WO2013150338A1

WO2013150338A1 - Stapled cell penetrating peptides for intracellular delivery of molecules

Info

Publication number: WO2013150338A1
Application number: PCT/IB2012/051668
Authority: WO
Inventors: Gilles Divita; Karidia KONATE; Sébastien DESHAYES; Gudrun ALDRIAN
Original assignee: Centre National De La Recherche Scientifique
Priority date: 2012-04-04
Filing date: 2012-04-04
Publication date: 2013-10-10

Abstract

The present invention pertains to the field of intracellular delivery of molecules such as nucleic acids and small hydrophobic molecules. In particular, the invention relates to new cell-penetrating peptides (CPP) carriers, which comprise a chemical staple between at least two amino acid residues and exhibit increased stability and efficacy.

Description

STAPLED CELL PENETRATING PEPTIDES FOR INTRACELLULAR

DELIVERY OF MOLECULES

Although small molecules remain the major drugs used in clinic, in numerous cases, their therapeutic impact has reached limitations such as insufficient capability to reach targets, lack of specificity, requirement for high doses leading to toxicity and major side effects. Over the past ten years, in order to circumvent limitations of small molecules and of gene-based therapies, a dramatic acceleration was observed in the discovery of larger therapeutic molecules such as proteins, peptides and nucleic acids which present a high specificity for their target but do not follow Lipinski's rules. Pharmaceutical potency of these molecules remains restricted by their poor stability in vivo and by their low uptake in cells. Therefore, "delivery" has become a central piece of the therapeutic puzzle and new milestones have been established to validate delivery strategies: (a) lack of toxicity, (b) efficacy at low doses in vivo, (c) easy to handle for therapeutic applications (d) rapid endosomal release and (e) ability to reach the target.

Cell Penetrating Peptides (CPP) are one of the most promising non-viral strategies currently explored. Although definition of CPPs is constantly evolving, they are generally described as short peptides of less than 30 amino acids either derived from proteins or from chimeric sequences. They are usually amphipathic and possess a net positive charge. CPPs are able to penetrate biological membranes, to trigger the movement of various biomolecules across cell membranes into the cytoplasm and to improve their intracellular routing, thereby facilitating interactions with the target. CPPs can be subdivided into two main classes, the first requiring chemical linkage with the cargo and the second involving the formation of stable, non-covalent complexes. CPPs from both strategies have been reported to favour the delivery of a large panel of cargos (plasmid DNA, oligonucleotide, siRNA, PNA, protein, peptide, liposome, nanoparticle...) into a wide variety of cell types and in vivo models [1].

Twenty years ago, the concept of protein transduction domain (PTD) was proposed, based on the observation that some proteins, mainly transcription factors, could shuttle within cells and from one cell to another [for review see ref. 1]. The first observation was made in 1988, by Frankel and Pabo. They showed that the transcription- transactivating (Tat) protein of HIV- 1 could enter cells and translocate into the nucleus. In 1991, the group of Prochiantz reached the same conclusions with the Drosophila Antennapedia homeodomain and demonstrated that this domain was internalized by neuronal cells. These works were at the origin of the discovery in 1994 of the first Protein Transduction Domain: a 16 mer-peptide derived from the third helix of the homeodomain of Antennapedia named Penetratin. In 1997, the group of Lebleu identified the minimal sequence of Tat required for cellular uptake and the first proofs-of-concept of the application of PTD in vivo, were reported by the group of Dowdy, for the delivery of small peptides and large proteins. Historically, the notion of Cell Penetrating Peptide (CPP) was introduced by the group of Langel, in 1998, with the design of the first chimeric peptide carrier, the Transportan, which derived from the N-terminal fragment of the neuropeptide galanin, linked to mastoparan, a wasp venom peptide. Transportan has been originally reported to improve the delivery of PNAs both in cultured cells and in vivo. In 1997, the group of Heitz and Divita proposed a new strategy involving CPP in the formation of stable but non-covalent complexes with their cargo [2]. The strategy was first based on the short peptide carrier (MPG) consisting of two domains: a hydrophilic (polar) domain and a hydrophobic (apolar) domain. MPG was designed for the delivery of nucleic acids [2]. The primary amphipathic peptide Pep-1 was then proposed for non-covalent delivery of proteins and peptides [3]. Then the groups of Wender and of Futaki demonstrated that polyarginine sequences (Arg8) are sufficient to drive small and large molecules into cells and in vivo. Ever since, many CPPs derived from natural or unnatural sequences have been identified and the list is constantly increasing. Peptides have been derived from VP22 protein of Herpes Simplex Virus, from calcitonin, from antimicrobial or toxin peptides, from proteins involved in cell cycle regulation, as well as from polyproline-rich peptides [review, 1].

The inventors previously demonstrated that structural polymorphism of amphipathic CPPs plays a major role in their efficacy and cellular uptake. They demonstrated that CPPs such as PEP-1 (NH₂-KETWWETWWTEWSQPKKKRKV-Cya, SEQ ID No: 1), MPG (NH₂-GALFLGFLGAAGSTMGAWSQPKKKRKV-Cya, SEQ ID No: 8), CADYl (NH₂-GL WRAL WRLLRS L WRLL WKA-Cya, SEQ ID No: 18), CADY-2 (NH₂-GL WWRL W WRLRS WFRL WFRA-Cya, SEQ ID No: 29), VEPEP-6 (NH₂- ALFLARWRLLRSLWRLLWK-Cya, SEQ ID No: 39) and derived sequences are able to adopt an helical conformation when interacting with their respective cargos or phospholipids. The helical motif of these peptides was identified by both molecular modelling and NMR. They have now done the same for novel CPPs such as MPG8 (NH₂- AFLGWLGAWGTMGWSPKSKRK, SEQ ID No: 13), PG9 (NH₂- GL WRAL WRAL WRSLWRLKRKV-Cya, SEQ ID No: 52), PG16 (NH₂- GLWRALWRGLRSLWRLLWKV-Cya, SEQ ID No: 56), PGW (NH₂- GL WRAL WRLWRSLWRLL WKA-Cya, SEQ ID No: 59) and CCR (NH₂- RELWRELWRLWRELWREWRV-Cya, SEQ ID No: 66). Recently, in a context completely different from CPPs, hydrocarbon alpha helix stapled peptides have been developed and reported to be more stable and able to enter the cell [4]. The a-helix features 3.6 residues per complete turn, which places the i, i+4, i+7, and i+11 side chains on the same face of the folded structure. In general, the first step in designing stapled peptides for macromolecular target is the identification of appropriate sites for incorporating the non natural amino acids used to form the hydrocarbon cross-link. Generally, residues which are not involved in the target recognition are chosen as potential sites for incorporation of olefin-bearing building blocks. These site are subsequently used to incorporate various suitable stapling systems such as i, i+3; i, i+4 or i, i+7.The classical strategy to stabilize the a-helical conformation in peptides employs covalent bonds between the i and i+3, i and i+4 or i and i+7 side chain groups (Figure 1 A, B). The i, i+4 is the optimal stabilized stapled peptides structure. The stapled are synthetized by solid-phase peptide synthesis (SPPS), using animo acids with acid-labile side chain protecting groups and a base labile fluorenylmetoxycarbonyl protecting group on the backbone amine (Figure 1 C, D).

The inventors have now demonstrated that stabilizing the helical core of amphipathic CPPs surprisingly improves both the stability of their interaction with their respective cargos and their uptake and delivery properties.

The present invention hence pertains to a cell-penetrating peptide comprising an amphipathic peptide moiety made of 15 to 27 amino acids, characterized in that it further comprises a hydrocarbon linkage (also designed as the "staple") between two residues of said peptide moiety separated by two (i, i+3), three (i, i+4) or six residues (i, i+7). Obviously, the "peptide moiety" designates not only molecules in which amino acid residues (in L or D configurations) are joined by peptide (-CO-NH-) linkages, but also synthetic pseudopeptides or peptidomimetics in which the peptide bond is modified, provided the immunogenicity and the toxicity of the CPP is not increased by this modification, and provided the CPP retains its ability to bind to its cargo and to form "cages" with the same or better affinity and stability. Of course, the binding of a hydrocarbon linkage (also called "stapling") implies the use of modified amino acid residues, some of which will be described in more details below. The hydrocarbon linkage can also be replaced by another chemical linkage.

