Fusion polypeptides for cell penetration and cell targeting

Abstract

The present invention relates to a fusion polypeptide comprising a cell penetrating domain and a substrate binding domain. In addition, the invention relates to genetically modified cells expressing these fusion polypeptides and the use of these fusion polypeptides for introducing a substance into a target cell.

Description

[0001] The present invention relates to fusion polypeptides comprising a cell penetrating domain and a substrate binding domain. In addition, the present invention relates to genetically modified cells expressing these fusion polypeptides and the use of these fusion polypeptides for introducing substances into target cells.

[0002] The delivery of substances to cells is the basis for successful drug targeting or specific epigenetic modifications, for example. For this purpose, active pharmaceutical ingredients or DNA / RNA sequences must be introduced into target cells. In order to achieve the introduction of sequences into cells, it is necessary to pass through the cell barrier that protects cells from environmental influences. In addition to protection from environmental influences, cells also need certain substances and signal molecules from other cells of the organism that need to enter the cell.

[0003] There are several mechanisms for material transport inside and outside the cell to maintain natural balance and provide the cell with the required substances. Independent and non-directional material transport modes entering the cell are diffusion and osmosis. These processes do not require energy and are therefore called passive material transport. Active material transport requires energy. Such active transport processes are, for example, membrane proteins, endocytosis and exocytosis.

[0004] Natural transport processes inside cells can be targeted to deliver active substances, such as, for example, delivering drugs to cancer cells or delivering genetic information for gene silencing.

[0005] Another way to introduce substances into cells is to use cell-penetrating peptides (CPP). These peptides allow for energy-independent pathways for delivering substances within cells, which are thought to be delivered almost only by energy-dependent pathways. CPP is associated with the substance, which is thought to be introduced into cells ("cargo") by covalent bonds or non-covalent interactions. CPP can be of different sizes, amino acid sequences and charges, but all CPPs have the ability to translocate plasma membranes and promote the delivery of a variety of molecular cargoes to the cytoplasm or organelles.

[0006] CPPs are specific to certain organisms or cell types, depending on the cell membrane composition. Plant cells require different CPPs from mammalian cells to introduce cargo into cells. Numata et al. ("Library screening of cell-penetrating peptide for BY-2 cells, leaves of Arabidopsis, tobacco, tomato, poplar and rice callus", (2018) 8: 10966, Nature scientific rtports) reported the screening of different CPPs for targeting different plant species. It is common that the results of different CPPs between different target plants are very different and depend on the secondary structure that the CPP is forming.

[0007] One of the challenges in utilizing CPPs is the dependency of transport efficiency on cargo. It would be preferable if a CPP were identified with good transport efficiency for a wide variety of different cargoes, e.g., transport of polypeptides as well as small molecules or DNA / RNA.

[0008] CPP, especially cationic peptides, are believed to be able to make fusion polypeptides pass through membranes through non-endosomal mechanisms. CPP Tat was compared with Arg9 to find out their ability to transduce polypeptides and nucleic acids, and it was found that most of the CPP-labeled cargo simply adhered to the cell surface without being transported into the cell, while the nucleic acid cargo was introduced ("Cell-penetrating peptides-A Reevaluation Of The Mechanism Of Cellular Uptake", Richard et al., The Journal of biological Chemistry, 278 (1) (585-590), 2003).

[0009] Therefore, it is desirable to have CPPs that can be used with different types of cargo without having to screen for the best for each type of cell and each type of cargo.

[0010] Another disadvantage of using CPPs to deliver cargo into cells is that it is difficult to produce cell penetrating peptides as transport molecules in amounts that achieve the concentrations required to deliver the appropriate amount of cargo into cells. In addition, the application of CPP-mediated transport is limited due to the high molecular weight and the large amount of negative charge required. Compared to the large amount of peptides required, the uptake of cargo via CPP-mediated transport is low. Fusion polypeptides can be a way to overcome this inefficiency problem, but covalent attachment of cargo can lead to inefficient action of the substance in the cell.

[0011] Therefore, a main object of the present invention is to provide a tool for targeted delivery of cargo inside cells, which tool is applicable to a variety of different cell types in different organisms and can be used with different types of cargo.

[0012] Another object of the present invention is to provide such a tool which can be produced economically and can be easily scaled up.

[0013] The main object of the present invention is solved by providing a fusion polypeptide comprising at least three functional domains or a fusion polypeptide consisting of at least three functional domains:

[0014] i) a substrate binding domain that non-covalently binds to a substrate, wherein the substrate is selected from a polypeptide, a polysaccharide, DNA, RNA, or a mixture of DNA and RNA;

[0015] ii) cell penetrating domain;

[0016] iii) signal sequence;

[0017] iv) optionally a linker domain; and

[0018] v) optionally a pH-sensitive autoprotease domain,

[0019] wherein the length of the cell-penetrating domain is 15 to 50 amino acid residues, preferably 20 to 40 amino acid residues and particularly preferably 24 to 34 amino acid residues, and

[0020] Wherein at least 70%, preferably at least 75% and particularly preferably 80% of the amino acid residues comprised in the cell-penetrating domain, based on the total length of the cell-penetrating domain, have a helix potential value >1.

