Molecules for organelle-specific protein delivery

Abstract

This invention provides an effective technology for delivering therapeutic proteins into cells while maintaining their appropriate intracellular localization or function. [Solution] A fusion protein useful for treating non-nuclear organelle-related disorders, such as hereditary disorders, such as Friedreich's ataxia, is provided. This fusion protein comprises a target protein to be delivered to a non-nuclear organelle, an organelle transfer sequence (OTS), a cell-permeable peptide (CPP), and a target-enhancing sequence (TES), wherein the CPP can interfere with the delivery of the target protein to the non-nuclear organelle, and the TES prevents interference by the CPP. This fusion protein may comprise a target protein to be delivered to a non-nuclear organelle, a CPP, and a TES. A method for treating non-nuclear organelle-related disorders by administering the fusion protein to a subject in need is also provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 000,138, filed on 26 March 2020. The entirety of this disclosure is incorporated herein by reference. [Background technology]

[0002] Cationic cell-permeable peptides (CPPs) have emerged as a promising means of delivering therapeutic proteins into cells. However, one potential drawback of attaching CPPs to therapeutic proteins is that the CPPs may interfere with the appropriate intracellular localization or function of the therapeutic protein. For example, certain cell-permeable peptides, such as HIV-TAT, have been shown to direct the delivery of therapeutic proteins to the nucleus, but the nucleus may not be the desired intracellular destination for the target therapeutic protein.

[0003] The delivery of therapeutic proteins to mitochondria in mammalian cells is being attempted, for example, using TAT-FXN fusion proteins. TAT-FXN fusion proteins contain TAT and frataxin (FXN). FXN is a mitochondrial protein associated with Friedreich's ataxia (FRDA), a genetic condition. FRDA patients suffer from progressive damage to the nervous system, resulting in muscle weakness and ultimately loss of motor control. FRDA is caused by transcriptional repression of the FRDA gene, resulting in the absence or minimal presence of hFXN protein in FRDA patients. [Overview of the project] [Problems that the invention aims to solve]

[0004] Improving effective techniques for delivering therapeutic proteins into cells while maintaining their appropriate intracellular localization or function, including in the context of organelle-specific therapeutic protein delivery, remains a highly demanded area in medicine. [Means for solving the problem]

[0005] This disclosure provides fusion proteins that can be efficiently delivered to cells, for example, at a higher level than previously achieved. This disclosure further provides fusion proteins that can efficiently deliver a target therapeutic protein to cells and achieve and maintain appropriate intracellular localization and / or function of the target therapeutic protein. For example, this disclosure provides fusion proteins that can be efficiently delivered to non-nuclear organelles. Exemplary fusion proteins provided by this disclosure include, in addition to the target protein to be delivered to cells, a cell-permeable peptide (CPP) and / or a target-enhancing sequence (TES). In the fusion proteins of this disclosure, the CPP can interfere with the delivery of the target protein to its appropriate intracellular localization and / or its function, and the TES prevents this interference by the CPP. Another exemplary fusion protein provided by this disclosure includes, in addition to the target protein to be delivered to non-nuclear organelles, an organelle-transfer sequence (OTS), a cell-permeable peptide (CPP), and / or a target-enhancing sequence (TES). In the fusion protein of this disclosure, the CPP can interfere with the delivery of the target protein to a non-nuclear organelle, and the TES prevents this interference by the CPP.

[0006] This disclosure is also based at least in part on the surprising discovery that by including a specific amino acid sequence (i.e., TES) in a fusion protein containing the target protein and CPP, the effective delivery of the target protein to cells is enhanced, resulting in, for example, an increase in the level of the target protein in cells treated with the fusion protein, and enabling proper intracellular localization and / or function of the target protein. In detail, the inventors found that cells treated with a fusion protein containing the target protein, CPP, and TES contained significantly higher amounts of the target protein than cells treated with a fusion protein containing the target protein and CPP but without TES. Thus, the inventors have surprisingly discovered that by introducing TES into a fusion protein containing CPP and the target protein, the amount of the target protein in cells in contact with the fusion protein can be significantly increased. The inventors have also discovered, surprisingly, that the fusion protein containing the target protein, CPP, and TES is correctly processed by cellular mechanisms, achieves appropriate cellular localization, and, for example, is targeted to non-nuclear organelles and delivered to cells, possesses the desired activity of the target protein.

[0007] In some exemplary fusion proteins provided by this disclosure, the TES comprises an amino acid sequence cleavable by an endogenous intracellular protease. In some embodiments, this TES is located adjacent to a CPP, which is located at the N-terminus of the fusion protein. The TES is cleaved by an intracellular nuclease as the fusion protein enters the cytoplasm. While we do not wish to be bound by any particular theory, it is thought that cleavage of the TES facilitates the removal of the CPP from the fusion protein, thereby preventing the CPP from facilitating the target protein's trans-plasma membrane and diffusion outside the cell. This allows for the intracellular accumulation of the target protein. This disclosure is further based, at least in part, on the surprising discovery that including a specific amino acid sequence (i.e., a TES) in a fusion protein, including the target protein, CPP, and OTS, facilitates the effective delivery of the target protein to non-nuclear organelles, such as mitochondria. In detail, the inventors discovered that a fusion protein containing the target protein and CPP, along with an organelle transfer sequence (OTS), such as a mitochondrial transfer sequence (MTS), but without TES, localizes to the cell nucleus rather than the mitochondria upon entering the cell, because the CPP interferes with the mitochondrial delivery of the target protein. The inventors further discovered that by including a specific amino acid sequence, such as TES, in the fusion protein containing the target protein, CPP, and OTS, such as MTS, the delivery of the target protein to the mitochondria is promoted upon the fusion protein's entry into the cell.

[0008] In the exemplary fusion proteins provided by this disclosure, the TES comprises an amino acid sequence cleavable by an endogenous intracellular protease. In some embodiments, this TES is located immediately adjacent to the CPP, which is located at the N-terminus of the fusion protein. The TES is cleaved by an intracellular nuclease as the fusion protein enters the cytoplasm. Cleavage of the TES facilitates the removal of the CPP from the fusion protein, preventing the CPP from interfering with the delivery of the fusion protein to the mitochondria. In other exemplary fusion proteins of this disclosure, the TES comprises a nuclear export signal peptide (NES) which prevents the CPP-facilitated delivery of the fusion protein to the nucleus and instead facilitates the delivery of the fusion protein to non-nuclear organelles, such as mitochondria.

[0009] In other exemplary fusion proteins of this disclosure, the TES comprises a nuclear export signaling peptide (NES) which prevents the accumulation of the target protein in the nucleus.

[0010] In some embodiments, the disclosure provides a fusion protein comprising a target protein to be delivered to a cell, a cell-permeable peptide (CPP), and a target-enhancing sequence (TES).

[0011] In some embodiments, the CPP is located at the N-terminus of the fusion protein, and the TES is fused to the C-terminus of the CPP. In some embodiments, the fusion protein comprises, or consists of, the CPP, the TES, and the target protein, starting from the N-terminus.

[0012] In some embodiments, CPP is located at the C-terminus of the fusion protein, and TES is fused to the N-terminus of CPP.

[0013] In some embodiments, the fusion protein includes, or comprises, the target protein, TES, and CPP, starting from the C-terminus.

[0014] In some embodiments, the target protein lacks OTS.

[0015] In other embodiments, the fusion protein further comprises an exogenous organelle targeting sequence (OTS) for the target protein.

[0016] In some embodiments, the target protein includes OTS.

[0017] In some embodiments, the OTS is heterogeneous for the target protein. In some embodiments, the OTS is endogenous for the target protein.

[0018] In some embodiments, the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and their variants or derivatives. In some embodiments, the target protein includes frataxin (FXN), PARK2 protein (PARKIN), or its variants or derivatives.

[0019] In some embodiments, CPP comprises peptides selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0. In some embodiments, CPP comprises peptides selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and their variants or derivatives. In some embodiments, CPP comprises HIV-TAT or its variants or derivatives.

[0020] In some embodiments, the TES is a nuclear export signal peptide. In some embodiments, the nuclear export signal peptide comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 36-43. In some embodiments, the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8, and variants or derivatives thereof. In some embodiments, the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1 and NES2, or variants or derivatives thereof.

[0021] In some embodiments, the TES is a protease-sensitive peptide. In some embodiments, the protease-sensitive peptide comprises a ubiquitin-like modifier. In some embodiments, the protease-sensitive peptide comprises a sequence having at least 85% sequence identity to any one of SEQ ID NOs: 18-31.

[0022] In some embodiments, the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, caspase cleavage domain, calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

[0023] In some embodiments, the fusion protein comprises an amino acid sequence having at least 85%, 90%, or 95% sequence identity to any of SEQ ID NOs: 55-61, 69-71, and 81.

[0024] In some aspects, the present disclosure provides a fusion protein comprising a protein to be delivered to a non-nuclear organelle, an organella targeting sequence (OTS), a cell-penetrating peptide (CPP), and a target facilitating sequence (TES), wherein the CPP can interfere with the delivery of the protein to be targeted to the non-nuclear organelle, and the TES prevents the interference by the CPP.

[0025] In some embodiments, the CPP is located at the N-terminus of the fusion protein, and the TES is fused to the C-terminus of the CPP.

[0026] In some embodiments, the fusion protein includes, or consists of, CPP, TES, OTS, and the target protein, starting from the N-terminus.

[0027] In some embodiments, the CPP is located at the C-terminus of the fusion protein, and the TES is fused to the N-terminus of the CPP. In some embodiments, the non-nuclear organelles are selected from the group consisting of mitochondria, cytosol, lysosomes, endoplasmic reticulum (ER), peroxisomes, and the Golgi apparatus. In some embodiments, the target protein in its native form is localized to mitochondria, cytosol, lysosomes, endoplasmic reticulum (ER), peroxisomes, or the Golgi apparatus.

[0028] In some embodiments, the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and variants or derivatives thereof. In some embodiments, the target protein is frataxin (FXN) or its variants or derivatives. In some embodiments, the target protein is pyruvate dehydrogenase (PDH) or its variants or derivatives.

[0029] In some embodiments, CPP comprises peptides selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0. In some embodiments, CPP comprises peptides selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and their variants or derivatives. In some embodiments, CPP comprises HIV-TAT or its variants or derivatives.

[0030] In some embodiments, TES is a nuclear export signal peptide. In some embodiments, the nuclear export signal peptide contains a sequence having at least 85% sequence identity with one of SEQ ID NOs: 36-43. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2 and its variants or derivatives.

[0031] In some embodiments, TES is a protease-sensitive peptide. In some embodiments, the protease-sensitive peptide includes a ubiquitin-like modifying factor. In some embodiments, the protease-sensitive peptide includes a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18-31. In some embodiments, the protease-sensitive peptide includes a peptide selected from the group consisting of ubiquitin, caspase cleavage domains, calpain cleavage domains, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and their variants or derivatives.

[0032] In some embodiments, the fusion protein of this disclosure further comprises a secretory signal (SS).

[0033] In some embodiments, the fusion protein of this disclosure further comprises an extracellular proteolytic site (EPS).

[0034] In some embodiments, the fusion protein delivers the target protein to a non-nuclear organelle. In some embodiments, the fusion protein delivers the target protein to a mitochondria.

[0035] In some embodiments, the fusion protein contains an amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

[0036] In some embodiments, the Disclosure also provides nucleic acids that encode the fusion proteins of the Disclosure.

[0037] In some embodiments, the Disclosure also provides expression vectors for introducing fusion proteins into cells, comprising nucleic acids of the Disclosure. In some embodiments, the expression vectors are selected from the group consisting of retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, phagemids, baculoviruses, and combinations thereof.

[0038] In some embodiments, the Disclosure provides a conjugate for intracellular delivery of a protein to a non-nuclear organelle, the fusion protein of the Disclosure and a portion linked to the fusion protein, wherein the portion is selected from the group consisting of radiolabeled, fluorescently labeled, small molecule, and polymer molecule. In some embodiments, the polymer molecule is polyethylene glycol (PEG).

[0039] In some embodiments, the Disclosure provides cells comprising the fusion protein of the Disclosure, nucleic acids of the Disclosure, expression vectors of the Disclosure, conjugates of the Disclosure, or combinations thereof.

[0040] In some embodiments, the cells are stem cells or iPS cells. In some embodiments, the cells are selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cell lines, or yeast cell lines.

[0041] In some embodiments, the Disclosure also provides a pharmaceutical composition comprising a fusion protein of the Disclosure, a conjugate of the Disclosure, or a combination thereof, and a pharmaceutically acceptable diluent, carrier, additive, or excipient.

[0042] In some embodiments, the Disclosure provides a method for delivering a target protein to a cell, comprising contacting the cell with a fusion protein, nucleic acid, vector, or conjugate of the Disclosure.

[0043] In some embodiments, the Disclosure also provides a method for intracellular delivery of a target protein to a non-nuclear organelle within a cell, comprising contacting the cell with a fusion protein of the Disclosure, a nucleic acid of the Disclosure, a vector of the Disclosure, or a conjugate of the Disclosure. In some embodiments, the non-nuclear organelle is a mitochondria.

[0044] In some embodiments, the Disclosure also provides therapeutic compounds for treating nonnuclear organelle-related disorders, comprising fusion proteins of the Disclosure, nucleic acids of the Disclosure, vectors of the Disclosure, or conjugates of the Disclosure. In some embodiments, the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, fibrosis, and PLA2G6-associated neurodegeneration (PLAN). In some embodiments, the nonnuclear organelle-related disorder is FDRA. In some embodiments, the nonnuclear organelle-related disorder is Parkinson's disease.

[0045] In some embodiments, the Disclosure also provides a method for treating a nonnuclear organelle-related disorder, comprising administering a fusion protein, nucleic acid, vector, conjugate, or pharmaceutical composition of the Disclosure to a subject in need so as to treat the nonnuclear organelle-related disorder.

[0046] In some embodiments, the non-nuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, and fibrosis. In some embodiments, the non-nuclear organelle-related disorder is FDRA. In some embodiments, the non-nuclear organelle-related disorder is Parkinson's disease.

[0047] In some embodiments, the subject is human.

[0048] In some embodiments, the Disclosure also provides a method for increasing the amount of a target protein delivered to cells, comprising the steps of: modifying the sequence of a fusion protein containing a target protein and a cell-permeable peptide (CPP) by introducing a target-promoting sequence (TES) into the fusion protein to generate a modified fusion protein; and contacting cells with the modified fusion protein, thereby increasing the amount of the target protein delivered to cells compared to the amount of the target protein delivered by a fusion protein without the TES.

[0049] In some embodiments, the modified fusion protein has CPP located at the N-terminus of the fusion protein and TES fused to the C-terminus of CPP.

[0050] In some embodiments, the modified fusion protein includes CPP, TES, and the target protein, starting from the N-terminus.

[0051] In some embodiments, the modified fusion protein includes CPP, TES, OTS, and the target protein, starting from the N-terminus.

[0052] In some embodiments, the modified fusion protein has CPP located at the C-terminus of the fusion protein and TES fused to the N-terminus of CPP. In some embodiments, the amount of target protein delivered to the target organelle by OTS is increased compared to the amount of target protein delivered to the organelle by the fusion protein without TES.

[0053] In some embodiments, the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, and type VI phospholipase A2, or their variants or derivatives. In some embodiments, the target protein is frataxin (FXN) or its variants or derivatives. In some embodiments, the target protein is PARK2 protein or its variants or derivatives.

[0054] In some embodiments, CPP comprises peptides selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0. In some embodiments, CPP comprises peptides selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and their variants or derivatives. In some embodiments, CPP comprises HIV-TAT or its variants or derivatives.

[0055] In some embodiments, TES is a nuclear export signal peptide. In some embodiments, the nuclear export signal peptide contains a sequence having at least 85% sequence identity with one of SEQ ID NOs: 36-43. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2 and its variants or derivatives.

[0056] In some embodiments, TES is a protease-sensitive peptide. In some embodiments, the protease-sensitive peptide includes a ubiquitin-like modifying factor. In some embodiments, the protease-sensitive peptide includes a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18-31. In some embodiments, the protease-sensitive peptide includes a peptide selected from the group consisting of ubiquitin, caspase cleavage domains, calpain cleavage domains, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and their variants or derivatives.

[0057] In some embodiments, the modified fusion protein contains an amino acid sequence having at least 85% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

[0058] In one embodiment, the present disclosure provides a fusion protein comprising a target protein to be delivered to a non-nuclear organelle, an organelle transfer sequence (OTS), a cell-permeable peptide (CPP), and a target-enhancing sequence (TES), wherein the CPP can interfere with the delivery of the target protein to the non-nuclear organelle, and the TES prevents such interference by the CPP.

[0059] In some embodiments, the CPP is located at the N-terminus of the fusion protein, and the TES is fused to the C-terminus of the CPP. In some embodiments, the disclosure provides a fusion protein comprising the CPP, TES, OTS, and the target protein, starting from the N-terminus.

[0060] In some embodiments, CPP is located at the C-terminus of the fusion protein, and TES is fused to the N-terminus of CPP.

[0061] In some embodiments, non-nuclear organelles are selected from the group consisting of mitochondria, cytosol, lysosomes, endoplasmic reticulum (ER), peroxisomes, and Golgi apparatus.

[0062] In some embodiments, the target protein in its native form is localized to mitochondria, cytosol, lysosomes, endoplasmic reticulum (ER), peroxisomes, or the Golgi apparatus.

[0063] In some embodiments, the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, type VI A2 phospholipase, transcription factors, and their variants or derivatives. In one embodiment, the target protein is frataxin (FXN) or its variant or derivative. In another embodiment, the target protein is pyruvate dehydrogenase (PDH) or its variant or derivative.

[0064] In some embodiments, CPP comprises peptides selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0. In yet other embodiments, CPP comprises peptides selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and their variants or derivatives. In some embodiments, CPP comprises HIV-TAT or its variants or derivatives.

[0065] In some embodiments, TES is a nuclear export signal peptide. In yet other embodiments, the nuclear export signal peptide contains a sequence having at least 85% sequence identity with one of SEQ ID NOs: 36-43. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives. In some embodiments, the nuclear export signal peptide includes a peptide selected from the group consisting of NES1, NES2 and their variants or derivatives.

[0066] In some embodiments, TES is a protease-sensitive peptide. In some embodiments, the protease-sensitive peptide includes a ubiquitin-like modifier. In some embodiments, the ubiquitin-like modifier includes a sequence having at least 85% sequence identity with one of SEQ ID NOs: 18-31.

[0067] In some embodiments, the protease-sensitive peptide includes a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and their variants or derivatives.

[0068] In some embodiments, the fusion protein provided by this disclosure includes a secretory signal (SS). In some embodiments, the fusion protein includes an extracellular proteolytic site (EPS).

[0069] In some embodiments, the fusion protein delivers the target protein to a non-nuclear organelle. In certain embodiments, the fusion protein delivers the target protein to a mitochondria.

[0070] In some embodiments, the fusion protein contains a sequence having at least 85% sequence identity with any of SEQ ID NOs. 55-61.

[0071] In another aspect, the Disclosure provides nucleic acids encoding the fusion protein of the Disclosure.

[0072] In one relevant aspect, the Disclosure provides an expression vector comprising the nucleic acid of the Disclosure for introducing a fusion protein into a cell.