The inventors have demonstrated (see experimental data below) that stapling improves the performances of amphipathic CPPs, whatever the CPP used. In particular, the present invention can advantageously be performed with CPPs derived from PEP-1 (KETWWETWWTEWSQPKKKRKV, SEQ ID No: 1), MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV, SEQ ID No: 8), CADY1 (GLWRALWRLLRSLWRLLWKA, SEQ ID No: 18), CADY-2 (GLWWRLWWRLRSWFRLWFRA, SEQ ID No: 29), VEPEP-6 (ALFLARWRLLRSLWRLLWK, SEQ ID No: 39).

According to a particular embodiment, the stapled CPP according to the present invention is derived from PEP- 1 (KETWWETWWTEWSQPK KR V, SEQ ID No: 1) and has, for example, the following sequence: KX₁X₂WWX₁TWWX₂X₁WX₃QX₄KKKRKV (stapled Pep-1, SEQ ID No: 7), wherein Xj is E or a non-natural amino acid used for the binding of a hydrocarbon staple, X₂ is T or a non-natural amino acid used for the binding of a hydrocarbon staple, X₃ is S or a non- natural amino acid used for the binding of a hydrocarbon staple and X4 is T or a non- natural amino acid used for the binding of a hydrocarbon staple. Of course, in this sequence, a non-null pair number of residues (generally two or four) will be non-natural amino residues used for the binding of a hydrocarbon staple. If the CPP comprises a unique staple, only two amino acids, separated by two, three or six amino acids, will be artificial amino acids designed for the binding of a hydrocarbon staple. If the CPP comprises two staples ("doubly-stapled CPP"), it will comprise two pairs of artificial amino acids designed for the binding of a hydrocarbon staple, wherein the two amino acids of each pair are separated by two, three or six amino acids. In a preferred embodiment of the doubly- stapled CPPs, the first amino acid of the second pair of amino acids bound by a staple is downstream the second amino acid of the first pair (for example, in the above sequence, amino acids in positions 2 and 5 can be bound by a first staple and amino acids in positions 9 and 12 can then be bound by a second staple). The same provisos apply to the other generic sequences described hereafter (SEQ ID Nos: 12, 17, 28, 35, 38, 49, 50, 51, 55, 58, 61, 63, 65 and 68), and will not be repeated. Preferred examples of stapled CPPs derived from Pep-1 are:

- SEQ ID No: 2 (Pep-stl) KEX]WWETWWXEWSQPKKKRKV

- SEQ ID No: 3 (Pep-st2) KETWWX^WWTEWXQPKKKRKV

- SEQ ID No: 4 (Pep-st3) KXTWWXTWWTEWSQPKKKRKV

- SEQ ID No: 5 (Pep-st4) KETWWETWWTXWSQXKKKRKV,

wherein X and X] designate non-natural amino acids which are linked by a hydrocarbon linkage, and

- SEQ ID No: 6 (Pep-st5) KXTWWXTWWTXWSQXKKKRKV,

wherein X designates non-natural amino acids used for the binding of a hydrocarbon staple and wherein the residues at positions 2 and 6 are linked by a first hydrocarbon linkage and the residues at positions 1 1 and 15 are linked by a second hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefin-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative R-configuration) as "Χ ' and an olefin- bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S-configuration) as "X". According to another particular embodiment, the stapled CPP according to the present invention is derived from MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV, SEQ ID No: 8) or its shortened version MPG8 (AFLGWLGAWGTMGWSPKSKRK, SEQ ID No: 13). In this embodiment, the peptide moiety of the stapled CPP can be, for example, GXLFLXFLXXAGSTMXAWSQPKKKRKV (stapled MPG, SEQ ID No: 12), wherein X is A, G or a non-natural amino acid used for the binding of a hydrocarbon staple, or AFX₁GWLX₂AWX₁X₃MGWX₄PKSKRK (stapled MPG8, SEQ ID No: 17), wherein Xj is L or a non-natural amino acid used for the binding of a hydrocarbon staple, X₂ is G or a non-natural amino acid used for the binding of a hydrocarbon staple, X₃ is T or a non- natural amino acid used for the binding of a hydrocarbon staple and X₄ is S or a non- natural amino acid used for the binding of a hydrocarbon staple. Preferred examples of stapled CPPs derived from MPG are:

- SEQ ID No: 9 (MPG-stl) GX^FLXFLGAAGSTMGAWSQPKKKRKV

- SEQ ID No: 10 (MPG-st2) GGLFLX _\ FLGXAGSTMGA WS QPKKKRKV

- SEQ ID No: 1 1 (MPG-st3) GGLFLGFLX^AGSTMXAWSQPKKKRKV

- SEQ ID No: 14 (MPG8-stl) AFLGWLG A WGX i MG WXPKSKRK

- SEQ ID No: 15 (MPG8-st2) AFX^WLGAWXTMGWSPKSKRK and

- SEQ ID No: 16 (MPG8-st3) AFXjGWLXAWGTMGWSPKSKRK, wherein X and X_\ designate non-natural amino acids which are linked by a hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefm-bearing non- natural amino acid (a-methyl, a-alkenyl glycine derivative R-configuration) as "Xi" and an olefm-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S- configuration) as "X".

According to another particular embodiment, the stapled CPP according to the present invention is derived from CADY (GLWRALWRLLRSLWRLLWKA, SEQ ID No: 18) or from its variants as described in EP1795539B1 , WO 2007/069090 and 12/346,000, as well as from other variants of CADY such as PG9 (GLWRALWRALWRSLWRLKRKV, SEQ ID No: 52), PG16 (GLWRALWRGLRSLWRLLWKV, SEQ ID No: 56) and PGW (GLWRALWRLWRSLWRLLWKA, SEQ ID No: 59). The peptide moiety of stapled CPPs according to this embodiment can be, for example,

GLX12X1AX2X12X1X6LX7X1X3X2X12X9X8X10X11X4X5 (stapled CADY, SEQ ID No: 28), wherein X} is R or a non-natural amino acid used for the binding of a hydrocarbon staple, X₂ is L or a non-natural amino acid used for the binding of a hydrocarbon staple, X₃ is R, S L or a non-natural amino acid used for the binding of a hydrocarbon staple, X is K, S or a non-natural amino acid used for the binding of a hydrocarbon staple and X₅ is A, V, Q or K, X₆ is A, L, G or a non-natural amino acid used for the binding of a hydrocarbon staple, X₇ is W or none, X₈ is K, L, S or a non-natural amino acid used for the binding of a hydrocarbon staple, X₉ is R or K, X₁₀ is L or K, Xn is R or W and X)₂ is W, F or Y, and more particularly GLWX₁AX₂WX₁X₂LX₁X₃X₂WRX₂LWX₄X₅ (stapled CADY1, SEQ ID No: 27), wherein Xj is R or a non-natural amino acid used for the binding of a hydrocarbon staple, X₂ is L or a non-natural amino acid used for the binding of a hydrocarbon staple, X₃ is R, S L or a non-natural amino acid used for the binding of a hydrocarbon staple, X₄ is K or a non-natural amino acid used for the binding of a hydrocarbon staple and X₅ is A or K, as well as GLWX!X₂LWX₃ALWX₃SLWRLKRKV (stapled PG9, SEQ ID No: 55), wherein X\ is R or A, X₂ is A if X[ is R and X₂ is a non-natural amino acid used for the binding of a hydrocarbon staple if Xj is A, and X₃ is R or a non-natural amino acid used for the binding of a hydrocarbon staple and GLWRALWXGLRXLWRLLWKV (stapled PG16, SEQ ID No: 58) or GLWRALWXLWRXLWRLLWKA (stapled PGW, SEQ ID No: 61),wherein X is a non-natural amino acid used for the binding of a hydrocarbon staple. Preferred examples of stapled CPPs derived from CADY are:

- SEQ ID No: 19 (CADYstl) GLWXiALWRLLXRLWRLLWKA

- SEQ ID No: 20 (CADYst2) GL WRAL WRLLRX _\ L WRLL WXK

- SEQ ID No: 21 (CADYst3) GL WRAX y WRLLRLX WRLL WKA

- SEQ ID No: 22 (CADYst4) GL WRAL WXLLRXL WRLL WKA

- SEQ ID No: 23 (CADYst5) GLWRAX! WRLLRLX WRLL WKA

- SEQ ID No: 24 (CADYst5a) GL WRAX iWRLLRSX WRLL WKA

- SEQ ID No: 25 (CADYst6) GLWRALWRXjLRSLWRXLWKA

- SEQ ID No: 26 (CADYst7) GL WRAL WRXLRSXWRLL WKA

- SEQ ID No: 53 (PG9-stl) GLWRALWXiALWXRLWRLKRKV

- SEQ ID No: 54 (PG9-st2) GL W AX [ L WRAL WXRL WRLKRK V

- SEQ ID No: 57 (PG16-stl) GLWRALWXi GLRXL WRLL WKV and

- SEQ ID No: 60 (PGW-stl) GL WRAL WX _\ LWRXL WRLL WKA, wherein X and Xi designate non-natural amino acids which are linked by a hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefin-bearing non- natural amino acid (a-methyl, a-alkenyl glycine derivative R-configuration) as '¾" and an olefin-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S- configuration) as "X".

