACTH secreting adenoma; Pituitary hormone deficiency; Pituitary tumor; Placental steroid sulfatase deficiency; Plasmin inhibitor deficiency; Plasminogen deficiency (types I and II); Plasminogen Tochigi disease; Platelet disorder; Platelet glycoprotein IV deficiency; Platelet-activating factor acetylhydrolase deficiency; Polycystic kidney disease; Polycystic lipomembranous osteodysplasia with sclerosing leukenencephalophathy; Polydactyl), postaxial; Polyposis; Popliteal pterygium syndrome; Porphyria (acute hepatic or acute intermittent or congenital erythropoietic); Porphyria cutanea tarda; Porphyria hepatoerythropoietic; Porphyria variegata; Prader-Willi syndrome; Precocious puberty; Premature ovarian failure; Progeria Typ I; Progeria Typ II; Progressive external ophthalmoplegia; Progressive intrahepatic cholestasis-2; Prolactinoma (hyperparathyroidism, carcinoid syndrome); Prolidase deficiency; Propionicacidemia; Prostate cancer; Protein S deficiency; Proteinuria; Protoporphyria (erythropoietic); Pseudoachondroplasia; Pseudohermaphroditism; Pseudohypoaldosteronism; Pseudohypoparathyroidism; Pseudovaginal perineoscrotal hypospadias; Pseudovitamin D deficiency rickets; Pseudoxanthoma elasticum (autosomal dominant; autosomal recessive); Pulmonary alveolar proteinosis; Pulmonary hypertension; Purpura fulminans; Pycnodysostosis; Pyropoikilocytosis; Pyruvate carboxylase deficiency; Pyruvate dehydrogenase deficiency; Rabson-Mendenhall syndrome; Refsum disease; Renal cell carcinoma; Renal tubular acidosis; Renal tubular acidosis with deafness; Renal tubular acidosis-osteopetrosis syndrome; Reticulosis (familial histiocytic); Retinal degeneration; Retinal dystrophy; Retinitis pigmentosa; Retinitis punctata albescens; Retinoblastoma; Retinal binding protein deficiency; Retinoschisis; Rett syndrome; Rh(mod) syndrome; Rhabdoid predisposition syndrome; Rhabdoid tumors; Rhabdomyosarcoma; Rhabdomyosarcoma (alveolar); Rhizomelic chondrodysplasia punctata; Ribbing-Syndrom; Rickets (vitamin D-resistant); Rieger anomaly; Robinow syndrome; Rothmund-Thomson syndrome; Rubenstein-Taybi syndrome; Saccharopinuria; Saethre-Chotzen syndrome; Salla disease; Sandhoff disease (infantile, juvenile, and adult forms); Sanfilippo syndrome (type A or type B); Schindler disease; Schizencephaly; Schizophrenia (chronic); Schwannoma (sporadic); SCID (autosomal recessive, T-negative/Bpositive type); Secretory pathway w/TMD; SED congenita; Segawa syndrome; Selective T-cell defect; SEMD (kistani type); SEMD (Strudwick type); Septooptic dysplasia; Severe combined immunodeficiency (B cellnegative); Severe combined immunodeficiency (T-cell negative, B-cell/natural killer cell-positive type); Severe combined immunodeficiency (Xlinked); Severe combined immunodeficiency due to ADA deficiency; Sex reversal (XY, with adrenal failure); Sezary syndrome; Shah-Waardenburg syndrome; Short stature; Shprintzen-Goldberg syndrome; Sialic acid storage disorder; Sialidosis (type I or type II); Sialuria; Sickle cell anemia; Simpson-Golabi-Behmel syndrome; Situs ambiguus; Sjogren-Larsson syndrome; Smith-Fineman-Myers syndrome; Smith-Lemli-Opitz syndrome (type I or type II); Somatotrophinoma; Sorsby fundus dystrophy; Spastic paraplegia; Spherocytosis; Spherocytosis-1; Spherocytosis-2; Spinal and bulbar muscular atrophy of Kennedy; Spinal muscular atrophy; Spinocerebellar ataxia; Spondylocostal dysostosis; Spondyloepiphyseal dysplasia tarda; Spondylometaphyseal dysplasia (Japanese type); Stargardt disease-1; Steatocystoma multiplex; Stickler syndrome; Sturge-Weber syndrorn; Subcortical laminal heteropia; Subcortical laminar heterotopia; Succinic semialdehyde dehydrogenase deficiency; Sucrose intolerance; Sutherland-Haan syndrome; Sweat chloride elevation without CF; Symphalangism; Synostoses syndrome; Synpolydactylyl-; Tangier disease; Tay-Sachs disease; T-cell acute lymphoblastic leukemia; T-cell immunodeficiency; T-cell prolymphocytic leukemia; Thalassemia (alpha-or delta-); Thalassemia due to Hb Lepore; Thanatophoric dysplasia (types I or II); Thiamine-responsive megaloblastic anemia syndrome; Thrombocythemia; Thrombophilia (dysplasminogenemic); Thrombophilia due to heparin cofactor II deficiency; Thrombophilia due to protein C deficiency; Thrombophilia due to thrombomodulin defect; Thyroid adenoma; Thyroid hormone resistance; Thyroid iodine peroxidase deficiency; Tietz syndrome; Tolbutamide poor metabolizer; Townes-Brocks syndrome; Transcobalamin II deficiency; Treacher Collins mandibulofacial dysostosis; Trichodontoosseous syndrome; Trichorhinophalangeal syndrome; Trichothiodystrophy; Trifunctional protein deficiency (type I or type II); Trypsinogen deficiency; Tuberous sclerosis-1; Tuberous sclerosis-2; Turcot syndrome; Tyrosine phosphatase; Tyrosinemia; Ulnar-mammary syndrome; Urolithiasis (2,8-dihydroxyadenine); Usher syndrome (type 1B or type 2A); Venous malformations; Ventricular tachycardia; Virilization; Vitamin K-dependent coagulation defect; VLCAD deficiency; Vohwinkel syndrome; von Hippel-Lindau syndrome; von Willebrand disease; Waardenburg syndrome; Waardenburg syndrome/ocular albinism; Waardenburg-Shah neurologic variant; Waardenburg-Shah syndrome; Wagner syndrome; Warfarin sensitivity; Watson syndrome; Weissenbacher-Zweymuller syndrome; Werner syndrome; Weyers acrodental dysostosis; White sponge nevus; Williams-Beuren syndrome; Wilms tumor (type1); Wilson disease; Wiskott-Aldrich syndrome; Wolcott-Rallison syndrome; Wolfram syndrome; Wolman disease; Xanthinuria (type I); Xeroderma pigmentosum; X-SCID; Yemenite deaf-blind hypopigmentation syndrome; ypocalciuric hypercalcemia (type I); Zellweger syndrome; Ziotogora-Ogur syndrome.
  • Diseases to be treated in the context of the present invention likewise also include diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws are preferably selected from the group consisting of autosomal-recessive inherited diseases, such as, for example, adenosine deaminase deficiency, familial hypercholesterolaemia, Canavan's syndrome, Gaucher's disease, Fanconi anaemia, neuronal ceroid lipofuscinoses, mucoviscidosis (cystic fibrosis), sickle cell anaemia, phenylketonuria, alcaptonuria, albinism, hypothyreosis, galactosaemia, alpha-1-anti-trypsin deficiency, Xeroderma pigmentosum, Ribbing's syndrome, mucopolysaccharidoses, cleft lip, jaw, palate, Laurence Moon Biedl Bardet sydrome, short rib polydactylia syndrome, cretinism, Joubert's syndrome, type II progeria, brachydactylia, adrenogenital syndrome, and X-chromosome inherited diseases, such as, for example, colour blindness, e.g. red/green blindness, fragile X syndrome, muscular dystrophy (Duchenne and Becker-Kiener type), haemophilia A and B, G6PD deficiency, Fabry's disease, mucopolysaccharidosis, Norrie's syndrome, Retinitis piyinentosa, septic granulomatosis, X-SCID, ornithine transcarbamylase deficiency, Lesch-Nyhan syndrome, or from autosomal-dominant inherited diseases, such as, for example, hereditary angiooedema, Marfan syndrome, neurofibromatosis, type I progeria, Osteogenesis imperfecta, Klippel-Trenaurnay syndrome, Sturge-Weber syndrome, Hippel-Lindau syndrome and tuberosis sclerosis.
  • The present invention also allows treatment of diseases, which have not been inherited, or which may not be summarized under the above categories. Such diseases may include e.g. the treatment of patients, which are in need of a specific protein factor, e.g. a specific therapeutically active protein as mentioned above. This may e.g. include dialysis patients, e.g. patients which undergo a (regular) a kidney or renal dialysis, and which may be in need of specific therapeutically active proteins as defined herein, e.g. erythropoietin (EPO), etc.
  • Likewise, diseases in the context of the present invention may include cardiovascular diseases chosen from, without being limited thereto, coronary heart disease, arteriosclerosis, apoplexy and hypertension, etc.
  • Finally, diseases in the context of the present invention may be chosen from neuronal diseases including e.g. Alzheimer's disease, amyotrophic lateral sclerosis, dystonia, epilepsy, multiple sclerosis and Parkinson's disease etc.
  • According to a final embodiment, the present invention also provides kits, particularly kits of parts, comprising as components alone or in combination with further ingredients at least one inventive polymeric carrier molecule according to generic formula (I) or (Ia) or according to any of subformulas thereof as defined herein, at least one inventive polymeric carrier cargo complex formed by the nucleic acid cargo and the inventive polymeric carrier molecule, at least one nucleic acid as defined herein, at least one pharmaceutical composition comprising same and/or kits comprising same, and optionally technical instructions with information on the administration and dosage of the inventive polymeric carrier molecule, the nucleic acid, the inventive polymeric carrier complex, and/or the inventive pharmaceutical composition. Such kits, preferably kits of parts, may be applied, e.g., for any of the above mentioned applications or uses. Such kits, when occurring as a kit of parts, may further contain each component in a different part of the kit.
  • FIGURES
  • The following Figures are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.
  • FIG. 1: shows the results of the confocal microscopy of L929 cells 5 minutes after transfection with fluorescence labelled RNA complexed with the peptide PB19 in a molar ratio of 1:500. As a result, several complexes are detectable in the cells already 5 minutes after transfection indicating a good transfection rate.
  • FIG. 2: shows the results of the confocal microscopy of L929 cells 1 hour after transfection with fluorescence labelled RNA complexed with the peptide PB19 in a molar ratio of 1:500. As a result, most of the particles were taken up in the cells 1 hour after transfection showing a good transfection rate.
  • FIG. 3: illustrates stability experiments with regard to the electrostatic displacement of the bound nucleic acid from the complex. As can be seen the addition of the anionic polymer heparin can not displace the RNA from the complex because it still migrates in the gel. This indicates that the complex binding is so strong that a competitive complex partner cannot displace the RNA from the complex.
  • FIG. 4: depicts a gel shift assay to examine the strength of complex binding. It could be shown that the addition of the anionic polymer heparin or the reducing agent DTT alone cannot display the RNA from the complex with the polymer according to the invention (PB19). Only together they are able to displace the RNA from the complex (lane 7). This indicates that the complex binding is so strong that neither a competitive complex partner nor a reducing agent can displace the RNA from the complex.
  • FIG. 5: shows a gel shift assay to examine the strength of complex binding. As can be seen, the addition of the anionic polymer heparin alone cannot displace the RNA from the complex with polymers according to the invention (PB22). In contrast mRNA could be readily displayed by heparin from PEI complexes.
  • FIG. 6: depicts the results from expression experiments with of luciferase encoding mRNA according to SEQ ID NO: 77 in HeLa cells. As can be seen, formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 77 with the P1319 peptide (molar ratio of RNA:PB19 1:1000, 1:500, 1:100) lead to expression of luciferase independently of the presence of serum containing medium. These results are unexpected because serum containing medium leads in general to a loss of transfection efficiency.
  • FIG. 7A: illustrates the results from expression experiments with luciferase encoding mRNA according to SEQ ID NO: 77 in BALB/c mice after intradermal injection. As a result, the formulation of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 77 with the PB19 polymer leads to expression of luciferase in the dermis of female BALB/c mice. Other transfection reagents known in the art did not showed any expression of the Luciferase protein (see FIG. 7A).
  • FIG. 7B: illustrates the results from expression experiments with luciferase encoding mRNA according to SEQ ID NO: 77 in BALB/c mice after intramuscular injection. As a result, formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 77 with the P1319 polymer leads to expression of luciferase in the m. tibialis of female BALB/c mice. Other transfection reagents known in the art did not showed any expression of the Luciferase protein.
  • FIG. 8: shows the expression of luciferase in BALB/c mice after intradermal injection of different formulations using formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 77 with two different polymers according to the invention [PB19 and PB48]. These formulations of mRNA coding for luciferase (luc-RNActive) according to SEQ ID NO: 77 with two different polymers according to the invention [PB19 and PB48] lead to expression of luciferase in the dermis of female BALB/c mice. The inventive polymeric carrier cargo complex formed by a peptide according to RPC CH6R4H6C (without PEGylation) and RNA in a molar ratio of 2500:1 procedure showed no expression of luciferase after intradermal injection of the complexed RNA.
  • FIG. 9: depicts the secretion of h IL-6 cytokine in hPBMCs. It could be shown that the polymers according to the invention (P819 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA and cationic peptides (H3R9H3) or PEGylated cationic peptides (which confers in general to subsequent hydrophilic coating of pre formed nucleic acid condensates). The polymers used were:
