Peptides that self-assemble into nanoparticles

Abstract

The present invention relates to polypeptides that self-assemble into nanoparticles. Specifically, the present invention relates to a polypeptide comprising amino acid sequence I (SEQ ID NO: 1), a nucleic acid sequence encoding the polypeptide, a nanoparticle comprising at least one polypeptide of the present invention, a complex comprising the nanoparticle and one or more cargo molecules, and a method for transfecting cells with the complex.

Description

[0001] The present invention relates to the field of polypeptides that self-assemble into nanoparticles. Specifically, the present invention relates to a polypeptide comprising amino acid sequence I (SEQ ID NO: 1), a nucleic acid sequence encoding the polypeptide, a nanoparticle comprising at least one polypeptide of the present invention, a complex comprising the nanoparticle and one or more negative cargo molecules, and a method for transfecting cells with the complex. Background Art

[0002] Although oligonucleotides (ONs) are a class of molecular drugs with many potential clinical applications, their suboptimal pharmacokinetic properties have severely hampered drug development (Whitehead, KA et al. Nat. Rev. Drug Discov. 2009, 8, 129-138). Specifically, because ONs are large, highly charged entities and do not diffuse into cells, intracellular delivery of ONs remains a significant obstacle (Lundin, KE et al. Oligonucleotide Therapies: The Past and the Present. Hum. Gene Ther. 2015, 26, 475-485). Additional issues regarding in vivo applications include susceptibility to nucleases present in biological serum, which results in rapid degradation and urinary excretion and undesirable off-target effects and activation of intracellular and extracellular immune responses.

[0003] In order to overcome these shortcomings, it is proposed to use oligonucleotide delivery systems, such as virus-like particles (VLPs) derived from bacteriophage P22 (Qazi, S et al., Mol. Pharm. 2016, 13, 1191-1196) or Q virus-like particles (VLP) functional nanoparticles (Fang, PY et al., Nucleic Acids Res. 2017, 45, 3519). However, the production of virus-like particles carrying the desired therapeutic cargo is complicated, expensive and still associated with safety issues.

[0004] Although a variety of materials have been proposed as oligonucleotide delivery carriers, simple, effective, safe and non-toxic delivery schemes are still being sought (Whitehead, KA et al., Knocking down barriers: advances in siRNA delivery. Nature Review: Drug Discovery 2009, 8, 129-138). Recently, the development of computer-designed proteins that can self-assemble into highly ordered and symmetrical cage-like nanostructures has been described (King, NP et al., Science, 2012, 336, 1171-1174; US9,630,994B2). In addition, the design of such self-assembled protein nanocages has been described recently, which refers to envelope protein nanocages (EPNs) that guide their own release from human cells into vesicles. One of the designed symmetric self-assembling scaffolds that supports EPN formation when fused to the N-terminal p6Gag peptide and the C-terminal PH domain was named O3-33 (King et al., 2012, loc. cit.; J. Votteler et al., Nature, 2016, 540, 292-295). Summary of the invention

[0005] The present invention provides designed and non-natural novel polypeptides, which can not only self-assemble into nanoparticles, but also can encapsulate negatively charged macromolecules (such as oligonucleotides with high binding affinity) and deliver the macromolecules in vitro and in vivo. It has been further found that preferred nanoparticles of the present invention are effectively absorbed by mammalian cells and can release their goods to induce, for example, RNA interference and knock down gene expression. Therefore, designed and non-natural novel polypeptides are engineered to produce nanoparticles that allow goods to be delivered (particularly, nucleic acid delivery), while minimizing or avoiding the undesirable characteristics of the delivery vectors of the prior art.

[0006] Importantly, nanoparticles of the present invention are easy to produce and purify with high yield and allow any macromolecule (particularly, any oligonucleotide) to be packaged in vitro into the nanoparticles. In addition, the nanoparticles of the present invention therefore provide a universal platform for delivering the multiple goods (such as treatment ON) in cells to, for example, regulate gene expression for various applications, such as gene interference. Therefore, its usefulness encompasses biotechnology applications and therapeutic applications. In addition, nanoparticles of the present invention generally have uniform size, and are particularly safe and well tolerated in vivo, and further generally biodegradable. Additionally, designed and non-naturally occurring novel polypeptides generally and preferably can be easily produced by recombinant expression, which is scalable, economical and allows both genetic modification and post-translational (chemical) modification. The adjustment of the characteristics of the nanoparticles of the present invention further allows the nanoparticles not only to enter the cell, and conditionally release the goods in the cytoplasm to control, for example, described gene expression.

[0007] Thus, the novel non-naturally occurring polypeptides developed by the inventors self-assemble into nanoparticles capable of encapsulating and delivering cargo (preferably, negatively charged macromolecules such as ON).

[0008] Therefore, in a first aspect, the present invention provides a polypeptide comprising an amino acid sequence I consisting of:

[0009] MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15 LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX 27 QXd 28 GX 29 E (SEQ ID NO: 1),

[0010] Where X1 to X 29 Any one of X1 to X6 is independently an amino acid, provided that at least 3 of X1 to X6 are independently positively charged amino acids, and wherein except for X1 to X6 in SEQ ID NO: 1 29 Optionally up to 5 amino acids at positions other than the indicated positions are exchanged with any amino acids.

[0011] In another aspect, the present invention provides a nucleic acid sequence encoding the polypeptide of the present invention.

[0012] In a further aspect, the present invention provides a nanoparticle comprising at least one polypeptide of the present invention.

[0013] In a further aspect, the present invention provides a complex comprising the nanoparticle of the present invention and one or more cargo molecules, wherein the one or more cargo molecules are encapsulated in the nanoparticle.

[0014] In a further aspect, the present invention provides a method for transfecting a cell, the method comprising the step of contacting the cell with the complex of the present invention.

[0015] In a further aspect, the present invention provides a method for producing the nanoparticle of the present invention, the method comprising the step of allowing the polypeptide of the present invention to self-assemble into the nanoparticle.

[0016] In a further aspect, the present invention provides a method for making a complex of the present invention, the method comprising the step of mixing the nanoparticle of the present invention with one or more negatively charged macromolecules.

[0017] In a further aspect, the present invention relates to the use of a complex of the present invention for transfecting cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 : (a) Surface model of an OP cage viewed from the 4-fold and 2-fold symmetry axes, with one trimer shown as a ribbon. Model generated from X-ray diffraction data. (b) Side view of one trimer, with arginine insertion sites shown as black spheres. (c) Luminal surface of a trimer, with arginine sites shown as black spheres. (d) Negative transmission transmission electron micrograph (TEM) of OP.

[0019] Figure 2: (a) Electrophoretic mobility shift assay of Atto488-labeled ssDNA in the presence of OP visualized by Atto488 fluorescence. (b) Absorbance spectra of SEC-purified OP:nucleic acid complexes. (c) k obtained from fluorescence quenching of Atto488-ssDNA cargo after addition of OP. obs Plotting of values ​​against [OP] to determine k on (d) Plot of fluorescence recovery of Atto488-ssDNA released from OP cages induced by addition of unlabeled competitor DNA.

[0020] Figure 3: (a) Cellular uptake of Atto488-DNA in the presence or absence of OP as determined by flow cytometry (n=3). (b) Confocal fluorescence microscopy of HeLa cells treated with Atto488-DNA or OP:Atto488-DNA (OP with Atto488-ssDNA cargo). Hoechst refers to nuclear staining. (c) Flow cytometric analysis of cellular uptake across different cell lines. Median fluorescence intensity of seven different cell types after treatment with 200 nM of Atto488-DNA or OP:Atto488-DNA for 18 hours. Median fluorescence intensity was obtained from 10,000 cells. MCF-7 - breast cancer, HepG2 - liver cancer, A431 - skin cancer, HT-29 - colon cancer, CHO-K1 - hamster ovary (non-cancerous), Vero - African green monkey kidney (non-cancerous).

[0021] Figure 4 : (a) GFP knockdown determined by flow cytometry (n=4). OP:scramble refers to scrambled siRNA that does not target GFP mRNA or any other endogenous sequence, and no His6 refers to siRNA packaged in an OP cage without a His6 tag. (b) Native AGE analysis of tRNA-induced dsDNA cargo release from OP over time. Visualized by Atto488 fluorescence. (c) Flow cytometric analysis of cellular uptake across different cell lines. Median fluorescence intensity of seven different cell types after treatment with 200nM Atto488-DNA or OP:Atto488-DNA for 18 hours. Median fluorescence intensity was obtained from 10,000 cells.

[0022] Figure 5 : Electrostatic potential surface. Coulomb surface comparison of the trimer subunits from the crystal structures of O3-33 and OP, showing both the inner and outer surfaces.

[0023] Figure 6: SDS-PAGE. Both proteins from crude cell lysate and products isolated from Ni-NTA are shown. The lower mobility of OP is due to additional positively charged amino acids rather than differences in molecular weight. The faint band above OP was removed after size exclusion chromatography. The gel was visualized with Coomassie blue. Typical yield of OP protein after Ni-NTA is 100 mg / L of E. coli culture.

[0024] Figure 7 : SEC of O3-33 and OP. Size exclusion chromatography of O3-33 and the positively charged caged nanoparticles OP of the present invention. Both analytical traces of the crude mixture and the purified assembly from Ni-NTA chromatography are shown. The dotted line corresponds to the absorbance at 260nm (nucleic acid), and the solid line corresponds to the absorbance at 280nm (protein). Based on TEM analysis, the peak of OP (Ni-NTA) at 12-14mL contains dimers, timers and higher-order aggregates of fully assembled cages, rather than alternative quaternary assemblies. The peak of OP (Ni-NTA) at 22mL contains nucleic acid fragments loaded during protein expression in E. coli and easily removed by SEC. The main peak at 16mL shows the collected and further characterized eluted fractions of the protein. The SEC of OP is representative, and OTR101 to OTR503 show a comparable elution volume of about 16mL.

[0025] Figure 8: DLS and AGE of O3-33 and OP (a) Dynamic light scattering (DLS) of original (O3-33) and positively charged (OP) cage variants. (b) Native agarose gel electrophoresis (AGE) (3%); channel 1: O3-33, channel 2: OP, stained with Coomassie blue. Given the SEC, DLS, TEM, and X-ray diffraction data, the reduced mobility of OP compared to O3-33 is due to increased positive charge rather than a change in size. (c) 3% AGE of OP variants, stained with Coomassie blue for proteins. The difference in mobility is due to a change in overall charge rather than size. The tailing is caused by the large amount of protein loaded, not by degradation of the cage. Channel 1: O3-33. Channel 2: OTR101. Channel 3: OTR201. Channel 4: OTR202. Channel 5: OTR203. Channel 6: OTR301. Channel 7: OTR302. Channel 8: OTR401. Channel 9: OTR402. Channel 10: OTR501. Channel 11: OTR502. Channel 12: OTR503. Channel 13: OP. All proteins have comparable electrophoretic mobilities; the increase in the number of positively charged arginine residues may cause a slight deviation, since both SEC and TEM show that the particles have the same size. Overall, these characterization methods support the hypothesis that the structure of all variants is identical to OP.

[0026] Fig. 9 : Transmission electron microscopy (TEM) images. The scale bars for OP and O3-33 are 100 nm, and the scale bar for OTR501 is 200 nm. Negative staining transmission electron microscopy images of OP and OTR101 to OTR503 were obtained. The SEC shown herein represents OP and all variants OTR101 to OTR503. These images show uniform distribution of the capsid and morphology of the variants consistent with OP.

[0027] Fig.10 : EMSA of O3-33 and OP in the presence of ssDNA. Electrophoretic mobility shift assay of ssDNA labeled with Atto488 in the presence of increasing equivalents of OP or O3-33. Lanes 1-7: Atto488-ssDNA+OP, Lane 8: Atto488-ssDNA; Lanes 9-15: Atto488-ssDNA+O3-33. Gels were visualized by Atto488 fluorescence.

[0028] Fig.11: EMSA of OP in the presence of dsDNA and dsRNA. Electrophoretic mobility shifts of (a) dsDNA and (b) dsRNA in the presence of increasing molar equivalents of OP cages, both of which are 21 bp in length. The faint band of free nucleic acid at 0.5 equivalents of OP is due to non-equilibrium conditions during electrophoresis and represents a small portion of the total nucleic acid. Since there are 144 arginine residues in each capsid, charge neutrality will be achieved after encapsulating only 144 nucleotides, which is equivalent to 3.6 duplexes (21 bp) per capsid. The maximum loading capacity of the two duplexes observed here can be rationalized based on the physical limitations imposed by RNA-RNA charge repulsion, the spherical container, and the need for available arginine residues to contribute to favorable binding interactions. Nucleic acids were stained with GelRed.

[0029] Fig.12 : SEC of nucleic acid encapsulation. Size exclusion chromatography of empty OP cages compared to OP after incubation with 5 equivalents of ssDNA, dsDNA or dsRNA (all 21 nt in length) per cage. The dashed line corresponds to the absorbance at 260 nm (nucleic acids) and the solid line corresponds to the absorbance at 280 nm (proteins). No nucleic acid-mediated aggregation was observed. All complexes showed the same retention volume as the empty OP cages, but the A260 / A280 ratio was increased, indicating internalization of the oligonucleotides.

[0030] Fig.13 : Kinetic measurements. Representative data set for determining the kinetics of 1:1 encapsulation of ssDNA in OP cages. As obtained from EMSA ( Fig.10 ) The band intensity seen in (a) is obvious, and encapsulation of Atto488-ssDNA by OP leads to quenching of the fluorophore, which is used for the time-resolved binding assay shown here. The Atto488 fluorophore was excited at 488 nm and the emission was measured at 515 nm. In each experiment (ae), the concentration of Atto488-ssDNA was 10 nM, and the concentration of OP added at t=0 was as shown on the plot. (f) Comparison of the single exponential fits of these five concentration points without any normalization (shown in ae).

[0031] Fig.14: Nuclease assay. Native agarose gel shows (a) free nucleic acids (R = dsRNA, ssD = ssDNA and dsD = dsDNA) treated with the indicated nucleases (Bzase = Benzonase) and (b) the same nucleic acids pre-packaged in OP cages, also treated with the indicated nucleases. Lane M contains 300nt ssRNA for reference. Samples were incubated with nucleases in PBS at 37°C for 18 hours. For DNA samples and single-stranded DNA with DNase I, there was some incomplete digestion in the case of Benzonase (a, top gel lanes 8, 11 and 12). However, comparison of the encapsulated samples with the fully digested samples clearly reveals that OP can protect its cargo from damage by the larger nucleases Benzonase and DNase I, while the smaller RNase A is able to enter the cage and digest the RNA cargo.

[0032] Fig.15 : Cytotoxicity of OP and Lipofectamine 2000 at different concentrations in HeLa cells after (a) 24 hours and (b) 48 hours of incubation. Control: PBS or 1% Triton X-100 surfactant (n=3).

[0033] Fig.16 : Qualitative assessment of GFP knockdown by fluorescence microscopy. Knockdown of GFP expression in HeLa cells is shown based on relative GFP content from live cell fluorescence microscopy. The trends seen here match well with quantitative flow cytometry analysis ( Figure 4 a). Sample names are shown above each panel pair, with bright field on the left and GFP fluorescence on the right (LED excitation at 470 nm, emission filter 535 / 50 nm). The siRNA concentration for each sample was 100 nM, and images were acquired 46 hours after addition of the siRNA sample. Scale bar is 100 μm.

[0034] Figure 17: Kinetics at higher DNA to OP ratios. At higher ratios of Atto488-DNA to OP, two effects consistent with loading of two ssDNA molecules per OP cage were observed. First, an increase in quenching was observed, evident from the endpoints in (a)-(c). This is most likely due to self-quenching of the fluorophore at the higher effective concentration in the small cavity of the OP. The second effect is a shift from single exponential decay to double exponential decay, which reveals two distinct binding events. For (b) and (c), the single exponential fit is shown as a solid line and the double exponential fit is shown as a dashed line.

[0035] Fig.18: Characterization of OP without His6 tag. (a) SDS-PAGE comparison of OP (channel 1) and OP-without His6 (channel 2). (b) SEC of OP-without His6 shows a similar retention volume (16 mL) as OP. (c) Negative stain TEM of OP-without His6 looks consistent with OP. (d) Channel 1: OP and channel 2: native AGE of OP-without His. Gels were visualized with Coomassie Blue for proteins. (e) Native AGE encapsulated with siRNA at a molar ratio of 1:1 of OP (channel 2) to OP-without His6 (channel 3). Channel 1 is a free siRNA control. Visualization was performed with GelRed for nucleic acids. (f) SEC of OP-without His6 showed the same retention volume as the empty cage (16 mL) after incubation with 5 equivalents of siRNA, but the A260 / A280 ratio increased, indicating internalization of the oligonucleotide. The peak at 19-21 mL corresponds to excess RNA.

[0036] Fig.19 : Encapsulation of tRNA. Native agarose gel analysis shows complete binding of tRNA in the presence of 1.2 equivalents of OP cage. Lane 1 - tRNA, Lane 2 - tRNA + OP. Staining with GelRed.

[0037] Fig. 20 : EMSA of O3-33 in the presence of oligonucleotides. The same native agarose gel was (a) stained with GelRed for nucleic acids and (b) stained with Coomassie Blue for proteins. Arrows: complexes (nanoparticles plus ssDNA). Unlabeled ssDNA, dsDNA and dsRNA were analyzed after incubation with two equivalents of O3-33. There was no interaction between the protein cage and any of the oligonucleotides.

[0038] Fig.21 : Loading of different DNA sequences. The same native agarose gel (3% native AGE) was (a) stained with GelRed for DNA and (b) stained with Coomassie Blue for proteins. Lower arrow: ssDNA, upper arrow: complex (nanoparticles plus ssDNA, dsDNA, dsRNA). Each sample contained a 2:1 ratio of ssDNA to capsid, where the oligonucleotides ranged from 18-26nt with random sequences (provided in Table 2 below). Lane 1 - 18nt ssDNA1, Lane 2 - 26nt ssDNA, Lane 3 - OP, Lanes 4-12 - OP + 2 equivalents of different ssDNA.