According to another particular embodiment, the stapled CPP according to the present invention is derived from CADY2 (GLWWRLWWRLRSWFRLWFRA, SEQ ID No: 29). The peptide moiety of stapled CPPs according to this embodiment can be, for example,

(stapled CADY-2, SEQ ID No: 35), wherein X] is L or a non-natural amino acid used for the binding of a hydrocarbon staple, X₂ is R or a non-natural amino acid used for the binding of a hydrocarbon staple, X₃ is L or none, is S or a non-natural amino acid used for the binding of a hydrocarbon staple and X₅ is none if X₃ is L and X₅ is A if X₃ is none, or GXWWRLWWXRLXWWRLWWXR (stapled CADY-2a, SEQ ID No: 38), wherein X is a non-natural amino acid used for the binding of a hydrocarbon staple, or GWWRLWXWRLRSWXRLWFRA (stapled SVPEP-3a, SEQ ID No: 63), or GLWWRLWWRLXSWFRSWXFA (stapled SVPEP-3b, SEQ ID No: 65), wherein X is a non-natural amino acid used for the binding of a hydrocarbon staple. Preferred examples of stapled CPPs derived from CADY2 are:

- SEQ ID No: 30 (CADY2 stl) GL WWX i L W WRLRX WFRLWFRA

- SEQ ID No: 31 (CADY2 st3) GL WWRL WWX j LLRX WFRL WFR

- SEQ ID No: 32 (CADY2 st4) GLWWRLWWXjLRSWFRXWFRA

- SEQ ID No: 34 (CADY2 st7) GLWWRXWWRXRSWFRLWFRA

- SEQ ID No: 62 (SVPEP-3a-stl) GWWRLWXjWRLRSWXRLWFRA and

- SEQ ID No: 64 (SVPEP-3b-stl)

wherein X and Xi designate non-natural amino acids which are linked by a hydrocarbon linkage, as well as

- SEQ ID No: 33 (CADY2 st5) GXiWWRLWWXLRXjWFRLWFXA

- SEQ ID No: 36 (CAD Y-2a-st6) GXWWRLWWXRLXWWRLWWXR and

- SEQ ID No: 37 (CADY-2a-st6) GXiWWRLWWXRLXiWWRLWWXR, wherein, wherein X and X\ designate non-natural amino acids used for the binding of a hydrocarbon staple and wherein the residues at positions 2 and 9 are linked by a first hydrocarbon linkage and the residues at positions 12 and 19 are linked by a second hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefin- bearing non-natural amino acid (oc-methyl, a-alkenyl glycine derivative R-configuration) as "Xi" and an olefin-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S-configuration) as "X".

According to yet another particular embodiment, the stapled CPP according to the present invention is derived from VEPEP-6 (ALFLARWRLLRSLWRLLWK, SEQ ID No: 39). In this embodiment, the peptide moiety of the stapled CPP can be, for example, X₁LX₂RALWX9LX₃X9X₄LWX9LX₅X₆X₇X₈ (stapled VEPEP-6a, SEQ ID No: 49), or XiLX₂LARWX₉LX₃X9X₄LWX9LX₅X₆X₇X₈ (stapled VEPEP-6b, SEQ ID No: 50), or X₁LX₂ARLWX₉LX₃X₉X₄LWX9LX₅X₆X₇X₈ (stapled VEPEP-6c, SEQ ID No: 51), wherein X! is beta-A or S, X₂ is F or W, X₃ is L, W, C or I, X₄ is S, A, N, T or a non-natural amino acid used for the binding of a hydrocarbon staple, X₅ is L or W, X₆ is W or R, X₇ is K or R, X₈ is A or none, and Xg is R or a non-natural amino acid used for the binding of a hydrocarbon staple. Preferred examples of stapled CPPs derived from CADY2 are:

- SEQ ID No: 40 (ST-VEPEP-6a) X₂LFRAL WX _\ LLRXL WRLL WK

- SEQ ID No: 41 (ST-VEPEP-6aa) XzLFLARWXj LLRXL WRLLWK - SEQ ID No: 42 (ST-VEPEP-6ab) X₂LFRALWXLLRXLWRLLWK

- SEQ ID No: 43 (ST-VEPEP-6ad) X₂LFLARWXLLRXLWRLLWK

- SEQ ID No: 44 (ST-VEPEP-6b) X₂LFRAL WRLLX i SLWXLLWK

- SEQ ID No: 45 (ST-VEPEP-6ba) X₂LFLARWRLLXi SLWXLLWK

- SEQ ID No: 46 (ST-VEPEP-6bb) X₂LFRALWRLLXSLWXLLWK

- SEQ ID No: 47 (ST-VEPEP-6bd) X₂LFL ARWRLLXS L WXLL WK and

- SEQ ID No: 48 (ST-VEPEP-6c)

wherein X and X are non-natural amino acids used for the binding of a hydrocarbon staple and X₂ is a beta-alanine or a serine, and wherein the non-natural residues are linked by said hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefin- bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative R-configuration) as "Xi" and an olefm-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S-configuration) as "X".

According to a particular embodiment, the stapled CPP according to the present invention is derived from CCR (RELWRELWRLWRELWREWRV, SEQ ID No: 66) and has, for example, the following sequence: RELWRXLWRLWRXLWREWRV (stapled CCR, SEQ ID No: 68), wherein X is a non-natural amino acid used for the binding of a hydrocarbon staple. A preferred CPP derived from CCR is:

- SEQ ID No: 67 (stapled CCR) REL WRX _\ L WRL WRXL WRE WRV, wherein X and Xi designate non-natural amino acids which are linked by a hydrocarbon linkage. The inventors have synthesized these stapled CPPs using an olefm-bearing non- natural amino acid (a-methyl, a-alkenyl glycine derivative R-configuration) as "X" and an olefm-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S- configuration) as "X".

According to a preferred embodiment, a cell-penetrating peptide of the present invention further comprises, covalently linked to the N-terminal end of the amino acid sequence, one or several chemical entities selected in the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, a nuclear export signal, an antibody, a polysaccharide and a targeting molecule.

In addition or alternatively, a cell-penetrating peptide according to the invention can comprise, covalently linked to the C-terminal end of its amino acid sequence, one or several groups selected in the group consisting of a cysteamide, a cysteine, a thiol, an amide, a nitrilotriacetic acid (NTA) optionally substituted, a carboxyl, a linear or ramified Ci-C₆ alkyl optionally substituted, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, nuclear export signal, an antibody, a polysaccharide and a targeting molecule. Another aspect of the present invention is a complex comprising a cell- penetrating peptide as described above and a cargo selected amongst proteins, peptides, nucleic acids and small molecules. Examples of nucleic acid cargoes are small single stranded RNA or DNA (size between 2 to 40 bases) and double stranded RNA or DNA (size up to 100 base pairs), in particular siRNA selected to silence a target mRNA and microRNAs (miR As), selected for their ability to affect expression of genes and proteins that regulate cell proliferation and/or cell death. Non-limitative examples of small molecules, especially small hydrophobic molecules which can be used include daunomycin, Paclitaxel, doxorubicin, AZT, porphyrin, fluorescently-labelled-nucleosides or nucleotides (FAM-Guanosine), hydrophobic maghemite (contrast agents or magnetic nanoparticles Fe₂ 0₃) and fluorescent dyes. Of course, in the complexes according to the present invention, the cargo and the CPP will be chosen so that the CPP efficiently binds to said cargo. Appropriate pairs of (cargo, CPP) are disclosed in Table 1 below:

Table 1 : the CPPs used in the present study and their respective cargoes

As disclosed in Example 3 below, the inventors have made complexes with a cargo and a mix of stapled and non-stapled CPPs. The results showed that complexes made with a mix comprising around 50% of stapled CPP and 50% of non- stapled CPPs are more effective as complexes comprising only stapled CPPs. In a preferred embodiment of the complexes according to the invention, these complexes hence further comprise non-stapled cell-penetrating peptides. More preferably, between 25 to 75% of the cell-penetrating peptides are stapled cell-penetrating peptides as described above.

