Novel transporter constructs and transporter cargo conjugate molecules   

Abstract: The present invention relates to novel transporter constructs of the generic formula (I) DlLLLxDm(LLLyDn)a and variants thereof. The present invention also refers to transporter cargo conjugate molecules, particularly of conjugates of the novel transporter constructs with a cargo moiety, e.g. proteins or peptides, nucleic acids, cytotoxic agents, organic molecules, etc. The present invention furthermore discloses (pharmaceutical) compositions comprising these conjugates and methods of treatment and uses involving such transporter constructs.

The present invention relates to novel transporter constructs of the generic formula (I) DlLLLxDm(LLLyDn)a and variants thereof. The present invention also refers to transporter cargo conjugate molecules, particularly of conjugates of the novel transporter constructs with a cargo moiety, e.g. proteins or peptides, nucleic acids, cytotoxic agents, organic molecules, etc. The present invention furthermore discloses (pharmaceutical) compositions comprising these conjugates and methods of treatment and uses involving such transporter constructs.

Techniques enabling efficient transfer of a substance of interest from the external medium into tissue or cells, and particularly to cellular nuclei, such as nucleic acids, proteins or cytotoxic agents, but also of other (therapeutically useful) compounds, are of considerable interest in the field of biotechnology. These techniques may be suitable for transport and translation of nucleic acids into cells in vitro and in vivo and thus for protein or peptide production, for regulation of gene expression, for induction of cytotoxic or apoptotic effects, for analysis of intracellular processes and for the analysis of the effect of the transport of a variety of different cargos into a cell (or cell nucleus), etc.

One important application of such a transfer of a cargo of interest from the external medium into tissue or cells is gene therapy, wherein the cargo is typically a nucleic acid or a gene. Although this technique has shown some rather promising developments in the last decades, gene transfer is typically limited by the inability of the gene transfer vectors to effectively transfer the biologically active cargo into the cytoplasm or nuclei of cells in the host to be treated without affecting the host genome or altering the biological properties of the active cargo.

In this respect, several techniques have been developed in an effort to more efficiently transfect e.g. nucleic acids, such as DNA or RNA, into cells. Transfection of nucleic acids into cells or tissues of patients by methods of gene transfer is a central method of molecular medicine and plays a critical role in therapy and prevention of numerous diseases.

Representative examples of gene transfer methods include general (physical or physico-chemical) methods such as coprecipitating nucleic acids with calcium phosphate or DEAE-dextran, a method which enables nucleic acids to penetrate the plasma membrane and then enter the cell and/or nucleus. However, this technique suffers from low transfer efficiency and a high percentage of cell death. Additionally, this method is restricted to in vitro or ex vivo methods, but is not applicable to in vivo situations due to its very nature.

The same holds for methods involving in vitro electroporation. In vitro electroporation is based on the use of high-voltage current to make cell membranes permeable to allow the introduction of new nucleic acids, e.g. DNA or RNA, into the cell. However, such methods are typically not suitable in vivo. Furthermore, this technique also suffers from low transfer efficiency and a high percentage of cell death.

Further well known physical or physico-chemical methods include (direct) injection of (naked) nucleic acids or biolistic gene transfer. Biolistic gene transfer (also known as biolistic particle bombardment) is a method developed at Cornell University that allows introducing genetic material into tissues or culture cells. Biolistic gene transfer is typically accomplished by surface coating metal particles, such as gold or silver particles, and shooting these metal particles, comprising the adsorbed DNA, into cells by using a gene gun. Similar as discussed above this method is restricted to in vitro or ex vivo methods, but is usually not applicable in in vivo situations.

Other methods utilize the transport capabilities of so called transporter molecules. Transporter molecules to be used in this context typically may be divided into viral vectors, i.e. transporter molecules, which involve viral elements, and nonviral vectors.

The most successful gene therapy strategies available today rely on viral vectors, such as adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses. These viral vectors typically employ a conjugate of a virus-related substance with a strong affinity for DNA and a nucleic acid. Due to their infection properties, viruses or viral vectors have a very high transfection rate. The viral vectors typically used are genetically modified in a way that no functional infectious particles are formed in the transfected cell. In spite of this safety precaution, however, there are many problems associated with viral vectors related to immunogenicity, cytotoxicity, and insertional mutagenesis. As an example, the risk of uncontrolled propagation of the introduced therapeutically active genes or viral genes cannot be ruled out, e.g., because of possible recombination events. Additionally, the viral conjugates are difficult to use and typically require a long preparation prior to treatment (see, e.g., U.S. Pat. No. 5,521,291).

