Cell membrane penetrating conjugates for gene editing

Abstract

A genome editing complex for modifying a target polynucleotide, comprising a recombinant beta helical protein linked to one or more genome editing system molecules or a plasmid encoding one or more genome editing system molecules, wherein the beta helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

Description

Technical Field

[0001] This disclosure broadly relates to the field of delivering one or more gene-editing molecules into the cytoplasm of cells, and specifically discloses a conjugate comprising a recombinant protein linked to one or more gene-editing molecules for penetrating cell membranes, a method for preparing said conjugate, and its use. This disclosure also relates to a conjugate comprising a recombinant protein linked to a nucleic acid or plasmid encoding one or more gene-editing molecules for penetrating cell membranes, a method for preparing said conjugate, and its use. Background Technology

[0002] The cell membrane is a semi-permeable membrane that separates the cell's internal environment from its external environment. While prokaryotic and eukaryotic cell membranes differ in some properties and composition, they both consist of a semi-permeable bilayer structure of phospholipids. The semi-permeable nature of the cell membrane makes it selective in terms of the types of molecules that can permeate it. Those molecules that can permeate the cell membrane hold promise for applications in cell labeling, cell penetration, cell delivery, drug uptake, gene therapy, and many other applications involving cell membrane penetration.

[0003] Certain peptides exist that can penetrate the cell membrane and translocate into the cytoplasm; these peptides are called cell-penetrating peptides (CPPs). CPP conjugates linked to one or more functional molecules have been studied as a means of transmembrane delivery of various bioactive molecules. For example, in Caco-2 cells, treatment with the CPP conjugate—CPP insulin—significantly increased insulin uptake (6-8 times) (Liang et al., Biochem. Biophys. Res. Commun.; 2005; 335(3):734-738). Conjugates including Tat peptides have shown similar results (ibid.). Another study has reported the use of Pep-1, a short amphiphilic peptide carrier that can penetrate membranes and deliver various peptides and proteins in several cell lines (Morris et al., Nature Biotechnol., 2001, 1173-1176).

[0004] CPP has also been used to study the efficient delivery of various anticancer drugs as drug-CPP conjugates, which can penetrate cell membranes more effectively than drugs alone due to their properties. One such study reported the use of Tat protein conjugated with a CK2 inhibitor (P15) for the treatment of solid tumors (Perea et al., Cancer Res. 2004, 7127-7129).

[0005] Several molecular transporters exist that can deliver molecules across cell membranes. Guanidinium-rich molecular transporters (GR-MoTrs), including peptide and non-peptide drugs, have been shown to penetrate cell membranes due to the number and spatial array of guanidinium groups. GR-MoTrs can enhance the delivery of various cargoes containing small molecules, metals, imaging agents, iron particles, and proteins within mammalian cells (Wender et al., *Advanced Drug Delivery Review*, 2008, 452-472; Wender et al., *Drug Discovery Today: Technology*, 2012, e49-e55).

[0006] US20130137644 discloses a conjugate composed of a cell-penetrating peptide, a nucleic acid, and a hydrophilic polymer, which allows the conjugate to penetrate cell membranes with increased efficiency. The nucleic acid used in the conjugate is described as preferably siRNA using polyethylene glycol (PEG) as the hydrophilic polymer.

[0007] US20040176282 discloses methods and uses of compositions for cellular delivery of nucleic acids, peptides, fluorophores, and molecular complexes. Intracellular release of bioactive molecules following cell penetration is stimulated by photoactivated dispersion of the complex. This system helps to inhibit the biological function of molecules while they are part of the complex, but once inside the cell and upon photoactivation, the molecules disperse and their biological activity can be restored.

[0008] Furthermore, and generally speaking, most mechanisms developed for cell delivery, which aim to deliver all functional molecules into the cell, rely on endocytosis-dependent mechanisms for entry into the cell. The efficiency of translocation, due to, for example, drug capture in endosomes or degradation in lysosomes, has become a major area of ​​concern in endocytosis-dependent pathways. Therefore, there is an urgent need to devise novel mechanisms for more reliable and efficient penetration of the cell membrane.

[0009] One example of a promising and widely used technology involving functional molecules crossing the cell membrane barrier is genome engineering, which involves editing DNA (i.e., inserting, deleting, modifying, or replacing one or more nucleotides) in the genome of a living organism.

[0010] One type of genome engineering is gene therapy. Gene therapy involves delivering the gene of interest into cells to compensate for abnormal gene activity or to provide beneficial proteins. Gene therapy has proven beneficial in treating diseases such as chronic lymphocytic leukemia, X-linked SCID, multiple myeloma, and hemophilia. Many life-threatening diseases have an underlying genetic origin, meaning the disease is caused by dysfunction or lack of proper function displayed by one or more related genes. Gene therapy has shown promise in treating such disease conditions. However, gene therapy still faces challenges in delivering the desired gene across the cell membrane. To date, two methods for delivering genes have been used—virus-based and non-virus-based.

[0011] Virus-based methods for gene therapy utilize attenuated viruses as vectors, within which a desired gene is cloned and transferred into the target cells via a process called transduction. The advantage of this method is the proper integration of the delivered gene into the cell's genome, but it also has other disadvantages, one of which is the tendency to induce cancer in cases of inappropriate genome integration. Non-viral methods involve injecting isolated DNA into cells and using cationic lipids to surround the plasmid DNA (lipid transfection). Non-viral methods do not require any integration of the gene into the genome and are inefficient in transferring the desired gene into other cells within the tissue. Therefore, while gene therapy is a promising and excellent technology for treating many life-threatening diseases, the problem of delivering the gene into cells remains.

[0012] For the most common inherited diseases, such as cystic fibrosis or muscular dystrophy, effective gene therapy may still be challenging due to the difficulty in delivering genetic material into cells. There is no simple way to deliver genes into a significant portion of the cells in tissues, such as the lung epithelium or skeletal muscle (Collins et al., Proceedings of the Royal Society B, Vol. 282, No. 1821. The Royal Society, 2015). Therefore, there is a need for efficient cell delivery mechanisms that could greatly enhance the benefits of gene therapy and pave the way for promising treatments for many life-threatening diseases.

[0013] Another type of genome engineering that requires functional molecules to cross the cell membrane barrier is gene or genome editing. Genome editing allows site-specific double-strand breaks to be created at desired locations in the genome, typically using engineered nucleases. These breaks can then be repaired by non-homologous end joining (NHEJ), homologous recombination (HR), or homologous directed repair (HDR), resulting in site-specific mutations or "editing" of the genome. Known engineered nucleases include zinc finger nucleases (ZFNs) and nucleases based on transcription activator-like effectors. Homing endonucleases (such as ARC nuclease) TM Or nucleic acid-guided endonucleases such as DNA-guided and RNA-guided endonucleases, such as the CRISPR system, which is primarily (but not entirely) based on clustered, regularly spaced short palindromic repeats.

[0014] Zinc finger nucleases (ZFNs) are engineered DNA-binding proteins that facilitate targeted genome editing by creating double-strand breaks in DNA at user-specified locations. Each ZFN consists of two functional domains: a) a DNA-binding domain containing two finger-like modules, each recognizing a unique hexameric (6 bp) DNA sequence. The two modules are spliced ​​together to form a zinc finger protein, with each module having a specificity of ≥24 bp. b) a DNA-cutting domain containing the Fok I nuclease domain. When the DNA-binding and DNA-cutting domains fuse together, a highly specific pair of “genome scissors” is created.

[0015] Transcription activator-like effector nucleases The technology utilizes artificial restriction enzymes created by fusing the DNA-binding domain of a TAL effector with a DNA-cutting domain. Restriction enzymes are enzymes that cleave DNA strands according to specific sequences. Transcription activator-like effectors... It can be quickly engineered to incorporate virtually any desired DNA sequence. By engineering this... Combined with DNA cleavage domains (which cut DNA strands), restriction enzymes can be engineered to specifically cleave any desired DNA sequence. When these restriction enzymes are introduced into cells, they can be used for gene editing or for in situ genome editing.

[0016] CRISPR gene editing utilizes a multi-component molecular system comprising a nuclease (typically a Cas or Cpf1 nuclease) and one or more guide RNA (gRNA) molecules capable of directing the nuclease to a specific genomic sequence. The nuclease, upon hybridization with the genomic sequence, cuts the DNA strand. By delivering a Cas9 nuclease complexed with synthetic gRNA into the cell, the cell's genome can be cut at the desired location, allowing for the removal of existing genes and / or the addition of new genes (Ledford, H., Nature, 2015, 522, 7554; Zetsche et al., Cell, 2015, 163(3), 759–771).

[0017] A key step in any CRISPR technology is the delivery of gRNA and a nuclease (such as Cas9) into the cytoplasm and / or nucleus of a target cell. Delivering nucleic acids into the cell is called transfection. gRNA and Cas9 can be introduced as DNA, RNA, or pre-complexed RNA and a protein called a ribonucleoprotein (RNP). Delivering foreign material across multiple cellular barriers (such as the plasma membrane and nuclear membrane) is a challenge.

[0018] Transfection methods can be broadly classified into physical, chemical, and virus-mediated categories. Each method has different advantages and disadvantages in terms of efficiency, yield, equipment, skill, and cost. The choice of transfection method also depends on the format of the CRISPR components.

[0019] The translocation of the CRISPR gene-editing mechanism, which involves a pre-formed ribonucleoprotein (CRISPR-RNP) consisting of the Cas9 enzyme and RNA, means that no further transcription or translation is required before gene editing. This allows for rapid editing due to the fewer steps involved. This can be useful in some cases, such as transient transfections that introduce CRISPR components into cells without incorporating the DNA encoding the guide RNA or Cas9 into the cell's genome. CRISPR-Cas9 can only cut the cell's genomic DNA within a limited timeframe. However, the translocation of such a large set of different macromolecules across the cell membrane presents unique challenges in terms of the efficiency of transmembrane transfer.

[0020] Adeno-associated virus particles are commonly used as gene delivery agents; however, viral vectors are non-ideal delivery media due to safety concerns and limited payload capacity (Swiech et al., Nature Biotechnol., 2015, 33, 102–106). Non-viral methods for delivering genome editing system molecules include lipid-based vectors, lipid nanoparticles, polymeric vectors, polyethyleneimine, and poly(L-lysine), but in vivo genome editing (e.g., using CRISPR tools) remains a challenge due to the limitations of currently used delivery methods (Li et al., Human Gene Therapy, 2015, 26(7), 452–462; Wang et al., Proceedings of the National Academy of Sciences, 2016, 113(11), 2868–2873).

[0021] Cell-penetrating peptides are also known to deliver the CRISPR gene-editing mechanism into cells. WO2017 / 205846 describes the use of cell-penetrating peptides VEPEP-3a / b and ADGN-100a / b to promote CRISPR-RNP transfection. However, it is evident that cell-penetrating peptides VEPEP-9 and CATY are only moderate binding agents, and cell-penetrating peptide VEPEP-6 interacts poorly with labeled Cas9 and the labeled Cas9-gRNA complex. Therefore, it is clear that the choice of CPP is a crucial determinant of the transfection efficiency of the gene-editing mechanism. The potential for in vivo use of cell-penetrating peptides used in the literature on gene editing is limited by immunoreactivity and the disruption of Cas9 reactivity due to the strong electrostatic interaction between positively charged CPPs and negatively charged Cas9-gRNA complexes. "Analytical Biochemistry", 345(2005)55–65, *International Journal of Molecular Sciences*, 2016, 17, 626; doi:10.3390 / ijms17050626

[0022] There are still improved or alternative effective means in this field to facilitate the transfer of gene editing mechanisms across various cell membranes. Summary of the Invention

[0023] These and other features, aspects, and advantages of this subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a series of concepts in a simplified form. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter.

[0024] In a first aspect, the present invention provides a genome editing complex for modifying target polynucleotides, the genome editing complex comprising a class of recombinant β-helical proteins linked to one or more genome editing system molecules, wherein the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0025] In a second aspect, the present invention provides a genome editing complex for modifying target polynucleotides, the genome editing complex comprising a recombinant β-helix protein linked to a plasmid encoding one or more genome editing system molecules, wherein the β-helix protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0026] In this embodiment, the one or more genome editing systems are selected from the group consisting of:

[0027] a. RNA-guided endonucleases and / or guide RNA (gRNA);

[0028] b. Zinc finger nucleases (ZFNs);

[0029] c. Transcription activator-like effector nucleases

[0030] d. DNA-guided endonucleases and / or guide DNA;

[0031] e. Homing endonucleases;

[0032] f. Integrase.

[0033] Suitablely, the genome editing system is CRISPR-Cas9.

[0034] In an embodiment, the gRNA has a sequence complementary to the target sequence in the target polynucleotide.

[0035] In the embodiments, the modifications resulting from genome editing are the addition, deletion, or substitution of one or more nucleotides in the target polynucleotide.

[0036] In an embodiment, the β-helical protein has a generally quadrilateral tip shape, the length of which is in the range of 5 nm to 25 nm and the width is in the range of 1 nm to 5 nm.

[0037] In an embodiment, the β-helical protein includes one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0038] In the embodiments, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamate sequence ladder comprises 10 to 40 glutamate residues.

[0039] In this embodiment, the total charge of the β-helical protein is less than zero.

[0040] In the embodiments, the total charge of the β-helical protein is -20 to -60.

[0041] In the embodiments, the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 Such as measurements obtained by atomic force microscopy.

[0042] In the embodiments, the β-helical protein is a pentapeptide repeat sequence protein.

[0043] In an embodiment, the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0044] In the embodiments, the β-helical protein is represented by sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0045] In an embodiment, the recombinant β-helical protein is linked to one or more genome editing system molecules or the plasmid via non-covalent interactions.

[0046] In the embodiments, the non-covalent interaction is selected from the group consisting of hydrogen bonding, electrostatic interaction, van der Waals interaction, hydrophobic interaction, or a combination thereof.

[0047] In an embodiment, the recombinant β-helical protein is linked to one or more genome editing system molecules or the plasmid via a linker molecule selected from the group consisting of: polyethylene glycol (PEG); ethylenediamine; peptides; metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0048] In an embodiment, when the linker molecule is a peptide, the peptide comprises an amino acid selected from the group consisting of aliphatic amino acids; aromatic amino acids; and combinations thereof.

[0049] In an embodiment, the adapter is connected to the recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

[0050] In the embodiments, the recombinant β-helical protein is linked to one or more genome editing system molecules or the plasmid via ester or amide bonds.

[0051] In an embodiment, the genome editing complex further includes a signal sequence, wherein the signal sequence directs the genome editing complex to a specific cell or a portion of a cell.

[0052] In an embodiment, the signal sequence is selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0053] In an embodiment, the genome editing complex further comprises a phosphatidylcholine molecule.

[0054] In an embodiment, the genome editing complex transfers one or more genome editing system molecules or the plasmid to a location selected from the group consisting of: organelles; nuclei; and P-cadherin overexpressing breast cancer cells.

[0055] In this embodiment, the genome editing complex is used for genome editing.

[0056] In a third aspect, the present invention provides a method for preparing a genome editing complex according to the first aspect of the present invention, the method comprising combining a cell-penetrating protein with one or more genome editing system molecules, thereby forming the genome editing complex.

[0057] In an embodiment of a third aspect of the invention, the one or more genome editing system molecules are in a reaction buffer containing molecules that stabilize the complex between the cell-penetrating protein and the genome editing system. The reaction buffer can be selected based on ionic strength to optimize the electrostatic interactions between the various components of the complex. The ionic strength can be varied by selecting components in the buffer solution, such as sodium chloride (NaCl).

[0058] Alternatively, in a fourth aspect, the present invention provides a method for preparing the genome editing complex of the second aspect of the invention, the method comprising combining a cell-penetrating protein with a plasmid, thereby forming the genome editing complex.

[0059] In a fifth aspect, the present invention provides a method for transferring one or more genome editing system molecules into cells, the method comprising:

[0060] a) Linking one or more genome editing system molecules with a recombinant β-helical protein to obtain a conjugate or genome editing complex;

[0061] b) Contact the conjugate or the genome editing complex with at least one cell;

[0062] In step (b), contact with the conjugate or the genome editing complex transfers one or more genome editing system molecules into the cell; and wherein the β-helix protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm. Suitably, the method includes detecting the transfer of the genome editing complex into the cell after step (b).

[0063] In a sixth aspect, the present invention provides a method for transferring one or more genome editing system molecules into cells, the method comprising:

[0064] d) Link the plasmid with the recombinant β-helical protein to obtain a conjugate or genome editing complex;

[0065] e) Contact the conjugate or the genome editing complex with at least one cell;

[0066] In step (d), contact with the conjugate or the genome editing complex transfers the plasmid into the cell; and the β-helix protein is in the range of 5 nm to 25 nm in length and 1 nm to 5 nm in width. Suitably, the method includes detecting the transfer of the genome editing complex into the cell after step (d).

[0067] In embodiments of the fifth and sixth aspects, the one or more genome editing systems are selected from the group consisting of:

[0068] a. RNA-guided endonucleases and / or guide RNA (gRNA);

[0069] b. Zinc finger nucleases (ZFNs);

[0070] c. Transcription activator-like effector nucleases

[0071] d. DNA-guided endonucleases and / or guide DNA;

[0072] e. Homing endonucleases;

[0073] f. Integrase.

[0074] In this embodiment, the genome editing system is CRISPR-Cas9.

[0075] In an embodiment, the gRNA has a sequence complementary to the target sequence in the target polynucleotide.

[0076] In an embodiment, the modification resulting from the genome editing is the addition, deletion, or substitution of one or more nucleotides in the target polynucleotide.

[0077] In an embodiment, step a) involves linking the one or more genome editing system molecules to a recombinant β-helix protein to obtain a genome editing complex in a reaction buffer containing molecules that stabilize the complex between the recombinant β-helix protein and the one or more genome editing system molecules; and / or transferring the one or more genome editing system molecules into cells in a serum-free culture medium.

[0078] In an embodiment, the β-helical protein has a generally quadrilateral tip shape, the length of which is in the range of 5 nm to 25 nm and the width is in the range of 1 nm to 5 nm.

[0079] In an embodiment, the β-helical protein includes one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0080] In an embodiment, when present, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamate sequence ladder comprises 10 to 40 glutamate residues.

[0081] In this embodiment, the total charge of the β-helical protein is less than zero.

[0082] In the embodiments, the total charge of the β-helical protein is -20 to -60.

[0083] In the embodiments, the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 For example, measurements can be made using atomic force microscopy.

[0084] In this embodiment, the β-helical protein is a pentapeptide repeat sequence protein.

[0085] In an embodiment, the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0086] In the embodiments, the β-helical protein is represented by sequences selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0087] In the embodiments, the genome editing complex is the genome editing complex described in the first or second aspect of the present invention.

[0088] In this embodiment, the cells are selected from the group consisting of eukaryotic cells, prokaryotic cells, and combinations thereof.

[0089] In the embodiments, the eukaryotic cells are mammalian cells, bacterial cells, yeast cells, plant cells, insect cells, or fish cells.

[0090] In a seventh aspect, the present invention provides a method for modifying a target polynucleotide in a cell, the method comprising contacting the cell with a genome editing complex of the first or second aspect, wherein the genome editing complex targets a sequence in the target polynucleotide.

[0091] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the genome editing complex of the first or second aspect of the present invention.

[0092] In a ninth aspect, the present invention provides a method for treating a disease in an individual, the method comprising administering to the individual an effective amount of the pharmaceutical composition of the seventh aspect of the present invention.

[0093] In a tenth aspect, the present invention provides the use of the genome editing complex of the first or second aspect of the present invention for gene editing.

[0094] In an eleventh aspect, the present invention provides the use of the genome editing complex of the first or second aspect of the present invention for gene therapy.

[0095] This article also discloses a cell-penetrating conjugate comprising at least one recombinant β-helical protein molecule linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0096] A method for transferring a functional molecule into a cell is further disclosed, the method comprising: (a) conjugating the functional molecule to a recombinant β-helical protein to obtain a conjugate; and (b) contacting the cell through the conjugate to at least one cell; wherein contacting the conjugate with the at least one cell transfers a nucleic acid molecule into the cell, and wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0097] This document also discloses the use of a cell-penetrating conjugate of the first aspect of the invention for delivering functional molecules into cells, wherein the functional molecules are selected from the group consisting of dyes, drugs, metals, drug-metal, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0098] This document further discloses the use of the cell-penetrating conjugate of the first aspect of the present invention for cell penetration.

[0099] In addition, this document discloses the use of the cell-penetrating conjugate of the first aspect of the present invention for cell labeling.

[0100] This document also discloses a conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0101] This document further discloses a method for transferring nucleic acid molecules into cells, the method comprising: (i) linking the nucleic acid molecule to at least one recombinant β-helical protein via at least one adapter to obtain a conjugate; and (ii) contacting the conjugate with at least one cell, wherein contacting the conjugate with the at least one cell transfers the nucleic acid molecule into the cell, and wherein the recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0102] In addition, this document discloses the use of the cell-penetrating conjugate of the sixth aspect of the present invention as a transfection agent.

[0103] This article further discloses the use of the cell-penetrating conjugate of the sixth aspect of the present invention for gene therapy. Attached Figure Description

[0104] The following drawings form part of and are included within this specification to further illustrate various aspects of this disclosure. A better understanding of this disclosure can be achieved by referring to the accompanying drawings in combination with the detailed description of the specific embodiments presented herein.

[0105] Figure 1 The CD (circular dichroism) spectrum of the isolated AlbG protein according to this disclosure is shown.

[0106] Figure 2 An agarose gel containing purified plasmids (containing the AlbG and EfsQNR genes) according to this disclosure is shown.

[0107] Figure 3 A polyacrylamide gel containing purified proteins (AlbG and EfsQNR) according to this disclosure is shown.

[0108] Figure 4 The MALDI-TOF spectroscopic analysis of EfsQNR and AlbG proteins is shown.

[0109] Figure 5 A graphical representation of the characterization of labeled proteins (AlbG-NHSC and EfsQNR-NHSC) according to an embodiment of the present disclosure by UV-Vis spectrophotometry is shown.

[0110] Figure 6 Differential labeling of HeLa cells by labeled proteins (conjugates) AlbG-NHSC, EfsQNR-NHSC, and TtCuA-NHSC is shown according to embodiments of the present disclosure.

[0111] Figure 7 The labeling of HeLa cells by EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0112] Figure 8 The labeling of HeLa cells by EfsQNR labeled with ATTO-390 (a commercially available blue fluorescent dye) is shown according to one embodiment of the present disclosure.

[0113] Figure 9 The illustration shows microglia labeled with EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) according to one embodiment of the present disclosure (inset A); and differentially labeled microglia with EfsQNR labeled with ATTO-520 (inset B).

[0114] Figure 10 The following illustration shows the labeling of keratinocytes by EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) according to one embodiment of the present disclosure (inset A); and the differential labeling of keratinocytes by EfsQNR labeled with ATTO-520 (inset B).