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • FIG. 10: illustrates the secretion of h TNFα Cytokine in hPBMCs. It could be shown that the polymers according to the invention (P819 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA and cationic peptides (H3R9H3) or such complexes coated with PEGylated peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). The polymers used were:
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • FIG. 11: illustrates the secretion of h TNFα Cytokine in hPBMCs. It could be shown that the polymers according to the invention (PB19 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA and cationic peptides (H3R9H3) or such complexes coated with PEGylated peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates). The polymers used were:
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • FIG. 12A: shows the secretion of h IFNa cytokine in hPBMCs in a comparison of cytokine stimulating properties of polymers according to the invention to state of the art transfection reagent like Lipofectamin or PEI. As can be seen, both transfection reagents Lipofectamin and PEI lead to a high amount of secretion of hIFNa, whereas polymers according to the invention do not.
  • FIG. 12B: shows the secretion of h TFNa cytokine secretion in hPBMCs in a comparison of cytokine stimulating properties of polymers according to the invention to state of the art transfection reagent like Lipofectamin or PEI. As can be seen, Lipofectamine leads to a high amount of secretion of h TFNa.
  • FIG. 13: shows the mRNA sequence encoding Photinus pyralis luciferase (SEQ ID NO: 77) in the mRNA construct pCV19-Pp luc(GC)-muag-A70-C30; which exhibits a length of 1857 nucleotides. The mRNA sequence contains following sequence elements:
      • the coding sequence encoding Photinus pyralis luciferase;
      • stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR));
      • 70×adenosine at the 3′-terminal end (poly-A-tail);
      • 30×cytosine at the 3′-terminal end (poly-C-tail).
      • The ORF is indicated in italic letters, muag (mutated alpha-globin-3′-UTR is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.
  • FIG. 14: shows the mRNA sequence encoding Ovalbumin; which exhibits a length of 1365 nucleotides. The mRNA sequence contains following sequence elements:
      • the coding sequence encoding Ovalbumin;
      • stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR));
      • 70×adenosine at the 3′-terminal end (poly-A-tail);
      • 30×cytosine at the 3′-terminal end (poly-C-tail).
      • The ORF is indicated in italic letters, muag (mutated alpha-globin-3′-UTA is indicated with a dotted line, the poly-A-tail is underlined with a single line and the poly-C-tail is underlined with a double line.
    EXAMPLES
  • The following examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.
  • 1. Preparation of DNA and mRNA Constructs Encoding Pp Luciferase (Photinus Pyralis)
  • For the present examples DNA sequences, encoding Photinus pyralis luciferase, and corresponding mRNA sequences encoding same, were prepared and used for subsequent in vitro transcription reactions and stability studies.
  • According to a first preparation, the DNA sequence and corresponding mRNA sequence termed pCV19-Ppluc(GC)-muag-A70-C30 sequence was prepared, which corresponds to the Photinus pyralis luciferase coding sequence. The constructs were prepared by modifying the wildtype Photinus pyralis luciferase encoding DNA sequence by introducing a GC-optimized sequence for a better codon usage and stabilization, stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTA)), a stretch of 70×adenosine at the 3′-terminal end (poly-A-tail) and a stretch of 30×cytosine at the 3′-terminal end (poly-C-tail), leading to SEQ ID NO: 77 (see FIG. 13). The sequence of the final DNA construct had a length of 1857 nucleotides and was termed “pCV19-Ppluc(GC)-muag-A70-C30”. In SEQ ID NO: 77 (see FIG. 13) the sequence of the corresponding mRNA is shown.
  • The sequence contains following sequence elements:
      • the coding sequence encoding Photinus pyralis luciferase;
      • stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR));
      • 70×adenosine at the 3′-terminal end (poly-A-tail);
      • 30×cytosine at the 3′-terminal end (poly-C-tail).
        2. Preparation of DNA and mRNA Constructs Encoding Ovalbumin
  • Furthermore, for the present examples DNA sequences, encoding Ovalbumin, and corresponding mRNA sequences encoding same, were prepared and used for subsequent in vitro transcription reactions and stability studies.
  • Thus, according to a second preparation, the DNA sequence and corresponding mRNA sequence termed CAP-GgOva(GC)-muag-A70-C30 DNA sequence was prepared, which corresponds to the Ovalbumin coding sequence. Therefore, a basic DNA construct was prepared termed CAP-GgOva(GC)-muag-A70-C30 by introducing into the underlying wildtype sequence construct stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR)), a stretch of 70×adenosine at the 3′-terminal end (poly-A-tail) and a stretch of 30×cytosine at the 3′-terminal end (poly-C-tail), leading to a sequence according to SEQ ID NO: 78 (see FIG. 14). SEQ ID NO: 78 (see FIG. 14) shows the corresponding mRNA sequence.
  • The sequence contains following sequence elements:
      • the coding sequence encoding Ovalbumin;
      • stabilizing sequences derived from alpha-globin-3′-UTR (muag (mutated alpha-globin-3′-UTR));
      • 70×adenosine at the 3′-terminal end (poly-A-tail);
      • 30×cytosine at the 3′-terminal end (poly-C-tail).
    3. In Vitro Transcription:
  • The respective DNA plasmids prepared according to Example 1 and 2 were transcribed in vitro using T7-Polymerase (T7-Opti mRNA Kit, CureVac, Tubingen, Germany) following the manufactures instructions. Subsequently the mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany).
  • 4. Reagents:
  • Peptides: The peptides used in the present experiments were as follows:
  • PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-
    S-PEG5000-OH
    (pegylated CH3R4H3C peptide polymer)
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
    (pegylated CH6R4H6C peptide polymer)
    PB48: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)3-
    S-PEG5000-OH
    H3R9H3: HHHRRRRRRRRRHHH
    CH6R4H6C: H-(S-CHHHHHHRRRRHHHHHHC-S)5-H
  • Further tranfection reagents used are:
  • Lipofectamine (Invitrogen)
  • PEI 25 kDa (branched) (Aldrich)
  • 4. Synthesis of the Polymers:
  • The condensation reaction was performed with the calculated amount of peptide which is dissolved in a mixture of a buffered aqueous solution at pH 8.5 with an additive 5% (v/v) Dimethylsulfoxide (DMSO) and stirred for 18 h at ambient temperature. Afterwards the calculated amount of a thiol group containing PEG derivative (dissolved in water) is added and the resulting solution is stirred for another 18 h. Subsequent lyophilisation and purification yield the desired polymer.
  • The condensation reaction in this reaction environment is reversible, therefore the chain length of the polymer is determined by the amount of the monothiol compound which terminates the polymerisation reaction. In summary the length of the polymer chain is determined by the ratio of oligo-peptide and monothiol component,
  • 4.1. 1. Step: Polymerization:
  • n HS-CHHHRRRHHHC-SH → H-(S-CHHHRRRRHHHC-S)n-H
  • 4.2. 2. Step: Stop Reaction:
  • H-(S-CHHHRRRRHHHC-S)n-H + 2 PEG-SH → PEG-S-
    (S-CHHHRRRRHHHC-S)n-S-PEG
  • 4.3. Exemplary Synthesis Reaction:
  • step 1)
    5 x HS-CHHHRRRHHHC-SH → H-(S-CHHHRRRRHHHC-S)5-H
    step 2)
    H-(S-CHHHRRRRHHHC-S)5-H + 2x PEG5000-SH → PEG5000-
    S-(S-CHHHRRRRHHHC-S)5-S-PEG5000
  • To achieve a polymer length of 5 a molar ratio of peptide:PEG of 5:2 was used.
  • 5. Complexation of RNA:
  • The mRNA constructs defined above in Examples 1 and 2 were complexed for the purposes of the present invention with the polymers, preferably as defined in Example 4. Therefore, 4 μg RNA coding for luciferase pCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive) according to SEQ ID NO: 77 were mixed in molar ratios as indicated with the inventive polymer (according to formula I) or a control, thereby forming a complex. Afterwards the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 minutes at room temperature.
  • The different polymers and the different ratios of polymer/RNA used in this experiment are shown in table 1.
  • Cationic
    Präfix Polymer Ratio AS N/P
    PB19 HO—PEG5000—S—(S—CHHHRRRRHHHC—S)5—S—PEG5000—OH 500 20 5.6
    PB19 HO—PEG5000—S—(S—CHHHRRRRHHHC—S)5—S—PEG5000—OH 250 20 2.8
    PB19 HO—PEG5000—S—(S—CHHHRRRRHHHC—S)5—S—PEG5000—OH  50 20 0.6
    PB48 HO—PEG5000—S—(S—CHHHRRRRHHHC—S)3—S—PEG5000—OH 250 12 1.67
    PB76 HO—PEG5000—S—(S—CHHHHHHRRRRHHHHHHC—S)10—S—PEG5000—OH 500 40 11.2
    PB76 HO—PEG5000—S—(S—CHHHHHHRRRRHHHHHHC—S)10—S—PEG5000—OH 250 40 5.6
    PB76 HO—PEG5000—S—(S—CHHHHHHRRRRHHHHHHC—S)10—S—PEG5000—OH  50 40 1.1
    Ratio = molar ratio of RNA: peptide
    cationic AS = cationic amino acids, which are positively charged at a physiological pH (i.e. not histidine (H) but e.g. arginine (R)) Whereas PB76 has a cationic insert that is double in size compared to PB19, therefore has double as much cationic amino acid residues. PB48 has a shorter cationic insert compared to PB19, therefore has less cationic amino acid residues per molecule polymer.
    N/P = is the ratio of basic nitrogen atoms to phosphate residues, considering that nitrogen atoms confer to positive charges and phosphate of the phosphate backbone of the nucleic acid confers to the negative charge. Histidine residues are counted neutral, because complex formation is done at physiological pH, therefore the imidazole residue is uncharged.
    N/P is calculated by the following formula:
    N / P = pmol [ RNA ] * ratio * cationic AS μg RNA * 3 * 1000 For the calculations RNA coding for luciferase pCV19-Ppluc(GC)-muag-A70-C30 (Luc-RNActive) according to SEQ ID NO: 77 was applied, which has a molecular weight of 660 kDa. Therefore 1 μg pCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 77 confers to 1.67 pmol pCV19-Ppluc(GC)-muag-A70-C30 RNA according to SEQ ID NO: 77.
  • 6. Size and Zetapotential Measurements:
  • The hydrodynamic diameters of polyplexes as prepared above were measured by dynamic light scattering using a Zetasizer Nano (Malvern Instruments, Malvern, UK) according to the SOPs distributed by Malvern. The measurements were performed at 25° C. in, the specified buffer analysed by a cumulant method to obtain the hydrodynamic diameters and polydispersity indices of the polyplexes.
  • The Zeta potential of the polyplexes was evaluated by the laser Doppler electrophoresis method using a Zetasizer Nano (Malvern Instruments, Malvern, UK). The measurement was performed at 25° C. and a scattering angle of 173° was used.
  • 7. Gel Shift Assay
  • Furthermore, mRNA coding for luciferase (Luc-RNActive) according to SEQ ID NO: 77 was formulated with the polymers as indicated and aliquots were incubated with either heparin (100 μg) or Dithiothreitol (DTT) for 15 Minutes at 37° C. Afterwards electrophoresis was done on agarose gel and the nucleic acids were visualized by ethidium bromide staining.
  • 8. Confocal Laser Scanning Microscopy
  • Confocal laser scanning microscopy was performed on an inverted LSM510 laser scanning microscope (Carl Zeiss, Germany) using a Plan-Apochromat 63x/1.4 N.A. lens. All analyses were performed with living, nonfixed cells grown in eight-well chambered cover slides (Nunc, Germany). For the detection of Alexa Fluor 647 labelled mRNA only the light of a 633-nm helium neon laser, directed over a UV/488/543/633 beam splitter in combination with a LP 650 long pass filter was used. Life cell microscopy was performed at room temperature.
  • For this purpose, L929 cells (25×103/well) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out. Cells were transfected with formulations containing 2 μg Alexa Fluor 647 labelled mRNA in 8 chamber well slides directly before conduction of the microscopy experiment.
  • 8. Cytokinstimulation in hPBMC
  • HPBMC cells from peripheral blood of healthy donors were isolated using a Ficoll gradient and washed subsequently with 1×PBS (phophate-buffered saline). The cells were then seeded on 96-well microtiter plates (200×103/well). The hPBMC cells were incubated for 24 h with 10 μl of the RNA/peptide complex in X-VIVO 15 Medium (BioWhittaker). As RNA, mRNA of either SEQ ID NO: 77 or 78 were used. The peptides were as shown above for generic formula (I). The immunostimulatory effect upon the hPBMC cells was measured by detecting the cytokine production (Interleukin-6; Tumor necrose factor alpha, Interferon alpha). Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated over night (o/n) with binding buffer (0.02% NaN3, 15 mM Na2CO3, 15 mM NaHCO3, pH 9.7), additionally containing a specific cytokine antibody. Cells were then blocked with 1×PBS, containing 1% BSA (bovine serum albumin). The cell supernatant was added and incubated for 4 h at 37° C. Subsequently, the microtiter plate was washed with 1×PBS, containing 0.05% Tween-20 and then incubated with a Biotin-labelled secondary antibody (BD Pharmingen, Heidelberg, Germany). Streptavidin-coupled horseraddish peroxidase was added to the plate. Then, the plate was again washed with 1×PBS, containing 0.05% Tween-20 and ABTS (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as a substrate. The amount of cytokine was determined by measuring the absorption at 405 nm (OD 405) using a standard curve with recombinant cytokines (BD Pharmingen, Heidelberg, Germany) with the Sunrise ELISA-Reader from Tecan (Crailsheim, Germany).
  • 9. Transfection of HeLa Cells:
