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{"words":"$head_words:人乳头瘤病毒+$head_words:\\ 晶体结构+$head_words:\\ 低温电镜单颗粒重构+$head_words:\\ 全原子结构+$head_words:\\ 宫颈癌+$head_words:\\ 组织特异性","themeword":"$head_words","params":"$title:人乳头瘤病毒59型L1病毒样颗粒\\(VLP\\)的全原子结构研究"}
&&&人乳头瘤病毒59型L1病毒样颗粒(VLP)的全原子结构研究
人乳头瘤病毒59型L1病毒样颗粒(VLP)的全原子结构研究
<input type="hidden" class="share_summary"
value="高危型人乳头瘤病毒(HPV)被证实是诱发女性宫颈癌的主要病原体。流行病学调查显示98％的宫颈癌都是由人乳头瘤病毒(HPV)感染引起，其中，HPV59型是引起宫颈癌的主要型别之一。HPV的主要衣壳蛋白L1与病毒的吸附、病毒基因型及诱导宿主的免疫应答等作用紧密相关，是HPV疫苗的主要成分。HPV病毒样颗粒(VLP)是由L1五聚体组成，同时保留了其天然的免疫表位。因此，开展对HPV59L1蛋白五聚体及其组成的VLP结构特征的研究对阐明HPV59型特异表位和VLP组装机制的结构基础等提供参考，并为HPV宿主及组织特异性研究奠定了基础。　　
首先，本研究利用大肠杆菌表达系统，成功的表达了HPV59L1蛋白，利用阳离子交换层析和羟基磷灰石层析等纯化方法，获得了纯度大于95％的L1蛋白。在进一步的组装研究中，考察了组装温度对HPV59L1 VLP组装的影响，研究中发现，在常温组装后置于37℃中温育的组装条件下，L1蛋白能形成均一的直径为60nm左右的二十面体VLP。接着，利用低温电镜单颗粒重构技术成功重建了HPV59L1 VLP的三维结构，分辨率为9.5(A)，从结构中，可以看出，HPV59L1 VLP表面五聚体相互之间的连接方式有三种:双弧线连接，单直线连接和双直线连接;从低温电镜表面密度值可以看出，HPV59L1 VLP表面的五邻体(5-coordinatedpentamer)和六邻体(6-coordinated pentamer)是存在结构差异的。　　
其次，本研究通过L1蛋白关键位点突变制备了稳定均一的HPV59L1五聚体。并将HPV59L1五聚体进行晶体培养，经培养条件的筛选和优化，获得了三个能够获得适合衍射晶体的结晶条件，其中一颗晶体并通过X-射线衍射实验收集了一套用于结构解析的衍射数据，接着使用HKL2000，CCP4和COOT软件，通过分子置换方法，解析了HPV59L1五聚体的晶体结构，分辨率达到3.7(A)。将该HPV59五聚体结构与已报道HPV11/16/18/35四型别的五聚体结构进行比较，发现HPV59的BC环与其他四型别差异较大，提示该区域可能存在较多地HPV59型特异的表位;而五个型别结构中对于颗粒组装具有关键作用的EF环没有体现出明显差异，提示HPV59与其他四型别具有相似的组装机制。　　
最后，通过同源模建的方法构建HPV59五聚体结构上缺失的α4环的结构，接着利用Fitting技术将完整的HPV59五聚体晶体结构与HPV59L1 VLP低温电镜三维结构结合，获得完整的HPV59L1 VLP原子模型。并进一步对该结构模型进行分析得出，五邻体与六邻体之间通过相邻单体上Cys175与Cys428之间相互作用，且分属于不同五聚体的相邻单体CD环上的Phe82、Gly83、Pro85、Thr88和Val89之间可能会通过强疏水作用相互作用;而六邻体与六邻体之间的相互作用仅由Cys175和Cys428的二硫桥维系。　　
综上所述，本研究成功地获得了HPV59T=7的二十面体VLP的全原子结构，从原子水平上阐明了HPV59L1蛋白组装的结构基础，从而为人乳头瘤病毒的生物学研究、型特异性表位和宿主研究提供指导。"/>
高危型人乳头瘤病毒(HPV)被证实是诱发女性宫颈癌的主要病原体。流行病学调查显示98％的宫颈癌都是由人乳头瘤病毒(HPV)感染引起，其中，HPV59型是引起宫颈癌的主要型别之一。HPV的主要衣壳蛋白L1与病毒的吸附、病毒基因型及诱导宿主的免疫应答等作用紧密相关，是HPV疫苗的主要成分。HPV病毒样颗粒(VLP)是由L1五聚体组成，同时保留了其天然的免疫表位。因此，开展对HPV59L1蛋白五聚体及其组成的VLP结构特征的研究对阐明HPV59型特异表位和VLP组装机制的结构基础等提供参考，并为HPV宿主及组织特异性研究奠定了基础。　　
首先，本研究利用大肠杆菌表达系统，成功的表达了HPV59L1蛋白，利用阳离子交换层析和羟基磷灰石层析等纯化方法，获得了纯度大于95％的L1蛋白。在进一步的组装研究中，考察了组装温度对HPV59L1 VLP组装的影响，研究中发现，在常温组装后置于37℃中温育的组装条件下，L1蛋白能形成均一的直径为60nm左右的二十面体VLP。接着，利用低温电镜单颗粒重构技术成功重建了HPV59L1 VLP的三维结构，分辨率为9.5(A)，从结构中，可以看出，HPV59L1 VLP表面五聚体相互之间的连接方式有三种:双弧线连接，单直线连接和双直线连接;从低温电镜表面密度值可以看出，HPV59L1 VLP表面的五邻体(5-coordinatedpentamer)和六邻体(6-coordinated pentamer)是存在结构差异的。　　
其次，本研究通过L1蛋白关键位点突变制备了稳定均一的HPV59L1五聚体。并将HPV59L1五聚体进行晶体培养，经培养条件的筛选和优化，获得了三个能够获得适合衍射晶体的结晶条件，其中一颗晶体并通过X-射线衍射实验收集了一套用于结构解析的衍射数据，接着使用HKL2000，CCP4和COOT软件，通过分子置换方法，解析了HPV59L1五聚体的晶体结构，分辨率达到3.7(A)。将该HPV59五聚体结构与已报道HPV11/16/18/35四型别的五聚体结构进行比较，发现HPV59的BC环与其他四型别差异较大，提示该区域可能存在较多地HPV59型特异的表位;而五个型别结构中对于颗粒组装具有关键作用的EF环没有体现出明显差异，提示HPV59与其他四型别具有相似的组装机制。　　
最后，通过同源模建的方法构建HPV59五聚体结构上缺失的α4环的结构，接着利用Fitting技术将完整的HPV59五聚体晶体结构与HPV59L1 VLP低温电镜三维结构结合，获得完整的HPV59L1 VLP原子模型。并进一步对该结构模型进行分析得出，五邻体与六邻体之间通过相邻单体上Cys175与Cys428之间相互作用，且分属于不同五聚体的相邻单体CD环上的Phe82、Gly83、Pro85、Thr88和Val89之间可能会通过强疏水作用相互作用;而六邻体与六邻体之间的相互作用仅由Cys175和Cys428的二硫桥维系。　　
综上所述，本研究成功地获得了HPV59T=7的二十面体VLP的全原子结构，从原子水平上阐明了HPV59L1蛋白组装的结构基础，从而为人乳头瘤病毒的生物学研究、型特异性表位和宿主研究提供指导。
摘要： 高危型人乳头瘤病毒(HPV)被证实是诱发女性宫颈癌的主要病原体。流行病学调查显示98％的宫颈癌都是由人乳头瘤病毒(HPV)感染引起，其中，HPV59型是引起宫颈癌的主要型别之一。HPV的主要衣壳蛋白L1与病毒的吸附、病毒基因型及诱导宿主的免疫应答等作用紧密相关，是HPV疫苗的主要成分。HPV病毒样颗粒(VLP)是由L1五聚体组成，同时保留了其天然的免疫表位。...&&
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我们会通过消息、邮箱等方式尽快将举报结果通知您。JC POLYOMAVIRUS VLP (VIRUS-LIKE PARTICLE) WITH A TARGETING PEPTIDE
United States Patent Application
The disclosure relates to a fusion protein comprising at least a first and a second peptide, wherein —the second peptide comprises a targeting region and a first and a second interaction region, —the second peptide is located on the surface o —the second peptide comprises at least two interaction pairs, wherein an interaction pair is formed by an amino acid of the first interaction region and an amino acid of the second interaction region, —the interaction between the amino acids of an interaction pair is covalent or non- and —at least one interaction pair is a covalent interaction pair in which the amino acids are covalently bound, and to virus like particles (VLP) comprising the fusion protein for use as drug delivery system. Also provided are polynucleotides encoding the fusion protein, suitable expression vectors, host cells, production methods for the fusion protein and the VLP comprising the fusion protein.