Although nonviral vectors are not as efficient as viral vectors, many have been developed to provide a safer alternative in gene therapy. Some of the most common nonviral vectors include polyethylenimine, dendrimers, chitosan, polylysine, and peptide based transporter systems, e.g. many types of peptides, which are generally cationic in nature and able to interact with nucleic acids such as plasmid DNA through electrostatic interactions.

For successful delivery, the nonviral vectors, particularly peptide based transporter systems must be able to overcome many barriers. Such barriers include protection of the cargo moiety, e.g. of DNA or other compounds, during transport and prevention of an early degradation or metabolisation of the cargo moiety in vivo. In case of nucleic acids, such as DNA and RNA molecules, the nonviral vectors must furthermore be capable to specifically deliver these molecules for efficient gene expression in target cells.

Particularly for nucleic acids such DNA and RNA molecules there are presently 4 barriers nonviral vectors must overcome to achieve successful gene delivery (see e.g. Martin et al., The AAPS Journal 2007; 9 (1) Article 3). The nonviral vector must be able to 1) tightly compact and protect the nucleic acids, 2) it must able to target specific cell-surface receptors, 3) the nonviral vector must be capable to disrupt the endosomal membrane, and 4) it has to deliver the nucleic acid cargo to the nucleus and allow translation of an encoded protein or peptide sequence.

Such nonviral vectors, particularly peptide-based nonviral vectors, are advantageous over other nonviral strategies in that they are in general able to achieve all 4 of these goals, however, with different efficiency regarding the different barriers.

As an example, cationic peptides rich in basic residues such as lysine and/or arginine are able to efficiently condense nucleic acids such as DNA into small, compact particles that can be stabilized in serum. Furthermore, attachment of a peptide ligand to the polyplex allows targeting to specific receptors and/or specific cell types. Polyplexes or cationic polymers as mentioned above typically form a complex with negatively charged nucleic acids leading to a condensation of nucleic acids and protecting these nucleic acids against degradation. Transport into cells using polyplexes (cationic polymers) typically occurs via receptor mediated endocytosis. Thereby, the DNA is coupled to a distinct molecule, such as Transferrin, via e.g. the polyplex poly-L-lysine (PLL), which binds to a surface receptor and triggers endocytosis. Polyplexes (cationic polymers) include e.g. poly-L-lysine (PLL), chitosan, polyethylenimine (PEI), polydimethylaminoethylmethacrylate (PD-MAEMA), polyamidoamine (PAMAM). Such effects are also known from nanoplexes (nanoparticular systems) or lipoplexes (liposomal systems). Nanoplexes (nanoparticular systems) typically involve the use of polyacrylates, polyamides, polystyrene, cyanoacrylates, polylactat (PLA), polylactic-co-glycolic acid) (PLGA), etc. Lipoplexes or liposomal systems typically involve the use of cationic lipids, which are capable to mimic a cell membrane. Thereby, the positively charged moiety of the lipid interacts with the negatively charged moiety of the nucleic acid and thus enables fusion with the cell membrane. Lipoplexes or liposomal systems include, e.g. DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, EDMPC, etc.

In this context, receptor-mediated endocytosis is also widely exploited in experimental systems for the targeted delivery of cargos such as nucleic acids or therapeutic agents into cells. During receptor-mediated endocytosis the cargo-containing complexes are either selectively internalized by receptors located in the cell membrane which are specific for the cargos, or by specific antibodies located in membrane constituents. Endocytotic activity has been described for many receptors including IgG Fc, somatostatin, insulin, IGF-I and -II, transferrin, EGF, GLP-1, VLDL or integrin receptors, etc.

Different peptide or protein sequences have been tested widely for their use in gene transfer methods via receptor-mediated endocytosis. Interestingly, the isolation of peptide sequences that direct efficient receptor-mediated endocytosis have been profoundly boosted by the use of phage display technologies. Phage display libraries are extremely powerful tools that provide for a practically unlimited source of molecular variants including modifications of natural ligands or cargo moieties to cell receptors and short peptides. Similar libraries have also been injected directly into mice and peptide sequences have been successfully isolated that show a 13-fold selectivity for brain and kidney.