[0073] In some embodiments, the expression vector is selected from the group consisting of retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, phagemids, baculoviruses, and combinations thereof.

[0074] In another embodiment, the present disclosure provides a protein conjugate for intracellular delivery of a protein to a nonnuclear organelle, comprising a fusion protein and a portion linked to the fusion protein, wherein the portion is selected from the group consisting of radiolabeled, fluorescently labeled, small molecule, and polymer molecule. In one embodiment, the polymer molecule is polyethylene glycol (PEG).

[0075] In another embodiment, the Disclosure provides cells comprising the fusion proteins, nucleic acids, expression vectors, or conjugates of the Disclosure, or combinations thereof. The cells may be transformed cells, for example, cells transformed with the fusion proteins, nucleic acids, expression vectors, or conjugates of the Disclosure, or combinations thereof. In some embodiments, the cells are stem cells or iPS cells. In some embodiments, the cells may be selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cells, or yeast cells.

[0076] In another embodiment, the Disclosure provides a pharmaceutical composition comprising a fusion protein or protein conjugate of the Disclosure, or a combination thereof, and a pharmaceutically acceptable diluent, carrier, additive, or excipient.

[0077] In another embodiment, the Disclosure provides a method for intracellular delivery of a target protein to a non-nuclear organelle within a cell, comprising contacting the cell with a fusion protein, nucleic acid, vector, or protein conjugate of the Disclosure. In one embodiment, the non-nuclear organelle is a mitochondria.

[0078] In another embodiment, the Disclosure provides therapeutic compounds for treating nonnuclear organelle-associated disorders, comprising fusion proteins, nucleic acids, vectors, or protein conjugates of the Disclosure. In some embodiments, the nonnuclear organelle-associated disorders are selected from the group consisting of Friedreich's ataxia (FDRA), Barth syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, fibrosis, and PLA2G6-associated neurodegenerative disorders (PLAN). In one embodiment, PLAN includes disorders selected from the group consisting of Parkinsonian syndromes, including infantile axonal dystrophy (INAD), atypical axonal dystrophy (ANAD), adult-onset dystonia-parkinsonism (DP), and autosomal recessive juvenile parkinsonism (AREP). In one embodiment, the nonnuclear organelle-associated disorder is FDRA.

[0079] In one relevant embodiment, the Disclosure provides a method for treating a nonnuclear organelle-related disorder, comprising administering a fusion protein, nucleic acid, vector, protein conjugate, or pharmaceutical composition of the Disclosure to a subject in need thereof, such that the nonnuclear organelle-related disorder is treated. In some embodiments, the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, and fibrosis. In one particular embodiment, the nonnuclear organelle-related disorder is FDRA.

[0080] In some embodiments, the subject is human.

[0081] This summary is provided to introduce in a simplified form the selected concepts that are further described in the embodiments for carrying out the invention described below. This summary is not intended to identify any important or essential features of the claimed subject matter, nor to limit the scope of the claimed subject matter.

[0082] Non-limiting examples of embodiments of the present disclosure are described below with reference to the figures accompanying this specification, which are listed after this paragraph. Identical features shown in multiple figures are generally marked with the same symbols in all figures in which they are shown. Symbols marking icons representing given features of embodiments of the present disclosure in the figures may be used to refer to the given features. The dimensions of features shown in the figures have been chosen for convenience and clarity in presentation and are not necessarily shown on a fixed scale. [Brief explanation of the drawing]

[0083] [Figure 1] This is a schematic diagram showing a specific fusion protein of the present disclosure, which includes a target-enhancing sequence (TES) having a protease cleavage site. [Figure 2] This is a schematic diagram showing the configuration of various domains in the fusion protein disclosed herein. [Figure 3] This is a photograph of a set of rat L6 myoblasts after treatment with TAT-GG-hFXN fusion protein (5 μM) and immunofluorescence labeling with anti-hFXN antibody (left panel) or mitochondrial-specific anti-TOMM20 antibody (center panel). The right panel shows the merge of anti-hFXN and anti-TOMM20 labeling. [Figure 4] This is a photograph of a set of rat L6 myoblasts transfected with plasmids encoding either hFXN (top panel) or TAT-GG-hFXN fusion protein (bottom panel) after immunofluorescence labeling. The myoblasts were labeled with anti-hFXN antibody (left panel) or anti-TOMM20 antibody (center panel). The right panel shows the merger of the two labels. [Figure 5]This is a schematic diagram illustrating the design of an exemplary fusion protein of the present disclosure, including hFXN. Panel A shows TAT-GG fused to the amino terminus of hFXN1-210 (TAT-GG-hFXN fusion protein). Panel B shows TAT-GG fused to a proteolytically sensitive amino acid sequence followed by hFXN1-210. Panel C shows, according to one embodiment of the present disclosure, TAT-GG fused to hFXN1-210 followed by a nuclear export signal (NES). [Figure 6-1] These are photographs of rat L6 myoblasts transfected with plasmids encoding hFXN (Figure 4A), TAT-GG-hFXN fusion protein (Figure 4B), TAT-GG-SUMO1-hFXN (Figure 4C), TAT-GG-LLVY-hFXN (Figure 4D), TAT-GG-hFXN-NES1 (Figure 4E), and TAT-GG-hFXN-NES2 (Figure 4F), and then immunofluorescently labeled. For immunofluorescence labeling, L6 myoblasts were stained with antibodies against hFXN (left panel) and TOMM20 (center panel). The right panel shows the merge of the two labels. [Figure 6-2] These are photographs of rat L6 myoblasts transfected with plasmids encoding TAT-GG-ubiquitin-hFXN (Figure 4G), TAT-GG-DEVD-hFXN (Figure 4H), and TAT-GG-EPLFAERK-hFXN (Figure 4I), and then immunofluorescently labeled. For immunofluorescence labeling, L6 myoblasts were stained with antibodies against hFXN (left panel) and TOMM20 (center panel). The right panel shows the merge of the two labels. [Figure 7]These are images of Western blot analysis of cell lysates obtained from rat L6 myoblasts transfected with the following hFXN variants: hFXN (lane 1), TAT-GG-hFXN fusion protein (lane 2), TAT-GG-SUMO1-hFXN (lane 3), TAT-GG-ubiquitin-hFXN (lane 4), TAT-GG-EPLFAERK-hFXN (lane 5), TAT-GG-LLVY-hFXN (lane 6), TAT-GG-hFXN-NES1 (lane 7), TAT-GG-hFXN-NES2 (lane 8), and a mock (lane 9). The blots were labeled with an anti-hFXN antibody, and β-actin was used as a control. [Figure 8] This is a schematic diagram of the structure of the hFXN fusion protein of this disclosure, which was tested for its cell transduction ability in Example 5. [Figure 9A] These are a series of photographs of Schwann cells treated with 0.25 μM, 0.5 μM, and 1 μM TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and a negative control medium. In the photographs, green corresponds to the mitochondrial marker TOMM20 signal, red to the hFXN signal, and blue to the nuclear marker Hoechst 33342 dye signal. [Figure 9B] This is a high-magnification image of cells treated with TAT-GG-ubiquitin-hFXN. This image shows the details of hFXN staining and its localization to mitochondria. [Figure 9C] This bar graph shows the ratio of the mean hFXN staining signal to the mean nuclear staining signal in cells treated with various concentrations of TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. [Figure 9D]This is a photograph of a nitrocellulose membrane after staining the total protein following transfer from an SDS-PAGE gel loaded with samples of TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN used in a transduction experiment. The photograph demonstrates that there was no degradation of the fusion proteins and that the same amount of each fusion protein was used in the transduction experiment. [Figure 10A] These are a series of representative images of Schwann cells and H9C2 cells treated with 1 μM hFXN fusion proteins TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN, and then stained with mitochondrial, nuclear, and anti-FXN stains. White arrows indicate the representative localization of hFXN to mitochondria. [Figure 10B] This graph shows the ratio of the mean anti-FXN staining signal to the mean anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in Schwann cells treated with TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. [Figure 10C] This graph shows the ratio of the mean anti-FXN staining signal to the mean anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in H9C2 cells treated with TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. [Figure 10D] This graph shows the ratio of the mean anti-FXN staining signal to the mean anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in Schwann cells treated with TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and TAT-GG-hFXN-NES1. [Figure 11] This is a Western blot image of total protein samples isolated from cells transfected with various hFXN fusion proteins and analyzed using anti-FXN antibodies. [Figure 12A]This is a photograph of a culture plate containing samples of LRPPRC KD cells and Scr-5 cells treated with the hFXN fusion protein of this disclosure. [Figure 12B] These are a series of photographs of LRPPRC KD and Scr-5 control cells treated with the highest concentration of each hFXN fusion protein or medium tested. [Figure 12C] This graph shows the amount of CYR61 in the culture medium of LRPPRC KD cells treated with hFXN fusion protein. [Figure 12D] This graph shows the amount of lactate in the culture medium of LRPPRC KD cells treated with hFXN fusion protein. [Figure 13] This is a schematic diagram of the structure of the PARKIN fusion protein disclosed herein. [Figure 14] This graph shows the ratio of PARKIN staining to mitochondrial staining as a function of PARKIN fusion protein concentration in cells treated with various concentrations of His6-SUMO-TAT-GG-PARKIN, TAT-GG-EPLFAERK-PARKIN, and TAT-GG-ubiquitin-PARKIN. [Figure 15] These are a series of images of L6 rat myoblasts treated with 0 μM or 0.5 μM TAT-GG-EPLFAERK-PARKIN in the absence of CCCP or in the presence of 10 μM CCCP. [Modes for carrying out the invention]

[0084] One aspect of the present disclosure relates to providing a fusion protein comprising a protein to be delivered to a cell. In some embodiments, the fusion protein comprises a protein to be delivered to a specific organelle, such as a non-nuclear organelle, such as a mitochondria. The fusion protein also comprises at least one cell-permeable peptide (CPP), at least one target-promoting sequence (TES), and optionally at least one organelle-transition signal (OTS).

[0085] In certain aspects of this disclosure, a CPP can interfere with the delivery of a target protein to an organelle, such as a non-nuclear organelle. For example, as demonstrated by certain experimental data contained herein, a CPP, such as HIV-TAT, can interfere with the delivery of a target protein, such as FXN, to mitochondria. Specifically, HIV-TAT promotes the delivery of FXN to the nucleus rather than to mitochondria. Thus, the fusion proteins of this disclosure include a TES that prevents or attenuates CPP interference with the delivery of a target protein to an appropriate cellular (e.g., non-nuclear) organelle.

[0086] For example, in some embodiments, the CPP may be located at the N-terminus of the fusion protein and the TES may be fused to the C-terminus of the CPP. Alternatively, the CPP may be located at the C-terminus of the fusion protein and the TES may be fused to the N-terminus of the CPP. The TES may include an intracellular protease recognition sequence that, upon entering a cell, promotes the cleavage of the fusion protein at the TES, thereby removing the CPP from the remainder of the fusion protein and preventing it from orienting the fusion protein toward the nucleus.

[0087] Alternatively, the TES may include nuclear export signals, such as signals that promote the translocation of the fusion protein out of the nucleus after it has been delivered to the nucleus via CPP.

[0088] Another aspect of this disclosure relates to providing a fusion protein comprising a target protein to be delivered to a cell. The fusion protein comprises at least one cell-permeable peptide (CPP), at least one target-enhancing sequence (TES), and the target protein.

[0089] In some embodiments of this disclosure, CPP can interfere with the delivery of the target protein to cells. For example, as demonstrated by certain experimental data contained herein, cells treated with a fusion protein containing the target protein, CPP, and TES contained significantly higher amounts of the target protein than cells treated with a fusion protein containing the target protein and CPP but without TES. Thus, unexpectedly, it was discovered that introducing TES into a fusion protein containing CPP and the target protein can significantly increase the amount of the target protein delivered to cells.

[0090] While we do not wish to be bound by any particular theory, it is thought that cleavage of the TES by an endogenous protease within the cell promotes the removal of CPP from the fusion protein, thereby preventing CPP from promoting the target protein's transplasma membrane and diffusion outside the cell. This enables the intracellular accumulation of the target protein. The removal of CPP from the fusion protein also prevents CPP from interfering with the proper intracellular localization of the target protein. Therefore, the fusion protein of this disclosure includes a TES that prevents or attenuates CPP interference with the delivery of the target protein to the cell. In some embodiments, the fusion protein of this disclosure includes a TES that prevents or attenuates CPP interference with the proper intracellular localization of the target protein to the cell.

[0091] In some embodiments, the CPP may be located at the N-terminus of the fusion protein and the TES may be fused to the C-terminus of the CPP. Alternatively, the CPP may be located at the C-terminus of the fusion protein and the TES may be fused to the N-terminus of the CPP. The TES may include an intracellular protease recognition sequence that, upon entering a cell, promotes the cleavage of the fusion protein at the TES, thereby removing the CPP from the remainder of the fusion protein and preventing it from directing the fusion protein out of the cell.

[0092] Alternatively, the TES may contain a nuclear export signaling peptide (NES). While we do not wish to be bound by any particular theory, it is thought that CPPs promote the transport of fusion proteins into the nucleus, and NESs, if present in the fusion protein, prevent its accumulation in the nucleus. By preventing the accumulation of fusion proteins in the nucleus, NESs promote the proper intracellular localization of the target protein contained in the fusion protein outside the nucleus, for example, in mitochondria.

[0093] In some embodiments, removal of CPP via cleavage of TES by endogenous proteases promotes the proper intracellular localization of the target protein. In some cases, the target protein, such as NF kappa b, androgen receptor, estrogen receptor, or aconitase, may typically localize to multiple cellular compartments, such as the nucleus and mitochondria. Removal of CPP via cleavage of TES by endogenous proteases prevents CPP from interfering with the proper intracellular localization of the target protein in response to endogenous stimuli.

[0094] Target protein The fusion proteins provided in this disclosure include a protein to be delivered to a cell. In some embodiments, the fusion proteins provided in this disclosure include a protein to be delivered to a specific organelle within the cell, such as a non-nuclear organelle. The target protein included in the fusion protein of this disclosure may be related to a non-nuclear organelle, for example, the target protein may be a non-nuclear organelle-specific protein within the cell. For example, the target protein may be localized to a non-nuclear organelle and / or perform or facilitate an intracellular function carried out by a non-nuclear organelle. Exemplary non-nuclear organelles include the cytosol, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and peroxisomes. In some embodiments, the target protein included in the fusion protein of the Disclosure may instead be related to the nucleus, or in some embodiments, to the nucleus and one or more non-nuclear organelles. For example, the target protein may be an intracellular protein localized to both the nucleus and other non-nuclear organelles (e.g., cytosol, mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus, and peroxisomes).

[0095] The target protein may be associated with a pathological condition of the subject. Such a pathological condition may result, for example, from a deficiency of the target protein in the subject, for example, from the possibility that the target protein has mutated in a way that reduces or eliminates its native activity, or from the complete absence of the target protein.

[0096] In certain aspects of this disclosure, the target proteins may be mitochondrial. Exemplary mitochondrial target proteins may include, for example, frataxin (FXN), tafadin, pyruvate dehydrogenase beta-2 (PDHB), leucine-rich PPR motif-containing protein (LRPPRC protein), SLIRP, parkin RBR E3 ubiquitin protein ligase (PARK2 protein or PARKIN), PTEN-induced kinase 1 (PINK1 or PARK6 protein), protein deglycase DJ-1 (PARK7 protein), and type VI A2 phospholipase.

[0097] In some embodiments, the target protein contains OTS. In some embodiments, OTS is endogenous to the target protein. In some embodiments, OTS is exogenous to the target protein.

[0098] In some embodiments, the target protein lacks OTS. In some embodiments, the target protein lacks OTS in its native form; that is, it does not contain OTS in nature. In some embodiments, the target protein lacking OTS is the mature form of the protein from which endogenous OTS has been removed.

[0099] In a particular embodiment, the target protein related to mitochondria may be frataxin (FXN), such as human FXN, associated with the pathological condition Friedreich's ataxia (FRDA). FRDA is a genetic, progressive neurodegenerative disorder caused by mutations in the gene encoding FXN. FXN is an essential and systematically conserved protein found in cells throughout the body, but at the highest levels in the heart, spine, liver, pancreas, and skeletal muscle. FXN is encoded in the nucleus, expressed in the cytoplasm, imported into mitochondria, where it is processed into its mature form. In humans, the full-length hFXN (hFXN) is 210 amino acids. 1-210The protein (23.1 kDa) contains a typical mitochondrial localization sequence (MTS) at its amino terminus, and upon importation into the mitochondrial matrix, it is processed by a two-step cleavage by mitochondrial matrix processing peptidases (MPPs). The resulting protein is a mature hFXN protein (hFXN) of 130 amino acids and 14.2 kDa. 81-210 The sequences of full-length hFXN and mature hFXN are shown in Table 1 below.

[0100] [Table 1]

[0101] Therefore, in some embodiments, the fusion protein provided in this disclosure is full-length, i.e., hFXN(hFXN) containing the MTS as the organelle transition sequence (OTS). 1-210 ) may include. In some embodiments, the fusion protein may contain an amino acid sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the full-length hFXN sequence, for example, SEQ ID NO: 1 as listed in Table 1.

[0102] In some embodiments, the fusion protein provided in this disclosure is mature hFXN (hFXN 81-210 ) may include. In some embodiments, the fusion protein may contain an amino acid sequence having at least 85%, for example, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the sequence of a mature hFXN, for example, SEQ ID NO: 2 listed in Table 1.

[0103] In other embodiments, exemplary target proteins that may be included in the fusion proteins of the present disclosure include tafadin, pyruvate dehydrogenase beta-2 (PDHB), leucine-rich PPR motif-containing protein (LRPPRC protein), SLIRP, parkin RBR E3 ubiquitin protein ligase (PARK2 protein or PARKIN), PTEN-induced kinase 1 (PINK1 or PARK6 protein), protein deglycase DJ-1 (PARK7 protein), and type VI A2 phospholipase.

[0104] Table 2 below lists exemplary target proteins, corresponding human sequences, and relevant pathological conditions that can be treated with the fusion proteins of this disclosure, including the aforementioned target proteins.

[0105] [Table 2] TIFF2026108757000004.tif236170TIFF2026108757000005.tif237170TIFF2026108757000006.tif233170TIFF2026108757000007.tif202170

[0106] In some embodiments, the target protein included in the fusion protein of the present disclosure may include or consist of any one of the sequences listed in Table 2, for example, SEQ ID NOs: 3-9 or 72-80. In some embodiments, the target protein included in the fusion protein of the present disclosure may include or consist of an amino acid sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of the amino acid sequences listed in Table 2, for example, SEQ ID NOs: 3-9 or 72-80.

[0107] In some embodiments, the target protein included in the fusion protein of this disclosure contains or consists of any one of the sequences listed in Table 2, for example, one of sequence numbers 3-9 or 72-80, and lacks an endogenous OTS, i.e., the endogenous OTS has been removed. The OTS of a particular protein will be readily recognizable to those skilled in the art.

[0108] In some aspects of this disclosure, the protein in question may be related to lysosomes. In some aspects of this disclosure, the protein in question may be related to the endoplasmic reticulum (ER). In some aspects of this disclosure, the protein in question may be related to the Golgi apparatus.