Inventors:
Franken, Sebastian (BONN, DE)
Glassmann, Alexander (BONN, DE)
Temme, Nadine (BONN, DE)
Application Number:
Publication Date:
01/04/2018
Filing Date:
12/08/2015
Export Citation:
LIFE SCIENCE INKUBATOR GMBH (BONN, DE)
International Classes:
C07K14/01; C07K2/00; C12N7/04
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Primary Examiner:
Attorney, Agent or Firm:
PATENT LAW OFFICES OF DR. NORMAN B. THOT (POSTFACH 10 17 56
A fusion protein comprising at least a first and a second peptide, wherein the second peptide comprises a targeting region and a first and a second interaction region, the second peptide is located on the surface o the second peptide comprises at least two interaction pairs, wherein an interaction pair is formed by an amino acid of the first interaction region and an amino acid of the second interaction region, the interaction between the amino acids of an interaction pair is covalent or non- and at least one interaction pair is a covalent interaction pair in which the amino acids are covalently bound.
The fusion protein according to claim 1, wherein the first peptide is a polyoma virus VP1, preferably a VP1 from JCV.
The fusion protein according to claim 1 or 2, wherein the covalent interaction pair is formed by a cysteine in the first interaction region and a cysteine in the second interaction region.
The fusion protein according to any of the preceding claims, wherein at least two interaction pairs are non-covalent interaction pairs in which the amino acids interact non-covalently.
The fusion protein according to claim 4, wherein the 2 to 6 acid/base interaction pairs, preferably 3 to 5 non-covalent interaction pairs are acid/base interaction pairs.
The fusion protein according to claim 5, wherein the acid/base interaction pairs are formed by acidic amino acids in the first interaction region and basic amino acids in the second interaction region.
The fusion protein according to claim 6, wherein the majority of the basic amino acids are arginine, preferably all basic amino acids are arginines.
The fusion protein according to any of claim 6 or 7, wherein the majority of the acidic amino acids are glutamic acid, preferably all acidic amino acids are glutamic acids.
The fusion protein according to any of the preceding claims, wherein the number of amino acids in the first and/or second interaction region between the at least one covalent interaction pair the closest non-covalent interaction pair is in the range from 1 to 6 preferably 1 to 4, more preferably 2.
The fusion protein according to any of the preceding claims, wherein the amino acid sequence of the targeting region is selected from the group consisting of Lyp-1, RGD, RGR, HER-2 binding peptide, CREKA peptide, NGR peptide CPP-2, CPP-44, F3, RMS-P3, F56, LTVSPWY-peptide, and WNLPWYYSVSPT-peptide.
A virus-like particle (VLP) comprising at least one fusion protein according to any of claims 1 to 10.
The VLP according to claim 11, wherein the second peptide is located on the outer surface of the VLP capsid.
The VLP according to claim 11 or 12, further comprising a second fusion protein comprising a VLP binding protein and an exogenous peptide.
The VLP according to any of claims 11 to 13, wherein the VLP comprises no cargo.
The VLP according to any of claims 11 to 13, wherein the VLP comprises a cargo selected from single-stranded or double-stranded DNA or RNA, preferably siRNA, oligopeptides, polypeptides, hormones, lipids, carbohydrates, other small organic compounds or mixtures thereof.
The VLP according to any of claims 11 to 15 for use in a method of treatment or in a diagnostic method.
The VLP according to any of claims 11 to 16 for use as drug delivery system.
A pharmaceutical composition comprising the VLP according to any of claims 11 to 17 and at least one pharmaceutically acceptable carrier.
An isolated polynucleotide comprising a nucleic acid sequence encoding a fusion protein according to any claims 1 to 10.
An expression vector comprising the polynucleotide according to claim 19.
A host cell comprising the expression vector according to claim 20.
A process of producing the VLP according to any of claims 11 to 17, comprising the steps of: a) introducing a polynucleotide according to claim 19 b) culturing the transformed host cell in a medium under conditions leading to a protein expression with the nuclei c) isolating the expression p and d) assembly of the expression product optionally with further viral proteins into the VLP.
The process according to claim 22, further comprising the steps: f) disassembly of the VLP g) mixture of the pentamers with wildtype VP1 and h) reassembly of VLPs from the pentamer mixture.