[0021] The substrate binding domain of the fusion polypeptide according to the present invention receives a substrate or cargo to be introduced into a cell. The fusion polypeptide according to the present invention is contacted with the cargo and the cargo is non-covalently bound to the substrate binding domain.

[0022] In the present invention, it is unexpectedly found that the combination of a substrate binding domain and a cell penetrating domain allows the complex of a fusion polypeptide and a substrate to penetrate the cell membrane. The type of nucleic acid is independent of the binding process and the transport process. Naturally occurring DNA or RNA binding domains are limited to binding specific substrates. The substrate binding domain of the present invention can bind to a very wide variety of different DNA and / or RNA sequences, regardless of the composition, length, circular or linear sequence, or DNA and RNA of the sequence. Unexpectedly, the substrate binding domain binds to DNA and RNA because the naturally occurring CBM of C. chromiireducens, on which the substrate binding domain is based, exhibits the function of a CBM at pH values ​​that destabilize DNA and RNA. The substrate binding domain of the present invention can bind to DNA and RNA at pH values ​​that ensure the stability of nucleic acid molecules.

[0023] Another advantage of the substrate binding domain compared to other reported DNA / RNA binding domains is that the sequence of the DNA / RNA molecule is not altered.

[0024] It has been found that in addition to receiving the cargo that should be introduced into the cell, the substrate binding domain also plays a role in the targeted delivery of cargo inside the cell, because the substrate binding domain can also bind to cell structures (e.g., DNA / RNA or actin cytoskeleton). When there is a cell structure composed of polysaccharides or a cell structure that exhibits a polysaccharide motif, the substrate binding domain has a higher affinity for such a substrate and releases the DNA / RNA cargo inside the cell.

[0025] The cell penetrating domain of the fusion polypeptide according to the invention allows the introduction of cargo into cells by triggering a membrane translocation mechanism. Preferably, the cell penetrating domain of the fusion polypeptide according to the invention triggers endocytosis-mediated translocation or translocation by forming a transient structure.

[0026] Surprisingly, it was found that the use of a cell-penetrating domain having a length of 15 to 50 amino acid residues, preferably 20 to 40 amino acid residues and particularly preferably 24 to 34 amino acid residues and comprising, based on the total length of the cell-penetrating domain, at least 70%, preferably at least 75% and particularly preferably 80% of the amino acid residues having a helical potential value > 1 allows for high efficiency of introduction of cargo into cells and compatibility with a variety of different organisms.

[0027] The helical potential (Pα) is a determined value of the frequency of an amino acid to be incorporated into an α-helical secondary structure. Amino acids with a Pα value>1 are helical forming amino acids, while amino acids with a Pα value<1 are helical destroying amino acids. Table 1 shows an overview of the helical potential of proteinogenic amino acids according to Stryer Biochemie, 5th edition, page 73, Table 3.3.

[0028] The cell-penetrating domain according to the present invention thereby forms a compact and stable secondary helical structure, resulting in an overall improvement in cell-penetrating performance.

[0029] Table 1: Overview of amino acids and their helical potential

[0030]

[0031]

[0032] The signal sequence of the fusion polypeptide according to the invention allows the fusion polypeptide to be directed to certain cell compartments. This is beneficial for the production of these fusion polypeptides in recombinant expression systems and for the cellular targeting of cargo bound to the substrate binding domain of the fusion polypeptide.

[0033] Preferably, the signal sequence of the fusion polypeptide according to the present invention is an inclusion body promoting sequence or a secretion sequence, preferably a secretion sequence of type IV or type II secretion of gram-negative bacteria. These sequences allow the fusion polypeptide according to the present invention to be produced in a gram-negative recombinant production system (e.g., such as intestinal bacteria (E.coli)).

[0034] Furthermore preferably, the signal sequence of the fusion polypeptide according to the invention is a known signal sequence, for example as described in Stryer Biochemie, 5. Auflage, Table 12.4.

[0035] Preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the cell penetrating domain comprises or consists of at least one amino acid sequence selected from the group consisting of SEQ ID NOs.: 1 to 24 or an amino acid sequence having greater than 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.: 1 to 24.

[0036] Particularly preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the cell penetrating domain comprises or consists of at least one amino acid sequence selected from the group consisting of SEQ ID NO.: 1, 2, 3, 17, 18, 19, 20 and 21, or an amino acid sequence having greater than 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO.: 1, 2, 3, 17, 18, 19, 20 and 21.