[0039] Fig. 22: Serum stability. The same native agarose gel was stained (a) with GelRed for RNA and (b) with Coomassie Blue for protein. Lower arrow: dsRNA, upper arrow: complex (nanoparticles plus dsRNA), middle arrow: BSA. There was no obvious interaction between the dsRNA-loaded OP cages and serum proteins and no displacement or degradation of the RNA cargo. Lane 1 - dsRNA, Lane 2 - OP:dsRNA, Lanes 3-7 - OP:dsRNA after incubation with 10% FBS at 37°C for 0.5, 1, 2, 4 and 7 hours, Lane 8 - 10% FBS alone.

[0040] Fig.23 : Small angle X-ray scattering. (a) One-dimensional scattering curves for OP, OP:dsDNA, and OP:dsRNA. The experimental data are shown as points, and the fit used to generate the distance distribution is shown as a gray line. (b) Pair distance distribution functions for OP, OP:dsDNA, and OP:dsRNA calculated from the above data using AUTOGNOM.2-3. The dashed lines correspond to the particle size obtained from the DLS experiment ( Figure 8 a), the tailing greater than 14 nm at some distances is due to transient intermolecular interactions between individual protein cages. After complexing with oligonucleotides, the outer diameter of the protein cages did not change and the density shifted from hollow spheroidal particles toward filled spheres. Both phenomena are consistent with the internalization of oligonucleotides within the cavity of OP.

[0041] Fig.24 : Circular dichroism spectra of O3-33 and OP. The solid lines are the mean values ​​and the error bars show the standard deviation of triplicate measurements for each protein.

[0042] Fig.25 : An overview of the preparation and use of the nanoparticles and composites of the present invention.

[0043] Fig.26 : ssDNA loading into nanoparticles (NPs) studied by electrophoretic shift assay (EMSA). (a) 2% native gel visualized with Atto488 absorbance by UV transilluminator. Channel 1: O3-33. Channel 2: OTR101. Channel 3: OTR201. Channel 4: OTR202. Channel 5: OTR203. Channel 6: OTR301. Channel 7: OTR302. Channel 8: OTR401. Channel 9: OTR402. Channel 10: OTR501. Channel 11: OTR502. Channel 12: OTR503. Channel 13: OP. Channel 14: Atto488-ssDNA. The broad band is caused by impurities of the DNA batch. (b) Proteins visualized with Coomassie blue.

[0044] Figure 27: Fluorescence quenching after ssDNA encapsulation. (a) Fluorescence decay of all variants at 50 nM protein concentration in 10 nM Atto-488 ssDNA. This indicates that the speed of this decay depends on the number and position of arginines. (b) Close-up view of the slow variants in (a). Here, it can be clearly seen that O3-33, OTR101, OTR201, OTR202, and OTR203 do not bind ssDNA.

[0045] Fig.28 : In vitro study of dsDNA loading into nanoparticles (NPs). (a) Native AGEs visualized for hpDNA by Atto-488 absorbance. (b) Same gel used for protein visualization after staining with Coomassie Blue. Channel 1: hpDNA. Channel 2: OTR301. Channel 3: OTR302. Channel 4: OTR401. Channel 5: OTR402. Channel 6: OTR501. Channel 7: OTR502. Channel 8: OTR503. Channel 9: OP.

[0046] Fig.29 : GFP knockdown determined by flow cytometry (n=3).

[0047] Fig.30 : Immunogenicity of OP cages in mice as determined by ELISA. As indicated by the arrows, OP-specific IgG1 (a), IgG2a (b), and IgG2b (c) antibody titers were determined in sera from BALB / c mice immunized with OP. Each point is the average IgG titer from 4 mice.

[0048] Fig.31 : Immunogenicity time course of OP cages in mice as determined by ELISA. OP-specific IgG1 (a), IgG2a (b), and IgG2b (c) antibody titers were determined in sera from BALB / c mice immunized with OP as indicated by arrows. Each point is the average IgG titer from 4 mice. DETAILED DESCRIPTION

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0050] Throughout this specification and the appended claims, unless the context requires otherwise, the word “comprise” or the word “include” and variations such as “comprises / includes” or “comprising / including” will be understood to imply the inclusion of an element, stated integer, step or group of elements, stated integers, steps but not the exclusion of any other element, stated integer, step or any other group of elements, stated integers, steps.

[0051] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0052] The term "about" or "approximately" when used in conjunction with a numerical value is intended to encompass a numerical value within a range where the lower limit is 0-10% less than the indicated numerical value and the upper limit is 0-10% greater than the indicated numerical value. The term "about" or "approximately" means preferably ±10%, more preferably ±5%, again more preferably ±3% or most preferably ±0% (respectively with reference to a given numerical value). In each of the embodiments of the present invention, "about" may be deleted. All ranges of values ​​disclosed herein shall refer to and include any and all values ​​falling within the range, including values ​​defining the range.

[0053] In a first aspect, the present invention relates to a polypeptide comprising an amino acid sequence I consisting of:

[0054] MX 13 QAIGILELX1SIAAGMELGDAMLKSAX 14 VX 15 LLVSKTISX2GKFLLMLGGDIX8AIX9X 12 AIX 10 TGTX 11 QAGX3LLVDSLVLAX 16 IHPSVLPAIX 17 GX 18 NX 19 VX 20 X7X 21 QAVGIVETX4SVAACISAADX 22 AVX 23 GSX 24 VTLVRVHMAX5GIGGKCYMVVAGDVSDVALAVTVASSSAGAYGX6LVYASLIPX 25 PHX 26 AMWX27 QXd 28 GX 29 E (SEQ ID NO: 1),

[0055] Where X1 to X 29 Any one of X1 to X6 is independently an amino acid, provided that at least 3 of X1 to X6 are independently positively charged amino acids, and wherein except for X1 to X6 in SEQ ID NO: 1 29 Optionally up to 5 amino acids at positions other than the indicated positions are exchanged with any amino acids.

[0056] In a preferred embodiment, the polypeptide comprises an amino acid sequence I consisting of SEQ ID NO: 1, wherein X1 to X 29 Any of X1 to X6 are independently amino acids, provided that at least 3 of X1 to X6 are independently positively charged amino acids.

[0057] As used herein, the term "polypeptide" refers to any peptide-bonded polymer of amino acids, regardless of size, length, secondary and tertiary structure, number of subunits or post-translational modifications. Therefore, the term "polypeptide" should be understood to encompass the terms "peptide", "protein", "amino acid chain", "amino acid sequence". The polypeptide according to the present invention can be an open linear peptide chain or a cyclic peptide; alternatively or additionally, the peptide of the present invention can contain at least one chemical modification, such as lipidation, glycosylation and phosphorylation. As understood herein, peptides (especially peptides of the present invention) are isolated or preferably can be produced by chemical synthesis, RNA translation and / or recombinant processes.

[0058] As used herein, the term "amino acid" refers to an organic compound containing the functional groups amine (-NH2) and carboxylic acid (-COOH) and its zwitterions, usually and preferably, it also contains a side chain specific for each amino acid. The term "amino acid" usually and preferably includes naturally occurring amino acids, such as proteinogenic amino acids (produced by RNA translation), non-proteinogenic amino acids (produced by other metabolic mechanisms (e.g., post-translational modifications), standard or typical amino acids (which are directly encoded by the codons of the genetic code) and non-standard or atypical amino acids (not directly encoded by the genetic code). Naturally occurring amino acids include non-eukaryotic and eukaryotic amino acids. As used herein, the term "amino acid" also includes chemically synthesized non-natural amino acids. In addition, the term encompasses amino acids such as α- (α-), β- (β-), γ- (γ-) and δ- (δ-) in any ratio and mixtures thereof, and, if applicable, any isomeric form of an amino acid in any ratio (preferably, a racemic ratio of 1:1), i.e., its D- and L-stereoisomers (alternatively, resolved by (R) and (S) nomenclature) and mixtures thereof. The amino acids in the present invention are usually and preferably in the L-configuration. The terms "D-stereoisomer", "L-stereoisomer", "D-amino acid" or "L-amino acid" refer to the chiral α carbon of an amino acid. Amino acids may contain modified and / or attached compounds and residues, such as residues used in peptide synthesis, such as Boc, Fmoc or both.

[0059] The term "X n To X m ” and “X n-m " are used interchangeably herein to refer to certain amino acid positions in SEQ ID NO:1.

[0060] Optionally, except for X in SEQ ID NO: 1 1-29 Up to 5 amino acids at positions other than the indicated positions can be exchanged by any amino acid. The terms "amino acid exchange" or "exchange by any amino acid" used interchangeably herein include or preferably refer to an exchange by a deletion of an amino acid or a single amino acid replaced by one or more amino acids (addition), more preferably by one, two or three amino acids. Most preferably, the term "amino acid exchange" refers to a deletion of a single amino acid or a single amino acid replaced by one, two or three amino acids. In a preferred embodiment, the amino acid exchange is a substitution. In another preferred embodiment, the amino acid exchange is a conservative amino acid substitution.

[0061] The term "conservative substitution" is an amino acid substitution that changes a given amino acid into a different amino acid with similar biochemical properties. Conservative substitutions include and preferably refer to isosteric substitutions and substitutions in which the amino acid maintains charged, polar, aromatic, aliphatic or hydrophobic properties. Conservative substitutions refer to substitutions that maintain the ability of the polypeptide of the present invention to self-assemble into nanoparticles (preferably, caged nanoparticles of the present invention).

[0062] As used herein, the term "positively charged" includes and preferably refers to a molecule having a positively charged group. More preferably, the positively charged molecule has a positively charged group at neutral or physiological pH.

[0063] In a preferred embodiment of the present invention, except for X in SEQ ID NO: 1 1-29 Optionally up to 4 amino acids, more preferably up to 3 amino acids, still more preferably up to 2 amino acids, most preferably 1 amino acid at positions other than the indicated positions are exchanged by any amino acid.

[0064] In preferred embodiments, the polypeptide is about 500 amino acids or less, preferably about 400 amino acids or less, more preferably 300 amino acids or less, again more preferably 250 amino acids or less, again more preferably 200 amino acids or less, again more preferably 188 to 230 amino acids in length, and most preferably 192 amino acids in length.

[0065] In another preferred embodiment, the polypeptide is an isolated polypeptide.

[0066] In another preferred embodiment, the polypeptide consists of an amino acid sequence consisting of SEQ ID NO: 1. In another preferred embodiment, the polypeptide consists of an amino acid sequence consisting of SEQ ID NO: 1, wherein except for X in SEQ ID NO: 1 1-29 Optionally up to 5 amino acids at positions other than the indicated positions are exchanged with any amino acids.

[0067] In another preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine or conservative substitutions of arginine. In another preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently selected from the group consisting of: arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine, thiolysine and histidine.

[0068] In another preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently selected from the group consisting of arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0069] In another more preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently histidine, arginine or lysine. In another more preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine or lysine. In another more preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine. In another more preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine. In another more preferred embodiment, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine. In one embodiment, each of the positively charged amino acids X1 to X6 is arginine. In one embodiment, each of the positively charged amino acids X1 to X6 is lysine.

[0070] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, the positively charged amino acids of at least 3 of X1 to X6 are independently arginine or lysine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, the positively charged amino acids of at least 3 of X1 to X6 are arginine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, the positively charged amino acids of at least 3 of X1 to X6 are lysine.

[0071] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein the positively charged amino acids of at least 3 of X1 to X6 are independently arginine or lysine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein the positively charged amino acids of at least 3 of X1 to X6 are arginine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein the positively charged amino acids of at least 3 of X1 to X6 are lysine.

[0072] X7 is cysteine, a conservative substitution thereof, or a positively charged amino acid.

[0073] In a preferred embodiment, the conservative substitution of cysteine ​​is selected from the group consisting of: methionine, homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. In a preferred embodiment, the conservative substitution of cysteine ​​is selected from the group consisting of: homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxypentanoic acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s, 3r)-2-amino-3-hydroxy-4-methylpentanoic acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. More preferably, the conservative substitution of cysteine ​​is selected from the group consisting of: homocysteine, selenocysteine ​​and serine. Again more preferably, the conservative substitution of cysteine ​​is homocysteine ​​or selenocysteine.

[0074] In another preferred embodiment, X7 is selected from the group consisting of homocysteine, selenocysteine, serine, arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0075] In another preferred embodiment, X7 is selected from the group consisting of homocysteine, selenocysteine, serine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy -1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine, arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine. In another preferred embodiment, the X7 is selected from arginine, lysine, serine, homocysteine ​​or cysteine. In another other preferred embodiment, the X7 is selected from arginine, lysine, homocysteine ​​or cysteine.

[0076] If X7 is cysteine ​​or a conservative substitution of cysteine, it can be used to append a positively charged group (eg, cysteamine, 1-(3-mercaptopropyl)guanidine, etc.) to a polypeptide of the invention via disulfide formation.

[0077] In one embodiment, the at least three of X1 to X6 are independently lysine or arginine, and X7 is selected from arginine, lysine, serine, homocysteine ​​or cysteine.

[0078] In the amino acid sequence I consisting of SEQ ID NO: 1, at least 3 of the amino acids X1 to X6 are independently positively charged amino acids. In a preferred embodiment, at least 4, more preferably at least 5, and even more preferably 6 (i.e., each) of the amino acids X1 to X6 are independently positively charged amino acids.

[0079] In a preferred embodiment, at least 4, preferably at least 5, more preferably 6 (i.e., each) of the amino acids X1 to X6 are independently positively charged amino acids, wherein the positively charged amino acids are arginine or lysine. In a preferred embodiment, at least 4, preferably at least 5, more preferably each of the amino acids X1 to X6 are independently positively charged amino acids, wherein the positively charged amino acids are arginine. In a preferred embodiment, at least 4, preferably at least 5, more preferably 6 of the amino acids X1 to X6 are independently positively charged amino acids, wherein the positively charged amino acids are lysine.

[0080] In a preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1 to X3. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2 and X4. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2 and X5. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2 and X6.

[0081] In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X2, X3 and X4. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X2, X3 and X5. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X2, X3 and X6.

[0082] In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X3, X4 and X5. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X3, X4 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X3, X4 and X1.

[0083] In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X4, X5 and X6. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X4, X5 and X1. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X4, X5 and X2.

[0084] In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X5, X6 and X1. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X5, X6 and X2. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X5, X6 and X3.

[0085] In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X3 and X5. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X3 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X2, X4 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X4 and X6.

[0086] In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2, X3 and X4. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2, X3 and X5. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X2, X3 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X2, X3, X4 and X5. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X2, X3, X4 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X3 to X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X2, X4, X5 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X4, X5 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X3, X5 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X1, X3, X4 and X6. In another preferred embodiment, the at least 3 of X1 to X6 that are independently positively charged amino acids are X2, X3, X4 and X6.

[0087] In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X 1-5 In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X 1-4 In another preferred embodiment, at least three of X1 to X6 that are independently positively charged amino acids are X 1-3 , X5 and X6. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X1, X2 and X4 to X6. In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X1 and X2. 3-6 In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X2 to X6.

[0088] In another preferred embodiment, the at least three of X1 to X6 that are independently positively charged amino acids are X1 to X6 that are independently positively charged amino acids.

[0089] More preferably, the at least three of X1 to X6 that are independently positively charged amino acids are X1 to X6, or X4 to X6, or X1, X2 and X5, or X1, X2, X4 and X5, or X1, X3, X4 and X6, or X1 and X4 to X6, or X1 to X3 and X6, or X1 to X5. More preferably, the at least three of X1 to X6 that are independently positively charged amino acids are X1 to X6, or X4 to X6, or X1, X2 and X5, or X1, X2, X4 and X5, or X1, X3, X4 and X6, or X1 and X4 to X6, or X1 to X3 and X6, or X1 to X5.

[0090] In a preferred embodiment, X4 to X6 are independently lysine or arginine. In another preferred embodiment, X1, X2 and X4 are independently lysine or arginine. In another preferred embodiment, X1, X2, X4 and X5 are independently lysine or arginine. In another preferred embodiment, X1, X3, X4 and X6 are independently lysine or arginine. In another preferred embodiment, X1 and X3 to X6 are independently lysine or arginine. In another preferred embodiment, X1 and X3 to X6 are independently lysine or arginine. 1-3 , X5 and X6 are independently lysine or arginine. In another preferred embodiment, X 1-5 In another preferred embodiment, X1 to X6 are independently lysine or arginine.

[0091] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein each of X1 to X6 is independently arginine or lysine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein each of X1 to X6 is arginine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 to 9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein each of X1 to X6 is lysine.

[0092] In another preferred embodiment, X1 to X6 are independently lysine or arginine, and X7 is selected from arginine, lysine or cysteine.

[0093] The inventors found that at positions X8 to X 12 The amino acids at positions X8 to X9 define the pore size of the nanoparticles formed by self-assembly of the polypeptides of the present invention. 12 Larger amino acids at these positions reduce the pore size, while smaller amino acids at these positions increase the pore size of the nanoparticles.