  • 4 μg RNA stabilized luciferase mRNA (Luc-RNActive) according to SEQ ID NO: 77 were mixed in molar ratios as indicated with the respectively polymer (according to formula I), thereby forming a complex. Afterwards the resulting solution was adjusted with water to a final volume of 50 μl and incubated for 30 minutes at room temperature. The used ratios are indicated in table 1 above.
  • Hela-cells (150×103/well) were seeded 1 day prior to transfection on 24-well microtiter plates leading to a 70% confluence when transfection was carried out. For transfection 50 μl of the RNA/(peptide)-solution were mixed with 250 μl serum free or FCS containing medium (as indicated in the provided table) and added to the cells (final RNA concentration: 13 μg/ml). Prior to addition of the serum free transfection solution the HeLa-cells were washed gently and carefully 2 times with 1 ml Optimen (Invitrogen) per well. Then, the transfection solution (300 μl per well) was added to the cells and the cells were incubated for 4 h at 37° C. Subsequently 300 μl RPMI-medium (Carnprex) containing 10% FCS was added per well and the cells were incubated for additional 20 h at 37° C. The transfection solution was sucked off 24 h after transfection and the cells were lysed in 300 μl lysis buffer (25 mM Tris-PO4, 2 mM EDTA, 10% glycerol, 1% Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO4, 5 mM ATP, 62.5 μM luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).
  • 10. Expression of Luciferase In Vivo:
  • 10 μg mRNA coding for luciferase (Luc-RNActive) were mixed in the indicated molar ratio with the respectively peptide (according to formula I), thereby forming a complex. Afterwards the resulting solution was adjusted with Ringer Lactate solution to a final volume of 100 μl und incubated for 30 minutes at room temperature, yielding a solution with a 0.1 g/l concentration of complexed RNA.
  • 100 μl (20 μI) of this solution was administrated intradermally (ear pinna or back) or intramuscularly (m. tibialis) of 7 week old BALB/c mice. After 24 h the mice were sacrificed and the samples (ear, skin from the back or muscle) were collected, frozen at −78° C. and lysed for 3 Minutes at full speed in a tissue lyser (Qiagen, Hilden, Germany). Afterwards 600 μl of lysis buffer were added and the resulting solutions were subjected another 6 minutes at full speed in the tissue lyser. After 10 minutes centrifugation at 13500 rpm at 4° C. the supernatants were mixed with luciferin buffer (25 mM Glycylglycin, 15 mM MgSO4, 5 mM ATP, 62.5 μM luciferin) and luminiscence was detected using a luminometer (Lumat LB 9507 (Berthold Technologies, Bad Wildbad, Germany)).
  • 11. Detection of Antigen-Specific Antibodies:
  • C57 BL/6 mice were vaccinated 2 times on day 1 and day 15 with 20 μg formulated RNActive-derived mRNA coding for Gallus gallus Ovalbumine in 100 μl RiLa solution. 1 week after the last vaccination blood samples were collected and expression of Ovalbumine-specific antibodies was determined.
  • MaxiSorb plates (Nalgene Nunc International) were coated with Antigen (Ovalbumine-specific peptide). After blocking with 1×PBS, 0.05% Tween und 1% BSA the plates were incubated with sera of the mice. Subsequently the biotin-coupled secondary antibody was added. After washing the plate was incubated with horseradish peroxidise and the enzyme activity was determined by measuring the conversion of the substrate ABTS substrate (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (OD 405 nm).
  • 12. Results: 12.1. DLS/Zetasizer Determinations:
  • The size and ζ-potential of the polyplexes according to the invention were evaluated in triplicates by dynamic light scattering (DLS) and laser-doppler electrophoresis and compared to different polyplexes known in the state of the art. Table 1 summarizes the cumulant diameters and the ζ-potential of the polyplexes.
  • Cumulant
    diameter in Cumulant diameter ζ-potential
    Polyplex N/P water (nm) in ringer lactate (nm) (mV)
    PB19 6 58 ± 2  81 ± 3  −9 mV
    PEI 10 88 ± 4 110 ± 2 +20 mV
    RPC like 5 170 ± 8  >1000 +39 mV
    polymer
    CH6R4H6C
  • The cumulant diameters of the polyplexes formed by polymers according to the invention showed that small, uniform complexes were formed. These complexes are stable against agglomeration in salt containing buffer and are less than 100 nm in size. In contrast polyplexes according to the RPC procedure are unstable in salt containing buffers, forming large aggregates. The polymers according to the invention also form complexes of low ζ-potential, which is linked to a low tendency in binding components of the serum, and therefore have a low tendency for opsonisation.
  • 12.2. Confocal Microscopy:
  • The transfection of L929 cells with AlexaFlour647 aminoallyl-labelled RNA complexed with the peptide PB19 after 5 minutes already led to detectable complexes in the cell. The results are shown in FIGS. 1 and 2. As can be seen in FIG. 1, confocal microscopy of L929 cells 5 minutes after transfection with fluorescence labelled RNA (SEQ ID NO: 77) complexed with the peptide PB19 in a molar ratio of 1:500 showed that already 5 minutes after transfection several complexes are detectable in the cells After 1 h confocal microscopy of L929 cells 1 h after transfection with fluorescence labelled RNA complexed with the peptide PB19 in a ratio of 1:500 revealed that after transfection most of the particles were taken up in the cells
  • 12.3. Stability Towards Electrostatic Displacement
  • Since the complexes of the resent invention, particularly the polymers according to formula (I) are unique with respect to their composition and their surface charge, unexpected results could be observed in gel shift assays. Normally, it is determined, as to whether a polyplex condenses the nucleic acid and thus prevents the nucleic acid to migrate in an electrical field. If a complex partner is added, which exhibits a stronger affinity for the cation than the nucleic acid, the nucleic acid is displaced from the complex and again can migrate in an electrical field. For this purpose, PEI may be used, which is known to exhibit extremely strong complexes with nucleic acid.
  • In a gel shift assay to examine the strength of complex binding (see FIG. 3) it could be shown that the addition of the anionic polymer heparin can not displace the RNA from the complex because it still migrates in the gel. This indicates that the complex binding is so strong that a competitive complex partner cannot displace the RNA from the complex.
  • In a further a gel shift assay to examine the strength of complex binding (see FIG. 4) it could be shown that the addition of the anionic polymer heparin or the reducing agent DTT alone cannot display the RNA from the complex with the polymer according to the invention (PB19). Only together they are able to display the RNA from the complex (lane 7). This indicates that the complex binding is so strong that neither a competitive complex partner nor a reducing agent can displace the RNA from the complex.
  • Contrary to PEI complexes, the RNA is not released from the complex upon addition of heparin. Only a combination of heparin and DTT releases the RNA. It is to be noted that DTT reduces disulfide bonds and thus destroys the conjugate. This imitates in vivo conditions, where the reducing conditions in the cell releases the RNA from the complex.
  • In comparison thereto, Gel shift assays with PEI complexes to examine the strength of complex binding show that the addition of the anionic polymer heparin alone cannot displace the RNA from the complex with polymers used according to the present invention (PB22). In contrast mRNA could be readily displayed by heparin from PEI complexes (see FIG. 5).
  • 12.4. Expression of Luciferase in HeLa Cells:
  • The expression of luciferase in HeLa cells was determined using a complex of a polymer according to formula (I) herein and an mRNA according to SEQ ID NO: 77 (mRNA coding for luciferase (luc-RNActive) with the PB19 peptide (molar ratio of RNA:PB19 1:1000, 1:500, 1:100). These formulations of mRNA coding for luciferase (luc-RNActive) with the PB19 peptide (molar ratio of RNA:PB19 1:1000, 1:500, 1:100) lead to expression of luciferase independently of the presence of serum containing medium. These results are unexpected because serum containing medium leads in general to a loss of transfection efficiency.
  • 12.5. Expression of Luciferase In Vivo:
  • Expression of luciferase in BALB/c mice was determined after intradermal injection. As can be seen in FIG. 7 a, formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 77) with the PB19 polymer leads to expression of luciferase in the dermis of female BALB/c mice. Other transfection reagents known in the art did not showed any expression of the Luciferase protein.
  • Furthermore, expression of luciferase in BALB/c mice after intramuscular injection was determined. As a result (see FIG. 7 b), formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 77) with the PB19 polymer leads to expression of luciferase in the m. tibialis of female BALB/c mice. Other transfection reagents known in the art did not showed any expression of the Luciferase protein.
  • Additionally, expression of luciferase in BALB/c mice after intradermal injection of different formulations was determined. As a result (see FIG. 8), formulations of mRNA coding for luciferase (luc-RNActive) (SEQ ID NO: 77) with two different polymers according to the present invention (polymers PB19 and PB48) lead to expression of luciferase in the dermis of female BALB/c mice. The inventive polymeric carrier cargo complex formed by a peptide according to RPC CH6R4H6C (without PEGylation) and RNA in a molar ratio of 2500:1 procedure showed no expression of luciferase after intradermal injection of the complexed RNA.
  • 12.6 Cytokinstimulation in hPBMC
  • Cytokinstimulation, particularly h IL-6 cytokine secretion in hPBMCs was measured. As a result (see FIG. 9) it could be shown that the complexes according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention (PB19 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA (SEQ ID NO: 77) and cationic peptides (H3R9H3) or PEGylated cationic peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates).
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • Furthermore, h TNFα cytokine secretion in hPBMCs was measured. The results show (see FIG. 10), that the complexes according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention (PB19 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA (SEQ ID NO: 77) and cationic peptides (H3R9H3) or such complexes coated with PEGylated peptides (which confers in general to subsequent hydrophilic coating of preformed nucleic acid condensates).
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • Additionally, h TNFα Cytokine secretion in hPBMCs was measured. The results (see FIG. 11) show that the complexes according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention (PB19 and PB22) do not induce the secretion of cytokines in hPBMCs in contrast to complexes consisting of RNA and cationic peptides (H3R9H3) or such complexes coated with PEGylated peptides (which confers in general to subsequent hydrophilic coating of pre-formed nucleic acid condensates).
  • E9-PEG5k: HO-PEG5000-EEEEEEEEE
    E9-PEG3k: HO-PEG3000-EEEEEEEEE
    R9-PEG3k: HO-PEG3000-RRRRRRRRR
    PB19: HO-PEG5000-S-(S-CHHHRRRRHHHC-S)5-S-PEG5000-
    OH
    PB22: HO-PEG5000-S-(S-CHHHHHHRRRRHHHHHHC-S)5-
    S-PEG5000-OH
  • Moreover, h IFNa cytokine secretion in hPBMCs was determined in a comparison of cytokine stimulating properties of complexes according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention to state of the art transfection reagent like Lipofectamin or PEI. As a result (see FIG. 11A) both Lipofectamin and PEI lead to a high amount of secretion of hIFNa, whereas the complex according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention did not (PB19 500).
  • Furthermore, h TFNa cytokine secretion in hPBMCs was measured in a comparison of cytokine stimulating properties of complexes according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to the invention to state of the art transfection reagent like Lipofectamin or PEI. As a result (see FIG. 11B) Lipofectamine lead to a high amount of secretion of h TFNa, whereas PEI and the complex according to the invention consisting of RNA (SEQ ID NO: 77) and polymers according to, the invention (PB19 500) did not.
  • Finally, induction of Ovalbumin-specific IgG1 antibodies was measured. According to the results, the formulations containing the complexing reagent according to the invention [PB19], particularly complexes according to the invention consisting of RNA (SEQ ID NO: 77) and the complexing reagent according to the invention [PB19], showed no induction of an antibody response after two vaccination cycles against OVA antigen. The cationic peptide [CH6R4H6C], polymerized according to the state of the art (RPC) shows a strong induction of Ovalbumin-specific IgG1 antibodies.
  • Furthermore, induction of Ovalbumin-specific IgG2 antibodies was measured. According to the results, the formulations containing the complexing reagent according to the invention [PB19], particularly complexes according to the invention consisting of RNA (SEQ ID NO: 77) and the complexing reagent according to the invention [PB19], show no induction of an antibody response after two vaccination cycles against OVA antigen. The cationic peptide [CH6R4H6C], polymerized according to the state of the art (RPC) shows a strong induction of Ovalbumin-specific IgG2 antibodies.