Description:
FIELD OF THE INVENTIONThe present invention relates to a fusion protein comprising a peptide with a targeting region and a first and a second interaction region for targeting the fusion protein and to virus like particles (VLP) comprising the fusion protein for use as drug delivery system.BACKGROUND OF THE INVENTIONIn the development of specific diagnostic or therapeutic procedures, the use of transfer systems (delivery systems) that allow a possible cell-specific transfer of substances and nucleic acids such as, markers or agents, is of great importance. For this cell-specific transfer systems based on virus-like particles (VLP) have been developed. One of these systems is the VLP from the human polyomavirus John-Cunningham virus (JCV) as described in WO 97/19174 and EP . Basis of this system is the ability of the VLP to package foreign cargo such as drugs or and nucleic acids instead of the viral DNA. As a VLP still has the ability to specifically recognize cells and to be internalized by the cells the VLP can be used to introduce a cargo of choice into specific cells. However, the VLPs of the state art interact with a very broad spectrum of cells. For certain applications it is desirable to be able to address only specific cell types.In view of this prior art it is an object of the present invention to improve VLPs for the use as a drug delivery system. In particular it is an object of the present invention to provide means to improve the cell specificity of VLPs.SUMMARY OF THE INVENTIONAccording to a first aspect, the invention provides a fusion protein comprising at least a first and a second peptide, wherein
the second peptide comprises a targeting region and a first and a second interaction region,the second peptide is located on the surface of the fusion protein,the second peptide comprises at least two interaction pairs, wherein an interaction pair is formed by an amino acid of the first interaction region and an amino acid of the second interaction region,the interaction region between the amino acid of an interaction pair is covalent or non-covalent, andat least one interaction pair is a covalent interaction pair in which the amino acids are covalently bound.
The second peptide according to the invention is a modular targeting peptide that has several advantages for targeting a protein of interest to which it is fused forming the fusion protein. The second peptide provides a self-forming secondary structure that presents a specific targeting sequence of choice in form of a loop spaced apart from the surface of the fusion protein. Due to the spacing of the targeting sequence away from the surface of the fusion protein, the second peptide provides an improved accessibility of the targeting sequence. The fusion protein may be used in particular in combination with a virus-like particle (VLP) cargo transport to provide to the VLP the ability to target specific predefined receptors/cell types.Thus, according to the second aspect, the invention provides a VLP comprising the fusion peptide of the first aspect of the invention.According to a third aspect, the invention provides a pharmaceutical composition comprising the VLP according to the second aspect and at least one pharmaceutically acceptable carrier.According to a fourth aspect, the invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a fusion protein according to the first aspect of the invention. According to a fifth aspect, the invention provides an expression vector comprising the nucleotide according to the fourth aspect of the invention.According to a sixth aspect, the invention provides a host cell comprising the expression vector according to the fifth aspect of the invention.According to a seventh aspect, the invention provides a process of producing the VLP according to the second aspect of the invention which comprises the steps of: a) introducing a polynucleotide according to the second aspect of the inventb) culturing the transformed host cell in a medium under conditions leading to a protein expression with the polynucleotide as a template,c) isolating the expression product from the cell andd) assembly of the expression product optionally with further viral proteins into the VLP BRIEF DESCRIPTION OF THE FIGURESFIG. 1 shows two specific embodiments of the second peptide according to the invention (a and b)FIG. 2 provides an overview of a specific embodiment for the production of VLPs comprising a fusion protein according to the invention.FIG. 3 shows histograms calculated from flow cytometry data obtained from MDA-MB-231 cells treated with VLPs. The histograms a) to c) correspond to the following VLPs:
a) VLP with VP1 and VP1-FITC, VLP with VP1-DE-Loop-Lyp1 and VP1-FITC, VLP with VP1-HI-Loop-Lyp1 and VP1-FITCb) VLP with VP1 and VP1-Dylight488, VLP with VP1-DE-Loop-Lyp1 and VP1-Dylight488, VLP with VP1-HI-Loop-Lyp1 and VP1-Dylight488c) VLP with VP1 and VP1-Atto647, VLP with VP1-DE-Loop-Lyp1 and VP1-Atto647, VLP with VP1-HI-Loop-Lyp1 and VP1-Atto647.The X-axis represents the measured intensity of the signal, the Y-axis the number of cells with the specific signal intensity.
FIG. 4 shows a column diagram representation of the flow cytometry data obtained from MDA-MB-231 cells treated with VLPs. Each column represents the percentage of cells—identified by a minimum signal strength—with a positive uptake of a specific type combination of VLP and fluorescent dye as indicated below the columns. Three different VLPs are tested: VLPs containing VP1-DE-Loop-Lyp1 or VP1-HI-Loop-Lyp1 VLPs from normal VP1. Also three different dyes are tested: FITC, Atto-488 and Atto 647.FIG. 5 shows the detection of VLP in spheroids of a breast cancer spheroid model. The following VLPs were tested: VP1, VP1-HI-Loop-linRGD, and VP1-HI-Loop-cycRGD, each labelled with Atto-488. A mock probe was used as a control.FIG. 6 study design of an orthotopic breast cancer tumor modelFIG. 7 shows the flow cytometry analysis of the tumor cells from the orthotopic breast cancer tumor model of FIG. 6. Nine animals had been treated with a VLP comprising VP1-HI-Loop-Lyp1 and three animals were used as control.DETAILED DESCRIPTION OF THE INVENTION1. DefinitionsA “peptide” according to the present invention may be composed of any number of amino acids of any type, preferably naturally occurring amino acids, which preferably are linked by peptide bonds. In particular, a peptide comprises at least 3 amino acids, preferably at least 5, at least 7, at least 9, at least 12 or at least 15 amino acids. Furthermore, there is no upper limit for the length of a peptide. However, preferably a peptide according to the invention does not exceed a length of 500 amino acids, more preferably, it does not exceed a length of 300 even more preferably, it is not longer than 250 amino acids. Thus, the term peptide includes oligopeptides, which usually refer to peptides with a length of 2 to 10 amino acids, and polypeptides, which usually refer to peptides with a length of more than 10 amino acids. The term “protein” refers to a peptide with at least 60, at least 80, preferably at least 100 amino acids.The term “fusion protein” according to the invention relates to proteins or peptides created through the joining of two or more genes that originally coded for separate proteins. The genes may be naturally occurring from the same organism or different organisms or may be synthetic polynucleotides.The term “exogenous” according to the invention relates to the property of a peptide or polynucleotide that it does not naturally occur in polyomaviruses.The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the degree of sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment-Total Number of Gaps in Alignment) For purposes of the present invention, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Desoxyribonucleotides×100)/(Length of Alignment-Total Number of Gaps in Alignment) The term “isolated” means a substance in a form or environment which does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.The term “operably linked” means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence. Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.The term “expression vector” means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to additional nucleotides that provide for its expression.The term “host cell” means any cell type that is susceptible to transformation, transfection, transduction, and the like, with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.The term “comprise”, as used herein, besides its literal meaning also includes and specifically refers to the expressions “consist essentially of” and “consist of”. Thus, the expression “comprise” refers to embodiments wherein the subject-matter which “comprises” specifically listed elements does not comprise further elements as well as embodiments wherein the subject-matter which “comprises” specifically listed elements may and/or indeed does encompass further elements. Likewise, the expression “have” is to be understood as the expression “comprise”, also including and specifically referring to the expressions “consist essentially of” and “consist of”.The term “carrier” applied to pharmaceutical compositions of the invention refers to a diluent, excipient, or vehicle with which the VLP of the invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical carriers are described in “Remington The Science and Practice of Pharmacy,” 21th edition, (David B. Troy ed., 2006, p. 745-775, p. 802-836 and p. 837-849).As used herein, the term “pharmaceutical composition” refers to any composition comprising at least the VLP with or without cargo and at least one other ingredient, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the term “pharmaceutical composition” as used herein may encompass, inter alia, any composition made by admixing a pharmaceutically active ingredient and one or more pharmaceutically acceptable carriers.The term “low stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 degrees centigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25 percent formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2 percent SDS at 50 degrees centigrade.The term “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 degrees centigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35 percent formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 55° C.The term “high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42 degrees centigrade in 5×SSPE, 0.3 percent SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50 percent formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C.The term “PBS” means phosphate buffered saline. It is a water-based salt solution containing sodium phosphate, sodium chloride and, in some formulations, potassium chloride and potassium phosphate. The osmolarity and ion concentrations of the solutions match those of the human body.2. Fusion ProteinAccording to a first aspect, the invention provides a fusion protein comprising at least a first and a second peptide, wherein:
the second peptide comprises a targeting region and a first and a second interaction region,the second peptide is located on the surface of the fusion protein,the second peptide comprises at least two interaction pairs, wherein an interaction pair is formed by an amino acid of the first interaction region and an amino acid of the second interaction region,the interaction region between the amino acid of an interaction pair is covalent or non-covalent, andat least one interaction pair is a covalent interaction pair in which the amino acids are covalently bound.