The size of the complexes described above is preferably between 50 and 300 nm, more preferably between 50 and 500 nm, more preferably 50 to 350 nm (the size of the complex herein designates its mean diameter).

In the complexes according to the invention, the CPP/cargo molar ratio depends on the nature and size of the cargo, but is generally comprised between 1/1 and 50/1. For siRNA cargoes, the cargo/CPP molar ratio preferably ranges from 10/1 to 30/1, more preferably from 15/1 to 25/1.

According to an advantageous embodiment of the complexes as described above, the CPPs comprise an acetyl group covalently linked to their N-terminus, and/or a cysteamide group covalently linked to their C-terminus.

The above complexes can be advantageously used as "core shells" for obtaining bigger complexes, or nanoparticles, by an additional step of coating the complex with another layer of cell-penetrating peptides, which can be different from the cell- penetrating peptides used to make the complexes. Non-limitative examples of such nanoparticles are ST-CADY/ST-VEPEP-6/siRNA and ST-MPG/ST-VEPEP-6/siRNA nanoparticles, it being understood that the following notation is used: "peripheral CPP/core shell CPP/cargo", and that "ST-CPP" can designate a mix of stapled and non-stapled CPPs.

Other nanoparticles according to the present invention comprise a core which comprises a cargo complexed to a first entity selected in the group consisting of cell-penetrating peptides (stapled or not), liposomes, polycationic structures and carbon nanoparticles, wherein said core is coated by a layer of peripheral a cell-penetrating peptides comprising at least 25% of stapled cell-penetrating peptides as described above.

In all the embodiments of nanoparticles according to the present invention, the layer of peripheral cell-penetrating peptides (outer layer) preferably comprises at least 25% of stapled cell-penetrating peptides as described above, preferably between 25 and 75%, more preferably around 50%.

In these nanoparticles, the ST-CPP/core molar ratio depends on the nature and size of the core, but is generally comprised between 1/1 and 50/1, it being understood that "ST-CPP" herein designates the CPPs of the outer layer, which can be a mix of stapled and non-stapled CPP (the ratio corresponds to the total number of outer CPPs, whether stapled or not, per cargo). For CPP/siRNA cores, the peripheral ST- CPP/cargo molar ratio preferably ranges from 10/1 to 30/1, more preferably from 15/1 to 25/1.

In a preferred embodiment of the nanoparticles according to the invention, the size of the nanoparticle is between 80 and 500 nm.

According to an advantageous embodiment of the complexes and particles according to the invention, the ST-CPP peptides forming the peripheral layer of the nanoparticles comprise an acetyl group covalently linked to their N-terminus, and/or a cysteamide group covalently linked to their C-terminus.

In particular embodiments of the complexes and nanoparticles according to the invention, at least part of the cell-penetrating peptides are bound to a targeting molecule. In the case of bi-layer nanoparticles, at least part of the cell-penetrating peptides which are at the periphery of the nanoparticle are preferentially bound to a targeting molecule. When the outer layer is made of a mix of stapled and non-stapled CPPs, the targeting molecules are preferably bound to the non-stapled CPPs. Examples of targeting molecules include: antibodies, Fc and FAB fragments, and ligands, especially targeting receptors which are over-expressed at the surface of certain cell-types, etc. Among the numerous molecules which can be targeted by antibodies, Fc and Fab fragments, one can cite the receptor tyrosine kinase HEK2 receptor, MUC1, the EGF receptor and CXCR4. Non-limitative examples of other ligands which can be used are: RGD-peptide, homing targeting peptides (brain NT1 peptide, Ganglion GM peptide), folic acid, polysaccharides, Matrix metalloprotease targeting peptide motif (MMP-9).

Another aspect of the present invention is a therapeutic composition comprising a complex or a nanoparticle as described above. For example, a composition comprising a complex or nanoparticle having an anti-cyclin Bl siRNA as a cargo, a mix of stapled and non-stapled VEPEP-6 as CPPs, and a targeting molecule specific for tumor cells (for example: RGD-peptide, folic acid, MUC-1 or HEK2 receptor antobodies ...), is part of the present invention. It is to be noted that complexes and nanoparticles according to the invention can also be advantageously used for formulating non-therapeutic compositions (for example, for research, imaging and/or diagnosis purposes).

Compositions according to the invention (therapeutic or non-therapeutic) can be formulated, for example, for intravenous, topical, intrarectal, intratumoral, intranasal or intradermal administration, as well as for administration via a mouth spray. Any formulation known in the pharmacologic field can be used, such as suppository, solutions, sprays, ointments, etc. Of course, another object of the present invention is a method for delivering a molecule to a patient in need thereof, comprising administrating to said patient a complex or nanoparticle according to the present invention, in particular through intrarectal, intranasal (or oral, with a spray) or intradermal routes.

The present invention also pertains to a method for delivering a molecule into a cell in vitro or ex vivo, comprising a step of putting said cell into contact with a complex comprising said molecule and stapled cell-penetrating peptides as described above.

The invention is further illustrated by the following figures and examples.

LEGENDS TO THE FIGURES

Figure 1: Illustration of stapled peptides. (A) The two types of all- hydrocarbon stapled peptides. a-Methyl, a-alkenylglycine cross-linking amino acids are incorporated during solid-phase peptide synthesis. An i, i+4 stapled peptide requires two units of S5 incorporated at the relative positions i and i+4. An i, i+7 stapled peptide requires one unit of R8 at the i position and one unit of S5 at the i+7 position (ST-VEPEP- 6C). Resin-bound peptide is treated with Grubbs I olefin metathesis catalyst to produce a cross-link between the two nonnatural amino acids, resulting in a stapled peptide that is braced in an a-helical conformation. (B) Schematic representation of three stapled peptides (Form Kim et al, 201 1). The nomenclature Rj, i₊₃ S(8) refers to an eight-carbon metathesized cross-link with R-configuration at i and S-configuration at i+3, The nomenclature Rj, j₊₄ S(8) refers to an eigth-carbon metathesized cross-link with S- configuration at i and S-configuration at i+4, The nomenclature Rj, j₊₇ S(l l) refers to 11- carbon metathesized cross-link with R-configuration at i and S-configuration at i+7 position. (C) Fmoc-based solid-phase peptide synthesis of hydrocarbon stapled peptides (from Verdine & Hilinski, 2012, [6]). (D) Olefin-bearing non-natural amino acids used for the synthesis of hydrocarbon stapled peptides.

Figure 2: Binding of various cargoes to stapled and non-stapled CPPs, as monitored by fluorescence spectroscopy. (A) Binding of a short peptide (12 mer) and protein (small nanobody, 21 kDa) to PEP-1 and ST-PEP as monitored by fluorescence spectroscopy using tryptophan -intrinsic of the PEP-1. A fixed concentration of PEP-1 (circle) or of ST-PEP (triangle) (100 nM) was titrated by increasing concentrations of peptide (open symbol) or protein (closed symbol). Changes in Tryptophan fluorescence were monitored at 340 nm upon excitation at 295 nm. Dissociation constants were calculated from data fitting using quadratic equation. (B) Binding of siRNA to CADY and ST-CADY as monitored by fluorescence spectroscopy using a fluorescently labelled double stranded siRNA (19/19) (fluorescein-labelled). A fixed concentration of siRNA (100 nM) was titrated by increasing concentrations of CADY (closed symbol) and ST- CADY (open symbol). Changes in FITC fluorescence was monitored at 512nm nm upon excitation at 490 nm. Dissociation constants were calculated from data fitting using quadratic equation. (C) Binding of a short peptide (12 mer) to CADY-2 and ST-CADY2 as monitored by fluorescence spectroscopy using tryptophan -intrinsic of the CADY2. A fixed concentration of CADY2 (open circle) or of ST-CADY2 (closed circle) (100 nM) was titrated by increasing concentrations of peptide. Changes in Tryptophan fluorescence were monitored at 340 nm upon excitation at 295 nm. Dissociation constants were calculated from data fitting using quadratic equation. (D) Binding of small nucleic acids to stapled VEPEP-6 Binding of nucleic acid to ST-VEPEP-6a was monitored by TRP- intrinsic fluorescence spectroscopy. Nucleic acids used: double stranded DNA (45/35 mer), siRNA (19/19 or 25/25) and single stranded DNA (19 or 36 mer). A fixed concentration of ST-VEPEP-6a (50 nM) was titrated by increasing concentrations of nucleic acid, then dissociation constants were calculated from data fitting using a quadratic equation.