[0109] In some embodiments, the target protein may be a protein localized to the nucleus. In some embodiments, the target protein may be a protein localized to the nucleus and one or more other cellular organelles. Those skilled in the art will understand that introducing a TES into a fusion protein containing CPP and such a target protein will eliminate CPP interference with the localization of the target protein, for example, preventing or reducing abnormal localization to the nucleus, thereby enabling innate regulatory pathways to direct the protein to proper cellular localization in response to endogenous signals and thereby improving the tendency to relocalize to the appropriate intracellular location. Such proteins may include, for example, NFK beta, STAT3, androgen receptor, estrogen receptor, and p53.

[0110] In some embodiments, the fusion proteins provided herein may include the subject protein described herein, or functional analogues, derivatives, or fragments of the subject protein. As used herein, the term “derivative” encompasses amino acid sequences (polypeptides) that differ from the polypeptides specifically defined herein, e.g., SEQ ID NOs: 3–9 or 72–80, through insertions, deletions, substitutions, and modifications of amino acids that do not alter the activity of the original polypeptide. As used herein, the terms “insertion,” “deletion,” or “substitution” are understood to encompass any addition, deletion, or substitution of 1–50 amino acid residues in a polypeptide, e.g., 1–5 amino acid residues, 1–10 amino acid residues, 5–15 amino acid residues, 10–20 amino acid residues, 25–40 amino acid residues, or 30–50 amino acid residues, respectively. More specifically, the insertion, deletion, or substitution may be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. It should be noted that insertions, deletions, or substitutions may occur at any position in the modified peptide, including at either the N-terminus or the C-terminus. In one embodiment, the target protein is FXN, for example, a derivative of human FXN. In one embodiment, the target protein is a derivative of tafazine. In one embodiment, the target protein is a derivative of PDHB. In one embodiment, the target protein is a derivative of LRPPRC. In one embodiment, the target protein is a derivative of SLIRP. In one embodiment, the target protein is a derivative of PARK2 protein. In one embodiment, the target protein is a derivative of PINK1 (or PARK6 protein). In one embodiment, the target protein is a derivative of PARK7. In one embodiment, the target protein is a derivative of type VI phospholipase A2 (e.g., variants 1, 2, 3, 4, 5, 6, 7, 8, or 9).

[0111] In some embodiments, the amino acid sequence of the protein in question within the context of this disclosure may differ from the amino acid sequence of a naturally occurring protein associated with any of the aforementioned pathological conditions. In some embodiments, the amino acid sequence of the protein in question may include conserved amino acid substitutions, i.e., substitutions by structurally similar amino acids, compared to the amino acid sequence of a naturally occurring protein associated with any of the pathological conditions. For example, structurally similar amino acids include (isoleucine (I) and leucine (L) and valine (V)), (phenylalanine (F) and tyrosine (Y)), (lysine (K) and arginine (R)), (glutamine (Q) and asparagine (N)), (aspartic acid (D) and glutamic acid (E)), and (glycine (G) and alanine (A)).

[0112] As used herein, the term “derivative” encompasses homologs, variants, and analogs of the original polypeptide, as well as covalent modifications of the original polypeptide. Any derivative, variant, or analog of any protein or peptide contained in the fusion protein provided herein will have the same biological activity as its native form. In a particular embodiment, the protein in question is an FXN homolog, variant, or analog of, for example, human FXN.

[0113] In some embodiments, the target protein that may be included in the fusion protein of this disclosure may be one that is naturally present in the subject. The subject may be a mammal, such as a mouse, rat, monkey, or human.

[0114] Organelle transition sequences (OTS) In some embodiments, the fusion proteins of this disclosure may also include an organelle transfer sequence (OTS). The terms “organelle transfer sequence” or “OTS,” as used herein, refer to any amino acid sequence, e.g., a protein, peptide, or consensus domain, that facilitates the delivery of a target protein to a specific non-nuclear organelle. For example, an OTS may be a mitochondrial transfer sequence (MTS) that facilitates the delivery of a target protein to mitochondria. An MTS may be a peptide of about 10 to 70 amino acids containing an alternating pattern of hydrophobic and positively charged amino acids forming an amphiphilic helix.

[0115] OTS may also be an endoplasmic reticulum (ER) transition sequence. The ER transition sequence (ER targeting sequence) may be a peptide of about 16 to 30 amino acids, containing a hydrophilic region, a hydrophobic domain, and a C-terminal region containing a cleavage site for an ER-specific signal peptidase.

[0116] OTS may also contain HEAT motifs, which can be found in naturally occurring proteins localized in the Golgi and endoplasmic reticulum (ER). HEAT is a protein tandem repeat structural motif consisting of two alpha helices linked by a short loop. HEAT repeats can form alpha solenoids, a type of solenoid protein domain found in some cytoplasmic proteins.

[0117] The OTS may also be a peroxisome translocation signal (PTS) that can be located at the N-terminus or C-terminus of the target protein. The N-terminal peroxisome translocation signal may have a consensus sequence of Arg-Leu-XXXXX-His / Gln-Leu, where X can be any amino acid. The C-terminal peroxisome translocation signal may contain a short Ser-Lys-Leu, but other variations exist, such as PTS2 and mPTS (membrane PTS). The mPTS may consist of a cluster of basic amino acids (arginine and lysine) in the protein loop (i.e., between the transmembrane segments).

[0118] The OTS may also be a sequence containing a lysosome-transition motif, such as Tyr-X-XO, where X is any amino acid and O is a bulky hydrophobic amino acid or a dileucine motif (Leu-Leu).

[0119] In the fusion proteins of this disclosure, the OTS may be a natural sequence; that is, the OTS may be naturally occurring in the target protein that is also present in the fusion protein, or it may be naturally occurring co-occurring. For example, the OTS and the target protein may be parts of the same naturally occurring polypeptide sequence. In one specific example, the fusion protein of this disclosure may contain mature hFXN and the MTS sequence MWTLTRGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRK (SEQ ID NO: 10), which are both parts of the same naturally occurring polypeptide sequence corresponding to full-length hFXN (SEQ ID NO: 1). After being synthesized in a cell, when SEQ ID NO: 1, a naturally occurring polypeptide sequence, is imported into the mitochondrial matrix, this polypeptide sequence is processed by a two-step cleavage by mitochondrial matrix processing peptidase (MPP). As a result, mature hFXN (SEQ ID NO: 2) without MTS is produced.

[0120] In other embodiments, the OTS contained in the fusion protein of this disclosure may be heterologous sequences, i.e., they do not naturally coexist with the target protein also contained in the fusion protein. Heterologous sequences of OTS may originate from a protein different from the target protein contained in the fusion protein. For example, the fusion protein of this disclosure may include FXN, e.g., mature hFXN, as the target protein and include MTS derived from citrate synthase or lipoamide dehydrogenase (LAD) as described in U.S. Patent No. 8,912,147, the entire content of which is incorporated herein by reference. Heterologous OTS may be generated by cleavage from another protein, or fragments obtained by recombination techniques, or they may be chemically synthesized and further fused to the target protein.

[0121] In some embodiments, the OTS may contain an amino acid sequence having at least 85%, for example, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a naturally occurring OTS domain of a native protein or a heterologous protein.

[0122] In some embodiments, the fusion protein of the Disclosure comprises an MTS, e.g., MWTLTLGRRAVAGLLASPSPAQAQTLTRVPRPAELAPLCGRRGLRTDIDATCTPRRASSNQRGLNQIWNVKKQSVYLMNLRK (SEQ ID NO: 10). In other embodiments, the fusion protein of the Disclosure comprises an amino acid sequence having at least 85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the fusion protein may comprise a functional analogue, derivative, or fragment of SEQ ID NO: 10 as defined above.

[0123] In some embodiments, the OTS may be naturally coexisting with the target protein, for example, the target protein in its naturally occurring form may contain the OTS as part of its sequence. An example of such a target protein is full-length hFXN (SEQ ID NO: 1), which contains MTS (SEQ ID NO: 10) and mature hFXN (SEQ ID NO: 2) as parts of its sequence. If the target protein containing the OTS as part of its sequence is included in the fusion protein of this disclosure, the fusion protein does not contain any other OTS besides the OTS present as part of the target protein.

[0124] In other embodiments, the OTS naturally coexisting with the target protein may be replaced with an OTS not naturally coexisting with the target protein. For example, the MTS that is part of a full-length hFXN and naturally coexists with hFXN (SEQ ID NO: 10) may be replaced with a different OTS, such as an MTS derived from citrate synthase or lipoamide dehydrogenase (LAD) as described in U.S. Patent No. 8,912,147. Thus, the fusion protein of this disclosure may contain a mature hFXN (SEQ ID NO: 2) and an MTS different from that of SEQ ID NO: 10, such as an MTS derived from LAD.

[0125] In some embodiments, OTS is not present in the fusion protein of the Disclosure. For example, the fusion protein of the Disclosure may comprise the target protein, a CPP, e.g., HIV-TAT, and a TES, and lack OTS. In some embodiments, the fusion protein of the Disclosure consists of the target protein, a CPP, e.g., HIV-TAT, and a TES.

[0126] Cell-permeable peptides (CPPs) The fusion proteins provided in this disclosure also include cell-permeable peptides (CPPs). Cell-permeable peptides (CPPs) are short peptide sequences, typically between 5 and 30 amino acids in length, that can facilitate cellular uptake of various molecular cargoes, such as proteins. CPPs may have an amino acid composition containing polycations, i.e., positively charged amino acids, such as lysine or arginine, in high relative abundances. CPPs may also have an amphiphilic sequence, i.e., a sequence containing alternating patterns of polar / charged amino acids and nonpolar, hydrophobic amino acids. CPPs may also be hydrophobic, i.e., containing only nonpolar residues with a small net charge, or having hydrophobic amino acid groups that are critically important for intracellular uptake.

[0127] A useful CPP in the context of this disclosure may be any CPP known to those skilled in the art. For example, a CPP included in the fusion protein of this disclosure may be any CPP listed in the cell permeable peptide database CPPsite2.0, the entire contents of which are incorporated herein by reference. For example, a useful CPP in the context of this disclosure may be selected from the group consisting of HIV-TAT (sometimes referred to herein as "TAT"), galanine, mastoparan, transportan, penetratin, polyarginine, or VP22. In one particular embodiment, the CPP is HIV-TAT. Table 4 below lists the amino acid sequences of exemplary CPPs.

[0128] [Table 4] TIFF2026108757000008.tif67170

[0129] In some examples, the fusion proteins of this disclosure may contain an amino acid sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of the amino acid sequences listed in Table 4, i.e., SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 83.

[0130] In one embodiment, the CPP contained in the fusion protein of the Disclosure is HIV-TAT, for example, SEQ ID NO: 11. In another specific example, the CPP contained in the fusion protein of the Disclosure is HIV-TAT (having an N-terminal methionine), for example, SEQ ID NO: 83. In yet another example, the fusion protein of the Disclosure contains a full-length FXN, for example, SEQ ID NO: 1, and contains HIV-TAT as the CPP. In yet another example, the fusion protein of the Disclosure contains a mature FXN, for example, SEQ ID NO: 2, as the protein of interest, and contains HIV-TAT as the CPP.

[0131] In some embodiments, while we do not wish to be bound by any particular theory, the CPPs contained in the fusion proteins of the Disclosure may be capable of interfering with the delivery of the target protein, also contained in the fusion protein, to organelles, such as non-nuclear organelles. For example, as shown in Example 1 of the Disclosure, the TAT-GG-hFXN fusion protein is localized primarily in the cytosol and nucleus, rather than within the mitochondria.

[0132] Target-enhancing sequences (TES) The fusion proteins of this disclosure also include target-enhancing sequences (TES). As used herein, the term “target-enhancing sequence” (TES) refers to an amino acid sequence that, in the presence of a fusion protein containing a CPP, prevents or attenuates interference by the CPP to the delivery of the target protein to an appropriate intracellular location. For example, in some embodiments, a TES refers to an amino acid sequence that, as used herein, promotes the effective delivery of the target protein to an intended organelle (e.g., a non-nuclear organelle, e.g., mitochondria) by eliminating or attenuating interference caused by the CPP to the delivery of the target protein to the intended organelle. Exemplary TES may include protease-sensitive sequences, also referred herein as “protease-cleavable domains,” “protease-sensitive domains,” “protease-sensitive proteins,” or “protease-sensitive peptides.” Exemplary TES may also include nuclear export signal peptides.

[0133] Protease-sensitive peptides are sometimes also referred to as "protease cleavage sites." A protease-sensitive peptide or protease cleavage site refers to a specific amino acid motif within an amino acid sequence that is recognized and cleaved by a particular intracellular cytosolic protease.

[0134] As described above, in some embodiments of the fusion proteins of this disclosure, the CPP may be able to interfere with the delivery of the target protein contained in the fusion protein to a non-nuclear organelle. The TES, which includes a protease cleavage site, facilitates the removal of the CPP from the fusion protein, thereby preventing interference by the CPP. For example, as shown in Figure 1, Panel A, in some exemplary fusion proteins of this disclosure, the CPP may be located at the N-terminus of the fusion protein, and the TES may be fused to the C-terminus of the CPP. When the fusion protein is exposed to an endogenous protease, the endogenous protease may cleave the TES at the protease cleavage site, thereby facilitating the removal of the CPP from the N-terminus of the fusion protein. Similarly, as shown in Figure 1, Panel B, in other exemplary fusion proteins of this disclosure, the CPP may be located at the C-terminus of the fusion protein, and the TES may be fused to the N-terminus of the CPP. When the fusion protein is exposed to an endogenous protease, the endogenous protease may cleave the TES at the protease cleavage site, thereby potentially removing CPP from the C-terminus of the fusion protein.

[0135] Non-limiting examples of protease-sensitive peptides or proteins that may be included in TES include ubiquitin-like modifiers, e.g., ubiquitin, caspase cleavage domains, calpain cleavage domains, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, and NEDD8.

[0136] Ubiquitin is highly conserved in eukaryotes, and the sequence of the human ortholog is MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Sequence ID 18).

[0137] Exemplary caspase cleavage domains may include DEVD (SEQ ID NO: 19).

[0138] Non-restrictive examples of the calpain cleavage domain include EPLFAERK (SEQ ID NO: 20) or LLVY (SEQ ID NO: 21).

[0139] Other exemplary protease cleavage sites may include any cleavage sites described in Waugh, Protein Expr. Purif. 2011, 80(2):283-293, the entire contents of which are incorporated herein by reference.

[0140] Exemplary proteases having specific protease cleavage sites include ubiquitinase, caspase, calpain, enterokinase (light chain), enteropeptidase, prescision protease, human rhinovirus protease (HRV 3C), TEV (tobacco etch virus) protease, TVMV (tobacco spot virus) protease, factor Xa protease, thrombin, and other proteases known to those skilled in the art. Table 5 below contains some examples of amino acid sequences of protease cleavage sites and their respective proteases.

[0141] [Table 5] TIFF2026108757000009.tif90170

[0142] SUMO (small ubiquitin-like modifier) ​​proteins are a family of small proteins (approximately 100 amino acids in length and 12 kDa in molecular weight) that modify the functions of other proteins in cells by covalently linking to them or detaching from them. The exact length and molecular weight vary among SUMO family members and depend on the organism from which the protein originates. Examples of SUMO proteins are SUMO1, SUMO2, SUMO3, and SUMO4, which have the amino acid sequences listed in Table 6 below.

[0143] [Table 6] TIFF2026108757000010.tif78170

[0144] In some embodiments, the TES contained in the fusion protein of the present disclosure may include a protease-sensitive peptide or protein. In some examples, the TES may include the sequences described herein, for example, one of the SEQ ID NOs listed in Table 5 (SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 84), or a sequence having at least 85%, for example, at least 90% or at least 95%, sequence identity with one of the SEQ ID NOs listed in Table 5 (SEQ ID NOs: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 84). In some examples, the fusion proteins of this disclosure may include a functional analogue, derivative, or fragment of the target protein of any one of the sequence numbers listed in Table 5 (Sequence Number 22, Sequence Number 23, Sequence Number 24, Sequence Number 25, Sequence Number 26, Sequence Number 27, Sequence Number 28, Sequence Number 29, Sequence Number 30, Sequence Number 31, or Sequence Number 84).

[0145] In other embodiments, the TES contained in the fusion protein of this disclosure may contain a ubiquitin-like modifier. In some examples, the TES may contain a sequence described herein, for example, one of the SEQ ID NOs listed in Table 6 (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35), or a sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with one of the SEQ ID NOs listed in Table 6 (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35). In some examples, the fusion protein of this disclosure may contain a functional analogue, derivative, or fragment of the target protein of one of the SEQ ID NOs listed in Table 6 (SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35).

[0146] Amino acid sequences having at least 85% sequence identity with any of sequence numbers 22-31 or 32-35 can be found in one or more of the following databases: GenBank, Protein Data Bank (PDB), SwissProt, Protein Information Resource (PIR), or Protein Research Foundation (PRF).

[0147] In some cases, the TES contained in the fusion protein of this disclosure may contain a nuclear export signal (NES). When present in a protein, the NES targets the protein to translocate from the cell nucleus to the cytoplasm. Nuclear export of proteins occurs via nuclear transport, through the nuclear pore complex. Therefore, in the context of this disclosure, when the CPP present in the fusion protein promotes the delivery of the target protein to the nucleus, the TES counteracts the effect of the CPP by promoting the nuclear export of the target protein to the cytoplasm. In this way, the TES increases the likelihood that the target protein will be delivered to a target non-nuclear organelle, namely the mitochondria.

[0148] In some cases, NES can be a peptide containing four hydrophobic residues that do not necessarily have to be in series. For example, an NES may be a peptide containing the amino acid sequence LXXXLXXLXL, where "L" is a hydrophobic residue (often leucine) and "X" is any amino acid other than leucine. While we do not wish to be bound by any particular theory, it is thought that the spacing of hydrophobic residues in NES can be explained by examining known structures containing NES, since critically important residues are usually on the same plane of adjacent secondary structures in a protein, thereby enabling them to interact with exportins.

[0149] Any NES known to those skilled in the art, including those enumerated in NESbase version 1.0, a database of nuclear export signals whose entire contents are incorporated herein by reference, are included in this disclosure. Table 7 lists specific exemplary NES and their corresponding sequences.

[0150] [Table 7] TIFF2026108757000011.tif60160

[0151] In some embodiments of this disclosure, the TES may include any of the sequences described herein, for example, any one of the sequence numbers listed in Table 7 (sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, or sequence number 43), or may be a sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of the sequence numbers listed in Table 7 (sequence number 36, sequence number 37, sequence number 38, sequence number 39, sequence number 40, sequence number 41, sequence number 42, or sequence number 43). In some examples, the fusion proteins of this disclosure may include a functional analogue, derivative, or fragment of the target protein of any one of the SEQ ID NOs listed in Table 7 (SEQ ID NOs: 36, 37, 38, 39, 40, 41, 42, or 43).

[0152] Amino acid sequences with at least 85% sequence identity to any of sequence numbers 36-43 may be found in one or more of the following databases: GenBank, Protein Data Bank (PDB), SwissProt, Protein Information Resource (PIR), or Protein Research Foundation (PRF).