The second peptide according to the invention, i.e. the targeting peptide, is based on a particular secondary structure resembling a hair pin known from single-stranded polynucleotides especially in RNA molecules. When folded into its secondary structure, the peptide preferably comprises two paired regions of the amino acid sequence, the first and second interaction region and an unpaired loop comprising the targeting region as shown schematically in FIGS. 1a and b. The targeting region according to the invention comprises an amino acid sequence—the targeting sequence—that is known to interact with a target of interest, in particular a cellular receptor. The secondary structure of the second peptide may also be described as a stem loop comprising a stem region and a loop region. Accordingly, the two interaction regions of the peptide preferably form the stem and the targeting region forms the loop (see FIGS. 1a and b). When located on the surface of the fusion protein, the stem, i.e. the first and second interaction region of the second peptide, lead to a sufficient spacing between the surface of the protein and the targeting region so that an interaction with a targeting recognizing means, in particular a cellular receptor, is possible without steric hindrance.The folding of the structure is based on the following theoretic principle. During protein folding, the amino acids on the first and second interaction region get into proximity.When two complementary amino acids of the two interaction regions get in proximity to each other, they will transiently bind to each other, i.e. interact non-covalently, and, thus, form a non-covalent interaction pair. The more interaction pairs are formed at a time, the higher is the binding strength and the longer the transient interaction of the two interaction regions. The interaction pairs of the second peptide are preferably set up such that the formation of these non-covalent interaction pairs brings the amino acids of the covalent interaction pair, in particular cysteines, into proximity to each other for a sufficient time so as to allow formation of a covalent bond, e.g. a disulfide-bridge. The formation of the covalent interaction pair leads to a further stabilization of the interaction of the first and second interaction region of the second peptide. Accordingly, the final secondary structure of the second peptide with a loop including the targeting region and a stem formed by the first and second interaction region is formed. The loop can be regarded as a circular peptide connected by a covalent interaction pair. It was shown for a variety of signaling/targeting peptides that a circular shape of the peptide improves its recognition by the specific receptor.Thus, according to one embodiment of the first aspect of the invention, the amino acid sequence of the targeting region is located between the amino acid sequences of the first and second interaction region. A location of the amino acid sequence of the targeting region between the first and second interaction region is required to obtain a targeting region that is located in the loop of the folded second peptide.The amino acid sequence of the targeting region may overlap with the amino acid sequences of the first and/or second interaction region. In particular, the amino acids forming the covalent interaction pair may be part of the targeting region.The amino acid sequence of the targeting region may be any sequence that is recognized or binds to a target molecule, in particular a cellular receptor. Non-limiting examples of such peptides are Lyp-1 (SEQ ID NO: 1), RGD (SEQ ID NO: 60, RGD), RGR, HER2 binding peptide (SEQ ID NO: 2), CREKA peptide (SEQ ID NO: 3), NGR peptide, CPP-2 (SEQ ID NO: 4), CPP-44 (SEQ ID NO: 5), F3 (SEQ ID NO: 6), RMS-P3 (SEQ ID NO: 7), F56 (SEQ ID NO: 8), LTVSPWY-peptide (SEQ ID NO: 9), WNLPWYYSVSPT-peptide (SEQ ID NO: 10), SP5-2 (SEQ ID NO: 11), heparan sulfate targeting peptide (SEQ ID NO: 61, CKNEKKNKIERNNKLKQPP), CGKRK-peptide (SEQ ID NO: 62, CGKRK), CSRPRRSEC-peptide (SEQ ID NO: 63, CSRPRRSEC), CREAGRKAC-peptide (SEQ ID NO: 64, CREAGRKAC), CAGRRSAYC-peptide (SEQ ID NO: 65, CAGRRSAYC), RMS-P3 (SEQ ID NO: 66, CMGTINTRTKKC), CKAAKN-peptide (SEQ ID NO: 67, CKAAKN), CSNRDARRC-peptide (SEQ ID NO: 68, CSNRDARRC), CGNSNPKSC-peptide (SEQ ID NO: 69, CGNSNPKSC), CSRESPHPC-peptide (SEQ ID NO: 70, CSRESPHPC), ASGALSPSRLDT-peptide (SEQ ID NO: 71, ASGALSPSRLDT), IL-4-receptor binding peptide (SEQ ID NO: 72, CRKRLDRNC), and PSP1 (SEQ ID NO: 73, CLSYYPSYC).Thus, according to one embodiment of the first aspect of the invention, the targeting region comprises a sequence selected from the group consisting of SEQ ID NO: 1, RGD, RGR, SEQ ID NO: 2, SEQ ID NO: 3, NGR peptide, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, or SEQ ID NO: 73. Preferably the amino acid sequence of the targeting region comprises SEQ ID NO: 1. In another preferred embodiment, the amino acid sequence of the targeting region comprises SEQ ID NO: 60.Lyp-1 is a tumor homing peptide that selectively binds the tumor-associated lymphatic vessels and tumor cells in certain tumors. The nine amino acid long peptide specifically recognizes the receptor P32. The RGD-peptide and NGR-peptide are tri-peptides composed of L-arginine-glycine-L-aspartic acid and L-asparagine-glycine-L-arginine, respectively. The sequences are common elements in cellular recognition. RGD peptides are implicated in cellular attachment via integrins. The HER2 binding peptide specifically targets the Human Epidermal Growth Receptor 2 (HER2). The CREKA peptide is a tumor homing peptide identified in phage display libraries consisting of the sequence Cys-Arg-Glu-Lys-Ala (see Simberg D, et al. Biomimetic amplification of nanoparticle homing to tumors. Proc Natl Acad Sci USA. 2007 Jan. 16; 104(3):932-6). CPP-2 and CPP-44 are tumor homing peptides described in Kondo et al. Tumourlineage-homing cell-penetrating peptides as anticancer molecular delivery systems. Nat Commun. 2012 Jul. 17; 3:951. F3 comprises amino acid sequences 17-48 of High Mobility Group Nucleosomal Binding Protein 2 (HMGN2) and was identified in a phage display cDNA library screen for peptides capable of homing to tumors, especially to their vascular endothelium (see (see Christian et al., Nucleolin expressed at the cell surface is a marker of endothelial cells in angiogenic blood vessels. J Cell Biol. 2003 Nov. 24; 163(4):871-8). RMS-P3 is a furin targeted peptide suitable for targeting Rhabdomyosarcoma (RMS) cells (see Hajdin K, et al. Furin targeted drug delivery for treatment of rhabdomyosarcoma in a mouse model. PLoS One. 2010 May 3; 5(5)). F56 specifically binds to VEGF receptor Fit-1 (see Herringson and Altin, Effective tumor targeting and enhanced anti-tumor effect of liposomes engrafted with peptides specific for tumor lymphatics and vasculature. Int J Pharm. 