Claims (18)

1. Polymeric carrier molecule according to generic formula (I):

L-P1—S—[S—P2—S]n—S—P3-L
wherein,
P1 and P3 represent a linear or branched hydrophilic polymer chain, each P1 and P3 exhibiting at least one —SH-moiety, capable to form a disulfide linkage upon condensation with component P2, the linear or branched hydrophilic polymer chain selected independent from each other from polyethylene glycol (PEG), poly-N-(2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl L-asparagine) or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 100 kDa;
P2 is a cationic or polycationic peptide or protein, having a length of about 3 to 100 amino acids, or
is a cationic or polycationic polymer, having a molecular weight of about 0.5 kDa to about 100 kDa,
each P2 exhibiting at least two —SH-moieties, capable to form a disulfide linkage upon condensation with component(s) P1 and/or P3;
—S—S— is a (reversible) disulfide bond;
L is an optional ligand, selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, or TAT;
n is an integer, selected from a range of about 1 to 50.
2. The polymeric carrier molecule according to claim 1, wherein the polymeric carrier molecule additionally contains a repetitive amino acid component (AA)x, wherein x is an integer selected from a range of about 1 to 10, and wherein AA is an amino acid selected from aromatic amino acids Trp, Tyr, or Phe, or from hydrophilic, non charged polar amino acids Thr, Ser, Asn or Gln, or from lipophilic amino acids Leu, Val, Ile, Ala, or Met, or is selected from weak basic amino acids histidine or aspartate (aspartic acid).
3. The polymeric carrier molecule according to claim 1, wherein repetitive amino acid component (AA)x occurs as a mixed repetitive amino acid component [(AA)x]z, wherein z is an integer selected from a range of about 1 to 30.
4. The polymeric carrier molecule according to claim 1, wherein formula (I) is modified with mixed repetitive amino acid component [(AA)x]z according to following formula (Ia)

L-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3-L,
wherein x, z, S, L, AA, P1, P2 and P3 are as defined herein and
a+b=n, wherein
n is as defined herein
a is an integer, selected independent from integer b from a range of about 1 to 50, and
b is an integer, selected independent from integer a from a range of about 0 to 50,
and wherein the single components [S—P2—S] and [S-(AA)x-S] occur in any order in the subformula {[S—P2—S]a[S-(AA)x-S]b}.
5. The polymeric carrier molecule according to claim 1, formula (I) or formula (Ia) are further modified with repetitive amino acid component (AA)x or with mixed repetitive amino acid component [(AA)x]z according to any of following subformulae