[0153] Fusion protein In one embodiment of the present disclosure, the fusion protein provided herein is a fusion protein comprising a target protein to be delivered to a non-nuclear organelle, an organelle transfer sequence (OTS), a cell-permeable peptide (CPP), and a target-enhancing sequence (TES), wherein the CPP may interfere with the delivery of the target protein to the non-nuclear organelle, and the TES may prevent such interference by the CPP. In some examples, the target protein, OTS, CPP, and TES may fuse directly with each other to form a single polypeptide chain. In other examples, the target protein, OTS, CPP, and TES may fuse with each other via spacers to form a single polypeptide chain.

[0154] In a particular embodiment, the target protein is FXN, for example, SEQ ID NO: 2. In a particular embodiment, OTS is MTS, for example, SEQ ID NO: 10. In a particular embodiment, CPP is HIV-TAT, for example, SEQ ID NO: 11. In a particular embodiment, TES may be a protease-sensitive peptide or protein, for example, SUMO1 (for example, SEQ ID NO: 32), ubiquitin (for example, SEQ ID NO: 18), a caspase cleavage site, for example, DEVD (for example, SEQ ID NO: 19), or a calpain cleavage site, for example, EPLFAERK (for example, SEQ ID NO: 20) or LLVY (for example, SEQ ID NO: 21). In a particular embodiment, TES may be an NES, for example, NES1 (for example, SEQ ID NO: 36) or NES2 (for example, SEQ ID NO: 37).

[0155] In one embodiment of this disclosure, the fusion protein provided herein may comprise a target protein to be delivered to a cell, a cell-permeable peptide (CPP), and a target-enhancing sequence (TES). In some embodiments, the CPP may interfere with the delivery of the target protein to the cell and / or cellular organelle, and the TES prevents such interference by the CPP. In some examples, the target protein, CPP, and TES may fuse directly with each other to form a single polypeptide chain. In other examples, the target protein, CPP, and TES may fuse with each other via spacers to form a single polypeptide chain.

[0156] In a particular embodiment, the target protein is PARKIN, for example, SEQ ID NO: 7. In a particular embodiment, CPP is HIV-TAT, for example, SEQ ID NO: 11. In a particular embodiment, TES may be a protease-sensitive peptide or protein, for example, SUMO1 (for example, SEQ ID NO: 32), ubiquitin (for example, SEQ ID NO: 18), a caspase cleavage site, for example, DEVD (SEQ ID NO: 19), or a calpain cleavage site, for example, EPLFAERK (SEQ ID NO: 20) or LLVY (SEQ ID NO: 21). In a particular embodiment, TES may be an NES, for example, NES1 (SEQ ID NO: 36) or NES2 (SEQ ID NO: 37).

[0157] In the fusion proteins provided by this disclosure, the relative positions of the target protein, OTS (if present), CPP, and TES to each other and to the N-terminus and C-terminus can vary. Exemplary fusion proteins, including various relative arrangements of the target protein, OTS, CPP, and TES, are shown in Figure 2. For example, as shown in Figure 2, panel A, the exemplary fusion protein may start from the N-terminus, containing the CPP, followed by the TES, followed by the OTS, and then the target protein at the C-terminus. In another example shown in Figure 2, panel B, the fusion protein may start from the N-terminus, containing the CPP, followed by the TES, followed by the target protein, and then the OTS at the C-terminus. In yet another example shown in Figure 2, panel C, the exemplary fusion protein may start from the N-terminus, containing the OTS, followed by the target protein, followed by the TES, and then the CPP at the C-terminus. In another example shown in panel D of Figure 2, the fusion protein may start at the N-terminus, contain the target protein, followed by the OTS, followed by the TES, and then the C-terminus followed by the CPP. In yet another example shown in panel E of Figure 2, the fusion protein may start at the N-terminus, contain the CPP, followed by the OTS, followed by the target protein, and then the TES. In yet another example shown in panel F of Figure 2, the fusion protein may start at the N-terminus, contain the TES, followed by the OTS, followed by the target protein, and then the CPP. In yet another example shown in panel G of Figure 2, the fusion protein may start at the N-terminus, contain the CPP, followed by the TES, and then the target protein. In yet another example shown in panel H of Figure 2, the fusion protein may start at the N-terminus, contain the target protein, followed by the TES, and then the CPP.

[0158] In some examples, in the fusion proteins of this disclosure, various domains (i.e., the protein of interest, CPP, OTS, and TES, if present) may fuse directly with each other to form a single polypeptide chain. As used herein, the term “directly” means that there are no interfering (intervening) amino acids between the C-terminal amino acid of the first domain and the N-terminal amino acid of the second domain that are directly fused with each other. That is, the (first or last) terminal (N or C-terminal) amino acid of the first domain may fuse with the (first or last) terminal (N or C-terminal) amino acid of the second domain to form a single polypeptide. In other words, in this embodiment, the last C-terminal amino acid of the first domain is directly covalently linked to the first N-terminal amino acid of the second domain, or the first N-terminal amino acid of the first domain is directly covalently linked to the last C-terminal amino acid of the second domain to form a single polypeptide.

[0159] In other examples, in the fusion proteins of this disclosure, various domains (i.e., the protein of interest, CPP, OTS, and TES, if present) may fuse together via spacers to form a single polypeptide chain. As used herein, the term “spacer” may be used interchangeably with the term “linker” and refers to a sequence of at least one amino acid that links two domains together to form a single polypeptide chain. When present in a fusion protein, spacers can prevent steric hindrance. In some examples, the length of a spacer or linker may vary from 2 to 31 amino acids, for example, from 2 to 10 amino acids, from 5 to 15 amino acids, from 10 to 25 amino acids, or from 12 to 31 amino acids. Those skilled in the art will be able to determine an appropriate linker or spacer length for each particular fusion protein such that the linker or spacer does not impose any constraints on the conformation of the fusion protein or on the interactions between the domains of the fusion protein. The linker or spacer may be an endogenous and naturally occurring linker or spacer as previously described, which separates domains within the protein or plays a role in dimerization. Alternatively, the linker or spacer may be an artificially designed linker or spacer useful in recombinant techniques for the generation of fusion proteins. Examples of linkers or spacers useful in the context of this disclosure are shown in Table 8 below.

[0160] [Table 8] TIFF2026108757000012.tif84170

[0161] In some embodiments, the fusion protein of the Disclosure may include a GG (Gly-Gly) linker. For example, the fusion protein of the Disclosure may include a GG linker that connects the CPP domain to a TES domain, such as a protease cleavage site. In another example, the fusion protein of the Disclosure may include a GG linker that connects the CPP domain to an OTS domain, such as MTS. In a particular embodiment, TAT-CPP is connected to MTS-FXN via a GG linker.

[0162] The exemplary fusion proteins of this disclosure and their corresponding sequences are listed in Table 9 below.

[0163] [Table 9] TIFF2026108757000013.tif154170TIFF2026108757000014.tif245168TIFF2026108757000015.tif103168

[0164] In some embodiments, the fusion protein provided in this disclosure includes or consists of any one of the sequences listed in Table 9, namely, SEQ ID NOs. 55-61, 69-71, or 81 (SEQ ID NOs. 55, 56, 57, 58, 59, 60, 61, 69, 70, 71, or 81). In some embodiments, the fusion protein provided by this disclosure contains or comprises an amino acid sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of the sequences listed in Table 9, for example, SEQ ID NOs. 55-61, 69-71, or 81 (SEQ ID NOs. 55, SEQ ID NOs. 56, SEQ ID NOs. 57, SEQ ID NOs. 58, SEQ ID NOs. 59, SEQ ID NOs. 60, SEQ ID NOs. 61, SEQ ID NOs. 69, SEQ ID NOs. 70, SEQ ID NOs. 71, or SEQ ID NOs. In some embodiments, the fusion protein of this disclosure may include or consist of any one of the sequences listed in Table 9, namely, a functional analogue, derivative, or fragment of any of SEQ ID NOs. 55-61 (SEQ ID NOs. 55, 56, 57, 58, 59, 60, 61, 69, 70, 71, or 81).

[0165] In one embodiment, the fusion protein contains or comprises TAT-GG-SUMO1-hFXN having the amino acid sequence described in SEQ ID NO: 55. In one embodiment, the fusion protein contains or comprises TAT-SS_SUMO-hFXN having the amino acid sequence described in SEQ ID NO: 81. In one embodiment, the fusion protein contains or comprises TAT-GG-ubiquitin-hFXN having the amino acid sequence described in SEQ ID NO: 56. In one embodiment, the fusion protein contains or comprises TAT-GG-DEVD-hFXN having the amino acid sequence described in SEQ ID NO: 57. In one embodiment, the fusion protein contains or comprises TAT-GG-EPLFAERK-hFXN having the amino acid sequence described in SEQ ID NO: 58. In one embodiment, the fusion protein contains or comprises TAT-GG-LLVY-hFXN having the amino acid sequence described in SEQ ID NO: 59. In one embodiment, the fusion protein contains or comprises TAT-GG-hFXN-NES1 having the amino acid sequence described in SEQ ID NO: 60. In one embodiment, the fusion protein comprises or consists of TAT-GG-hFXN-NES2 having the amino acid sequence described in SEQ ID NO: 61. In one embodiment, the fusion protein comprises or consists of TAT-GG-ubiquitin-PARKIN having the amino acid sequence described in SEQ ID NO: 69. In one embodiment, the fusion protein comprises or consists of TAT-GG-EPLFAERK-PARKIN having the amino acid sequence described in SEQ ID NO: 70. In one embodiment, the fusion protein comprises or consists of TAT-PARKIN-NES1 having the amino acid sequence described in SEQ ID NO: 71.

[0166] In some embodiments, the fusion protein provided in this disclosure comprises: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the full-length hFXN (SEQ ID NO: 1) of the protein in question; 2) any CPP as defined herein; and 3) any TES as defined herein.

[0167] In some embodiments, the fusion protein provided in this disclosure comprises 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the full-length hFXN (SEQ ID NO: 1), 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83), and 3) any TES as described herein.

[0168] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein, full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with ubiquitin (SEQ ID NO: 18).

[0169] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein, full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the calpain cleavage domain of SEQ ID NO: 20.

[0170] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0171] In some embodiments, the fusion protein provided in this disclosure comprises 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (SEQ ID NO: 7), 2) any CPP as described herein, and 3) any TES as described herein.

[0172] In some embodiments, the fusion protein provided herein comprises 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (also referred to herein as "PARK2 protein," SEQ ID NO: 7), 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11), and 3) any TES as described herein.

[0173] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with ubiquitin (SEQ ID NO: 18).

[0174] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the calpain cleavage domain of SEQ ID NO: 20.

[0175] For example, the fusion protein of this disclosure may include: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% with TAT-HIV (SEQ ID NO: 11 or SEQ ID NO: 83); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% with any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0176] In some embodiments, one or more of the fusion proteins provided in this disclosure may also include post-translational modifications characteristic of eukaryotic cells, such as mammalian cells, such as human cells. In some embodiments, the fusion protein may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) post-translational modifications, such as glycosylation, phosphorylation, acetylation, or a combination thereof. In some embodiments, glycosylation may include the addition of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan. Glycosylation may include, for example, O-linked glycosylation or N-linked glycosylation. The level of glycosylation of the fusion proteins of this disclosure may be evaluated in vitro using SDS-PAGE gel and Western blotting, and a modified periodate Schiff (PAS) method. The cellular localization of fusion proteins that may involve glycosylation can be achieved using lectin fluorescent conjugates known in the art. Phosphorylation that may be present in the fusion proteins of this disclosure may be evaluated by Western blotting using phosphorylation-specific antibodies.

[0177] Post-translational modifications that may be present in the fusion proteins of this disclosure include binding to hydrophobic groups (e.g., myristoylation, palmitoylation, isoprenylation, prenylation, or GPIation), binding to cofactors (e.g., lipoylation, flavin moiety (e.g., FMN or FAD), heme C binding, phosphopantetheinylation, or retinilidensch base formation), diphthamide formation, ethanolamine phosphoglycerol binding, hypsin formation, acylation (e.g., O-acylation, N-acylation, or S-acylation), formylation, acetylation, alkylation (e.g., methylation or ethylation), amidation, butyrylation, gamma-carboxylation, and malo This may also include nylation, hydroxylation, iodization, nucleotide addition, e.g., ADP-ribosylation, oxidation, phosphate ester (O-linked) or phosphoramidate (N-linked) formation (e.g., phosphorylation or adenylation), propionylation, pyroglutamate formation, S-glutathioneation, S-nitrosylation, succinylation, sulfation, ISG formation, SUMO formation, ubiquitination, NEDD formation, or chemical modification of amino acids (e.g., citrullination, deamide, eliminylation, or carbamylation), disulfide crosslinking, and racemization (e.g., racemization of proline, serine, alanine, or methionine).

[0178] nucleic acid This disclosure also provides nucleic acids encoding the aforementioned fusion protein or any variant thereof.

[0179] As used herein, the term “nucleic acid” is interchangeable with the term “polynucleotide” and generally refers to any polyribonucleotide or polydeoxyribonucleotide that may be unmodified RNA or DNA, modified RNA or DNA, or any combination thereof. The term “nucleic acid” includes, non-limitingly, single-stranded nucleic acids and double-stranded nucleic acids.

[0180] This disclosure provides fusion proteins or variants thereof as described herein. The variants provided herein may include conservatively substituted variants applicable to both amino acid sequences and nucleic acid sequences. With respect to nucleic acid sequences, a conservatively modified variant refers to a nucleic acid that codes for the same or essentially the same amino acid sequence. Specifically, degenerate codon substitution can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Therefore, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described herein without changing the coded polypeptide. Such nucleic acid variations are silent variations, which are a type of conservatively modified variation. Any nucleic acid sequence described herein that codes for a polypeptide also describes all possible silent variations of the nucleic acid. Those skilled in the art will recognize that by modifying each codon in a nucleic acid (with the exception of AUG, usually the sole codon of methionine, and TGG, usually the sole codon of tryptophan), it is possible to encode functionally identical polypeptides. Therefore, each silent variation of the nucleic acid encoding a polypeptide is latent in each described sequence.

[0181] With regard to conserved substitutions of amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences that modify, add, or remove a single amino acid or a small proportion of amino acids in the encoded sequence are conserved variants if the modification results in a substitution of one amino acid with a chemically similar amino acid. Conservative substitution tables showing functionally similar amino acids are well known in the art. Such conserved variants are additions to, and not exclusions of, the polymorphic variants, interspecific homologs, and alleles of this disclosure.

[0182] The following groups each contain amino acids that are conserved substitutions with each other. 1) Alanine (A), Glycine (G), 2) Serine (S), threonine (T), 3) Aspartic acid (D), glutamic acid (E), 4) Asparagine (N), glutamine (Q), 5) Cysteine ​​(C), Methionine (M), 6) Arginine (R), Lysine (K), Histidine (H), 7) Isoleucine (I), leucine (L), valine (V), 8) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0183] Table 10 below lists specific exemplary nucleic acids that encode the fusion proteins of this disclosure.

[0184] [Table 10] TIFF2026108757000016.tif254170TIFF2026108757000017.tif246166TIFF20261087570 00018.tif249166TIFF2026108757000019.tif249166TIFF2026108757000020.tif177166

[0185] In some embodiments, the nucleic acid sequence encoding the fusion protein of the Disclosure includes one of the sequences listed in Table 10, i.e., any of SEQ ID NOs. 62-68 (SEQ ID NOs. 62, 63, 64, 65, 66, 67, 68, 86, 87, or 88). In some embodiments, the nucleic acid sequence encoding the fusion protein provided by the Disclosure includes a sequence having at least 85%, for example, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any of SEQ ID NOs.

[0186] In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-SUMO1-hFXN has the sequence described in SEQ ID NO: 62. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-ubiquitin-hFXN has the sequence described in SEQ ID NO: 63. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-DEVD-hFXN has the sequence described in SEQ ID NO: 64. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-EPLFAERK-hFXN has the sequence described in SEQ ID NO: 65. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-LLVY-hFXN has the sequence described in SEQ ID NO: 66. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-hFXN-NES1 has the sequence described in SEQ ID NO: 67. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-hFXN-NES2 has the sequence described in SEQ ID NO: 68. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-ubiquitin-PARKIN has the sequence described in SEQ ID NO: 86. In one embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-EPLFAERK-PARKIN has the sequence described in SEQ ID NO: 87. In another embodiment, the nucleic acid sequence encoding the fusion protein TAT-GG-PARKIN-NES1 has the sequence described in SEQ ID NO: 88.

[0187] This disclosure also provides expression vectors for generating the fusion proteins defined herein in cells, such as mammalian cells, bacterial cells, or fungal cells. The expression vectors of this disclosure may contain one of the nucleic acids encoding the fusion proteins of this disclosure, for example, SEQ ID NOs. 62-68. For example, the expression vectors may be retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, phagemids, baculoviruses, or any combination thereof.

[0188] vector A vector can enable the integration of a DNA fragment or nucleic acid sequence into a host genome, or enable the expression of an unintegrated genetic element. A vector is typically a self-replicating DNA or RNA construct containing a desired nucleic acid sequence and ligably linked to a gene regulatory element that is recognized in a suitable host cell and results in the translation of a desired spacer. Generally, the gene regulatory element may include a prokaryotic promoter system or a eukaryotic promoter expression regulatory system. Such systems typically include transcription promoters and transcription enhancers for increasing the level of RNA expression. A vector usually contains a replication origin that allows the vector to replicate independently of the host cell. Therefore, the terms regulatory and regulatory elements include promoters, terminators, and other expression regulatory elements. Such regulatory elements have been described in the art and are known to those skilled in the art. For example, any of the broad range of expression regulatory sequences that control the expression of a DNA sequence when ligably linked to it can be used in these vectors to express a DNA sequence encoding any desired fusion protein described herein.

[0189] The vector may further include appropriate restriction sites, antibiotic resistance, or other markers for selecting vector-containing cells. Plasmids are the most commonly used vector form, but other forms of vectors known or to be known in the art that perform equivalent functions are also suitable for use here.

[0190] A vector may also be referred to as a construct or recombinant nucleic acid. Where used herein, the terms “recombinant DNA,” “recombinant nucleic acid sequence,” or “recombinant gene” refer to a nucleic acid comprising an open reading frame (ORF) encoding a fusion protein as described herein. A construct comprising an ORF encoding a fusion protein as described herein may optionally further include additional elements, such as promoters, regulatory and control elements, translation, expression, and other signals, that are operablely ligated to the nucleic acid sequence as described herein. As used herein, “operable ligation” means that the elements are ligated in such a way that the transgene can be transcribed. As used herein, “encodes” means that the nucleic acid of interest can be transcribed in a suitable expression system and translated into a desired polypeptide or protein of interest, for example, that the nucleic acid of interest is ligated to a suitable regulatory sequence, such as a promoter and enhancer element, in a suitable vector (e.g., an expression vector) and that the vector is introduced into a suitable system or cell.

[0191] According to some embodiments, the recombinant DNA encoding the fusion protein of the Disclosure may be cloned into a vector, such as a lentiviral vector plasmid that can be incorporated into cells. According to some embodiments, the recombinant DNA encoding one or more fusion proteins may be cloned into a plasmid DNA construct encoding a selectable trait, such as an antibiotic resistance gene. According to some embodiments, the recombinant DNA encoding the fusion protein may be cloned into a plasmid construct adapted to stably express the fusion protein in cells.