[0115] Figure 11 The labeling of SH-SY5Y cells with EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0116] Figure 12 The labeling of mouse ES cells by EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0117] Figure 13 The labeling of E. coli cells by EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0118] Figure 14The labeling of yeast (Kluyveromyces) cells by EfsQNR labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0119] Figure 15 shows the results of standard FACS sorting of HeLa cells treated with a conjugate labeled with EfsQNR-ATTO-647N for 10 minutes (inset B), 1 hour (inset C), and 3 hours (inset D) compared to untreated cells (inset A).

[0120] Figure 16 The percentage of cellular uptake of a drug as part of a conjugate according to one embodiment of the present disclosure is shown compared to the unconjugated drug.

[0121] Figure 17 This demonstrates a chemotherapy drug, according to an embodiment of the present disclosure, used as part of a conjugate with the protein EfsQNR (labeled CYDD), compared to an unconjugated drug. Percentage of cell (HeLa and HepG2) survival after treatment.

[0122] Figure 18 A plasmid vector carrying the mcherry gene is shown according to one embodiment of the present disclosure.

[0123] Figure 19 A process for preparing a conjugate according to an embodiment of the present disclosure is shown.

[0124] Figure 20 A representation of transfection using a conjugate according to an embodiment of the present disclosure is shown.

[0125] Figure 21 The image shown is a confocal micrograph of HeLa cells after transfection with a conjugate according to the present invention; the conjugate comprises the mcherry gene encoding RFP (red fluorescent protein) linked to EfsQNR protein via copper[II]phenanthroline.

[0126] Figure 22 This demonstrates real-time direct penetration of the cell membrane via the EfsQNR-ATTO 520 conjugate according to the present invention.

[0127] Figure 23 The labeling of Arabidopsis thaliana cells by CPP labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0128] Figure 23The labeling of Arabidopsis thaliana cells by CPP labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0129] Figure 24 The following labeling of Arabidopsis thaliana cells according to one embodiment of the present disclosure is shown: A) CPP labeled with ATTO-520 (a commercially available green fluorescent dye); B) CPP labeled with ATTO-594 (a commercially available red fluorescent dye); and C) CPP labeled with ATTO-390 (a commercially available blue fluorescent dye).

[0130] Figure 25 The labeling of yeast cells with CPP labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0131] Figure 26 The labeling of bacterial cells with CPP labeled with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0132] Figure 27 The illustration shows the labeling of Drosophila embryonic cells by CPP labeled with ATTO-520 (a commercially available green fluorescent dye) according to one embodiment of the present disclosure.

[0133] Figure 28 Labeling of CPP zebrafish embryonic cells with ATTO-520 (a commercially available green fluorescent dye) is shown according to one embodiment of the present disclosure.

[0134] Figure 29 The results of gene editing performed on HEK293 cells expressing eGFP after CPP-assisted delivery of CRISPR-Cas9 and eGFP-specific gRNA are shown, as observed by a decrease in fluorescence intensity induced by eGFP (as measured by a fluorescent plate reader).

[0135] Figure 30 The results of transfection experiments performed in MCF7 cells using the following: (A) the conjugate (EfsQNR) (SEQ ID NO:2) obtained in Example 1 and a 2.7 kb red-labeled circular plasmid DNA (product code MIR 7904) commercially available from Mirus Bio; and (B) a control red-labeled plasmid used as above in the absence of the conjugate, (i) showing the maximum projection of the cells after culture; (ii) an image corresponding to the intensity map of the cells after culture; and (iii) showing the intensity map of the cells after culture.

[0136] Figure 31 The results of transfection experiments in MCF-7 cells using the conjugate (EfsQNR) obtained in Example 1 and the Cas9 endonuclease tagged with either (i) red fluorescent protein or (ii) green fluorescent dye Atto520 are shown. (A) shows the maximum projection of the cells after culture; (B) shows the intensity map of the cells after culture; and (C) shows a 3D depth image of the cells.

[0137] Figure 32 The results of transfection experiments performed in MCF-7 cells are shown: (A) the conjugate (EfsQNR) obtained in Example 1 and gRNA tagged with the green fluorescent dye MFP488; and (B) the control gRNA tagged with the green fluorescent dye MFP488. (i) shows the maximum projection of the cells after culture; (ii) shows a 3D depth image of the cells; (iii) shows an image corresponding to the intensity map of the cells after culture; and (iv) shows the intensity map of the cells after culture. Detailed Implementation

[0138] Those skilled in the art will recognize that variations and modifications can be made to this disclosure in addition to those specifically described. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all such steps, features, compositions, and compounds individually or collectively referenced or indicated in this specification, as well as any and all combinations of any or more of such steps or features.

[0139] definition

[0140] For convenience, certain terms and examples used in this specification are described before further description of this disclosure. These definitions should be read and understood by those skilled in the art in light of the remainder of this disclosure. The terms used herein have meanings that are generally accepted and known to those skilled in the art; however, for convenience and completeness, specific terms and their meanings are set forth below.

[0141] Unless otherwise stated, the present invention is practiced using conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology and chemical methods, which are within the capabilities of a person skilled in the art. Such techniques are explained in the following literature, for example: MR Green and J. Sambrook, 2012, *Molecular Cloning: A Laboratory Manual*, 4th edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, FM et al. (1995 and regular supplements); *Current Protocols in Molecular Biology*, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, *DNA Isolation and Sequencing: Essential Techniques*, John Wiley & Sons; JMPolak and James O'D. McGee, 1990, *In Situ Hybridization: Principles and Practice*. Hybridisation: Principles and Practice, Oxford University Press; MJ Gait (ed.), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and DMJ Lilley and JED Ahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts is incorporated herein by reference.

[0142] The articles “a,” “an,” and “the” are used to refer to one or more (i.e., at least one) grammatical objects of the article.

[0143] As used herein, the term "comprising" means that any of the elements set forth must be included, and other elements may optionally be included. "Substantially consisting of" means that any of the elements set forth must be included, excluding elements that would substantially affect the essential and novel characteristics of the listed elements, and other elements may optionally be included. "Constitutes of" means excluding all elements other than those listed. Embodiments defined by each of these terms are within the scope of this invention.

[0144] The term "including" is used to mean "includes but is not limited to". "Includes" and "includes but is not limited to" are used interchangeably.

[0145] As used herein, the term "cell membrane" is a biological membrane present in both prokaryotic and eukaryotic cells that separates the cell's internal and external environments. The cell membrane acts as a semi-permeable barrier, typically formed by a phospholipid bilayer, to inspect the transport of substances into and out of the cell. The membrane also acts as a support structure and helps maintain the cell's shape and structure.

[0146] As used herein, the term "β-helical protein" refers to a protein that forms the β-helix secondary structure. β-helical proteins are formed by the normally parallel association between adjacent β chains of a polypeptide chain. β-helical proteins can be right-handed or left-handed β-helices, depending on the orientation of the helical structure.

[0147] As used herein, the term "pentapeptide repeat protein (PRP)" refers to a β-helical protein composed of tandemly repeating pentapeptides. In the examples, the tandemly repeating pentapeptides have a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5. The PRP family has well over 500 members in the prokaryotic and eukaryotic kingdoms.

[0148] As used herein, the term "functional molecule" is any molecule that has an effect within cells. Examples of functional molecules suitable for use in this invention include dyes, drug molecules, proteins, enzymes, antibodies, and nucleic acids.

[0149] As used herein, the term "cell-penetrating peptide" or "CPP" refers to peptide sequences that facilitate cellular uptake / recombination of various functional molecules. Cell-penetrating peptides typically deliver functional molecules directly across the cell membrane, thereby avoiding the need for endocytosis-mediated pathways for cell entry.

[0150] As used in this article, the term "P-cadherin" refers to a cell-cell adhesion molecule that has a homeostatic function in normal tissues. Overexpression of this molecule is associated with significant tumor-promoting effects in breast, ovarian, prostate, endometrial, skin, gastric, pancreatic, and colon tumors.

[0151] As used herein, the term "NHS coumarin" or "NHSC" refers to a fluorescent dye widely used in cell biology techniques. It is the common name for 7-(diethylamino)coumarin-3-carboxylic acid N-succinimide ester, with a molecular weight of 358.35 g / mol. NHS coumarin (NHSC) has an excitation wavelength of 445 nm and an emission wavelength of 482 nm. Under fluorescence microscopy, it emits green fluorescence, indicating the position and quantification of molecules conjugated with this dye.

[0152] As used herein, the term "phosphatidylcholine" defines a class of phospholipid molecules incorporating choline as a head group. Phosphatidylcholine can be used as a signaling molecule that promotes selective binding and connection to cell membranes.

[0153] As used herein, the term "Hoechst 33342" refers to a fluorescent dye solution used in cell imaging techniques for both staining of fixed cells and live cells for DNA and nuclei. Hoechst 33342 is a cell-permeable DNA dye with an excitation wavelength of 460 nm and an emission wavelength of 490 nm, and it preferentially binds to the adenine (A)-thymine (T) region of DNA.

[0154] As used herein, the terms “ATTO 520” or “A-520”, “ATTO 390” or “A-390”, and “ATTO 647N” or “A-647N” refer to fluorescent dyes developed by ATTO-Tec GmbH and commercially available from Sigma Aldrich.

[0155] As used herein, the term "ruthenium metal complex" refers to a coordination complex of ruthenium metal known to possess anticancer activity. Octahedral ruthenium(III) and ruthenium(II) complexes have shown antitumor activity against many experimental tumors. Ruthenium metal complexes are considered an excellent alternative to avoid the side effects of platinum-based compounds. A non-limiting example of a ruthenium metal complex that has been shown to be usable in this invention is tricarbonyl dichlororuthenium(II) (formerly Sigma-Aldrich).

[0156] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. Polynucleotides can consist of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can comprise DNA and RNA and are generally manufactured synthetically, but can also be isolated from natural sources. Nucleic acids may further comprise modified DNA or RNA, such as DNA or RNA that has been methylated or chemically modified, for example, by 5'-capping with 7-methylguanosine, 3'-processing such as cleavage and polyadenylation, and splicing or labeling with fluorophores or other compounds. Nucleic acids may also comprise synthetic nucleic acids (XNAs), such as hexetol nucleic acids (HNAs), cyclohexene nucleic acids (CeNAs), threonine nucleic acids (TNAs), glycerol nucleic acids (GNAs), locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). Therefore, in the context of the use of the terms "DNA" and "RNA" herein, it should be understood that these terms are not limited to encompassing only naturally occurring nucleotides. The size of nucleic acids, also referred to as "polynucleotides" in this article, is usually expressed as the number of base pairs (bp) in double-stranded polynucleotides, or as the number of nucleotides (nt) in the case of single-stranded polynucleotides. One thousand bp or nt equals one thousand bases (kb). Polynucleotides with a length of less than approximately 100 nucleotides are usually referred to as "oligonucleotides".

[0157] As used herein, the terms “3’” (“3-primer”) and “5’” (“5-primer”) take their usual meaning in the art, namely, distinguishing the ends of polynucleotides. Polynucleotides have a 5’ end and a 3’ end, and polynucleotide sequences are conventionally written in the 5’ to 3’ direction.

[0158] The term “amino acid” in the context of this invention is used in its broadest sense and is intended to include naturally occurring Lα-amino acids or residues. Commonly used single-letter and three-letter abbreviations for naturally occurring amino acids are as follows: A = Ala; C = Cys; D = Asp; E = Glu; F = Phe; G = Gly; H = His; I = Ile; K = Lys; L = Leu; M = Met; N = Asn; P = Pro; Q = Gln; R = Arg; S = Ser; T = Thr; V = Val; W = Trp; and Y = Tyr (Lehninger, AL, (1975) Biochemistry, 2nd ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not typically incorporated into proteins (such as ortholeucine), and chemically synthesized compounds that have properties known in the art as amino acids, such as β-amino acids. For example, analogs or mimics of phenylalanine or proline that allow conformational restrictions to the same peptide compounds as natural Phe or Pro are included within the definition of an amino acid. Such analogs and mimics are referred to herein as “functional equivalents” of the corresponding amino acids. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, edited by Gross and Meiehofer, Vol. 5, p. 341, Academic Press Ltd., New York (NY), 1983, which is incorporated herein by reference.

[0159] A "polypeptide" is a polymer of amino acid residues linked by peptide bonds, whether naturally occurring or synthesized in vitro. Polypeptides with a length of less than about 12 amino acid residues are generally called "peptides," and polypeptides with a length between about 12 and about 30 amino acid residues can be called "oligopeptides." As used herein, the term "polypeptide" refers to naturally occurring polypeptides, precursor forms, or preprotein products. Polypeptides can also undergo maturation or post-translational modifications, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipolysis, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" is used herein to refer to macromolecules comprising one or more polypeptide chains.

[0160] The term "isolated," when applied to polynucleotide or protein sequences, indicates that the sequence has been removed from its natural source organism and therefore does not contain foreign or unwanted coding or regulatory sequences. Isolated sequences are suitable for the assembly of the compositions and nanostructures of the present invention. Such isolated sequences can comprise cDNA and RNA.

[0161] According to the present invention, homology with the nucleic acid or protein sequences described herein is not limited to 100% sequence identity. Any nucleic acid or protein sequence closely related to those specified herein that exhibits functional and / or biochemical equivalence is considered to be within the scope of the invention as defined by the claims.

[0162] In the context of this invention, the term "signal sequence" refers to the nuclear localization or recognition sequence of different organelles or nucleic acid binding domains, such as zinc finger binding proteins.

[0163] As used in this article, the term "carrier" refers to a substance that acts as a mechanism for improving drug delivery and efficacy.

[0164] As used in this article, the term "diluent" (also known as filler, diluent, or thinner) refers to a diluent agent.

[0165] As used herein, the term "excipient" refers to an inactive substance that acts as a medium or agent for a drug or other active substance. Excipients include colorants, humectants, preservatives, emollients, and combinations thereof.

[0166] As used herein, the term "RNA-guided endonuclease" refers to a polypeptide whose activity and specificity depend on its association with at least one guide RNA molecule. An example of an RNA-guided endonuclease is Cas9, which is part of the Cas9 / CRISPR system.

[0167] As used herein, the term "CRISPR" (clustered, regularly spaced short palindromic repeats) refers to a family of DNA sequences present in the genomes of prokaryotes such as bacteria. The term can also be used to refer to gene editing technologies that rely on CRISPR-associated proteins, such as Cas-9 (CRISPR-associated protein 9), that can be associated with and then used as guides by a CRISPR sequence to recognize and cut specific DNA strands complementary to the CRISPR sequence. The Cas9 nuclease, together with the CRISPR sequence, forms the basis of CRISPR-Cas9 gene editing technology.

[0168] As used herein, the term "Cas9" refers to the Cas9 endonuclease protein. Also known as Csn1 (COG3513) Cas9, it is a large protein involved in crRNA biogenesis and the disruption of invading DNA. The term "Cas9" is also intended to encompass engineered endonucleases or homologs of Cas9 capable of processing target nucleic acid sequences. Cas9 can induce cleavage in the target nucleic acid sequence, which can correspond to double-strand breaks or single-strand breaks. The term Cas9 is intended to include variants of Cas9 endonucleases that are not naturally occurring and are obtained through protein engineering or random mutagenesis, provided that such Cas9 variants remain functional, i.e., they retain the ability to process target nucleic acid sequences.

[0169] As used herein, the term "CRISPR-RNP" refers to a CRISPR ribonucleotide comprising a CRISPR endonuclease protein and one or more guide RNAs. The term "CRISPR-RNP-CPP" as used herein refers to a conjugate of the CRISPR-RNP complex with a cell-penetrating peptide from an embodiment of the present invention.

[0170] As used herein, the term "guide RNA" or "gRNA" refers to an RNA polynucleotide sequence that includes a guide sequence. In use, the guide RNA may be associated with a nuclease that acts as a cleavage enzyme at the site defined by the gRNA. The term "guide sequence" refers to a sequence that is preferably longer than 8 nucleotide bases, more preferably longer than 10 nucleotide bases, and even more preferably longer than 12 nucleotide bases, capable of specifying a target sequence in the genome. Typically, this RNA molecule is capable of hybridizing to the target sequence and mediating the nuclease activity of the nuclease.

[0171] As used herein, the term "wild type" refers to the typical or naturally occurring form of an organism, strain, gene, or trait that is distinguishable from "mutant" or "variant" forms.

[0172] As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably. When referring to a nucleic acid molecule or polypeptide, the term means that the nucleic acid molecule or polypeptide differs from those found in nature and / or at least substantially lacks at least one other component naturally associated with it in nature.

[0173] As used herein, the term "complementarity" refers to the ability of a nucleic acid to be associated with another nucleic acid sequence through conventional Watson-Crick base pairing or other non-conventional means, such as non-covalent interactions via hydrogen bonds. The complementarity percentage indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 50%, 60%, 70%, 80%, 90%, and 100% complementarity out of 10).

[0174] As used herein, the term “expression” refers to the process of transcribing polynucleotides from a DNA template (such as into mRNA or other RNA transcripts) and / or the subsequent translation of the transcribed mRNA into peptides, polypeptides, or proteins.

[0175] Unless otherwise defined, 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 disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0176] sequence

[0177] SEQ ID NO:1 depicts the amino acid sequence of the AlbG protein.

[0178] MPAKTLESKDYCGESFVSEDRSGQSLESIRFEDCTFRQCNFTEAELNRCKFRECEFVDCNLSLISIPQTSFMEVRFVDCKMLGVNWTSAQWPSVKMEGALSFERCILNDSLFYGLYLAGVKMVECRIHDANFTEADCEDADFTQSDLKGSTFHNTKLTGASFIDAVNYHIDIFHNDIKRARFSLPEAASLLNSLDIELSD

[0179] SEQ ID NO:2 depicts the amino acid sequence of the EfsQNR protein.

[0180] GSHMKITYPLPPNLPEQLPLLTNCQLEDEAILENHLYQQIDLPNQEVRNLVFRDAVFDHLSLANGQFASFDCSNVRFEACDFSNVEWLSGSFHRVTFLRCNLTGTNF ADSYLKDCLFEDCKADYASFRFANFNLVHFNQTRLVESEFFEVTWKKLLLEACDLTESNWLNTSLKGLDFSQNTFERLTFSPNYLSGLKVTPEQAIYLASALGLVIT

[0181] SEQ ID NO:3 depicts the amino acid sequence of an antifreeze protein from the mealworm (Tenebrio molitor). QCTGGADCTSCTGACTGCGNCPNAVTCTNSQHCVKANTCTGSTDCNTAQTCTNSKDCFEANTCTDSTNCYKATACTNSSGCPGH

[0182] SEQ ID NO:4 depicts the amino acid sequence of an antifreeze protein from the pine bark beetle (Rhagium inquisitor). GYSCRAVGVDGRAVTDIQGTCHAKATGAGAMASGTSEPGSTSTATATGRGATARSTSTGRGTATTTATGTASATSNAIGQGTATTTATGSAGGRATGSATTSSSASQPTQTQTITGPGFQTAKSFARNTATTTVTASHHHHHH

[0183] SEQ ID NO:5 depicts the amino acid sequence of an antifreeze protein from the spruce leafroller (Choristoneura fumiferana).

[0184] DGSCTNTNSQLSANSKCEKSTLTNCYVDKSEVYGTTCTGSRFDGVTITTSSTGSRISGPGCKISTCIITGGVPAPSAACKISGCTFSAN

[0185] SEQ ID NO:6 depicts the amino acid sequence of the QNRB1 protein.

[0186] GSHMALALVGEKIDRNRFTGEKIENSTFFNCDFSGADLSGTEFIGCQFYDRESQKGCNFSRAMLKDAIFKSCDLSMADFRNSSALGIEIRHCRAQGADFRGASFMNMI TTRTWFCSAYITNTNLSYANFSKVVLEKCELWENRWIGAQVLGATFSSGSDLSGGEFSTFDWRAANFTHCDLTNSELGDLDIRGVDLQGVKLDNYQASLLMERLGIAVIG

[0187] SEQ ID NO:7 describes the amino acid sequence of the UDP-N-acetylglucosamine acyltransferase protein.

[0188] MIDKSAFVHPTAIVEEGASIGANAHIGPFCIVGPHVEIGEGTVLKSHVVVNGHTKIGRDNEIYQFASIGEVNQDLKYAGEPTRVEIGDRNRIRESVTIHRGTVQGGGLTKVGSDNLLMINAHIAHDCTVGN RCILANNATLAGHVSVDDFAIIGGMTAVHQFCIIGAHVMVGGCSGVAQDVPPYVIAQGNHATPFGVNIEGLKRRGFSREAITAIRNAYKLIYRSGKTLDEVKPEIAELAETYPEVKAFTDFFARSTRGLIR

[0189] SEQ ID NO:8 depicts the amino acid sequence of the NP275 protein from *Nostoc punctiforme*.

[0190] SEQ ID NO:9 depicts the amino acid sequence of pectic acid lyase C.

[0191] ATDTGGYAATAGGNVTGAVSKTATSMQDIVNIIDAARLDANGKKVKGGAYPLVITYTGNEDSLINAAAANICGQWSKDPRGVEIKEFTKGITIIGANGSSANFGIWIKKSSDVVVQNMRIGYLPGGAKDGDMIRVDDSPNVWVDHNELFAANHECDGTPD NDTTFESAVDIKGASNTVTVSYNYIHGVKKVGLDGSSSDTGRNITYHHNYYNDVNARLPLQRGGLVHAYNNLYTNITGSGLNVRQNGQALIENNWFEKAINPVTSRYDGKNFGTWVLKGNNITKPADFSTYSITWTADTKPYVNADSWTSTGTFPTVAY NYSPVSAQCVKDKLPGYAGVGKNLATLTSTACK

[0192] SEQ ID NO:10 depicts the amino acid sequence of a pectic acid lyase from the pyrolytic cellulosic bacterium (Caldicellulosiruptor bescii).

[0193] VGTNTGGVLVITDTIIVKSGQTYDGKGIKIIAQGMGDGSQSENQKPIFKLEKGANLKNVIIGAPGCDGIHCYGDNVVENVVWEDVGEDALTVKSEGVV EVIGGSAKEAADKVFQLNAPCTFKVKNFTATNIGKLVRQNGNTTFKVVIYLEDVTLNNVKSCVAKSDSPVSELWYHNLNVNNCKTLFEFPSQSQIHQY

[0194] SEQ ID NO:11 depicts the amino acid sequence of carbonic anhydrase from Methanosarcina thermophila.

[0195] QEITVDEFSNIRENPVTPWNPEPSAPVIDPTAYIDPQASVIGEVTIGANVMVSPMASIRSDEGMPIFVGDRSNVQDGVVLHALETINEEGEPIEDNIVEVDGKEYA VYIGNNVSLAHQSQVHGPAAVGDDTFIGMQAFVFKSKVGNNCVLEPRSAAIGVTIPDGRYIPAGMVVTSQAEADKLPEVTDDYAYSHTNEAVVYVNVHLAEGYKETS

[0196] SEQ ID NO:12 depicts the amino acid sequence of pectin lyase A protein from Aspergillus niger.