L-P1—S—S-(AA)x-S-{[S—P2—S]a[S-(AA)x-S]b}—S-(AA)x-S—S—P3-L, or

L-P1—S—[S-(AA)x-S]z—{[S—P2—S]a[S-(AA)x-S]b}-[S-(AA)x-S]z—S—P3-L, or

L-(AA)x-P1—S-{[S—P2—S]a[S-(AA)x-S]b}S—P3-(AA)x-L, or

L-[(AA)x]zP1—S-{[S—P2—S]a[S-(AA))x—S]b}—S—P3-[(AA)x]z-L, or

L-(AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x-S—S-L, or

L-S—S-(AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x-S—S-L, or

L-S—[S-(AA)x-S]z-S—P1—S-{[S—P2—S]a[S-(AA)-S]b}—S—P3—S—[S-(AA)x-S]z-S-L, or

(AA)x-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P8-(AA)x, or

[(AA)x]z-P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—[(AA)x]z, or

(AA)x-S—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—S-(AA)x, or

H—[S-(AA)x-S]z—S—P1—S-{[S—P2—S]a[S-(AA)x-S]b}—S—P3—S—[S-(AA)x-S]z—H,
wherein a, b, n, x, z, S, L, AA, P1, P2 and P3 are as defined herein.
6. The polymeric carrier molecule according to claim 1, wherein component P2 is a cationic or polycationic peptide selected from protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, such as Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, e.g. Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, pIsl, etc., antimicrobial-derived CPPs e.g. Buforin-2, Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, MAP, KALA, PpTG20, Proline-rich peptides, Loligomere, Arginine-rich peptides, Calcitonin-peptides, FGF, Lactoferrin, poly-L-Lysine, poly-Arginine, histones, VP22 derived or analog peptides, HSV, VP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs, PpT620, prolin-rich peptides, arginine-rich peptides, lysine-rich peptides, Pep-1, L-oligomers, or from Calcitonin peptide(s).
7. The polymeric carrier molecule according to claim 1, wherein the —SH-moiety in component(s) in P1, P2 and P3 is provided by a cysteine.
8. The polymeric carrier molecule according to claim 1, wherein component P2 is selected from a peptide comprising a cationic peptide of formula (IIa):

Cys((Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x)Cys,
wherein
l+m+n+o+x=8−15, and l, m, n or o independently of each other may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overall content of Arg, Lys, His and Orn represents at least 10% of all amino acids of the oligopeptide; and Xaa may be any amino acid selected from native (=naturally occurring) or non-native amino acids except of Arg, Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14, provided, that the overall content of Xaa does not exceed 90% of all amino acids of the oligopeptide.
9. Polymeric carrier cargo complex formed of a polymeric carrier molecule according to claim 1 and a nucleic acid.
10. The polymeric carrier cargo complex of a polymeric carrier molecule according to claim 9, wherein the nucleic acid is provided in a molar ratio of about 10 to 10000 of inventive polymeric carrier molecule: nucleic acid.
11. The polymeric carrier cargo complex of a polymeric carrier molecule according to claim 9, wherein the nucleic acid is a DNA or a RNA.
12. The polymeric carrier cargo complex of a polymeric carrier molecule according to claim 9, wherein the nucleic acid is a coding RNA selected from an mRNA, or an siRNA.
13. The polymeric carrier cargo complex of a polymeric carrier molecule according to claim 9, wherein the nucleic acid encodes a therapeutically active protein or peptide, an antigen, including tumor antigens, pathogenic antigens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens, autoimmune antigens, allergens, antibodies, immunostimulatory proteins or peptides, or antigen-specific T-cell receptors.
14. Method of preparing the polymeric carrier according to claim 1 comprising following steps:
a. providing at least one cationic or polycationic protein or peptide and/or at least one cationic or polycationic polymer as component P2 as defined according to claim 1 and optionally a repetitive amino acid component (AA)x or a mixed repetitive amino acid component [(AA)x]z as defined according to claim 1, in the ratios indicated by formulae (I) or (Ia), mixing these components for a time of at least about 5 hours and thereby condensing and thus polymerizing these components with each other via disulfide bonds in a polymerization condensation or polycondensation to obtain a repetitive component H—[S—P2—S]n—H or H{[S—P2—S]a[S-(AA)x-S]b}H, as defined according to claim 1;
b. providing a hydrophilic polymer P1 and/or P3 as defined according to claim 1, optionally modified with a ligand L and/or a repetitive amino acid component (AA)x as defined according to claim 1;
c. mixing the hydrophilic polymer P1 and/or P3 according to step b) with the repetitive component H—[S—P2—S]n—H or H{[S—P2—S]a[S-(AA)x-S]b}H obtained according to step a) in a ratio of about 2:1, and thereby terminating the polymerization condensation or polycondensation reaction and obtaining the inventive polmeric carrier according to formula (I) or (Ia);
d. optionally purifying the inventive polymeric carrier obtained according to step c);
e. optionally adding a nucleic acid as defined herein to the polymeric carrier obtained according to step c) or d) and complexing the nucleic acid with the polymeric carrier obtained according to step c) or d) to obtain a polymeric carrier cargo complex as defined according to claim 9.
15. Pharmaceutical composition, comprising the polymeric carrier cargo complex according to claim 9 and optionally a pharmaceutically acceptable carrier and/or vehicle.
16. Use of the polymeric carrier molecule according to claim 1 and a nucleic acid as defined according to claim 9 or use of the polymeric carrier cargo complex according to claim 9 as a medicament.
17. Use of the polymeric carrier molecule according to any of claims 1 to 8 and a nucleic acid as defined according to claim 11, or of the polymeric carrier cargo complex according to claim 9, for the preparation of a pharmaceutical composition for the prophylaxis, treatment and/or amelioration of diseases as defined herein, selected from cancer or tumor diseases, infectious diseases, including viral, bacterial or protozoological infectious diseases, autoimmune diseases, allergies or allergic diseases, monogenetic diseases, including hereditary diseases or genetic diseases, diseases which have a genetic inherited background and which are typically caused by a single gene defect and are inherited according to Mendel's laws, cardiovascular diseases, and neuronal diseases.
18. Kit, including kit of parts, comprising as components at least one polymeric carrier molecule according to claim 1 and at least one nucleic acid according to claim 10, provided as a polymeric carrier cargo complex according to claim 10, optionally with information on the administration and dosage of the polymeric carrier complex.
US12/553,559 2009-09-03 2009-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids Abandoned US20110053829A1 (en)

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US12/553,559 US20110053829A1 (en) 2009-09-03 2009-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
MX2012002502A MX2012002502A (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids.
EP10749816.4A EP2331138B1 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
AU2010291499A AU2010291499B2 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
KR1020127006279A KR20120102586A (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
KR1020157014584A KR101790236B1 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
BR112012008142A BR112012008142A8 (en) 2009-09-03 2010-09-03 peptide conjugates / disulfide linked polyethylene for the transfection of nucleic acids
RU2012112640/04A RU2552352C2 (en) 2009-09-03 2010-09-03 Disulphide-linked polyethylene glycol/peptide conjugates for nucleic acid transfection
CN201080039095.8A CN102573919B (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
ES10749816.4T ES2526055T3 (en) 2009-09-03 2010-09-03 Conjugates polyethylene glycol / peptide disulfide bonded to transfect nucleic acids
JP2012527240A JP5813642B2 (en) 2009-09-03 2010-09-03 Polyethylene glycol / peptide conjugates disulfide bonds for nucleic acid transfection
PCT/EP2010/005438 WO2011026641A1 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
CA2765421A CA2765421C (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
EP20140002542 EP2810661A3 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
KR1020147016368A KR101572249B1 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
US13/378,241 US8703906B2 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
DK10749816.4T DK2331138T3 (en) 2009-09-03 2010-09-03 Disulfide-linked polyethylene glycol / peptide conjugates for transforming the infection of nucleic acids
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US14/195,864 US9314535B2 (en) 2009-09-03 2014-03-04 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
US15/065,769 US9907862B2 (en) 2009-09-03 2016-03-09 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
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US14/195,864 Active US9314535B2 (en) 2009-09-03 2014-03-04 Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
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Cited By (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130142818A1 (en) * 2010-07-30 2013-06-06 Curevac Gmbh Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
EP2623121A1 (en) * 2012-01-31 2013-08-07 Bayer Innovation GmbH Pharmaceutical composition comprising a polymeric carrier cargo complex and an antigen
WO2013113326A1 (en) * 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
US20130259879A1 (en) * 2011-02-21 2013-10-03 Curevac Gmbh Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
US8664194B2 (en) 2011-12-16 2014-03-04 Moderna Therapeutics, Inc. Method for producing a protein of interest in a primate
WO2014051318A2 (en) * 2012-09-28 2014-04-03 한양대학교 산학협력단 Short interference rna gene delivery system for systemic circulation
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
WO2014072999A1 (en) * 2012-11-07 2014-05-15 Council Of Scientific And Industrial Research Nanocomplex containing cationic peptide for biomolecule delivery
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
CN104530438A (en) * 2014-12-11 2015-04-22 华南理工大学 Cholesterol modification based pH response polypeptide polymer as well as preparation method and application thereof
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
CN104870023A (en) * 2012-09-28 2015-08-26 汉阳大学校产学协力团 Short interference RNA gene delivery system for systemic circulation
EP2872160A4 (en) * 2012-04-17 2015-11-25 Univ Brown Neuroprotective composition and method of use
US9234013B2 (en) 2010-08-13 2016-01-12 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded protein
US9238064B2 (en) 2008-03-03 2016-01-19 University Of Miami Allogeneic cancer cell-based immunotherapy
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US9303079B2 (en) 2012-03-30 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9352028B2 (en) 2007-10-09 2016-05-31 Curevac Ag Composition for treating lung cancer, particularly of non-small lung cancers (NSCLC)
US9402887B2 (en) 2007-10-09 2016-08-02 Curevac Ag Composition for treating prostate cancer (PCa)
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9447431B2 (en) 2012-02-15 2016-09-20 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US9616084B2 (en) 2009-12-09 2017-04-11 Curevac Ag Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US9623095B2 (en) 2011-03-02 2017-04-18 Curevac Ag Vaccination in newborns and infants
US9669089B2 (en) 2012-02-15 2017-06-06 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen
US9683233B2 (en) 2012-03-27 2017-06-20 Curevac Ag Artificial nucleic acid molecules for improved protein or peptide expression
US9688729B2 (en) 2013-08-21 2017-06-27 Curevac Ag Respiratory syncytial virus (RSV) vaccine
US9737595B2 (en) 2010-12-29 2017-08-22 Curevac Ag Combination of vaccination and inhibition of MHC class I restricted antigen presentation
US9796762B2 (en) 2013-04-10 2017-10-24 4D Pharma Research Limited Polypeptide and immune modulation
US9872900B2 (en) 2014-04-23 2018-01-23 Modernatx, Inc. Nucleic acid vaccines
US9890391B2 (en) 2012-03-27 2018-02-13 Curevac Ag RNA vector with an open reading frame, an albumin 3′-UTR, and a histone stem loop
US9937211B2 (en) 2011-10-07 2018-04-10 4D Pharma Research Limited Composition of Roseburia hominis
US9974845B2 (en) 2013-02-22 2018-05-22 Curevac Ag Combination of vaccination and inhibition of the PD-1 pathway
US10010592B2 (en) 2012-02-15 2018-07-03 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen
US10047375B2 (en) 2013-12-30 2018-08-14 Curevac Ag Artificial nucleic acid molecules
US10080809B2 (en) 2012-03-27 2018-09-25 Curevac Ag Artificial nucleic acid molecules comprising a 5′TOP UTR
US10111967B2 (en) 2007-09-04 2018-10-30 Curevac Ag Complexes of RNA and cationic peptides for transfection and for immunostimulation
US10232024B2 (en) 2012-02-15 2019-03-19 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded allergenic antigen or an autoimmune self-antigen