Proprotein convertases may serve as an example of peptide or protein sequences that may be used for transport of molecules into cells. Proprotein convertases are an example of a cell surface receptor which gets internalized through receptor mediated endocytosis. These proteins have been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms. All cleavage sites for the proprotein convertase family obey to the consensus R—X—X—R. The mammalian proprotein convertases can be classified into three groups on the basis of their tissue distribution. Furin, PACE4, PC5/PC6, and LPCIPC7/PC8/SPC7 are expressed in a broad range of tissues and cell lines. In contrast, expression of PC2 and PC1/PC3 is limited to neuroendocrine tissues, such as pancreatic islets, pituitary, adrenal medulla and many brain areas. Expression of PC4 is highly restricted to testicular spermatogenic cells. The neuroendocrine-specific convertases, PC2 and PC1/PC3, are mainly localized in secretory granules. PC5/PC6A has also been reported to be localized to secretory granules. Furthermore, indirect evidence has suggested that a proportion of proprotein convertases molecules is present on the cell surface, and it has been shown that furin cycles between the TGN and the cell surface. Taken together, these properties indicate that proprotein convertases transport extracellular ligands into the intracellular space.

Advantageous are also so called translocatory proteins or of protein transduction domains (PTDs). Peptide sequences derived from translocatory proteins or protein transduction domains (PTDs) are typically able to selectively lyse the endosomal membrane in its acidic environment leading to cytoplasmic release of the polyplex. Translocatory proteins are considered as a group of peptides capable of effecting transport of macromolecules between cells (translocatory proteins), such as HIV-1 TAT (HIV), antennapedia (Drosophila antennapedia), HSV VP22 (Herpes simplex), FGF or lactoferrin, etc. In contrast, protein transduction domains (PTDs) are considered as a group of peptides capable of directing proteins and peptides covalently bound to these sequences into a cell via the cell membrane (Leifert and Whitton: Translocatory proteins and protein transduction domains: a critical analysis of their biological effects and the underlying mechanisms. Molecular Therapy Vol. 8 No. 1 2003). Common to translocatory proteins as well as to PTDs is a basic region, which is regarded as mainly responsible for transport of the fusion peptides since it is capable of binding polyanions such as nucleic acids. Without being bound thereto, PTDs may act similar to cationic transfection reagents using receptor dependent non-saturatable adsorptive endocytosis. PTDs are typically coupled to proteins or peptides in order to effect or enhance a CTL response when administering a peptide based vaccine (see review: Melikov and Chemomordik, Arginine-rich cell penetrating peptides: from endosomal uptake to nuclear delivery, Cell. Mol. Life. Sci. 2005).

Unfortunately, peptide based transporter systems typically undergo proteolytic degradation in vivo due to peptidases leading to truncated transporter (and/or cargo) sequences. Such peptidases may be distinguished into exopeptidases and endopeptidases, which are both enzymes capable of catalysing the splitting of proteins into smaller peptide fractions and even into single amino acids by a process known as proteolysis. In this context, endopeptidases are typically proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule). Endopeptidases are typically specific for certain amino acids. Examples of endopeptidases include e.g. trypsin, chymotrypsin, elastase, thermolysin, pepsin and endopeptidase V, etc. Trypsin is known to cut after Arg or Lys, unless followed by a Pro. Chymotrypsin is known to cut after Phe, Trp, or Tyr, unless followed by a Pro. Chymotrypsin cuts more slowly after Asn, His, Met or Leu. Elastase cuts after Ala, Gly, Ser, or Val, unless followed by a Pro. Thermolysin is a heat stable endoprotease, which cuts before Ile, Met, Phe, Trp, Tyr, or Val, unless preceded by Pro. Thermolysin sometimes cuts after Ala, Asp, His or Thr. Pepsin is known to cut before Leu, Phe, Trp or Tyr, unless preceded by Pro. Finally, endopeptidase V8 is known to cut after Glu. In contrast to endopeptidases exopeptidases are enzymes that catalyse the removal of an amino acid from the end of a polypeptide chain and thus cleave the end of said polypeptide chain. Exopeptidases may be distinguished from their cleavage site into aminopeptidases and carboxypeptidases. Aminopeptidases are typically zinc-dependent enzymes and are produced by glands of the small intestine. Aminopeptidases usually cleave a single amino acid from the amino-terminal end of a peptide or protein sequence. Carboxypeptidases are typically enzymes that hydrolyze the carboxy-terminal (C-terminal) end of a peptide bond. Humans, animals, and plants contain several types of carboxypeptidases with diverse functions ranging from catabolism to protein maturation, which is a digestive enzyme present in pancreatic juice, will cleave a single amino acid from the carboxylic end of the peptide. A particular example is Carboxypeptidase N(CPN), a plasma zinc metalloprotease comprised of two small subunits that have enzymatic activity, and two large subunits, which protect the enzyme from degradation. CPN cleaves the carboxyl-terminal amino acids arginine and lysine from biologically active peptides such as complement anaphylatoxins, kinins, and fibrinopeptides.