[0197] VGVSGSAEGFAKGVTGGGSATPVYPDTIDELVSYLGDDEARVIVLTKTFDFTDSEGTTTGTGCAPWGTASACQVAIDQDDWCENYEPDAPSVSVEYYNAGTLGITVTSNKSLIGEGSSGAIKGKGLRIVSGAENIIIQNIAVTDINPKYVWGGDAITLDD CDLVWIDIHVTTARIGRQHYVLGTSADNRVSLTNNYIDGVSDYSATCDGYHYWAIYLDGDADLVTMKGNYIYHTSGRSPKVQDNTLLHAVNNYWYDISGHAFEIGEGGYVLAEGNVFQNVDTVLETYEGEAFTVPSSTAGEVCSTYLGRDCVINGFGSSGT FSEDSTSFLSDFEGKNIASASAYTSVASRVVANAGQGNL

[0198] SEQ ID NO:13 depicts the amino acid sequence of the TtCuA protein.

[0199] AYTLATHTAGVIPAGKLERVDPTTVRQEGPWADPAQAVVQTGPNQYTVYVLAFAFGYQPNPIEVPQGAEIVFKITSPDVIHGFHVEGTNINVEVLPGEVSTVRYTFKRPGEYRIICNQYCGLGHQNMFGTIVVKE

[0200] SEQ ID NO:14 describes the signal sequence used for targeting the nucleus.

[0201] PAAKRVKCD

[0202] SEQ ID NO:15 depicts a signaling sequence for targeting the endoplasmic reticulum of cells.

[0203] YPYDVPDYAKDEL

[0204] SEQ ID NO:16 depicts a signaling sequence for targeting mitochondria in cells.

[0205] MLSLRQSIRFFKPATRTLCSSRYLL

[0206] SEQ ID NO:17 depicts a signaling sequence for targeting P-cadherin overexpressing breast cancer cells.

[0207] LSTAADMQGVVTDGMASGLDKDYLKPDD

[0208] SEQ ID NO:18 depicts a common sequence in a pentapeptide repeat protein.

[0209] (STAV)1(DN)2(LF)3(STR)4(G)5

[0210] SEQ ID NO:19 depicts the nucleic acid sequence of the AlbG gene.

[0211] ATGCCGGCGAAAACCCTGGAAAGCAAAGATTATTGCGGCGAAAGCTTTGTGAGCGAAGATCGCAGCGGCCAGAGCCTGGAAAGCATTCGCTTTGAAGATTGCACCTTTCGCCAGTGCAACTTTACCGAAGCGGAACTGAACCGCT GCAAATTTCGCGAATGCGAATTTGTGGATTGCAACCTGAGCCTGATTAGCATTCCGCAGACCAGCTTTATGGAAGTGCGCTTTGTGGATTGCAAAATGCTGGGCGTGAACTGGACCAGCGCGCAGGCGGGCGCGCTGAGCTTTGAA CGCTGCATTCTGAACGATAGCCTGTTTTATGGCCTGTATCTGGCGGGCGTGAAAATGGTGGAATGCCGCATTCATGATGCGAACTTTACCGAAGCGGATTGCGAAGATGCGGATTTTACCCAGAGCGATCTGAAAGGCAGCACCT TTCATAACACCAAACTGACCGGCGCGAGCTTTATTGATGCGGTGAACTATCATATTGATATTTTTCATAACGATATTAAACGCGCGCGCTTTAGCCTGCCGGAAGCGGCGAGCCTGCTGAACAGCCTGGATATTGAACTGAGCGAT

[0212] SEQ ID NO:20 depicts the nucleic acid sequence of the EfsQNR gene.

[0213] GGCAGCCATATGAAAATTACCTATCCGCTGCCGCCGAACCTGCCGGAACAGCTGCCGCTGCTGACCAACTGCCAGCTGGAAGATGAAGCGATTCTGGAAAACCATCTGTATCAGCAGATTGATCTGCCGAACCAGGAAGTGCGCAACCTGGTGTTTCGCGATGCGGTGTTTGATCATCTGAGCCTGGCGAACGGCCAGTTTGCGAGCTTTGATTGCAGCAACGTGCGCTTTGAAGCGTGCGATTTTAGCAACGTGGAATGGCTGAGCGGCAGCTTTCATCGCGTGACCTTTCTGCGCTGCAACCTGACCGGCACCAACTTTGCGGATAGCTATCTGAAAGATTGCCTGTTTGAAGATTGCAAAGCGGATTATGCGAGCTTTCGCTTTGCGAACTTTAACCTGGTGCATTTTAACCAGACCCGCCTGGTGGAAAGCGAATTTTTTGAAGTGACCTGGAAAAAACTGCTGCTGGAAGCGTGCGATCTGACCGAAAGCAACTGGCTGAACACCAGCCTGAAAGGCCTGGATTTTAGCCAGAACACCTTTGAACGCCTGACCTTTAGCCCGAACTATCTGAGCGGCCTGAAAGTGACCCCGGAACAGGCGATTTATCTGGCGAGCGCGCTGGGCCTGGTGATTACC

[0214] SEQ ID NO: 21 depicts the nucleic acid sequence of the TtCuA gene.

[0215] GCGTATACCCTGGCGACCCATACCGCGGGCGTGATTCCGGCGGGCAAACTGGAACGCGTGGATCCGACCACCGTGCGCCAGGAAGGCCCGTGGGCCGGATCCGGCGCAGGCGGTGGTGCAGACCGGCCCGAACCAGTATACCGTGTATGTGCTGGCGTTTGCGTTTGGCTATCAGCCGAACCCGATTGAAGTGCCGCAGGGCG CGGAAATTGTGTTTAAAATTACCAGCCCGGATGTGATTCATGGCTTTCATGTGGAAGGCACCAACATTAACGTGGAAGTGCTGCCGGGCGAAGTGAGCACCGTGCGCTATAACCTTTAAACGCCCGGGCGAATATCGCATTATTTGCAACCAGTATTGCGGCCTGGGCCATCAGAACATGTTTGGCACCATTGTGGTGAAAGAA

[0216] SEQ ID NO:22 depicts a signaling sequence for targeting actin in cells.

[0217] GDVQKKRWLFETKPLD

[0218] SEQ ID NO:23 depicts a signaling sequence for targeting tubulin in cells.

[0219] VQSKCGSKDNIKHVPGGG

[0220] SEQ ID NO.24: The amino acid sequence of the zinc finger protein is drawn.

[0221] MERPYACPVESCDRRFSDSSNLTRHIRIHTGQKPFQCRICMRNFSRSDHLTTHIRTHTGEKPFACDICGRKFARSDERKR HTKIHLRQKD

[0222] SEQ ID NO.25: Depicts the nucleic acid sequence of the mcherry gene.

[0223] GTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAGTAATCTAGAGGGCCCTATTCTATAGTGTCACC.

[0224] SEQ ID NO. 26: depicts the nucleic acid sequence of a primer used for PCR amplification of the AlbG gene.

[0225] ATCCCGCTCATATGCCGGCCAAGACCCTTG

[0226] SEQ ID NO. 27: depicts the nucleic acid sequence of a primer used for PCR amplification of the AlbG gene.

[0227] ATCCCGCTCTCGAGTCAATCGGACAGCTCGATATC

[0228] SEQ ID NO.28: Depicts the nucleic acid sequence of the primers used for PCR amplification of the EfsQNR gene.

[0229] ATCCCGCTCATATGAAAATAACTTATCCCTTGCCA

[0230] SEQ ID NO.29: Depicts the nucleic acid sequence of the primers used for PCR amplification of the EfsQNR gene.

[0231] ATCCCGCTCTCGAGTTAGGTAATCACCAAACCAAGT

[0232] This invention also provides conjugates comprising recombinant proteins and functional molecules for penetrating cell membranes, and uses of conjugates thereof with sequence identity or homology substantially similar to that of SEQ ID NO:1 to 12. The term “substantially similar sequence identity” is used herein to indicate a level of sequence similarity of about 50%, 60%, 70%, 80%, 90%, 95% to about 99%. The percentage of sequence identity can be determined using conventional methods, such as those described for nucleic acids and for proteins in Henikoff and Henikoff, Proceedings of the National Academy of Sciences 1992;89:10915, and Altschul et al., Nucleic Acids Res. 1997;25:3389-3402, using methods such as… The comparison will be determined after the system performs the comparison.

[0233] Cell-penetrating conjugates with functional molecules

[0234] Identifying novel drugs for the treatment or prevention of life-threatening diseases remains a highly active area of ​​research. Most such drugs tend to have intracellular targets, and to reach these targets, the drugs must cross semi-permeable membranes, which in many cases is neither direct nor effective. Therefore, developing novel mechanisms for penetrating cell membranes remains desirable. On the other hand, scientific investigations and research aimed at unraveling various cellular mechanisms also seek to find novel methods for penetrating cell membranes, which could help label cells and their individual organelles. Similarly, for scientific experiments, such methods would be highly applicable for delivering desired materials within cells. Most cell-penetrating molecules described in recent reports involve endocytosis, a mechanism of cell entry.

[0235] To avoid the drawbacks of endocytosis-induced molecule intake, such as the retention and degradation of drugs like lysosomes in different types of endosome compartments that ultimately fuse with the cell's degradation compartments, this paper discloses a cell membrane permeation conjugate that can permeate the cell membrane to obtain a pathway into the cell and can also be used for intracellular delivery of various cargoes containing intracellular dyes, drugs, proteins, enzymes, antibodies, and nucleic acids.

[0236] The scope of this disclosure is not limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly apparent within the scope of this disclosure as described herein.

[0237] This invention discloses a conjugate capable of penetrating cell membranes. The conjugate comprises a recombinant β-helical protein or a recombinant β-helical protein moiety linked to a functional molecule, wherein the longest dimension of the protein is defined as its length, in the range of 5 nm to 25 nm, and the width or diameter is defined as a dimension of the protein structure substantially perpendicular to its length, in the range of 1 nm to 5 nm. The size of the protein is defined as measured in a solid state (characterized by X-ray crystallography or atomic force microscopy) or in a solution state (measured by dynamic light scattering).

[0238] In embodiments of the invention, the longest dimension or length of the protein structure of the conjugate can be greater than 5 nm, 7.5 nm, 10 nm, 11 nm, 12 nm, 13 nm, or 14 nm. In embodiments, the longest dimension or length of the protein structure of the conjugate can be less than 25 nm, 20 nm, 17.5 nm, 15 nm, 14 nm, 13 nm, 12 nm, or 11 nm. Suitably, the length ranges from 5 nm to 25 nm, more suitably from 10 nm to 15 nm, and even more suitably from 11 nm to 14 nm or from 12 nm to 13 nm. In embodiments, the width or diameter of the protein structure of the conjugate in dimensions substantially perpendicular to its length is at least 1 nm, 1.1 nm, 1.2 nm, 1.3 nm, 1.4 nm, 1.5 nm, 1.6 nm, 1.7 nm, 1.8 nm, 1.9 nm, or 2.0 nm. In embodiments, the longest dimension or length of the protein structure of the conjugate can be less than 5.0 mm, 4.5 nm, 4.0 nm, 3.5 nm, 3.0 nm, 2.9 nm, 2.8 nm, 2.7 nm, 2.6 nm, 2.5 nm, 2.4 nm, 2.3 nm, 2.2 nm, 2.1 nm, or 2.0 nm. Suitably, the width ranges from 1 nm to 5 nm, more suitably from 1 nm to 3 nm, and even more suitably from 1.5 nm to 2.5 nm. In one embodiment, the protein structure of the conjugate of the present invention can have a pointed shape with a cross-section that is generally quadrilateral (four sides) along the longitudinal axis. Suitably, the shape is rectangular. Suitably, the tip or end of the protein structure is quadrilateral. The tip can be generally rectangular, i.e., without precise right angles or the corners can be somewhat rounded. Suitably, the length of the tip is in the range of 5 nm to 25 nm, and the width is in the range of 1 nm to 5 nm. In embodiments, the size of the tip can be as defined above for protein conjugates.

[0239] The physical size of the protein moiety of the conjugates of the present invention is defined as a combination of its β-helix structure and its molecular weight. Definitions of protein size based on its physical size or its molecular weight are used interchangeably. In embodiments of the invention, the molecular weight of the protein moiety may be at least 30 kDa. Suitably, the molecular weight of the protein moiety may be at least 35 kDa, 40 kDa, 45 kDa, or 50 kDa. In embodiments of the invention, the molecular weight of the protein moiety may be at most 100 kDa. Suitably, the molecular weight of the protein moiety may be at most 90 kDa, 80 kDa, 70 kDa, 60 kDa, 55 kDa, or 50 kDa. Suitably, the molecular weight of the protein moiety of the conjugates of the present invention ranges from 30 to 100 kDa, more suitably from 40 to 60 kDa, and even more suitably from 48 to 55 kDa.

[0240] Not wanting to be bound by theory, it is envisioned that the beneficial cell-penetrating properties demonstrated by the conjugates of this invention are due to the physical size of the protein portion as defined by the size or molecular weight as outlined above. Another characteristic of proteins is the stiffness primarily derived from the uncommon β-helical secondary structure. Appropriately, proteins can be more rigid than the membrane to be penetrated. Typical cell membrane stiffness ranges from 0.005 to 0.02 N / m. 2 (As measured by atomic force microscopy; Hayashi, "Tensile Properties and Local Stiffness of Cells"; Mechanics of Biological Tissue, pp. 137-152), this depends on the cell type. Appropriately, the stiffness or high stiffness parameter (K) of the proteins of this invention is 0.7 to 12 N / m. 2 The β-helix is ​​typically (Keten et al., Cell and Molecular Bioengineering, 2009; 2; 66-74, which is incorporated herein by reference).

[0241] Another feature considered to influence the cell penetration efficiency of the conjugates of the present invention is the charge spectrum and arrangement of amino acids in the β-helix structure of the protein. Specifically, the presence of charged lysine, arginine asparagine, aspartic acid, and / or glutamate "sequence ladders" in the β-helix protein structure promotes cell membrane penetration and the total negative charge of the protein sequence.

[0242] The term "sequence ladder" for amino acid residues in the structure of β-helical proteins is defined as the alternating arrangement of positively charged residues (lysine, arginine, and / or asparagine) and negatively charged residues (aspartic acid and / or glutamic acid) along the length of the protein surface.

[0243] Without being bound by theory, the presence of a charged “sequence ladder” structure in the β-helix structure of the protein according to the present invention can promote the interaction between the protein and lipid molecules of the cell membrane (e.g., in terms of forming hydrogen bonds with the hydroxyl groups of lipid molecules) and / or the protein first attaches to the negatively charged cell membrane.

[0244] According to the invention, the total negative charge of the protein can promote repulsion that causes the protein to move angularly, so that it stands upright on the membrane and punctures the membrane.

[0245] In embodiments of the invention, the protein of the conjugate comprises a sequence ladder of alternately arranged, surface-exposed positively and negatively charged amino acid residues. In embodiments, the protein comprises at least one of the following: an arginine sequence ladder (10-30 Arg residues), a lysine sequence ladder (10-30 Lys residues), an asparagine sequence ladder (10-40 Asn residues), aspartic acid (10-40 Asp residues), and glutamic acid (10-40 Glu residues).

[0246] In embodiments of the invention, the total formal charge of the protein may be zero, or it may be non-zero. Suitably, the total formal charge is non-zero; more suitably, the total formal charge is below zero (negative). In embodiments, the total formal charge of the protein is below (i.e., more negative) -10. Suitably, the total formal charge of the protein is below -20, -25, -30, -35, -40, -45, or -50. More suitably, the total formal charge is below -20. In embodiments, the total formal charge of the protein is above (i.e. less negative) -80. Suitably, the total formal charge of the protein is above -70, -65, -60, -55, -50, -45, -40, -35, or -30. More suitably, the total formal charge is above -60. In embodiments, the total formal charge of the protein is in the range of -10 to -80, more suitably -20 to -60.

[0247] The efficiency and mechanism of cell penetration can be modulated by optimizing the total formal charge of the conjugate by increasing the number of positively charged residues (e.g., arginine) along the surface of the protein, and the sequence can be mutated at the N-terminus along with a signal transduction sequence (SEQ ID NO: 14, 15, 16 or 17 and / or phosphatidylcholine) to obtain organelle specificity or specific cancer cell specificity.

[0248] In one embodiment, the protein of the conjugate of the present invention, suitable for direct cell penetration (i.e., non-endocytosis), may have one or more of the following structural parameters:

[0249] The hardness parameter K(β-helix) of the β-helix structure ranges from 0.2 to 12 N / m. 2 ;

[0250] - Length is between 5nm and 25nm;

[0251] - The diameter is between 1nm and 5nm;

[0252] - Molecular weight is between 25 kDa and 100 kDa;

[0253] - A sequence ladder of positively and negatively charged residues arranged along the length of the protein surface: arginine sequence ladder (10-30 Arg residues), lysine sequence ladder (10-30 Lys residues), asparagine sequence ladder (10-40 Asn residues), aspartic acid (10-40 Asp residues), and glutamic acid (10-40 Glu residues);

[0254] - The total formal charge is (-20 to -60).

[0255] In embodiments of the invention, the linker between the β-helical protein and the functional molecule can be formed by a direct connection between the β-helical protein and the functional molecule, for example, through covalent linkage of an amide (or peptide) or ester or through metal coordination; or the linker can take the form of a linker molecule. Suitablely, the connection is achieved through covalent or non-covalent bonds or interactions. When the linker is a linker molecule, the linker molecule can take any suitable form that reversibly connects the β-helical protein and the functional molecule. In some embodiments, the linker molecule can be selected from the group consisting of: peptides or proteins, PEG (polyethylene glycol) linkers, organic molecules, metal conjugates, drug-metal conjugates, nucleic acid binding domains, and nucleic acid intercalation molecules.

[0256] In one embodiment, the present invention discloses a conjugate having a recombinant β-helical protein with a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm, wherein the β-helical protein has a specific sequence for linking to a functional molecule. Suitably, the specific sequence is a pentapeptide repeat sequence. In one embodiment, the common sequence of the recombinant β-helical protein linked to the functional molecule is (STAV)1(DN)2(LF)3(STR)4(G)5. Examples of pentapeptide repeat sequence proteins are SEQ ID Nos: 1, 2, 6, and 8.

[0257] In embodiments of this disclosure, the recombinant β-helical protein is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0258] In embodiments of the present invention, the functional molecule can be any organic molecule (anticancer drug, antibiotic, NSAID, analgesic or any other drug molecule, fluorescent dye, insecticide, pesticide, etc.), drug-metal complex, metal, antibody, protein, polysaccharide, nucleic acid, peptide, nuclear localization signal, quantum dot and nanoparticle.

[0259] The conjugates can be used to facilitate the transfer of functional molecules within cells through their ability to penetrate the cell membrane. Utilizing appropriate localization signals associated with or integrated with the conjugate or the functional molecule, or both, the conjugates of the present invention can be used to target functional molecules to specific parts of the cell interior, such as organelles present within the cell.

[0260] In one embodiment, the present invention also discloses a method for transferring functional molecules within cells using the cell-penetrating conjugate of the present invention. The disclosed method can be further used for cell labeling, cell penetration, and targeting any functional molecule to organelles.

[0261] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0262] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0263] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0264] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0265] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the β-helical protein is AlbG having the sequence as described in SEQ ID NO:1.

[0266] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the β-helical protein is an EfsQNR having the sequence described in SEQ ID NO:2.

[0267] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having the sequence as described in SEQ ID NO:3.

[0268] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having the sequence as described in SEQ ID NO:4.

[0269] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having the sequence as described in SEQ ID NO:5.

[0270] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is QNRB1 having the sequence as described in SEQ ID NO:6.

[0271] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a UDP N-acetylglucosamine acyltransferase protein having the sequence as described in SEQ ID NO:7.

[0272] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is NP275 having the sequence as described in SEQ ID NO:8.

[0273] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectic acid lyase C having the sequence as described in SEQ ID NO:9.

[0274] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectic acid lyase having the sequence as described in SEQ ID NO: 10.

[0275] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a carbonic anhydrase having the sequence as described in SEQ ID NO: 11.

[0276] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectin lyase A having the sequence as described in SEQ ID NO: 12.

[0277] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0278] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a dye.

[0279] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a drug.

[0280] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a metal.

[0281] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a drug-metal complex.

[0282] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a protein.

[0283] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is an enzyme.

[0284] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is an antibody.

[0285] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a nucleic acid.

[0286] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a polysaccharide.

[0287] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a nuclear localization signal.

[0288] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein the functional molecule is a nanoparticle.

[0289] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein a functional molecule is linked to a recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

[0290] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein a functional molecule is covalently linked to a recombinant β-helical protein.

[0291] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein a functional molecule is linked to a recombinant β-helical protein via a non-covalent bond.

[0292] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence.

[0293] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a phospholipid molecule.

[0294] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0295] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a phosphatidylcholine molecule.

[0296] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence as described in SEQ ID NO: 14.

[0297] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence as described in SEQ ID NO: 15.

[0298] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence as described in SEQ ID NO: 16.

[0299] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule as described herein, wherein the conjugate further comprises a signal sequence as described in SEQ ID NO: 17.

[0300] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the nucleus of a cell, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO: 14.

[0301] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the nucleus of a cell as described herein, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and combinations thereof, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:14.

[0302] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the nucleus of a cell as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0303] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the endoplasmic reticulum of a cell, wherein the β-helical protein has a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:15.

[0304] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the endoplasmic reticulum of a cell as described herein, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:15.

[0305] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the endoplasmic reticulum of a cell as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0306] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the mitochondria of a cell, wherein the β-helical protein has a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:16.

[0307] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the mitochondria of a cell as described herein, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:16.

[0308] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into the mitochondria of a cell as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0309] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule to P-cadherin-overexpressing breast cancer cells, wherein the β-helical protein has a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:17.

[0310] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a functional molecule linked to a recombinant β-helical protein for transferring the functional molecule to P-cadherin-overexpressing breast cancer cells as described herein, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the conjugate further comprises a signal sequence as described in SEQ ID NO:17.

[0311] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a functional molecule linked to a recombinant β-helical protein for transferring the functional molecule to P-cadherin-overexpressing breast cancer cells as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0312] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule into a cell membrane, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the conjugate further comprises a phosphatidylcholine molecule.

[0313] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule to the membrane of a cell as described herein, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and combinations thereof, and wherein the conjugate further comprises a phosphatidylcholine molecule.

[0314] In one embodiment of this disclosure, a cell-penetrating conjugate is provided, the cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule for transferring the functional molecule to the membrane of a cell as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0315] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell is provided, the method comprising: (a) conjugating the functional molecule to a recombinant β-helical protein to obtain a conjugate; and (b) contacting the conjugate with at least one cell, wherein contacting the conjugate transfers the functional molecule into the cell, and wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the method further comprises, after step (c), a step (d) of detecting the transfer of the conjugate within the cell.

[0316] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0317] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0318] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0319] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is AlbG having the sequence shown in SEQ ID NO:1.

[0320] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein is an EfsQNR having the sequence shown in SEQ ID NO:2.

[0321] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:3.

[0322] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:4.

[0323] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:5.

[0324] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is QNRB1 having the sequence shown in SEQ ID NO:6.

[0325] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is a UDP-N-acetylglucosamine acyltransferase protein having the sequence shown in SEQ ID NO:7.

[0326] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is NP275 having the sequence shown in SEQ ID NO:8.