2011 Jun. 15; 411(1-2):206-14). LTVSPWY-peptide and WNLPWYYSVSPT-peptide specifically bind to breast cancer cells (see Shadidi and Sioud, Identification of novel carrier peptides for the specific delivery of therapeutics into cancer cells, FASEB J. 2003 F 17(2):256-8). SP5-2 specifically binds to non-small cell lung cancer (see Chang D K, et al. A Novel Peptide Enhances Therapeutic Efficacy of Liposomal Anti-Cancer Drugs in Mice Models of HumanLung Cancer, PLoS ONE 2009 (1):e4171).According to one embodiment of the first aspect of the invention, the loop between the first and second interaction region which comprises the targeting region has a number of amino acids in the range from 3 to 50 amino acids. The number of amino acids of the loop is counted from the covalent interaction pair “closing” the loop and consequently includes the amino acids of the covalent interaction pair. Accordingly a number of loop amino acids of 2 only includes the covalent interaction pair. Thus, the minimal number of amino acids in the loop is 3. The maximum length of the loop is in principle limited by the influence of the peptide on the folding of the fusion protein and the tendency for aggregation with higher length. Thus, the maximum number of amino acids in the loop is preferably 25, more preferably 20, most preferably 15 amino acids. According to a particularly preferred embodiment, the number of amino acids in the loop is in the range from 5 to 15 amino acids.The covalent interaction pair may be formed by any two amino acids, the side chains of which may form a covalent bond. These may be in particular cysteines or seleno cysteines which form disulfide bridges. According to one embodiment of the first aspect of the invention, the covalent interaction pair is formed by a cysteine in the first interaction region and by a cysteine in the second interaction region. A fusion protein according to the invention may comprise more than one covalent interaction pair. For example, the fusion protein according to the invention may comprise 7 or less, 6 or less, 5, or less, 4 or less, 3 or less, 2 or less interaction pairs. The covalent interaction pairs may be located in sequence or spaced apart. Preferably, the fusion protein according to the invention comprises one covalent interaction pair.According to one embodiment of the first aspect of the invention, at least two interaction pairs are non-covalent interaction pairs in which the amino acids interact non-covalently. Preferably, the second peptide comprises at least 3 non-covalent interaction pairs, more preferably at least 4 non-covalent interaction pairs. In principal, the higher the number of interaction pairs, the stronger the interaction of the first and second interaction region of the second peptide. The number of non-covalent interaction pairs also depends on the type of interaction of the amino acids. The non-covalent interaction may be by hydrogen bridges, van der Waal forces, hydrophobic interactions or acid-base interactions.Preferably, at least a part of the non-covalent interaction pairs are acid-base interaction pairs formed by an acidic amino acid in one interaction region and a basic amino acid in the other interaction region. At a neutral pH, these amino acids are charged negatively and positively, respectively. The contrary charges of the amino acids lead to an attraction of these amino acids and consequently of the interaction regions. Moreover, the contrary charges provide a tight binding of binding.According to one embodiment, the second peptide comprises 2 to 20 acid-base interaction pairs, preferably 2 to 10 acid-base interaction pairs, more preferably 2 to 6 acid-base interaction pairs, most preferably 3 to 5 acid-base interaction pairs. The higher the number of acid-base interaction pairs, the higher the attraction of the first and second interaction region. However, a number more than 20 acid-base interaction pairs will be problematic for the folding of the fusion protein. A number of more than 10 acid-base interaction pairs renders cloning more problematic as very long primers have to be used. Moreover, it is assumed that the use of more than 6 acid-base interaction pairs does not further significantly increase the interaction of the first and second interaction region. With regard to ease of cloning and optimal strength of interaction of the non-covalent interaction pairs, a number of 3 to 5 acid-base interaction pairs are preferred. In a particularly preferred embodiment of the first aspect of the invention the second peptide comprises 4 acid-base interaction pairs.Basic amino acids according to the invention can be arginine, lysine or histidine. Acidic amino acids according to the invention can be glutamic acid or aspartic acid.The first and second interaction region may comprise both acidic and basic amino acids, only acidic amino acids or only basic amino acids. The basic and acid amino acids in one interaction region may be alternating or form clusters. Non-limiting examples of alternating sequences are: EERR (SEQ ID NO: 45), ERER (SEQ ID NO: 46), EERREE (SEQ ID NO: 47), RREERR (SEQ ID NO: 48). Examples of clusters are EEERRR (SEQ ID NO: 49), DDERKK (SEQ ID NO: 50), DDDRR (SEQ ID NO: 51). However, it is preferred that one of the interaction regions comprises a majority of acidic amino acids and the other, consequently, a majority of basic amino acids. For example, the first inter action region comprises the mainly basic sequence RRRRE (SEQ ID NO: 52) and the second interaction region comprises the mainly acidic sequence EEEER (SEQ ID NO: 53).According to a preferred embodiment of the first aspect of the invention, the non-covalent interaction pairs are acid-base interaction pairs and formed by an acidic amino acid in the first interaction region and basic amino acid in the second region. The first interaction region may comprise at least 2, at least 3, at least 4, at least 5, at least 6 acidic amino acids. Also, the second interaction region may comprise at least 2, at least 3, at least 4, at least 5, at least 6 basic amino acids. Preferably, the first interaction region comprises at least 4 acidic amino acids and the second interaction region comprises at least 4 basic amino acids. More preferably, the first interaction region comprises at least 4 consecutive acidic amino acids and the second interaction region comprises at least 4 consecutive basic amino acids.According to one embodiment of the first aspect of the invention, the majority of the basic amino acids are arginine. In an alternative embodiment, the majority of the basic amino acids are lysines. Preferably, all basic amino acids in the interaction region are arginines.The charged amino acids in the interaction regions, i.e. the acidic and basic amino acids of the first and second interaction region may be directly in sequence or contain non-charged amino acids as spacers in between. Accordingly, two charged amino acids in the interaction region may be directly connected or may be separated by one or more non-charged amino acids. The non-charged amino acids as spacer between the charged amino acids are preferably selected from the group consisting of serine and glycine. The number of non-charged amino acids, i.e. the spacing, between two charged amino acids may be for example 0, 1, 2, 3 or 4. According to one embodiment of the invention, the spacing of the charged amino acids within the amino acid sequence of the first interaction region is 0 or 1. According to one embodiment of the invention the spacing of the charged amino acids in the second interaction region is 0 or 1. According to one embodiment the spacing of the charged amino acids in the first and/or second interaction region is preferably 0. Thus, the charged amino acids in the first and/or second interaction region are directly