Figure 3: (A) Dissociation of siRNA/ST- VEPEP-6, siRNA/VEPEP-6, siRNA ST-CADY, siRNA/CADY complexes in the presence of heparan sulphate. Dissociation of siRNA/ST-CPP and siRNA/CPP complexes formed at 100 nM at a molar ratio of 20/1 was monitored by fluorescence spectroscopy, using FITC-labelled siRNA associated to ST-VEPEP-6a, VEPEP-6a, ST-CADY or CADY. Preformed complexes were incubated with increasing concentrations of Heparan sulphate and dissociation was measured at 520 nM upon excitation at 492 nm. Dissociation constants were calculated from data fitting. (B) Acetonitrile mediated dissociation of the preformed Peptide/ST- CADY-2 and Peptide/ST-PEP complexes. A fixed concentration of CY5-labelled peptide associated to ST-CADY-2 (open circle), CADY-2 (closed circle) or PEP (closed triangle) or St-PEP (open triangle) at a molar ratio 20/1. Dissociated was triggered by adding increasing acetonitrile concentrations ranging from 1 to 30%. Dissociation was monitored by following CY5-peptide fluorescence at 560 nm upon excitation at 520 nm.

Figure 4: ST-PEP- 1 and ST-CADY2 mediated delivery of peptide inhibitors in different cell lines. (A) ST-PEP- 1 and ST-CADY2 peptides have been applied for the delivery of peptide inhibitor PC4 targeting cdk2/Cyclin A association on two cancer cell lines, PC3 (A) and PANl . (B) PC-4 peptide was complexed with CADY2 (open circle), ST-CADY2 (closed circle), PEP- 1 (open triangle) and ST-PEP 1 (closed triangle). Cells were treated with increasing concentrations of CPP/PC-4 complexes on Day 1, maintained at 37°C for 4 days and cell proliferation was quantified by FACS counting. Data correspond to an average of three different experiments.

Figure 5: ST-MPG-mediated delivery of plasmid in cultured cells. 5 μg of luciferase plasmid were associated with MPG or ST-MPG peptides at two different charge ratio (4/1 and 8/1), cells were transfected with complexes and incubated for 24 hrs at 37°C. Luciferase expression was measured by luminometry.

Figure 6: ST-MPG-mediated delivery of plasmid in cultured cells. 5 μg of luciferase plasmid were associated with mixed MPG/ST-MPG peptides at ¼ charge ratio. Nanoparticles were formed with five different ratios of ST-MPG (0, 25, 50, 75, 100%), cells were transfected with complexes and incubated for 24 hrs at 37°C. Luciferase expression was measured by luminometry.

Figure 7: ST-PG16/9 mediated protein in cultured cells. ST-PG16 (Panel A) and ST-PG9 (Panel B) peptides have been applied for the delivery of small fluorescently labelled antibody in cultured Hela cell. Cy-5 labelled anti-GST antibodies were associated with ST-PG16 and ST-PG9 at molar ratio 1/20 and complexes were overlaid onto cultured cell. Afterlhr incubation the localization of the Cy-5-antibody was monitored by fluorescence microscopy.

Figure 8: ST-VEPEP-6 Cyclin Bl siRNA delivery upon systemic injection. Athymic female nude mice were subcutaneously inoculated into the flank with 1 x 10⁶ HT29. When tumour size reached about 100 mm³, animals were treated by intravenous injections, every 4 days, with a solution either saline buffer solution, free Cyc- Bl siRNA (open square), or Cyc-Bl siRNA at 3 μg (open symbol) or 5 μg(close symbol) complexed with VEPEP-6 (circle) or ST-VEPEP-6a or ST-VEPEP-6b (triangle) at a 1/20 molar ratio. Tumour diameter was measured in two directions at regular intervals using a digital calliper and tumour volume was calculated as length x width x height x 0.52. Curves show the mean value of tumour size in a cohort of three animals and neither animal death nor any sign of toxicity were observed. (A) Increase of tumor volume upon treatment. (B) Target cyclin Bl mRNA level. After 48 days, HT29 tumors were removed, and Cyclin Bl mRNA levels were evaluated by Quantigen technology and normalized to cyclophilin levels. Control (black), 5 μg siRNA-cyc-Bl (grey) complexed with VEPEP-6 or ST-VEPEP-6a at a 1/20 molar ratio.

EXAMPLES

Example 1: Materials and Methods

Synthesis protocol (Figure 1)

The stapled peptides were synthesized according to the protocols described by Young- Woo Kim et al. (Nature protocols, 2011, ref [5]), and the notations R5, S5 etc. which follow have the same meaning as in this article. Briefly, the construction of the peptide is carried out using Fmoc based solid phase synthesis. During the synthesis, the two alpha methyl, a-alkenyl amino acids are incorporated at positions separated by two, three or six intervening amino acids residues: R5 at i and S5 at i+3, or two S5 residues at both i and i+4 for one helical turn, or R5 at i and S5 at i+7 for two helical turns. The assembled peptides are then subjected in ruthenium catalyzed RMCb to form the macrocyclic hydrocarbon cross-link.

The following stapled CPPs have been synthesized:

Pep-1 derived sequences

Pep-1 CPPs showed a primary amphipatic property and adopted a helical structure in their N-terminal domain. Helix structure of Pep-1 has been solved by NMR and covered residues from 2 to 12. The derived peptides are stapled in locations 2 to 11, in i, i+4 and i , i+7.

Original sequence Pep-1: NH₂-KETWWETWWTEWSQPKKKRKV-Cya (SEQ ID No: 1)

Pep-stl : NH₂-KER₅WWETWWS₅EWSQPKKKRKV-Cya (SEQ ID No: 2)

Pep-st2: NH₂-KETWWR₅TWWTEWS₅QPKKKRKV-Cya (SEQ ID No: 3)

Pep-st3: NH₂-KS₅TWWS₅TWWTEWSQPKKKRKV-Cya (SEQ ID No: 4)

Pep-st4: NH₂-KETWWETWWTS₅WSQS₅KKKRKV-Cya (SEQ ID No: 5)

Pep-st5: NH₂-KS₅TWWS₅TWWTS₅WSQS₅KKKRKV-Cya (SEQ ID No: 6) CADY derived sequences

CADY CPPs are secondary amphipathic, with a tri-partite helical structure in central, N- and C-terminal positions. The derived peptides are stapled in locations 4 to 12, in i, i+4 and i , i+7. Residues indicated in bold are those which are bound by the hydrocarbon molecule.

Original CADY: NH₂-GLWRALWRLLRSLWRLLWKA- Cya (SEQ ID No: 18)

CADYstl : NH₂-GLWRALWRLLSRLWRLLWKA- Cya (SEQ ID No: 19)

CADYst2: NH₂-GLWRALWRLLRRLWRLLWSK- Cya (SEQ ID No: 20)

CADYst3: NH₂-GLWRARWRLLRLSWRLLWKA- Cya (SEQ ID No: 21)

CADYst4: NH₂-GLWRALWSLLRSLWRLLWKA- Cya (SEQ ID No: 22)

CADYst5: NH₂-GLWRARWRLLRLSWRLLWKA- Cya (SEQ ID No: 23)

CADYst5a: NH₂-GL WRARg WRLLRS S5 WRLL WKA- Cya (SEQ ID No: 24)

CADYst6: NH₂-GLWRALWRR8LRSLWRS₅LWKA- Cya (SEQ ID No: 25)

CADYst7: NH₂-GLWRALWRS₅LRSS₅ WRLL WKA- Cya (SEQ ID No: 26)

Non-stapled PG09: NH₂-GLWRALWRALWRSLWRLKRKV-ONH₂(Cya) (SEQ ID No: 52) PG09 stl: NH₂-GLWRALWRALWSRLWRLKRKV-ONH₂(Cya) (SEQ ID No: 53)

PG09 st2: NH₂-GLWARLWRALWSRLWRLKRKV-ONH₂(Cya) (SEQ ID No: 54)

Non-stapled PG16: NH₂-GLWRALWRGLRSLWRLLWKV-ONH₂ (SEQ ID No: 56)

PG16 stl: NH₂-GLWRALWRGLRSLWRLLWKV-ONH₂ (SEQ ID No: 57)

Non-stapled PGW: NH₂-GLWRALWRLWRSLWRLLWKA-ONH₂ (SEQ ID No:59)

PGW stl: NH₂-GLwTlALWRLWRSLWRLLWKA-ONH₂ (SEQ ID No: 60)

MPG-derived sequences

The peptides derived from MPG are stapled in locations 2 to 11, in i, i+4 and i , i+7. Residues indicated in bold are those which are bound by the hydrocarbon molecule.