[0037] Whenever the disclosure relates to the identity percentage between nucleic acid or amino acid sequence, these values ​​are defined as those values ​​obtained by using EMBOSS Water paired sequence alignment (nucleotide) program or using EMBOSS Water paired sequence alignment (polypeptide) program to amino acid sequence.Comparison as used in this article or sequence comparison refer to the comparison on the total length of two sequences compared to each other.Those tools for local sequence alignment provided by European Molecular Biology Laboratory (European Molecular Biology Laboratory, EMBL) European Bioinformatics Institute (European Bioinformatics Institute, EBI) use modified Smith-Waterman algorithm (see Smith, TF&Waterman, MS " Identification of common molecular subsequences " Journal of Molecular Biology, 1981 147 (1): 195-197).When comparing, the default parameters limited by EMBL-EBI are used. Those parameters are (i) for amino acid sequences: matrix = BLOSUM62, gap open penalty = 10, and gap extension penalty = 0.5, or (ii) for nucleic acid sequences: matrix = DNAfull, gap open penalty = 10, and gap extension penalty = 0.5. The skilled person will be aware of the fact that a sequence encoding a polypeptide may be "codon-optimized" if, for example, the corresponding sequence is to be used in another organism than the original organism from which the molecule was derived.

[0038] Preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the substrate binding domain comprises at least one of the amino acid motif sequences IHYWN and VYVKV.

[0039] In terms of the present invention, it was found that a preferred amino acid motif sequence has a significant influence on the binding properties of the substrate binding domain and is found in all substrate binding domains according to the present invention.

[0040] Furthermore, it is preferred if two, three, four, five or more repetitions of these two amino acid motif sequences are present in the substrate binding domain according to the invention. Preferably, the repetitions are incorporated into the substrate binding domain in consecutive order or separated by short sequences of hydrophobic amino acids. For the purposes of the present invention, it is preferred if the maximum length of the short sequence of hydrophobic amino acids is 20 amino acids, of which at least 50%, preferably 60%, particularly preferably 70% of the amino acids, depending on the length of the short sequence, are hydrophobic amino acids.

[0041] For the purposes of the present invention, a hydrophobic amino acid is an amino acid selected from the group consisting of alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan and methionine.

[0042] Preferably, the present invention also relates to a fusion polypeptide according to the invention, wherein the substrate binding domain comprises or consists of an amino acid sequence of 200 to 600, preferably 250 to 550 amino acids in length.

[0043] Furthermore, preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the substrate binding domain comprises or consists of at least one amino acid sequence selected from the group consisting of SEQ ID Nos.: 25 and 26 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID Nos.: 25 and 26.

[0044] Preferably, the present invention relates to a fusion polypeptide according to the invention, wherein the signal sequence is an inclusion body promoting sequence or a secretion sequence, preferably a secretion sequence of type IV or type II secretion of Gram-negative bacteria.

[0045] Furthermore, preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the signal sequence comprises or consists of at least one amino acid sequence selected from the group consisting of SEQ ID Nos.: 27 and 28 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID Nos.: 27 and 28.

[0046] Preferably, the fusion polypeptide according to the present invention comprises a linker domain and preferably, the linker domain comprises an N-terminal alpha helix and / or a C-terminal sequence of a random coil structure, preferably, wherein the linker domain comprises or consists of at least one amino acid sequence selected from SEQ ID No.: 29 and 30 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or preferably at least 99% sequence identity with an amino acid sequence selected from SEQ ID No.: 29 and 30.

[0047] A linker sequence in the context of the present invention means a sequence between functional domains. The length of the linker is preferably 1 to 50 or more than 50 amino acids.

[0048] Preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the substrate non-covalently bound to the substrate binding domain is a DNA or RNA with a length of 3 to 3,000,000 base pairs, preferably a length of 3,000 to 30,000 base pairs, particularly preferably a length of 30,000 to 300,000 base pairs.

[0049] Likewise preferably, the substrate binding domain of the fusion polypeptide according to the invention binds to a polypeptide having an amino acid sequence length of 5 to 250 amino acids, preferably 20 to 200 amino acids and particularly preferably 50 to 150 amino acids.

[0050] Furthermore preferably, the substrate binding domain of the fusion polypeptide according to the invention binds to a polypeptide comprising a polysaccharide tag of minimum length of a disaccharide, preferably a pullulan tag or a dextrin tag.

[0051] Preferably, the present invention relates to a fusion polypeptide according to the present invention, wherein the fusion polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID No.: 32 to 99 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID No.: 32 to 99.

[0052] SEQ ID NO.: 32 to 99 are sequences of fusion polypeptides having functional domains according to the present invention. These fusion polypeptides are suitable for transferring a wide variety of different substrates into cells.