connected.In a preferred embodiment, the first interaction region comprises four consecutive arginines. According to one embodiment of the first aspect of the invention, the majority of the acidic amino acids are glutamic acids. According to an alternative embodiment, the majority of acidic amino acids are aspartic acids. Preferably, all acidic amino acids in the second peptide are glutamic acids. In particular the first interaction region comprises the sequence EEEE (SEQ ID NO: 54) and the second interaction region comprises the sequence RRRR (SEQ ID NO: 55). In a further preferred embodiment, the fusion protein comprises a first interaction region comprising the sequence RRRRSGC (SEQ ID NO: 74) and a second interaction region comprising the sequence CSGEEEE (SEQ ID NO: 75) as depicted in FIG. 1A. In a further preferred embodiment, the fusion protein comprises a first interaction region comprising the sequence RRRRC (SEQ ID NO: 76) and a second interaction region comprising the sequence CEEEE (SEQ ID NO: 77) as depicted in FIG. 1B.According to one embodiment the first interaction comprises the sequence EGEGEGE (SEQ ID NO: 56) and the second interaction region comprises the sequence RGRGRGR (SEQ ID NO: 57). According to the one embodiment the first interaction comprises the sequence ESESESE (SEQ ID NO: 58) and the second interaction region comprises the sequence RSRSRSR (SEQ ID NO: 59).The spacing region between the covalent interaction pair or pairs and the non-covalent interaction pair or pairs has an influence on the formation of the hair pin-like structure of the second peptide. If the number of amino acids forming the spacer is too high, the effect of bringing the amino acids of the covalent interaction pair proximity by means of the binding of the one or more non-covalent interaction pairs may be lost. In contrast, a too short distance may be problematic for steric reasons. For example, the size of the side chains of the acidic and basic amino acids is bigger than the size of the side chain of cysteines. Accordingly, if the cysteines are directly adjacent to the charged amino acids in the second peptide a disulfide bridge might not form. Thus, according to one embodiment of the first aspect of the invention, the number of amino acids in the first and second interaction region between the at least one covalent interaction pair and the closest non-covalent interaction pair is in the range from 1 to 6, preferably 1 to 4, more preferably 1 to 3. Most preferably, the spacers in both interaction regions between the at least one covalent interaction pair and the closest non-covalent interaction pair is 2 amino acids.In addition, the type of amino acids forming the spacer between the at least one covalent interaction pair and the closest non-covalent interaction pair influences the formation of the covalent bond. For the spacers, polar uncharged amino acids, with short side chains are preferred such as glycine, serine or alanine. More preferably the amino acids of the spacers between the at least one covalent interaction pair and the closest non-covalent interaction pair are glycine and serine. According to a particularly preferred embodiment of the invention, the spacers between the covalent interaction pair and the non-covalent interaction pair consist of one glycine and one serine.According to one embodiment of the fusion protein the first interaction region is Lyp-1 interaction region 1 as defined by SEQ ID NO: 20. According to one embodiment of the fusion protein the second interaction region is Lyp-1 interaction region 2 as defined by SEQ ID NO: 21. According to one embodiment the amino acid sequence of the first interaction region is defined as Lyp-1 interaction region 1 and the amino acid sequence of the second interaction region is defined as Lyp-1 interaction region 2.Preferably, the second peptide is introduced into a region of the first peptide that is not essential for folding so that the second peptide does not interfere with the folding of the first peptide. Moreover, it is preferred that the second peptide is introduced into a region of the first peptide that is located on the surface of the first peptide when folded. The skilled person knows how to determine suitable positions within an amino acid sequence. Suitable positions are preferably determined from crystal structures of the protein or related proteins. Preferably, the second peptide is located in a loop of the first peptide of the fusion protein. More preferably in a loop on the surface of the first peptide.The second peptide is particularly useful as a targeting peptide for targeting virus-like particles (VLPs). VLPs may comprise cargo that is useful only in specific cell types or toxic and must therefore address only specific cell types. Accordingly, it is preferred that the first peptide is a protein forming the capsid of a VLP.According to a preferred embodiment, the first peptide is a polyoma virus VP1. “VP1” or “virus protein 1” according to the invention refers to a protein which is identical to or derived from the natural VP1 of the JC virus, having the amino acid sequence according to SEQ ID NO: 12. A protein derived from the natural VP1 of the JC virus preferably has an amino acid sequence homology or identity with the amino acid sequence according to SEQ ID NO: 12 of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 97%, of at least 98%, or of at least 99%, or with a sequence of at least 100 contiguous amino acids, preferably of at least 150, of at least 200, of at least 250, of at least 300 contiguous amino acids. Most preferably, the amino acid sequence homology or identity is calculated over the entire length of the natural JCV-VP1. The terms “VP1 derived from the natural VP1 of the JC virus” and “VP1 derived from JC virus” in particular also include VP1, which is identical to the natural VP1 of the JC virus. The term “VP” according to the invention also encompasses fractions and derivatives of the natural VP1, which are capable of assembling into VLP. Preferably, said fractions and derivatives of VP1 at least comprise amino acids 32 to 316 of the amino acid sequence according to SEQ ID NO: 9 or a derivative thereof. Having a homology or identity with the amino acid sequence from amino acid position 32:316 of SEQ ID NO: 9 of at least 80%, of at last 85%, of at least 90%, of at least 95%, of at least 97%, of at least 98%, or of at least 99%.Preferably, the first peptide is a VP1 from JCV. According to X-ray crystallography analysis the folded VP1 (e.g. PDB entry 3NXD) contains three loops on the surface of the protein. Two of these loops, the DE-loop (aa 120-137) and the HI-loop (aa 262-272) are known to be eligible for the introduction of exogenous peptide structures as an exogenous structure introduced into the loop is accessible and in general does not interfere with folding of the VP1 protein. Thus, according to one embodiment, the second peptide is located in the DE-loop or the HI-loop of VP1. Preferably, the peptide is located between amino acid 120 and 137 (DE-loop) or 262 and 272 (HI-loop) of VP1. More preferably between amino acid 129 and 132 (DE-loop) or 265 and 268 (HI-loop) of VP1. In particular, the second peptide substitutes amino acid 267 of the HI-loop. As described below, different expression systems are suitable for the expression of