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WO 2010/072406 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 1 July 2010 (01.07.2010) WO 2010/072406 Al (51) International Patent Classification: AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, C07K 14/16 (2006.01) A61K 47/48 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/EP2009/009229 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 22 December 2009 (22.12.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, PCT/EP2008/0 11003 GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, 22 December 2008 (22.12.2008) EP ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, PCT/EP2009/003927 2 June 2009 (02.06.2009) EP TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (71) Applicant (for all designated States except US): XIGEN MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, SM, S.A. [CH/CH]; Rue des Terreaux 17 (4eme etage), TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, CH- 1003 Lausanne (CH). ML, MR, NE, SN, TD, TG). (72) Inventor; and Published: (75) Inventor/Applicant (for US only): BONNY, Christophe — with international search report (Art. 21(3)) [CH/CH]; 37c, chemin du Trabandan, CH-1006 Lausanne (CH). — before the expiration of the time limit for amending the claims and to be republished in the event of receipt of (74) Agents: GRAF VON STOSCH, Andreas et al; GRAF amendments (Rule 48.2(h)) VON STOSCH, Patentanwaltsgesellschaft MBH, Prinzre- gentenstrasse 22, 80538 Munchen (DE). — with sequence listing part of description (Rule 5.2(a)) (81) Designated States (unless otherwise indicated, for every kind of national protection available): AE, AG, AL, AM, (54) Title: NOVEL TRANSPORTER CONSTRUCTS AND TRANSPORTER CARGO CONJUGATE MOLECULES (57) Abstract: The present invention relates to novel transporter constructs of the generic formula (I) DiLLL Dm(LLLyDn) and x 11 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. Novel Transporter Constructs and Transporter Cargo Conjugate Molecules The present invention relates to novel transporter constructs of the generic formula (I) D LLLxDm(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 ox 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 ox 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., US Patent 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.
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