Non-stapled MPG: NH₂-GALFLGFLGAAGSTMGAWSQPKKKRKV-Cya (SEQ ID No: 8)

MPG stl : NH₂-GRLFLSFLGAAGSTMGAWSQPKKKRKV-Cya (SEQ ID No: 9) MPG st2: NH₂-GGLFLRFLGSAGSTMGAWSQPKKKRKV-Cya (SEQ ID No: 10) MPG st3 : NH₂-GGLFLGFLRAAGSTMSAWSQPKKKRKV-Cya (SEQ ID No: 11) Non-stapled MPG8: NH₂-AFLGWLGAWGTMGWSPKSKRK-Cya (SEQ ID No: 13) MPG8 stl : NH₂-AFLGWLGAWGRMGWSPKSKRK-Cya (SEQ ID No: 14)

MPG8 st2: NH₂-AFRGWLGAWSTMGWSPKSKRK-Cya (SEQ ID No: 15)

MPG8 st3 : NH₂-AFRGWLSAWGTMGWSPKSKRK-Cya (SEQ ID No: 16) CADY-2-derived sequences

CADY CPPs are secondary amphipathic, with a tri-partite helical structure in central, N- and C-terminal positions. The derived peptides are stapled in locations 2 to 12, in i, i+4 and i , i+7. Residues indicated in bold are those which are bound by the hydrocarbon molecule. Non-stapled CADY2: NH₂-GLWWRLWWRLRSWFRLWFRA- Cya (SEQ ID No: 29) SVPEP-3a stl : NH₂-GWWRLWRWRLRSWSRLWFRA- Cya (SEQ ID No: 62)

SVPEP-3b stl : NH₂-GLWWRLWWRLRSWFRSWRFA- Cya (SEQ ID No: 64)

CADY2 stl : NH₂-GLWWRLWWRLRSWFRLWFRA- Cya (SEQ ID No: 30)

CADY2 st3 : NH₂-GLWWRLWWRLLRSWFRLWFR- Cya (SEQ ID No: 31)

CADY2 st4: NH₂-GLWWRLWWRLRSWFRSWFRA- Cya (SEQ ID No: 32)

CADY2 st5: NH₂-GRWWRLWWSLRRWFRLWFSA- Cya (SEQ ID No: 33)

CADY2 st6 NH2-GSWWRLWWSRLSWWRLWWSR- Cya (SEQ ID No: 36)

CADY2 st6 NH2-GRWWRLWWSRLRWWRLWWSR- Cya (SEQ ID No: 37)

CADY2 st7: NH₂-GLWWRS₅WWRS₅RSWFRLWFRA- Cya (SEQ ID No: 34)

Non-stapled CCR: NH₂-RELWRELWRLWRELWREWRV-ONH₂ (SEQ ID No: 66) CCR st2: NH₂-RELWRRLWRLWRSLWREWRV-ONH₂ (SEQ ID No: 67)

VEPEP-6-derived sequences

In the following sequences, X_\ is a beta-alanine or a serine

ST-VEPEP-6a: Ac-X i LFRAL WRLLRSL WRLL WK-cy steamide (SEQ ID No: 40)

ST-VEPEP-6aa Ac-XiLFLARWRLLRSLWRLLWK-cysteamide (SEQ ID No: 41)

ST-VEPEP-6ab Ac-XiLFRALWSLLRSLWRLLWK-cysteamide (SEQ ID No: 42)

ST-VEPEP-6ad Ac-XiLFLARWSLLRSLWRLLWK-cysteamide (SEQ ID No: 43)

ST-VEPEP-6b: Ac-XjLFRALWRLLRSLWSLLWK-cysteamide (SEQ ID No: 44)

ST-VEPEP-6ba: Ac-XiLFLARWRLLRSLWSLLWK-cysteamide (SEQ ID No: 45)

ST-VEPEP-6bb Ac-XiLFRALWRLLSSLWSLLWK-cysteamide (SEQ ID No: 46)

ST-VEPEP-6bd Ac-XiLFLARWRLLSSLWSLLWK-cysteamide (SEQ ID No: 47)

ST-VEPEP-6c: Ac-X i LFARL WRLLRSL WRLL WK-cysteamide (SEQ ID No: 48)

Example 2: Stapling increases the interaction with cargoes

The impact of the stabilizing the helical structure of the different CPPs on their ability to interact with their respective cargoes was evaluated. Since the binding of cargoes to cell-penetrating peptides implies a certain flexibility of the CPP, the increased rigidity of the stapled CPP could lead to a decrease affinity for the cargo. PEP-1. Binding of peptide (12 mer) and protein (small nanobody) to ST- PEP-1 were monitored by fluorescence spectroscopy using TRP-intrinsic PEP-1. Dissociation constants were calculated from data fitting. Peptide and protein used: a 12- mer short peptides (PC4: TYTKKQVLRMAHL, SEQ ID No: 69) and a small protein nanobody (21 KdA). Figure 2A shows that ST-PEP strongly interacted with peptide and protein with dissociation constant in the low nanomolar range, 12-fold lower that the one obtained for the non modified peptide.

CADY: Binding of nucleic acid to ST-CADY was monitored by fluorescence spectroscopy using both TRP-intrinsic and fluorescently labelled nucleic acids. Dissociation constants were calculated from data fitting. Nucleic acids used: double stranded siRNA (19/19 or 25/25) and single stranded DNA (36 mer). Figure 2B reports that ST-CADY strongly interacted with single and double stranded nucleic acids with dissociation constant of 5 and 3 nM, 3 -fold lower that the one obtained for the non modified CADY.

CADY2: Binding of peptide to ST-CADY2 was monitored by fluorescence spectroscopy using both TRP-intrinsic and fluorescently labelled peptides. Dissociation constants were calculated from data fitting. Peptide used: a 12 and 17 mer peptides. Figure 2C shows that ST-CADY2 strongly interacted with short peptide and small molecules with dissociation constant 5 to 12-fold lower that the one obtained for the non modified peptide.

VEPEP-6 Binding of nucleic acid to ST-VEPEP-6a was monitored by fluorescence spectroscopy using both TRP-intrinsic and fluorescently labelled nucleic acids. Dissociation constants were calculated from data fitting. Nucleic acids used: double stranded siRNA (19/19 or 25/25) and single stranded DNA (36 mer). Figure 2D shows that ST- VEPEP-6 strongly interacted with single and double stranded nucleic acids with dissociation constant of 4 and 7 nM, 2-fold lower that the one obtained for the non modified peptide.

The dissociation of the preformed siRNA/ST- VEPEP-6 and siRNA/ST- CADY complexes in the presence of heparan sulphate was then analysed. Heparan sulphate constitutes one of major components of the proteoglycan. Kinetic of complexes dissociation is reported in figure 3 A, using FITC-labelled siRNA associated to ST-VEPEP- 6, ST-CADY, CADY or VEPEP-6. Dissociated was triggered by adding increasing Heparan sulphate concentrations. Data demonstrated that ST- VEPEP -/siRNA and ST- CADY/siRNA complexes are 5 to 10 fold more stable than complexes formed with unstapled peptides. The dissociation of the preformed Peptide/ST-CADY-2 and Peptide/ST- PEP complexes in the presence of chaotropic agents (acetonitrile) was then analysed. Kinetic of complexes dissociation is reported in figure 3B, using CY5-labelled peptide associated to ST-CADY-2 or St-PEP. Dissociation was triggered by adding increasing acetonitrile concentrations. Data demonstrated that Peptide/ST-CADY-2 and Peptide/ST- PEP complexes are dissociated with a concentration of acetonitrile 10-fold higher than in the case of unmodified peptide.