Families Citing this family (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
US20110053829A1 (en) 2009-09-03 2011-03-03 Curevac Gmbh Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
EP2680881B1 (en) 2011-03-02 2017-04-05 CureVac AG Vaccination in elderly patients
WO2013021056A1 (en) 2011-08-10 2013-02-14 Ludwig-Maximilians-Universität München Method for the controlled intracellular delivery of nucleic acids
JP6202707B2 (en) * 2011-10-13 2017-09-27 国立大学法人鳥取大学 New cell penetrating peptide
EP2854857B1 (en) * 2012-05-25 2018-11-28 CureVac AG Reversible immobilization and/or controlled release of nucleic acid containing nanoparticles by (biodegradable) polymer coatings
EP2875828A1 (en) * 2012-07-20 2015-05-27 Diater, Laboratorio de Diagnóstico y Aplicaciones Terapéuticas, S.A. Composition for the intradermal administration of allergoids
CN103396471B (en) * 2013-07-30 2015-01-14 武汉大学 Metal beta-lactamase inhibitory peptide and application thereof
AU2014361806A1 (en) * 2013-12-12 2016-07-28 Life Technologies Corporation Membrane-penetrating peptides to enhance transfection and compositions and methods for using same
GB201322396D0 (en) 2013-12-18 2014-02-05 Univ Nottingham Transduction
SG11201707663SA (en) 2015-04-17 2017-11-29 Curevac Ag Lyophilization of rna
AU2016251687A1 (en) 2015-04-22 2017-08-24 Curevac Ag RNA containing composition for treatment of tumor diseases
CA2975816A1 (en) 2015-05-15 2016-11-24 Curevac Ag Prime-boost regimens involving administration of at least one mrna construct
CA2998810A1 (en) 2015-09-17 2017-03-23 Modernatx, Inc. Compounds and compositions for intracellular delivery of therapeutic agents
WO2017066791A1 (en) 2015-10-16 2017-04-20 Modernatx, Inc. Sugar substituted mrna cap analogs
WO2017066781A1 (en) 2015-10-16 2017-04-20 Modernatx, Inc. Mrna cap analogs with modified phosphate linkage
JP2018530587A (en) 2015-10-16 2018-10-18 モデルナティエックス インコーポレイテッドModernaTX,Inc. The method of mRNA cap analogs and mRNA capping
WO2017066789A1 (en) 2015-10-16 2017-04-20 Modernatx, Inc. Mrna cap analogs with modified sugar
WO2017066782A1 (en) 2015-10-16 2017-04-20 Modernatx, Inc. Hydrophobic mrna cap analogs
EP3364950A1 (en) 2015-10-22 2018-08-29 ModernaTX, Inc. Tropical disease vaccines
WO2017070626A2 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Respiratory virus vaccines
AU2016342045A1 (en) 2015-10-22 2018-06-07 Modernatx, Inc. Human cytomegalovirus vaccine
WO2017112865A1 (en) 2015-12-22 2017-06-29 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2017182634A1 (en) 2016-04-22 2017-10-26 Curevac Ag Rna encoding a tumor antigen
EP3452493A1 (en) 2016-05-04 2019-03-13 CureVac AG Nucleic acid molecules and uses thereof
EP3452101A2 (en) 2016-05-04 2019-03-13 CureVac AG Rna encoding a therapeutic protein
CA3025757A1 (en) * 2016-05-27 2017-11-30 Orum Therapeutics Inc. Cytosol-penetrating antibody and use thereof
WO2017212008A1 (en) * 2016-06-09 2017-12-14 Curevac Ag Hybrid carriers for nucleic acid cargo
KR20190029576A (en) 2016-06-09 2019-03-20 큐어백 아게 Nucleic acid hybrid car hire carriers
WO2017218704A1 (en) 2016-06-14 2017-12-21 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
WO2018033254A2 (en) 2016-08-19 2018-02-22 Curevac Ag Rna for cancer therapy
WO2018041921A1 (en) 2016-08-31 2018-03-08 Curevac Ag Mixing device for the production of a liquid nucleic acid composition
WO2018089540A1 (en) 2016-11-08 2018-05-17 Modernatx, Inc. Stabilized formulations of lipid nanoparticles
WO2018104540A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rnas for wound healing
WO2018104538A1 (en) 2016-12-08 2018-06-14 Curevac Ag Rna for treatment or prophylaxis of a liver disease
WO2018115507A2 (en) 2016-12-23 2018-06-28 Curevac Ag Henipavirus vaccine
WO2018115525A1 (en) 2016-12-23 2018-06-28 Curevac Ag Lassa virus vaccine
WO2018115527A2 (en) 2016-12-23 2018-06-28 Curevac Ag Mers coronavirus vaccine
WO2018172556A1 (en) 2017-03-24 2018-09-27 Curevac Ag Nucleic acids encoding crispr-associated proteins and uses thereof
WO2018211038A1 (en) 2017-05-17 2018-11-22 Curevac Ag Method for determining at least one quality parameter of an rna sample
WO2018232120A1 (en) 2017-06-14 2018-12-20 Modernatx, Inc. Compounds and compositions for intracellular delivery of agents
WO2019036638A1 (en) 2017-08-18 2019-02-21 Modernatx, Inc. Methods of preparing modified rna
WO2019038332A1 (en) 2017-08-22 2019-02-28 Curevac Ag Bunyavirales vaccine