[0327] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein is a pectic acid lyase C having the sequence shown in SEQ ID NO:9.

[0328] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is a pectic acid lyase having the sequence shown in SEQ ID NO:10.

[0329] In one embodiment of this disclosure, a method for transferring a functional molecule into a cell as described herein is provided, wherein the β-helical protein is a carbonic anhydrase having the sequence shown in SEQ ID NO:11.

[0330] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein has the sequence described in SEQ ID NO:12.

[0331] In one embodiment of this disclosure, a method is provided for transferring functional molecules into cells as described herein, wherein the functional molecules are selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0332] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is a dye.

[0333] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into cells as described herein, wherein the functional molecule is a drug.

[0334] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is a metal.

[0335] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into cells as described herein, wherein the functional molecule is a drug-metal complex.

[0336] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is a protein.

[0337] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is an enzyme.

[0338] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into cells as described herein, wherein the functional molecule is an antibody.

[0339] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is a nucleic acid.

[0340] In one embodiment of this disclosure, a method for transferring a functional molecule, as described herein, into cells, wherein the functional molecule is a polysaccharide.

[0341] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is a nuclear localization signal.

[0342] In one embodiment of this disclosure, a method is provided for transferring functional molecules into cells as described herein, wherein the functional molecules are nanoparticles.

[0343] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the functional molecule is linked to a recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

[0344] In one embodiment of this disclosure, a method for transferring functional molecules into cells as described herein is provided, wherein the cells are selected from the group consisting of eukaryotic cells, prokaryotic cells, and combinations thereof.

[0345] In one embodiment of this disclosure, a method for transferring functional molecules into cells as described herein, wherein the cells are prokaryotic cells.

[0346] In one embodiment of this disclosure, a method for transferring functional molecules into cells as described herein, wherein the cells are eukaryotic cells.

[0347] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into cells as described herein, wherein the β-helical protein is represented by a sequence as shown in SEQ ID NO:1, and the functional molecule is an NHS coumarin dye.

[0348] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into cells as described herein, wherein the β-helical protein is represented by a sequence as shown in SEQ ID NO:2, and the functional molecule is an NHS coumarin dye.

[0349] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the β-helical protein is represented by a sequence as described in SEQ ID NO:2, and the functional molecule is a ruthenium metal complex.

[0350] In one embodiment of this disclosure, a method for transferring functional molecules into cells as described herein is provided, wherein the method is used for cell labeling.

[0351] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a cell as described herein, wherein the method is used to deliver the functional molecule into the cell.

[0352] In one embodiment of this disclosure, a method for transferring a functional molecule into organelles of a cell is provided, the method comprising: (a) conjugating the functional molecule to a recombinant β-helical protein to obtain a conjugate; (b) further incorporating the conjugate with any one signal sequence selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; (c) contacting the conjugate of step (b) with at least one cell; wherein contacting the conjugate in step (b) transfers the functional molecule into the organelle within the cell, wherein the organelle is selected from the group consisting of the nucleus, endoplasmic reticulum, and mitochondria, and wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the method further comprises, after step (c), a step (d) of detecting the transfer of the conjugate into the cell.

[0353] In one embodiment of this disclosure, a method for transferring a functional molecule into an organelle of a cell as described herein is provided, wherein the recombinant β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0354] In one embodiment of this disclosure, a method for transferring functional molecules into the nucleus of a cell as described herein is provided, wherein the signal sequence is shown in SEQ ID NO:14.

[0355] In one embodiment of this disclosure, a method for transferring functional molecules into the endoplasmic reticulum of cells as described herein is provided, wherein the signal sequence is shown in SEQ ID NO:15.

[0356] In one embodiment of this disclosure, a method for transferring functional molecules into the mitochondria of cells as described herein is provided, wherein the signal sequence is shown in SEQ ID NO:16.

[0357] In one embodiment of this disclosure, a method is provided for transferring functional molecules into organelles of cells as described herein, wherein the functional molecules are selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0358] In one embodiment of this disclosure, a method for transferring a functional molecule to P-cadherin-overexpressing breast cancer cells is provided, the method comprising: (a) linking the functional molecule to a recombinant β-helical protein to obtain a complex; (b) further incorporating the complex with a signal sequence as shown in SEQ ID NO:17; and (c) contacting the complex of step (b) with at least one P-cadherin-overexpressing breast cancer cell; wherein contacting the complex in step (b) transfers the functional molecule to the P-cadherin-overexpressing breast cancer cell, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the method further comprises, after step (c), a step (d) of detecting the transfer of the complex within the cells.

[0359] In one embodiment of this disclosure, a method for transferring a functional molecule to P-cadherin-overexpressing breast cancer cells as described herein is provided, wherein the recombinant β-helical protein having the sequence is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0360] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into P-cadherin-overexpressing breast cancer cells as described herein, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal complexes, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0361] In one embodiment of this disclosure, a method as described herein is provided, wherein the method is used for targeted delivery of the functional molecule into the cell.

[0362] In one embodiment of this disclosure, a method as described herein is provided, wherein the method is used to label the cells.

[0363] In one embodiment of this disclosure, a method as described herein is provided, wherein the method is used for targeted delivery of a drug in cells.

[0364] In one embodiment of this disclosure, a cell-penetrating complex as described herein is provided, wherein a recombinant β-helical protein is used to deliver a functional molecule into a cell, and wherein said functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal conjugates, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0365] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into an actin protein present in a cell, the method comprising: (a) conjugating the functional molecule to a recombinant β-helical protein to obtain a conjugate; (b) further incorporating the conjugate with a signal sequence as shown in SEQ ID NO:22; and (c) contacting the conjugate of step (b) with at least one cell; wherein contacting the conjugate in step (b) transfers the functional molecule into the cell, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the method further comprises, after step (c), a step (d) of detecting the transfer of the conjugate within the cell.

[0366] In one embodiment of this disclosure, a method is provided for transferring a functional molecule into a tubulin protein present in a cell, the method comprising: (a) conjugating the functional molecule to a recombinant β-helical protein to obtain a conjugate; (b) further incorporating the conjugate with a signal sequence as shown in SEQ ID NO:23; and (c) contacting the conjugate of step (b) with at least one cell; wherein contacting the conjugate in step (b) transfers the functional molecule into the P-cadherin-overexpressing breast cancer cells, wherein the β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the method further comprises, after step (c), a step (d) of detecting the transfer of the conjugate within the cell.

[0367] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein recombinant β-helical protein is used for cell penetration.

[0368] In one embodiment of this disclosure, a cell-penetrating conjugate as described herein is provided, wherein recombinant β-helical protein is used for cell labeling.

[0369] Cell-penetrating molecules containing nucleic acids

[0370] In one embodiment, the present invention provides a solution to the problem of delivering nucleic acid fragments as functional molecules in gene therapy or gene editing treatment. This document discloses a conjugate comprising a recombinant β-helical protein linked to a nucleic acid molecule (e.g., a plasmid, such as an RNA or DNA plasmid capable of penetrating a cell membrane). In one embodiment, the linked recombinant β-helical protein may be linked to one or more nucleic acid molecules, appropriately a single nucleic acid molecule or plasmid, via suitable interaction forces (e.g., electrostatic or hydrophobic forces) or via a linker element, molecule, portion, or moiety.

[0371] A conjugate comprising a recombinant β-helical protein, a linker, and a plasmid has been shown to successfully penetrate the cell membrane to establish the expression of a gene that forms part of the plasmid. The conjugate bypasses endocytosis and crosses the cell membrane directly. Therefore, the conjugate overcomes the challenges of entering via endocytosis while simultaneously and efficiently penetrating the cell membrane.

[0372] In one embodiment, the present invention discloses a conjugate capable of penetrating a cell membrane. In some embodiments, the conjugate comprises a recombinant β-helical protein linked to a nucleic acid molecule via a linker, having a length in the range of 5 nm-25 nm, suitably 10 nm-15 nm, and a width in the range of 1 nm-5 nm, suitably 1 nm-3 nm. In one embodiment, the recombinant β-helical protein may be a pentapeptide repeat sequence protein having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5. In another embodiment of the present disclosure, the recombinant β-helical protein is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0373] According to this disclosure, in one embodiment, the recombinant β-helical protein is linked to a nucleic acid molecule via a linker element, molecule, portion or portion. In embodiments, the linker element can be a direct link via a covalent or non-covalent bond. When the linker is a linker molecule, it can be selected from the group consisting of proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, and nucleic acid intercalation molecules. In one embodiment, the nucleic acid molecule comprising a portion of the conjugate includes at least one gene of interest. In one embodiment, the nucleic acid molecule is linked to a complex comprising the recombinant β-helical protein and the linker to form a conjugate as disclosed in this invention. In one embodiment, the conjugate is capable of entering the cell by penetrating the cell membrane, and the gene comprising a portion of the nucleic acid molecule is capable of being expressed within the cell.

[0374] The present invention also discloses a method for transferring nucleic acid molecules within cells using the conjugates of the present invention. In one embodiment, the disclosed process can be further used in gene therapy or gene editing techniques to facilitate non-viral methods for delivering genes of interest within cells, thereby effectively enabling intracellular editing of defective genes or compensating genes or proteins.

[0375] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0376] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0377] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0378] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein comprises a tandem repeating pentapeptide having a concordant sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0379] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the adapter is an adapter molecule selected from the group consisting of: proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0380] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the adapter is an adapter molecule selected from the group consisting of: proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0381] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the adapter is connected to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0382] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the adapter is an adapter molecule selected from the group consisting of: proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof, and wherein the adapter is connected to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0383] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the nucleic acid molecule comprises at least one gene of interest.

[0384] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the nucleic acid molecule comprises at least one gene of interest.

[0385] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the adapter is an adapter molecule selected from the group consisting of: proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof, and wherein the nucleic acid molecule comprises at least one gene of interest.

[0386] In one embodiment, the present invention provides a solution to the problem of transmembrane delivery of gene-editing molecules and their entry into the cell and / or its compartments by providing a method of transfecting plasmids or nucleic acids encoding one or more genome editing system molecules into the cell. Once the plasmids or nucleic acids are in the cell, the one or more genome editing system molecules entering the cell can be prepared using cellular processes and optionally combined with other desired components of a genome editing system to provide a complete gene-editing system within the cell.

[0387] In one embodiment of this disclosure, the gene of interest in a nucleic acid or plasmid encodes one or more molecules of a genome editing system. Suitable, the one or more molecules of the genome editing system are selected from, but not limited to, the group consisting of: RNA-guided endonucleases and / or guide RNA (gRNA), such as Cas9; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases. DNA-guided endonucleases and / or guide DNA; homing endonucleases; integrases; and any combination thereof.

[0388] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is AlbG having the sequence shown in SEQ ID NO:1.

[0389] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an EfsQNR having the sequence shown in SEQ ID NO:2.

[0390] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:3.

[0391] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:4.

[0392] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is an antifreeze protein having a sequence as shown in SEQ ID NO:5.

[0393] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is QNRB1 having the sequence shown in SEQ ID NO:6.

[0394] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a UDP-N-acetylglucosamine acyltransferase protein having the sequence shown in SEQ ID NO:7.

[0395] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is NP275 having the sequence shown in SEQ ID NO:8.

[0396] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectic acid lyase C having the sequence shown in SEQ ID NO:9.

[0397] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectic acid lyase having the sequence shown in SEQ ID NO: 10.

[0398] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a carbonic anhydrase having the sequence shown in SEQ ID NO: 11.

[0399] In one embodiment of this disclosure, a conjugate is provided, the conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm, and wherein the β-helical protein is a pectin lyase A having the sequence shown in SEQ ID NO: 12.

[0400] In one embodiment of this disclosure, a method for transferring a nucleic acid molecule or plasmid into a cell is provided, the method comprising: (i) conjugating the nucleic acid molecule to a complex comprising: (a) at least one recombinant β-helical protein; and (b) at least one adapter for obtaining the conjugate; and (ii) contacting the conjugate with at least one cell, wherein contacting the conjugate transfers the nucleic acid molecule into the cell, and wherein the recombinant β-helical protein has a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm.

[0401] In one embodiment of this disclosure, a method for transferring a nucleic acid molecule or plasmid into a cell as described herein is provided, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0402] In one embodiment of this disclosure, a method for transferring nucleic acid molecules into cells as described herein is provided, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0403] In one embodiment of this disclosure, a method is provided for transferring nucleic acid molecules into cells as described herein, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0404] In one embodiment of this disclosure, a method is provided for transferring nucleic acid molecules into cells as described herein, wherein the adapter is an adapter molecule selected from the group consisting of proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0405] In one embodiment of this disclosure, a method for transferring a nucleic acid molecule into a cell as described herein is provided, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof, and wherein the adapter is an adapter molecule selected from the group consisting of: proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0406] In one embodiment of this disclosure, a method for transferring nucleic acid molecules into cells as described herein is provided, wherein the adapter is linked to a recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

[0407] In one embodiment of this disclosure, a method is provided for transferring nucleic acid molecules into cells as described herein, wherein the adapter is an adapter molecule selected from the group consisting of proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof, and wherein the adapter is linked to a recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

[0408] In one embodiment of this disclosure, a method is provided for transferring nucleic acid molecules into cells as described herein, wherein the cells are prokaryotic or eukaryotic cells.

[0409] In one embodiment of this disclosure, a method is provided for transferring nucleic acid molecules into cells as described herein, wherein the gene of interest in the nucleic acid or plasmid encodes one or more molecules of a genome editing system. Suitably, the one or more molecules of the genome editing system are selected from, but not limited to, the group consisting of: RNA-guided endonucleases and / or guide RNA (gRNA), such as Cas9; zinc finger nucleases (ZFNs); transcription activator-like effector nucleases (…). DNA-guided endonucleases and / or guide DNA; homing endonucleases; integrases; and any combination thereof.

[0410] Cell-penetrating peptides for efficient transmembrane transfer of gene-editing molecules

[0411] This invention provides a solution to the problem of transmembrane delivery of gene-editing molecules and their entry into cells and / or their compartments. This document discloses a conjugate comprising a recombinant β-helical protein linked to at least one gene-editing molecule or gene-editing complex that can efficiently penetrate the cell membrane. In one embodiment, the recombinant β-helical protein can be linked to an endonuclease and one or more gRNA molecules via non-covalent interactions. In another embodiment, the recombinant β-helical protein can be linked to a CRISPR gene-editing molecule, such as the Cas9 endonuclease or gRNA, and / or to a complex of Cas9 and one or more gRNA molecules via non-covalent interactions.

[0412] Conjugates comprising recombinant β-helical protein and CRISPR gene-editing molecules have been shown to successfully penetrate cell membranes for site-specific genome editing. Without being bound by theory, it is assumed that the conjugates bypass endocytosis and cross the cell membrane directly. Therefore, these conjugates overcome the challenges of entering via endocytosis while simultaneously and effectively penetrating the cell membrane.

[0413] In some embodiments, the conjugate comprises a recombinant β-helical protein linked to a nucleic acid molecule via a linker, having a length in the range of 5 nm to 25 nm, suitably 10 nm to 15 nm, and a width in the range of 1 nm to 5 nm, suitably 1 nm to 3 nm. In one embodiment, the recombinant β-helical protein may be a pentapeptide repeat sequence protein having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5. In another embodiment of this disclosure, the recombinant β-helical protein is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0414] According to this disclosure, in one embodiment, the recombinant β-helical protein is linked to a nucleic acid molecule via a linker element, molecule, or part thereof. In embodiments, the linker element may be a direct link via a covalent or non-covalent bond. When the linker is a linker molecule, it may be selected from the group consisting of proteins, metal conjugates, drug-metal conjugates, DNA-binding domains, and nucleic acid intercalation molecules.

[0415] In one embodiment, the conjugate is capable of penetrating the cell membrane to enter the cell and migrating to the relevant genome where site-specific editing may occur.

[0416] The present invention also discloses a method for transferring CRISPR-RNPs within cells using the conjugates of the present invention. In one embodiment, the disclosed method can be further used to facilitate gene editing techniques using non-viral methods to allow the editing, removal of existing genes, and / or the addition of new genes.

[0417] In one embodiment of this disclosure, a method is provided for transferring CRISPR-RNP into cells as described herein, wherein the cells are prokaryotic or eukaryotic cells.

[0418] In one embodiment of this disclosure, a conjugate as described herein is provided, wherein the conjugate is used as a transfection agent.

[0419] In one embodiment of this disclosure, a conjugate as described herein is provided, wherein the conjugate is used for gene therapy.

[0420] Example - Cell-penetrating molecules with functional molecules

[0421] This disclosure will now be illustrated using working examples, intended to explain the work of this disclosure and not intended to restrictively imply any limitation on the scope of this disclosure. Unless otherwise defined, 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 disclosure pertains. Although similar or equivalent methods and materials to those described herein may be used in the practice of the disclosed methods and compositions, exemplary methods, apparatus, and materials are described herein. It should be understood that this disclosure is not limited to the specific methods and described experimental conditions, as such processes and conditions can vary.

[0422] In the following examples, methods and protocols for performing plasmid and protein studies are provided. Protocols for obtaining conjugates, labeling proteins, and performing cell penetration studies using protein-drug conjugates and protein-labeled conjugates are also provided. The Results section specifically describes proof-of-concept studies of the cell penetration capabilities of the conjugates disclosed herein. In vitro labeling of different cell lines has been performed using the conjugates of this invention according to the disclosed methods. Assays using drug-protein conjugates have also been performed to investigate the ability of the conjugates to enhance drug uptake through cell penetration.

[0423] Materials and methods

[0424] Hoechst 33342 dye was purchased from Invitrogen; NHS coumarin and ATTO 520-NHS, ATTO 390-NHS, and ATTO 647N-NHS were purchased from Sigma-Aldrich. Organic solvents and reagents for UV-Vis spectrophotometry and CD spectroscopy were purchased from Sigma-Aldrich and Merck. Reagents for studying plasmid expression and protein purification were purchased from Sigma-Aldrich and Merck. Reagents required for MTT assays were purchased from MP Biomedicals. Ruthenium metal complexes were purchased from Sigma-Aldrich.

[0425] Example 1

[0426] Plasmid / protein research - A plasmid containing the AlbG gene as shown in SEQ ID NO:19 was obtained by the method previously disclosed (Vetting et al., Acta Crystallographica Sinica Series F: Structural Biology and Crystallography Communications, 2011:67(3):296-302, the contents of which are incorporated herein by reference and specific details are provided below).

[0427] The open reading frame of AlbG was amplified using standard PCR techniques using X albilineans (ATCC 29184; Pieretti et al., BMC Genomics, 2009, 10:616, 1-15) chromosomal DNA as a template. Oligonucleotides AlbGF (5'-ATCCCGCTCATATGCCGGCCAAGACCCTTG-3') and AlbGR (5'-ATCCCGCTCTCGAGTCAATCGGACAGCTCGATATC-3'), containing NdeI and XhoI restriction sites, respectively, were used. The PCR fragments were cloned into pET-28a(+), and recombinant AlbG with a thrombin-cleavable N-terminal His6 tag was expressed in Escherichia coli strain BL21(DE3). For shake-flask growth, 10 ml of overnight culture was inoculated into 1 liter of Luria broth supplemented with kanamycin (35 μg / ml), and incubated at 37°C. The culture was allowed to grow to mid-log phase (A0). 600 Cells were cooled at 20°C, induced with 0.5 mM IPTG, and further cultured overnight at 20°C. All purification procedures were performed at 4°C. Cells were collected by centrifugation at 3000 g, resuspended in 50 mM Tris-HCl pH 7.8 containing 300 mM NaCl, protease inhibitors, lysozyme (5 μg / ml), and DNase I (0.1 μg / ml) and stirred for 20 min. Cells were then lysed by sonication and cell debris was removed by centrifugation at 10,000 g for 30 min. The supernatant was loaded into a nickel-nitrotriacetic acid (Ni-NTA) column pre-equilibrated with buffer A and washed with ten column volumes of the same buffer. Bound proteins were eluted with a linear 0 to 0.3 M imidazole gradient, and peak fractions were pooled and concentrated.

[0428] The plasmid containing the EfsQNR gene as shown in SEQ ID NO:2 was obtained by the previously disclosed method (Hegde et al., Antimicrob. Agents Chemother, January 2011; 55(1):110–117, the contents of which are incorporated herein by reference and specific details are provided below).

[0429] The open reading frame of EfsQNR was amplified using standard PCR techniques with *Enterococcus faecalis* V583 (ATCC 700802; ENTFA226185) chromosomal DNA as a template. Oligonucleotides containing NdeI and XhoI restriction sites (5'-ATCCCGCTCATATGAAAATAACTTATCCCTTGCCA-3') and (5'-ATCCCGCTCTCGAGTTAGGTAATCACCAAACCAAGT-3'), respectively, were used. The PCR fragment was cloned into pET-28a(+), and recombinant EfsQNR with a thrombin-cleavable N-terminal His6 tag was expressed in *Escherichia coli* strain BL21(DE3). For shake-flask growth, 10 ml of overnight culture was inoculated into 1 L of Luria broth supplemented with kanamycin (35 μg / ml) and incubated at 37°C. The cultures were allowed to grow to metaphase I (A1-A2). 600 The sample was cooled at 20°C, induced with 0.5 mM isopropyl-β-d-thiogalactoside (IPTG), and further cultured overnight at 20°C.

[0430] Cells were collected by centrifugation at 1,200 g and resuspended in buffer A (50 mM Tris-HCl [pH 7.8], 300 mM NaCl) containing protease inhibitors, lysozyme (5 μg / ml), and DNase I (0.1 μg / ml), with the mixture stirred for 20 min. Cells were then lysed by sonication, and cell debris was removed by centrifugation at 10,000 g for 30 min. The supernatant was loaded into a Ni-NTA column pre-equilibrated with buffer A and washed with 10 column volumes of the same buffer. Bound proteins were eluted using a linear 0–0.3 M imidazole gradient, with fractions combined and concentrated.

[0431] As a comparative example, a plasmid containing the TtCuA gene as shown in SEQ ID NO:13 was obtained using the method described in Biochemistry, 2008, 47, 1309-1318, which is incorporated herein by reference.

[0432] Example 2

[0433] Toxicity testing– An industry-standard MTT assay (formerly Sigma-Aldrich) was performed to analyze the toxicity of β-helical protein-cytotoxic drug conjugates to mammalian cells, such as HeLa cells. Cells were cultured using a standard protocol. One million cells were seeded in confocal plates (1 cm disks) and grown for 6–8 hours. A mixture of the cytotoxic drug and EfsQNR protein (mixed at a 2:1 ratio) was added to the cells and incubated at room temperature for 15 min to 24 to 72 hours. After incubation, the cells were washed with PBS and treated with MTT, and further incubated for 24 hours. The cells were then washed and analyzed for absorbance values ​​at 570 nm. Metabolically active live cells convert MTT into a purple formazan product, where the absorbance is near its maximum at 570 nm. Dead cells cannot convert MTT into formazan; therefore, by analyzing the absorbance values ​​at 570 nm, the percentage of live cells for a given protein or any other molecule can be calculated.