In order to modify proteolytic cleavage by peptide based transporter systems as defined above, the peptide based transporter systems may be composed entirely of D-amino acids, thereby forming “retro-inverso peptide sequences”. The term “retro-inverso (peptide) sequences” refers to an isomer of a linear peptide sequence in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted (see e.g. Jameson et al., Nature, 368, 744-746 (1994); Brady et al., Nature, 368, 692-693 (1994)). The advantage of combining D-enantiomeric amino acids and reverse synthesis is that the positions of carbonyl and amino groups in each amide bond are exchanged, while the position of the side-chain groups at each alpha carbon is preserved. Due to the conformational change of the naturally occurring L-enantiomeric amino acids of the peptide sequence of a peptide based transporter to D-enantiomeric amino acids the risk of proteolytic cleavage in vivo is eliminated, being advantageous and highly efficient for the purpose of transfection of a cargo moiety into a cell. In contrast, the term “reverse sequence” refers to a sequence in which the direction of the sequence is reversed (but the chirality of each amino acid residue is not inverted (e.g. D-Arg-L-Arg-L-Arg→L-Arg-L-Arg-D-Arg).

However, though efficiently working as a transporter molecule as defined above the conformational change of the naturally occurring L-enantiomeric amino acids of the peptide sequence of such a peptide based transporter to D-enantiomeric amino acids entails the risk of a predominant accumulation of these transporters in the cell during the whole lifetime of a cell or even longer in the (surrounding) tissue or organism. Accordingly, such transporters, even if the attached cargo moiety is cleaved off or is metabolised in the meantime, may remain in the cell and participate in further inter- and intracellular processes leading to unknown and unwanted side effects.

Accordingly, there is a need in the art to provide alternative nonviral transporter molecules, preferably peptide based transporter systems as defined above, which avoid such an unwanted accumulation in the cell or tissue but nevertheless allow an efficient transfer of cargo moieties into cells.

The above object is solved by the subject matter as defined in the claims attached hereto, particularly by a novel transporter construct and its conjugates (transporter cargo conjugate molecule) as defined in the claims. The above object is furthermore solved by methods and uses employing the novel transporter construct and its conjugates as defined in the claims.

According to a first aspect of the present invention, the object of the present invention is solved by a novel transporter construct comprising or consisting of at least one sequence of the generic formula (I) (SEQ ID NO: 1):

DlLLLxDm(LLLyDn)a wherein: D is a D-amino acid; L is a L-amino acid; a is 0-3, preferably 0-2, more preferably 0, 1, 2 or 3, even more preferably 0, 1, or 2 and most preferably 1; l, m and n are independently from each other 1 or 2, preferably 1; x and y are independently from each other 0, 1 or 2, preferably 1.

As used herein, the term “transporter construct” refers to an amino acid containing compound, which is capable of translocation across biological membranes. As used herein, the term “trafficking sequence” (or “transporter sequence”) refers to a sequence of amino acids providing translocation across biological membranes. Accordingly, the transporter constructs according to the present invention comprise a trafficking sequence which allow the transporter construct to translocate across biological membranes. Thus, the novel transporter constructs according to generic formula (I) effectively allow and may provide for the transport of cargo moieties, e.g. of proteins or peptides, of nucleic acids, of small organic molecules, of antigens, of cytotoxic agents, etc., into an organism, a tissue, a cell (e.g. to be treated), a cellular subcompartiment and/or into the nucleus of a cell.

Advantageously, the inventive transporter construct according to generic formula (I) is stable enough to prevent degradation by proteases prior to transport of the cargo moiety to its target site. On the other hand side, the inventive transporter constructs according to generic formula (I) are not permanently persistent in the cell and may be degraded by proteases within a considerable time limit so as to avoid negative side effects such as unwanted accumulation of the novel inventive transporter or its conjugate in the cell. As surprisingly found by the inventors, such advantageous properties may be conferred to a trafficking sequence, particularly to any trafficking sequence known in the art only by the inventive pattern (herein also described as D-/L-pattern) of the above defined generic formula (I), the specific content and position of the D-amino acids in alteration with the specific content of L-amino acids as defined in generic formula (I). This inventive D-/L-pattern allows a skilled person to define the in vivo or in vitro persistence of the inventive novel transporter construct as defined above in the cell precisely enough as a time sufficiently long to ensure administration and entering of the inventive novel transporter construct into the cell or nucleus prior to degradation of the inventive novel transporter construct by proteases within a considerable time limit. This in vivo or in vitro persistence of the inventive novel transporter construct in the cell is in fact dependent on the specific content and position of the D-amino acids in alteration with the specific content of L-amino acids as defined in generic formula (I). Furthermore, a transporter construct exhibiting the inventive D-/L-pattern of the above defined generic formula (I) is short enough to avoid a sterical hindrance of a cargo moiety by the inventive novel transporter construct in a transporter cargo conjugate molecule such as defined below. It also allows a cost efficient preparation of such inventive novel transporter constructs. Additionally, a conjugate of the transporter peptide or protein of the above defined generic formula (I) with either proteins or peptides, nucleic acids such as DNA and RNA molecules or with cytotoxic agents or even small organic molecules, etc., may be formed easily.