Family Cites Families (116)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3906092A (en) 1971-11-26 1975-09-16 Merck & Co Inc Stimulation of antibody response
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4401796A (en) 1981-04-30 1983-08-30 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
DE3314999A1 (en) 1983-04-26 1985-03-14 Behringwerke Ag Use of the diterpene forskolin derivative for immunostimulation
US5663163A (en) 1987-09-07 1997-09-02 Fujisawa Pharmaceutical Co., Ltd. Cephem compounds and processes for preparation thereof
CA1320446C (en) 1988-06-20 1993-07-20 William A. Carter Modulation of lymphokine-resistant cellular states by dsrnas
AT147630T (en) 1989-10-11 1997-02-15 Hem Pharma Corp Protection against shock due to damage by double-stranded rna's
AT138384T (en) 1993-01-25 1996-06-15 Hybridon Inc Olionukleotidalkylphosphonate and - phosphonothioate
CA2153243A1 (en) 1993-01-27 1994-08-18 Harold E. Selick Compositions and methods for transdermal drug delivery
US5516652A (en) 1993-10-06 1996-05-14 Merck Frosst Canada Inc. DNA encoding prostaglandin receptor IP
JP3482209B2 (en) 1994-03-18 2003-12-22 ジェロン・コーポレーション Oligonucleotides n3 '→ p5' phosphoramidate Synthesis and compounds; hybridization and nuclease resistance properties
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US5874560A (en) 1994-04-22 1999-02-23 The United States Of America As Represented By The Department Of Health And Human Services Melanoma antigens and their use in diagnostic and therapeutic methods
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
ES2267100T5 (en) 1994-07-15 2011-04-08 Coley Pharmaceutical Group, Inc. Immunomodulatory oligonucleotides.
US7148205B2 (en) 1995-12-13 2006-12-12 Mirus Bio Corporation Intravascular delivery of non-viral nucleic acid
US7371387B2 (en) 2001-02-21 2008-05-13 Genitrix Llc Vaccine compositions and methods of modulating immune responses
US6090391A (en) 1996-02-23 2000-07-18 Aviron Recombinant tryptophan mutants of influenza
ES2241042T3 (en) 1996-10-11 2005-10-16 The Regents Of The University Of California Conjugates immunostimulatory polynucleotide / immunomodulatory molecule.
EP0839912A1 (en) 1996-10-30 1998-05-06 Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof
GB9623051D0 (en) * 1996-11-06 1997-01-08 Schacht Etienne H Delivery of DNA to target cells in biological systems
EP0855184A1 (en) 1997-01-23 1998-07-29 Klaus Prof.-Dr. Heeg Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination
US6689759B1 (en) 1998-02-12 2004-02-10 G. D. Searle & Co. Methods of Treating hepatitis virus infections with N-substituted-1,5-dideoxy-1,5-imino-d-glucitol compounds in combination therapy
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
WO1998047913A2 (en) 1997-04-18 1998-10-29 The University Of Medicine And Dentistry Of New Jersey Inhibition of hiv-1 replication by a tat rna-binding domain peptide analog
ES2326848T3 (en) 1997-06-06 2009-10-20 Dynavax Technologies Corporation Inhibitors activity immunostimulatory DNA sequences.
EP1374894A3 (en) 1997-06-06 2004-09-22 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US6589940B1 (en) 1997-06-06 2003-07-08 Dynavax Technologies Corporation Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
US20040006034A1 (en) 1998-06-05 2004-01-08 Eyal Raz Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof
EP1009413B1 (en) 1997-09-05 2007-02-14 The Regents Of The University Of California Use of immunostimulatory oligonucleotides for preventing or treating asthma
US6096307A (en) 1997-12-11 2000-08-01 A. Glenn Braswell Compositions for immunostimulation containing Echinacea angustofolia, bromelain, and lysozyme
EP1067956B1 (en) 1998-04-03 2007-03-14 University Of Iowa Research Foundation Methods and products for stimulating the immune system using immunotherapeutic oligonucleotides and cytokines
WO1999053961A1 (en) 1998-04-23 1999-10-28 The Regents Of The University Of Michigan Peptides for efficient gene transfer
WO2000075304A1 (en) 1999-06-08 2000-12-14 Aventis Pasteur Immunostimulant oligonucleotide
CA2312385A1 (en) 1999-06-22 2000-12-22 Daiso Co., Ltd. Process for producing erythro-3-amino-2-hydroxybutyric acid derivatives
US6514948B1 (en) 1999-07-02 2003-02-04 The Regents Of The University Of California Method for enhancing an immune response
US6770740B1 (en) 1999-07-13 2004-08-03 The Regents Of The University Of Michigan Crosslinked DNA condensate compositions and gene delivery methods
WO2001004135A2 (en) 1999-07-13 2001-01-18 The Regents Of The University Of Michigan Crosslinked dna condensate compositions and gene delivery methods
US20030104622A1 (en) 1999-09-01 2003-06-05 Robbins Paul D. Identification of peptides that facilitate uptake and cytoplasmic and/or nuclear transport of proteins, DNA and viruses
DE69923840T2 (en) 1999-09-09 2006-04-06 Curevac Gmbh Transfer of mRNAs using polycationic compounds
AT409085B (en) 2000-01-28 2002-05-27 Cistem Biotechnologies Gmbh A pharmaceutical composition for immunomodulation and preparation of vaccines
CA2398756A1 (en) 2000-01-31 2001-08-02 Eyal Raz Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen
KR20080023768A (en) 2000-03-30 2008-03-14 막스-플랑크-게젤샤프트 츄어 푀르더룽 데어 비쎈샤프텐 에.파우. Rna sequence-specific mediators of rna interference
WO2001093902A2 (en) 2000-06-07 2001-12-13 Biosynexus Incorporated Immunostimulatory rna/dna hybrid molecules
DE60139689D1 (en) 2000-06-22 2009-10-08 Univ Iowa Res Found Combination of CpG and antibodies to CD19, CD20, CD22 or CD40 in the prevention or treatment of cancer.
AT343589T (en) 2000-06-23 2006-11-15 Wyeth Corp Modofiziertes morbillivirus v proteins
US6376704B1 (en) 2000-06-28 2002-04-23 3M Innovative Properties Company Naphthyoxyalkyl(meth)acrylates with high refractive indices and low glass transition temperatures
US6716434B1 (en) 2000-09-19 2004-04-06 Daniel R. Ansley Composition and method for immunostimulation in non- mammalian vertebrates
GB0025577D0 (en) 2000-10-18 2000-12-06 Smithkline Beecham Biolog Vaccine
EP1383924B1 (en) 2001-04-02 2010-02-10 University of South Florida Lps-responsive chs1/beige-like anchor gene and therapeutic applications thereof
EP1392341B1 (en) 2001-06-05 2005-03-30 Curevac GmbH Pharmaceutical composition containing a stabilised mrna which is optimised for translation in the coding regions thereof
US7785610B2 (en) 2001-06-21 2010-08-31 Dynavax Technologies Corporation Chimeric immunomodulatory compounds and methods of using the same—III
CA2451091A1 (en) 2001-06-25 2003-01-03 Yissum Research Development Company Of The Hebrew University In Jerusale M A method for preparation of vesicles loaded with biological material and different uses thereof
AR045702A1 (en) 2001-10-03 2005-11-09 Chiron Corp Adjuvant compositions.
DE10148886A1 (en) 2001-10-04 2003-04-30 Avontec Gmbh Inhibition of STAT-1
WO2003035836A2 (en) 2001-10-24 2003-05-01 Hybridon Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5' ends
DE10162480A1 (en) 2001-12-19 2003-08-07 Ingmar Hoerr The application of mRNA for use as a therapeutic agent against tumor diseases
AU2003235707A1 (en) 2002-01-18 2003-07-30 Curevac Gmbh Immunogenic preparations and vaccines on the basis of mrna
JP2005526497A (en) 2002-02-04 2005-09-08 ビオミラ,インコーポレーテッド Immunostimulatory, covalent lipid oligonucleotide
WO2003074551A2 (en) 2002-03-01 2003-09-12 The Administrators Of The Tulane Educational Fund Conjugates of therapeutic or cytotoxic agents and biologically active peptides
NZ573064A (en) 2002-04-04 2011-02-25 Coley Pharm Gmbh Immunostimulatory G,U-containing oligoribonucleotides
DE10229872A1 (en) 2002-07-03 2004-01-29 Curevac Gmbh Immunostimulation by chemically modified RNA
EP1393745A1 (en) 2002-07-29 2004-03-03 Hybridon, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5'ends
CA2521662A1 (en) 2003-04-10 2005-01-06 3M Innovative Properties Company Methods and compositions for enhancing immune response
US7276489B2 (en) 2002-10-24 2007-10-02 Idera Pharmaceuticals, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends
EP1625140A4 (en) 2002-12-23 2008-06-18 Dynavax Tech Corp Branched immunomodulatory compounds and methods of using the same
CA2512484A1 (en) 2003-01-16 2004-05-08 Hybridon, Inc. Modulation of immunostimulatory properties of oligonucleotide-based compounds by utilizing modified immunostimulatory dinucleotides
WO2004092329A2 (en) 2003-04-08 2004-10-28 Galenica Pharmaceuticals, Inc. Semi-synthetic saponin analogs with carrier and immune stimulatory activities for dna and rna vaccines
CA2530613A1 (en) 2003-06-30 2005-01-06 Universite De Lausanne Rasgap derived peptide for selectively killing cancer cells
AT494910T (en) 2003-07-10 2011-01-15 Cytos Biotechnology Ag Composition packaged virus-like particles comprising for enhancing an immune response
DE10335833A1 (en) 2003-08-05 2005-03-03 Curevac Gmbh Transfection of blood cells with mRNA for immune stimulation and gene therapy
US7807801B2 (en) 2003-08-05 2010-10-05 Avi Biopharma Inc. Oligonucleotide analog and method for treating flavivirus infections
EP1663316A2 (en) 2003-09-25 2006-06-07 Coley Pharmaceutical Group, Inc. Nucleic acid lipophilic conjugates
DE10346721A1 (en) 2003-10-08 2005-05-04 Holger Kalthoff New oligonucleotides, useful for treating cancer, especially of the pancreas, are not species specific but induce apoptosis or inhibit proliferation
EP1699814A4 (en) 2003-12-08 2009-01-14 Hybridon Inc Modulation of immunostimulatory properties by small oligonucleotide-based compounds
AT487792T (en) 2003-12-22 2010-11-15 Univ Pennsylvania Methods and compositions for the identification of RNA-binding proteins
AU2005230938A1 (en) 2004-02-19 2005-10-20 Coley Pharmaceutical Gmbh Immunostimulatory viral RNA oligonucleotides
WO2006080946A2 (en) 2004-06-08 2006-08-03 Coley Pharmaceutical Gmbh Abasic oligonucleotide as carrier platform for antigen and immunostimulatory agonist and antagonist
WO2006046978A2 (en) 2004-06-28 2006-05-04 Argos Therapeutics, Inc. Cationic peptide-mediated transformation
DE102004035227A1 (en) 2004-07-21 2006-02-16 Curevac Gmbh mRNA mixture for vaccination against tumors
DE102004042546A1 (en) 2004-09-02 2006-03-09 Curevac Gmbh Combination therapy for immune stimulation
WO2006029223A2 (en) 2004-09-08 2006-03-16 Children's Medical Center Corporation Method for stimulating the immune response of newborns
US20080248067A1 (en) 2005-04-14 2008-10-09 The University Of Queensland Immunomodulating Compositions and Uses Therefor
US20060241076A1 (en) 2005-04-26 2006-10-26 Coley Pharmaceutical Gmbh Modified oligoribonucleotide analogs with enhanced immunostimulatory activity
DK2027158T3 (en) 2006-05-02 2013-01-14 Carviar Aps Method for immunizing an avian species
DE102005023170A1 (en) 2005-05-19 2006-11-23 Curevac Gmbh Optimized formulation for mRNA
US9505867B2 (en) 2005-05-31 2016-11-29 Ecole Polytechmique Fédérale De Lausanne Triblock copolymers for cytoplasmic delivery of gene-based drugs
EP1764107A1 (en) 2005-09-14 2007-03-21 Gunther Hartmann Compositions comprising immunostimulatory RNA oligonucleotides and methods for producing said RNA oligonucleotides
WO2007031322A1 (en) 2005-09-14 2007-03-22 Gunther Hartmann Compositions comprising immunostimulatory rna oligonucleotides and methods for producing said rna oligonucleotides
JP2009511034A (en) 2005-10-12 2009-03-19 キャンサー・リサーチ・テクノロジー・リミテッドCancer Research Technology Limited Methods and compositions for the treatment of immune disorder
US8101741B2 (en) 2005-11-02 2012-01-24 Protiva Biotherapeutics, Inc. Modified siRNA molecules and uses thereof
US7470674B2 (en) 2005-11-07 2008-12-30 Idera Pharmaceuticals, Inc. Immunostimulatory properties of oligonucleotide-based compounds comprising modified immunostimulatory dinucleotides
EP1957647B1 (en) 2005-11-25 2015-03-04 Zoetis Belgium S.A. Immunostimulatory oligoribonucleotides
EP1968643A2 (en) 2005-12-16 2008-09-17 Diatos Cell penetrating peptide conjugates for delivering of nucleic acids into a cell
DE102006007433A1 (en) 2006-02-17 2007-08-23 Curevac Gmbh Immunostimulant adjuvant useful in vaccines against cancer or infectious diseases comprises a lipid-modified nucleic acid
JP2010507361A (en) 2006-07-31 2010-03-11 キュアバック ゲーエムベーハーCurevac Gmbh Specifically as immunostimulant / adjuvant of the general formula (I): GlXmGn or formula (II),: nucleic acid represented by ClXmCn
DE102006035618A1 (en) 2006-07-31 2008-02-07 Curevac Gmbh New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria
EP2051965A2 (en) 2006-08-18 2009-04-29 Nastech Pharmaceutical Company Inc. Dicer substrate rna peptide conjugates and methods for rna therapeutics
DE102006051516A1 (en) 2006-10-31 2008-05-08 Curevac Gmbh (Base-) modified RNA to increase in expression of a protein
DE102006061015A1 (en) 2006-12-22 2008-06-26 Curevac Gmbh A method for purifying RNA on a preparative scale by means of HPLC
US20100189729A1 (en) 2007-01-09 2010-07-29 Curvac Gmbh Rna-coded antibody
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
WO2009046738A1 (en) 2007-10-09 2009-04-16 Curevac Gmbh Composition for treating lung cancer, particularly of non-small lung cancers (nsclc)
WO2009046739A1 (en) 2007-10-09 2009-04-16 Curevac Gmbh Composition for treating prostate cancer (pca)
WO2009053700A1 (en) 2007-10-23 2009-04-30 Cancer Research Technology Limited Modification of nucleic acid-containing biological entities
WO2009086640A1 (en) 2008-01-10 2009-07-16 Nventa Biopharmaceuticals Corporation Adjuvant compositions comprising poly-ic and a cationic polymer
JP5721441B2 (en) 2008-01-31 2015-05-20 キュアバック ゲーエムベーハーCurevac Gmbh Expression as immunostimulant / adjuvant (I) (NuGlXmGnNv) nucleic acid molecules and derivatives thereof represented by a
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
US20110053829A1 (en) 2009-09-03 2011-03-03 Curevac Gmbh Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids
WO2011069529A1 (en) 2009-12-09 2011-06-16 Curevac Gmbh Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
EP2955230A1 (en) 2010-07-30 2015-12-16 CureVac AG Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
WO2012113413A1 (en) 2011-02-21 2012-08-30 Curevac Gmbh Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
WO2013113326A1 (en) 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
WO2013113325A1 (en) 2012-01-31 2013-08-08 Curevac Gmbh Negatively charged nucleic acid comprising complexes for immunostimulation
EP2854857B1 (en) 2012-05-25 2018-11-28 CureVac AG Reversible immobilization and/or controlled release of nucleic acid containing nanoparticles by (biodegradable) polymer coatings