[0192] Conjugate This disclosure also provides conjugates for intracellular delivery of target proteins to non-nuclear organelles. The conjugates of this disclosure may comprise at least one of the fusion proteins as defined in this disclosure and labels, such as radiolabels, fluorescent labels, chromophore labels, enzyme labels, peptide labels, or protein labels, small molecules, or polymers (e.g., polyethylene glycol, PEG).

[0193] cell This disclosure also provides cells containing any one of the fusion proteins, nucleic acids, vectors, conjugates, or any combination thereof as described herein. In some embodiments, the cells may be stem cells or iPS cells. In other embodiments, the cells may be selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cells, or yeast cells. For example, the cells may be any one of muscle or muscle progenitor cells, fibroblasts, epidermal cells, cardiac cells, stem cells, embryonic stem cells, pluripotent cells, neurons, bone marrow stem cells, and also include yeast cell lines or bacterial cell lines.

[0194] In certain embodiments, cells comprising a fusion protein, nucleic acid, vector, conjugate, or any combination thereof include long-term hematopoietic stem cells, short-term hematopoietic stem cells, pluripotent progenitor cells, lineage-restricted progenitor cells, lymphocyte progenitor cells, myeloid progenitor cells, myeloid common progenitor cells, erythrocyte progenitor cells, megakaryocyte-erythroblast progenitor cells, retinal cells, photoreceptor cells, rod cells, cone cells, retinal pigment epithelial cells, trabecular reticular cells, inner ear hair cells, outer hair cells, inner hair cells, lung epithelial cells, bronchial epithelial cells, alveolar epithelial cells, lung epithelial progenitor cells, rhabdomyomyocytes, cardiomyocytes, muscle satellite cells, neurons, neuronal stem cells, mesenchymal stem cells, induced pluripotent stem (iPS) cells, embryonic stem cells, fibroblasts, and monocyte-derived cells. The cells may be selected from the group consisting of clophages or dendritic cells, megakaryocytes, neutrophils, eosinophils, basophils, mast cells, cells, B cells, such as progenitor B cells, pre-B cells, pro-B cells, memory B cells, plasma B cells, gastrointestinal epithelial cells, bile duct epithelial cells, pancreatic duct epithelial cells, intestinal stem cells, hepatocytes, hepatic stellate cells, Kupffer cells, osteoblasts, osteoclasts, adipocytes, pre-adipocytes, islet cells (such as beta cells, alpha cells, delta cells), pancreatic exocrine cells, Schwann cells, and oligodendrocytes.

[0195] In some embodiments, the cells containing the fusion protein are dividing cells. In other embodiments, the cells containing the fusion protein are non-dividing cells.

[0196] In some embodiments, cells containing the fusion protein of this disclosure may be among subjects, for example, subjects to whom the fusion protein is administered, for example, to treat a pathological condition, for example, a genetic disorder. In some examples, the pathological condition may be a nonnuclear organelle-related disorder. In a particular example, the pathological condition is Friedreich's ataxia.

[0197] In a specific case, any of the fusion proteins of this disclosure, e.g., SEQ ID NOs. 55-61, may be administered to treat Friedreich's ataxia. In other cases, the fusion proteins of this disclosure may be administered to treat mitochondrial disorders, e.g., mitochondrial myopathy, diabetes mellitus, and deafness, Leber's hereditary optic neuropathy, neuropathy, retinitis pigmentosa, ptosis (NARP), myoneurogenic gastrointestinal encephalopathy, myoclonic epilepsy, Alzheimer's disease, muscular dystrophy, Lou Gehrig's disease, and cancer. In further examples, the fusion proteins of this disclosure may be administered to treat lysosomal disorders, such as lipid storage disorders of the sphingolipidosis group (e.g., Niemann-Pick disease, Fabry disease, Krabbe disease, Gaucher disease, Tay-Sachs disease, or metachromatic leukodystrophy), Hunter syndrome, I-cell disease, multiple sulfatase deficiency, mucolipidosis types II and IIIA, galactosialidosis, GM2-AP deficiency, SAP deficiency, and Salah disease. In yet another example, any of the fusion proteins provided in this disclosure, e.g., SEQ ID NOs. 69-71, may be administered to treat Parkinson's disease.

[0198] In other embodiments, cells containing the fusion protein of this disclosure may be cells that have been engineered to express the fusion protein. In some examples, such engineered cells may be obtained from a subject, for example, isolated cells. Methods for engineering cells to express the fusion protein of this disclosure are known to those skilled in the art and include the methods described herein in the section titled “Methods for Intracellular Delivery.”

[0199] Pharmaceutical composition This disclosure also provides pharmaceutical compositions comprising the above-mentioned fusion protein or conjugate and pharmaceutically acceptable diluents, carriers, additives, and / or excipients.

[0200] The preparation of pharmaceutical compositions is discussed, for example, in Hoover, John E. (ed.), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, USA, 18th edition (1990), the full contents of which are incorporated herein by reference, and in Liberman, HA and Lachman, L. (eds.), Pharmaceutical Dosage Forms, Marcel Decker, New York, New York, USA (1989).

[0201] The pharmaceutical composition may be delivered to cells in vitro, for example, by contacting the cells with the pharmaceutical composition. Alternatively, for in vivo delivery, the pharmaceutical composition provided herein may be administered orally, parenterally, by inhalation spray, rectally, intradermally, transdermally, or topically in a dosage unit formulation containing a preferred conventional non-toxic, pharmaceutically acceptable carrier, adjuvant, and medium. Topical administration may also involve transdermal administration, such as the use of a transdermal patch or ion electrophoresis device. As used herein, the term parenteral includes subcutaneous, intravenous, intramuscular, intrasternal injection, or infusion techniques.

[0202] Pharmaceutical compositions for injection, such as sterile aqueous or oily suspensions for injection, can be formulated by techniques known in the art using suitable dispersants or wetting agents and suspending agents. Sterile pharmaceutical compositions for injection may also be sterile solutions or suspensions for injection with non-toxic, parenterally acceptable diluents or solvents, such as 1,3-butanediol. Acceptable media and solvents include water, Ringer's solution, and isotonic salines. In addition, sterile non-volatile oils are conventionally used as solvents or suspension media. Any non-irritating non-volatile oil, including synthetic monoglycerides or diglycerides, can be used for this purpose. Furthermore, fatty acids, such as oleic acid, are useful in the preparation of injections. Surfactants, including dimethylacetamide, ionic and non-ionic surfactants, and polyethylene glycol can be used. Solvents and wetting agents, such as mixtures of those discussed above, are also useful.

[0203] Pharmaceutical compositions for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injectable solutions or suspensions. These solutions and suspensions may be prepared from sterile powders or granules having one or more carriers or diluents as referred to for use in oral formulations. Fusion proteins or compositions can be dissolved in water, polyethylene glycol, propylene glycol, ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium chloride, and / or various buffers. Other adjuvants and methods of administration are widely known in the pharmaceutical art.

[0204] Examples of liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing an inert diluent, such as water, which is commonly used in the art. Such pharmaceutical compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, as well as sweeteners, flavoring agents, and fragrance agents.

[0205] Examples of solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the fusion protein may be mixed with lactose, sucrose, sucrose powder, cellulose esters of alkanates, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfate, gelatin, acacia gum, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, and then compressed into convenient tablets or capsules for administration. Such capsules or tablets may contain controlled-release formulations, such as those in which the active compound is dispersed in hydroxypropyl methylcellulose. In the case of capsules, tablets, and pills, the dosage forms may also contain buffers, such as sodium citrate, magnesium, or calcium carbonate or calcium bicarbonate. Tablets and pills may be further prepared by enteric coating.

[0206] The suppositories for rectal administration of the fusion proteins discussed herein can be prepared by mixing the active agent with a suitable non-irritating excipient, such as cocoa butter, synthetic monoglycerides, diglycerides, or triglycerides, fatty acids, or polyethylene glycol, which are solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum and release the drug.

[0207] The fusion proteins of this disclosure can be administered in a variety of ways, for example, orally, enterally, mucosally, percutaneously, or parenterally. Parenteral administration is particularly preferred by intravenous, intramuscular, subcutaneous, intradermal, intra-articular, subarachnoid, and intraperitoneal injection or injection, including continuous or intermittent infusion using a pump available to those skilled in the art. Alternatively, the fusion proteins may be administered in formulations encapsulated in microcapsules, such as liposome-based capsules.

[0208] Methods for intracellular delivery In some embodiments, the disclosure also provides a method for intracellular delivery of the target protein to a non-nuclear organelle within a cell. This method, as used herein, involves contacting a cell with the fusion protein, nucleic acid, vector, or conjugate described above. In one example, the non-nuclear organelle is a mitochondria. In one example, the target protein is FXN, for example, human FXN.

[0209] Contacting cells with the fusion protein of this disclosure may occur in the context of administering the fusion protein, nucleic acid, vector, conjugate, pharmaceutical composition, or any combination thereof to a subject, for example, a human. The subject may have a pathological condition, such as a genetic disorder, such as a nonnuclear organelle-related disorder. In a particular example, the pathological condition is Friedreich's ataxia.

[0210] In some embodiments, a method for intracellular delivery of the target protein to a non-nuclear organelle may involve contacting cells, such as isolated cells, with a nucleic acid encoding the fusion protein of the Disclosure or a vector containing the nucleic acid of the Disclosure. In some embodiments, the cells are stem cells or iPS cells. In other embodiments, the cells may be selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cells, or yeast cells. In some embodiments, the cells may be muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cell lines, or yeast cell lines having the nucleic acid encoding the fusion protein of the Disclosure. The method may further include additional steps to facilitate intracellular delivery of the nucleic acid or vector of the Disclosure, such as electroporation, chemical or polymer translocation, viral transduction, mechanical membrane disruption, or other methods known to those skilled in the art.

[0211] In some embodiments, the Disclosure also provides a method for increasing the amount of target protein delivered to cells. As demonstrated by specific experimental data included in the Disclosure, for example in Example 6, cells treated with a fusion protein containing the target protein, CPP, and TES contained significantly higher amounts of target protein than cells treated with a fusion protein containing the target protein and CPP but without TES. Thus, the experimental data indicate that TES promotes the delivery and / or accumulation of the target protein within cells. Therefore, introducing TES into a fusion protein containing CPP and the target protein significantly increases the amount of target protein within cells.

[0212] While we do not wish to be bound by any particular theory, it is thought that cleavage of TES by endogenous proteases within the cell promotes the elimination of CPP from the fusion protein, preventing CPP from promoting the target protein's crossing of the plasma membrane and diffusion outside the cell. This allows for the intracellular accumulation of the target protein, resulting in an increase in the amount of target protein delivered to the cell.

[0213] In some embodiments, the cells may be present in the subject, for example, in a human. Therefore, methods for increasing the amount of the target protein delivered to the cells may allow for a reduction in the dose of the fusion protein delivered to the subject as part of a treatment, for example, a treatment to address a non-nuclear organelle-related disorder.

[0214] In some embodiments, the target protein may be FXN. Therefore, in some embodiments, the fusion protein may include CPP (e.g., SEQ ID NO: 11 or SEQ ID NO: 83), TES, and FXN (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), and the subject may have a disorder associated with FXN deficiency, such as Friedreich's ataxia. In other embodiments, the fusion protein may include CPP (e.g., SEQ ID NO: 11 or SEQ ID NO: 83), TES, and FXN (e.g., SEQ ID NO: 1 or SEQ ID NO: 2), and the subject may have a disorder not associated with FXN deficiency, such as French-Canadian type Leigh syndrome.

[0215] In some embodiments, the target protein may be PARKIN. Therefore, in some embodiments, the fusion protein may include CPP (e.g., SEQ ID NO: 11 or SEQ ID NO: 83), TES, and PARKIN (e.g., SEQ ID NO: 7), and the subject may have Parkinson's disease associated with a mutation in the PARK2 gene encoding PARKIN. In other embodiments, the fusion protein may include CPP (e.g., SEQ ID NO: 11 or SEQ ID NO: 83), TES, and PARKIN (e.g., SEQ ID NO: 7), and the subject may have Parkinson's disease not associated with a mutation in the PARK2 gene encoding PARKIN, for example, Parkinson's disease associated with a mutation in the PINK1 (PARK6) gene.

[0216] In some embodiments, the method provided in this disclosure for increasing the amount of target protein delivered to cells includes the steps of: modifying the sequence of a fusion protein containing the target protein and a cell-permeable peptide (CPP) by introducing a target-enhancing sequence (TES) into the fusion protein to generate a modified fusion protein; and contacting cells with the modified fusion protein to increase the amount of target protein delivered to the cells.

[0217] The CPP may be located at the N-terminus of the modified fusion protein, and the TES may be fused to the C-terminus of the CPP. For example, the modified fusion protein may include the CPP, TES, and the target protein, starting from the N-terminus. In another example, the modified fusion protein may include the CPP, TES, OTS, and the target protein, starting from the N-terminus.

[0218] The CPP may also be located at the C-terminus of the modified fusion protein, and the TES may be fused to the N-terminus of the CPP. For example, the modified fusion protein may include, starting from the N-terminus, the target protein, the TES, and the CPP. In another example, the modified fusion protein may include, starting from the N-terminus, the target protein, the OTS, the TES, and the CPP. In yet another example, the modified fusion protein may include, starting from the N-terminus, the OTS, the target protein, the TES, and the CPP.

[0219] In some embodiments, the target protein may be any target protein described herein, for example, any target protein listed in Table 2 herein. For example, the target protein may be selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, and their variants or derivatives. In one example, the target protein is frataxin (FXN) or its variant or derivative. In another example, the target protein is PARK2 protein (PARKIN) or its variant or derivative.

[0220] In some embodiments, CPP may be any CPP described herein, for example, any CPP listed in Table 4 herein. For example, CPP may contain peptides selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0. For example, CPP may contain peptides selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and their variants or derivatives. In one embodiment, CPP may contain HIV-TAT or its variants or derivatives.

[0221] In some embodiments, the TES contained in the modified fusion protein may be any TES described herein. For example, the TES may contain a sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with one of the nuclear export signal peptides, e.g., SEQ ID NOs. 36-43. In some examples, the nuclear export signal peptide may contain a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives. In one example, the nuclear export signal peptide may contain NES1 or its variants or derivatives. In another example, the nuclear export signal peptide may contain NES or its variants or derivatives.

[0222] The TES contained in the modified fusion protein may also be a protease-sensitive peptide. For example, the TES may contain a ubiquitin-like modifier, and may contain a sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any one of SEQ ID NOs. 18-31.

[0223] In other examples, the TES may include a protease-sensitive peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, and SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and their variants or derivatives.

[0224] In some cases, the modified fusion protein may contain an amino acid sequence that has at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with any of SEQ ID NOs. 55-61 and 69-71.

[0225] Treatment method Therapeutic compounds for treating non-nuclear organelle-related disorders in subjects requiring such treatment are provided herein. In some embodiments, the therapeutic compounds may comprise fusion proteins, nucleic acids, vectors, or conjugates of the Disclosure. In some embodiments, non-limiting examples of non-nuclear organelle-related disorders may include mitochondrial disorders and lysosomal disorders. In some embodiments, the non-nuclear organelle-related disorders are hereditary disorders.

[0226] This disclosure also provides a method for treating a nonnuclear organelle-related disorder, comprising administering a fusion protein, nucleic acid, vector, conjugate, cell, or pharmaceutical composition described herein to a subject requiring such treatment. In some embodiments, the nonnuclear organelle-related disorder is a mitochondrial disorder. In a particular example, the mitochondrial disorder is Friedreich's ataxia.

[0227] In some cases, non-nuclear organelle-related disorders may also be lysosomal disorders, i.e., disorders related to lysosomal dysfunction.

[0228] In some embodiments, non-nuclear organelle-related disorders may be selected from the group consisting of Friedreich ataxia (FRDA), Barth syndrome, Parkinson's disease, Leigh syndrome, e.g., French-Canadian Leigh syndrome, and pyruvate dehydrogenase deficiency.

[0229] Subjects who have or are at risk of developing one or more of the disorders disclosed herein may be selected for treatment based on a multifactorial basis, including the presentation of symptoms and / or the identification of markers (e.g., gene mutations) that give the subject a predisposition to the disease or disorder.

[0230] As used herein, the term “subject” includes human or non-human animals, preferably vertebrates, more preferably mammals. Transgenic organisms may also be included as subjects. Most preferably, the subject is a human, for example, a human with or predisposed to developing a non-nuclear organelle-related disorder, such as Friedreich's ataxia or Parkinson's disease.

[0231] As used herein, the term “treat a nonnuclear organelle-related disorder” includes reducing, improving, or eliminating one or more symptoms associated with a nonnuclear organelle-related disorder, such as Friedreich’s ataxia or Parkinson’s disease. The term “treat a nonnuclear organelle-related disorder” also includes partially or substantially achieving one or more of the following: improving or improving symptoms or indicators associated with a nonnuclear organelle-related disorder; halting the progression or worsening of at least one symptom of a nonnuclear organelle-related disorder. The term “treat a nonnuclear organelle-related disorder” also includes preventing the onset of a nonnuclear organelle-related disorder, delaying the onset of at least one symptom thereof, or reducing the onset of at least one symptom of a nonnuclear organelle-related disorder to a lower severity than the same symptom would develop without treatment.

[0232] How to treat Friedreich's ataxia (FRDA) In one embodiment, the Disclosure provides a method for treating Friedreich's ataxia (FRDA), comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating FRDA, comprising administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to a full-length hFXN (SEQ ID NO: 1), which is the protein of interest; 2) any CPP as described herein; and 3) any TES as described herein.

[0233] In some embodiments, a method for treating FRDA includes administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1), 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11), and 3) any TES as described herein.

[0234] In some embodiments, a method for treating FRDA includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to ubiquitin (SEQ ID NO: 18).

[0235] In some embodiments, a method for treating FRDA includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to the calpain cleavage domain of SEQ ID NO: 20.

[0236] In some embodiments, a method for treating FRDA includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0237] In some embodiments, a method for treating FRDA includes administering a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 56, 57, 58, 59, 60, 61, and 81 to a subject requiring it.

[0238] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 56.

[0239] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 57.

[0240] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 58.

[0241] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 59.

[0242] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 60.

[0243] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 61.

[0244] In one embodiment, a method for treating FRDA includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 81.