the protein. According to a preferred embodiment, the fusion protein is expressed in E. coli. According to one embodiment of the fusion protein the first peptide is a VP1 from JCV and the second peptide comprises the Lyp-1 peptide. According to one embodiment the first peptide is a VP1 from JCV and the second peptide comprises the amino acid sequence with a sequence homology or identity with the amino acid sequence according to SEQ ID NO: 13 of at least 95%. According to one embodiment the first peptide is a VP1 from JCV and the second peptide comprises a amino acid sequence according to SEQ ID NO: 13. According to one embodiment the first peptide is a VP1 from JCV, the second peptide comprises a amino acid sequence according to SEQ ID NO: 13 and the second peptide is integrated into the DE-loop of the VP1. According to one embodiment the first peptide is a VP1 from JCV, the second peptide comprises an amino acid sequence according to SEQ ID NO: 13 and the second peptide is integrated into the HI-loop of the VP1. The fusion protein may have an amino acid sequence with a sequence homology or identity with the amino acid sequence according to SEQ ID NO: 14 of at least 95%. Alternatively, the fusion protein may have an amino acid sequence with a sequence homology or identity with the amino acid sequence according to SEQ ID NO: 15 of at least 95%.3. Virus-Like ParticleAccording to a second aspect, the invention provides a virus-like particle (VLP) which comprises at least one fusion protein according to a first aspect of the invention. Preferably, the VLP is a polyoma virus VLP and the first peptide of the fusion protein is a polyoma virus VP1.Non-limiting examples for viruses of the polyoma family are: B-lymphotropic polyomavirus (formerly known as African green monkey polyomavirus, AGMPyV) (LPyV), Baboon polyomavirus 1 (SA12), Bat polyomavirus (formerly known as Myotis polyomavirus, MyPyV; BatPyV) BK polyomavirus (BKPyV), Bornean orang-utan polyomavirus (OraPyV1), Bovine polyomavirus (BPyV), California sea lion polyomavirus (SLPyV), Hamster polyomavirus (HaPyV), JC polyomavirus (JCPyV), Merkel Cell polyomavirus (MCPyV), Murine pneumotropic virus (formerly known as Kilham strain of polyomavirus, Kilham virus, K MPtV), Murine polyomavirus (MPyV), Simian virus 40 (formerly known as Simian vacuolating virus 40; SV40), Squirrel monkey polyomavirus (SqPyV), Sumatran orang-utan polyomavirus (OraPyV2), Trichodysplasia spinuolsa-associated polyomavirus (TSPyV), Human polyomavirus 6 (HPyV6), Human polyomavirus 7 (HPyV7), KI polyomavirus (formerly known as Karolinska Institute polyomavirus, KIPyV), WU polyomavirus (formerly known as Washington University polyomavirus, (WUPyV), Avian polyomavirus (formerly known as Budgerigar Fledgling disease polyomavirus, BFPyV, APyV), Canary polyomavirus (CaPyV), Crow polyomavirus (CPyV), Finch polyomavirus (FPyV), Goose Hemorrhagic polyomavirus (GHPyV), Athymic rat polyomavirus (RatPyV), Baboon polyomavirus 2 (BPyV2), Cynomolgus polyomavirus (CyPV), Gorilla gorilla gorilla polyomavirus 1 (GggPyV1), Human polyomavirus 9 (HPyV9), Trichodysplasia spinulosa-associated polyomavirus (TSV) Mastomys polyomavirus (multimammate mouse—Mastomys species), Pan troglodytes verus polyomavirus 1a (PtvPyV1a), Pan troglodytes verus polyomavirus 2c (PtvPyV2c), Rabbit kidney vacuolating virus (RKV).Preferably the VLP is derived from a human polyoma virus comprising Human polyomavirus 6 (HPyV6), Human polyomavirus 7 (HPyV7), Human polyomavirus 9 (HPyV9), BK polyomavirus (BKPyV), JC polyomavirus (JCPyV), Merkel Cell polyomavirus (MCPyV), KI polyomavirus (formerly known as Karolinska Institute polyomavirus, KIPyV), WU polyomavirus (formerly known as Washington University polyomavirus, (WUPyV), Trichodysplasia spinulosa-associated polyomavirus (TSV), human polyoma virus 10 (HPyV10), MW polyomavirus and MX polyomavirus. In a more preferred embodiment of the invention, the VLP is derived from the human polyoma virus JCV.VLPs are multi-protein structures that mimic the organization and conformation of authentic native viruses but lack the viral genome.The virus-like particle according to the invention is preferably derived from human polyomavirus. In the context of the invention, the term “from human polyomavirus” refers to a VLP with structural proteins that can be isolated or extracted from polyomaviruses or which can be generated by recombinant expression of a polyoma structural protein or a modified form of said structural protein.The capsids of all polyomaviruses have a similar structural set-up including the proteins VP1, VP2, VP3, and agnoprotein. The icosahedral virus capsid is formed by 72 VP1 pentamers. In the center of each of the pentamers, facing to the inside of the capsid, a VP2 or VP3 protein is located. VP3 is identical to the C-terminal two-thirds of VP2. This shared region comprises inter alia the nuclear localization signal (NLS), the DNA-binding domain (DBD), and the VP1 interacting domain (VID).“VP2” or “virus protein 2” according to the invention refers to a protein which is identical to or derived from the natural VP2 of the JC virus, having the amino acid sequence according to SEQ ID NO: 22. A protein derived from the natural VP2 of the JC virus preferably has an amino acid sequence homology or identity with the amino acid sequence according to SEQ ID NO: 22 of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 97%, of at least 98%, or of at least 99%, or with a sequence of at least 100 contiguous amino acids, preferably of at least 150, of at least 200, of at least 250, of at least 300 contiguous amino acids. Most preferably, the amino acid sequence homology or identity is calculated over the entire length of the natural JCV-VP2.“VP3” or “virus protein 3” according to the invention refers to a protein which is identical to or derived from the natural VP3 of the JC virus, having the amino acid sequence according to SEQ ID NO: 23. A protein derived from the natural VP3 of the JC virus preferably has an amino acid sequence homology or identity with the amino acid sequence according to SEQ ID NO: 23 of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 97%, of at least 98%, or of at least 99%, or with a sequence of at least 100 contiguous amino acids, preferably of at least 150, of at least 200, of at least 250, of at least 300 contiguous amino acids. Most preferably, the amino acid sequence homology or identity is calculated over the entire length of the natural JCV-VP3.With the integration of a fusion protein according to the first aspect of the invention, the VLP may be targeted to a cell of interest. Polyomavirus VLPs are formed up to 72 pentamers. Each of these pentamers consists of 5 copies of VP1. Preferably, at least one pentamer of the icosahedral VLP capsid is formed by 1 to 5 copies of the fusion protein of the first aspect of the invention. Pentamers of the VLP may comprise 1, 2, 3, 4 or 5 copies of the fusion protein according to the first aspect. As the pentamers form upon expression and are stable also upon disassembly of the VLP, recombinant expression of only the fusion protein in a host cell generally leads to the formation of pentamers with 5 copies of the fusion protein.According to one embodiment of the second aspect of the invention the ratio of pentamers formed by the fusion protein and pentamers