According to the above defined generic formula (I), the inventive novel transporter construct comprises L-amino acids and D-amino acids according to the specific D-/L-pattern as set forth in generic formula (I).

In the context of the present invention L-amino acids, also termed L-enantiomeric amino acids, are preferably amino acids selected from natively occurring amino acids or their derivatives. Naturally occurring amino acids are typically selected from the standard (proteinogenic) amino acids alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutaminic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, proline, serine, threonine, tryptophane, tyrosine, and valine, as well as from non-standard amino acids such as ornithine, citrulline, homocysteine, S-adenosyl methionione, hydroxyproline, selenocysteine, pyrrolysine, lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, etc.

Derivatives from such L-amino acids or L-enantiomeric amino acids typically comprise any naturally or non-naturally occurring derivative of these amino acids, including, without being limited thereto, amino acids as defined above comprising post-translational modifications or synthetic modifications, including acetylation (at the N-terminus of the peptide sequence, at lysine residues, etc.), deacetylation, alkylation, such as methylation, ethylation, etc. (preferably at lysine or arginine residues within the peptide sequence), dealkylation, such as demethylation, deethylation, etc., amidation (preferably at the C-terminus of the peptide sequence), formylation, gamma-carboxylation, glutamylation, glycosylation (preferably at asparagine, lysine, hydroxylysine, serine or threonine residues, etc., within the peptide sequence), addition of a heme or haem moiety, hydroxylation, iodination, isoprenylation addition of an isoprenoid moiety such as farnesyl or geranylgeraniol, etc.), lipoylation (attachment of lipoate functionality), such as prenylation, formation of a GPI anchor, including myristoylation, farnesylation, geranylgernaylation, etc., oxidation, phosphorylation (e.g. to a serine, tytosine, threonine or a histidine moiety, etc., within the peptide sequence), sulfation (e.g. of tyrosine), selenoylation, sulfation, etc.

Derivatives of L-amino acids also include, without being limited thereto, modified L-amino acids, which have been modified by introducing one of the following labels: (i) radioactive labels, i.e. radioactive phosphorylation or a radioactive label with sulphur, hydrogen, carbon, nitrogen, etc.; (ii) colored dyes (e.g. digoxygenin, etc.); (iii) fluorescent groups (e.g. fluorescein, rhodamine, fluorochrome proteins as defined below, etc.); (iv) chemoluminescent groups; (v) a combination of labels of two or more of the labels mentioned under (i) to (iv).

Particularly specific examples of derivatives of L-amino acids include, without being limited thereto, AMC (aminomethylcoumarin), Dabcyl (dimethylaminophenylazobenzoyl), Dansyl (dimethylaminonaphtalenesulfonyl), FAM (carboxyfluoroscein), Mca (methoxycoumarin acetyl), Xan (xanthyl), Abu (aminobutyric acid), Beta-Ala (beta-alanine), E-Ahx (6-aminohexanoic acid), Alpha-Aib (alpha-aminoisobutyric acid), Ams (aminoserine), Cha (cyclohexylamine), Dab (diaminobutyric acid), Hse (homoserine), Hyp (hydroxyproline), Mpr (mercaptopropionic acid), NaI (naphtylalanine), Nva (Norvaline), Orn (ornithine), Phg (phenylglycine), Sar (sarcosine), Sec (selenocysteine), Thi (thienylalanine), etc.