Example 3: Stapling increases the efficacy of CPPs for in cellulo cargo delivery

ST-VEPEP-6-mediated delivery of siRNA in different cell lines

ST-VEPEP-6 peptides have been used for the delivery of siRNA into three different cell lines, including adherent (Hela, HEK), suspension and challenging cell lines such as primary (Jurkat) and stem cell lines (mES). Stock solutions of ST-VEPEP- 6/siRNA particles were prepared by complexing 100 nM siRNA with ST-VEPEP-6 peptides at a molar ratio of 1/20 for 30 min at 37°C. Lower concentrations of ST-VEPEP- 6-carrier/siRNA (from 20 nM to 0.125 nM) were obtained by serial dilutions of the stock complexes in PBS, in order to preserve the same ST-VEPEP-6-carrier/siRNA ratio. Dose- response experiments performed on different cultured cells revealed that ST-VEPEP-6- mediated delivery of siRNA (GAPDH) induced a robust downregulation (EC50) of GAPDH mRNA level measured by quantigen technology (Table 2). ST-VEPEP-6 are between 2 to 6 fold more efficient than unmodified peptides and exhibit a 2-fold lower toxicity (EC50) as measured by MTT assays.

Table 2

ST-CADY and ST-PGW mediated delivery of siRNA in different cell lines

ST-CADY and ST-PGW peptides have been used for the delivery of siRNA into three different cell lines, including adherent (Hela, HEK), suspension and challenging cell lines such as primary (Jurkat) and stem cell lines (mES). Stock solutions of ST-CADY/siR A and ST-PGW/siRNA particles were prepared by complexing 100 nM siRNA with ST-CADY or ST-PGW peptides at a molar ratio of 1/20 and 1/10, respectively, for 30 min at 37°C. Lower concentrations of ST-carrier / siRNA (from 20 nM to 0.125 nM) were obtained by serial dilutions of the stock complexes in PBS, in order to preserve the same ST-peptide-carrier/siRNA ratio. Dose-response experiments performed on different cultured cells revealed that ST-CADY ST-PGW -mediated delivery of siRNA (GAPDH) induced a robust downregulation (EC50) of GAPDH mRNA level measured by quantigen technology. ST-CADY or ST-PGW are between 5 to 10 fold more effective that unmodified peptides and exhibit a 3 -fold lower toxicity (EC50) as measured by MTT assays (Tables 3 and 4).

Table 3

Table 4

ST-PEP-1 and ST-CADY2 mediated delivery of peptide inhibitors into different cell lines

ST-PEP-1 and ST-CADY2 peptides form stable particles with small peptides (Figures 1A and 1C). ST-PEP-1 peptides have been used for the delivery of peptide inhibitors targeting cdk2/Cyclin A association on three cancer cell lines (PC3/HT29/PAN1). The inventors have previously described a short peptide PC-4 that blocks Cdk2/Cyclin A association in vitro and prevents cell proliferation (Gondeau C, et al, 2005, ref [7]). They have now demonstrated that delivery of peptide inhibitors using ST- CADY2 and ST-PEP-1 inhibits cancer cell proliferation (Figure 4). Data demonstrated that stapling of the peptide improves cellular response by a factor of 5 to 10 and allows the use of lower concentrations of small peptide (Table 5).

Table 5

ST-MPG-mediated plasmid DNA/nucleic acid analog delivery into cultured cells

ST-MPG peptides form stable complexes with plasmid DNA. ST-MPG peptides have been used for the delivery of plasmid DNA (encoding luciferase) into different cell lines, including challenging cell lines. Delivery of plasmid with ST-MPG leads to a strong expression of luciferase (Figure 5, Table 6). 5 μg of luciferase plasmid were associated with MPG or ST-MPG peptides at two different charge ratio (4/1 and 8/1), cells were transfected with complexes and incubated for 24 hrs at 37°C. Luciferase expression was measured by luminometry. Data demonstrated that stapling of the peptide improves expression by a factor of 20.

Table 6: Level of expression of luciferase in the different cell line after 48hr

The impact of the ratio of stapled peptides within the particle was then investigated. Nanoparticles were formed with different ratios of ST-MPG versus MPG (0, 25, 50, 75, 100%), cells were transfected with complexes and incubated for 48 hrs at 37°C. Luciferase expression was measured by luminometry. Data demonstrated that the ratio of ST-peptide plays a major role in the biological response and that the best ratio MPG/st- MPG is around 50% of each peptide (Figure 6 and Table 7). MPG/ST- luciferase

MPG expression

0% 15000

25% 44000

50% 1800000

75% 780000

100% 600000

Table 7

ST-PG16/9 mediated protein delivery in cultured cells

ST-PG16 and ST-PG9 peptides form stable particles with proteins. ST- PG16 and ST-PG9 peptides have been used for the delivery of small fiuorescently labelled antibody in cultured cell lines (Hela). Cy-5 and Cy-3 labelled anti-GST antibodies were associated with PG16 and PG9 at molar ratios 1/10 and 1/20 and complexes were overlaid onto cultured cells. Data demonstrated that stapling of the peptide improves cellular delivery of large proteins (Figure 7).

Example 4: Stapling increases the efficacy of CPPs for in vivo cargo delivery

ST-VEPEP-6 mediated delivery of siRNA in vivo

ST-VEPEP-6 Cyclin Bl /siRNA delivery was assessed upon systemic injection. Athymic female nude mice were subcutaneously inoculated into the flank with 1 x 10⁶ HT-29 (lung-tumor). 20 days after tumour implant, when tumour size reached about 100 mm³, animals were treated by intravenous injections, every 4 days, with either saline buffer solution (PBS), free siRNA, Cyc-Bl siRNA at 3μg and 5μg complexed with ST-VEPEP-6 or VEPEP-6 at a 1/20 molar ratio (Figure 8). Curves show the mean value of tumor size in a cohort of three animals and neither animal death nor any sign of toxicity were observed. Data demonstrated that stapling of the peptide improves in vivo response by a factor of 2 and allows the use of lower concentrations of siRNA. REFERENCES

[1] F. Heitz, MC. Morris, G. Divita, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics; British Journal of Pharmacology 157 (2009) 195-206.

[2] MC. Morris, P. Vidal, L. Chaloin, F. Heitz, G Divita A new peptide vector for efficient delivery of oligonucleotides into mammalian cells, Nucleic Acids Res. 25 (1997) 2730-2736.

[3] MC. Morris, J. Depollier, J. Mery, F. Heitz, G. Divita A peptide carrier for the delivery of biologically active proteins into mammalian cells, Nat. Biotechnol. 19 (2001) 1173-1176.

[4] Zhang H, Curreli F, Zhang X, Bhattacharya S, Waheed AA, Cooper A, Cowburn D, Freed EO, Debnath AK. (201 1) Antiviral activity of a-helical stapled peptides designed from the HIV-1 capsid dimerization domain. Retrovirology. 2011 May 3; 8:28.

[5] Young- Woo Kim, Grossmann, T. N., and Verdine, G.L. (2011), Synthesis of all-hydrocarbon stapled α-helical peptides by ring-closing olefin methathesis. Nature protocols, vol. 6, No. 6, p. 761-771.

[6] Verdine, G.L. and Hilinski, G.J. (2012), Stapled peptides for intracellular drug targets. Methods in Enzymology, vol 503, p3-33.

[7] Gondeau C, Gerbal-Chaloin S, Bello P, Aldrian-Herrada G, Morris MC, Divita G. Design of a novel class of peptide inhibitors of cyclin-dependent kinase/cyclin activation. J Biol Chem. (2005) 280(14): 13793-800.

Claims

1. A cell-penetrating peptide comprising an amphipathic peptide moiety made of 15 to 27 amino acids, characterized in that it further comprises a hydrocarbon linkage between two residues of said peptide moiety separated by two, three or six residues.