[0245] As used herein, the term “FRDA” encompasses any disease, disorder, or condition associated with frataxin deficiency. As used herein, the term “disease, disorder, or condition associated with FRDA” encompasses any disease, disorder, or condition that is secondary to and / or caused by FRDA, i.e., if present in the subject, it occurs concurrently with FRDA and is not present in the subject in the absence of FRDA. Non-limiting examples of diseases, disorders, or conditions associated with FRDA include FRDA-associated pneumonia, FRDA-associated hypertrophic cardiomyopathy, and FRDA-associated diabetes. Other non-limiting examples of diseases, disorders, or conditions associated with FRDA include, but are not limited to, (1) Neurological disorders including, but not limited to, one or more of the following: lack of proprioception, loss of reflexes, loss of walking ability, loss of the ability to stare; (2) Dysphagia and / or progressive loss of swallowing ability, progressive hearing loss, (3) Progressive visual acuity loss due to retinal degeneration resulting from FXN deficiency, (4) progressive aphasia; (5) Metabolic syndrome including, but not limited to, elevated triglycerides, decreased high-density lipoprotein (HDL) cholesterol, and elevated low-density lipoprotein (LDL) cholesterol. (6) Scoliosis requiring surgery for correction, and / or combination thereof This includes diseases, disorders, or conditions associated with FRDA characterized by the following:

[0246] In some embodiments, administration of the fusion protein of this disclosure to a subject may treat FRDA, including, for example, a disease, disorder, or condition associated with FRDA. As used herein, “treating FRDA” includes achieving relief, improvement, or reduction of the severity of FRDA, including, for example, a disease, disorder, or condition associated with FRDA. For example, “treating FRDA” includes achieving relief, improvement, or reduction of at least one symptom or indicator associated with FRDA. As used herein, “treating FRDA” also includes delaying the progression of FRDA, including, for example, a disease, disorder, or condition associated with FRDA, delaying the onset of, for example, at least one symptom or indicator associated with FRDA, or preventing an increase in the severity of at least one symptom or indicator associated with FRDA.

[0247] In some embodiments, the term “treating FRDA” also includes achieving an extension of survival (e.g., life expectancy) of subjects with FRDA, including, for example, diseases, disorders, or conditions associated with FRDA, such as humans. For example, treating FRDA may result in an extension of expected life expectancy of subjects with FRDA, including, for example, diseases, disorders, or conditions associated with FRDA, such as humans. In some embodiments, treatment with FRDA in the context of this disclosure may result in an extension of the expected life expectancy of subjects by more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 100%, more than 110%, more than 120%, more than 130%, more than 140%, more than 150%, more than 160%, more than 170%, more than 180%, more than 190%, or more than 200% compared to the average expected life expectancy of one or more untreated control individuals with a similar disease.

[0248] In some embodiments, treatment of FRDA, including, for example, a disease, disorder, or condition associated with FRDA in the context of this disclosure, may extend the expected life expectancy of a subject by more than 6 months, more than 8 months, more than 10 months, more than 12 months, more than 2 years, more than 4 years, more than 6 years, more than 8 years, or more than 10 years compared to the average expected life expectancy of one or more untreated control individuals with a similar disease. In some embodiments, treatment of FRDA, including, for example, a disease, disorder, or condition associated with FRDA in the context of this disclosure, may result in long-term survival of a subject having FRDA, including, for example, a disease, disorder, or condition associated with FRDA, such as a human. As used herein, the term “long-term survival” means a period of survival or expected life expectancy longer than approximately 40 years, 45 years, 50 years, 55 years, 60 years, or more.

[0249] For example, clinical assessments known to those skilled in the art may be used to evaluate FRDA, including diseases, disorders, or conditions associated with FRDA, to determine the severity of FRDA, and / or to determine the effects of administering the fusion protein of this disclosure to a subject. Examples of methods for clinical assessment of FRDA, including assessment of the severity of FRDA, are described, for example, in Paap et al., "Standardized Assessment of Hereditary Ataxia Patients in Clinical Studies", Mov Disord Clin Pract. 2016, 3(3):230-240, and in Patel et al., "Progression of Friedreich ataxia: quantitative characterization over 5 years", Ann Clin Transl Neurol 2016, 3(9):684-694, the entire contents of which are incorporated herein by reference.

[0250] The Timed 25-Foot Walk (T25-FW) is a quantitative performance test of mobility and lower limb function that measures the time required to complete a 25-foot walk. In some embodiments, administration of the fusion protein of the Disclosure to a subject may result in a reduction in the severity of FRDA as measured, for example, by the time required to complete a 25-foot walk. For example, administration of the fusion protein of the Disclosure to a subject may result in a reduction in the time required to complete a 25-foot walk, for example, by at least about 5%, at least about 10%, at least about 25%, or at least about 50%, compared to the time required to complete a 25-foot walk measured in the subject before administration of the fusion protein of the Disclosure, or compared to a baseline value. The baseline value may be the time required to complete a 25-foot walk measured before administration of the fusion protein of the Disclosure.

[0251] In other embodiments, administration of the fusion protein of the Disclosure to a subject may delay the progression of FRDA in the subject, as measured, for example, by the time required to complete a 25-foot walk. For example, administration of the fusion protein of the Disclosure to a subject may cause the time required to complete a 25-foot walk to be substantially the same as, or substantially the same as, the baseline value, i.e., the time required to complete a 25-foot walk measured in the subject before administration of the fusion protein of the Disclosure, or there may be substantially no increase in the time required to complete a 25-foot walk (e.g., an increase of less than 20%, less than 10%, or less than 5%).

[0252] The modified Friedreich Ataxia Rating Scale (mFARS) is a test-based assessment scale for evaluating the severity of FRDA, as described, for example, in Burk et al., "Monitoring progression in Friedreich ataxia (FRDA): the use of clinical scales", J of Neurochemistry 2013, 126(suppl. 1):118-124, and Rummey et al., "Psychometric properties of the Friedreich's Ataxia Rating Scale", Neurol Genet 2019, 5:e371, the full contents of which are incorporated herein by reference.

[0253] In some embodiments, the mFARS score may include at least one of the following subscores: a) a score based on the Functional Impairment Scale (FARS-FDS, 0-6 scale, assessment usually performed by a neurologist), b) a score based on the Activities of Daily Living (FARS-ADL, 0-36 scale, assessment performed by the patient or caregiver), and c) a score based on the Neurological Assessment Scale (FARS-neuro, 0-125 scale, assessment performed by a neurologist). In some examples, the FARS_ADL score is a FARS assessment scale with a score range of 0-36 that evaluates the subject's ability to complete ADLs (e.g., speech, cutting food, dressing, and personal hygiene). Respondents may be the subject, a combination of the subject and family, or, in the case of subjects unable to complete the test, a family member, spouse, or caregiver.

[0254] In some embodiments, scores based on neurological assessment scales may include modified scoring of neurological assessment scales (mFARS-neuro, 0-99 scale) that target specific areas affected by FRDA, such as the medulla oblongata, upper extremities, lower extremities, and standing stability, with the direct participation of the subject. mFARS-neuro is derived from the neurological assessment scale of the FARS questionnaire by removing the first two questions of subscale D (peripheral nervous system) and subscale A (medulla oblongata).

[0255] In some embodiments, the mFARS score may be based on two subscores derived from the complete FARS questionnaire, namely the mFARS-neuro and FARS_ADL described above.

[0256] In some embodiments, administration of the fusion protein of the Disclosure to a subject may result in a reduction in the severity of FRDA as measured, for example, by the mFARS score or at least one of the mFARS subscores described herein. For example, administration of the fusion protein of the Disclosure to a subject may result in a reduction of the mFARS score or at least one mFARS subscore compared to a baseline value, i.e., the mFARS score or at least one mFARS subscore measured in the subject before administration of the fusion protein of the Disclosure.

[0257] In other embodiments, administration of the fusion protein of the Disclosure to a subject may delay the progression of FRDA in the subject as measured, for example, by the mFARS score or at least one mFARS subscore as disclosed herein. For example, administration of the fusion protein of the Disclosure to a subject may cause the mFARS score or at least one mFARS subscore to become substantially the same as, or substantially eliminate, the increase in the mFARS score or at least one mFARS subscore compared to, or compared to, the baseline value, i.e., the mFARS score or at least one mFARS subscore measured in the subject before administration of the fusion protein of the Disclosure.

[0258] The Nine-Hole Peg Test (9HPT) can be used to measure the finger dexterity of subjects with FRDA. In this test, subjects are asked to take pegs one by one from a container as quickly as possible and place them into nine holes on a board. They must then remove the pegs one by one from the holes and return them to the container. The score is based on the time, recorded in seconds, it takes to complete the test activity.

[0259] In some embodiments, administration of the fusion protein of the present disclosure to a subject can result in a reduction in the severity of FRDA, measured, for example, by the 9HPT score. For example, administration of the fusion protein of the present disclosure to a subject can result in a reduction in the 9HPT score expressed as the time to complete the activities of the test (e.g., at least about 5%, 10%, 25%, or 50% reduction in the 9HPT score expressed as the time to complete the activities of the test) compared to the baseline value, i.e., the 9HPT score measured in the subject prior to administration of the fusion protein of the present disclosure.

[0260] In other embodiments, administration of the fusion protein of the present disclosure to a subject can delay the progression of FRDA in the subject, measured, for example, by the 9HPT score. For example, administration of the fusion protein of the present disclosure to a subject can result in the 9HPT score expressed as the time to complete the activities of the test being substantially the same or no substantial increase in the 9HPT score in comparison to the baseline value, i.e., the 9HPT score measured in the subject prior to administration of the fusion protein of the present disclosure.

[0261] In some embodiments, administration of the fusion protein of the present disclosure to a subject results in an increase in the level of hFXN in at least one tissue or biological fluid of the subject compared to the baseline level, i.e., the level of hFXN in the subject prior to administration of the fusion protein of the present disclosure. In some embodiments, the increase in the level of hFXN in at least one tissue or biological fluid of the subject caused by administration of the fusion protein of the present disclosure to the subject is sufficient to have a therapeutic effect, i.e., sufficient to treat FRDA in the subject.

[0262] In some embodiments, administration of the fusion protein of the Disclosure to a subject having FRDA may result in an hFXN level in at least one tissue or biofluid of the subject that is lower than the hFXN level in at least one tissue or biofluid of a subject without FRDA (e.g., a normal, healthy subject), but still sufficient to have a therapeutic effect, i.e., sufficient to treat the FRDA in the subject. For example, after administration of the fusion protein of the Disclosure to a subject having FRDA, the hFXN level in at least one tissue or biofluid of the subject may be about 10% to about 50%, about 20% to about 60%, or about 30% to about 80% of the hFXN level in at least one tissue or biofluid of a subject without FRDA (e.g., a normal, healthy subject), but the hFXN level is still sufficient to have a therapeutic effect, i.e., sufficient to treat the FRDA in the subject.

[0263] In some embodiments, administration of the fusion protein of the present disclosure to a subject having FRDA may result in an increase of at least about 5%, about 10%, about 25%, about 50%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or about 600% of the hFXN level in at least one tissue or biofluid of the subject compared to the hFXN level in at least one tissue or biofluid of the subject before administration of the fusion protein of the present disclosure, or compared to a baseline level.

[0264] In some embodiments, administration of the fusion protein of the Disclosure to a subject having FRDA may result in an increase of approximately 5% to approximately 30%, approximately 10% to approximately 50%, approximately 25% to approximately 100%, approximately 50% to approximately 150%, approximately 100% to approximately 300%, approximately 50% to approximately 150%, approximately 100% to approximately 300%, approximately 50% to approximately 250%, approximately 150% to approximately 500%, or approximately 200% to approximately 700% of the hFXN level in at least one tissue or biofluid of the subject compared to or compared to baseline levels. In some embodiments, administration of the fusion protein of the Disclosure to a subject may result in an increase of at least approximately 2-fold, approximately 3-fold, approximately 4-fold, or approximately 5-fold of the hFXN level in at least one tissue or biofluid of the subject compared to or compared to baseline levels. In some embodiments, administration to a subject of a pharmaceutical composition comprising the TAT-FXN fusion polypeptide of the Public Disclosure and / or the TAT-FXN fusion polypeptide of the Public Disclosure may result in an increase of between approximately 2 and 5 or between approximately 2 and 10 times the hFXN level in at least one tissue or biological fluid of the subject, compared to or compared to a baseline level, compared to the hFXN level in at least one tissue or biological fluid of the subject before administration of the fusion protein of the Public Disclosure.

[0265] In some embodiments, the tissue of which hFXN levels can be measured and / or increased may be any tissue that can be biopsied. In some embodiments, the tissue may include bronchoalveolar tissue (e.g., which can be sampled by bronchoalveolar brushing), mucosa (e.g., nasal mucosa, which can be sampled by nasal brushing), hair follicles, skin tissue, or buccal tissue. In some embodiments, the tissue may include skin tissue or buccal tissue.

[0266] In some embodiments, the biological fluids in which the hFXN level can be measured and / or increased may be blood or blood components (e.g., serum, plasma, platelets, or any other blood components), urine, or saliva.

[0267] Pneumonia associated with FRDA Individuals diagnosed with FRDA suffer from neurodegenerative disease of the dorsal root ganglia that causes progressive ataxia. This typically results in a progressive loss of the ability to walk, eat independently, speak, and swallow, as well as aspiration. Aspiration events can lead to pneumonia, frequent hospitalizations, and ultimately death over a period of 10 to 15 years from the date of diagnosis.

[0268] Therefore, administration of the fusion protein of this disclosure may be effective as a protein replacement therapy to prevent aspiration and thereby prevent aspiration-associated pneumonia in FXN-deficient subjects diagnosed with FRDA. Accordingly, this disclosure provides a method for treating FRDA-associated pneumonia in a subject, comprising administering the fusion protein of this disclosure to a subject in need, thereby treating the subject's FRDA-associated pneumonia.

[0269] Hypertrophic cardiomyopathy associated with FRDA Hypertrophic cardiomyopathy (FRDA) is a condition in which the heart muscle thickens, making it difficult for the heart to pump blood through the circulatory system. This condition may be caused by a deficiency of FXN in the mitochondria of cardiac cells. In subjects diagnosed with FRDA, progressive hypertrophic cardiomyopathy progresses to heart failure and death in approximately 50% of cases. Protein replacement therapy with the fusion protein disclosed herein can replenish the underlying FXN deficiency in hypertrophic cardiomyopathy.

[0270] Administration of the fusion protein of this disclosure may therefore be effective as protein replacement therapy in subjects with FXN deficiency diagnosed with both FRDA and hypertrophic cardiomyopathy. Accordingly, this disclosure provides a method for treating FRDA-associated hypertrophic cardiomyopathy in a subject, comprising administering the fusion protein of this disclosure to a subject in need, thereby treating FRDA-associated hypertrophic cardiomyopathy in the subject.

[0271] diabetes The hallmark of diabetes is the inability to properly regulate blood glucose levels, which leads to elevated blood glucose levels. In subjects diagnosed with FRDA, diabetes often manifests as a result of FXN-deficient mitochondria in the pancreas. Protein replacement therapy with the fusion protein of this disclosure can replenish the underlying FXN deficiency in diabetes.

[0272] Administration of the fusion protein of this disclosure may therefore be effective as protein replacement therapy in subjects diagnosed with FXN deficiency and diabetes mellitus. Accordingly, this disclosure provides a method for treating FRDA-associated diabetes mellitus in a subject, comprising administering the fusion protein of this disclosure to a subject in need, thereby treating FRDA-associated diabetes mellitus in the subject.

[0273] Other FRDA-related illnesses / disorders Subjects diagnosed with FRDA often experience other disorders associated with FXN deficiency. Such FRDA-associated disorders include, but are not limited to, neurological disorders including proprioceptive deficiency, loss of reflexes, loss of gait, and loss of the ability to maintain gaze; dysphagia and / or progressive loss of swallowing ability; progressive hearing loss; progressive visual impairment due to retinal degeneration resulting from FXN deficiency; progressive aphasia; metabolic syndrome including, but not limited to, elevated triglycerides, decreased high-density lipoprotein (HDL) cholesterol, and elevated low-density lipoprotein (LDL) cholesterol; scoliosis requiring surgery for correction; and / or combinations thereof. Protein replacement therapy with the fusion protein of this disclosure can replenish the FXN deficiency underlying these FRDA-associated diseases / disorders.

[0274] Administration of the fusion protein of this disclosure may therefore be effective as protein replacement therapy in subjects of FXN deficiency experiencing FXN deficiency diagnosed with FRDA and non-limited neurological disorders including proprioceptive deficiency, anorexia, gait impairment, and loss of ability to maintain gaze; dysphagia and / or progressive loss of swallowing ability; progressive hearing loss; progressive visual impairment due to retinal degeneration resulting from FXN deficiency; progressive aphasia; metabolic syndrome including non-limited elevated triglycerides, decreased HDL cholesterol, and elevated LDL cholesterol; scoliosis requiring surgery for correction; and / or a combination thereof.

[0275] Accordingly, the present disclosure provides a method for treating a disease, disorder, or condition associated with FRDA, comprising administering the fusion protein of the present disclosure to a subject in need thereof, wherein the disease, disorder, or condition associated with FRDA is selected from among neurological disorders including, but not limited to, lack of proprioception, loss of reflexes, loss of gait, and loss of the ability to maintain gaze; dysphagia and / or progressive loss of swallowing ability; progressive hearing loss; progressive visual impairment due to retinal degeneration resulting from FXN deficiency; progressive aphasia; metabolic syndrome including, but not limited to, elevated triglycerides, decreased HDL cholesterol, and elevated LDL cholesterol; and scoliosis requiring surgery for correction.

[0276] In certain embodiments, treatment of a subject requiring the fusion protein, pharmaceutical composition, or therapeutic compound of the Disclosure, such as FXN, increases the cellular level of FXN in the subject. In some embodiments, the method of the Disclosure increases the cellular level of FXN by at least about 10%, for example, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more than about 95%, compared to the level of FXN in the subject before treatment. In other embodiments, the method of the Disclosure increases the cellular level of FXN by at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, or at least about 10 times. In some embodiments, the increase is measured by comparing the cellular level of FXN in the subject before and after administration of the fusion protein of the Disclosure.

[0277] The cellular levels of FXN in a subject can be measured before, during, and after administration of the fusion proteins, pharmaceutical compositions, and / or therapeutic compounds of this disclosure using biological samples, such as blood, serum, plasma, urine, ascites, cerebrospinal fluid, feces, intestinal mucosal swabs, tissue samples, and / or samples taken from the contents of one or more of the stomach, duodenum, jejunum, ileum, cecum, colon, rectum, and anal canal. In some embodiments, the method may include administering the fusion protein disclosed herein to increase the cellular levels of FXN in a subject. In some embodiments, the method may include administering the fusion protein disclosed herein to increase the cellular levels of FXN by more than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the cellular level of FXN in the subject before treatment. The cellular level of FXN can be measured by methods known in the art. For example, the cellular level of FXN can be measured by collecting samples, such as skin biopsies (punch biopsies), oral swabs, or platelets, and analyzing them for FXN using ELISA, hybrid LBA-LC-MS / MS, or other mass spectrometry-related methods.

[0278] How to treat Leigh syndrome, French-Canadian type (LSFC) The present disclosure also provides a method of treating Leigh syndrome French-Canadian type (LSFC), comprising administering to a subject in need thereof a fusion protein of the present disclosure such that the subject's LSFC is treated. Surprisingly, it has been discovered that mitochondrial defects in LRPPRC-deficient cells, demonstrated by acidification of the cell growth medium, are reduced by treatment of the cells with the hFXN fusion protein of the present disclosure. Surprisingly, it has also been discovered that the amount of CYR61 protein secreted into the cell growth medium by LRPPRC-deficient cells is reduced by treatment of the cells with the hFXN fusion protein of the present disclosure. Further, surprisingly, it has been discovered that the hFXN fusion protein of the present disclosure comprising TES is more effective than the hFXN fusion protein without TES in reducing medium acidification and secretion of CYR61 from LRPPRC-deficient cells.