Furthermore, L-enantiomeric amino acids selected for the inventive novel transporter construct as defined above furthermore may be selected from specific combinations of the above defined L-enantiomeric amino acids or derivatives thereof. Such combinations may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or even more of the above defined L-enantiomeric amino acids or derivatives thereof. Combinations are also possible between any of the above defined L-enantiomeric amino acids or derivatives thereof and any the above defined D-enantiomeric amino acids or derivatives thereof within the definitions of generic formula (I) or of any of subformulas as defined herein. Such specific combinations of amino acids may exhibit a higher or a lower stability towards peptidases and thus may provide a further possibility to render the in vivo or in vitro stability of the inventive novel transporter construct as defined above towards a higher or a lower stability. As an example, the inventive novel transporter construct may contain the dipeptide sequence Arg-Lys in D- and/or L-form (i.e. both as D-enantiomeric amino acids or as L-enantiomeric amino acids or mixed D- and L-enantiomeric amino acids), preferably in L-form, which exhibits a lower stability towards pepidases and thus may be used to destabilize the peptide sequence of the inventive novel transporter construct and therefore to decrease its half life in vivo to a further extent.

In the context of the present invention D-amino acids, also termed D-enantiomeric amino acids, are preferably non-native (non-proteinogenic) “retro-inverso” amino acids, wherein these non-native (non-proteinogenic) “retro-inverso” amino acids are preferably derived from naturally occurring L-amino acids and/or their derivatives as defined above. In this context, the term “retro-inverso” refers to an isomer of a naturally occurring L-amino acid as defined above (and peptides made therefrom) in which the chirality of the naturally occurring L-amino acid residue is inverted in the corresponding D-amino acid (see e.g. Jameson et al., Nature, 368, 744-746 (1994); Brady et aZ, Nature, 368, 692-693 (1994)). In other words, in the peptide bonds of D-amino acids the positions of carbonyl and amino groups are exchanged, while the position of the side-chain groups at each alpha carbon is preserved. Accordingly, D-amino acids may be inserted into a peptide sequence consisting of or comprising L-amino acids and therefore may be conjugated with L-amino acids as defined above by methods known in the art. Such methods known in the art include e.g., without being limited thereto, liquid phase peptide synthesis methods or solid peptide synthesis methods, e.g. solid peptide synthesis methods according to Merrifield, t-Boc solid-phase peptide synthesis, Fmoc solid-phase peptide synthesis, BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate) based solid-phase peptide synthesis, etc. The content of D-amino acids in the inventive novel transporter constructs according to the D-/L-pattern of generic formula (I) above additionally provides a further variety of useful properties. For example, such novel transporter constructs enter cells more efficiently and are more stable (especially in vivo) and show lower immunogenicity than corresponding L-amino-acid-sequence based transporter constructs. However, they are not as persistent in the cell as transporter constructs entirely made of D-amino acids, particularly due to the fact that almost all decomposition enzymes, like proteases or peptidases, cleave peptide bonds between adjacent L-amino acids. Consequently, peptides composed of D-enantiomeric amino acids and L-enantiomeric amino acids are largely resistant towards a fast proteolytic breakdown without leading to an accumulation in the cell due to a missing degradation by proteases.

The above defined inventive novel transporter construct according to generic formula (I), preferably comprises L-amino acids and D-amino acids or their derivatives as defined above. Such derivatives may be contained in the entire inventive novel transporter construct in a content of about 0%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%. In other words, the entire inventive novel transporter peptide may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or even more, of such derivatives, wherein the maximum number of possible derivatives is, of course, limited by the maximum number of amino acids as contained in the above defined inventive novel transporter construct according to generic formula (I).

According to the above defined generic formula (I), the inventive novel transporter construct comprises a specific D-/L-pattern of L-amino acids and D-amino acids, which is defined by integers a, l, m, n, x and y.

According to the above definition of generic formula (I), a is a determinant defining the number of repetitions of the subgroup (LLLyD) as defined in the generic formula (I) DlLLLxDm(LLLyDn)a. According to the definition above, a may be any number selected from the range 0-3, preferably selected from the range 0-2, more preferably selected from the range 0-1, or may be selected from individual numbers 0, 1, 2 or 3, even more preferably from individual numbers 0, 1, or 2 and most preferably a=1. According to the specific number of repetitions of a=0, 1, 2 or 3, the inventive novel transporter construct according to generic formula (I) DlLLLxDm(LLLyDn)a, may consist or comprise at least one of the following subformulas (Ia) to (Id):

(SEQ ID NO: 2) (Ia): DlLLLxDm; (SEQ ID NO: 3) (Ib): DlLLLxDmLLLyDn; (SEQ ID NO: 4) (Ic): DlLLLxDmLLLyDnLLLyDn or (SEQ ID NO: 5) (Id): DlLLLxDmLLLyDnLLLyDnLLLyDn.