[0434] Example 3

[0435] Proteins were labeled with fluorescent dyes. Fluorescent dyes are considered examples of functional molecules used to study the cell membrane penetration ability of conjugates. Recombinant proteins were labeled with dyes by performing a series of reactions under dark conditions. The proteins to be labeled were collected in PBS buffer (1× and pH 7.3) at a concentration two to three times higher than the protein concentration. The protein was added to 0.1 M sodium carbonate buffer (pH 8.5), followed by the dye. The dye was added very slowly (3 μl at a time) to the protein-containing buffer, kept on ice with occasional shaking. The resulting solution was wrapped in aluminum foil to protect it from any light. The solution was stirred at room temperature for 1 hour and then purified by gel filtration, either through a desalting column or a PD 10 column with 1× PBS buffer. The resulting labeled proteins were characterized using UV-Vis spectrophotometry to determine the dye:protein ratio.

[0436] Example 4

[0437] Cellular uptake of labeled proteins- The uptake of labeled proteins was examined by treating mammalian cells with different labeled proteins, such as AlbG (SEQ ID NO:1), EfsQNR (SEQ ID NO:2), and TtCuA (SEQ ID NO:13) as a comparative example. TtCuA is a cytochrome oxidase c protein from the thermophilic bacterium *Thermus thermophilus*. It is represented by the amino acid sequence shown in SEQ ID NO:13. Cells were seeded at a concentration of one million in confocal plates (1 cm disks) and allowed to grow for 6–8 hours. Subsequently, labeled proteins were added to the cells at different concentrations and cultured under standard conditions of 5% CO2 and 37°C. Cells were then washed twice with HBSS (Hanks blank salt solution) or PBS after 3 and 24 hours and observed under fluorescence microscopy and / or confocal laser scanning microscopy.

[0438] The uptake of labeled proteins was examined by treating *E. coli* and yeast (Kluveromyces) cells with different labeled proteins, such as AlbG (SEQ ID NO:1) and EfsQNR (SEQ ID NO:2). *E. coli* or *Kluveromyces* cells were inoculated and grown overnight until the OD reached 0.6. The cells were diluted 50-fold and 3 μmol of the labeled protein (conjugate) was added. The cells were further grown under standard growth conditions (37°C with shaking) for 24 hours. The treated cells were then centrifuged and washed 3–4 times in PBS. The cells were then dispersed in 100 μL of PBS and imaged under a fluorescence / confocal microscope.

[0439] In addition, different proteins labeled with the green fluorescent dye (A-520), such as AlbG (SEQ ID NO:1) and EfsQNR (SEQ ID NO:2), were found to be absorbed into antibiotic-resistant strains observed by confocal laser scanning microscopy, including bacteria (Vibrio pseudovibrio Ad37 / 5 / 13 / 14 and Enterobacteriaceae, among which Enterobacteriaceae strains are resistant to antibiotics such as erythromycin, chloramphenicol, tetracycline, nalidixic acid, kanamycin, and penicillin), yeast cells (Saccharomyces cerevisiae, Kluveromyces marxianus, and Candida albicans), plant cells (Arabidopsis thaliana), zebrafish embryos, and Drosophila embryos.

[0440] Protocols for intake by bacteria and yeast cells:

[0441] • Inoculate the corresponding bacterial and yeast strains the day before the experiment and allow them to grow overnight;

[0442] • Centrifuge the cells and add 2 ml of fresh culture medium;

[0443] • Add 100-200 μL of cells to the mixture;

[0444] • Add 10 μM of green-labeled CPP;

[0445] • Add fresh culture medium to bring the volume to 1 mL;

[0446] • After 24 hours, the treated cells were centrifuged and washed 3-4 times in PBS;

[0447] • The cells were dispersed in 100 μL of PBS and then imaged using a fluorescence / confocal microscope. Figure 25 and 26 ).

[0448] A protocol for plant cell uptake (Arabidopsis thaliana):

[0449] • The plants were grown for 7 days before the experiment;

[0450] • 20 μM of different cell-penetrating proteins labeled with green fluorescent dye (A-520), such as AlbG (SEQ ID NO:1) and EfsQNR (SEQ ID NO:2), were added to plants in buffer [three plant species grown in 500 μL PBS for 7 days];

[0451] • Place the mixture in a growth chamber and keep it static for 24 hours under standard growth conditions (keeping it static in a room with continuous light and a temperature of 23°C);

[0452] Wash the plant three times with PBS, then add fresh PBS and examine the cells under a microscope. Select colorless cells, such as those in the roots and stems, for visualization to avoid interference. Figure 23 and 24 ).

[0453] Protocols for the intake of zebrafish embryos and fruit fly embryos:

[0454] • Collect zebrafish and fruit fly embryos and wash them in PBS.

[0455] • Add 10 μM of different cell-penetrating proteins labeled with green fluorescent dye (A-520), such as AlbG (SEQ ID NO:1) and EfsQNR (SEQ ID NO:2), to a vial containing embryos and incubate for 2 hours;

[0456] • Collect embryos, wash them three times with PBS and disperse them in PBS, then place them on a glass coverslip and examine them under a microscope. Figure 27 and 28 ).

[0457] These results provide indications that the CPP of the present invention can transfer cargo into the cytoplasm of various cell types by crossing the cell membrane and / or cell wall. This opens the prospect of conjugating CPP with antibiotics such as ciprofloxacin and penicillin, or any other antibiotics, by using activated acid coupling chemistry (EDC / NHS) to conjugate the carboxyl group (-COOH) of the drug to the amino group (from the lysine residue) of the CPP. These reactions are well known in the literature and texts (reference: Greg T Hermansen, Bioconjugate Techniques, pp. 264-265). Conjugated CPPs are conjugated with antibiotics using linkers such as adipic acid dihydrazide, succinimide-6-hydrazine nicotinate acetone hydrazide, C6-succinimide-6-hydrazine nicotinate acetone hydrazide, and succinimide-hydrazine nicotinate hydrochloride. These linkers are known to be hydrolyzed under intracellular conditions to release the antibiotic intracellularly.

[0458] Compared to unconjugated antibiotics, antibiotic-CPP conjugates will improve the efficiency of cellular uptake and reduce or eliminate drug excretion through cellular mechanisms such as glycoproteins. Glycoprotein binding sites are in the <1 kDa region, while CPP drug conjugates are 48 kDa in size. This can help improve older antibiotics and contribute to addressing the problem of antimicrobial resistance. Similarly, this technology can be used to improve the efficiency of antibiotic / pesticide / nutrient delivery in fish and plants.

[0459] Example 5

[0460] cellular uptake of drugsHeLa cells were used to determine the enhancement of drug uptake using the cell-penetrating conjugate of the present invention. HeLa cells were seeded in 96-well plates at a concentration of 10,000 and allowed to grow for 6–8 hours. A ruthenium metal complex and EfsQNR were mixed at different ratios of 1:1, 1:2, and 1:3 to form conjugates, which were then added to the cells. The conjugate formed by ruthenium and EfsQNR at a 1:2 ratio produced the enhanced results. Cell death after 24 hours was monitored by MTT assay as previously described and correlated with the percentage of ruthenium-EfsQNR conjugate uptake by HeLa cells.

[0461] Results of Examples 1 to 5

[0462] Characterization of proteins and conjugates

[0463] Structural specificity – The size of the AlbG protein was determined from the publicly available crystal structure pvd id: 2xt2.pdb. This was achieved by measuring… The end-to-end atomic distance of the dimer structure was determined to be 10.7 nm in length and 2.6 nm in width (diameter). The total formal charge is -27. Alternating positive and negative charged residues are present: an arginine sequence ladder (20 Arg residues in total), lysine (14 residues), aspartic acid (30 Asp residues), and glutamic acid (31 Glu residues). Asparagine (20 Asn residues) forms a sequence ladder along the protein surface.

[0464] The size of the EfsQNR protein was determined from the publicly available crystal structure pvd id: 2w7z.pdb. This was achieved by measuring... The end-to-end atomic distance of the dimer structure determined the protein's length to be 10.9 nm and its width (diameter) to be 2.8 nm. The total formal charge is -41. Alternating positive and negative charged residues are present: an arginine sequence ladder (16 Arg residues in total), lysine (12 residues), aspartic acid (25 Asp residues), and glutamic acid (33 Glu residues). Asparagine (33 Asn residues) forms a sequence ladder along the protein surface.

[0465] For comparative purposes, the size of the TtCuA protein was determined from the publicly available crystal structure pvd id: 2CuA.pdb. This was achieved by measuring... The end-to-end atomic distance of the structure is determined to be 3.3 nm in length and 1.9 nm in width (diameter). The structure comprises 5 arginine residues, 4 lysine residues, 9 glutamic acid residues, and 6 asparagine residues, with a total charge of -3.

[0466] Figure 1The CD spectrum of the purified AlbG protein (SEQ ID NO:1) used in the study is shown. The protein was dialyzed in PBS, and CD spectra in the 200–300 nm region were measured using concentrations of 1–5 μmol. Additional dilutions with PBS were performed as needed. Negative CD values ​​at 220 nm indicate the arrangement of β-sheets (β-barrel or β-helical structures).

[0467] Figure 2 The purified plasmids used to express β-helical protein, AlbG, and EfsQNR are shown, each showing the same results in triplicate (AlbG-1 to AlbG-3 and EfsQNR-1 to EfsQNR-3).

[0468] Figure 3 The purified protein bands of AlbG and EfsQNR proteins are shown in a polyacrylamide gel. Following SDS gel electrophoresis, the molecular weight of both proteins in monomeric form was noted to be approximately 30 kDa. The approximate molecular weight of the dimer forms of AlbG and EfsQNR was approximately 48 kDa. Proteins AlbG and EfsQNR were used to form conjugates with functional molecules, which were then used to study cell penetration efficacy. Any cytotoxicity of the proteins was tested using HeLa cells to determine the study process (see “Cytotoxicity Studies” below).

[0469] Figure 4 The results of MALDI TOF mass spectrometry analysis of EfsQNR and AlbG proteins are shown. For this experiment, the proteins were dialyzed in water, and samples were prepared using sinapic acid, 0.1% TFA, and acetonitrile as the matrix. 1–10 μmol of protein was used. The results show that the molecular weight of EfsQNR protein is approximately 26.5 kDa, and the molecular weight of AlbG protein is approximately 23 kDa.

[0470] Cytotoxicity studies

[0471] The toxicity of AlbG and EfsQNR proteins to HeLa cells was investigated using the MTT assay as previously described. For the toxicity assay, samples of placebo, control, AlbG (30 μM), and EfsQNR (30 μM) were used in triplicate. Table 1 depicts the absorbance values ​​at 570 nm for each sample after MTT treatment. It is understood from the observed values ​​that cells treated with 30 μM AlbG and 30 μM EfsQNR showed 95% viability compared to control cells. This clearly demonstrates that the tested proteins at the tested concentrations are non-toxic to HeLa cells and are therefore safe concentrations for use in further experiments.

[0472] Table 1 presented in this article describes the absorbance values ​​at 570 nm in the MTT assay used to assess protein toxicity:

[0473] Table 1:

[0474]

[0475] The placebo in this experiment was 20 mM Tris buffer at pH 8.0; however, the control considered was only HeLa cells without any added proteins or other substrates.

[0476] Marker research

[0477] Figure 4 The UV-Vis spectrophotometry of the labeled AlbG and EfsQNR conjugates is illustrated using the procedure previously mentioned (see “Labeling Proteins with Fluorescent Dyes” above). UV-Vis spectra were analyzed to calculate the labeling ratio of the two conjugates. The labeling ratio represents the amount of dye attached to each protein molecule. This is calculated by measuring the ratio between the absorbance value of the dye (at a wavelength depending on the dye used for labeling) and the absorbance of the protein at 280 nm. The dye used for labeling in this experiment was NHS coumarin. In the case of AlbG protein, a dye:protein molecule ratio of 1.95 was observed. In the case of EfsQNR, a dye:protein molecule ratio of 5.5 was found. UV-Vis spectroscopy confirmed the labeling of both AlbG and EfsQNR proteins with NHS coumarin dye. Labeled proteins were further used in this study to establish cell penetration.

[0478] Cell penetration study

[0479] Figure 5 Fluorescence micrographs of HeLa cells treated with NHS-C-labeled AlbG, EfsQNR, and TtCuA proteins (conjugates) are shown. The uptake of the labeled proteins (conjugates) was compared with untreated HeLa cells, which served as the control group for this study. Hoechst Blue is a nuclear labeling dye used in conjunction with the NHS-C dye used to label the proteins. Proteins AlbG and EfsQNR are β-helical proteins with lengths ranging from 5 nm to 25 nm, suitably 10 nm to 15 nm, and widths ranging from 1 nm to 5 nm, suitably 1 nm to 3 nm, representing the recombinant β-helical proteins of this invention. However, TtCuA is a protein with a length of approximately 4 nm and consisting of β chains forming β barrels, thus representing a comparative example of the recombinant β-helical protein of this invention. The use of these two different classes of proteins in this study was considered to establish the superior cell penetration ability of the conjugates of this invention compared to different types of β-helical proteins.

[0480] In Figure 5 , panel A shows control cells treated only with the nuclear staining dye (Hoechst Blue), meaning the lighter dye is absent, panel B shows cells treated with Hoechst Blue dye and the protein (conjugate) labeled with TtCuA-NHSC, panel C shows cells treated with Hoechst Blue dye and the protein (conjugate) labeled with AlbG-NHSC, and panel D shows cells treated with Hoechst Blue dye and the protein (conjugate) labeled with EfsQNR-NHSC. Upon observation of said figure, it can be understood that, as shown in panel B, significantly less fluorescence is visible inside the cells, meaning less TtCuA-NHSC conjugate is present inside these cells, whereas more fluorescence is visible inside the cells as shown in panel C and panel D, meaning more AlbG-NHSC conjugate and EfsQNR-NHSC conjugate are present inside the cells. Therefore, it can be understood that, compared with the AlbG-NHSC conjugate and the EfsQNR-NHSC conjugate, the TtCuA-NHSC conjugate cannot effectively penetrate the cell membrane to enter the cell.

[0481] When comparing panel C and panel D, it can be observed that, compared with the AlbG-NHSC conjugate, the EfsQNR-NHSC conjugate can penetrate the membrane more effectively. Therefore, the cell penetration ability of the three conjugates can be summarized in the order of increasing cellular uptake as TtCuA-NHSC < AlbG-NHSC < EfsQNR-NHSC. This further demonstrates the cell penetration ability of conjugates comprising recombinant beta-helix proteins that have a length ranging from 5 nm to 25 nm, suitably from 10 nm to 15 nm, and a width ranging from 1 nm to 5 nm, suitably from 1 nm to 3 nm, respectively. Conjugates independently comprising AlbG and EfsQNR can exhibit enhanced cell penetration compared with conjugates comprising TtCuA.

[0482] Since the conjugate comprising EfsQNR protein shows the highest ability to penetrate cell membranes, further research was conducted on the enhanced labeling of mammalian cells by EfsQNR protein using different dyes.

[0483] Figure 22 shows real-time cell entry via direct interaction of the conjugate with the cell membrane. For viewing cells under fluorescence microscopy, cells were stained with Hoechst 33342, a nuclear-binding dye (blue, shown as dark color in Figure 22 it is shown as dark) and Alexa 594-WGA, a plasma membrane-binding dye (red, in Figure 22Cells were treated with EfsQNR-ATTO-520NHS (shown as lighter in color), indicating that EfsQNR-ATTO-520NHS (green, in...) was treated with lighter color in color. Figure 22 (Displayed as an aperture) It effectively penetrates into the cell through a direct mechanism that avoids any endocytosis.

[0484] Markers of mammalian cells

[0485] Figure 7 The ability of HeLa cells to be labeled with conjugates including dye-conjugated EfsQNR was described. Figure 6 Cells labeled with EfsQNR-ATTO-520NHS, prepared according to the labeling method described above, are depicted. The dye Hoechst 33342 (blue) is used. Figure 7 (Displayed as dark in the center) combines with the core, and the dye Alexa 594-WGA (red, in) Figure 7 (Appearing as lighter in color) it binds to the plasma membrane. This is achieved by binding the EfsQNR protein with the green fluorescent dye ATTO-520NHS (in...). Figure 7 The lightest dye shown in the image forms a conjugate. It can be clearly observed that the EfsQNR-ATTO-520NHS conjugate penetrates HeLa cells, resulting in effective cell labeling compared to control cells (Figure A, showing untreated HeLa cells) (Figure B).

[0486] Figure 8 Similar results were depicted in HeLa cells treated with a conjugate formed by linking the EfsQNR protein to the blue fluorescent dye ATTO-390NHS (in... Figure 8 (The image shows a lighter shade).

[0487] Figure 9 Similar results were depicted for microglia labeled with EfsQNR-ATTO-520NHS prepared according to the labeling method described above. Inset A shows the significant fluorescence of the dye from within the cells. Inset B shows additional results using the nuclear-binding dye Hoechst 33342 (blue, in...). Figure 9 (shown as dark in the middle) and the dye Alexa 594-WGA (red, in the middle) that binds to the plasma membrane. Figure 9 The microglia (shown as having a lighter treatment) indicate that EfsQNR-ATTO-520NHS has effectively penetrated into the cells.

[0488] Figure 10 Similar results were depicted in keratinocytes labeled with the conjugate EfsQNR-ATTO-520NHS prepared according to the labeling method described above. Inset A shows the significant fluorescence of the dye from within the cells. Inset B shows additional results using the nucleus-binding dye Hoechst 33342 (blue, in...) Figure 10 The keratinocytes treated (shown in dark) indicate that EfsQNR-ATTO-520NHS has effectively penetrated into the cell interior to be present near the nucleus.

[0489] Figure 11 Similar results were described for SH-SY5Y cells labeled with the conjugate EfsQNR-ATTO-520NHS prepared according to the above labeling method.

[0490] Figure 12 Similar results were described for mouse ES cells labeled with EfsQNR-ATTO-520NHS prepared according to the labeling method described above. Figure 12 The image shows the use of the nucleus-binding dye Hoechst 33342 (blue, in...) Figure 12 (shown as dark in the middle) and the dye Alexa 594-WGA (red, in the middle) that binds to the plasma membrane. Figure 12 The images show microglia mouse ES cells treated with a lighter dose (as shown in the images), indicating that EfsQNR-ATTO-520NHS has effectively penetrated into the cells.

[0491] Markers of non-mammalian cells

[0492] Figures 13 to 14 Figures 21 to 28 describe the ability of conjugates of dye-conjugated EfsQNR to label bacterial cells (Escherichia coli and Vibrio AD37), yeast cells (Kluyveromyces and Saccharomyces cerevisiae), plant cells (Arabidopsis thaliana), insect cells (Drosophila embryos), and fish cells (zebrafish embryos). Figures 13 to 14 Images 21 to 28 depict EfsQNR-ATTO-520NHS-labeled E. coli, Pseudomonas AD37, Kluyveromyces, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila embryonic cells, and zebrafish embryonic cells prepared according to the above labeling method.

[0493] FACS sorting of HeLa cells treated with conjugates

[0494] Figure 15 shows the results of standard FACS sorting of HeLa cells treated with a conjugate labeled with EfsQNR-ATTO-647N. Treatment of cells for only 10 minutes (inset B) resulted in significant dye uptake compared to the control (inset A), which continued to increase at 1 hour (inset C) until it reached its maximum at 3 hours (inset D).

[0495] Drug uptake research

[0496] Figure 16The cellular uptake of the ruthenium metal complex Ru(CO)3Clglycine in HeLa cells was depicted. The ruthenium metal complex conjugated with the EfsQNR protein to form a conjugate consisting of the ruthenium metal complex EfsQNR. HeLa cells were treated with the ruthenium metal complex-EfsQNR conjugate as described above (see “Cell Penetration Study” above), and the uptake of the complex was detected by examining cell viability using the MTT assay as previously described. (See Figure 1 for details.) Figure 14 It is understood that when treated with the ruthenium metal complex alone, cellular uptake was only 0.5%, while when treated with the ruthenium metal complex-EfsQNR protein conjugate, cellular uptake of the complex increased to 24.4%. Therefore, it can be determined that the conjugate including the EfsQNR protein promotes 50-fold higher cellular uptake compared to the ruthenium metal complex alone. This enhanced uptake can be further utilized by delivering many life-saving drugs to their targets using the conjugates of the present invention, thereby reducing the dosage of the drug and subsequently reducing side effects.

[0497] Figure 17 It shows the use of including and in Figure 17 Chemotherapy drugs labeled "CYDD" Results of a viability study of mammalian cells (HeLa and HepG2) cultured with conjugates of cisplatin or cisdichlorodiammineplatin(II) (CDDP) linked to the EfsQNR protein. The mammalian cells were cultured using a standard protocol (HeLa: inset A; or HepG2: inset B). One million cells were seeded in confocal plates (1 cm dishes) and grown for 8 hours. Then, cells containing... The conjugates of EfsQNR (prepared according to the above method at a molar ratio of 2:1) were used, and the cultures were kept at room temperature for 15 minutes. Cells were maintained under standard growth conditions for 24 and 72 hours. Cells were then washed with PBS and MTT solution was added. After the formation of formazan dye, it was dissolved in DMSO, and the absorbance at 550 nm was measured. The results clearly show that, in both cell types tested, Similar toxic effects are demonstrated when the drug is delivered at a lower dose in the form of a conjugate according to the invention.

[0498] It should be noted that similar results were obtained in all the experiments tested when the AlbG protein was used instead of the EfsQNR protein.

[0499] advantage

[0500] In summary, it can be concluded that conjugates and functional molecules comprising recombinant β-helical proteins of specific lengths and widths can act as highly efficient cell membrane-penetrating conjugates. The ability of these conjugates to directly penetrate the cell membrane offers an advantage over endocytic entry mechanisms. Working examples demonstrating the cell membrane-penetrating capabilities of AlbG-NHSC and EfsQNR-NHSC conjugates are provided in this disclosure. EfsQNR dye conjugates have shown enhanced labeling capabilities across a variety of mammalian and non-mammalian cells due to their efficient cell membrane penetration. Conjugates including EfsQNR and / or AlbG have been shown to increase the inclusion of ruthenium metal complexes and... The drug was observed to be absorbed by cells in HeLa and HepG2 cells. This ability could be further utilized to enhance the uptake of different anticancer drugs by cancer cells, which could facilitate treatment by selectively targeting cancer cells in an effective manner while simultaneously reducing the need for the drug. Reducing the dosage of anticancer drugs could also avoid the side effects associated with the administration of such drugs.

[0501] Example – Cell-penetrating molecules with nucleic acids

[0502] The following paragraphs provide working examples for intracellular delivery of nucleic acids, such as plasmids, containing genes of interest, via cell membrane penetration. Delivery is made possible by the use of conjugates that further comprise nucleic acid molecules or plasmids (such as cell-penetrating peptides described herein). The examples also depict the expression of the gene of interest after penetrating the cell membrane to enter the cell.

[0503] The first example describes an embodiment of transfecting a gene of interest into cells using the conjugate of the present invention, wherein the gene of interest is mcherry encoding RFP (red fluorescent protein). A plasmid containing the mcherry gene is linked to a protein to form a conjugate, wherein the conjugate comprises the EfsQNR protein and copper[II]phenanthroline. The conjugate is used to transfect HeLa cells. Successful expression of the mcherry gene in HeLa cells is shown as a proof of concept for establishing the ability of the conjugate to penetrate the cell membrane and transfect cells.