[0094] Thus, in a preferred embodiment, positions X8 to X 12 The amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the cargo can be loaded into the nanoparticles comprising the polypeptide without decomposition and released from the nanoparticles. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the cargo can be loaded into the nanoparticles comprising the polypeptide without decomposition and released from the nanoparticles in vivo. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the cargo can be loaded into the nanoparticles comprising the polypeptide in vitro without decomposition and released from the nanoparticles in vivo. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the cargo can be loaded into the nanoparticles comprising the polypeptide outside the cell without decomposition and released from the nanoparticles outside the cell, preferably to the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, more preferably RNA selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0095] In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include a hole with a size of 3-4 nm, and the cargo can be loaded into the nanoparticles comprising the polypeptide without decomposition. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include a hole with a size of 3-4 nm, and the cargo can be loaded into the nanoparticles comprising the polypeptide without decomposition and released from the nanoparticles. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include a hole with a size of 3-4 nm, and the cargo can be loaded into the nanoparticles comprising the polypeptide in vitro and released from the nanoparticles in vivo without decomposition. In another preferred embodiment, the amino acids at positions X8 to X12 in the polypeptide of the invention are selected so that the nanoparticles comprising at least one polypeptide of the invention comprise pores of 3-4 nm in size, and the cargo can be loaded into the nanoparticles comprising the polypeptide and released from the nanoparticles extracellularly without decomposition, preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0096] In a preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline, glutamine; asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising at least one polypeptide of the present invention comprises a pore with a size of 3-4 nm, wherein the cargo can be loaded into and released from the nanoparticle without decomposition.In a preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2 -aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention comprise pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cells. In another preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include pores with a size of 3-4 nm, wherein the cargo can be loaded into and released from the nanoparticles comprising the polypeptide without decomposition. In another preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptide extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell.In a further preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that a nanoparticle comprising at least one polypeptide of the present invention comprises a pore having a size of 3-4 nm, wherein cargo can be loaded into and released from the nanoparticle comprising the polypeptide without decomposition. In a further preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that a nanoparticle comprising at least one polypeptide of the present invention comprises a pore having a size of 3-4 nm, wherein cargo can be loaded into and released from the nanoparticle comprising the polypeptide without decomposition outside the cell, more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, most preferably siRNA.

[0097] In a preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-Hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising at least one polypeptide of the present invention comprises a pore with a size of 3-4 nm, wherein the cargo can be loaded into and released from the nanoparticle without decomposition.In a preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxy Asparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino -malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention comprise pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cells. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include pores with a size of 3-4 nm, wherein the cargo can be loaded into and released from the nanoparticles comprising the polypeptide without decomposition.In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptide extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising at least one polypeptide of the present invention include pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptide and released from the nanoparticles without decomposition. In a further preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the invention are selected so that the nanoparticle comprising at least one polypeptide of the invention comprises a pore of 3-4 nm in size, wherein the cargo can be loaded into the nanoparticle comprising the polypeptide and released from the nanoparticle extracellularly without decomposition, more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0098] In a preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyasparagine ... -hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that a nanoparticle comprising 6 of the polypeptides comprises 6 pores of a size of 3-4 nm, wherein cargo can be loaded into and released from the nanoparticle without decomposition.In a preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxy Asparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino- Malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 of the polypeptides comprises 6 pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticle comprising the polypeptide extracellularly without decomposition and released from the nanoparticle extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 of the polypeptides comprises 6 pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticle comprising the polypeptide and released from the nanoparticle without decomposition.In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles comprising 6 of the polypeptides include pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticles comprising the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 of the polypeptides comprises 6 pores with a size of 3-4 nm, wherein the cargo can be loaded into the nanoparticle comprising the polypeptide and released from the nanoparticle without decomposition. In a further preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the invention are selected so that a nanoparticle comprising 6 of the polypeptides comprises 6 pores of size 3-4 nm, wherein the cargo can be loaded into the nanoparticle comprising the polypeptides and released from the nanoparticles extracellularly without decomposition, more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0099] In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, wherein the nanoparticles comprising at least one polypeptide of the present invention comprise pores with a size of 3-4 nm, wherein the pore size is selected so that the cargo can be loaded into and released from the nanoparticles comprising the polypeptide without decomposition. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, wherein the nanoparticles comprising at least one polypeptide of the present invention comprise pores with a size of 3-4 nm, wherein the pore size is selected so that the cargo can be loaded into the nanoparticles comprising the polypeptide extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, wherein the nanoparticles comprising at least one polypeptide of the present invention comprise pores having a size of 3-4 nm, wherein the pore size is selected so that the cargo can be loaded into and released from the nanoparticles comprising the polypeptide without decomposition. In a further preferred embodiment, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, wherein the nanoparticle comprising at least one polypeptide of the present invention comprises a pore of 3-4 nm in size, wherein the pore size is selected so that the cargo can be loaded into the nanoparticle comprising the polypeptide and released from the nanoparticle extracellularly without decomposition, more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0100] In a preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline, glutamine; asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartate serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine; and a nanoparticle comprising 6 of the polypeptides of the present invention comprises 6 pores with a pore size of 3-4 nm, and cargo can be loaded into and released from the nanoparticles without decomposition.In a preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-propanediol Acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the nanoparticles comprising 6 of the polypeptides of the present invention comprise 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into the nanoparticles extracellularly without decomposition and released from the nanoparticles extracellularly, preferably into the cytoplasm of the cells. In another preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the nanoparticles comprising 6 of the polypeptides of the present invention include 6 pores with a pore size of 3-4 nm, and cargo can be loaded into and released from the nanoparticles without decomposition. In another preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the nanoparticles comprising 6 of the polypeptides of the present invention include 6 pores with a pore size of 3-4 nm, wherein cargo can be loaded into and released from the nanoparticles extracellularly without decomposition, preferably into the cytoplasm of the cell.In another preferred embodiment, each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, the nanoparticle comprising 6 of the polypeptides of the invention comprises 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into the nanoparticles and released from the nanoparticles extracellularly without decomposition, preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0101] In another preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homocysteine, thiazolyl acetate ... Leucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5 -o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 of the polypeptides of the present invention comprises 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into and released from the nanoparticle comprising the polypeptide without decomposition.In another preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, thiazolyl glycolate ... Leucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-Hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine The amino acids are selected from positions X8 to X12 in the polypeptide of the present invention, and the amino acids are selected so that the nanoparticle comprising 6 of the polypeptides of the present invention comprises 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into the nanoparticle comprising the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 of the polypeptides of the present invention comprises 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into and released from the nanoparticle comprising the polypeptide without decomposition.In another preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles including 6 of the polypeptides of the present invention include 6 pores with a size of 3-4 nm, and the cargo can be loaded into the nanoparticles including the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. In another preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticle comprising 6 polypeptides of the polypeptide of the present invention includes 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into the nanoparticle comprising the polypeptide and released from the nanoparticle without decomposition. In another preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus of the polypeptide, X1 to X6 are independently lysine or arginine; X7 is selected from arginine, lysine or cysteine; each of X8, X9, X10, X11 and X12 is independently selected from the group consisting of: serine, glycine, glutamine and glutamate, and the amino acids at positions X8 to X12 in the polypeptide of the present invention are selected so that the nanoparticles including 6 polypeptides of the polypeptide of the present invention include 6 pores with a pore size of 3-4 nm, and the cargo can be loaded into the nanoparticles including the polypeptides extracellularly without decomposition and released from the nanoparticles extracellularly, more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, most preferably siRNA.More preferably, the three or more consecutive histidines are 3-9 his tags.

[0102] In a preferred embodiment, the X8 is glycine or its conservative substitution. Preferably, the conservative substitution of glycine is selected from the group consisting of: alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, allo-isoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline. More preferably, the conservative substitution of glycine is selected from the group consisting of: alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, allo-isoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline. In another preferred embodiment, the X8 is selected from the group consisting of: glycine, alanine, leucine and valine. In a preferred embodiment, the X8 is glycine.

[0103] In another preferred embodiment, the X9 and X 12 are independently glutamine or a conservative substitution thereof. Preferably, the conservative substitution of glutamine is selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s, 4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine. In another preferred embodiment, X9 and X 12 Independently of each other is glutamine or asparagine.

[0104] In another preferred embodiment, the X9 and X 12 Both are glutamine. In another preferred embodiment, X9 and X 12 In another preferred embodiment, X9 or X 12 In another preferred embodiment, X9 is glutamine. In another preferred embodiment, X9 is glutamine. 12 It's glutamine.

[0105] In another preferred embodiment, the X 10 is glutamate or a conservative substitution thereof. Preferably, the conservative substitution of glutamate is selected from the group consisting of glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid. In another preferred embodiment, the X 10 In another preferred embodiment, X 10 It's glutamate.

[0106] In another preferred embodiment, the X 11 Selected from the group consisting of: serine or its conservative substitution. Preferably, the conservative substitution of serine is selected from the group consisting of: cysteine, methionine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s, 3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. More preferably, the conservative substitution of serine is selected from the group consisting of: cysteine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. Again more preferably, the conservative substitution of serine is selected from the group consisting of hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. Again more preferably, the conservative substitution of serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine.

[0107] In another preferred embodiment, the X 11 is selected from the group consisting of: serine, homoserine and threonine. In another preferred embodiment, the X 11 It's serine.

[0108] In a preferred embodiment, X8 is selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline; X9 and X 12 are independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine; X 10 is selected from the group consisting of glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methyl-aspartic acid; and X 11 Selected from the group consisting of: serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine.

[0109] In another preferred embodiment, the X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine.

[0110] In another preferred embodiment, X8 is glycine, X9 and X 12 It is glutamine, X 10 is glutamate, and X 11 It's serine.

[0111] In a preferred embodiment, X8, X9, X 10 , X 11 and X 12Each of the amino acids is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine.

[0112] In another preferred embodiment, X8, X9, X 10 , X 11 and X 12 Each of the amino acids is independently selected from the group consisting of glycine, alanine, leucine, valine; glutamate, aspartate; glutamine, asparagine; serine, threonine and homoserine.

[0113] In another preferred embodiment, X8, X9, X 10 , X 11 and X 12 Each of the amino acids is independently selected from the group consisting of serine, glycine, glutamine, and glutamate.

[0114] In another preferred embodiment, X 1-6 At least three of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 is selected from the group consisting of: serine, threonine and homoserine. In another preferred embodiment, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine.

[0115] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, and X8, X9, X 10 , X 11 and X 12 Each of the amino acids is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate Acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine. In a preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of 3-9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, and X8, X9, X 10 , X 11 and X 12Each of the amino acids is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine, norvaline; glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid, n-methyl-asparagine; glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate acid, 4-hydroxy-glutamic acid, 6-carboxylysine, β-hydroxyaspartic acid, 2-amino-malonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid, 3-methyl-aspartic acid; serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allo-threonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allo-threonine.

[0116] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 At least three of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 is selected from the group consisting of serine, threonine and homoserine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3-9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 At least three of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine.

[0117] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 is selected from the group consisting of serine, threonine and homoserine. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3-9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 is selected from the group consisting of: serine, threonine and homoserine. In another preferred embodiment, X 1-6 At least three of X8 and X9 are independently lysine or arginine, X8 is glycine, and X9 and X 12 Independently of each other are glutamine, X 10 is glutamate, and X 11 In another preferred embodiment, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, and X8 is glycine, X9 and X 12 Independently of each other are glutamine, X 10 is glutamate, and X 11 In another preferred embodiment, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, and X8 is glycine, X9 and X 12 Independently of each other are glutamine, X 10 is glutamate, and X 11 It's serine.

[0118] In one embodiment, the X 1-6At least three of X7 are independently lysine or arginine, X8 is glycine, X9 and X10 are selected from arginine, lysine or cysteine, 12 Independently of each other are glutamine, X 10 is glutamate, and X 11 The polypeptide comprises a polyhistidine, which is an amino acid sequence comprising two or more continuously connected histidines, preferably, the polyhistidine is a histidine (His) tag, more preferably a His6 tag. A histidine tag is defined herein as an amino acid sequence (His tag) consisting of two or more continuously connected histidines. A His6 tag (His6) is defined herein as an amino acid sequence consisting of 6 continuously connected histidines.

[0119] In another preferred embodiment, X1 to X6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X 12 Independently of each other are glutamine, X 10 is glutamate, and X 11 is serine, and the polypeptide includes polyhistidine, which is an amino acid sequence including two or more consecutively linked histidines. Preferably, the polyhistidine is a histidine (His) tag, more preferably a His6 tag.

[0120] In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein in the sequence I, X 1-6 is lysine or arginine, X7 is selected from arginine, lysine or cysteine, and X8 is glycine, X9 and X 12 Independently of each other are glutamine, X 10 is glutamate, and X 11is serine. In a preferred embodiment, the nanoparticle or the complex comprising the polypeptide of the present invention is capable of endosomal escape. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the nanoparticle or the complex comprising the polypeptide of the present invention is capable of endosomal escape. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or the N-terminus, and the nanoparticle or the complex comprising the polypeptide of the present invention is capable of endosomal escape. In a preferred embodiment, the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I; in sequence I, X1-6 are lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is glycine, X9 and X12 are independently glutamine, X10 is glutamate, and X11 is serine, and the nanoparticle or the complex of the present invention comprising the polypeptide of the present invention is capable of endosomal escape.

[0121] When the polypeptide of the present invention forms nanoparticles by self-assembly, amino acid X 13 To X 29 are exposed on the outer surface of the nanoparticle and are therefore most susceptible to mutations while still retaining the ability to self-assemble into cage-like nanoparticles.

[0122] Therefore, in a preferred embodiment, the amino acid X exposed on the outer surface of the nanoparticle 13 To X 29 is any amino acid.

[0123] In a preferred embodiment, X 13 , X 17 and X 19Each of the amino acids is independently serine or a conservative substitution thereof. Preferably, the conservative substitution of serine is selected from the group consisting of cysteine, methionine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s, 3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. More preferably, the conservative substitution of serine is selected from the group consisting of: cysteine, homocysteine, selenocysteine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. Again more preferably, the conservative substitution of serine is selected from the group consisting of hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxy-alanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine, homoserine, 3-hydroxy-1-valine, 4,5-dihydroxy-isoleucine, 6-hydroxy-1-norleucine, s-(2-hydroxyethyl)-1-cysteine, phosphoserine, 4-hydroxy-1-threonine, threonine and phosphothreonine. Again more preferably, the conservative substitution of serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine.

[0124] In another preferred embodiment, X 13 , X 17 and X 19 is independently selected from the group consisting of: serine, homoserine and threonine. In another preferred embodiment, X 13 , X 17 and X 19 Independently of each other are serine.

[0125] In another preferred embodiment, X 14 , X 16 and X 24 Each is independently asparagine or a conservative substitution thereof. Preferably, the conservative substitution of asparagine is selected from the group consisting of glutamine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s, 4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine.

[0126] In another preferred embodiment, X 14 , X 16 and X 24 In another preferred embodiment, X 14 , X 16 and X 24 Independently of each other is asparagine or glutamine.

[0127] In another preferred embodiment, X 15 , X 20 , X 26 and X 28 Each of the amino acids is independently aspartate, glutamate or a conservative substitution thereof. Preferably, the conservative substitution of aspartate or glutamate is selected from the group consisting of: 2s, 4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid.

[0128] In another preferred embodiment, X 15 and X 20 In another preferred embodiment, X 26 and X 28 are independently glutamate. In another preferred embodiment, X 15 , X 20 , X 26 and X 28 In another preferred embodiment, X 15 , X 20 , X 26 and X 28Independently selected from the group consisting of: glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid.

[0129] In another preferred embodiment, X 18 and X 29 Each of the amino acids is independently leucine or a conservative substitution thereof. Preferably, the conservative substitution of leucine is selected from the group consisting of glycine, alanine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline.

[0130] In another preferred embodiment, X 18 and X 29 are independently leucine. In a preferred embodiment, the X 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine and valine. 18 and X 29 Each is independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline.

[0131] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 Independently of each other are positively charged amino acids.

[0132] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27Each of the arginine, lysine or its conservative substitution is independently selected from the group consisting of arginine or leucine. Preferably, the conservative substitution of arginine or leucine is selected from the group consisting of: histidine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine. More preferably, the conservative substitution of arginine or leucine is selected from the group consisting of: 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0133] In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 are independently histidine, arginine or lysine. In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27 Independently of each other are 6 lysines.

[0134] In another preferred embodiment, X 21 , X 22 , X 25 and X 27 In another preferred embodiment, X 23 In another preferred embodiment, X 21 , X 22 , X 23 , X 25 and X 27Each of the amino acids is independently selected from the group consisting of arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0135] In another preferred embodiment, X 13 , X 17 and X 19 are independently serine; X 14 , X 16 and X 24 Independently of each other are asparagine; X 15 and X 20 are independently aspartic acid ester; X 18 and X 29 are independently leucine; X 26 and X 28 are independently glutamate; X 21 , X 22 , X 25 and X 27 are independently arginine; and X 23 It's lysine.

[0136] In another preferred embodiment, X 13 , X 17 and X 19 are independently selected from the group consisting of: serine, homoserine and threonine; X 14 , X 16 and X 24 Independently of each other is aspartate or glutamate; X 15 , X 20 , X 26 and X 28 are independently asparagine or glutamine; said X 18 and X 29 are independently selected from the group consisting of: glycine, alanine, leucine and valine; X 21 , X 22 , X 23 , X 25 and X 27 independently of each other is arginine or lysine.

[0137] In another preferred embodiment, X 13 , X 17 , and X 19are independently selected from the group consisting of: serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine;

[0138] X 14 , X 16 and X 24 independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine;

[0139] X 15 , X 20 , X 26 and X 28 independently selected from the group consisting of glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid;

[0140] X 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline; and

[0141] X 21 , X 22 , X 23 , X 25 and X 27 Each of the amino acids is independently selected from the group consisting of arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0142] In another preferred embodiment, X8 is glycine or a conservative substitution thereof, wherein preferably, the conservative substitution of glycine is selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline;

[0143] X9 and X 12 are independently glutamine or a conservative substitution thereof, wherein preferably, the conservative substitution of glutamine is selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine;

[0144] X 10 is glutamate or a conservative substitution thereof, wherein preferably, the conservative substitution of glutamate is selected from the group consisting of aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid; and

[0145] X 11 is serine or a conservative substitution thereof, wherein preferably, the conservative substitution of serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine;

[0146] More preferably, X8 is glycine, X9 and X 12 independently of each other is glutamine or asparagine, X 10 is glutamate or aspartate, and X 11 It's serine.