Furthermore, according to the above definition of generic formula (I), l, m and n are integers defining the number of D-amino acids occurring in generic formula (I), but also in the subformulas (Ia) to (Id) as defined herein. Integers l, m and n may be selected independently from each other. This particularly holds for determinant n, which may occur several times in generic formula (I) or subformulas (Ia) to (Id) as defined herein, i.e. if n occurs several times, each n may be selected independently from each other. According to the definition above, integers l, m and n independently of each other may be any number selected from the range 1-2, or may be selected from individual numbers 1 or 2, even more preferably l, m and/or n=1.

Additionally, according to the above definition of generic formula (I), x and y are integers defining the number of L-amino acids occurring in the generic formula (I), but also in the subformulas (Ia) to (Id) as defined herein. Integers x and y may be selected independently from each other. According to the definition above, integers x and y independently of each other may be any number selected from the range 0-2, preferably selected from the range 0-1, or may be selected from individual numbers 0, 1 or 2, even more preferably from individual numbers 0 or 1 and most preferably x and/or y=1.

According to one particularly preferred embodiment, the object of the present invention is solved by a novel transporter construct comprising or consisting at least one sequence of the specific subformula (Ie):

(SEQ ID NO: 6) DLLLD(LLLD)a; wherein D, L, and a are as defined above for generic formula (I) or subformulas (Ia) to (Id).

According to another particularly preferred embodiment, the object of the present invention is solved by a novel transporter construct comprising or consisting at least one sequence of the specific subformula (If):

(SEQ ID NO: 7) DLLLDLLLD; wherein D and L are as defined above for generic formula (I) or subformulas (Ia) to (Id).

The inventive novel transporter construct according to generic formula (I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), particularly transporter constructs comprising the novel D-/L-pattern may be used with or be applied to any trafficking sequence known in the art, wherein the selected number of contiguous amino acids of those trafficking sequences is determined by the number of amino acids as defined by generic formula (I) or any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If). Such trafficking sequences typically direct the transport of a cargo moiety into a cell or the nucleus or a further specific target region and may comprise, without being limited thereto, translocatory proteins as defined above, e.g. derived from HIV TAT (HIV), e.g. native proteins such as e.g. the TAT protein (e.g. as described in U.S. Pat. Nos. 5,804,604 and 5,674,980, each of these references being incorporated herein by reference), e.g. derived from HIV tat (HIV), HSV VP22 (Herpes simplex) (described in e.g. WO 97/05265; Elliott and O\'Hare, Cell 88: 223-233 (1997)), non-viral proteins (Jackson et al, Proc. Natl. Acad. Sci. USA 89: 10691-10695 (1992)), trafficking sequences derived from Antennapedia, particularly from Drosophila antennapedia (e.g. the antennapedia carrier sequence thereof), FGF, lactoferrin, etc. or derived from basic peptides, e.g. peptides having a length of 5 to 15 amino acids, preferably 10 to 12 amino acids and comprising at least 80%, more preferably 85% or even 90% basic amino acids, such as e.g. arginine, lysine and/or histidine, or may be selected from e.g. arginine rich peptide sequences, such as R9, R8, R7, R6, R5, etc., from VP22, from PTD-4 derived proteins or peptides, from RGD-K16, from PEPT1/2 or PEPT1/2 derived proteins or peptides, from SynB3 or SynB3 derived proteins or peptides, from PC inhibitors, from P21 derived proteins or peptides, or from JNKI derived proteins or peptides. Furthermore, variants, fragments and derivatives of one of the native proteins used as trafficking sequences are disclosed herewith.