[0504] Materials and methods

[0505] The copper phenanthrene-roline complex was commercially procured.

[0506] Example 6

[0507] Protein research– The study on the expression, culture conditions, and purification of the dominant EfsQNR plasmid was followed up as previously published (Hegde et al., Antimicrob. Agents and Chemother. 2011:55(1):110-7, which is incorporated herein by reference) to obtain the EfsQNR protein. SDS-PAGE (polyacrylamide gel electrophoresis) was performed using the Bio-Rad kit, and protein bands were viewed using a 12% polyacrylamide gel.

[0508] Example 7

[0509] Preparation of plasmid conjugates - copper[II]phenanthroline bond

[0510] As disclosed in this invention, a conjugate for transfecting cells was prepared, the conjugate comprising EfsQNR (SEQ ID NO:2), copper[II]phenanthroline, and a plasmid containing the gene of interest.

[0511] Figure 18 A vector diagram of the plasmid carrying the mcherry gene (SEQ ID NO:25) used to prepare the conjugate in this example is depicted. The EfsQNR protein (SEQ ID NO:2) is a β-helical protein having a pentapeptide repeat sequence (as depicted in SEQ ID NO:18) with a length in the range of 5 nm–25 nm, suitably 10 nm–15 nm, and a width in the range of 1 nm–5 nm, suitably 1 nm–3 nm. Copper[II]phenanthroline is the nucleic acid intercalating agent used as the linker in this example.

[0512] The EfsQNR protein, obtained by controlling the expression of the EfsQNR plasmid, was conjugated with a copper[II]phenanthroline complex to obtain a complex comprising EfsQNR and copper[II]phenanthroline. The obtained complex was further reacted with a plasmid containing the mcherry gene to obtain a conjugate according to an embodiment of the invention. The obtained conjugate was used for transfection of HeLa cells.

[0513] Figure 19 Two schemes for preparing conjugates suitable for transfection are described. Scheme 1 demonstrates the use of copper[II]phenanthroline as a linker to form a complex with a protein. The resulting complex is further reacted with a plasmid containing the gene of interest to obtain the conjugate to be used for transfection.

[0514] Scheme 2 demonstrates the use of a DNA-binding protein as a linker to form a complex with a protein. The resulting complex is further reacted with a plasmid containing the gene of interest to obtain a conjugate for transfection.

[0515] In this example, copper[II]phenanthroline was used as a linker for forming the complex, and recombinant β-helical protein –EfsQNR (SEQ ID NO:2) was used with a plasmid containing the mcherry gene. The complex and conjugate were prepared using the following protocol:

[0516] The protein EfsQNR was mixed with copper[II]phenanthroline at a 1:2 molar ratio and incubated at room temperature for 30 minutes to obtain a complex. A plasmid containing the mcherry gene was mixed with the complex obtained in the previous step and incubated at 4°C for 30 minutes to obtain a conjugate. The conjugate thus obtained was used for transfection of HeLa cells.

[0517] Example 8

[0518] HeLa cells transfected using the conjugate from Example 7

[0519] Figure 20 A protocol for transfection experiments using the conjugate obtained in Example 7 is described. In this example, the following conditions are used to perform the transfection experiment.

[0520] The conjugate including the mcherry plasmid, as obtained in Example 7(A), was added to HeLa cells (8 hours post-inoculation). Cell growth was allowed by incubation at 37°C and 5% CO2 for 48 hours. After incubation, the cells were observed under confocal microscopy.

[0521] Figure 21 Confocal microscopy images of HeLa cells after transfection experiments were depicted. RFP expression was observed as a lighter (red) dot within HeLa cells, demonstrating the cell penetration and transfection capabilities of the conjugate disclosed in this document.

[0522] Example 9

[0523] Preparation of plasmid conjugates – ethylenediamine bond

[0524] As further disclosed in this invention, a conjugate for transfecting cells with a plasmid, typically encoding one or more gene-editing molecules, was prepared. The conjugate comprises EfsQNR (SEQ ID NO: 2), an ethylenediamine linker, and a plasmid. The conjugate was prepared according to a known protocol (see: *Bioconjugate Techniques*, Greg T. Hermansen; Academic Press; 3rd ed.; 2013; pp. 264-265, which is incorporated herein by reference). The plasmid used was a 2.7 kb red-labeled circular plasmid DNA (labeled from MirusBio). plasmid sent for control (Product code MIR 7904)).

[0525] Example 10

[0526] Transfection of MCF7 cells using the conjugate from Example 9

[0527] This will include plasmids [labeled] as obtained in Example 9. plasmid sent for control The conjugate (product code MIR 7904) was added to MCF-7 cells (8 hours post-inoculation). Cell growth was allowed by incubation at 37°C and 5% CO2 for 12–16 hours. Cells were then observed under confocal microscopy after incubation.

[0528] Figure 30 The results of transfection experiments performed in MCF7 cells using the following: (A) the conjugate (EfsQNR) (SEQ ID NO:2) obtained in Example 1 and a 2.7 kb red-labeled circular plasmid DNA (product code MIR 7904) commercially available from Mirus Bio; and (B) a control red-labeled plasmid used as described above in the absence of the conjugate. (i) The maximum projection of the cells after culture is shown; (ii) an image corresponding to the intensity map of the cells after culture; and (iii) an intensity map of the cells after culture.

[0529] In the maximum projection image (i), it can be clearly seen that the plasmid is able to spread better throughout the cell when conjugated with the EfsQNR protein. This demonstrates the efficiency of the conjugate in carrying and localizing target nucleic acids such as plasmids within the cell.

[0530] It is evident from intensity diagram (iii) that plasmids are able to enter cells with the aid of proteins, and that diffusion is homogeneous and widespread.

[0531] Although the examples presented herein illustrate the use of protein EfsQNR (SEQ ID NO:2) in the preparation of conjugates for transfection, proteins having amino acid sequences as depicted in SEQ ID NO:1 and SEQ ID NO:3 to SEQ ID NO:12 can also be used to efficiently form conjugates. Similarly, recombinant β-helical proteins having a pentapeptide repeat sequence as depicted in SEQ ID NO:18 and a length in the range of 5 nm–25 nm, suitably 10 nm–15 nm, and a width in the range of 1 nm–5 nm, suitably 1 nm–3 nm, can be used to prepare conjugates as described in this disclosure. Likewise, this example depicts the use of copper[II]phenanthroline or ethylenediamine as a linker, but other molecules such as DNA-binding proteins, metal conjugates, drug-metal conjugates, and other nucleic acid intercalation molecules can also be used to form conjugates to be effectively used as transfection agents. One class of DNA-binding proteins, namely zinc finger proteins with a molecular weight less than 12 kDa, can also be used to form the conjugates. One such zinc finger protein that can be used as a linker has been described in SEQ ID NO:24. In this disclosure, a plasmid carrying the mcherry gene has been used ( Figure 18 A conjugate for transfection was prepared using 2.7 kb of red-labeled circular plasmid DNA, and expression of the mcherry gene has been demonstrated as a proof of concept. It is conceivable that virtually any nucleic acid or plasmid, including the gene of interest for transfection purposes, can be complexed with the conjugates disclosed herein to form conjugates for gene therapy or gene editing.

[0532] advantage

[0533] This disclosure provides conjugates comprising nucleic acids or plasmids containing a gene of interest, said conjugates being capable of penetrating cell membranes and expressing the gene of interest. Therefore, said conjugates can be used for transfection and have great potential for gene therapy and gene editing. It is well known that the delivery of nucleic acids and plasmids containing genes into cells is a major challenge in the fields of gene therapy and gene editing, and the disclosed conjugates open new avenues in this field. The disclosed conjugates are easy to prepare and can be complexed with a variety of nucleic acids and plasmids containing genes to be used in gene therapy and gene editing.

[0534] Example 11

[0535] Transfection of gene editing molecular mechanisms using the CPP of this invention

[0536] Transfection experiments were performed in mammalian cells using the conjugate obtained in Example 1 (EfsQNR: SEQ ID No. 2) and the CRISPR-RNP gene editing complex based on the Cas9 endonuclease. The results are shown in Table 2. Figure 29 The description was provided. Based on the results of Example 4 above, it is envisioned that the technology can be applied to a variety of cells, including bacterial cells, yeast cells, plant cells, insect cells, and fish cells.

[0537] Table 2:

[0538] Comparison PBS containing CRISPR-RNP-CPP Reaction buffer containing CRISPR-RNP-CPP 1 0.99 0.9 1 0.94 0.7

[0539] In one embodiment of the present invention, the following conditions were used to perform the transfection experiment.

[0540] Strategy: Gene editing in cells has been reported through several different approaches. A particularly relevant example is the delivery of a ribonuclease (such as Cas9) and gRNA ribonucleoprotein (RNP) complex into cells. This approach is particularly attractive because it does not require the intracellular synthesis of intermediates of the gene editing mechanism.

[0541] Several current methods, such as electroporation, microinjection, nanoparticles, and cationic lipid-mediated delivery, have been used to introduce gene editing mechanisms into the cytoplasm of cells, from which the gene editing mechanisms can migrate to the genome.

[0542] In one embodiment, the present invention utilizes the electrostatic interaction between Cas9, gRNA, and CPP to carry Cas9-gRNARNP (CRISPR-RNP) into cells. The use of conjugates in which the various components are linked by covalent and / or non-covalent interactions is also envisioned.

[0543] eGFP-expressing cells (HEK 293) were transfected with a CRISPR-RNP containing a gRNA specific for eGFP. CRISPR-RNP uptake is mediated by the CPP of this invention, which efficiently delivers the cargo into the cells (Example 4). Following cellular uptake, whether isolated from or uninhibited by the CPP, Cas9 is guided by the gRNA to its target DNA sequence, eGFP. This gene knockout and the resulting loss of fluorescence in the cells demonstrate an efficient and effective delivery method for the gene-editing complex.

[0544] To obtain stable eGFP-expressing cell lines, a selection method using the antibiotic G418 was employed. Cells expressing eGFP and thus taking up the eGFP plasmid will also be resistant to the G418 antibiotic. Cells that do not take up the eGFP plasmid are not resistant to G418 and therefore will die within a certain period of time when stored in the selection medium.

[0545] Experiment: HEK293 cells expressing eGFP (enhanced green fluorescent protein) were obtained by transfecting them with eGFP using a commercially available lipid-based transfectant (lipofectamine) reagent. Cells were grown for 48 hours to obtain 70-80% eGFP transfection. Cells were then passaged and grown in selective medium containing G418 antibiotic to ensure stable eGFP expression before gene editing experiments using HEK293 cells.

[0546] Approximately 5,000 cells were seeded into 96-well plates and allowed to grow overnight on day 1.

[0547] On day 2, the Cas9-gRNA-CPP (CRISPR-RNP-CPP) complex is formed in two steps:

[0548] Step 1: Mix Cas9 with gRNA in: i) phosphate-buffered saline (PBS); or ii) a reaction buffer containing commercially available Cas9 (Genaxxon Biosciences: Cas9-NLS-tagRFP Streptococcus pyogenes (S.) Cas9 protein NLS with a C-terminal red fluorescent protein (tagRFP) tag sequence; Product No.: S5306.0010; HS No.: 35040090). The reaction buffer serves to stabilize the Cas9 and RNP complex. The mixture is then incubated at room temperature for 25 minutes to form the CRISPR-RNP complex.

[0549] Step 2: Add PBS containing 1 μmol CPP to the Cas9-gRNA mixture obtained in Step 1 and incubate at room temperature for another 30 minutes to form the CRISPR-RNP-CPP complex;

[0550] Step 3: Add the CRISPR-RNP-CPP complex obtained in Step 2 to cell-containing medium (Dulbecco's Modified Eagle Medium (DMEM) with or without serum; e.g., Sigma) and culture the cells for 4 hours. After 4 hours, add medium (DMEM with serum) to the cells and maintain growth under standard cell growth conditions (37°C and 5% CO2) for 48 hours. After 48 hours, wash the cells with PBS and add fresh medium (DMEM with serum), and measure the fluorescence intensity induced by eGFP using a fluorescent plate reader.

[0551] Results of mammalian kidney HEK293 cells

[0552] The CPP-assisted delivery of CRISPR-RNP into cells is efficient according to this invention. Following cellular uptake, Cas9 is guided by gRNA to its target DNA sequence for eGFP. Knockout of this gene by Cas9 results in loss of fluorescence in the cell.

[0553] Using the serum-free medium in step 3 and the reaction buffer in step 1 (such as the commercially available buffer for dissolving GenaxonCas9 and gRNA; product number S5306.0010) resulted in a 30% reduction in fluorescence, indicating that CRISPR-RNPs were efficiently translocated into cells.

[0554] No significant change in fluorescence intensity was observed when the CRISPR-RNP complex was dissolved in PBS (phosphate-buffered saline).

[0555] To avoid being bound by theory, it is assumed that PBS cannot stabilize the complex between CRISPR-RNP and CPP.

[0556] Example 12

[0557] Transfection of fluorescently labeled Cas9 with the recombinant β-helical protein of this invention

[0558] Figure 31 The results of transfection experiments in MCF-7 cells using the conjugate obtained in Example 1 (EfsQNR: SEQ ID No. 2) and the endonuclease cas9 tagged with either (i) red fluorescent protein or (ii) green fluorescent dye Atto520 are depicted.

[0559] The conjugate obtained in Example 1 (EfsQNR: SEQ ID No. 2) was mixed with cas9 (e.g., Euheria Biotech) conjugated with red fluorescent protein or green fluorescent dye ATTO 520 (Sigma product 77810) at a ratio of 1:1 and incubated at room temperature for 30 minutes.

[0560] Add the obtained solution to the cells and incubate with the cells for 12–16 hours.

[0561] Figure 31 The images show (A) the maximum projection of the cells after culture; (B) the intensity map of the cells after culture; and (C) the 3D depth image of the cells after culture.

[0562] In the two maximum projection images (A), it can be clearly seen that Cas9 was successfully transfected and able to diffuse throughout the cell, regardless of the labeling dye. This demonstrates the efficiency of the conjugates of the present invention in carrying and localizing target proteins in cells.

[0563] As clearly shown in intensity map (B), Cas9 can enter cells using conjugates regardless of the accompanying dye. It diffuses throughout the cell and is homogeneous.

[0564] The 3D depth image (C) shows the Cas9 cargo spreading throughout the cell. Depth analysis and 3D representation show its presence in all planes.

[0565] Example 13

[0566] Transfection of fluorescently labeled gRNA with recombinant β-helical protein of the present invention

[0567] As further disclosed in this invention, a conjugate for transfecting cells with gRNA, the gRNA being typical of gene editing using the CRISPR-Cas9 gene editing system, is prepared. The conjugate comprises EfsQNR (SEQ ID NO:2), an ethylenediamine linker, and gRNA tagged with the green fluorescent dye MFP488 (e.g., Euphelia Biotech: esiCRISPR Kit-wt). The conjugate is prepared according to a known protocol (Reference: Bioconjugation Technology: Greg T. Hermansen; Academic Press; 3rd ed.; 2013; pp. 264-265, which is incorporated herein by reference).

[0568] Figure 32 The results of transfection experiments performed in MCF-7 cells using the following: (A) the conjugate (EfsQNR) obtained in Example 1 and gRNA tagged with the green fluorescent dye MFP488; and (B) control gRNA tagged with the green fluorescent dye MFP488 in the absence of the conjugate. (i) shows the maximum projection of the cells after culture; (ii) shows a 3D depth image of the cells; (iii) an image corresponding to the intensity map of the cells after culture; and (iv) an intensity map of the cells after culture.

[0569] The images clearly show that the intensity and homogeneity of gRNA in control cells were significantly reduced compared to cells cultured with gRNA and the conjugate. This indicates that the conjugate is able to deliver gRNA into the cells. Maximum projection, intensity, and 3D depth images of the control cells show that most gRNA is localized in the cell membrane, while with the conjugate, its distribution is clearly observed to be primarily in the cytoplasm.

[0570] The CRISPR components were obtained from two different commercial sources, Euheria Biotech and Genaxxon.

[0571] Genaxxon Biosciences: CRISPR GFP-targeting guide RNA; Product No.: P2008.0010, purified gRNA sequence encoding enhanced GFP; HS-No.: 29349990.

[0572] Genaxxon Biosciences: Cas9-NLS-tagRFP Streptococcus pyogenes (S.) Cas9 protein NLS with a C-terminal red fluorescent protein (tagRFP) tag sequence; Product No.: S5306.0010; HS No.: 35040090.

[0573] Eupheria Biotech: esiCRISPR Kit-wt – a kit comprising:

[0574] • 20 μg Streptococcus pyogenes Cas9-NLS (650 ng / μl)

[0575] • 15 μg custom guide (g)RNA (400 ng / μl)

[0576] • 35 μl CRISPRfection™ Transfection Reagent (A dedicated lipid-based transfection reagent suitable for delivering signal-guided (g)RNA-loaded Cas9 protein to most adherent cells)

[0577] • 1 ml CRISPRfection buffer (CRISPRfection diluent)

[0578] • 5 μg of target-free gRNA (human, mouse, rat) as a negative control

[0579] • Reaction buffer used to perform indel analysis of PCR products derived from DNA isolated from transfected cells.

[0580] • Positive control reagents used to confirm the performance of Cas9-NLS protein and gRNA: control target DNA, control gRNA, and reaction buffer.

[0581] • Includes an application manual with protocols for transfection, indel analysis, and Cas9 activity assays.

[0582] [https: / / www.eupheria.com / products / crisprcas9 / esicrispr-kits / ; Accessed February 25, 2019]

[0583] Strategies to confirm that cells still retain plasmids but have edited non-fluorescent eGFP

[0584] This plasmid expresses an “m-cherry” structure containing eGFP and RFP—green and red fluorescence, respectively. Transfection of mammalian cells with this plasmid will allow determination of whether cells express eGFP and whether delivery has been successful. Cells emitting red fluorescence instead of green fluorescence will indicate the absence of eGFP following delivery of gene-edited CRISPR-RNP-CPP.

[0585] Strategies to enhance cellular uptake and gene editing (deletion, substitution, or mutation).

[0586] Several strategies can be used to optimize the conditions for successful delivery of the CPP-Cas9-gRNA complex:

[0587] Strategy 1: It is assumed that a specifically tailored buffer can enhance the interaction between CPP and CRISPR-RNP. The following changes are envisioned to improve the efficiency of intracellular delivery:

[0588] i) Add crowding agent (PEG 4000, cholesterol) to promote the formation of CRISPR-RNP-CPP complex;

[0589] ii) Add specific small lipids (1,2-dioleoyl-3-trimethylaminopropane, 1-palmitoyl-2-oleoyl-sn-propanetriyl-3-phosphocholine) to promote the formation of the CRISPR-RNP-CPP complex by enhancing hydrophobic interactions;

[0590] iii) Small molecules such as phorbol-12-myristate-13-acetate (to enhance micropinocytosis in cells).

[0591] iv) Alternating pH and ionic strength (to drive electrostatic interactions between CPP-Cas9-gRNA)

[0592] Strategy 2: CPP can be functionalized using 3-(N-succinimide-oxoglutarate)aminopropyl, polyethylene glycol-carbamoyl distearate phosphatidyl-ethanolamine, and / or N-(aminopropyl polyethylene glycol)carbamoyl distearate phosphatidyl-ethanolamine. This functionalization can enhance the interaction between CPP and cell membrane lipid molecules and promote cell entry of the CRISPR-RNP-CPP complex of the embodiments of the present invention.

[0593] Strategy 3: Assume that the same method used with CRISPR endonucleases can be used with ZFN and... It can be used in conjunction with other gene-editing endonuclease systems.

[0594] It should be understood that the different embodiments of the invention described herein can be appropriately combined, and the features of the embodiments of the invention can be used interchangeably with other embodiments where appropriate.

[0595] Although specific embodiments of the invention have been disclosed in detail herein, they are merely illustrative and for purposes of explanation only. The above embodiments are not intended to limit the scope of the appended claims. The inventors envision various substitutions, changes, and modifications that can be made to the invention without departing from the scope of the invention as defined by the claims.

[0596] Table 3: Sequence List

[0597]

[0598]

[0599]

[0600]

[0601] Alternative expressions of the inventive concept are set forth in the following clauses:

[0602] 1. A cell-penetrating conjugate comprising a recombinant β-helical protein linked to a functional molecule, wherein the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0603] 2. The conjugate according to Clause 1, wherein the length of the β-helical protein is in the range of 10 nm to 15 nm and the width is in the range of 1 nm to 3 nm.

[0604] 3. The conjugate according to Clause 1 or Clause 2, wherein the β-helical protein comprises one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0605] 4. The conjugate according to Clause 3, wherein, when present, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamate sequence ladder comprises 10 to 40 glutamate residues.

[0606] 5. The conjugate according to any one of clauses 1 to 4, wherein the total charge of the β-helical protein is less than zero.

[0607] 6. The conjugate according to Clause 5, wherein the total charge of the β-helical protein is -20 to -60.

[0608] 7. The conjugate according to any one of clauses 1 to 6, wherein the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 Such as measurements obtained by atomic force microscopy.

[0609] 8. The conjugate according to any one of clauses 1 to 7, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0610] 9. The conjugate according to Clause 8, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0611] 10. The conjugate according to any one of clauses 1 to 7, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0612] 11. The conjugate according to any one of clauses 1 to 10, wherein the recombinant β-helical protein is linked to the functional molecule via a linker molecule selected from the group consisting of: polyethylene glycol (PEG); peptides; metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0613] 12. The conjugate according to Clause 11, wherein when the linker molecule is a peptide, the peptide comprises an amino acid selected from the group consisting of: aliphatic amino acids; aromatic amino acids; and combinations thereof.

[0614] 13. The conjugate according to any one of clauses 1 to 12, wherein the linker is connected to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0615] 14. The conjugate according to any one of clauses 1 to 13, wherein the recombinant β-helical protein is linked to a functional molecule via an ester bond or an amide bond.

[0616] 15. The conjugate according to any one of clauses 1 to 14, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal conjugates, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0617] 16. The conjugate according to any one of clauses 1 to 15, wherein the conjugate further comprises a signal sequence, wherein the signal sequence directs the conjugate to a particular cell or a portion of a cell.

[0618] 17. The conjugate according to Clause 16, wherein the signal sequence is selected from the group consisting of: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0619] 18. The conjugate according to any one of clauses 1 to 17, wherein the conjugate further comprises a phosphatidylcholine molecule.

[0620] 19. The conjugate according to any one of clauses 16 to 18, wherein the conjugate transfers the functional molecule to a location selected from the group consisting of: organelles; nucleus; endoplasmic reticulum; mitochondria; cell membrane; and P-cadherin overexpressing breast cancer cells.

[0621] 20. The conjugate according to Clause 19, wherein the organelle is selected from the group consisting of actin filaments, Golgi apparatus, cell membrane, and tubulin.

[0622] 21. The conjugate according to any one of clauses 1 to 20, wherein the conjugate is used as a cell marker.

[0623] 22. The conjugate according to any one of clauses 1 to 20, wherein the conjugate is used to transfer the functional molecule into cells.