[0279] The discoveries described above indicate that certain molecular and cellular changes associated with the deletion of LRPPRC may be alleviated and / or reversed by administering the hFXN fusion protein of the present disclosure. The discoveries described above also indicate that LSFC associated with the deletion of LRPPRC may be treated by administering the hFXN fusion protein of the present disclosure. Further, the discoveries described above indicate that the hFXN fusion protein of the present disclosure comprising TES is more effective than the hFXN fusion protein without TES in treating LSFC and / or lactic acidosis in a subject having LSFC.

[0280] In some embodiments, the present disclosure provides a method of treating LSFC in a subject in need thereof, comprising administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the full-length hFXN (SEQ ID NO: 1) which is the protein of interest, 2) any CPP described herein, and 3) any TES described herein.

[0281] In some embodiments, a method for treating an LSFC includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the full-length hFXN (SEQ ID NO: 1), which is the protein of interest; 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES as described herein.

[0282] In some embodiments, a method for treating LSFCs includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to ubiquitin (SEQ ID NO: 18).

[0283] In some embodiments, a method for treating LSFCs includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to the calpain cleavage domain of SEQ ID NO: 20.

[0284] In some embodiments, a method for treating LSFCs includes administering a fusion protein to a subject requiring it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0285] In some embodiments, a method for treating LSFCs includes administering a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 56, 57, 58, 59, 60, 61, and 83 to a subject requiring it.

[0286] In one embodiment, a method for treating LSFCs includes administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 56.

[0287] In one embodiment, a method for treating LSFCs involves administering a fusion protein containing an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 57 to a subject requiring it.

[0288] In one embodiment, a method for treating LSFCs involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 58.

[0289] In one embodiment, a method for treating LSFCs involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 59.

[0290] In one embodiment, a method for treating LSFCs involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 60.

[0291] In one embodiment, a method for treating LSFCs involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 61.

[0292] In one embodiment, a method for treating LSFCs involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 83.

[0293] In some embodiments, the Disclosure provides a method for treating lactic acidosis in a subject having an LSFC, comprising administering the fusion protein of the Disclosure to a subject in need so that the lactic acidosis of the subject is treated. For example, the Disclosure provides a method for treating lactic acidosis in a subject having an LSFC, comprising administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the full-length hFXN (SEQ ID NO: 1), which is the protein of interest; 2) any CPP as described herein; and 3) any TES as described herein.

[0294] In some embodiments, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1), 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11), and 3) any TES as described herein.

[0295] In some embodiments, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to ubiquitin (SEQ ID NO: 18).

[0296] In some embodiments, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to the calpain cleavage domain of SEQ ID NO: 20.

[0297] In some embodiments, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein full-length hFXN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0298] In some embodiments, a method for treating lactic acidosis in a subject having an LSFC includes administering to the subject a fusion protein comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs. 56, 57, 58, 59, 60, 61, and 83.

[0299] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 56.

[0300] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 57.

[0301] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 58.

[0302] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 59.

[0303] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 60.

[0304] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 61.

[0305] In one embodiment, a method for treating lactic acidosis in a subject having LSFCs includes administering to the subject a fusion protein containing or comprising an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 83.

[0306] As used herein, the term “to treat LSFC” includes achieving relief, improvement, or reduction in the severity of LSFC, for example, relief, improvement, or reduction of at least one symptom or indicator associated with LSFC in the subject. As used herein, “to treat LSFC” also includes delaying the progression of LSFC, for example, delaying the onset of at least one symptom or indicator associated with LSFC, or preventing an increase in the severity of at least one symptom or indicator associated with LSFC in the subject. In some embodiments, the at least one symptom or indicator associated with LSFC may be selected from the group consisting of growth retardation, ataxia, hypotonia, brain damage, coma, abnormal respiratory patterns, epileptic seizures, stroke-like episodes, and lactic acidosis.

[0307] Methods of treating Parkinson's disease In one embodiment, the Disclosure provides a method for treating Parkinson's disease (PD), comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating Parkinson's disease, comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating Parkinson's disease, comprising administering to a subject in need a fusion protein comprising 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PINK1 (SEQ ID NO: 8), 2) any CPP as described herein, and 3) any TES as described herein.

[0308] In some embodiments, a method for treating Parkinson's disease includes administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PINK1 (SEQ ID NO: 8); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES described herein, such as ubiquitin (SEQ ID NO: 18) or a calpain cleavage domain (SEQ ID NO: 20).

[0309] In some embodiments, a method for treating Parkinson's disease includes administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PINK1 (SEQ ID NO: 8); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to ubiquitin (SEQ ID NO: 18).

[0310] In one embodiment, the present disclosure provides a method for treating Parkinson's disease, comprising administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PARKIN (SEQ ID NO: 7); 2) any CPP as specified herein; and 3) any TES as specified herein.

[0311] In some embodiments, a method for treating Parkinson's disease includes administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES described herein.

[0312] In some embodiments, a method for treating Parkinson's disease involves administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to ubiquitin (SEQ ID NO: 18).

[0313] In some embodiments, a method for treating Parkinson's disease includes administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PARKIN (SEQ ID NO: 7); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% to the calpain cleavage domain of SEQ ID NO: 20.

[0314] In some embodiments, a method for treating Parkinson's disease includes administering a fusion protein to a subject in need of it, comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with the target protein PARKIN (SEQ ID NO: 1); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% with TAT-HIV (SEQ ID NO: 11); and 3) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% with any one of SEQ ID NOs: 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31, 32, 33, 34, and 35.

[0315] In some embodiments, a method for treating Parkinson's disease involves administering a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71 to a subject in need of it.

[0316] In one embodiment, a method for treating Parkinson's disease involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 69.

[0317] In one embodiment, a method for treating Parkinson's disease involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 70.

[0318] In one embodiment, a method for treating Parkinson's disease involves administering a fusion protein to a subject in need of it, which contains or comprises an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with SEQ ID NO: 71.

[0319] As used herein, the term “Parkinson’s disease” encompasses both sporadic and familial forms of Parkinson’s disease. Familial forms of Parkinson’s disease account for approximately 5–10% of cases, and mutations in approximately 20 genes have been associated with Parkinson’s disease. In some embodiments, familial forms of Parkinson’s disease may be associated with mutations in one or more genes selected from the group consisting of PARK2 (PRKN), PINK1 (PRKN6), PARK7, SNCA, and LRRK2. In one embodiment, familial forms of Parkinson’s disease may be associated with a mutation in PARK2 (PRKN). In another embodiment, familial forms of Parkinson’s disease may be associated with a mutation in PINK1 (PRKN6). In one embodiment, Parkinson’s disease is not sporadic forms of Parkinson’s disease.

[0320] In one embodiment, the present disclosure provides a method for treating Parkinson's disease associated with a mutation in the PARK2 gene, comprising administering to a subject in need of such treatment a fusion protein of the present disclosure containing PARKIN (SEQ ID NO: 7), for example, a fusion protein having an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with any one of SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71.

[0321] In one embodiment, the present disclosure provides a method for treating Parkinson's disease associated with mutations in the PINK1 (PRKN6) gene, comprising administering to a subject in need of such treatment a fusion protein of the present disclosure containing PARKIN (SEQ ID NO: 7), for example, SEQ ID NO: 69, SEQ ID NO: 70, or SEQ ID NO: 71, which has an amino acid sequence having at least about 85%, about 90%, about 95%, or about 99% sequence identity with any one of these.

[0322] In some embodiments, Parkinson's disease is characterized by at least one symptom selected from the following: a) Tremors or shaking, usually beginning in the limbs, often in the hands or fingers. Tremors associated with Parkinson's disease may include the pill-rolling tremor, where the thumb and index finger are rubbed back and forth, and tremors of the hands when relaxed (at rest). b) Slow movements (slow actions), c) muscular rigidity; d) Postural balance disorders, e) Loss of automatic motion

[0323] In addition, signs or symptoms of "non-motor symptoms" or "dopamine unresponsiveness" are also commonly seen in individuals with Parkinson's disease, and these may include the following: f) Cognitive impairment, g) Mood disorders, e.g., depression and anxiety, h) Sleep disorders, including REM sleep disorders, in which individuals act out their dreams. i) Orthostatic hypotension, j) constipation; k) Speech and swallowing disorders, l) Unexplained pain, salivation, and loss of smell.

[0324] As used herein, the term “to treat Parkinson’s disease” encompasses the reduction, alleviation, or improvement of one or more of the signs or symptoms of Parkinson’s disease described above, a reduction in the severity of Parkinson’s disease, maintenance of stability (i.e., no worsening) of Parkinson’s disease, or improvement or alleviation of the condition. Treatment of Parkinson’s disease may include the reduction, alleviation, or improvement of one or more of the principal or non-motor symptoms of PD described above, such as the reduction, alleviation, or improvement of tremor, bradykinesia, or muscle rigidity, or the reduction of speech and swallowing difficulties. In one embodiment, treatment may also include inhibiting and slowing the progression of Parkinson’s disease.

[0325] Treatment does not need to be curative. Treatment outcomes do not need to be quantitatively determined. However, in certain embodiments, treatment outcomes can be quantified by tracking the course of Parkinson's disease over time using, for example, the Unified Parkinson's Disease Rating Scale (UPDRS) or the modified UPDRS, known as the MDS-UPDRS. The UPDRS is the most commonly used scoring system for the clinical assessment of Parkinson's disease. The UPDRS consists of 42 items that are assessed through interviews and clinical findings of the subject. The UPDRS scale has a possible total score of 199, where 199 represents the worst impairment and 0 represents no impairment. The UPDRS consists of the following sections: Part I: Assessment of mental functions, behavior, and mood. Part II: Self-assessment of activities of daily living (ADL), including speech, swallowing, writing, dressing, hygiene, falls, drooling, turning over in bed, walking, and cutting food. Part III: Exercise assessment by tests scored by clinicians, Part IV: Complications of treatment, Part V: Hoehn and Yahr's classification of Parkinson's disease severity, and Part VI: ADL scales in Schwab and England.

[0326] These are assessed through interviews and clinical findings. In some sections, it is necessary to assign multiple grades to each limb. The revised UPDRS includes a reorganization of various subscales but retains the four-scale structure of the original UPDRS. The titles of each scale are as follows: (1) Non-motor activities in daily life (13 items), (2) Motor activities in daily life (13 items), (3) Motor tests (18 items), and (4) Motor complications (6 items). Each subscale has a score of 0 to 4, where 0 = normal, 1 = mild, 2 = mild, 3 = moderate, and 4 = severe.

[0327] UPDRS or MDS-UPDRS can be used to track the progression of Parkinson's disease in a person or to measure the benefits of a treatment, such as administering the fusion protein of this disclosure.

[0328] In some embodiments, administration of the fusion protein of the Disclosure to a subject by the method described herein results in inhibition or deceleration of the progression of Parkinson's disease in the subject. Specifically, in some embodiments, administration of the fusion protein of the Disclosure to a subject results in a substantially elimination of the increase in the subject's UPDRS score over a period of time, for example, one week, two weeks, three weeks, one month, two months, three months, six months, nine months, one year, two years, three years, or five years. In some embodiments, administration of the fusion protein of the Disclosure to a subject results in a reduction of the subject's UPDRS score over a period of time, for example, one week, two weeks, three weeks, one month, two months, three months, six months, nine months, one year, two years, three years, or five years.

[0329] Treatment methods for Barth syndrome and familial isolated dilated cardiomyopathy In one embodiment, the Disclosure provides a method for treating Baars syndrome and familial isolated dilated cardiomyopathy, comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating Baars syndrome and familial isolated dilated cardiomyopathy, comprising administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein tafadine (SEQ ID NO: 3); 2) any CPP as described herein; and 3) any TES as described herein.

[0330] In some embodiments, a method for treating Barth syndrome and familial isolated dilated cardiomyopathy includes administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein tafadine (SEQ ID NO: 3); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES described herein, such as ubiquitin (SEQ ID NO: 18) or a calpain cleavage domain (SEQ ID NO: 20).

[0331] Treatment methods for pyruvate dehydrogenase E1-beta deficiency In one embodiment, the Disclosure provides a method for treating pyruvate dehydrogenase E1-beta deficiency, comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating pyruvate dehydrogenase E1-beta deficiency, comprising administering to a subject in need a fusion protein comprising 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PDHB (SEQ ID NO: 4), 2) any CPP as described herein, and 3) any TES as described herein.

[0332] In some embodiments, a method for treating pyruvate dehydrogenase E1-beta deficiency includes administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein PDHB (SEQ ID NO: 4); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES described herein, such as ubiquitin (SEQ ID NO: 18) or a calpain cleavage domain (SEQ ID NO: 20).

[0333] How to treat Leigh syndrome, French-Canadian type In one embodiment, the Disclosure provides a method for treating the French-Canadian type of Leigh syndrome, comprising administering the fusion protein of the Disclosure to a subject in need thereof. For example, the Disclosure provides a method for treating the French-Canadian type of Leigh syndrome, comprising administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein LRPPRC (SEQ ID NO: 5) or SLIRP (SEQ ID NO: 6); 2) any CPP as described herein; and 3) any TES as described herein.

[0334] In some embodiments, a method for treating Leigh syndrome France-Canada type includes administering to a subject in need a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to the target protein LRPPRC (SEQ ID NO: 5) or SLIRP (SEQ ID NO: 6); 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NO: 11); and 3) any TES described herein, such as ubiquitin (SEQ ID NO: 18) or calpain cleavage domain (SEQ ID NO: 20).

[0335] Treatment methods for PLA2G6-associated neurodegeneration (PLAN) In one embodiment, the present disclosure provides a method for treating PLA2G6-associated neurodegenerative disorders (PLANs). In some embodiments, the term “PLA2G6-associated neurodegenerative disorders (PLANs)” may include one or more Parkinsonian syndromes, namely, infantile axonal dystrophy (INAD), atypical axonal dystrophy (ANAD), adult-onset dystonic parkinsonism (DP), and autosomal recessive juvenile parkinsonism (AREP).

[0336] In some embodiments, a method for treating PLAN includes administering to a subject requiring it a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any variant 1-9 (SEQ ID NOs. 72-80) of type VI phospholipase A2 listed in Table 2, which is the protein of interest; 2) any CPP as specified herein; and 3) any TES as specified herein.

[0337] In some embodiments, a method for treating PLAN includes administering to a subject requiring it a fusion protein comprising: 1) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to any variant 1-9 (SEQ ID NOs. 72-80) of type VI phospholipase A2 listed in Table 2, which is the protein of interest; 2) an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to TAT-HIV (SEQ ID NOs. 11); and 3) any TES listed herein, such as ubiquitin (SEQ ID NOs. 18) or a calpain cleavage domain (SEQ ID NOs. 20). Unless otherwise stated or made clear from the context, the term “about” as used herein is understood to mean within the normal tolerance range in the art, e.g., within a mean of 2 standard deviations. "Approximately" is understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise evident from the context, all numerical values ​​presented herein may be modified by the term "approximately."

[0338] As used herein, the terms “administer,” “give delivery,” or “give delivery” include any method of delivery of the fusion proteins, pharmaceutical compositions, or therapeutic compounds of this disclosure. In certain embodiments, the fusion proteins, pharmaceutical compositions, or therapeutic compounds of this disclosure may be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. In certain embodiments, the fusion proteins, pharmaceutical compositions, or therapeutic compounds of this disclosure may be administered subcutaneously. The administration of fusion proteins, pharmaceutical compositions, or therapeutic compounds may be carried out by several people working in coordination. Administering a drug may include, for example, prescribing the drug to be administered and / or directing, or otherwise, to take a particular drug by self-delivery, such as oral delivery, subcutaneous delivery, or intravenous delivery through a central line, or by delivery by a trained professional, such as intravenous delivery or intramuscular delivery.

[0339] [Examples] [Example 1] Comparison of intracellular localization of hFXN and TAT-GG-hFXN fusion protein Determination of intracellular localization of hFXN in rat L6 myoblasts treated with TAT-GG-hFXN fusion protein by immunofluorescence staining. Human frtaxin protein (hFXN 1-210 ) is encoded in the nucleus and is synthesized as a 210-amino acid precursor protein having the amino acid sequence shown in SEQ ID NO: 1 (Table 1). After its expression, the full-length hFXN 1-210 This protein is directed to mitochondria by an 80aa mitochondrial translocation sequence (MTS) at its N-terminus, where it is actively imported and processed by proteolysis to produce the 130aa mature form of this protein, i.e., the active fragment (hFXN), which has a predicted molecular weight of 14.2 kDa and the sequence shown in Sequence ID No. 2 (Table 1). 81-210 It produces FXN. The mature form of FXN eventually resides within the mitochondrial matrix. Sequence ID 1: TIFF2026108757000021.tif23160 Sequence ID 2: TIFF2026108757000022.tif18160

[0340] The TAT-GG-hFXN fusion protein was developed for use in protein replacement therapy for the treatment of Friedreich's ataxia (FRDA). The TAT-GG-hFXN fusion protein has been shown to rescue FRDA-related disease phenotypes in cells and animals. When intracellular, the TAT-GG-hFXN fusion protein is thought to localize to mitochondria.

[0341] The intracellular localization of hFXN in rat L6 myoblasts treated with the TAT-GG-hFXN fusion protein was evaluated by immunofluorescence microscopy (Figure 3). Briefly, rat L6 myoblasts were treated with TAT-GG-hFXN fusion protein (5 μM) in serum-free medium for 2 hours. After 2 hours, the serum-free medium containing the TAT-GG-hFXN fusion protein was replaced with complete medium, and the cells were incubated. As shown in Figure 3, hFXN was strongly detected in small punctate intracellular vesicles and nuclei (see Figure 3, "hFXN" and "Merge" panels). The observed hFXN-containing intracellular vesicles did not show significant overlap with the mitochondrial membrane marker TOMM20 (shown in the "Merge" panel of Figure 3). These vesicles may represent the transport of the TAT-GG-hFXN fusion protein via the endosomal / lysosomal system after uptake by cells, but further research is needed to validate this hypothesis. Furthermore, these data demonstrate that hFXN can be detected intracellularly and that it is strongly detected in the nucleus after treatment with the TAT-GG-hFXN fusion protein. While low levels of hFXN may be present in mitochondria after treatment with the TAT-GG-hFXN fusion protein under the described conditions, mitochondrial hFXN is practically impossible to detect in rat L6 myoblasts by immunofluorescence staining.