[0053] Preferably, the substrate is selected from the following: antibodies such as bermekimab or clazakizumab, and bermekimab or clazakizumab labeled with polysaccharides, small molecules such as adamantane or fullerene, and adamantane or fullerene labeled with polysaccharides, antibiotics such as chloramphenicol or kanamycin, and chloramphenicol and kanamycin labeled with polysaccharides, biological agents such as insulin or human growth factor, and insulin or human growth factor labeled with polysaccharides, polypeptides such as melatonin or lipocalin, and melatonin or lipocalin labeled with polysaccharides, transcription factors such as zinc finger polypeptides, DNA (such as plasmid DNA, viral double-stranded DNA or linear cDNA), RNA (such as siRNA, mRNA, or double-stranded RNA), or a mixture of DNA and RNA.

[0054] Preferably, the present invention relates to a fusion polypeptide, wherein the fusion polypeptide comprises or consists of the five functional domains i) to v), i.e. domains iv) and v) are present in such case and are not optional,

[0055] Preferably, the pH-sensitive autoprotease domain comprises or consists of at least one amino acid sequence according to SEQ ID NO.: 31 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with the amino acid sequence according to SEQ ID NO.: 31.

[0056] The pH sensitive autoprotease is capable of releasing peptides in response to the intracellular pH. Preferably, the pH sensitive autoprotease is active at a pH of 6.8 to 7.2.

[0057] Another aspect of the present invention relates to a recombinant nucleic acid molecule encoding a fusion polypeptide according to the present invention.

[0058] Preferably, the cell penetrating peptide domain of the fusion polypeptide according to the present invention is encoded by a nucleic acid molecule selected from SEQ ID NO.: 100 to 123 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with a nucleic acid molecule selected from SEQ ID NO.: 100 to 123.

[0059] Preferably, the substrate binding domain of the fusion polypeptide according to the present invention is encoded by a nucleic acid molecule selected from SEQ ID NO.: 124 and 125 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with a nucleic acid molecule selected from SEQ ID NO.: 124 and 125.

[0060] Preferably, the signal sequence domain of the fusion polypeptide according to the present invention is encoded by a nucleic acid molecule selected from SEQ ID NO.: 126 and 127 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with a nucleic acid molecule selected from SEQ ID NO.: 126 and 127.

[0061] Preferably, the fusion polypeptide according to the present invention comprises a linker domain, which is encoded by a nucleic acid molecule selected from SEQ ID NO.: 128 and 129 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with a nucleic acid molecule selected from SEQ ID NO.: 128 and 129.

[0062] Preferably, the fusion polypeptide according to the present invention comprises a pH-sensitive autoprotease domain, which is encoded by a nucleic acid molecule according to SEQ ID NO.: 130 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with the nucleic acid molecule according to SEQ ID NO.: 130.

[0063] Preferably, the fusion polypeptide according to the present invention is encoded by a nucleic acid molecule selected from SEQ ID NO.: 131 to 250 or a nucleic acid molecule having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with a nucleic acid molecule selected from SEQ ID NO.: 131 to 250.

[0064] Another aspect of the present invention relates to a genetically modified cell comprising a recombinant nucleic acid molecule according to the present invention, wherein the cell is capable of expressing a fusion polypeptide according to the present invention, preferably, wherein the cell is selected from Escherichia coli, Vibrio natrigens, Saccheromyces cerevisiae, Aspergillus niger, green algae, microalgae, HEK T293 and Chinese hamster ovary cell (CHO).

[0065] Yet another aspect of the present invention relates to a method for producing a fusion polypeptide according to the present invention, comprising the following steps:

[0066] a) providing a genetically modified cell according to the invention;

[0067] b) culturing cells under conditions suitable for the expression of the fusion polypeptide according to the present invention;

[0068] c) obtaining a fusion polypeptide and optionally, unfolding the obtained fusion polypeptide and directionally refolding the fusion polypeptide.

[0069] Step (a) comprises providing a genetically modified cell expressing a fusion polypeptide according to the present invention. Such a cell can be obtained by introducing a nucleic acid molecule (preferably in the form of a vector) comprising a sequence encoding a fusion polypeptide according to the present invention into the cell by known methods (e.g., such as by transfection or transformation).

[0070] In step (b), the cells are cultured under conditions suitable for expression of the fusion polypeptide according to the invention, preferably in high density culture. Culture conditions and in particular conditions for achieving high density culture and corresponding culture media are well known to those skilled in the art. Preferably, expression of the fusion polypeptide is achieved using a suitable signal sequence according to the invention followed by transport to inclusion bodies.

[0071] If the fusion polypeptide is present in inclusion bodies, step (c) comprises obtaining the fusion polypeptide from the culture broth and optionally unfolding the obtained fusion polypeptide and subjecting the fusion polypeptide to directed refolding.

[0072] The solubilization conditions for treating the inclusion bodies and the conditions for directional refolding are well known in the art. Preferably, the inclusion bodies are solubilized by using 6M guanidine chloride, 8M urea or 2% sodium dodecyl sulfate and refolded under neutral or weakly alkaline conditions.

[0073] Preferably, the fusion polypeptide is produced as described in application PCT / EP2023 / 053466.