[0147] In another preferred embodiment, X 13 , X 17 and X 19are independently serine or a conservative substitution thereof, wherein preferably, the conservative substitution of serine is selected from the group consisting of homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine;

[0148] X 14 , X 16 and X 24 Each is independently asparagine, glutamine or a conservative substitution thereof, wherein preferably, the conservative substitution of asparagine or glutamine is selected from the group consisting of: β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine;

[0149] X 15 , X 20 , X 26 and X 28 are independently aspartate, glutamate or conservative substitutions thereof, wherein preferably, the conservative substitution of aspartate or glutamate is selected from the group consisting of: 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid; and

[0150] X 18 and X 29 are independently leucine or a conservative substitution thereof, wherein preferably, the conservative substitution of leucine is selected from the group consisting of glycine, alanine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline; and

[0151] X 21 , X 22 , X 23 , X 25 and X 27Each is independently arginine, lysine or a conservative substitution thereof, wherein preferably, the conservative substitution of arginine or leucine is selected from the group consisting of: histidine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, diaminobutyric acid, 2,3-diaminopropionic acid, (2S)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0152] In another preferred embodiment, the positively charged amino acids of the at least three of X1 to X6 are independently selected from the group consisting of arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine;

[0153] X7 is cysteine, a conservative substitution thereof or a positively charged amino acid; preferably, X7 is selected from the group consisting of homocysteine, cysteine, selenocysteine, arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine, thiolysine and serine;

[0154] X8 is glycine or a conservative substitution thereof selected from the group consisting of alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline;

[0155] X9 and X 12 Each is independently glutamine or a conservative substitution thereof selected from the group consisting of asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine;

[0156] X 10 is glutamate or a conservative substitution thereof selected from the group consisting of aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid; and

[0157] X 11is serine or a conservative substitution thereof, wherein the conservative substitution is selected from the group consisting of homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine;

[0158] X 13 , X 17 , and X 19 are independently selected from the group consisting of: serine, homoserine, threonine, 4-hydroxy-1-threonine, 6-hydroxy-1-norleucine, 4,5-dihydroxy-isoleucine, 3-hydroxy-1-valine, hydroxynorvaline, 2-amino-5-hydroxyvaleric acid, allothreonine, 3,3-dihydroxyalanine, 4-hydroxy-L-isoleucine, (2s,3r)-2-amino-3-hydroxy-4-methylvaleric acid, β-hydroxyleucine and allothreonine;

[0159] X 14 , X 16 and X 24 independently selected from the group consisting of glutamine, asparagine, β-hydroxyasparagine, 3-methyl-1-glutamine, (2s,4s)-2,5-diamino-4-hydroxy-5-oxopentanoic acid and n-methyl-asparagine;

[0160] X 15 , X 20 , X 26 and X 28 independently selected from the group consisting of glutamate, aspartate, 2s,4r-4-methylglutamate, (3s)-3-methyl-1-glutamate, (3r)-3-methyl-1-glutamate, 5-o-methyl-glutamate, 4-hydroxy-glutamate, 6-carboxylysine, β-hydroxyaspartic acid, 2-aminomalonic acid, 3,3-dimethylaspartic acid, 2-aminoadipic acid and 3-methylaspartic acid;

[0161] X 18 and X 29 are independently selected from the group consisting of glycine, alanine, leucine, valine, tert-leucine, homoleucine, isoleucine, alloisoleucine, 2-aminobutyric acid, diethylalanine, norleucine and norvaline; and

[0162] X 21 , X 22 , X 23 , X 25and X 27 Each of the amino acids is independently selected from the group consisting of arginine, 5-methyl-arginine, c-γ-hydroxyarginine, 2-amino-4-guanidinobutyric acid, 2-amino-3-guanidinopropionic acid, canavanine, homoarginine, lysine, diaminobutyric acid, 2,3-diaminopropionic acid, (2s)-2,8-diaminooctanoic acid, ornithine and thiolysine.

[0163] In another preferred embodiment, the at least three positively charged amino acids in X1 to X6 are independently arginine or lysine, X7 is selected from the group consisting of homocysteine, cysteine, arginine and lysine, X8 is selected from the group consisting of glycine, alanine, leucine and valine, X9 and X10 are selected from the group consisting of arginine, lysine and arginine, and X11 is selected from the group consisting of arginine, lysine and arginine. 12 independently of each other is glutamine or asparagine, X 10 Is glutamate or aspartate, X 11 , X 13 , X 17 and X 19 are independently selected from the group consisting of: serine, threonine and homoserine, X 14 , X 16 and X 24 are, independently of each other, asparagine or glutamine, X 15 , X 20 , X 26 and X 28 Independently of each other, is aspartate or glutamate, X 18 and X 29 are independently selected from the group consisting of leucine, glycine, alanine and valine, and X 21 , X 22 , X 23 , X 25 and X 27 independently of each other is arginine or lysine.

[0164] In another preferred embodiment, the at least three positively charged amino acids in X1 to X6 are independently arginine or lysine, X7 is selected from the group consisting of homocysteine, cysteine, arginine and lysine, X8 is glycine, X9 and X10 are each independently arginine or lysine, X11 is selected from the group consisting of homocysteine, cysteine, arginine and lysine, X12 is glycine, X13 is glycine, X14 is glycine, X15 is glycine, X16 is glycine, X17 is glycine, X18 is glycine, X19 is glycine, X20 is glycine, X21 is glycine, X22 is glycine, X23 is glycine, X24 is glycine, X25 is glycine, X26 is glycine, X27 is glycine, X28 is glycine, X29 is glycine, X30 is glycine, X31 is glycine, X32 is glycine, X33 is glycine, X34 is glycine, X35 is glycine, X36 is glycine, X37 is glycine, X38 is glycine, X39 is glycine, X40 is glycine, X41 is glycine, X42 is glycine, X43 is glycine, X44 is glycine, X45 is glycine, X46 is glycine, X47 is glycine, X48 is glycine, X49 is glycine, X50 is glycine, X51 is glycine, X52 is glycine, X53 is glycine, X54 is glycine, X55 is gly 12 independently of each other is glutamine or asparagine, X 10 Is glutamate or aspartate, X 11 is serine, X 13 , X 17 and X 19 Independently of each other, serine, X 14 , X 16 and X 24 Independently of each other are asparagine, X15 , X 20 , X 26 and X 28 Independently of each other, is aspartate or glutamate, X 18 and X 29 are independently leucine, and X 21 , X 22 , X 23 , X 25 and X 27 independently of each other is arginine or lysine.

[0165] In another preferred embodiment, X 1-6 At least three of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine; X 18 and X 29 are independently leucine; X 15 and X 20 are independently aspartic acid ester; X 26 and X 28 are independently glutamate; X 14 , X 16 and X 24 are independently asparagine; X 13 , X 17 and X 19 are independently serine; X 21 , X 22 , X 25 and X 27 are independently arginine; and X 23 It's lysine.

[0166] In another preferred embodiment, X 1-6 At least three of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine; X 13 , X 17 and X 19are independently selected from the group consisting of: serine, homoserine and threonine; X 14 , X 16 and X 24 independently of each other is asparagine or glutamine; X 15 , X 20 , X 26 and X 28 are independently aspartate or glutamate; said X 18 and X 29 are independently selected from the group consisting of: glycine, alanine, leucine and valine; X 21 , X 22 , X 23 , X 25 and X 27 independently of each other is arginine or lysine.

[0167] In another preferred embodiment, X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X 12 independently of each other is glutamine or asparagine; X 10 Is glutamate or aspartate; X 11 Selected from the group consisting of: serine, threonine and homoserine; X 13 , X 17 and X 19 are independently selected from the group consisting of: serine, homoserine and threonine; X 14 , X 16 and X 24 independently of each other is asparagine or glutamine; X 15 , X 20 , X 26 and X 28 are independently aspartate or glutamate; said X 18 and X 29 are independently selected from the group consisting of: glycine, alanine, leucine and valine; X 21 , X 22 , X 23 , X 25 and X 27 independently of each other is arginine or lysine.

[0168] In another preferred embodiment, X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine; X 13 , X 17 and X 19 are independently serine; X 14 , X 16 and X 24 are independently asparagine; X 15 and X 20 are independently aspartic acid ester; X 18 and X 29 are independently leucine; X 26 and X 28 are independently glutamate; X 21 , X 22 , X 25 and X 27 are independently arginine; and X 23 It's lysine.

[0169] In another preferred embodiment, X 1-6 At least three or at least four of X7 are independently lysine or arginine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X10 are selected from the group consisting of arginine, lysine and cysteine; 12 independently of each other is glutamine or asparagine; X 10 is glutamate or aspartate; and X 11 Selected from the group consisting of: serine, threonine and homoserine; X 13 , X 17 and X 19 are independently selected from the group consisting of: serine, homoserine and threonine; X 14 , X 16 and X 24 independently of each other is asparagine or glutamine; X 15 , X 20 , X 26 and X 28 are independently aspartate or glutamate; said X 18 and X 29 are independently selected from the group consisting of: glycine, alanine, leucine and valine; X 21 , X 22 , X 23 , X 25 and X 27are independently arginine or lysine, and the polypeptide comprises polyhistidine, which is an amino acid sequence comprising two or more consecutively linked histidines. Preferably, the polyhistidine is a histidine (His) tag, more preferably a His6 tag.

[0170] In another preferred embodiment, X 1-6 are independently lysine or arginine, X7 is selected from arginine, lysine or cysteine, X8 is selected from the group consisting of glycine, alanine, leucine and valine; X9 and X 12 independently of each other is glutamine or asparagine; X 10 Is glutamate or aspartate; X 11 Selected from the group consisting of: serine, threonine and homoserine; X 13 , X 17 and X 19 are independently selected from the group consisting of: serine, homoserine and threonine; X 14 , X 16 and X 24 independently of each other is asparagine or glutamine; X 15 , X 20 , X 26 and X 28 are independently aspartate or glutamate; said X 18 and X 29 are independently selected from the group consisting of: glycine, alanine, leucine and valine; X 21 , X 22 , X 23 , X 25 and X 27 are independently arginine or lysine, and the polypeptide comprises polyhistidine, which is an amino acid sequence comprising two or more consecutively linked histidines. Preferably, the polyhistidine is a histidine (His) tag, more preferably a His6 tag.

[0171] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16.

[0172] In a preferred embodiment, the amino acid sequence I is a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16, 37 and 38. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16, 37 and 38.

[0173] In a preferred embodiment, the amino acid sequence I is SEQ ID NO:2. In a preferred embodiment, the amino acid sequence I is SEQ ID NO:3. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:4. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:5. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:10. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:11. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:12. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:13. In a preferred embodiment, the amino acid sequence I is SEQ ID NO:14. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:15. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:16. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:37. In another preferred embodiment, the amino acid sequence I is SEQ ID NO:38.

[0174] In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:2. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:3. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:4. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:5. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:10. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:11. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:12. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:13. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:14. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO:15. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 16. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 37. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 38. In a preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3, wherein the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence 1. In another preferred embodiment, the polypeptide of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3, wherein the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3-9 consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence 1. In a preferred embodiment, the polypeptide of the present invention comprises an additional tag (i.e., a peptide or non-peptide tag), preferably a peptide tag (i.e., a functional amino acid sequence). In a preferred embodiment, the tag is located at the C-terminus or N-terminus of the polypeptide of the present invention.

[0175] In a preferred embodiment, the tag is a non-peptide tag, preferably polyethylene glycol (PEG). Preferably, the PEG is coupled to the amino acid sequence 1 via the amino acid serine or cysteine. PEG coupled to the polypeptide of the invention increases stability and immunogenicity.

[0176] In another preferred embodiment, the tag is selected from the group consisting of: polyhistidine, His tag (ie, an amino acid sequence consisting of two or more consecutively linked histidines), degradation tag, targeting tag, cell permeation tag and endosomal escape tag.

[0177] In a preferred embodiment, the polypeptide of the present invention comprises polyhistidine. In a preferred embodiment, the polyhistidine is an amino acid sequence comprising two or more histidines. In a preferred embodiment, the polyhistidine is an amino acid sequence comprising two or more histidines connected in succession. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising 3 or more histidines connected in succession. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising 3 to 9 histidines connected in succession. In another preferred embodiment, the polyhistidine is an amino acid sequence comprising 3, 6 or 9 histidines connected in succession.

[0178] In a preferred embodiment, the polypeptide of the present invention includes a histidine tag (His tag) consisting of continuously linked histidines (His tag). Preferably, the His tag consists of 3 or more continuous histidines. Preferably, the His tag consists of 3 to 9 continuous histidines. Preferably, the His tag consists of 3, 6 or 9 continuous histidines. Preferably, the His tag consists of 3 continuous histidines. Preferably, the His tag consists of 9 continuous histidines. Preferably, the His tag consists of 6 continuous histidines (i.e., His6 tags). In a preferred embodiment, the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I. In a preferred embodiment, the His6 tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I. Preferably, the His tag consists of 3 or more continuous histidines and is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I. Preferably, the His tag consists of 3 to 9 consecutive histidines, and the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence 1. Preferably, the His tag consists of 3 to 9 consecutive histidines, and the His tag is attached to the C-terminus of the amino acid sequence 1. Preferably, the His tag consists of 3, 6 or 9 consecutive histidines, and is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence 1. Preferably, the His tag consists of 3 consecutive histidines (i.e., His3 tags), and the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence 1. Preferably, the His tag consists of 9 consecutive histidines (i.e., His9 tags), and the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence 1.

[0179] In a preferred embodiment, the His tag (preferably the His6 tag) comprises a halogenated (preferably fluorinated) histidine. In a preferred embodiment, the His tag comprises a fluorophore. In a preferred embodiment, the polypeptide of the present invention comprises a fluorophore.

[0180] In a preferred embodiment, the polypeptide of the present invention comprises a degradation tag. Preferably, the degradation tag functions in mammalian cells. In a preferred embodiment, the degradation tag is the C-terminal sequence of ornithine decarboxylase (cODC) (preferably, SEQ ID NO: 17) or a (poly)ubiquitin comprising or consisting of at least two consecutively linked ubiquitins. In another preferred embodiment, the degradation tag consists of the cODC of SEQ ID NO: 17 (EFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV). In another preferred embodiment, the degradation tag comprises at least two consecutively linked ubiquitins. More preferably, the degradation tag consists of at least two consecutively linked ubiquitins.

[0181] In a preferred embodiment, the polypeptide of the present invention includes a targeting tag, which is a peptide targeting tag or a non-peptide targeting tag. In Zhao et al. (Tumor-targeting Peptides: Ligands for Molecular Imaging and Therapy, Anticancer Agents Med Chem. 2018, 18 (1): 74-86), a preferred peptide targeting tag is mentioned. Preferably, the targeting tag is bound to a cancer target and is therefore referred to herein as a cancer targeting tag. The cancer target includes a receptor whose expression level is increased in certain tumor cells and tumor antigens. In a preferred embodiment, the cancer targeting tag is a peptide ligand, a peptidomimetic or an antibody bound to a cancer target. Preferably, the cancer targeting tag is folic acid. For example, folic acid can be conjugated to the polypeptide of the present invention through a peptide bond.

[0182] In a further embodiment, the polypeptide of the present invention includes an endosome escape peptide or a cell penetrating peptide (CPP). An endosome escape peptide is, for example, a TAT or thiol group connected by a dimerized disulfide bond. As used herein, the term "cell penetrating peptide" or CPP refers to a group of peptides having the ability to penetrate the plasma membrane to deliver cargo into a cell. Preferably, the CPP used in the polypeptide of the present invention is a hydrophilic peptide or a cationic peptide. In another embodiment, the CPP is selected from amphipathic peptides, anionic peptides or hydrophobic peptides. A database of more than 1,600 CPPs is described by Agrawal et al. (Agrawal P, Bhalla S, Usmani SS, Singh S, CHaudhary K, Raghava GPS et al. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucleic Acids Research 2016, 44: D1098-D103).

[0183] In a preferred embodiment, the additional tag comprised in the polypeptide of the invention is attached via a releasable bond (such as a photodegradable bond) or via a reversible coupling.

[0184] The polypeptide of the present invention can self-assemble into nanoparticles, preferably cage-shaped nanoparticles. Further, the mutant protein of SEQ ID NO: 1 can also self-assemble into nanoparticles, preferably cage-shaped nanoparticles.

[0185] In a further aspect, the invention relates to a nucleic acid encoding a polypeptide according to the invention.

[0186] In a further aspect, the present invention relates to nanoparticles comprising at least one polypeptide according to the present invention.

[0187] In a preferred embodiment, each of the at least one polypeptide of the nanoparticle of the present invention comprises a histidine tag consisting of at least 3 consecutively linked histidines, wherein each of the histidine tags is attached to the C-terminus or N-terminus, preferably the C-terminus, of the at least one polypeptide.