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Publication number Priority date Publication date Assignee Title
US10111967B2 (en) 2007-09-04 2018-10-30 Curevac Ag Complexes of RNA and cationic peptides for transfection and for immunostimulation
US9352028B2 (en) 2007-10-09 2016-05-31 Curevac Ag Composition for treating lung cancer, particularly of non-small lung cancers (NSCLC)
US9402887B2 (en) 2007-10-09 2016-08-02 Curevac Ag Composition for treating prostate cancer (PCa)
US9238064B2 (en) 2008-03-03 2016-01-19 University Of Miami Allogeneic cancer cell-based immunotherapy
US9616084B2 (en) 2009-12-09 2017-04-11 Curevac Ag Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US20130142818A1 (en) * 2010-07-30 2013-06-06 Curevac Gmbh Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
US8968746B2 (en) * 2010-07-30 2015-03-03 Curevac Gmbh Complexation of nucleic acids with disulfide-crosslinked cationic components for transfection and immunostimulation
US9181319B2 (en) 2010-08-06 2015-11-10 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9937233B2 (en) 2010-08-06 2018-04-10 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9447164B2 (en) 2010-08-06 2016-09-20 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US8822663B2 (en) 2010-08-06 2014-09-02 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9234013B2 (en) 2010-08-13 2016-01-12 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded protein
US9839697B2 (en) 2010-08-13 2017-12-12 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein
US9334328B2 (en) 2010-10-01 2016-05-10 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US10064959B2 (en) 2010-10-01 2018-09-04 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9701965B2 (en) 2010-10-01 2017-07-11 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9657295B2 (en) 2010-10-01 2017-05-23 Modernatx, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9737595B2 (en) 2010-12-29 2017-08-22 Curevac Ag Combination of vaccination and inhibition of MHC class I restricted antigen presentation
US20130259879A1 (en) * 2011-02-21 2013-10-03 Curevac Gmbh Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
US9421255B2 (en) * 2011-02-21 2016-08-23 Curevac Ag Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
US9623095B2 (en) 2011-03-02 2017-04-18 Curevac Ag Vaccination in newborns and infants
US10172935B2 (en) 2011-03-02 2019-01-08 Curevac Ag Vaccination in newborns and infants
US8710200B2 (en) 2011-03-31 2014-04-29 Moderna Therapeutics, Inc. Engineered nucleic acids encoding a modified erythropoietin and their expression
US9533047B2 (en) 2011-03-31 2017-01-03 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US9950068B2 (en) 2011-03-31 2018-04-24 Modernatx, Inc. Delivery and formulation of engineered nucleic acids
US10022425B2 (en) 2011-09-12 2018-07-17 Modernatx, Inc. Engineered nucleic acids and methods of use thereof
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US9428535B2 (en) 2011-10-03 2016-08-30 Moderna Therapeutics, Inc. Modified nucleosides, nucleotides, and nucleic acids, and uses thereof
US9937211B2 (en) 2011-10-07 2018-04-10 4D Pharma Research Limited Composition of Roseburia hominis
US8680069B2 (en) 2011-12-16 2014-03-25 Moderna Therapeutics, Inc. Modified polynucleotides for the production of G-CSF
US9186372B2 (en) 2011-12-16 2015-11-17 Moderna Therapeutics, Inc. Split dose administration
US8664194B2 (en) 2011-12-16 2014-03-04 Moderna Therapeutics, Inc. Method for producing a protein of interest in a primate
US8754062B2 (en) 2011-12-16 2014-06-17 Moderna Therapeutics, Inc. DLIN-KC2-DMA lipid nanoparticle delivery of modified polynucleotides
US9271996B2 (en) 2011-12-16 2016-03-01 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
US9295689B2 (en) 2011-12-16 2016-03-29 Moderna Therapeutics, Inc. Formulation and delivery of PLGA microspheres
WO2013113736A1 (en) * 2012-01-31 2013-08-08 Bayer Innovation Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and an antigen
WO2013113501A1 (en) * 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or pepide antigen
WO2013113326A1 (en) * 2012-01-31 2013-08-08 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
EP2623121A1 (en) * 2012-01-31 2013-08-07 Bayer Innovation GmbH Pharmaceutical composition comprising a polymeric carrier cargo complex and an antigen
AU2013214572B2 (en) * 2012-01-31 2017-08-17 Curevac Gmbh Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen
RU2617058C2 (en) * 2012-01-31 2017-04-19 Кьюрвак Аг Pharmaceutical composition containing complex of polymer carrier and carried substance and at least one protein or peptide antigen
US10166283B2 (en) 2012-02-15 2019-01-01 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen
US10232024B2 (en) 2012-02-15 2019-03-19 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded allergenic antigen or an autoimmune self-antigen
US10111968B2 (en) 2012-02-15 2018-10-30 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
US9447431B2 (en) 2012-02-15 2016-09-20 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
US9669089B2 (en) 2012-02-15 2017-06-06 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen
US10010592B2 (en) 2012-02-15 2018-07-03 Curevac Ag Nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal for increasing the expression of an encoded tumour antigen
US9683233B2 (en) 2012-03-27 2017-06-20 Curevac Ag Artificial nucleic acid molecules for improved protein or peptide expression
US10080809B2 (en) 2012-03-27 2018-09-25 Curevac Ag Artificial nucleic acid molecules comprising a 5′TOP UTR
US9890391B2 (en) 2012-03-27 2018-02-13 Curevac Ag RNA vector with an open reading frame, an albumin 3′-UTR, and a histone stem loop
US9303079B2 (en) 2012-03-30 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9061059B2 (en) 2012-04-02 2015-06-23 Moderna Therapeutics, Inc. Modified polynucleotides for treating protein deficiency
US9192651B2 (en) 2012-04-02 2015-11-24 Moderna Therapeutics, Inc. Modified polynucleotides for the production of secreted proteins
US9216205B2 (en) 2012-04-02 2015-12-22 Moderna Therapeutics, Inc. Modified polynucleotides encoding granulysin
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
US9221891B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. In vivo production of proteins
US9587003B2 (en) 2012-04-02 2017-03-07 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides
US9220755B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9149506B2 (en) 2012-04-02 2015-10-06 Moderna Therapeutics, Inc. Modified polynucleotides encoding septin-4
US8999380B2 (en) 2012-04-02 2015-04-07 Moderna Therapeutics, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9233141B2 (en) 2012-04-02 2016-01-12 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders
US9114113B2 (en) 2012-04-02 2015-08-25 Moderna Therapeutics, Inc. Modified polynucleotides encoding citeD4
US9220792B2 (en) 2012-04-02 2015-12-29 Moderna Therapeutics, Inc. Modified polynucleotides encoding aquaporin-5
US9675668B2 (en) 2012-04-02 2017-06-13 Moderna Therapeutics, Inc. Modified polynucleotides encoding hepatitis A virus cellular receptor 2
US9254311B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides for the production of proteins
US9050297B2 (en) 2012-04-02 2015-06-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator
US9107886B2 (en) 2012-04-02 2015-08-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding basic helix-loop-helix family member E41
US9301993B2 (en) 2012-04-02 2016-04-05 Moderna Therapeutics, Inc. Modified polynucleotides encoding apoptosis inducing factor 1
US9095552B2 (en) 2012-04-02 2015-08-04 Moderna Therapeutics, Inc. Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1
US9255129B2 (en) 2012-04-02 2016-02-09 Moderna Therapeutics, Inc. Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1
US9782462B2 (en) 2012-04-02 2017-10-10 Modernatx, Inc. Modified polynucleotides for the production of proteins associated with human disease
US9878056B2 (en) 2012-04-02 2018-01-30 Modernatx, Inc. Modified polynucleotides for the production of cosmetic proteins and peptides
US9814760B2 (en) 2012-04-02 2017-11-14 Modernatx, Inc. Modified polynucleotides for the production of biologics and proteins associated with human disease
US9828416B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of secreted proteins
US9827332B2 (en) 2012-04-02 2017-11-28 Modernatx, Inc. Modified polynucleotides for the production of proteins
US9089604B2 (en) 2012-04-02 2015-07-28 Moderna Therapeutics, Inc. Modified polynucleotides for treating galactosylceramidase protein deficiency
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
EP2872160A4 (en) * 2012-04-17 2015-11-25 Univ Brown Neuroprotective composition and method of use
US10040822B2 (en) 2012-04-17 2018-08-07 National Institutes of Health, U.S. Dept. of Health and Human Services, NIH Division of Extramural Inventions and Technology Resources Neuroprotective composition and method of use
US20150290340A1 (en) * 2012-09-28 2015-10-15 Industry-University Cooperation Foundation Hanyang University Short interference rna gene delivery system for systemic circulation
WO2014051318A2 (en) * 2012-09-28 2014-04-03 한양대학교 산학협력단 Short interference rna gene delivery system for systemic circulation
CN104870023A (en) * 2012-09-28 2015-08-26 汉阳大学校产学协力团 Short interference RNA gene delivery system for systemic circulation
WO2014051318A3 (en) * 2012-09-28 2014-05-22 한양대학교 산학협력단 Short interference rna gene delivery system for systemic circulation
US9713645B2 (en) * 2012-09-28 2017-07-25 Industry-University Cooperation Foundation Hanyang University Short interference RNA gene delivery system for systemic circulation
US9572893B2 (en) 2012-11-07 2017-02-21 Council Of Scientific And Industrial Research Nanocomplex containing cationic peptide for biomolecule delivery
WO2014072999A1 (en) * 2012-11-07 2014-05-15 Council Of Scientific And Industrial Research Nanocomplex containing cationic peptide for biomolecule delivery
US9597380B2 (en) 2012-11-26 2017-03-21 Modernatx, Inc. Terminally modified RNA
US10117920B2 (en) 2013-02-22 2018-11-06 Curevac Ag Combination of vaccination and inhibition of the PD-1 pathway
US9974845B2 (en) 2013-02-22 2018-05-22 Curevac Ag Combination of vaccination and inhibition of the PD-1 pathway
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
US9796762B2 (en) 2013-04-10 2017-10-24 4D Pharma Research Limited Polypeptide and immune modulation
US9688729B2 (en) 2013-08-21 2017-06-27 Curevac Ag Respiratory syncytial virus (RSV) vaccine
US10150797B2 (en) 2013-08-21 2018-12-11 Curevac Ag Respiratory syncytial virus (RSV) vaccine
US10047375B2 (en) 2013-12-30 2018-08-14 Curevac Ag Artificial nucleic acid molecules
US10022435B2 (en) 2014-04-23 2018-07-17 Modernatx, Inc. Nucleic acid vaccines
US9872900B2 (en) 2014-04-23 2018-01-23 Modernatx, Inc. Nucleic acid vaccines
CN104530438A (en) * 2014-12-11 2015-04-22 华南理工大学 Cholesterol modification based pH response polypeptide polymer as well as preparation method and application thereof

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