[0074] One aspect of the invention relates to a method for introducing a substrate into a cell, comprising the steps of:

[0075] a) providing a fusion polypeptide according to the invention, preferably obtained by a method according to the invention;

[0076] b) contacting the fusion polypeptide according to the invention with a substrate to be introduced into the cell;

[0077] c) obtaining a fusion polypeptide having a substrate, wherein the substrate is non-covalently bonded to the substrate binding domain of the fusion polypeptide;

[0078] d) contacting the fusion polypeptide / substrate complex obtained in step c) with cells into which the substrate is to be introduced.

[0079] e) Obtaining cells with the introduced substrate.

[0080] Preferably, the method is performed ex vivo.

[0081] Preferably, the method comprises the following steps:

[0082] (i) culturing a genetically modified cell according to the present invention, which expresses a fusion polypeptide according to the present invention;

[0083] (ii) purifying the expressed fusion polypeptide obtained in step (i);

[0084] (iii) contacting the expressed fusion polypeptide with a substance to be introduced, preferably DNA, RNA or a mixture thereof;

[0085] (iv) contacting the mixture obtained in step (iii) with cells into which the substance is to be introduced;

[0086] (v) obtaining a cell, preferably a transformed cell, with the introduced substance,

[0087] Wherein steps (i) to (iv) are performed ex vivo, preferably in vitro.

[0088] Preferably, the contacting step b) or iv) is carried out at a temperature between 20°C and 37°C and a pH between 6 and 9 in the presence of a buffer selected from TRIS, HEPES, PIPES or a buffer comprising sodium acetate or arginine and sucrose.

[0089] Also preferably, if plant cells are to be transformed or substances are to be introduced into plant cells, the contacting step d) or iv) is carried out using a low pressure spray method (LPS). Preferably, if the substances are to be introduced into organisms other than plants, the mixture obtained in step (iii) is contacted with the cells in the presence of a buffer, which is ideal for the target cells.

[0090] Those skilled in the art are aware of appropriate buffers for use with specific cells and organisms.

[0091] Another aspect relates to a kit for transforming cells, comprising at least one fusion polypeptide according to the invention.

[0092] Preferably, the kit is a transformation kit and further comprises buffers and reagents suitable for transformation of the target cells. Suitable buffers and reagents are known to those skilled in the art.

[0093] Another aspect of the present invention relates to the non-therapeutic use of the fusion polypeptide according to the present invention, which is used to introduce a substance into a target cell, wherein the substance is selected from antibodies, small molecules, antibiotics, biological agents, polypeptides, transcription factors or DNA, RNA, or a mixture of DNA and RNA.

[0094] Preferably, the substrate is selected from the following: antibodies such as bemakizumab or clazakizumab and bemakizumab or clazakizumab labeled with polysaccharides, small molecules such as adamantane or fullerene and adamantane or fullerene labeled with polysaccharides, antibiotics such as chloramphenicol or kanamycin and chloramphenicol and kanamycin labeled with polysaccharides, biological agents such as insulin or human growth factor and insulin or human growth factor labeled with polysaccharides, polypeptides such as melatonin or lipocalin and melatonin or lipocalin labeled with polysaccharides, transcription factors such as zinc finger polypeptides, DNA (such as plasmid DNA, viral double-stranded DNA or linear cDNA), RNA (such as siRNA, mRNA, or double-stranded RNA), or a mixture of DNA and RNA.

[0095] Another aspect of the present invention relates to non-therapeutic uses of the fusion polypeptide according to the present invention, wherein the target cell is selected from the group consisting of plant cells, bacterial cells, mammalian cells and yeast cells, preferably plant cells.

[0096] Further aspects and advantages of the invention result from the following description of preferred embodiments.

[0097] Sequence Description

[0098] SEQ ID NO.: 1 to SEQ ID NO. 24 describe amino acid sequences encoding artificial cell-penetrating peptides 1 to 24.

[0099] SEQ ID NOs.: 25 and 26 depict the amino acid sequences encoding the binding domains.

[0100] SEQ ID NOs.: 27 and 28 depict the amino acid sequences encoding the signal sequences.

[0101] SEQ ID NOs.: 29 and 30 depict the amino acid sequences encoding the linker domain.

[0102] SEQ ID NO.: 31 depicts the amino acid sequence encoding a pH-sensitive autoprotease.

[0103] SEQ ID NO.: 32 to 99 describe the amino acid sequences encoding the fusion polypeptides according to the present invention.

[0104] SEQ ID NOs.: 100 to 123 describe nucleic acid sequences encoding artificial cell penetrating peptides 1 to 24.

[0105] SEQ ID NO.: 124 and 125 describe nucleic acid sequences encoding the binding domains.

[0106] SEQ ID NO.: 126 and 127 depict nucleic acid sequences encoding linker domains.

[0107] SEQ ID NOs.: 128 and 129 depict nucleic acid sequences encoding linker domains.

[0108] SEQ ID NO.: 130 depicts a nucleic acid domain encoding a pH sensitive autoprotease.