[0624] 23. A method for transferring a functional molecule into a cell, the method comprising:

[0625] a) Link the functional molecule to recombinant β-helical protein to obtain a conjugate;

[0626] b) Contact the conjugate with at least one cell;

[0627] In step (b), contacting the conjugate transfers the functional molecule into the cell; and the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0628] 24. The method according to Clause 23, wherein the method includes, after step (b):

[0629] c) Detect the transfer of the conjugate within the cells.

[0630] 25. The method according to Clause 23 or Clause 24, wherein the length of the β-helical protein is in the range of 10 nm to 15 nm and the width is in the range of 1 nm to 3 nm.

[0631] 26. The method according to any one of clauses 23 to 25, wherein the β-helical protein comprises one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0632] 27. The method according to Clause 26, wherein, when present, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamate sequence ladder comprises 10 to 40 glutamate residues.

[0633] 28. The method according to any one of clauses 23 to 27, wherein the total charge of the β-helical protein is less than zero.

[0634] 29. The method according to Clause 28, wherein the total charge of the β-helical protein is -20 to -60.

[0635] 30. The method according to any one of clauses 23 to 29, wherein the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 Such as measurements obtained by atomic force microscopy.

[0636] 31. The method according to any one of clauses 23 to 30, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0637] 32. The method according to Clause 31, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0638] 33. The method according to any one of clauses 23 to 30, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0639] 34. The method according to any one of clauses 23 to 33, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal conjugates, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0640] 35. The method according to any one of clauses 23 to 34, wherein the functional molecule is linked to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0641] 36. The method according to any one of clauses 23 to 35, wherein the cell is selected from the group consisting of eukaryotic cells, prokaryotic cells, and combinations thereof.

[0642] 37. The method according to any one of clauses 23 to 36, wherein the method is used for cell labeling.

[0643] 38. Use of the cell-penetrating conjugate according to any one of clauses 1 to 20 for delivering a functional molecule into a cell, wherein the functional molecule is selected from the group consisting of dyes, drugs, metals, drug-metal, proteins, enzymes, antibodies, nucleic acids, polysaccharides, nuclear localization signals, nanoparticles, and combinations thereof.

[0644] 39. Use of the cell-penetrating conjugate according to any one of clauses 1 to 20, for cell penetration.

[0645] 40. Use of the cell-penetrating conjugate according to any one of clauses 1 to 20 for cell labeling.

[0646] 41. A conjugate comprising: (a) at least one recombinant β-helical protein; (b) at least one adapter; and (c) at least one nucleic acid molecule, wherein the at least one recombinant β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0647] 42. The conjugate according to Clause 41, wherein the length of the at least one recombinant β-helical protein is in the range of 10 nm to 15 nm and the width is in the range of 1 nm to 3 nm.

[0648] 43. The conjugate according to Clause 41 or Clause 42, wherein the β-helical protein comprises one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0649] 44. The conjugate according to Clause 43, wherein, when present, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamic acid sequence ladder comprises 10 to 40 glutamic acid residues.

[0650] 45. The conjugate according to any one of clauses 41 to 44, wherein the total charge of the β-helical protein is less than zero.

[0651] 46. ​​The conjugate according to Clause 45, wherein the total charge of the β-helical protein is -20 to -60.

[0652] 47. The conjugate according to any one of clauses 41 to 46, wherein the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 Such as measurements obtained by atomic force microscopy.

[0653] 48. The conjugate according to any one of clauses 41 to 47, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0654] 49. The conjugate according to Clause 48, wherein the β-helical protein comprises a tandem repeating pentapeptide having a concordant sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0655] 50. The conjugate according to any one of clauses 41 to 47, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0656] 51. The conjugate according to any one of clauses 41 to 50, wherein the linker is a linker molecule selected from the group consisting of: polyethylene glycol (PEG); peptides; metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0657] 52. The conjugate according to clause 51, wherein when the linker molecule is a peptide, the peptide comprises an amino acid selected from the group consisting of: aliphatic amino acids; aromatic amino acids; and combinations thereof.

[0658] 53. The conjugate according to any one of clauses 41 to 52, wherein the connector is linked to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0659] 54. The conjugate according to Clause 53, wherein the linker is connected to the recombinant β-helical protein via an ester bond or an amide bond.

[0660] 55. The conjugate according to any one of clauses 41 to 54, wherein the nucleic acid molecule comprises at least one gene of interest.

[0661] 56. The conjugate according to Clause 55, wherein the nucleic acid molecule is a plasmid.

[0662] 57. The conjugate according to Clause 56, wherein the plasmid is a DNA plasmid or an RNA plasmid.

[0663] 58. The conjugate according to any one of clauses 55 to 57, wherein the gene of interest encodes one or more molecules of a genome editing system.

[0664] 59. The conjugate according to clause 58, wherein the genome editing system is selected from the group consisting of:

[0665] a. RNA-guided endonucleases and / or guide RNA (gRNA);

[0666] b. Zinc finger nucleases (ZFNs);

[0667] c. Transcription activator-like effector nucleases

[0668] d. DNA-guided endonucleases and / or guide DNA;

[0669] e. Homing endonucleases;

[0670] f. Integrase.

[0671] 60. The conjugate according to any one of clauses 55 to 59, wherein the gene of interest encodes an RNA-guided endonuclease and / or guide RNA (gRNA);

[0672] 61. The conjugate according to Clause 60, wherein the RNA-guided endonuclease is Cas9.

[0673] 62. A method for transferring nucleic acid molecules into cells, the method comprising:

[0674] a) linking the nucleic acid molecule to at least one recombinant β-helical protein via at least one adapter to obtain a conjugate; and

[0675] b) Contact the conjugate with at least one cell;

[0676] Contact with the conjugate transfers the nucleic acid molecule into the cell, and the recombinant β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0677] 63. The method according to clause 62, wherein the method includes, after step (b):

[0678] c) Detect the transfer of the conjugate within the cells.

[0679] 64. The method according to Clause 62 or Clause 63, wherein the recombinant β-helical protein has a length in the range of 10 nm to 15 nm and a width in the range of 1 nm to 3 nm.

[0680] 65. The method according to any one of clauses 62 to 64, wherein the β-helical protein comprises one or more amino acid sequence ladder structures selected from the group consisting of: arginine sequence ladder; lysine sequence ladder; asparagine sequence ladder; aspartic acid sequence ladder; and glutamate sequence ladder.

[0681] 66. The method according to clause 65, wherein, when present, the arginine sequence ladder comprises 10 to 20 arginine residues; the lysine sequence ladder comprises 10 to 30 lysine residues; the asparagine sequence ladder comprises 10 to 40 asparagine residues; the aspartic acid sequence ladder comprises 10 to 40 aspartic acid residues; and the glutamate sequence ladder comprises 10 to 40 glutamate residues.

[0682] 67. The method according to any one of clauses 62 to 66, wherein the total charge of the β-helical protein is less than zero.

[0683] 68. The method according to Clause 67, wherein the total charge of the β-helical protein is -20 to -60.

[0684] 69. The method according to any one of clauses 62 to 68, wherein the β-helical protein has a β-helical structure, and the stiffness parameter K (β-helix) of the helical structure is 0.2 to 12 N / m. 2 Such as measurements obtained by atomic force microscopy.

[0685] 70. The method according to any one of clauses 62 to 69, wherein the β-helical protein is a pentapeptide repeat sequence protein.

[0686] 71. The method according to Clause 70, wherein the β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0687] 72. The method according to any one of clauses 62 to 70, wherein the β-helical protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0688] 73. The method according to any one of clauses 62 to 72, wherein the adapter is selected from the group consisting of: polyethylene glycol (PEG); peptides, metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

[0689] 74. The method according to clause 73, wherein when the linker molecule is a peptide, the peptide comprises an amino acid selected from the group consisting of: aliphatic amino acids; aromatic amino acids; and combinations thereof.

[0690] 75. The method according to any one of clauses 62 to 74, wherein the adapter is connected to the recombinant β-helical protein by covalent bonds, non-covalent bonds, and combinations thereof.

[0691] 76. The method according to Clause 75, wherein the linker is connected to the recombinant β-helical protein via an ester bond or an amide bond.

[0692] 77. The method according to any one of clauses 62 to 76, wherein the cells are selected from the group consisting of eukaryotic cells or prokaryotic cells.

[0693] 78. The method according to any one of clauses 62 to 77, wherein the nucleic acid molecule comprises at least one gene of interest.

[0694] 79. The method according to Clause 78, wherein the nucleic acid molecule is a plasmid.

[0695] 80. The method according to Clause 79, wherein the plasmid is a DNA plasmid or an RNA plasmid.

[0696] 81. The method according to any one of clauses 78 to 80, wherein the gene of interest encodes one or more molecules of the genome editing system.

[0697] 82. The method according to clause 81, wherein the genome editing system is selected from the group consisting of:

[0698] a. RNA-guided endonucleases and / or guide RNA (gRNA);

[0699] b. Zinc finger nucleases (ZFNs);

[0700] c. Transcription activator-like effector nucleases

[0701] d. DNA-guided endonucleases and / or guide DNA;

[0702] e. Homing endonucleases;

[0703] f. Integrase.

[0704] 83. The method according to any one of clauses 78 to 82, wherein the gene of interest encodes an RNA-guided endonuclease and / or guide RNA (gRNA);

[0705] 84. The method according to Clause 83, wherein the RNA-guided endonuclease is Cas9.

[0706] 85. A genome editing complex for modifying target polynucleotides, the genome editing complex comprising a recombinant β-helical protein linked to a plasmid encoding one or more genome editing system molecules, wherein the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm.

[0707] 86. The genome editing complex according to Clause 85, wherein the recombinant β-helical protein comprises a tandem repeating pentapeptide having a common sequence (STAV)1(DN)2(LF)3(STR)4(G)5.

[0708] 87. The genome editing complex according to clause 85, wherein the recombinant β-helix protein is represented by a sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and combinations thereof.

[0709] 88. A genome editing complex according to any one of clauses 85 to 87, wherein the plasmid comprises at least one gene of interest.

[0710] 89. A genome editing complex according to any one of clauses 85 to 88, wherein the plasmid is a DNA plasmid or an RNA plasmid.

[0711] 90. A genome editing complex according to any one of clauses 85 to 89, wherein the genome editing system is selected from the group consisting of:

[0712] a. RNA-guided endonucleases and / or guide RNA (gRNA);

[0713] b. Zinc finger nucleases (ZFNs);

[0714] c. Transcription activator-like effector nucleases

[0715] d. DNA-guided endonucleases and / or guide DNA;

[0716] e. Homing endonucleases;

[0717] f. Integrase.

[0718] 91. The genome editing complex according to any one of clauses 85 to 90, wherein the one or more genome editing system molecules are RNA-guided endonucleases and / or guide RNA (gRNA).

[0719] 92. The genome editing complex according to Clause 91, wherein the RNA-guided endonuclease is Cas9.

[0720] 93. Use of a conjugate according to any one of Clauses 41 to 61 or a genome editing complex according to any one of Clauses 85 to 92, as a transfection agent.

[0721] 94. Use of a conjugate according to any one of Clauses 41 to 61 or a genome editing complex according to any one of Clauses 85 to 92 for use in gene therapy.