[0188] In a preferred embodiment, each of the at least one polypeptide of the nanoparticle of the present invention comprises a histidine tag consisting of at least 3 consecutively linked histidines, wherein each of the histidine tags is attached to the C-terminus or N-terminus, preferably the C-terminus, of the at least one polypeptide, wherein the nanoparticle can load and unload cargo without decomposing the nanoparticle. In a preferred embodiment, each of the at least one polypeptide of the nanoparticle of the present invention comprises a histidine tag consisting of at least 3 consecutively linked histidines, wherein each of the histidine tags is attached to the C-terminus or N-terminus, preferably the C-terminus, of the at least one polypeptide, wherein the nanoparticle can load and unload cargo in vivo and in vitro without decomposing the nanoparticle. In a preferred embodiment, each of the at least one polypeptide of the nanoparticle of the present invention comprises a histidine tag consisting of at least 3 consecutively linked histidines, wherein each of the histidine tags is attached to the C-terminus or N-terminus, preferably the C-terminus, of the at least one polypeptide, wherein the nanoparticle can load cargo in vitro and unload cargo in vivo without decomposing the nanoparticle. In a preferred embodiment, each of the at least one polypeptide of the nanoparticle of the present invention comprises a histidine tag consisting of at least 3 consecutively linked histidines, wherein each of the histidine tags is attached to the C-terminus or N-terminus, preferably the C-terminus, of the at least one polypeptide, wherein the nanoparticle is capable of endosomal escape and can load and unload cargo without disassembling the nanoparticle. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0189] In a preferred embodiment, the polypeptide of the present invention included in the nanoparticle of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3, wherein the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein the nanoparticle can load and unload cargo without decomposing the nanoparticle. In a preferred embodiment, the polypeptide of the present invention included in the nanoparticle of the present invention consists of a sequence selected from the group consisting of SEQ ID NO: 3, wherein the polypeptide of the present invention comprises a histidine tag (His tag) consisting of 3 or more consecutive histidines, wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, and wherein the nanoparticle can load cargo in vitro and unload cargo in vivo without decomposing the nanoparticle. In a preferred embodiment, the nanoparticle of the present invention is contained in a composition, preferably in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0190] As used herein, the term "nanoparticle" refers to particles in the nanometer size range (ie, about 1 nm to about 1000 nanometers) that are capable of encapsulating molecules. The nanoparticle has a surface of a regular array of linked molecules that self-assemble to form the nanoparticle.

[0191] The nanoparticle of the present invention has a positively charged cavity. Therefore, it can encapsulate goods, such as oligonucleotides (ON). Further, the nanoparticle can enter cells and conditionally release goods in cells, preferably in the cytoplasm. Therefore, nanoparticles can be used, for example, to deliver ON in cells so as to control gene expression by antisense ON, for vaccination or immune system stimulation.

[0192] In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo, preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, most preferably siRNA, and are capable of delivering the encapsulated cargo to a cell. Preferably, the cargo is delivered intracellularly. In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo, preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably RNA selected from the group consisting of: antisense oligonucleotides (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, most preferably siRNA, and are capable of releasing the encapsulated cargo intracellularly. In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo outside the cell, are capable of entering the cell together with the encapsulated cargo, and are capable of releasing the cargo intracellularly. In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo outside the cell, are capable of entering the cell together with the encapsulated cargo, and are capable of releasing the cargo into the cytoplasm of the cell. In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo in vitro, and are capable of releasing the cargo in vivo (preferably, in the cell), more preferably into the cytoplasm. In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating cargo in vitro, are capable of entering the cell together with the encapsulated cargo, and are capable of releasing the cargo in vivo (preferably, in the cell), more preferably into the cytoplasm of the cell. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs, most preferably siRNAs.

[0193] In a preferred embodiment, the nanoparticles of the present invention can load and unload cargo without decomposing the nanoparticles. In a preferred embodiment, the nanoparticles of the present invention can load cargo outside the cell and unload it inside the cell without decomposing the nanoparticles. In a preferred embodiment, the nanoparticles of the present invention allow loading and unloading of cargo without decomposing the nanoparticles. In a preferred embodiment, the nanoparticles of the present invention allow loading of cargo outside the cell and unloading of cargo inside the cell without decomposing the nanoparticles. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, again more preferably an RNA selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, most preferably siRNA.

[0194] In a preferred embodiment, the nanoparticles of the present invention can be loaded with cargo in vivo and in vitro and unloaded with cargo in vivo and in vitro without decomposing the nanoparticles. In a preferred embodiment, the nanoparticles of the present invention can be loaded in vitro and unloaded in vivo without decomposing the nanoparticles.

[0195] In a further embodiment, the nanoparticle comprises at least one subunit, wherein the subunit comprises at least one polypeptide according to the present invention. As used herein, the term "subunit" refers to a building block that assembles and forms a nanoparticle of the present invention. A subunit comprises at least one polypeptide of the present invention. Preferably, the subunit comprises three polypeptides of the present invention, preferably exactly three peptides of the present invention. More preferably, the subunit consists of three polypeptides of the present invention. In another preferred embodiment, the subunit comprises three identical peptides of the present invention, preferably exactly three identical peptides of the present invention. More preferably, the subunit consists of three identical polypeptides of the present invention. In another more preferred embodiment, the subunit comprises or more preferably consists of three polypeptides of the present invention, and the subunit has C3 symmetry.

[0196] In a preferred embodiment, the nanoparticle comprises exactly 24 polypeptides of the present invention. In a preferred embodiment, the nanoparticle comprises exactly 8 subunits. In another preferred embodiment, the nanoparticle has an octahedral geometry (octahedral point group symmetry). In a preferred embodiment of the present invention, the nanoparticle has a quaternary structure of multiple subunits (preferably, 8 subunits each comprising 3 polypeptides of the present invention).

[0197] In a preferred embodiment, the nanoparticles of the present invention include a central cavity (also referred to herein as a cavity, an inner cavity, or an inner cavity). The nanoparticles including a central cavity are also referred to herein as "caged nanoparticles" or "cages". In a preferred embodiment, the caged nanoparticles include (i) an outer support and (ii) a central cavity. Preferably, the outer support surrounds the central cavity, i.e., is assembled around the central cavity. The cavity may be empty or it may contain cargo.

[0198] The nanoparticles of the present invention have a positively charged interior. The caged nanoparticles of the present invention are positively charged on the cavity surface. The positive source is from a plurality of positively charged amino acids, such as arginine or lysine. Therefore, the nanoparticles of the present invention have a very strong affinity to encapsulate negatively charged macromolecules.

[0199] In a preferred embodiment, the diameter of the cavity is about 6.5nm to about 8nm. In a preferred embodiment, the caged nanoparticle has a porous structure, that is, the nanoparticle comprises holes. Preferably, the outer support of the caged nanoparticle comprises holes connected to the cavity of the nanoparticle. Holes are defined as openings or gaps in the nanoparticle and in the outer support of the nanoparticle, respectively, in this article.

[0200] The loading (or encapsulation) and unloading (or release) of cargo in nanoparticles are carried out through the pores. The term loading or encapsulation refers to any absorption of cargo into the nanoparticle (including the cavity, scaffold and porous structure of the nanoparticle). The term unloading (or release) refers to the partial or complete (preferably complete) release or replacement of cargo by another cargo. Preferably, unloading occurs under competitive conditions (i.e., conditions in which the cargo to be unloaded competes with another cargo to be loaded).

[0201] In a preferred embodiment, the caged nanoparticle comprises six holes. Preferably, the caged nanoparticle comprises six holes connected to the cavity. Preferably, the external support comprises six holes connected to the cavity of the nanoparticle.

[0202] In another preferred embodiment, the diameter of the pores is about 3 nm to about 4 nm. In another preferred embodiment, the nanoparticles comprise 6 pores with a diameter of 3-4 nm.

[0203] In another preferred embodiment, the outer diameter of the nanoparticles is at most about 50 nm. In a very preferred embodiment, the outer diameter of the nanoparticles is about 13 nm.

[0204] In another further preferred embodiment, the nanoparticle comprises 6 pores with a diameter of about 3-4 nm, the diameter of the inner cavity is about 8 nm, and the outer diameter of the nanoparticle is about 13 nm.

[0205] In a preferred embodiment, the nanoparticles of the present invention are capable of encapsulating and releasing cargo without decomposing the nanoparticles. Preferably, the cargo is a negatively charged macromolecule, more preferably RNA, and again more preferably an RNA selected from the group consisting of: antisense oligonucleotides (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA.

[0206] Due to the multimeric nature of the nanoparticles, modification of the polypeptides of the invention with a functional moiety (eg, a peptide tag or a chemical group such as PEG) provides for multivalent display of this functional moiety on the surface of the nanoparticle.

[0207] In a preferred embodiment, the nanoparticles (especially caged nanoparticles) include a functional portion (also referred to herein as a portion). Preferably, the portion is positioned on the outer surface of the nanoparticle. More preferably, the additional portion is attached to the nanoparticle, preferably to the outer surface of the nanoparticle.

[0208] In a preferred embodiment, the moiety is attached to the nanoparticle or its outer surface via a covalent or non-covalent bond (preferably, via a covalent bond). The covalent bond comprises and is preferably a peptide bond or a disulfide bond.

[0209] In another preferred embodiment, the moiety attached to the nanoparticle is a peptide tag or a non-peptide tag of the polypeptide of the present invention.

[0210] In another preferred embodiment, the moiety is selected from the group consisting of: polyhistidine, His tag (ie, an amino acid sequence consisting of two or more consecutively linked histidines), degradation tag, targeting tag, cell permeation tag and endosomal escape tag.

[0211] Preferably, the targeting tag binds to a cancer target, i.e., the targeting tag is a cancer targeting tag. The cancer target comprises a receptor whose expression level is increased in / on certain tumor cells and tumor antigens. In a preferred embodiment, the cancer targeting tag is a peptide ligand, peptidomimetic or antibody that binds to a cancer target. Preferably, the cancer targeting tag is folic acid.

[0212] In a preferred embodiment, the nanoparticles of the invention display at least 72 histidyl residues on the outer surface of the nanoparticle. In a further preferred embodiment, the nanoparticles of the invention display at least 72-216 histidyl residues on the outer surface of the nanoparticle.

[0213] In a preferred embodiment, the nanoparticles of the invention display at least 72 histidyl residues on the outer surface of the nanoparticle, and wherein the histidyl residue is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I, wherein the nanoparticles can be loaded and unloaded with cargo in vivo and in vitro without decomposition. In a preferred embodiment, the nanoparticles of the invention display at least 72 histidyl residues on the outer surface of the nanoparticle, and wherein the histidyl residue is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I. In a further preferred embodiment, the nanoparticles of the invention display at least 72-216 histidyl residues on the outer surface of the nanoparticle, and wherein the histidyl residue is attached to the C-terminus or N-terminus, preferably the C-terminus, of the amino acid sequence I.

[0214] In a preferred embodiment, the nanoparticles of the invention display at least 96 histidyl residues, preferably 144 histidyl residues, on the outer surface of the nanoparticles. In another preferred embodiment, the nanoparticles of the invention display 3 to 50 (preferably 24) histidine-rich amino acid sequences on their outer surface, wherein each of the histidine-rich amino acid sequences is independently attached to the N-terminus or C-terminus of the polypeptide of the invention and each of the histidine-rich amino acid sequences contains at least 5 (preferably 6) histidyl residues. Preferably, the length of the histidine-rich amino acid sequence is 30 amino acids or less, preferably 25 amino acids or less, more preferably 20 amino acids or less, again more preferably 15 amino acids or less, again more preferably 10 or less, again more preferably 5 or 6 amino acids.

[0215] In another preferred embodiment, the nanoparticles of the invention display at least 72, preferably at least 96, more preferably at least 120, and again more preferably at least 144 histidyl residues on their outer surface. In another preferred embodiment, the nanoparticles of the invention display about 144 histidyl residues on their outer surface.

[0216] In a more preferred embodiment, the nanoparticle of the present invention displays 24 His tags on its outer surface, wherein each of the His tags consists of 3 or more continuously linked histidines. In another preferred embodiment, the nanoparticle of the present invention displays 24 His tags on its outer surface, wherein each of the His tags consists of 3-9 continuously linked histidines. In a more preferred embodiment, the nanoparticle of the present invention displays 24 His tags on its outer surface, wherein each of the His tags consists of 3 or more continuously linked histidines, and wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I. In another preferred embodiment, the nanoparticle of the present invention displays 24 His tags on its outer surface, wherein each of the His tags consists of 3-9 continuously linked histidines, and wherein the His tag is attached to the C-terminus or N-terminus, preferably the C-terminus, of amino acid sequence I.

[0217] In another preferred embodiment, the nanoparticles of the present invention display 3 to 50 (preferably 24) His6 tags on their outer surface, wherein each of the His6 tags consists of 6 consecutively connected histidines. In a preferred embodiment, the nanoparticles of the present invention display 3 to 50 (preferably 24) amino acid sequences including His6 tags (which are defined herein as sequences consisting of 6 consecutively connected histidines (i.e., high six histidines)) on their outer surface, wherein each of the His6 tags including the sequence is independently attached to the N-terminus or C-terminus of the polypeptide of the present invention. Preferably, the length of the sequence including the His6 tag is 30 amino acids or less, preferably 25 or less, more preferably 20 or less, again more preferably 15 or less, again more preferably 10 or less. In a preferred embodiment, the nanoparticles of the present invention display 3 to 50 (preferably 24) His6 tags on their outer surface, wherein each of the His6 tags is independently attached to the N-terminus or C-terminus of the polypeptide of the present invention.

[0218] These histidine-rich peptides may be crucial for delivery to the cytoplasm of target cells due to endosomal escape properties. It has been reported that histidine residues can play an important role in the endosomal escape of synthetic peptides and some viruses. However, the use of multimeric protein assemblies to present multiple copies of functional histidine-rich peptides has not been demonstrated and represents a new approach for cytoplasmic delivery of protein nanoparticles.

[0219] In preferred embodiments, the nanoparticles of the invention are modified genetically (for direct fusion of peptides) or chemically after production.

[0220] Nanoparticles of the present invention only show negligible cytotoxicity.Use WST-8 to measure the in vitro cytotoxicity of HeLa cells in 24 hours with quantitative dehydrogenase activity (see materials and methods, cytotoxicity below).Nanoparticles of the present invention reduce vigor to about 95% at maximum.In the protein (400nM cage) of concentration from 12 μg / mL to 193 μg / mL, vigor is reduced by about 0% to about 5%.By contrast, Lipofectamine 2000 reduces cell viability to about 35-45% with 19 times lower mass concentration (10 μg / mL) under the same assay conditions.

[0221] In another aspect, the invention relates to a complex comprising the nanoparticle of the invention and one or more cargo molecules.

[0222] In a preferred embodiment, the complex comprises the caged nanoparticle of the present invention and one or more cargo molecules encapsulated in the cavity of the caged nanoparticle.

[0223] In a preferred embodiment, the complex of the present invention is capable of inducing an immune response in an organism. Preferably, the organism is an animal, more preferably a mammal, still more preferably a mouse or a human, most preferably a human.

[0224] Preferably, the cargo molecule of the complex of the present invention is a macromolecule. In a further preferred embodiment, the macromolecule is negatively charged.

[0225] As used herein, the term "negatively charged" includes and preferably refers to molecules having positively charged groups. More preferably, the negatively charged molecules have negatively charged groups at neutral or physiological pH.

[0226] Since the interior of the nanoparticles of the present invention derived from arginine residues is highly positively charged, the nanoparticles have a strong affinity to encapsulate negatively charged macromolecules, such as ON. Treating cells with the complex of the present invention results in intracellular delivery of the complex and release of its functional negatively charged macromolecules. Therefore, the complex of the present invention is able to enter cells, escape from endosomes and release negatively charged macromolecules in cells. For example, due to the cytoplasmic delivery and release of ON, the complex comprising ON provides selective knockdown of desired genes.

[0227] The complex of the present invention is further able to enter cells, and it is also able to release negatively charged macromolecules (such as ON) within the cell, preferably to the cytoplasm. Therefore, the complex of the present invention is conditionally stable with respect to negatively charged macromolecules (such as ON). The positively charged interior of the cage provides a strong driving force for the encapsulation of negatively charged macromolecules (such as ON), thereby producing a complex that is stable for several weeks in standard buffer at room temperature.

[0228] In the presence of high concentrations of competitive guest molecules, encapsulation is reversible, and the complex of the present invention is stable outside the cell. In the presence of high concentrations of negatively charged competitive factors (e.g., nucleic acids found in the cytoplasm of cells), negatively charged macromolecules (such as ON) are gradually released and can perform their biological functions. The concept of position-specific release of negatively charged macromolecules (such as ON) induced by this competitive factor is unique and may be modified by changing the number and position of arginine residues in the interior of the protein cage. Therefore, in a preferred embodiment, the nanoparticles of the present invention can reversibly encapsulate and release cargo. In another preferred embodiment, the nanoparticles of the present invention can reversibly encapsulate and release cargo without decomposing the nanoparticles. In another preferred embodiment, the nanoparticles of the present invention can encapsulate and release cargo without decomposing the nanoparticles. The cargo is a negatively charged macromolecule, more preferably RNA, and again more preferably selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs.

[0229] In another embodiment, the nanoparticles of the present invention are capable of encapsulating cargo outside the cell, preferably, the cargo is a negatively charged macromolecule, more preferably RNA, and again more preferably an RNA selected from the group consisting of: antisense oligonucleotides (ASO), small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA) and anti-miRNA, and the nanoparticles encapsulating the cargo are capable of releasing the cargo inside the cell.

[0230] In a preferred embodiment, each of the one or more negatively charged macromolecules has a diameter of about 3.5 nm or less. In a further preferred embodiment, each of the one or more negatively charged macromolecules has a diameter of about 2 nm or less. In a preferred embodiment, each of the one or more negatively charged macromolecules has a diameter of about 2 nm and a length of about 8 nm. Preferably, the macromolecule is a straight chain of monomers, preferably with a diameter of about 2 nm and a length of about 8 nm.

[0231] In a preferred embodiment, the negatively charged macromolecule is a nucleic acid or a polypeptide.

[0232] In another preferred embodiment, the cargo molecule is an oligonucleotide (ON).