[0109] SEQ ID No.: 131 to 250 describe nucleic acid sequences encoding the fusion polypeptides according to the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1 : A graph showing the average of the GFP silencing ratios of all silenced leaves relative to the total leaf area from the apical meristem of the treated lines. The results of the GFP silencing assay using the transporter-22nt siRNA, R9-transporter-22nt siRNA, R5A4-transporter-22nt siRNA, S4 13 PV-22nt siRNA, R9-S4 13 PV-22nt siRNA and KLAVH 16-22nt siRNA complexes with LPS treatment, and total results (combined). 10% CPD-22nt siRNA complex controls are shown under the category "combined". The number of leaves evaluated depended on the individual and ranged from 1 to 6.

[0112] Figure 2 : Number of GFP-silenced flowers developed in N. benthamiana 16C of the first progeny (F1) treated with CPP-22nt siRNA complex LPS. The concentration of siRNA applied was 14 ng μl-1.

[0113] Figure 3 : Relative amount of silenced progeny in F1 generation relative to parental plants. Each column (sample) is derived from a single treated maternal plant. The number at the end of the cell line name indicates the molar siRNA / CPP ratio applied (e.g. 50=50:1). Seeds were germinated on 0.5×MS medium under sterile conditions, n=200.

[0114] Figure 4 Figure A shows the colony growth of E. coli BL21 on an agar plate. No Figure B shows the colony growth on an agar plate of E. coli BL21, which was incubated with a fusion polypeptide according to the invention having a binding domain according to SEQ ID NO.: 25 and a cell penetrating peptide according to SEQ ID NO.: 18, with the application of heat shock. Figure C shows the colony growth on an agar plate of E. coli BL21 after incubating the cells with the plasmid without applying heat shock. Figure C shows the colony growth on an agar plate of E. coli BL21 after incubating the cells with the plasmid with the application of heat shock. The agar plate contains the antibiotics kanamycin and chloramphenicol, wherein the plasmid carries genes that convey resistance to kanamycin and chloramphenicol.

[0115] Figure 5 : Gel showing digestion of plasmids present in E. coli cells grown under conditions supplemented with antibiotics Figure 4 agar plates. F1 shows the digestion of the plasmid found in the colonies according to panel A, F2 shows the digestion of the plasmid found in the colonies according to panel B, F3 shows the digestion of the plasmid found in the colonies according to panel C, and F4 shows the digestion of the plasmid found in the colonies according to panel D. The band on the right shows the 1 kb DNA marker. Example

[0116] Example 1: Treatment of plants with siRNA fusion polypeptide complexes

[0117] The fusion polypeptides according to SEQ ID NO.: 67, 68, 69, 52, 53, 48 and 72 were subjected to DNase I digestion at 37°C for 15 minutes to eliminate the remaining DNA from the subsequent purification step. DNase I was inactivated at 65°C for 10 minutes. Thereafter, siRNA was contacted with the fusion polypeptide samples. The siRNA concentration was 14 ng / μl and the molar ratio of siRNA to CPP carrying the fusion polypeptide was 5:1, 10:1 and 50:1. In the presence of 0.1% In the case of L-77, they were mixed at 1100 rpm for 2 hours at room temperature.

[0118] In one aspect, low pressure spray (LPS) is used for plant treatment. The method involves spraying siRNA: fusion polypeptide complexes at 1.3 bar at nanomolar amounts / 100 μL to 500 μL buffer (1:10 siRNA: fusion polypeptide ratio) on Nicotiana benthamiana plants. Nicotiana benthamiana plants express a GFP transgene that is silenced by the introduced siRNA.

[0119] Prepare the plants by incubating them in a highly humidified environment at 60% to 100% humidity for one hour. TM HP-200 spray gun sprays the leaves and / or stems of the plants. The siRNA: fusion polypeptide mixture is applied using young plants that are two to fourteen days after the end of the embryonic stage as the object of treatment. The young plants are treated. The organs treated are meristems, leaves themselves, and leaf edges.

[0120] The siRNA used to generate the results was an RNA sequence of 22 amino acids in length. The fusion polypeptides used were designed with the following cell penetrating peptides: transporter-(SEQ ID No.: 16), R9-transporter (SEQ ID No.: 17), R5A4-transporter (SEQ ID NO.: 18), S4 13 PV (SEQ ID NO.: 1), R9-S4 13 PV (SEQ ID NO.: 2), and KLAVH 16 (SEQ ID NO.: 21). Therefore, fusion polypeptides having SEQ ID NO.: 67, 68, 69, 52, 53, 48 and 72 were used in this experiment.

[0121] The results were measured as the GFP silencing ratio of all silenced leaves originating from the top medical stem. The results were compared with the method of introducing siRNA into cells using carbon polymer dots (as described in Schwartz et al., 2020, "Carbon Dots for Efficient Small Interfering RNA Delivery and Gene Silencing in Plants").