formed by other VP1 is at least 1:71, at least 1:35, at least 1:23, at least 1:17, at least 1:11, at least 1:8, at least 1:7, at least 1:5, at least 1:3 at least 1:2. In this regard other VP1 proteins are VP1 constructs that do not include a fusion protein according to the invention. Preferably the ratio is at least 1:5. The higher the number of the fusion protein according to the invention in a VLP the higher the chance that the VLP will interact with a cell type specific for the targeting region of the fusion protein. However, the integration of a peptide into the structure of the VP1 may interfere with assembly of the VLP. Thus, it is preferred that at least a fraction of the pentamers is formed by VP1 without a peptide insertion, preferably by a VP1 with a sequence identity of at least 90%, of at least 95%, of at least 97%, of at least 98%, or of at least 99% to SEQ ID NO: 9.The VLP according to the second aspect of the invention may comprise more than one type fusion protein according to the first aspect of the invention. For example, the VLP may comprise one type of fusion protein with a targeting region specific for a first target and a second type of fusion protein with a targeting region specific for a second target. Such a VLP is able to target to different targets. The two targets may for example be epitopes located on different cell types. Accordingly, at least two different types of cells can be targeted. Alternatively, the two targets may be located on one type of cells. In this scenario the two targeting regions increase the chance of affecting the specific cell type by the VLP. The VLP with more than one fusion protein according to the first aspect of the invention may comprise a first fusion protein with the Lyp1-peptide as targeting region and a second fusion protein with the LTVSPWY-peptide as targeting region. The VLP with more than one fusion protein according to the first aspect of the invention may further comprise a first fusion protein with the Lyp1-peptide as targeting region and a second fusion protein with the F3-peptide as targeting region. This VLP is particularly useful for targeting breast tumor cells. Moreover, the VLP with more than one fusion protein according to the first aspect of the invention may comprise a first fusion protein with the SP5-2-peptide as targeting region and a second fusion protein with the F3-peptide as targeting region.The VLP according to the second aspect may further comprise minor capsid proteins such as VP2, VP3 or agno protein. The VLP preferably comprises a fusion protein comprising a VP1 binding protein, which preferably comprises the VP1 interacting domain of VP2 and an exogenous peptide, preferably selected from a cargo binding peptide (CBP) and endosomal translocating peptide (ETP).According to one embodiment of the second aspect, the VLP comprises a second fusion protein comprising a VP1 binding protein and an exogenous peptide, wherein the exogenous peptide comprises a cargo-loading peptide and/or an endosomal translocating peptide (ETP).The term “VP1 binding protein” refers to any peptide that has the ability to bind to the major capsid protein VP1 of a polyomavirus. In particular, the VP1 binding protein is a peptide comprising the VP1 interacting domain (VID) of a polyomavirus VP2/VP3 protein.The VID may be derived from a VP2 or VP3 or differently termed functional equivalent thereof from any known polyomavirus.The VP1 binding protein according to the invention comprises the VP1 interacting domain of VP2 and allows a positioning of the fusion protein and, in particular, the exogenous peptide within a VLP derived from a polyomavirus. Preferably, the VP1 interacting domain has an identity of at least 90%, preferably at least 95%, more preferably at least 98% to SEQ ID NO: 24. Thus, the VP1 binding protein preferably comprises at least a sequence with an identity of at least 90%, preferably at least 95%, more preferably at least 98% to SEQ ID NO: 24.The VP1 binding protein is preferably a full length polyomavirus VP2 or VP3. These proteins are naturally adapted for the interaction with the VP1. Accordingly, in one embodiment of the first aspect of the invention, the VP1 binding protein comprises an amino acid sequence that has an identity of at least 80%, preferably at least 90%, more preferably at least 95% to SEQ ID NO: 22 or SEQ ID NO: 23.However, any fragment or sub-structure of VP2 or VP3 may be sufficient for a tight interaction with VP1 as long as it contains a functional VP1 interacting domain of VP2/VP3. For example, the VP1 binding protein according to the invention may include or exclude the DNA-binding domain. For example, the VP1 binding protein may comprise the VID and the NLS of VP2. Further examples are a VP1 binding protein comprising the VID and the NLS of VP2, a VP1 binding protein comprising the VID and the DBD of VP2, and VP1 binding protein comprising the VID, DBD and the NLS of VP2.The VP1 binding protein may be a modified version of VP2 or VP3, e.g. mutated by insertion, deletion, or amino-acid replacement with respect to SEQ ID NO: 22 or 23. However, the VP1 binding protein may only be modified to the point that the VP1 interacting domain is still functional, i.e. still binds to a polyomavirus VP1.The exogenous peptide may be located at any position of the second fusion protein, i.e. at the C-terminus, at the N-terminus, or at any position within the amino acid sequence of the fusion protein. The location of the exogenous peptide is preferably on the surface of the folded protein. The exogenous peptide is further preferably freely accessible when the second fusion protein is bound to the VLP capsid. The skilled person knows how to determine positions within the amino acid sequences that fulfill these prerequisites. The structure predictions of VP2 or VP3 show that the N-terminus and the C-terminus are located on the surface of VP2 and VP3, and oriented to the inside of the polyoma virus when VP2 or VP3 is bound to a VP1 pentamer.In one embodiment of the second aspect of the invention the exogenous peptide forms the C-terminus or the N-terminus of the second fusion protein. A second fusion protein containing the exogenous peptide on the C-terminus or N-terminus of the protein has the further advantage of an easier construction of the polynucleotide encoding the fusion protein. The C-terminus is particularly preferred as the location for the exogenous peptide because it is the part of the protein that is the last to be translated. Thus, an exogenous peptide on the C-terminus has the lowest influence on protein folding. According to one embodiment of the first aspect, the endosomal translocating peptide, preferably the CPP, is located on the C-terminus of the protein. Alternatively, the endosomal translocating peptide, preferably the CPP, forms the N-terminus of the second fusion protein. According to an alternative embodiment, the cargo loading peptide, in particular the cargo binding peptide, forms the C-terminus of the second fusion protein. Alternatively, the cargo binding peptide may form the C-terminus.The exogenous peptide preferably has a percentage of basic amino acids of at least 25%, more preferably of at least 30%.According to one embodiment of the first aspect of the invention, the exogenous peptide comprises a cargo loading peptide. A cargo lo