[0233] As used herein, the term "oligonucleotide" refers to a compound including 2 or more nucleotides connected to each other by nucleoside linkage. Oligonucleotide is a polyribonucleotide or a polydeoxyribonucleotide or a mixture thereof, and preferably, selected from single-stranded or double-stranded (a) unmodified (i.e., naturally occurring) RNA or DNA, (b) modified RNA or DNA or (c) RNA or DNA mimics, such as locked nucleic acid (LNA) or peptide nucleic acid (PNA). Preferably, oligonucleotide is selected from the group consisting of: (a) single-stranded and double-stranded DNA, (b) DNA that is a mixture of single-stranded region and double-stranded region, (c) single-stranded and double-stranded RNA, (d) RNA that is a mixture of single-stranded region and double-stranded region and (e) including a hybrid molecule that is a mixture of single-stranded or double-stranded DNA and RNA or single-stranded region and double-stranded region. In other preferred embodiments, in other embodiments, oligonucleotide is a triple-stranded region and includes a higher order structure of RNA or DNA or both RNA and DNA. In other embodiments, oligonucleotide is synthetic, genomic or recombinant. In one embodiment, an oligonucleotide is a DNA or RNA that (a) contains at least one modified nucleotide or at least one nucleotide analog or (b) has a backbone modified for stability or other reasons.

[0234] As used herein, the term "modified RNA" or "modified DNA" refers to an oligonucleotide having at least one modified internucleoside bond and / or at least one sugar modification and / or at least one base modification as defined above and compared to a naturally occurring oligonucleotide based on a ribonucleotide or deoxyribonucleotide. The modified internucleoside bond indicates the presence of a modified version of a phosphodiester that is not naturally occurring in RNA and DNA. Examples of internucleoside bond modifications known to those skilled in the art and compatible with the present invention include and specifically include aminophosphorothioates, diaminophosphorothioates, phosphorodithioates, H-phosphonates, methylphosphonates, and methylthiophosphorothioates. Sugar modification indicates the presence of a modified version (i.e., furanose moiety) of a ribosyl moiety naturally occurring in RNA and DNA, such as bicyclic sugars, tetrahydropyrans, 2'-modified sugars, 3'-modified sugars, 4'-modified sugars, 5'-modified sugars, or 4'-substituted sugars. Examples of suitable sugar modifications are known to those skilled in the art and include, but are not limited to, 2'-O-modified RNA nucleotide residues, such as 2'-O-alkyl or 2'-O-(substituted)alkyl, for example 2'-O-methyl, 2'-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-(haloalkoxy)methyl, for example 2'-O-(2-chloroethoxy)methyl methyl (MCEM), 2'-O-(2,2-dichloroethoxy)methyl (DCEM), 2'-O-alkoxycarbonyl, for example 2'-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), 2'-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DMCE), specifically 2'-O-methyl modification or 2'-O-(2-methoxy)ethyl (2'-MOE). Another important modification comprises a sugar moiety that is bridged or modified by a bicyclic nucleic acid (BNA), such as found in, for example, locked nucleic acid (LNA), xylose-LNA, α-L-LNA, β-D-LNA, cEt (2'-0,4'-C constrained ethyl) LNA, cMOEt (2'-0,4'-C constrained methoxyethyl) LNA, ethylene bridged nucleic acid (ENA), hexitol nucleic acid (HNA), fluorinated HNA (F-HNA), pyranosyl-RNA (p-RNA), 3'-deoxypyranosyl-DNA (p-DNA); or other modified sugar moieties, such as morpholino (PMO), cationic morpholino (PMOPlus) or PMO-X, all of which are known to those skilled in the art. As used herein, the term "base modification" refers to the modification of naturally occurring bases (i.e., pyrimidine or purine bases) in RNA and / or DNA.Base modifications are known to those skilled in the art and include, but are not limited to, modified versions of natural purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine and thymine), such as hypoxanthine, pseudouracil, pseudothymine, 2-mercaptopyrimidine (e.g., 2-thiouracil, 2-thiothymine), 2,6-diaminopurine, 5-substituted pyrimidines (e.g., 5-halouracil, 5-methyluracil, 5-methylcytosine), 7-deazaguanine, 7-deazapurine, 7-aza-2,6-diaminopurine, 8-aza7-deazaguanine, 8-aza7-deazapurine or 8-aza7-deaza-2,6-diaminopurine. The present invention also encompasses oligonucleotides comprising more than one identical or different internucleoside linkage modification, sugar modification and / or base modification. Therefore, the oligonucleotide referred to in the present invention may be composed of any combination of the above-described nucleotides and modifications thereof.

[0235] More preferably, the one or more negatively charged macromolecules are one or more oligoribonucleotides selected from the group consisting of: antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), microRNAs (miRNAs) and anti-miRNAs. Further preferred are oligonucleotides capable of stimulating IFN-α production in cells (preferably dendritic cells).

[0236] In a preferred embodiment, the one or more negatively charged macromolecules are one or more ONs, wherein the total number of nucleotides of the one or more ONs is about 300 or less. In another preferred embodiment, the total number of nucleotides of the one or more ONs is about 10 to about 300, preferably about 20 to about 300, more preferably about 40 to about 300, again more preferably about 50 to about 300, again more preferably about 100 to about 300, again more preferably about 200 to about 300.

[0237] In another preferred embodiment, the total number of nucleotides of the one or more ONs is from about 10 to about 300, preferably from about 10 to about 200, more preferably from about 10 to about 50, again more preferably from about 10 to about 40, again more preferably from about 10 to about 30.

[0238] In another preferred embodiment, the total number of nucleotides of the one or more ONs is from about 10 to about 300, preferably from about 20 to about 200, more preferably from about 40 to about 100.

[0239] In a preferred embodiment, the total number of nucleotides of the one or more ONs is about 300 or less, and the ON is RNA, preferably ssRNA.

[0240] In another preferred embodiment, the one or more ONs are single-stranded ONs (ssONs), preferably ssRNA or ssDNA, wherein the ssONs collectively have a length of about 40 nts or longer, preferably about 40 to about 300 nts, more preferably about 40 to about 200 nts, again more preferably about 40 to about 100 nts, again more preferably about 40 nts. In another preferred embodiment, the one or more ONs are double-stranded ONs (dsONs), preferably dsRNA or dsDNA, wherein the dsONs collectively have a length of about 40 bps or longer, preferably about 40 to about 300 bps, more preferably about 40 to about 200 bps, again more preferably about 40 to about 100 bps, again more preferably about 40 bps.

[0241] In a preferred embodiment, the one or more macromolecules are one or more oligonucleotides, wherein the one or more oligonucleotides are encapsulated in the nanoparticle and are not prone to DNA enzymatic hydrolysis. Therefore, in a preferred meaning, the term "encapsulated" indicates that one or more oligonucleotides in an encapsulated state are not prone to DNA enzymatic hydrolysis. More preferably, the term "encapsulated" indicates that one or more oligonucleotides in an encapsulated state are not prone to DNA enzymatic hydrolysis, wherein the DNA enzyme is DNA enzyme I or Benzonase. Again more preferably, the term "encapsulated" indicates that one or more oligonucleotides in an encapsulated state are not prone to Benzonase hydrolysis.

[0242] The negatively charged macromolecule is preferably encapsulated within the nanoparticle via non-covalent bonds. Preferably, the non-covalent bonds are electrostatic bonds.

[0243] Other optional agents, such as adjuvants, may be included within the nanoparticles.

[0244] In another preferred embodiment, the negatively charged macromolecule has an association rate constant k on ((6.6±0.6)×10 5 M -1 s -1 )ON( Figure 2 c). Binding kinetics were determined by measuring fluorescence quenching and fluorimetry. The data were fitted to a single exponential decay (with a linear fit to the rate constant (k) plotted against ON concentration). obs )).

[0245] In another preferred embodiment, the negatively charged macromolecule has a dissociation constant K d ((2.4±0.3)×10 -10 M) and the dissociation rate constant k off ((1.6±0.1)×10 -4 s-1 ) of ON dissociated from the nanoparticles. To determine the dissociation rate, a 100-fold excess of unlabeled DNA was used to induce a slow release of the encapsulated Atto488-ssDNA cargo ( Figure 2 d). The data were fitted to a single exponential decay to provide the dissociation rate constant koff ((1.6±0.1)×10 -4 s -1 The association rate constant and dissociation rate constant are used to calculate the equilibrium dissociation constant K d ((2.4±0.3)×10 -10 M).

[0246] In a preferred embodiment, the negatively charged macromolecule contained in the complex is a nucleic acid (preferably, ON), and the nanoparticle of the complex of the present invention is capable of protecting the nucleic acid (preferably, the ON) from nuclease degradation. In a preferred embodiment, the molecular weight of the nuclease is more than 14kD, preferably more than 20kD, more preferably more than 30kD, more preferably more than 40kD, more preferably more than 50kD and more preferably 60kD or more. In a preferred embodiment, the molecular weight of the nuclease is more than 14kD. In a preferred embodiment, the molecular weight of the nuclease is more than 60kD or more.

[0247] In a preferred embodiment, the complexes or nanoparticles of the present invention show about 30-fold increase in cellular uptake compared to a control of naked (ie, free) Atto488-ssDNA.

[0248] In a further aspect, the present invention provides a method for producing the nanoparticle of the present invention, the method comprising the step of allowing the polypeptide of the present invention to self-assemble into the nanoparticle.

[0249] In a preferred embodiment, the method for making the nanoparticles of the present invention comprises the following steps: (i) producing a polypeptide of the present invention, and (ii) allowing the polypeptide of the present invention to self-assemble into the nanoparticles. The polypeptide for making the nanoparticles of the present invention is preferably produced by recombinant expression. In a preferred embodiment, the polypeptide is recombinantly expressed in a bacterial cell (preferably, in an Escherichia coli cell). After expression, the polypeptide of the present invention self-assembles into well-defined nanoparticles that can be isolated.

[0250] In a preferred embodiment, the method for making nanoparticles further comprises a step of isolating the nanoparticles. The isolating step is preferably affinity chromatography, more preferably fixed metal affinity chromatography. In a preferred embodiment, the isolating step is affinity chromatography using a support comprising nickel, cobalt or copper. Most preferably, the isolating step is Ni-NTA affinity chromatography.

[0251] Since the interior of the nanoparticles of the present invention is positively charged, it may contaminate nucleic acids derived from host cells expressing the polypeptides of the present invention. These undesirable cargoes can be removed with high ionic strength to weaken electrostatic interactions and with RNase A to digest contaminant RNA.

[0252] Therefore, in a preferred embodiment, the method for making nanoparticles further comprises the step of treating the nanoparticles with a high ionic strength buffer and / or an RNase. In a preferred embodiment, the ionic strength of the high ionic strength buffer is 0-2M. In another preferred embodiment, the method for making the nanoparticles of the present invention comprises the following steps: (i) producing the polypeptide of the present invention by recombinant expression, (ii) treating the expressed polypeptide in a buffer having an ionic strength of 0-2M and comprising an RNase and / or a DNase, and allowing the polypeptide of the present invention to self-assemble into the nanoparticles. Preferably, the high ionic strength of 0-2M of the buffer is adjusted by adding NaCl. Preferably, in addition to an RNase and / or a DNase, the high ionic strength buffer also comprises a sodium phosphate buffer, NaCl, imidazole and optionally lysozyme. The buffer comprises, for example, about 50mM sodium phosphate buffer (pH 7.4), about 1000mM NaCl and imidazole supplemented with lysozyme, DNase I and RNase A. The high ionic strength buffer contains at least 20mM imidazole.

[0253] In another aspect, the present invention provides a method for making the complex of the present invention, the method comprising the step of mixing the nanoparticle of the present invention with one or more negatively charged macromolecules. In a preferred embodiment, the mixing step occurs in an aqueous buffer (preferably, at room temperature).

[0254] In a further aspect, the present invention provides a method for transfecting a cell, the method comprising the step of contacting the cell with the complex of the present invention.

[0255] As used herein, the term "transfection" refers to the process of introducing negatively charged macromolecules, preferably nucleic acids, more preferably ONs, into cells. Preferably, the method for transfecting cells is an in vitro process.

[0256] In a preferred embodiment, the cell is a eukaryotic cell. More preferably, the cell is an animal cell, and even more preferably a mammalian cell.

[0257] In a further aspect, the present invention relates to the use of a complex of the present invention for transfecting cells.

[0258] Examples

[0259] Example 1 - Materials and Methods

[0260] Materials and instruments

[0261] All enzymes used for molecular cloning were purchased from New England BioLabs (USA). All synthetic oligonucleotides were purchased from Microsynth AG (Switzerland). Plasmid microprep kits and DNA purification kits were purchased from Zymo Research (USA). Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Fluorochem (UK). Lysozyme was purchased from PanReac Axon Lab AG (Switzerland). To isolate His-tagged proteins, Ni-NTA agarose from Qiagen (Germany) was used. DNase I was from Roche (Switzerland) and RNase A was from Merck (Germany). Benzonase was purchased from Merck-Millipore (USA). GelRed was purchased from Biotium, Inc. (USA). Plasmid pET29b(+)-03-332 was kindly provided by Professor David Baker.

[0262] DNA and protein quantification was performed using a NanoDrop 2000c spectrophotometer from ThermoFisher Scientific Inc. (USA). All size exclusion chromatography was performed at NGC TM The chromatography was performed on a medium pressure chromatography system from Bio-Rad Laboratories, Inc. (USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run and developed on a PhastGel system from GE Healthcare (USA). Agarose gel electrophoresis (AGE) and denaturing polyacrylamide gel electrophoresis (PAGE) were performed on CellGT and Mini-PROTEAN systems were performed, both from Bio-Rad Laboratories, Inc. (USA). Gel images were captured using an EOS 1100D from Canon (Japan). Transmission electron microscopy (TEM) images were obtained on a Morgagni 268 from FEI (USA). Fluorescence microscopy images were obtained on an Eclipse-Ti inverted microscope from Nikon (Japan). Confocal fluorescence microscopy images were obtained on an SP8-AOBS from Leica (Germany). Flow cytometry was performed on an LSRFortessa from BD Biosciences (USA).

[0263] Molecular modeling

[0264] X-ray diffraction data of the original O3-33 assembly (PDB: 3VCD) (King, NP et al., Science 2012, 336, 1171-1174) were used to select sequence positions for mutation to arginine, which were obtained by UCSF Chimera (Pettersen, EF et al., J. Comput. Chem. 2004, 25, 1605-1612) using the Dynameomics backbone-independent rotamer library (Scouras, AD et al., Protein Sci. 2011, 20, 341-352).

[0265] Molecular cloning

[0266] The gene of OP was purchased from ATG: biosynthetics (Germany) and obtained as plasmid pGH-OP. The OP gene insert was excised from pGH-OP and cloned into the vector pET29b (+) -03-33 vector (King et al., 2012, cited above) using XhoI and NdeI restriction sites to replace the O3-33 gene and obtain the plasmid pET29b (+) -OP. The OP gene without the His6 tag was prepared by cloning in a DNA box encoding GS** at the XhoI site immediately upstream of the His6 tag. The plasmid sequence was confirmed by Sanger DNA sequencing performed by Microsynth AG (Switzerland).

[0267] Protein expression

[0268] Proteins were expressed in E. coli strain BL21-Gold (DE3) transformed with pET29b(+)-O3-33, pET29b(+)-OP or pET29b(+)-OP_no His6. Cells were grown in LB medium containing kanamycin sulfate (86 μM) at 37°C until OD 600 The protein expression was induced by IPTG (0.1 mM) until the p-value reached 0.6-0.8. After culturing at 25°C for about 18 hours, the cells were harvested by centrifugation (5,000×g) at 4°C for 15 minutes. The cell pellet was stored at -20°C until purification.

[0269] Cell lysis, Ni-NTA and RNA removal of OP

[0270] Since OP can be loaded with small RNA molecules during expression in E. coli, an optimized protocol was developed to isolate empty cages of good purity. The optimized protocol includes the use of high ionic strength buffers to weaken interactions with nucleic acids and prolonged incubation with RNase A. Each cell pellet from 800 mL of culture was resuspended in 10 mL of lysis buffer (50 mM sodium phosphate buffer (pH 7.4), 1000 mM NaCl, 20 mM imidazole), supplemented with lysozyme (0.1 mg / mL), DNase I (10 U / mL), RNase A (5 U / mL) and a protease inhibitor cocktail (Sigma), and incubated at 37°C for 1.5 hours.

[0271] After lysis, ultrasound and centrifugation (10,000 × g) at 25 ° C for 25 minutes, the supernatant was loaded onto a 5mL gravity flow column containing Ni-NTA resin and incubated for 15 minutes. After multiple washings with lysis buffer containing 20mM and 40mM imidazole, the target protein was eluted with elution buffer (50mM sodium phosphate buffer (pH 7.4), 300mM NaCl, 500mM imidazole). Typically, 10-20mL is collected and supplemented with 2U / mL RNase A, 5mM EDTA and a mixture of protease inhibitors, and incubated overnight at 37 ° C to digest any contaminant E. coli RNA that was not removed during Ni-NTA purification. Then, the protein is ready for SEC. Thereafter, unless otherwise stated, the storage of the protein and all experiments were performed at room temperature.

[0272] Pyrolysis of O3-33 and Ni-NTA

[0273] The separation of O3-33 followed the same steps as OP, but the conditions were less stringent because nucleic acid contamination was not a problem. For lysis, sonication, and Ni-NTA, the buffers used had a lower ionic strength (50 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, and 20 mM imidazole). After separation from Ni-NTA with elution buffer (50 mM sodium phosphate buffer (pH 7.4), 300 mM NaCl, 500 mM imidazole), the protein was ready for SEC.

[0274] Size Exclusion Chromatography (SEC)

[0275] Prior to SEC, the protein sample was exchanged into 20 mM sodium phosphate buffer (pH 7.4) with 200 mM NaCl and 5 mM EDTA using an Amicon Ultra-15 centrifugal filter unit (30k MWCO) (Merck Millipore, USA). This process also removes some RNase A and digested nucleic acid fragments from OP. The fully assembled 24-mer cages were then separated by SEC at a flow rate of 1.0 ml / min at room temperature using a Superose 6 Increase 10 / 300GL column from GE Healthcare (USA). Both the O3-33 cage and the OP cage were eluted with 20 mM sodium phosphate buffer (pH 7.4) containing 200 mM NaCl and 5 mM EDTA at 15-16 mL. The purified protein was stored in SEC elution buffer at room temperature. The protein was quantified by absorbance at 280 nm. Additionally, the protein was quantified according to A 260 / A 280 The ratio was calculated and the removal of all RNA was verified by native agarose gel stained with GelRed, which is a sensitive nucleic acid stain. Protein purity was verified by SDS-PAGE stained with Coomassie blue.