[0122] exist Figure 1 The results of the gene silencing experiment of the parental generation are shown. ... 13 PV-22nt siRNA, R9-S4 13 PV-22nt siRNA and KLAVH 16 -22nt siRNA complexes with LPS treatment, and total results (combined). Results for 10% CPD (carbon polymer dot)-22nt siRNA complex controls are shown under the bar "combined" because only one fixed ratio was applied. The number of leaves evaluated depended on the individual and ranged from 1 to 6.

[0123] The GFP silencing of the first progeny (F1) of the treated plants was also studied. It was found that silencing was observed in the case where the fusion polypeptide according to the present invention designed with a cell-penetrating peptide was used to carry the 22nt siRNA inside the target cell, and silencing also occurred in the F1 generation (first progeny). The applied concentration of siRNA was 14ngμl -1 .

[0124] The results are Figure 2 , and shows that several plants (Nicotiana benthamiana) also showed silenced GFP levels in the first progeny F1 while maintaining seed production capacity. The relative amount of silenced plants in the F1 generation relative to the parental generation was Figure 3 The numbers at the end of the sample names indicate the molar siRNA / CPP ratio applied (eg 50=50:1). Seeds were germinated on 0.5×MS medium under sterile conditions, and the sample size was 200.

[0125] Example 2: Transfection of tobacco leaves for gene silencing

[0126] Any of the fusion polypeptides according to SEQ ID NO.: 32 to 99 can be used as a transport vector for introducing DNA into plant leaves. In the following, fusion polypeptides according to SEQ ID NO.: 67, 68, 69, 52, 53, 48 and 72 were used.

[0127] Step 1) The pH of the fusion polypeptide sample is adjusted to pH 7.7 to pH 8.3.

[0128] Step 2) Add 80 μl aliquots of 1000 U DNase I to samples of more than one gram of fusion polypeptide to eliminate excess DNA from subsequent purification steps. Gently stir the mixture at 37°C for 30 minutes. Then, allow the sample to relax at room temperature for at least 30 minutes. Then, incubate DNase I at 65°C for 30 minutes.

[0129] Step 3) Determine the concentration of the fusion polypeptide photometrically and mix the fusion polypeptide with the desired siRNA at the desired ratio in a buffer with a pH greater than 7.2. Incubate the sample at room temperature without stirring for at least ten minutes.

[0130] Step 4) Spray the mixture on growing plants grown on a suitable medium and observe for a few days.

[0131] This treatment resulted in systemic gene silencing, which means that the effect was not restricted to penetration sites. Example 3: Using root Transformation of Escherichia coli with the fusion polypeptides of the present invention (SEQ ID NO.: 67, 68, 69, 52, 53, 48 and 72)

[0132] The method using the fusion polypeptide according to the invention was compared with the known heat shock protocol for transformation of E. coli cells:

[0133] Step 1) Four 100 μl calcium competent BL21 E. coli cells were thawed in a refrigerator at 4° C. for 15 minutes. Four LB agar plates with 15 g / L agarose, 30 mg / L kanamycin and 30 mg / L chloramphenicol were incubated at 37° C. with 4 mL SOC (Super optimal broth with catabolite repression) medium.

[0134] Step 2) After thawing, the cells were contacted with 500 ng of pure plasmid DNA or 500 ng of plasmid DNA with fusion polypeptides according to SEQ ID NO.: 67, 68, 69, 52, 53, 48 and 72, which additionally carried the autologous protease domain according to SEQ ID NO.: 31, which were mixed with the plasmid DNA at a ratio of 1:10 in favor of the DNA as a carrier of known protein concentration. The plasmid contained resistance to chloramphenicol and kanamycin.

[0135] Step 3) Incubate the cells from step 2) at 4°C.

[0136] Step 4) After incubation at 4° C., each of the two vials with cells applied with different DNA according to step 2 was heat shocked at 42° C. for one minute and then relaxed at 4° C. for 2 minutes. The other two vials were placed at 4° C. for the same amount of time.

[0137] Step 5) After heat shock treatment or no heat shock treatment, respectively, all four vials were treated with 900 μl of warm SOC medium and stirred at 37° C. for one hour.

[0138] Step 6) Transfer an aliquot of cells to warm LB agar plates and incubate them at 37°C overnight.

[0139] Step 7) The growth on all plates was evaluated and can be seen as shown in Table 1 and Figure 4 As summarized in .

[0140] Table 1: Summary of comparison of transformation protocols

[0141]

[0142] The results show that the fusion polypeptide system is able to a) take up plasmid DNA and b) upon penetrating the membrane, be actively transported into the cell without utilizing the cell's transport system.

[0143] The presence of the resistance gene in all growing colonies ( Figure 5 ), wherein the highest amount of resistance genes can be extracted from cells which have been transformed by using the fusion polypeptide according to the invention.