2. The cell-penetrating peptide of claim 1, characterized in that the peptide moiety is selected in the group consisting of stPEP-1 (SEQ ID No: 7), stMPG (SEQ ID No: 12), stMPG8 (SEQ ID No: 17), stCADY (SEQ ID No: 28), stCADY-2 (SEQ ID No: 35), stCADY-2a (SEQ ID No: 38), stVEPEP-6a (SEQ ID No: 49), stVEPEP-6b (SEQ ID No: 50), stVEPEP-6c (SEQ ID No: 51), stPG9 (SEQ ID No: 55), stPG16 (SEQ ID No: 58), stPGW (SEQ ID No: 61), stSVPEP-3a (SEQ ID No: 63), stSVPEP-3b (SEQ ID No: 65) and stCCR (SEQ ID No: 68).

3. The cell-penetrating peptide of claim 1 or claim 2, characterized in that the peptide moiety is selected in the group consisting of:

SEQ ID No: 2 (Pep-stl) KEXi WWETWWXEWSQPKKKRKV

SEQ ID No: 3 (Pep-st2) KETWWX j TWWTE WXQPKKKRKV

SEQ ID No: 4 (Pep-st3) KXTWWXTWWTEWSQPKKKRKV

SEQ ID No: 5 (Pep-st4) KETWWETWWTXWSQXKKKRKV

SEQ ID No: 9 (MPG-stl) GXi LFLXFLGAAGSTMGAWSQPKKKRKV

SEQ ID No: 10 (MPG-st2) GGLFLXiFLGXAGSTMGAWSQPKKKRKV

SEQ ID No: 11 (MPG-st3) GGLFLGFLXiAAGSTMXAWSQPKKKRKV

SEQ ID No: 14 (MPG8-stl) AFLGWLGAWGXiMGWXPKSKRK

SEQ ID No: 15 (MPG8-st2) AFXi GWLGAWXTMGWSPKSKRK

SEQ ID No: 16 (MPG8-st3) AFX_! GWLX A WGTMGWSPKSKRK

SEQ ID No: 19 (CADYstl) GL WX i AL WRLLXRL WRLL WKA

SEQ ID No: 20 (CADYst2) GL WRAL WRLLRX _\ L WRLL WXK

SEQ ID No: 21 (CADYst3) GLWRAXi WRLLPvLX WRLL WKA

SEQ ID No: 22 (CADYst4) GL WRAL WXLLRXL WRLL WKA

SEQ ID No: 23 (CADYst5) GL WRAX i WRLLRLX WRLL WKA

SEQ ID No: 24 (CADYst5a) GLWRAXi WRLLRSXWRLL WKA

SEQ ID No: 25 (CADYst6) GL WRAL WRXi LRSLWRXL WKA

SEQ ID No: 26 (CADYst7) GL WRAL WRXLRSXWRLL WKA

SEQ ID No: 30 (CADY2 stl) GL W WX i L W WRLRX WFRL WFRA

SEQ ID No: 31 (CADY2 st3) GLWWRLWWXiLLRXWFRLWFR

SEQ ID No: 32 (CADY2 st4) GL W WRL W WX i LRS WFRX WFRA

SEQ ID No: 34 (CADY2 st7) GLWWRXWWRXRS WFRL WFRA

SEQ ID No: 40 (ST-VEPEP-6a) X₂LFRAL WX i LLRXL WRLL WK - SEQ ID No: 41 (ST-VEPEP-6aa)

- SEQ ID No: 42 (ST-VEPEP-6ab)

- SEQ ID No: 43 (ST-VEPEP-6ad)

- SEQ ID No: 44 (ST-VEPEP-6b) X2LFRALWRLLX1 SLWXLLWK

- SEQ ID No: 45 (ST-VEPEP-6ba)

- SEQ ID No: 46 (ST-VEPEP-6bb)

- SEQ ID No: 47 (ST-VEPEP-6bd)

- SEQ ID No: 48 (ST-VEPEP-6c) X₂LFAX₁LWRLLRXLWRLLWK

- SEQ ID No: 53 (PG9-stl) GL WRAL WX , AL WXRL WRLKRK V

- SEQ ID No: 54 (PG9-st2) GL WAX 1 LWRAL WXRL WRLKRK V

- SEQ ID No: 57 (PG16-stl) GLWRALWXi GLRXL WRLLWKV

- SEQ ID No: 60 (PGW-stl) GL WRALWXi LWRXL WRLLWKA

- SEQ ID No: 62 (SVPEP-3a-stl) GWWRLWXj WRLRS WXRLWFRA

- SEQ ID No: 64 (SVPEP-3b-stl) GLWWRLWWRLX^WFRSWXiFA and

- SEQ ID No: 67 (stapled CCR) RELWRXi L WRL WRXL WRE WRV, wherein X and X\ are non-natural amino acids used for the binding of a hydrocarbon staple and X₂ is a beta-alanine or a serine, and wherein the non-natural residues are linked by said hydrocarbon linkage.

4. The cell-penetrating peptide of claim 1 or claim 2, characterized in that the peptide moiety is selected in the group consisting of:

- SEQ ID No: 6 (Pep-st5) KXTWWXTWWTXWSQXKKKRKV

- SEQ ID No: 33 (CADY2 st5)

- SEQ ID No: 36 (CADY-2a-st6) GXWWRLWWXRLXWWRLWWXR and

- SEQ ID No: 37 (CADY-2a-st6a)

wherein X and Xi are non-natural amino acids used for the binding of a hydrocarbon staple and wherein the two first non-natural residues are linked by a first hydrocarbon linkage and the two other non-natural residues are linked by a second hydrocarbon linkage.

5. The cell-penetrating peptide of claim 3 or claim 4, characterized in that Xi is an olefin-bearing non-natural amino acid (oc-methyl, -alkenyl glycine derivative R-configuration) and X is an olefin-bearing non-natural amino acid (a-methyl, a-alkenyl glycine derivative S -configuration).

6. The cell-penetrating peptide of any of claims 1 to 5, further comprising, covalently linked to the N-terminal end of the amino acid sequence, one or several chemical entities selected in the group consisting of an acetyl, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, nuclear export signal, an antibody, a polysaccharide and a targeting molecule.

7. The cell-penetrating peptide of any of claims 1 to 6, further comprising, covalently linked to the C-terminal end of said amino acid sequence, one or several groups selected in the group consisting of a cysteamide, a cysteine, a thiol, an amide, a nitrilotriacetic acid optionally substituted, a carboxyl, a linear or ramified Ci-C₆ alkyl optionally substituted, a primary or secondary amine, an osidic derivative, a lipid, a phospholipid, a fatty acid, a cholesterol, a poly-ethylene glycol, a nuclear localization signal, nuclear export signal, an antibody, a polysaccharide and a targeting molecule.

8. A complex comprising a cell -penetrating peptide according to any of claims 1 to 7 and a cargo selected amongst proteins, peptides, nucleic acids and small hydrophobic molecules.

9. The complex of claim 8, further comprising non-stapled cell- penetrating peptides.

10. The complex of claim 9, wherein 25 to 75% of the cell-penetrating peptides are stapled cell-penetrating peptides according to any of claims 1 to 7.

11. The complex according to claim 10, wherein the size of the complex is between 50 and 500 nm.

12. A nanoparticle comprising a complex according to any of claims 8 to 11, coated by a layer of peripheral cell-penetrating peptides.

13. The nanoparticle of claim 12, wherein the layer of peripheral cell- penetrating peptides comprises at least 25%o of cell-penetrating peptides according to any of claims 1 to 6.

14. A nanoparticle comprising a core which comprises a cargo complexed to a first entity selected in the group consisting of cell-penetrating peptides, liposomes, polycationic structures and carbon nanoparticles, wherein said core is coated by a layer of peripheral a cell-penetrating peptides comprising at least 25% of cell-penetrating peptides according to any of claims 1 to 7.

15. The nanoparticle of any of claims 11 to 13, wherein the size of the nanoparticle is between 80 and 500 nm.

16. The complex of any of claims 8 to 11 , or the nanoparticle of any of claims 12 to 15, wherein the cell-penetrating peptide according to any of claims 1 to 7 comprises an acetyl group covalently linked to its N-terminus, and/or a cysteamide group covalently linked to its C-terminus.

17. The complex or nanoparticle of any of claims 8 to 16, wherein at least part of the cell-penetrating peptides are bound to a targeting molecule.

18. A therapeutic or diagnostic composition comprising a complex or a nanoparticle according to any of claims 8 to 17.