[0276] General characterization of protein cages

[0277] All native gel electrophoresis was performed using 2% or 3% (w / v) agarose gel in Tris-acetate-EDTA buffer (40 mM Tris-HCl, 19 mM acetic acid, 1 mM EDTA, pH 8.3). The gel was cast with GelRed to detect unlabeled nucleic acids as needed and stained with Coomassie blue for protein visualization. In a typical experiment for analyzing proteins, 10 μL of buffer containing about 100 or 200 pmol of capsid (relative to monomer) was loaded per channel with an additional 2 μL of 70% (v / v) glycerol aqueous solution. The gel was run at 100 V for 30 minutes, stained with Coomassie blue to visualize the protein, and detected ON with Atto488 fluorescence.

[0278] SEC analysis was performed under the same conditions as for cage separation. Negatively stained transmission electron microscopy (TEM) was performed as previously reported (Beck, T. et al., Angew. Chem. Int. Ed. 2015, 54, 937-940). For all TEM experiments, storage buffer (20 mM sodium phosphate buffer (pH 7.4), 200 mM NaCl, and 5 mM EDTA) containing 3-5 μM (monomer) protein samples was used. DLS measurements were performed on a Zetasizer Nano (Malvern Instruments, UK) at 25°C using a 0.22 μm filtered storage buffer containing 150-250 μM (monomer) samples.

[0279] Melting temperature

[0280] Using Protein Thermal Shift TM Melting temperatures were obtained using a dye kit (Thermo Fisher Scientific, USA) on a StepOnePlus real-time PCR system (Applied Biosystems, USA). Proteins were measured in triplicate at 5, 10 and 20 μM in a buffer containing 25 mM Tris-HCl (pH 8.0), 200 mM NaCl. All data were averaged to generate values.

[0281] Table 4: Melting temperature

[0282] protein Tm(℃) O3 96.08±0.09 O3+siRNA 95.98±0.28 OP 94.42±0.33 OP+siRNA 95.80±0.63

[0283] Table 4 shows the melting temperatures, Tm, of O3-33 and OP proteins in the presence and absence of two equivalents of siRNA duplexes.

[0284] Protein crystallography

[0285] Sitting drop vapor diffusion experiments were set up in an Intelli-Plate R96-3LV using a Phoenix crystallization robot (Art Robbins Instruments). Initial crystallization attempts were performed at 20°C. Hole-diffracting OP crystals were obtained at 20°C using a sitting drop vapor diffusion method with a 3 mg / mL enzyme solution in 2M (NH4)2SO4 and 5% 2-propanol. The crystals were transferred to a reservoir solution with 20% (v / v) glycerol as a cryoprotectant and then flash cooled at -173°C in a stream of nitrogen. X-ray diffraction data sets were collected at X06SA at the Swiss Light Source (Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland) using an EIGER X16M detector. wavelengths for data collection.

[0286] The diffraction data of OP were analyzed using the XDS package (Kabsch W., Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 125-132). The initial phase was determined by molecular replacement using the structure of O3-33 (PDB code: 3VCD) as a search model. Molecular replacement was performed using Phaser (McCoy, AJ et al., J. Appl. Crystallogr. 2007, 40, 658-674). The structure was manually modified using Coot (Emsley, P. et al., Acta Crystallogr. D Biol. Crystallogr. 2004, 60, 2126-2132) and analyzed using PHENIX (Adams, P.D et al., Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 213-221). The final crystal data and intensity statistics are summarized in Table 5.

[0287] Electrophoretic mobility shift assay (EMSA)

[0288] All EMSAs were performed on 3% (w / v) agarose gels as described above. For Atto488-labeled ssDNA, 10 pmol of DNA was used per sample (= one gel lane) and incubated at room temperature with different molar equivalents of protein in a total volume of 10 μL of PBS. For unlabeled dsDNA and dsRNA, 40 pmol of duplex was used per sample under the same incubation conditions and the gels were cast with GelRed for visualization.

[0289] Cargo loading capacity

[0290] Pure OP protein (1 nmol) was incubated overnight at room temperature with 5 equivalents of ssDNA, dsDNA or dsRNA in 200 μL of SEC buffer (20 mM sodium phosphate buffer (pH 7.4), 200 mM NaCl and 5 mM EDTA). The mixture was purified by SEC and absorbance spectra were obtained. All absorbance spectra were corrected for light scattering using a|e UV-Vis-IR software. The concentrations of proteins and nucleic acids were determined based on the absorbance values ​​at 280 and 260 nm using the equation described by Porterfield and Zlotnick (Porterfield, JZ et al., Virology 2010, 407, 281-288).

[0291] Encapsulation kinetics

[0292] All kinetic measurements were performed in PBS at room temperature. on For each experiment, the fluorescence emission of 800 μL of a solution of Atto488-labeled DNA (10 nM) was measured at 515 nm (excitation, 488 nm), and this value was taken as t = 0 sec. Next, a small volume of concentrated OP solution was added to a final concentration of 5-125 nM, and the emission decrease over time was monitored at 10 sec intervals. Each measurement of emission versus time was fitted to a single exponential, providing k for each OP concentration. obs Then, these k obs The values ​​were plotted against [OP] and k was obtained from the slope of the linear fit on Since there are one or two guest DNA molecules per cage, k obs The values ​​of are unreliable (Figure 17). Therefore, only k values ​​in the range of 25-125 nM were used. obs To determine k on , and fix the y-intercept of the linear fit to a certain k off The value is 1.6×10-4 seconds.

[0293] To determine k off 800 μL of a solution of Atto488-labeled DNA (10 nM) and OP (5 nM) were incubated until no further change in fluorescence was observed. This provided an OP cage containing two ssDNA molecules, which would be removed when the second DNA molecule was encapsulated in a 1:1 complex. offThe measurement was simplified to only one rate constant. Due to the high stability of the OP:DNA complex, an excess of unlabeled ssDNA was used to displace the encapsulated Atto488-DNA and restore fluorescence upon its release from the protein cage. After the addition of 1 μM unlabeled ssDNA, the change in fluorescence emission at 515 nm was monitored at 60 s intervals for 16 h. The data were fitted to a single exponential to generate k off .

[0294] Nuclease digestion assay

[0295] Complexes of OP with ssDNA, dsDNA or dsRNA (1: 1 molar ratio) were prepared by incubation in PBS at room temperature for 1 hour. Equivalent stock solutions containing only nucleic acids were also prepared. The stock solutions were then divided into aliquots to provide samples for each gel lane. Each sample contained 24 pmol of OP / nucleic acid in a total volume of 10 μL PBS supplemented with MgCl2 (2.5 mM), CaCl2 (0.5 mM). The control samples remained as is, and 0.2 U of RNase A, DNase I or Benzonase was supplemented to the nuclease samples. All samples were then incubated at 37°C for 18 hours ( 4% ) before being analyzed by staining with GelRed and then with Coomassie blue stained native agarose gel. Fig.14 ).

[0296] Cell culture

[0297] HeLa cells and A431 cells were maintained in Dulbecco's modified Eagle medium (high glucose), MCF-7 cells and Vero cells were maintained in Iscove's modified Dulbecco medium, HepG2 and HT29 were maintained in RPMI-1640 medium, and CHO was maintained in Ham's F-12K medium. In all cases, 10% fetal bovine serum (FBS), 2mM L-glutamine, 2mM GlutaMAX and 1μg / mL gentamicin were supplemented to the medium. Cells were cultured at 37°C in 5% CO2 and usually split at a ratio of 1:4 every 3 days. HeLa-GFP cells were maintained in the same manner as HeLa cells, and 0.1mM non-essential amino acids (NEAA) and 10μg / mL blasticidin (select antibiotics) were added to the medium. Because NEAA and blasticidin were found to have no effect within those time frames, they were not added to the medium. Cytotoxicity

[0298] The cytotoxicity of OP and Lipofectamine 2000 (Thermo Fisher Scientific, USA) was assessed using a WST-8-based cell counting kit 8 from Sigma according to the manufacturer's instructions. HeLa cells were seeded in 96-well plates at a density of 5,000 cells per well in 100 μL of culture medium and allowed to recover for 24 h at 37 °C and 5% CO2. OP (pre-sterilized by 0.22 μm membrane filtration) and Lipofectamine 2000 samples were prepared by serial dilution in sterile PBS. A total volume of 10 μL of sample was added to each well to provide Fig.15 As a negative control, 10 μL of 10% Triton X-100 in PBS was used per well. The positive control was PBS alone. Cells were incubated at 37°C in 5% CO2 for 24 h or 48 h before 10 μL of CKK-8 reagent was added to each well. Plates were then incubated at 37°C and 5% CO2 for 4 h before measuring the absorbance at 450 nm. The absorbance of CKK-8 in medium without cells was used for background subtraction and samples were normalized to untreated cells to provide Fig.15 Values ​​shown in . Samples were measured in triplicate.

[0299] Cellular uptake by flow cytometry

[0300] HeLa cells were seeded in 24-well plates at a density of 30,000 cells per well in 500 μL of culture medium and allowed to recover at 37°C and 5% CO2 for 24 hours to reach 70-90% confluence. Both OP protein and Atto488-DNA were sterilized by 0.22 μm membrane filtration and stock solutions were prepared in sterile PBS. For each well, 20 μL of PBS containing the sample was added to 200 μL of culture medium to give a final concentration of Atto488-DNA of 200 nM. Cells were incubated at 37°C in 5% CO2 for 24 hours before being washed with PBS and subjected to 4 minutes of trypsin digestion (0.05% trypsin-EDTA (Thermo Fisher Scientific, USA)) at 37°C. Cells were collected in cold culture medium and washed twice with cold PBS before being resuspended in flow cytometry buffer (PBS with 5% FBS and 10 μg / mL propidium iodide).

[0301] Cellular uptake examined by confocal fluorescence microscopy

[0302] HeLa cells were seeded in μ slide 8-well compartment coverslips with ibiTreat surface from ibidi GmbH (Germany) at a density of 15,000 cells per well. Before adding the sample, the cells were incubated in 200 μL of culture medium at 37°C and 5% CO2 for 24 hours. Both OP protein and Atto488-DNA were sterilized by 0.22 μm membrane filtration, and stock solutions were prepared in sterile PBS. For each well, 10 μL of PBS containing the sample was added to 100 μL of culture medium so that the final concentration of Atto488-DNA was 200 nM. Before washing with PBS and performing nuclear staining with 100 μL of Hoechst 33342 solution (PBS containing 5 μg / mL) at 37°C for 10 minutes, the cells were incubated in 5% C CO2 O2 at 37°C for 24 hours. Then, the cells were washed twice with PBS and examined under a microscope at 37°C in PBS containing 5% FBS.

[0303] GFP knockdown

[0304] HeLa-GFP cells were seeded in 24-well plates in culture medium at a density of 30,000 cells per well in 500 μL of culture medium, and allowed to recover at 37°C and 5% CO2 for 24 hours to reach about 70% confluence. Sterile protein, RNA and Lipofectamine 2000 samples were prepared in PBS. Except for Lipofectamine 2000 control, all samples (PBS containing 20 μL) were added to the cells in 200 μL OptiMEM before adding 300 μL of full culture medium, and incubated at 37°C and 5% CO2 for 4 hours. Then, the cells were further incubated at 37°C and 5% CO2, and a total of 48 hours were performed between sample addition and flow cytometry analysis. Although the knockdown efficiency of Lipofectamine 2000 was negligible under the conditions used for all other samples, toxicity was a problem. Therefore, without compromising the knockdown efficiency, the necessary minimum amount of Lipofectamine 2000 was used, which minimized toxicity. The minimum amount was determined to be 1 μL of Lipofectamine 2000 per 40 pmol of siRNA, which is also the minimum amount recommended by the manufacturer. Then, using the manufacturer's recommended protocol, and before flow cytometry analysis, cells were incubated with siRNA liposomes (pre-prepared in PBS) in culture medium for 48 hours at 37°C and 5% CO2.

[0305] Fluorescence microscopy was performed directly on the 24-well plates prior to flow cytometry analysis to obtain a directional measure of GFP expression levels and assess cell health ( Fig.16). Then, the cells were washed with PBS and trypsinized. The cells were collected in cold culture medium and washed twice with cold PBS before resuspending in flow cytometry buffer (PBS with 5% FBS and 10 μg / mL propidium iodide). Untreated HeLa-GFP cells (with matching culture medium and incubation conditions as samples) were used as positive controls, and HeLa cells that do not express GFP were used as negative controls for GFP fluorescence to optimize the cytometer settings. In many cases, samples were measured at least in triplicate with different batches of protein and siRNA.

[0306] Purification of OP without His6 tag

[0307] As previously reported, ammonium sulfate precipitation was used (Beck, T. et al., Angew. Chem. Int. Ed. Engl., 2015, 54, 937-940), but modified to optimize the removal of small RNA molecules. Each cell pellet from 800 mL of culture was resuspended in 10 mL of buffer (25 mM Tris-HCl buffer (pH 8.0), 1000 mM NaCl), supplemented with lysozyme (0.1 mg / mL), DNase I (10 U / mL), RNase A (5 U / mL) and protease inhibitor cocktail (Sigma), and incubated at 37°C for 1.5 hours. After lysis, the suspension was sonicated and centrifuged (10,000 × g, 25°C, 25 minutes). Next, the supernatant was heated to 65°C for 30 minutes and centrifuged (10,000 × g, 4°C, 25 minutes) to precipitate denatured E. coli proteins. Ammonium sulfate was added to the supernatant to 70% of its saturation concentration, and the suspension was centrifuged (10,000 × g, 4°C, 20 minutes). The protein precipitate was resuspended in 20 mL of buffer (25 mM Tris-HCl buffer (pH 8.0), 1000 mM NaCl), and ammonium sulfate precipitation was performed twice again. Finally, the precipitated protein was resuspended in low salt buffer (25 mM Tris-HCl buffer (pH 8.0), 200 mM NaCl), supplemented with 2 U / mL RNase A, 5 mM EDTA and a protease inhibitor cocktail, and then incubated overnight at 37°C to digest any contaminant E. coli RNA. The protein was then purified by SEC in a buffer containing 25 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.5), 1000 mM NaCl, and 250 mM MgSO4 to weaken the interaction between any remaining guest molecules and the protein cage. Thereafter, the protein was stored in PBS buffer at room temperature.

[0308] Cargo replacement by tRNA

[0309] First, the ability of OP to encapsulate tRNA was determined by EMSA (Figure 17). Samples were prepared with 20 pmol of tRNA (transfer RNA from wheat germ of Fluka, Germany) in 10 μL of PBS with and without OP (1.2 molar equivalents) and analyzed by native agarose gel stained with GelRed. The intracellular concentration of tRNA was calculated based on the average volume of HeLa cells, the quality of RNA isolated from individual cells, and the percentage of total RNA (which is tRNA) (Luby-Phelps, K. International Review of Cytology, ed.: Walter, H., Brooks, DE, Srere, PA, Academic Press: 1999; Vol. 192, pp. 189-221; Lodish H, BA, Zipursky SL Molecular Cell Biology, 4th ed.; WH Freeman: New York, 2000). This estimate does not take into account the effects of molecular crowding within the cytoplasm, the relative fraction of free tRNA, or other molecules that may also be encapsulated by OP. For the displacement assay, complexes of OP and Atto488-DNA duplexes were prepared by incubation in PBS for 1 hour at room temperature (1:1 molar ratio). The stock solution was then divided into aliquots to provide samples for each gel lane. Each sample contained 10 pmol of OP / nucleic acid in a total volume of 10 μL of PBS. Next, 2 μL of concentrated tRNA stock solution was added to the samples at different time points so that the final [tRNA] was 26 μM, and the samples were incubated at 37°C. The samples were then analyzed by native agarose gel, which was visualized by Atto488 fluorescence ( Figure 4 b).

[0310] Serum stability

[0311] Complexes of OP and siRNA (1:1 molar ratio) were prepared by incubation in PBS at room temperature for 1 hour. The stock solution was then divided into aliquots to provide samples for each gel lane. Each sample contained 10 pmol of OP / nucleic acid in a total volume of 5 μL of PBS. At different time points, 5 μL of PBS containing 20% ​​fetal bovine serum was added to the samples, and the samples were incubated at 37°C. The samples were then analyzed by native agarose gel, which was visualized by GelRed and Coomassie Blue ( Fig. 22).

[0312] Small angle X-ray scattering

[0313] SAXS data were acquired at beamline P12 of the PETRA-III synchrotron (DESY, Hamburg, Germany) with an X-ray wavelength of 1.24A. The Pilatus 1M detector was positioned 2 m from the sample to collect scattering vectors in the range of 0.028–6.7 nm-1. Samples were measured at different concentrations (between 0.5-5 mg / mL protein) in SAXS buffer (25 mM Tris-HCl buffer (pH 8.0), 200 mM NaCl, 5 mM EDTA, and 10% glycerol), and data from samples showing the least aggregation and noise were used for further analysis. All samples were dialyzed against SAXS buffer to provide a well-matched buffer blank. For OP:dsDNA and OP:dsRNA, the protein cages were incubated overnight with 2 equivalents of duplex before dialysis against SAXS buffer. Scattering curves were analyzed using PRIMUS16 and pair distance distributions were calculated using AUTOGNOM.