Claims

1. A fusion polypeptide comprising or consisting of at least three functional domains: i) a substrate binding domain that non-covalently binds to a substrate, wherein the substrate is selected from a polypeptide, a DNA, an RNA, or a mixture of a DNA and an RNA, and Wherein the substrate binding domain comprises or consists of: at least one amino acid sequence selected from the group consisting of SEQ ID NOs.: 25 and 26, or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs.: 25 and 26; ii) cell penetrating domain; iii) signal sequence; iv) optionally a linker domain; and v) optionally a pH-sensitive autoprotease domain, wherein the length of the cell-penetrating domain is 15 to 50 amino acid residues, preferably 20 to 40 amino acid residues and particularly preferably 24 to 34 amino acid residues, and Wherein, based on the total length of the cell-penetrating domain, at least 70%, preferably at least 75% and particularly preferably at least 80% of the amino acid residues comprised by the cell-penetrating domain have a helical potential value >1.

2. The fusion polypeptide according to claim 1, wherein the cell penetrating domain comprises or consists of: At least one amino acid sequence selected from SEQ ID NO.: 1 to 24 or an amino acid sequence having greater than 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from SEQ ID NO.: 1 to 24.

3. The fusion polypeptide according to any one of the preceding claims, wherein the signal sequence is an inclusion body promoting sequence or a secretion sequence, preferably a secretion sequence of type IV or type II secretion of Gram-negative bacteria.

4. A fusion polypeptide according to any one of the preceding claims, wherein the signal sequence comprises or consists of: At least one amino acid sequence selected from SEQ ID NO.: 27 and 28 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from SEQ ID NO.: 27 and 28.

5. A fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises a linker domain, and wherein The linker domain comprises an N-terminal α-helix and / or a C-terminal sequence of a random coil structure, preferably, wherein the linker domain comprises or consists of: At least one amino acid sequence selected from SEQ ID NO.: 29 and 30 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98% or preferably at least 99% sequence identity with an amino acid sequence selected from SEQ ID NO.: 29 and 30.

6. A fusion polypeptide according to any one of the preceding claims, wherein the substrate non-covalently bound to the substrate binding domain is a DNA or RNA with a length of 3 to 3,000,000 base pairs, preferably a length of 3,000 to 30,000 base pairs, and particularly preferably a length of 30,000 to 300,000 base pairs.

7. A fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises or consists of: An amino acid sequence selected from the group consisting of SEQ ID NOs.: 32 to 99, or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NOs.: 32 to 99.

8. The fusion polypeptide according to any one of the preceding claims, wherein the fusion polypeptide comprises or consists of five functional domains i) to v), Preferably, the pH-sensitive autoprotease domain comprises or consists of: At least one amino acid sequence according to SEQ ID NO.: 31 or an amino acid sequence having at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity with the amino acid sequence according to SEQ ID NO.:

31.

9. A recombinant nucleic acid molecule encoding a fusion polypeptide according to any one of the preceding claims.

10. A genetically modified cell comprising the recombinant nucleic acid molecule according to claim 9, wherein the cell is capable of expressing the fusion polypeptide according to any one of claims 1 to 8, preferably, wherein the cell is selected from Escherichia coli, Vibrio natrigens, Saccheromyces cerevisiae, Aspergillus niger, green algae, microalgae, HEK T293 and Chinese hamster ovary cells (CHO).

11. A method for introducing a substance into a cell, preferably DNA, RNA or a mixture thereof, the method comprising the steps of: (i) culturing the genetically modified cell according to claim 10, wherein the genetically modified cell expresses the fusion polypeptide according to any one of claims 1 to 8; (ii) purifying the expressed fusion polypeptide obtained in step (i); (iii) contacting the expressed fusion polypeptide of step (ii) with a substance to be introduced, preferably DNA, RNA or a mixture thereof; (iv) contacting the mixture obtained in step (iii) with cells into which the substance is to be introduced; (v) obtaining a cell, preferably a transformed cell, with the introduced substance, Wherein steps (i) to (iv) are performed ex vivo, preferably in vitro.

12. The method according to claim 11, wherein the cells into which the substance is introduced are selected from eukaryotic and prokaryotic organisms, preferably selected from microorganisms, yeast cells, fungal cells, plant cells, green algae, microalgae, HEK T293 and Chinese hamster ovary cells (CHO), and preferably, wherein the contacting in step (iv) is carried out at a temperature of 2°C to 40°C for a period of 10 minutes to 360 minutes.

13. A kit for transforming cells, comprising at least one fusion polypeptide according to any one of claims 1 to 8.

14. Non-therapeutic use of the fusion polypeptide according to any one of claims 1 to 8 for introducing a substance into a target cell, wherein the substance is selected from antibodies, small molecules, antibiotics, biological agents, polypeptides, transcription factors or DNA, RNA, or a mixture of DNA and RNA.

15. The non-therapeutic use of the fusion polypeptide according to claim 14, wherein the target cell is selected from the group consisting of plant cells, bacterial cells, mammalian cells and yeast cells, preferably plant cells.

Images