Particular examples of trafficking sequences forming a basis for the novel transporter construct according to generic formula (I) or to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, may be selected from, without being limited thereto, a so-called TAT cell permeation sequence derived from the basic trafficking sequence of the HIV-1 TAT protein. Preferably, the basic trafficking sequence of the HIV-1 TAT protein may include sequences from the human immunodeficiency virus HIV-1 TAT protein, e.g. as described in, e.g., U.S. Pat. Nos. 5,804,604 and 5,674,980, each incorporated herein by reference. In this context, the full-length HIV-1 TAT protein has 86 amino acid residues [SEQ ID NO: 8] encoded by two exons of the HIV TAT gene. TAT amino acids 1-72 are encoded by exon 1, whereas amino acids 73-86 are encoded by exon 2. The full-length TAT protein is characterized by a basic region which contains two lysines and six arginines (amino acids 49-57) and a cysteine-rich region which contains seven cysteine residues (amino acids 22-37). The basic region (i.e., amino acids 49-57) was thought to be important for nuclear localization. Ruben, S. et al., J. Virol. 63: 1-8 (1989); Hauber, J. et al., J. Virol. 63 1181-1187 (1989). The cysteine-rich region mediates the formation of metal-linked dimers in vitro (Frankel, A. D. et al, Science 240: 70-73 (1988); Frankel, A. D. et al., Proc. Natl. Acad. Sci. USA 85: 6297-6300 (1988)) and is essential for its activity as a transactivator (Garcia, J. A. et al., EMBO J. 7: 3143 (1988); Sadaie, M. R. et al., J. Virol. 63:1 (1989)). As in other regulatory proteins, the N-terminal region may be involved in protection against intracellular proteases (Bachmair, A. et al., Cell 56: 1019-1032 (1989)). Preferred TAT trafficking sequences utilized with a to generic formula (I) or any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), are preferably characterized by the presence of the TAT basic region amino acid sequence (amino acids 49-57 of naturally-occurring tat protein); the absence of the TAT cysteine-rich region amino acid sequence (amino acids 22-36 of naturally-occurring TAT protein) and the absence of the TAT exon 2-encoded carboxy-terminal domain (amino acids 73-86 of naturally-occurring TAT protein).

According to a more preferred embodiment the trafficking sequences forming a basis for the novel transporter construct according to generic formula (I) or to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, may be selected from an amino acid sequence containing TAT residues 48-57 or 49 to 57, and most preferably a TAT sequence according to any of SEQ ID NOs: 8 to 14, or from a generic TAT sequence NH2—Xnb-RKKRRQRRR-Xnb—COOH (L-generic-TAT (s)) [SEQ ID NO: 16] and/or XXXXRKKRRQ RRRXXXX (L-generic-TAT) [SEQ ID NO: 15]. In this context, each X typically represents an amino acid residue, preferably selected from any (naturally occurring) amino acid residue as defined herein. Furthermore, each Xnb may be selected from any amino acid residue as defined herein, wherein n (the number of repetitions of X) is 0-5, 5-10, 10-15, 15-20, 20-30 or more. Preferably, Xnb represents a contiguous stretch of peptide residues derived from the sequence according to SEQ ID NO: 8 (TAT (1-86)). Alternatively, the trafficking sequences forming a basis for the novel transporter construct according to generic formula (I) or to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, may be selected from, e.g., a peptide containing the amino acid sequence NH2-GRKKRRQRRR-COOH (L-TAT (s1a)) [SEQ ID NO: 17] or the amino acid sequence NH2-RKKRRQRRR-COOH (L-TAT (s1b)) [SEQ ID NO: 18].

The person skilled in the art will understand that phrases like that a sequence according to generic formula (I) or according to any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If) may be used with or be applied to a particular (trafficking) sequence or may form a basis for a transporter peptide construct to generic formula (I) or any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), etc. is intended to illustrate that a sequence is claimed which exhibits certain characterisitics with regard to: i) the sequence of side chain residues characterizing specific amino acid entities, and ii) the sequence of D- and L-amino acids in said sequence.

To give an illustrative example: If subformula (If) is used with or applied to TAT (1-86) (SEQ ID NO:8), the sequence of the side chain residues (i) is as indicated in SEQ ID NO: 8. However, this claimed sequence is not a pure L-amino acid sequence, but comprises somewhere the motif of subformula (If). An example for such embodiment would be the following sequence (D-amino acids indicated in small letters, L amino acids indicated in capital letters):

Since SEQ ID NO:8 comprises 86 amino acids, there are of course several further possibilities of placing the motif of subformula (If) elsewhere in this sequence. It is also envisioned that the sequence may comprise in particular embodiments more than one motif of the generic formula and/or subformulas.

Particular preferred examples of trafficking sequences forming a basis for a transporter peptide construct to generic formula (I) or any of subformulas (Ia), (Ib), (Ic), (Id), (Ie), or (If), as defined above, may be selected, without being limited thereto, from sequences or a part thereof as defined according to Table 1 below, or any fragment or variant or derivative thereof (as long as it retains the function of translocating across a biological membrane).

TABLE 1 SEQUENCE/PEPTIDE SEQ NAME ID NO AA SEQUENCE TAT (1-86)   8 86 MEPVDPRLEP WKHPGSQPKT ACTNCYCKKC CFHCQVCFIT KALGISYGRK KRRQRRRPPQ GSQTHQVSLS KQPTSQSRGD

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