[0314] Dissociation of double-stranded DNA

[0315] To verify that the hpDNA does not dissociate, the fluorescence spectra of 800 μL of 10 nM Atto488-hpDNA solution in PBS were measured from 450 nm to 600 nm. Subsequently, 50 nM OTR501 was added to each sample and the fluorescence decay was followed for 600 seconds before recording another spectrum. The same procedure was applied to 10 nM solutions of dsDNA (Atto-488ssDNA and Luc-g), 10 nM solutions of dsDNA with 8 M urea, and 10 nM solutions of Atto-488ssDNA and GFP-g.

[0316] Example 2 - Protein and Nucleic Acid Sequences

[0317] The protein kinetics at 280 nm (ε) was calculated using the SIB Bioinformatics Resource Portal tool (http: / / web.expasy.org / protparam / ). 280 ,M -1 cm -1 )'s molecular weight (MW), isoelectric point (pI) and extinction coefficient.

[0318] Table 1: Peptide sequences at 280nm (ε 280 ) and its molecular weight (MW) and extinction coefficient

[0319]

[0320]

[0321]

[0322]

[0323] SDS-PAGE and Coomassie blue visualization showed different mobilities, which were caused by the different mobilities of proteins OP and OTR101 to OTR503. This proved that all proteins had the same weight of about 20 kDa and all variants had appropriate purity.

[0324] Table 2: Oligonucleotide sequences

[0325]

[0326] Capital letters denote deoxy and lower case letters denote ribonucleotides, respectively. A488 refers to the fluorophore Atto488 phosphoramidite coupled during solid phase synthesis.

[0327] Example 3 - Structural Characterization of Nanoparticles

[0328] As a starting scaffold, the inventors chose O3-33, a self-assembling protein cage computationally designed by Baker and colleagues (King, NP et al., Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy. Science 2012, 336, 1171-1174), which is a non-functional protein cage and a simple computationally designed scaffold without the ability to encapsulate or deliver macromolecules within cells.

[0329] To allow cargo loading, the inventors introduced binding interactions with O3-33. Amino acids Thr11, Pro39, Glu66, Trp103, Phe130, and Leu163 were replaced with arginine to create an O3-33 variant (referred to herein as OP) with a highly positively charged internal cavity ( Figure 1 b, 5). OP proteins can be overexpressed in E. coli cells and isolated by Ni-NTA affinity chromatography via the C-terminal His6 tag ( Figure 6). Since the internal cavity is highly positively charged, endogenous E. coli RNA causes serious contamination. However, these RNA contaminants are easily removed using high ionic strength buffers to weaken electrostatic interactions and RNase A to digest contaminant RNA (see Materials and Methods, Cell Lysis, Ni-NTA and RNA Removal in OP).

[0330] The formation of the desired cage-like quaternary structure ( Figure 1 a) ability to assess ( Figure 7 , 8 All data are consistent with the formation of assemblies with the same dimensions as the O3-33 cage. Furthermore, OP and O3-33 cannot be distinguished by TEM ( Figure 1 c, 9), and X-ray diffraction confirmed that the addition of 144 arginines to the ~8 nm diameter cavity had negligible effects on monomer folding and cage structure ( Figure 1 d). The RMS deviation of all Cα atoms of each monomer is

[0331] Furthermore, characterization of OP variants OTR101 to OTR503 by SEC, AGE, and TEM showed that the structures of all OP variants were identical to OP.

[0332] Example 4 - Nucleic Acid Cargo Loading

[0333] Given the structural integrity of the empty protein cages established, nucleic acid cargo loading was investigated in vitro. DNA and RNA duplexes with a diameter of 2 nm were able to pass through the pores with a diameter of approximately 3.5 nm, leading to encapsulation. Electrophoretic mobility shift assays (EMSA) revealed that the parent O3-33 cage had no effect on the electrophoretic mobility of Atto488-ssDNA (single-stranded (ss) DNA labeled with 21 nt Atto488) Fig.10 In contrast, colocalization of protein and DNA bands of OP was observed ( Figure 2 a). The binding of double-stranded (ds) DNA and dsRNA to OP was also evaluated ( Fig.11 ), providing similar results to ssDNA. In all cases, nearly complete binding of the oligonucleotide was observed after the addition of 0.5 equivalents of OP, indicating two guests per cage. 260 / A 280 The ratio confirms this stoichiometry ( Figure 2 b and 12, Table 3).

[0334] Table 3: Absorbance of SEC-purified OP:nucleic acid complexes and calculated number of guests per cage

[0335] sample <![CDATA[A 260 / A 280 Ratio]]> Objects per cage OP 0.64 - / - OP:ssDNA 1.21 2.05 OP:dsDNA 1.29 1.98 OP:dsRNA 1.40 1.97

[0336] Example 5 - Binding Kinetics

[0337] The encapsulation-induced fluorescence quenching of the DNA-conjugated fluorophore Atto488 was used to determine the binding kinetics by fluorimetry ( Fig.13 ). Association rate constant k on =(6.6±0.6)×10 5 M -1 s -1 ( Figure 2 c) and the dissociation rate constant k off =(1.6±0.1)×10 -4 s -1 ( Figure 2 d) Used to calculate the dissociation constant of this protein-DNA complex Kd = (2.4 ± 0.3) x 10 -10M This Kd value is two orders of magnitude lower than that reported for RNA-protein interactions in viruses (10 -8 -10 -7 M) and is within the range of the tightest binding transcription factors (10 -15 -10 -10 M). It must be noted that a key distinction of this artificial system is the lack of sequence specificity, which is advantageous for a delivery device as it allows for the packaging of any desired nucleotide sequence and retention of negatively charged oligonucleotide chemical modifications.

[0338] An important role of any nucleic acid delivery system is to protect its cargo from nuclease degradation. Therefore, digestion assays with three nucleases were performed: DNase I (30 kDa), RNase A (14 kDa), and benzonase 50 (60 kDa). Fig.14 ) revealed protection from the larger enzyme. Only RNase A was able to enter the cage and digest the cargo. These nuclease assay data corroborate kinetic studies, indicating that the guest is tightly bound in the lumen with a negligible dissociation rate.

[0339] With many nucleic acid delivery vehicles (such as cationic lipids and polymers), a compromise must be made between transfection efficiency and cytotoxicity. In vitro cytotoxicity of HeLa cells was measured over 24 hours using WST-8 to quantify dehydrogenase activity ( Fig.15, Table 6). OP showed negligible toxicity up to 400 nM cage (193 μg / mL protein). These results compare favorably with the commonly used transfection reagent Lipofectamine 2000 (cationic lipid), which reduced cell viability to 35%-45% at a 19-fold lower mass concentration (10 μg / mL) under the assay conditions described above.

[0340] Table 6: Cytotoxicity of different concentrations of OP and Lipofectamine 2000 in HeLa cells

[0341] sample Concentration (μg / mL) vitality(%) PBS n / a 100.0±0.8 Triton n / a 7.4±0.2 Lipofectamine 0.6 98.1±8.7 Lipofectamine 1.3 91.2±1.4 Lipofectamine 2.5 76.8±8.1 Lipofectamine 5 51.0±5.9 Lipofectamine 10 34.2±0.9 OP 12 96.7±5.4 OP 24 99.0±10.9 OP 48 96.9±2.0 OP 96 94.6±3.2 OP 193 99.5±4.8

[0342] Example 6 - Cellular Uptake and Cargo Release

[0343] In addition, the ability of the nanoparticles of the present invention to enter cells and deliver their nucleic acid cargo was investigated using flow cytometry and confocal fluorescence microscopy (CFM) with the Atto488-ssDNA cargo as a probe. Seven different cell lines (including HeLa cells) were treated with 200 nM of free Atto488-ssDNA or Atto488-ssDNA packaged in OP cages, and cell fluorescence was measured by flow cytometry. Plots of median fluorescence intensity show the effective cellular uptake of OP cages in six of the seven cell lines tested ( Figure 3a -c). In HeLa cells, the cellular uptake of ssDNA by these cells was shown to be increased by almost 30-fold compared to background and delivered to more than 90% of HeLa cells. Even greater effects were observed for Vero cells and HepG2 cells. To confirm this finding, CFM was performed on cells treated with free Atto488-ssDNA or Atto488-ssDNA loaded OP cages ( Fig.16 Cells treated with free Atto488-DNA showed no obvious fluorescence, whereas the intracellular fluorescence of cells treated with OP:Atto488-DNA was localized in many intense foci, confirming the high uptake efficiency ( Figure 3b ). These punctate structures indicate endocytosis of nanoparticles typically observed in this size range.

[0344] Example 7 - Regulation of gene expression

[0345] There are few reports of non-viral delivery systems that can deliver functional nucleic acid cargo without the need for additional transfection agents. Confirming the high cellular uptake of OP cages, the ability of the cages to deliver siRNA and modulate gene expression was tested using HeLa cells stably expressing green fluorescent protein (GFP) as a model to measure protein expression. Using Lipofectamine 2000 as a positive control, HeLa cells expressing GFP were treated with naked siRNA, OP-encapsulated siRNA, empty OP cages, and OP cages loaded with scrambled sequence siRNA. To determine the gene knockdown efficiency, fluorescence microscopy ( Fig.16 ) and were qualitatively measured by flow cytometry ( Figure 4 a) Quantitative measurement of GFP fluorescence. After treatment with 20 pmol of OP-packaged siRNA and incubation for 48 hours, a decrease in GFP fluorescence of approximately 70% was observed, with efficacy within the range of Lipofectamine 2000. In addition, due to the significantly reduced toxicity of OP, cell viability was significantly reduced relative to the commercial reagent (2.5 μg / mL) in these assays. Fig.16 The lack of effect of OP cages loaded with scrambled sequence siRNAs confirmed that the expression levels were not due to induction of antiviral interferon pathway mechanisms but were target mRNA specific.

[0346] Example 8 - His6 tag and high cytosolic concentration of RNA contribute to ON delivery efficiency

[0347] Successful induction of RNAi requires delivery of siRNA to the cytoplasm. Therefore, the data mentioned above suggest that OP cages can achieve endosomal escape and release their cargo. Without being bound by any theory, the inventors propose two explanations:

[0348] First, the ability of histidine-rich peptides to promote endosomal escape has been well documented due to the proton sponge effect and the ability of protonated histidine to permeabilize endosomal membranes. Since each OP monomer has a His6 tag, the complete 24-mer cage presents a total of 144 histidyl residues on its exterior. To test whether this affects siRNA delivery, the inventors generated variants of OP without the His6 tag and assayed their RNAi activity (Materials and Methods, Purification of OP without the His6 tag). Under the same experimental conditions, only a decrease of about 40% in GFP fluorescence was observed ( Figure 4 a), indicating that the His6 tag does contribute to delivery efficiency.

[0349] Second, the release of cargo molecules in the cytoplasm can be Figure 2The kinetic parameters shown in d are explained from the perspective of OP-nucleic acid complexes. Although OP-nucleic acid complexes are stable, encapsulation is reversible in the presence of high concentrations of competing guest molecules. Therefore, the inventors expected that high cytosolic concentrations of tRNA could provide a site-specific release mechanism for the siRNA cargo, thereby releasing it for RNAi-induced gene knockdown. This hypothesis was verified in vitro: OP cages containing A488-dsDNA were treated with intracellular concentrations of tRNA (26 μM) in PBS at 37 °C, and time-dependent release of the cargo was observed over 6 h ( Figure 4 b and 19). For oligonucleotides targeting mRNA in the cytoplasm, exploiting the physicochemical properties of the cytosol provides a simple solution for cargo release.

[0350] In contrast, the same experiment performed with 10% fetal bovine serum showed no release or degradation of the cargo ( Fig. 22 ).

[0351] Example 9 - Protein Crystallography of Nanoparticles

[0352] Table 5: Data collection and refinement statistics

[0353]

[0354] *One crystal was used for data collection. *Values ​​in brackets are for the highest resolution shell.

[0355] Example 10-OP and its variants OTR101 to OTR503

[0356] In vitro studies of single-stranded DNA loading

[0357] To test whether the different variants of OP, OTR101 to OTR503, can load ssDNA, their interaction with ssDNA was investigated in an electrophoretic shift assay (EMSA). Thus, it was expected that colocalization of DNA and protein bands would be observed for the binding variants, while the ssDNA band mobility would not be affected by the non-binding variants. Fig.26 As seen in Figure 3, this colocalization was observed for all studied variants containing a minimum of three arginine mutations per monomer, whereas all studied variants containing one or two arginines per monomer as well as O3-33 did not result in any shift in the Atto488-ssDNA band.

[0358] Binding kinetics of variants compared with OP

[0359] It was investigated whether the results obtained from EMSA could be validated within the framework of a fluorescence assay. As shown in Figure 27, it can indeed be concluded that variants with two or fewer arginines do not bind to ssDNA. Furthermore, it can be seen that the relative decrease in fluorescence corresponding to the quenching effect of the capsid increases with the increase in the electrostatic contribution.

[0360] The kinetic parameters for binding to ssDNA for all variants having three or more arginine mutations per monomer are shown in Table 7 below.

[0361] Table 7: Summary of kinetic parameters for binding to ssDNA

[0362]

[0363] In vitro studies of double-stranded DNA loading

[0364] It has been shown that OP protein variants with three, four or five arginines can load ssDNA, especially because double-stranded siRNA is more relevant to biomedical applications, so the loading capacity of dsDNA will be of interest. For all dsDNA measurements, hairpin DNA (hpDNA) containing a loop region of four nucleobases between the two complementary strands was used. The hpDNA was selected to avoid the dissociation of the two complementary strands in the dsDNA.

[0365] Prior to the development of a fluorescence-based assay, cargo loading of all variants binding to ssDNA was investigated by EMSA ( Fig.28 ). Variants OTR402, OTR501, OTR502, OTR503 and OP were found to bind hpDNA, whereas OTR301, OTR302 and OTR401 did not. -10 M -1 The capsid variant with the greatest binding affinity can also bind dsDNA / hpDNA.

[0366] The kinetic parameters for binding to hpDNA for all variants having 5 or more arginine mutations per monomer are shown in Table 8 below.

[0367] Table 8: Summary of kinetic parameters. Association rate constant k on and the dissociation rate constant k off To determine the dissociation constant K d .

[0368]

[0369] Example 11 - Variation of Externally Displayed Histidine-Rich Peptides

[0370] OP cages carrying one hexa-histidine tag per monomeric protein at the C-terminus have been shown to effectively escape endosomal entrapment and thereby successfully release their siRNA cargo into the cytoplasm of cells for gene knockdown. Variants of OP cages carrying histidine tags containing 3, 6, or 9 histidines fused to the C-terminus were generated and assayed for gene knockdown activity with reference to OP cages containing a 6-histidine tag. Fig.29 As shown, all three variants showed the same ability to deliver siRNA into mammalian cells and reduce the expression level of the target protein (in this case GFP). Additionally, the importance of the position of the hexa-histidine tag was investigated by testing the gene silencing activity of the variants OP-cODC. After the hexa-histidine tag, OP-cODC contains a 38 amino acid C-terminal appendage. Fig.29 As shown in , this abolished the gene knockdown ability of siRNA containing the OP cage, possibly by interfering with the endosomal escape mechanism of the hexahistidine tag.

[0371] Example 12 - Immunogenicity of OP Cages in Mice

[0372] Two groups of four BALB / c mice received 1 or 10 μg of PBS containing OP particles by tail vein injection on days 0, 16 and 38, and blood was collected on days 0, 14, 29, 52 and 73. Evaluation of OP-specific IgG1, IgG2a and IgG2b in serum was performed by enzyme-linked immunosorbent assay (ELISA). Briefly, two-fold dilution series of mouse serum samples were applied to OP-coated multiwell immunoplates (Sigma M9410) and then detected by biotinylated anti-mouse antibodies against different IgG isotypes (Abcam ab97238 / ab97243 / ab97248). Colorimetric development was performed using streptavidin-HRP (Biolegend 405210) and 3,3',5,5'-tetramethylbenzidine (TMB, Biolegend 77247 / 77248) as chromogenic substrates. The optical density of the chromogenic substrate was measured at 450 nm and plotted against the dilution of the serum sample ( Fig.30 The 1 / 2000 dilution was selected for comparison of immune responses at each time point ( Fig.31 After all injections, elevated anti-OP IgG titers and dose-dependent responses were determined ( Fig.31 ). On day 72, one month after the second booster immunization, the IgG titer showed a slight retraction. This study shows that OP particles induce a specific immune response.

Claims

1. A polypeptide comprising an amino acid sequence I, wherein the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16, 37 and 38.

2. The polypeptide according to claim 1, wherein the amino acid sequence I is an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5 and SEQ ID NOs: 10 to 16.

3. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

2.

4. The polypeptide according to claim 1, wherein the amino acid sequence I is the amino acid sequence of SEQ ID NO:

4.

5. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

5.

6. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

12.

7. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

13.

8. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

14.

9. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

15.

10. The polypeptide according to claim 1, wherein the amino acid sequence 1 is the amino acid sequence of SEQ ID NO:

16.

11. The polypeptide according to claim 1, wherein the polypeptide further comprises a histidine tag, wherein the histidine tag consists of at least two consecutively linked histidines. The polypeptide according to claim 11 , wherein the histidine tag consists of 6 consecutively linked histidines.

13. A nucleic acid sequence encoding the polypeptide according to any one of claims 1 to 12.

14. A nanoparticle comprising at least one polypeptide according to any one of claims 1 to 12.

15. A complex comprising the nanoparticle according to claim 14 and one or more cargo molecules, wherein the one or more cargo molecules are encapsulated in the nanoparticle.

16. The complex of claim 15, wherein the one or more cargo molecules are one or more oligonucleotides.

17. The complex of claim 16, wherein the one or more oligonucleotides are RNAs selected from the group consisting of antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), micro RNAs (miRNAs), and anti-miRNAs.

18. The complex of claim 16, wherein the one or more oligonucleotides encapsulated in the nanoparticle are not susceptible to DNase hydrolysis.

19. A method for transfecting a cell, said method comprising the step of contacting said cell with a complex according to claims 15 to 18.

Images