[0722] 95. Use of a conjugate according to any one of Clauses 41 to 61 or a genome editing complex according to any one of Clauses 85 to 92 for gene editing. sequence list <110> CyCa OncoSolutions Limited <120> Cell membrane permeation conjugate <130> P50168GB / DJC / JG <160> 29 <170> BiSSAP 1.3.6 <210> 1 <211> 200 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of AlbG protein <400> 1 Met Pro Ala Lys Thr Leu Glu Ser Lys Asp Tyr Cys Gly Glu Ser Phe 1 5 10 15 Val Ser Glu Asp Arg Ser Gly Gln Ser Leu Glu Ser Ile Arg Phe Glu 20 25 30 Asp Cys Thr Phe Arg Gln Cys Asn Phe Thr Glu Ala Glu Leu Asn Arg 35 40 45 Cys Lys Phe Arg Glu Cys Glu Phe Val Asp Cys Asn Leu Ser Leu Ile 50 55 60 Ser Ile Pro Gln Thr Ser Phe Met Glu Val Arg Phe Val Asp Cys Lys 65 70 75 80 Met Leu Gly Val Asn Trp Thr Ser Ala Gln Trp Pro Ser Val Lys Met 85 90 95 Glu Gly Ala Leu Ser Phe Glu Arg Cys Ile Leu Asn Asp Ser Leu Phe 100 105 110 Tyr Gly Leu Tyr Leu Ala Gly Val Lys Met Val Glu Cys Arg Ile His 115 120 125 Asp Ala Asn Phe Thr Glu Ala Asp Cys Glu Asp Ala Asp Phe Thr Gln 130 135 140 Ser Asp Leu Lys Gly Ser Thr Phe His Asn Thr Lys Leu Thr Gly Ala 145 150 155 160 Ser Phe Ile Asp Ala Val Asn Tyr His Ile Asp Ile Phe His Asn Asp 165 170 175 Ile Lys Arg Ala Arg Phe Ser Leu Pro Glu Ala Ala Ser Leu Leu Asn 180 185 190 Ser Leu Asp Ile Glu Leu Ser Asp 195 200 <210> 2 <211> 214 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> EfsQNR protein sequence of amino acid sequence (Amino acid sequence of EfsQNR protein) <400> 2 Gly Ser His Met Lys Ile Thr Tyr Pro Leu Pro Pro Asn Leu Pro Glu 1 5 10 15 Gln Leu Pro Leu Leu Thr Asn Cys Gln Leu Glu Asp Glu Ala Ile Leu 20 25 30 Glu Asn His Leu Tyr Gln Gln Ile Asp Leu Pro Asn Gln Glu Val Arg 35 40 45 Asn Leu Val Phe Arg Asp Ala Val Phe Asp His Leu Ser Leu Ala Asn 50 55 60 Gly Gln Phe Ala Ser Phe Asp Cys Ser Asn Val Arg Phe Glu Ala Cys 65 70 75 80 Asp Phe Ser Asn Val Glu Trp Leu Ser Gly Ser Phe His Arg Val Thr 85 90 95 Phe Leu Arg Cys Asn Leu Thr Gly Thr Asn Phe Ala Asp Ser Tyr Leu 100 105 110 Lys Asp Cys Leu Phe Glu Asp Cys Lys Ala Asp Tyr Ala Ser Phe Arg 115 120 125 Phe Ala Asn Phe Asn Leu Val His Phe Asn Gln Thr Arg Leu Val Glu 130 135 140 Ser Glu Phe Phe Glu Val Thr Trp Lys Lys Leu Leu Leu Glu Ala Cys 145 150 155 160 Asp Leu Thr Glu Ser Asn Trp Leu Asn Thr Ser Leu Lys Gly Leu Asp 165 170 175 Phe Ser Gln Asn Thr Phe Glu Arg Leu Thr Phe Ser Pro Asn Tyr Leu 180 185 190 Ser Gly Leu Lys Val Thr Pro Glu Gln Ala Ile Tyr Leu Ala Ser Ala 195 200 205 Leu Gly Leu Val Ile Thr 210 <210> 3 <211> 84 <212> PRT <213> Tenebrio molitor <220> <221> <222> <223> Amino acid sequence of anti-freeze protein from Tenebrio molitor. <400> 3 Gln Cys Thr Gly Gly Ala Asp Cys Thr Ser Cys Thr Gly Ala Cys Thr 1 5 10 15 Gly Cys Gly Asn Cys Pro Asn Ala Val Thr Cys Thr Asn Ser Gln His 20 25 30 Cys Val Lys Ala Asn Thr Cys Thr Gly Ser Thr Asp Cys Asn Thr Ala 35 40 45 Gln Thr Cys Thr Asn Ser Lys Asp Cys Phe Glu Ala Asn Thr Cys Thr 50 55 60 Asp Ser Thr Asn Cys Tyr Lys Ala Thr Ala Cys Thr Asn Ser Ser Gly 65 70 75 80 Cys Pro Gly His <210> 4 <211> 143 <212> PRT <213> Pine bark beetle (Rhagium inquisitor) <220> <221> <222> <223> Acid sequence of anti-freeze protein from Rhagium inquisitor. <400> 4 Gly Tyr Ser Cys Arg Ala Val Gly Val Asp Gly Arg Ala Val Thr Asp 1 5 10 15 Ile Gln Gly Thr Cys His Ala Lys Ala Thr Gly Ala Gly Ala Met Ala 20 25 30 Ser Gly Thr Ser Glu Pro Gly Ser Thr Thr Ala Thr Ala Thr Gly 35 40 45 Arg Gly Ala Thr Ala Arg Ser Thr Ser Thr Gly Arg Gly Thr Ala Thr 50 55 60 Thr Thr Ala Thr Gly Thr Ala Ser Ala Thr Ser Asn Ala Ile Gly Gln 65 70 75 80 Gly Thr Ala Thr Thr Thr Ala Thr Gly Ser Ala Gly Gly Arg Ala Thr 85 90 95 Gly Ser Ala Thr Thr Ser Ser Ser Ala Ser Gln Pro Thr Gln Thr Gln 100 105 110 Thr Ile Thr Gly Pro Gly Phe Gln Thr Ala Lys Ser Phe Ala Arg Asn 115 120 125 Thr Ala Thr Thr Thr Val Thr Ala Ser His His His His His His 130 135 140 <210> 5 <211> 90 <212> PRT <213> Choristoneura fumiferana <220> <221> <222> <223> Amino acid sequence of anti-freeze protein from Spruce Budworm. <400> 5 Asp Gly Ser Cys Thr Asn Thr Asn Ser Gln Leu Ser Ala Asn Ser Lys 1 5 10 15 Cys Glu Lys Ser Thr Leu Thr Asn Cys Tyr Val Asp Lys Ser Glu Val 20 25 30 Tyr Gly Thr Thr Cys Thr Gly Ser Arg Phe Asp Gly Val Thr Ile Thr 35 40 45 Thr Ser Thr Ser Thr Gly Ser Arg Ile Ser Gly Pro Gly Cys Lys Ile 50 55 60 Ser Thr Cys Ile Ile Thr Gly Gly Val Pro Ala Pro Ser Ala Ala Cys 65 70 75 80 Lys Ile Ser Gly Cys Thr Phe Ser Ala Asn 85 90 <210> 6 <211> 217 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of QNRB1 protein <400> 6 Gly Ser His Met Ala Leu Ala Leu Val Gly Glu Lys Ile Asp Arg Asn 1 5 10 15 Arg Phe Thr Gly Glu Lys Ile Glu Asn Ser Thr Phe Phe Asn Cys Asp 20 25 30 Phe Ser Gly Ala Asp Leu Ser Gly Thr Glu Phe Ile Gly Cys Gln Phe 35 40 45 Tyr Asp Arg Glu Ser Gln Lys Gly Cys Asn Phe Ser Arg Ala Met Leu 50 55 60 Lys Asp Ala Ile Phe Lys Ser Cys Asp Leu Ser Met Ala Asp Phe Arg 65 70 75 80 Asn Ser Ser Ala Leu Gly Ile Glu Ile Arg His Cys Arg Ala Gln Gly 85 90 95 Ala Asp Phe Arg Gly Ala Ser Phe Met Asn Met Ile Thr Thr Arg Thr 100 105 110 Trp Phe Cys Ser Ala Tyr Ile Thr Asn Thr Asn Leu Ser Tyr Ala Asn 115 120 125 Phe Ser Lys Val Val Leu Glu Lys Cys Glu Leu Trp Glu Asn Arg Trp 130 135 140 Ile Gly Ala Gln Val Leu Gly Ala Thr Phe Ser Gly Ser Asp Leu Ser 145 150 155 160 Gly Gly Glu Phe Ser Thr Phe Asp Trp Arg Ala Ala Asn Phe Thr His 165 170 175 Cys Asp Leu Thr Asn Ser Glu Leu Gly Asp Leu Asp Ile Arg Gly Val 180 185 190 Asp Leu Gln Gly Val Lys Leu Asp Asn Tyr Gln Ala Ser Leu Leu Met 195 200 205 Glu Arg Leu Gly Ile Ala Val Ile Gly 210 215 <210> 7 <211> 262 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of UDP-N-acetylglucosamine acyltransferase protein <400> 7 Met Ile Asp Lys Ser Ala Phe Val His Pro Thr Ala Ile Val Glu Glu 1 5 10 15 Gly Ala Ser Ile Gly Ala Asn Ala His Ile Gly Pro Phe Cys Ile Val 20 25 30 Gly Pro His Val Glu Ile Gly Glu Gly Thr Val Leu Lys Ser His Val 35 40 45 Val Val Asn Gly His Thr Lys Ile Gly Arg Asp Asn Glu Ile Tyr Gln 50 55 60 Phe Ala Ser Ile Gly Glu Val Asn Gln Asp Leu Lys Tyr Ala Gly Glu 65 70 75 80 Pro Thr Arg Val Glu Ile Gly Asp Arg Asn Arg Ile Arg Glu Ser Val 85 90 95 Thr Ile His Arg Gly Thr Val Gln Gly Gly Gly Leu Thr Lys Val Gly 100 105 110 Ser Asp Asn Leu Leu Met Ile Asn Ala His Ile Ala His Asp Cys Thr 115 120 125 Val Gly Asn Arg Cys Ile Leu Ala Asn Asn Ala Thr Leu Ala Gly His 130 135 140 Val Ser Val Asp Asp Phe Ala Ile Ile Gly Gly Met Thr Ala Val His 145 150 155 160 Gln Phe Cys Ile Ile Gly Ala His Val Met Val Gly Gly Cys Ser Gly 165 170 175 Val Ala Gln Asp Val Pro Pro Tyr Val Ile Ala Gln Gly Asn His Ala 180 185 190 Thr Pro Phe Gly Val Asn Ile Glu Gly Leu Lys Arg Arg Gly Phe Ser 195 200 205 Arg Glu Ala Ile Thr Ala Ile Arg Asn Ala Tyr Lys Leu Ile Tyr Arg 210 215 220 Ser Gly Lys Thr Leu Asp Glu Val Lys Pro Glu Ile Ala Glu Leu Ala 225 230 235 240 Glu Thr Tyr Pro Glu Val Lys Ala Phe Thr Asp Phe Phe Ala Arg Ser 245 250 255 Thr Arg Gly Leu Ile Arg 260 <210> 8 <211> 201 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Aminoacid sequence of NP275 protein from Nostoc punctiforme. <400> 8 Met Gly Ser Ser His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Asp Val Glu Lys Leu Arg Gln Leu Tyr Ala Ala 20 25 30 Gly Glu Arg Asp Phe Ser Ile Val Asp Leu Arg Gly Ala Val Leu Glu 35 40 45 Asn Ile Asn Leu Ser Gly Ala Ile Leu His Gly Ala Met Leu Asp Glu 50 55 60 Ala Asn Leu Gln Gln Ala Asn Leu Ser Arg Ala Asp Leu Ser Gly Ala 65 70 75 80 Thr Leu Asn Gly Ala Asp Leu Arg Gly Ala Asn Leu Ser Lys Ala Asp 85 90 95 Leu Ser Asp Ala Ile Leu Asp Asn Ala Ile Leu Glu Gly Ala Ile Leu 100 105 110 Asp Glu Ala Val Leu Asn Gln Ala Asn Leu Lys Ala Ala Asn Leu Glu 115 120 125 Gln Ala Ile Leu Ser His Ala Asn Ile Arg Glu Ala Asp Leu Ser Glu 130 135 140 Ala Asn Leu Glu Ala Ala Asp Leu Ser Gly Ala Asp Leu Ala Ile Ala 145 150 155 160 Asp Leu His Gln Ala Asn Leu His Gln Ala Ala Leu Glu Arg Ala Asn 165 170 175 Leu Thr Gly Ala Asn Leu Glu Asp Ala Asn Leu Glu Gly Thr Ile Leu 180 185 190 Glu Gly Gly Asn Asn Asn Leu Ala Thr 195 200 <210> 9 <211> 353 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of pectatelyase C protein. <400> 9 Ala Thr Asp Thr Gly Gly Tyr Ala Ala Thr Ala Gly Gly Asn Val Thr 1 5 10 15 Gly Ala Val Ser Lys Thr Ala Thr Ser Met Gln Asp Ile Val Asn Ile 20 25 30 Ile Asp Ala Ala Arg Leu Asp Ala Asn Gly Lys Lys Val Lys Gly Gly 35 40 45 Ala Tyr Pro Leu Val Ile Thr Tyr Thr Gly Asn Glu Asp Ser Leu Ile 50 55 60 Asn Ala Ala Ala Ala Asn Ile Cys Gly Gln Trp Ser Lys Asp Pro Arg 65 70 75 80 Gly Val Glu Ile Lys Glu Phe Thr Lys Gly Ile Thr Ile Ile Gly Ala 85 90 95 Asn Gly Ser Ser Ala Asn Phe Gly Ile Trp Ile Lys Lys Ser Ser Asp 100 105 110 Val Val Val Gln Asn Met Arg Ile Gly Tyr Leu Pro Gly Gly Ala Lys 115 120 125 Asp Gly Asp Met Ile Arg Val Asp Asp Ser Pro Asn Val Trp Val Asp 130 135 140 His Asn Glu Leu Phe Ala Ala Asn His Glu Cys Asp Gly Thr Pro Asp 145 150 155 160 Asn Asp Thr Thr Phe Glu Ser Ala Val Asp Ile Lys Gly Ala Ser Asn 165 170 175 Thr Val Thr Val Ser Tyr Asn Tyr Ile His Gly Val Lys Lys Val Gly 180 185 190 Leu Asp Gly Ser Ser Ser Ser Asp Thr Gly Arg Asn Ile Thr Tyr His 195 200 205 His Asn Tyr Tyr Asn Asp Val Asn Ala Arg Leu Pro Leu Gln Arg Gly 210 215 220 Gly Leu Val His Ala Tyr Asn Asn Leu Tyr Thr Asn Ile Thr Gly Ser 225 230 235 240 Gly Leu Asn Val Arg Gln Asn Gly Gln Ala Leu Ile Glu Asn Asn Trp 245 250 255 Phe Glu Lys Ala Ile Asn Pro Val Thr Ser Arg Tyr Asp Gly Lys Asn 260 265 270 Phe Gly Thr Trp Val Leu Lys Gly Asn Asn Ile Thr Lys Pro Ala Asp 275 280 285 Phe Ser Thr Tyr Ser Ile Thr Trp Thr Ala Asp Thr Lys Pro Tyr Val 290 295 300 Asn Ala Asp Ser Trp Thr Ser Thr Gly Thr Phe Pro Thr Val Ala Tyr 305 310 315 320 Asn Tyr Ser Pro Val Ser Ala Gln Cys Val Lys Asp Lys Leu Pro Gly 325 330 335 Tyr Ala Gly Val Gly Lys Asn Leu Ala Thr Leu Thr Ser Thr Ala Cys 340 345 350 Lys <210> 10 <211> 196 <212> PRT <213> BESC (Caldicellulosiruptor bescii) <220> <221> <222> <223> Amino acid sequence of pectate lyase from Caldicellulosiruptor bescii (BESC) <400> 10 Val Gly Thr Asn Thr Gly Gly Val Leu Val Ile Thr Asp Thr Ile Ile 1 5 10 15 Val Lys Ser Gly Gln Thr Tyr Asp Gly Lys Gly Ile Lys Ile Ile Ala 20 25 30 Gln Gly Met Gly Asp Gly Ser Gln Ser Glu Asn Gln Lys Pro Ile Phe 35 40 45 Lys Leu Glu Lys Gly Ala Asn Leu Lys Asn Val Ile Ile Gly Ala Pro 50 55 60 Gly Cys Asp Gly Ile His Cys Tyr Gly Asp Asn Val Val Glu Asn Val 65 70 75 80 Val Trp Glu Asp Val Gly Glu Asp Ala Leu Thr Val Lys Ser Glu Gly 85 90 95 Val Val Glu Val Ile Gly Gly Ser Ala Lys Glu Ala Ala Asp Lys Val 100 105 110 Phe Gln Leu Asn Ala Pro Cys Thr Phe Lys Val Lys Asn Phe Thr Ala 115 120 125 Thr Asn Ile Gly Lys Leu Val Arg Gln Asn Gly Asn Thr Thr Phe Lys 130 135 140 Val Val Ile Tyr Leu Glu Asp Val Thr Leu Asn Asn Val Lys Ser Cys 145 150 155 160 Val Ala Lys Ser Asp Ser Pro Val Ser Glu Leu Trp Tyr His Asn Leu 165 170 175 Asn Val Asn Asn Cys Lys Thr Leu Phe Glu Phe Pro Ser Gln Ser Gln 180 185 190 Ile His Gln Tyr 195 <210> 11 <211> 213 <212> PRT <213> Methanosarcina thermophila <220> <221> <222> <223> Amino acid sequence of carbonic anhydrase from Methanosarcina thermophila. <400> 11 Gln Glu Ile Thr Val Asp Glu Phe Ser Asn Ile Arg Glu Asn Pro Val 1 5 10 15 Thr Pro Trp Asn Pro Glu Pro Ser Ala Pro Val Ile Asp Pro Thr Ala 20 25 30 Tyr Ile Asp Pro Gln Ala Ser Val Ile Gly Glu Val Thr Ile Gly Ala 35 40 45 Asn Val Met Val Ser Pro Met Ala Ser Ile Arg Ser Asp Glu Gly Met 50 55 60 Pro Ile Phe Val Gly Asp Arg Ser Asn Val Gln Asp Gly Val Val Leu 65 70 75 80 His Ala Leu Glu Thr Ile Asn Glu Glu Gly Glu Pro Ile Glu Asp Asn 85 90 95 Ile Val Glu Val Asp Gly Lys Glu Tyr Ala Val Tyr Ile Gly Asn Asn 100 105 110 Val Ser Leu Ala His Gln Ser Gln Val His Gly Pro Ala Ala Val Gly 115 120 125 Asp Asp Thr Phe Ile Gly Met Gln Ala Phe Val Phe Lys Ser Lys Val 130 135 140 Gly Asn Asn Cys Val Leu Glu Pro Arg Ser Ala Ala Ile Gly Val Thr 145 150 155 160 Ile Pro Asp Gly Arg Tyr Ile Pro Ala Gly Met Val Val Thr Ser Gln 165 170 175 Ala Glu Ala Asp Lys Leu Pro Glu Val Thr Asp Asp Tyr Ala Tyr Ser 180 185 190 His Thr Asn Glu Ala Val Val Tyr Val Asn Val His Leu Ala Glu Gly 195 200 205 Tyr Lys Glu Thr Ser 210 <210> 12 <211> 359 <212> PRT <213> Aspergillus niger <220> <221> <222> <223> Amino acid sequence of pectin lyase A protein from Aspergillus niger. <400> 12 Val Gly Val Ser Gly Ser Ala Glu Gly Phe Ala Lys Gly Val Thr Gly 1 5 10 15 Gly Gly Ser Ala Thr Pro Val Tyr Pro Asp Thr Ile Asp Glu Leu Val 20 25 30 Ser Tyr Leu Gly Asp Asp Glu Ala Arg Val Ile Val Leu Thr Lys Thr 35 40 45 Phe Asp Phe Thr Asp Ser Glu Gly Thr Thr Thr Gly Thr Gly Cys Ala 50 55 60 Pro Trp Gly Thr Ala Ser Ala Cys Gln Val Ala Ile Asp Gln Asp Asp 65 70 75 80 Trp Cys Glu Asn Tyr Glu Pro Asp Ala Pro Ser Val Ser Val Glu Tyr 85 90 95 Tyr Asn Ala Gly Thr Leu Gly Ile Thr Val Thr Ser Asn Lys Ser Leu 100 105 110 Ile Gly Glu Gly Ser Ser Gly Ala Ile Lys Gly Lys Gly Leu Arg Ile 115 120 125 Val Ser Gly Ala Glu Asn Ile Ile Ile Gln Asn Ile Ala Val Thr Asp 130 135 140 Ile Asn Pro Lys Tyr Val Trp Gly Gly Asp Ala Ile Thr Leu Asp Asp 145 150 155 160 Cys Asp Leu Val Trp Ile Asp His Val Thr Thr Ala Arg Ile Gly Arg 165 170 175 Gln His Tyr Val Leu Gly Thr Ser Ala Asp Asn Arg Val Ser Leu Thr 180 185 190 Asn Asn Tyr Ile Asp Gly Val Ser Asp Tyr Ser Ala Thr Cys Asp Gly 195 200 205 Tyr His Tyr Trp Ala Ile Tyr Leu Asp Gly Asp Ala Asp Leu Val Thr 210 215 220 Met Lys Gly Asn Tyr Ile Tyr His Thr Ser Gly Arg Ser Pro Lys Val 225 230 235 240 Gln Asp Asn Thr Leu Leu His Ala Val Asn Asn Tyr Trp Tyr Asp Ile 245 250 255 Ser Gly His Ala Phe Glu Ile Gly Glu Gly Gly Tyr Val Leu Ala Glu 260 265 270 Gly Asn Val Phe Gln Asn Val Asp Thr Val Leu Glu Thr Tyr Glu Gly 275 280 285 Glu Ala Phe Thr Val Pro Ser Ser Thr Ala Gly Glu Val Cys Ser Thr 290 295 300 Tyr Leu Gly Arg Asp Cys Val Ile Asn Gly Phe Gly Ser Ser Gly Thr 305 310 315 320 Phe Ser Glu Asp Ser Thr Ser Phe Leu Ser Asp Phe Glu Gly Lys Asn 325 330 335 Ile Ala Ser Ala Ser Ala Tyr Thr Ser Val Ala Ser Arg Val Val Ala 340 345 350 Asn Ala Gly Gln Gly Asn Leu 355 <210> 13 <211> 135 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of TtCuA protein <400> 13 Ala Tyr Thr Leu Ala Thr His Thr Ala Gly Val Ile Pro Ala Gly Lys 1 5 10 15 Leu Glu Arg Val Asp Pro Thr Thr Val Arg Gln Glu Gly Pro Trp Ala 20 25 30 Asp Pro Ala Gln Ala Val Val Gln Thr Gly Pro Asn Gln Tyr Thr Val 35 40 45 Tyr Val Leu Ala Phe Ala Phe Gly Tyr Gln Pro Asn Pro Ile Glu Val 50 55 60 Pro Gln Gly Ala Glu Ile Val Phe Lys Ile Thr Ser Pro Asp Val Ile 65 70 75 80 His Gly Phe His Val Glu Gly Thr Asn Ile Asn Val Glu Val Leu Pro 85 90 95 Gly Glu Val Ser Thr Val Arg Tyr Thr Phe Lys Arg Pro Gly Glu Tyr 100 105 110 Arg Ile Ile Cys Asn Gln Tyr Cys Gly Leu Gly His Gln Asn Met Phe 115 120 125 Gly Thr Ile Val Val Lys Glu 130 135 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting the cell nucleus. <400> 14 Pro Ala Ala Lys Arg Val Lys Cys Asp 1 5 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting the endoplasmic reticulum of the cell. <400> 15 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Lys Asp Glu Leu 1 5 10 <210> 16 <211> 25 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting the mitochondria of the cell. <400> 16 Met Leu Ser Leu Arg Gln Ser Ile Arg Phe Phe Lys Pro Ala Thr Arg 1 5 10 15 Thr Leu Cys Ser Ser Arg Tyr Leu Leu 20 25 <210> 17 <211> 28 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting the P-cadherin-over expressing breast cancer cells. <400> 17 Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala 1 5 10 15 Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp 20 25 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Consensus sequence in apentapeptide-repeat protein. <220> <221> UNSURE <222> (1)..(1) <223> Ser or Thr or Ala or Val <220> <221> UNSURE <222> (2)..(2) <223> Asp or Asn <220> <221> UNSURE <222> (3)..(3) <223> Leu or Phe <220> <221> UNSURE <222> (4)..(4) <223> Ser or Thr or Arg <400> 18 Xaa Xaa Xaa Xaa Gly 1 5 <210> 19 <211> 582 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of AlbG gene <400> 19 atgccggcga aaaccctgga aagcaaagat tattgcggcg aaagctttgt gagcgaagat 60 cgcagcggcc agagcctgga aagcattcgc tttgaagatt gcacctttcg ccagtgcaac 120 tttaccgaag cggaactgaa ccgctgcaaa tttcgcgaat gcgaatttgt ggattgcaac 180 ctgagcctga ttagcattcc gcagaccagc tttatggaag tgcgctttgt ggattgcaaa 240 atgctgggcg tgaactggac cagcgcgcag gcgggcgcgc tgagctttga acgctgcatt 300 ctgaacgata gcctgtttta tggcctgtat ctggcgggcg tgaaaatggt ggaatgccgc 360 attcatgatg cgaactttac cgaagcggat tgcgaagatg cggattttac ccagagcgat 420 ctgaaaggca gcacctttca taacaccaaa ctgaccggcg cgagctttat tgatgcggtg 480 aactatcata ttgatatttt tcataacgat attaaacgcg cgcgctttag cctgccggaa 540 gcggcgagcc tgctgaacag cctggatatt gaactgagcg at 582 <210> 20 <211> 642 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of EfsQNR gene <400> 20 ggcagccata tgaaaattac ctatccgctg ccgccgaacc tgccggaaca gctgccgctg 60 ctgaccaact gccagctgga agatgaagcg attctggaaa accatctgta tcagcagatt 120 gatctgccga accaggaagt gcgcaacctg gtgtttcgcg atgcggtgtt tgatcatctg 180 agcctggcga acggccagtt tgcgagcttt gattgcagca acgtgcgctt tgaagcgtgc 240 gattttagca acgtggaatg gctgagcggc agctttcatc gcgtgacctt tctgcgctgc 300 aacctgaccg gcaccaactt tgcggatagc tatctgaaag attgcctgtt tgaagattgc 360 aaagcggatt atgcgagctt tcgctttgcg aactttaacc tggtgcattt taaccagacc 420 cgcctggtgg aaagcgaatt ttttgaagtg acctggaaaa aactgctgct ggaagcgtgc 480 gatctgaccg aaagcaactg gctgaacacc agcctgaaag gcctggattt tagccagaac 540 acctttgaac gcctgacctt tagcccgaac tatctgagcg gcctgaaagt gaccccggaa 600 caggcgattt atctggcgag cgcgctgggc ctggtgatta cc 642 <210> 21 <211> 405 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> TtCuA gene (Nucleic acid sequence of TtCuA gene) <400> 21 gcgtataccc tggcgaccca taccgcgggc gtgattccgg cgggcaaact ggaacgcgtg 60 gatccgacca ccgtgcgcca ggaaggccg tgggcggatc cggcgcaggc ggtggtgcag 120 accggcccga accagtatac cgtgtatgtg ctggcgtttg cgtttggcta tcagccgaac 180 ccgattgaag tgccgcaggg cgcggaaatt gtgtttaaaa ttaccagccc ggatgtgatt 240 catggctttc atgtggaagg caccaacatt aacgtggaag tgctgccggg cgaagtgagc 300 accgtgcgct atacctttaa acgcccgggc gaatatcgca ttatttgcaa ccagtattgc 360 ggcctgggcc atcagaacat gtttggcacc attgtggtga aagaa 405 <210> 22 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting actin in the cell. <400> twenty two Gly Asp Val Gln Lys Lys Arg Trp Leu Phe Glu Thr Lys Pro Leu Asp 1 5 10 15 <210> twenty three <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Signal sequence for targeting tubulin in the cell. <400> twenty three Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly 1 5 10 15 Gly Gly <210> twenty four <211> 90 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Amino acid sequence of a zinc finger protein <400> twenty four Met Glu Arg Pro Tyr Ala Cys Pro Val Glu Ser Cys Asp Arg Arg Phe 1 5 10 15 Ser Asp Ser Ser Asn Leu Thr Arg His Ile Arg Ile His Thr Gly Gln 20 25 30 Lys Pro Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Arg Ser Asp 35 40 45 His Leu Thr Thr His Ile Arg Thr His Thr Gly Glu Lys Pro Phe Ala 50 55 60 Cys Asp Ile Cys Gly Arg Lys Phe Ala Arg Ser Asp Glu Arg Lys Arg 65 70 75 80 His Thr Lys Ile His Leu Arg Gln Lys Asp 85 90 <210> 25 <211> 740 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of mcherry gene <400> 25 gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg cttcaaggtg 60 cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc 120 ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc cctgcccttc 180 gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt gaagcacccc 240 gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg 300 atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctccct ccaggacggc 360 gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg 420 cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga ggacggcgcc 480 ctgaagggcg agatcaagca gaggctgaag ctgaaggacg gcggccacta cgacgctgag 540 gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta caacgtcaac 600 atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgaacgc 660 gccgagggcc gccactccac cggcggcatg gacgagctgt acaagtagta atctagaggg 720 ccctattcta tagtgtcacc 740 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of the AlbG gene (PCR primer). <400> 26 atcccgctca tatgccggcc aagacccttg 30 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of the AlbG gene (PCR primer). <400> 27 atcccgctct cgagtcaatc ggacagctcg atatc 35 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of a PCR primer EfsQNR gene. <400> 28 atcccgctca tatgaaaata acttatccct tgcca 35 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <221> <222> <223> Nucleic acid sequence of a PCR primer EfsQNR gene. <400> 29 atcccgctct cgagttaggt aatcaccaaa ccaagt 36

Claims

1. A genome editing complex for modifying a target polynucleotide, the genome editing complex comprising a recombinant β-helical protein linked to one or more genome editing system molecules, wherein the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm; and wherein the recombinant β-helical protein is represented by a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

12.

2. A genome editing complex for modifying target polynucleotides, the genome editing complex comprising a recombinant β-helical protein linked to a plasmid encoding one or more genome editing system molecules, wherein the β-helical protein has a length in the range of 5 nm to 25 nm and a width in the range of 1 nm to 5 nm; and wherein the recombinant β-helical protein is represented by a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO:

12.

3. The genome editing complex according to claim 1 or claim 2, wherein the one or more genome editing system molecules are selected from the group consisting of: a. RNA-guided endonucleases and / or guide RNA (gRNA); b. Zinc finger nucleases (ZFNs); c. Transcription activator-like effector nucleases (TALEN®); d. DNA-guided endonucleases and / or guide DNA; e. Homing endonucleases; f. Integrase.

4. The genome editing complex according to claim 3, wherein the one or more genome editing system molecules are RNA-guided endonucleases and / or guide RNA (gRNA).

5. The genome editing complex according to claim 3, wherein the RNA-guided endonuclease is Cas9.

6. The genome editing complex of claim 3, wherein the gRNA has a sequence complementary to the target sequence in the target polynucleotide.

7. The genome editing complex according to claim 1 or claim 2, wherein the modification is the addition, deletion, or substitution of one or more nucleotides in the target polynucleotide.

8. The genome editing complex according to claim 1 or claim 2, wherein the β-helical protein has a generally quadrilateral tip shape, the length of which is in the range of 5 nm to 25 nm and the width is in the range of 1 nm to 5 nm.

9. The genome editing complex according to claim 1 or claim 2, wherein the total charge of the β-helix protein is less than zero.

10. The genome editing complex of claim 9, wherein the total charge of the β-helical protein is -20 to -60.

11. The genome editing complex according to claim 1 or claim 2, wherein the β-helix protein has a β-helix structure, and the stiffness parameter K (β-helix) of the helix structure is 0.2 to 12 N / m as measured by atomic force microscopy. 2 .

12. The genome editing complex according to claim 1 or claim 2, wherein the recombinant β-helix protein is linked to one or more genome editing system molecules or plasmids via non-covalent interactions.

13. The genome editing complex of claim 12, wherein the non-covalent interaction is selected from the group consisting of hydrogen bonding, electrostatic interactions, van der Waals interactions, hydrophobic interactions, or combinations thereof.

14. The genome editing complex of claim 1 or claim 2, wherein the recombinant β-helix protein is linked to one or more genome editing system molecules or plasmids via a linker molecule, the linker molecule being selected from the group consisting of: polyethylene glycol (PEG); ethylenediamine; peptides; metal conjugates, drug-metal conjugates, DNA-binding domains, nucleic acid intercalation molecules, and combinations thereof.

15. The genome editing complex of claim 14, wherein when the adapter molecule is a peptide, the peptide comprises amino acids selected from the group consisting of: aliphatic amino acids; aromatic amino acids; and combinations thereof.

16. The genome editing complex of claim 14, wherein the adapter molecule is linked to the recombinant β-helical protein via covalent bonds, non-covalent bonds, and combinations thereof.

17. The genome editing complex of claim 1 or claim 2, wherein the recombinant β-helix protein is linked to one or more genome editing system molecules or plasmids via ester or amide bonds.

18. The genome editing complex of claim 1 or claim 2, wherein the genome editing complex further comprises a signal sequence, wherein the signal sequence directs the genome editing complex to a specific cell or a portion of a cell.

19. The genome editing complex of claim 18, wherein the signal sequence is selected from the group consisting of: SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:

17.

20. The genome editing complex according to claim 1 or claim 2, wherein the genome editing complex further comprises a phosphatidylcholine molecule.

21. The genome editing complex of claim 18, wherein the genome editing complex transfers one or more genome editing system molecules or plasmids to a location selected from the group consisting of: organelles; cell nuclei; and P-cadherin overexpressing breast cancer cells.

22. The genome editing complex according to claim 1 or claim 2, wherein the genome editing complex is used for genome editing.

23. A method for preparing the genome editing complex according to claim 1, the method comprising combining a recombinant β-helix protein with one or more genome editing system molecules.

24. The method of claim 23, wherein the one or more genome editing system molecules are in a reaction buffer containing molecules that stabilize the complex between the recombinant β-helix protein and the one or more genome editing system molecules.

25. A method for preparing the genome editing complex according to claim 2, the method comprising combining recombinant β-helical protein with a plasmid.

26. A method for modifying a target polynucleotide in a cell, the method comprising contacting the cell with a genome editing complex according to any one of claims 1 to 22, wherein the genome editing complex targets a sequence in the target polynucleotide.

27. A pharmaceutical composition comprising the genome editing complex according to any one of claims 1 to 22.

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