[0342] Comparison of the intracellular localization of hFXN between rat L6 myoblasts after transfection with hFXN or TAT-GG-hFXN fusion protein Under the above conditions, most of the hFXN was detected in the nuclei of rat L6 myoblasts after treatment with the TAT-GG-hFXN fusion protein (Figure 3). The N-terminal TAT-GG sequence added to the full-length hFXN may be able to direct the therapeutic protein to the nucleus after it is taken up into the cell. To more carefully evaluate this effect of TAT-GG on hFXN, the intracellular localization of the intracellularly expressed TAT-GG-hFXN fusion protein was detected and compared by immunofluorescence microscopy with hFXN in rat L6 myoblasts. Using a lipid-based transfection reagent known to those skilled in the art of transfection, such as the L6 cell Avalanche (trademark) system (sold by EZ Biosystems), the TAT-GG-hFXN fusion protein or hFXN 1-210 expressing plasmids containing the gene construct encoding it were used to transfect rat L6 myoblasts. After 24 hours, the transfected cells were fixed using 4% paraformaldehyde, stained using an antibody against hFXN or the mitochondrial membrane marker TOMM20, and Hoechst 33342 nuclear staining was also performed. As expected, hFXN 1-210Rat L6 myoblasts transfected with construct were positive for the presence of mitochondrial hFXN, as evidenced by clear co-localization with the mitochondrial membrane marker TOMM20 (Figure 4, upper panel, "Merge"). In contrast, rat L6 myoblasts transfected with the TAT-GG-hFXN fusion protein were positive for hFXN, staining throughout the cytosol and strongly in the nucleus (Figure 4, lower panel, "Merge"). In a very small percentage of cells transfected with the TAT-GG-hFXN fusion protein, hFXN was also detected in the mitochondria (Figure 4, lower panel, "Merged" - very slight co-localization staining). In summary, these data demonstrate that the intracellular localization of the TAT-GG-hFXN fusion protein differs from the innate intracellular localization of hFXN, primarily from mitochondria (hFXN) to the cytosol and strongly to the nucleus (TAT-GG-hFXN fusion protein).

[0343] [Example 2] Design of a novel TAT-GG-hFXN construct to enhance hFXN delivery to mitochondria. Figures 3 and 4, which show the fluorescence co-localization results in Example 1 described above, indicate that the majority of TAT-GG-hFXN fusion protein molecules in cells do not specifically localize to mitochondria, which may be due to the presence of the TAT-GG sequence attached to the amino terminus of this molecule. To overcome this obstacle, several novel frataxin fusion proteins were designed and their mitochondrial delivery yields were tested. Improving the mitochondrial delivery yield of therapeutic fusion proteins that act in mitochondria would also provide better treatment for mitochondrial diseases, such as FRDA. To achieve this goal, novel fusion proteins were designed. Schematic diagrams of these proteins are shown in panels a-c of Figure 5.

[0344] A group of proteins were designed to contain a protease-cleavable domain, and a library of constructs encoding a "protease-activatable" cell-permeable hFXN fusion protein was constructed. These constructs contain a cell-permeable peptide (CPP), such as the HIV-TAT (YGRKKRRQRRR; SEQ ID NO: 11) peptide, fused to an amino acid sequence that can be cleaved by an endogenous intracellular protease, immediately followed by the target protein, in this case the full-length precursor hFXN. The dipeptide GG was used as a linker between the CPP and the proteolytic cleavage domain. When TAT-GG is cleaved from the fusion protein in the cytosol, a TAT-free hFXN protein is produced, which can then be imported into the mitochondria by the endogenous MTS. For example, the cleavable intracellular protease-sensitive proteins used were SUMO-1 (cleaved by SUMO protease) and ubiquitin (cleaved by ubiquitinase), and the proteolytically sensitive peptides were DEVD (caspase cleavage site, SEQ ID NO: 19), EPLFAERK (SEQ ID NO: 20), and LLVY (calpain cleavage site, SEQ ID NO: 21).

[0345] The alternative proteins were designed to include nuclear export signals (NES) and to avoid accumulation of the target protein in the cell nucleus. Specifically, the constructs were designed to include CPPs linked to the target protein and linked to the NES. For example, the carboxyl terminus of the TAT-GG-hFXN fusion construct was directly fused to the NES domain derived from nuclear export signals (NES), such as PKIα (NES1) and MAPKK (NES2).

[0346] Schematic diagrams of these fusion proteins are shown in Figure 5, panels a-c, and an overview of the fusion protein constructs is shown in Table 11.

[0347] [Table 11] TIFF2026108757000023.tif64163

[0348] [Example 3] Intracellular expression of hFXN fusion proteins - preliminary screening for mitochondrial transport Gene constructs encoding various hFXN variants, namely hFXN, TAT-GG-hFXN (TAT-hFXN fusion protein), TAT-GG-SUMO1-hFXN, TAT-GG-ubiquitin-hFXN, TAT-GG-DEVD-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-LLVY-hFXN, TAT-GG-hFXN-NES1, and TAT-GG-hFXN-NES2, were cloned into expression vectors. As an example, the plasmid vector pcDNA3.1(+) (Genscript®, Piscataway, New Jersey, USA) was used. All protein-encoding sequences were codons optimized for mammalian (human) expression. The amino acid sequences of the variants and the nucleic acid sequences encoding each of them are shown in Tables 9 and 10, which are found elsewhere in this disclosure.

[0349] Using lipid-based translocation reagents known to those skilled in the field of translocation, rat L6 myoblasts were translocated with these expression plasmids, for example, using the L6 cell Avalanche® system (EZ Biosystems). Intracellular localization of hFXN was detected by immunofluorescence microscopy as described in Example 1. Translocation cells were cultured for 24 hours, then fixed with 4% paraformaldehyde and stained with an antibody against hFXN and the mitochondrial membrane marker TOMM20. In addition, the cell nuclei were stained using Hoechst33342 nuclear stain.

[0350] The results of immunolabeling are shown in Figure 6, panels a-i, and summarized in Table 12.

[0351] [Table 12] TIFF2026108757000024.tif69161

[0352] As demonstrated in Example 1, when expressed in rat L6 myoblasts, hFXN localized to mitochondria, but the TAT-GG-hFXN fusion protein was detected primarily in the cytosol and nucleus. In cells transfected with TAT-GG-SUMO1-hFXN and TAT-GG-ubiquitin-hFXN, hFXN was detected in mitochondria. In cells transfected with TAT-GG-DEVD-hFXN, hFXN was detected in the cytosol and nucleus, and no significant mitochondrial localization was observed. In cells transfected with TAT-GG-EPLFAERK-hFXN and TAT-GG-LLVY-hFXN, hFXN was detected in mitochondria as well as in the cytosol and nucleus. In summary, these data demonstrate that intracellular SUMO proteases, dubiquitinases, and intracellular calpain can cleave the fusion protein at their respective cleavage sites, producing mitochondrially localized hFXN variants. Similar to the TAT-GG-hFXN fusion protein, TAT-GG-DEVD-hFXN was not detected in mitochondria. This suggests that caspase-mediated cleavage of DEVD (SEQ ID NO: 19) was inefficient under current conditions.

[0353] In cells transfected with TAT-GG-hFXN-NES1 and TAT-GG-hFXN-NES2, hFXN was detected in mitochondria, cytosol, and nucleus. These data also demonstrate that nuclear export of the hFXN fusion protein leads to improved mitochondrial importation of hFXN.

[0354] [Example 4] Assessment of hFXN maturity by Western blot analysis In its natural state, the full length hFXN 1-210 However, it is directed to the mitochondria by the N-terminal mitochondrial transition sequence (MTS), where it is imported and becomes the mature form of this protein, hFXN. 81-210The cells undergo a two-step proteolytic processing that produces fragments. To evaluate the degree of hFXN maturation by proteolytic processing of novel hFXN variants, Western blot analysis was performed on hFXN variants generated in rat L6 myoblasts transfected with expression plasmids containing the genes encoding hFXN, TAT-GG-hFXN fusion protein, TAT-GG-SUMO1-hFXN, TAT-GG-ubiquitin-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-LLVY-hFXN, TAT-GG-hFXN-NES1, and TAT-GG-hFXN-NES2. Cells were harvested 72 hours after transfection and lysed in ice-cold radioimmunoprecipitation (RIPA) buffer supplemented with protease inhibitors and EDTA. Cell lysates were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), transferred to a nitrocellulose membrane, and analyzed by Western blotting using a commercially available antibody against hFXN (purchased from AbCam) and an antibody against β-actin, which served as a loading control.

[0355] The results are shown in Figure 7. Fully mature hFXN (lane 1, band below the 15 kDa marker, approximately 14.2 kDa) was detected in lysates obtained from cells transfected with the hFXN construct. The band corresponding to mature hFXN (approximately 14.2 kDa) was also detected in lysates obtained from cells transfected with plasmids encoding the TAT-GG-hFXN fusion protein (lane 2) and TAT-GG-SUMO1-hFXN (lane 3). Mature hFXN was detected in greater quantities in lysates obtained from cells transfected with TAT-GG-ubiquitin-hFXN (lane 4), TAT-GG-EPLFAERK-hFXN (lane 5), and TAT-GG-hFXN-NES1 (lane 7; in this lane, the addition of NES1 increases the molecular weight by approximately 1 kDa, so hFXN-NES1 migrated more slowly in SDS-PAGE). Bands corresponding to mature hFXN were also detected for TAT-GG-LLVY-hFXN (lane 6) and TAT-GG-hFXN-NES2 (lane 8). These results indicate that the novel constructs TAT-GG-ubiquitin-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-hFXN-NES1 undergo proteolytic processing in cells, producing a greater amount of mature hFXN than the TAT-GG-hFXN fusion protein.

[0356] [Example 5] Transduction of Schwann cells using hFXN fusion protein The goal of this experiment was to determine and compare the ability of various hFXN fusion proteins to transduce cells.

[0357] This experiment used the TAT-GG-hFXN fusion protein described in Example 1, as well as novel TAT-FXN fusion proteins, namely TAT-GG-ubiquitin-hFXN (SEQ ID NO: 56) and TAT-GG-EPLFAERK-hFXN (SEQ ID NO: 58). In TAT-GG-EPLFAERK-hFXN (SEQ ID NO: 58), EPLFAERK (SEQ ID NO: 20) is the calpain 1 sensitive sequence. The schematic structures of the fusion proteins used in the experiment are shown in Figure 8.

[0358] The experiment used Schwann cells because they grow relatively quickly and are easily transduced. Furthermore, the endogenous levels of hFXN in these cells are below the detection limit and therefore are not expected to interfere with the signal generated by the TAT-GG-hFXN fusion protein.

[0359] Schwann cells were plated into 96-well plates (Corning 3904) at a seeding density of 8,000 cells per well and incubated overnight at 37°C. On day 1, solutions containing 1.25 μM, 1 μM, and 0.5 μM of TAT-GG-hFXN fusion protein were prepared in transduction medium containing DMEM, 1% heat-inactivated FBS, and 20 mM glycerol, respectively. Cells were washed with 150 μL of PBS per well, and then 60 μL of either the TAT-GG-hFXN solution or the transduction medium for negative control was added per well. Cells were incubated at 37°C for 2 hours, and then 60 μL of complete medium containing DMEM, 10% FBS, and 1% antibiotic:antifungal substance (Gemini Bio-Products 400-101) was added per well, and the cells were incubated overnight at 37°C.

[0360] The same procedure was repeated on day 2. On day 3, the cells in each well were washed with 150 μL of PBS, and then the cells were trypsinized by adding 50 μL of TrypLE (Gibco® 12604021) to each well and incubating at 37°C for 5 minutes. The trypsinization reaction was then stopped by adding 50 μL of complete medium to each well. The cells were resuspended and transferred to fibronectin-coated glass-bottom plates (Corning 4584) containing 40 μL of complete DMEM per well. The cells were deposited overnight at 37°C.

[0361] On day 4, the cells were washed with PBS, and then 50 μL of freshly prepared 4% paraformaldehyde solution was added to each well. The cells were incubated at room temperature for 10 minutes. Subsequently, the cells were washed twice with 150 μL of PBS per well, and then 50 μL of blocking buffer (PBS containing 0.3% Triton-X100 and 5% normal goat serum) was added to each well. The cells were incubated at room temperature for 1 hour, then the blocking buffer was removed, and 50 μL of primary antibodies (abcam anti-frataxin antibody ab110328 and abcam anti-TOMM20 antibody ab78547), diluted in blocking buffer, were added to each well. The anti-frataxin antibody was diluted 1:600, and the TOMM20 antibody was diluted 1:300. The cells were incubated overnight at 4°C.

[0362] On day 5, the cells were washed twice with 120 μL of PBS per well, and 50 μL of secondary antibodies (ab150116, anti-mouse IgG AlexaFluor594, from abcam, and ab150077, anti-rabbit IgG AlexaFluor488, from abcam) diluted in blocking buffer were added to each well. Each antibody was diluted 1:1000. The cells were incubated at room temperature for 1 hour, washed with 150 μL of PBS per well, stained by adding 50 μL of 300 nM Hoescht 33342 stain per well, and incubated at room temperature for 3 minutes. Subsequently, the cells were washed with PBS.

[0363] Cells were imaged using a Lionheart microscope. Human FXN was detected using an Alexa-594-labeled goat anti-mouse IgG secondary antibody that binds to a mouse anti-hFXN monoclonal primary antibody. Mitochondrial membranes were detected using an Alexa-488-labeled goat anti-rabbit IgG secondary antibody that binds to an anti-TOMM20 polyclonal primary antibody.

[0364] The experimental results are shown in Figure 9. In detail, Figure 9, panel A, is a series of photographs of Schwann cells treated with 0.25 μM, 0.5 μM, and 1 μM TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and a negative control medium. In the photographs, green corresponds to the mitochondrial marker TOMM20 signal, red corresponds to the hFXN signal, and blue corresponds to the nuclear marker Hoechst33342 dye signal. Under these imaging conditions, endogenous frataxin levels were undetectable in Schwann cells. As demonstrated in Figure 9, panel A, cells transduced with the medium showed only nuclear and mitochondrial staining and no hFXN staining. Cells transduced with TAT-GG-hFXN showed some degree of hFXN staining. In contrast, cells transduced with TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN showed higher levels of hFXN staining at corresponding concentrations.

[0365] Figure 9, panel B, shows high-magnification images of cells treated with the TAT-GG-ubiquitin-hFXN fusion protein. This image shows details of hFXN staining and its localization to mitochondria. This image shows that hFXN localizes to mitochondria in cells treated with the novel hFXN fusion protein.

[0366] The amount of hFXN signal in the photograph shown in Figure 9 was quantified by determining the average intensity of the Texas Red channel used to detect Alexa594-related signals. The amount of nuclear signal was quantified by determining the average intensity of the DAPI channel used to detect signals related to the Hoechst 33342 dye.

[0367] Figure 9, Panel C is a bar graph showing the ratio of the mean hFXN staining signal to the mean nuclear staining signal in cells treated with various concentrations of TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. The results shown in Figure 9, Panel C, indicate that the amount of hFXN detected in cells treated with the novel fusion proteins of this disclosure, namely TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN, is more than two orders of magnitude greater than the amount of hFXN detected in cells treated with TAT-GG-hFXN at all concentrations studied.

[0368] To further ensure the accuracy of hFXN quantification in microscopy experiments, purified recombinant hFXN fusion proteins used in cell transduction experiments were separated using SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were stained with total protein stain and visualized.

[0369] Figure 9, Panel D shows photographs of nitrocellulose membranes after transfer and total protein staining from SDS-PAGE gels loaded with samples of TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN used in transduction experiments. The results shown in Figure 9, Panel D demonstrate that similar amounts of TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN were found in the samples used for transduction. These results demonstrate the absence of fusion protein degradation and confirm that the same amount of each fusion protein was used in the transduction experiment. These results also demonstrate that the quantification results observed in the microscopic experiments in Figure 9, Panels A and B are not due to differences in the amount of hFXN fusion protein added to the cells.

[0370] The results shown in Figure 9, panels A-D, demonstrate that the amount of target protein, e.g., hFXN, detected in cells transduced with the novel fusion protein of this disclosure containing CPP and TES is significantly higher than the amount of target protein, e.g., hFXN, detected in cells transduced with a fusion protein containing CPP but not TES. The results indicate that introducing TES into a CPP-containing fusion protein, such that TES is positioned between CPP and the target protein, can significantly increase the amount of target protein in cells.

[0371] [Example 6] Transduction of Schwann cells and H9C2 cells using hFXN fusion protein The goal of this experiment was to determine and compare the ability of hFXN to localize in mitochondria after transduction of cells with various hFXN fusion proteins.

[0372] In this experiment, Schwann cells and H9C2 myoblasts were used, along with the same hFXN fusion proteins used in Example 5 and shown in Figure 8: TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. The cells were treated with hFXN fusion proteins at concentrations of 0.00625 μM, 0.125 μM, 0.25 μM, and 0.5 μM using the same procedure as described in Example 5. The cells were then stained using anti-FXN staining, nuclear staining, and mitochondrial staining, and analyzed using the microscopy method described in Example 5.

[0373] Figure 10, Panel A, shows a series of representative images of Schwann cells and H9C2 cells treated with 1 μM hFXN fusion protein and stained with mitochondrial staining, nuclear staining, and anti-FXN staining. White arrows indicate representative localization of high levels of hFXN to mitochondria. The results shown in Figure 10, Panel A, show that the amount of hFXN in cells treated with 1 μM TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN is greater than the amount of hFXN in cells treated with TAT-GG-hFXN. The results also show that in cells treated with 1 μM TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN, hFXN is localized to mitochondria (see white arrows), but in cells treated with 1 μM TAT-GG-hFXN, the observed mitochondrial localization of hFXN is less.

[0374] For each sample, the mean values ​​of the anti-FXN staining signal and the mean values ​​of the anti-mitochondrial staining signal were determined, and the ratio of the mean value of the anti-FXN staining signal to the mean value of the anti-mitochondrial staining signal (TOMM20) was calculated. Figure 10, Panel B is a graph showing the ratio of the mean value of the anti-FXN staining signal to the mean value of the anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in Schwann cells. Figure 10, Panel C is a graph showing the ratio of the mean value of the anti-FXN staining signal to the mean value of the anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in H9C2 cells. The results shown in Figure 10, Panels B and C demonstrate that Schwann cells and H9C2 cells treated with GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN localize a greater amount of hFXN to mitochondria compared to Schwann cells and H9C2 cells treated with TAT-GG-hFXN. Furthermore, the amount of mitochondrial localization in cells treated with GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN is dose-dependent.

[0375] Another cell translocation experiment was performed using Schwann cells and hFXN fusion proteins including TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and TAT-GG-hFXN NES1 (SEQ ID NO: 60, shown in Figure 8). The experimental protocol used was the same as described above. Figure 10, panel D is a graph showing the ratio of the mean anti-FXN staining signal to the mean anti-mitochondrial staining signal as a function of the hFXN fusion protein concentration in Schwann cells. The results shown in Figure 10, panel D demonstrate that Schwann cells treated with TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and TAT-GG-hFXN-NES1 localize a greater amount of hFXN to mitochondria compared to Schwann cells and H9C2 cells treated with TAT-GG-hFXN. The results also demonstrate that, at a fusion protein concentration of 0.5 μM, the amount of hFXN localized in mitochondria after treatment with TAT-GG-hFXN-NES1 was significantly higher than the amount of hFXN localized in mitochondria after treatment with TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN. Furthermore, the amount of mitochondrial localization in cells treated with the novel hFXN fusion protein was dose-dependent.

[0376] The results shown in Figure 10, panels A-D, demonstrate that cells treated with the novel fusion proteins of this disclosure—namely TAT-GG-EPLFAERK-hFXN, TAT-GG-ubiquitin-hFXN, and TAT-GG-hFXN-NES1—are detected in greater quantities of hFXN compared to cells treated with TAT-GG-hFXN. The results also demonstrate that hFXN in cells treated with the novel hFXN fusion proteins of this disclosure is localized to mitochondria.

[0377] [Example 7] Confirmation of intracellular processing of novel hFXN fusion proteins. The goal of this experiment was to confirm that the novel hFXN fusion proteins of this disclosure are correctly processed by cellular mechanisms to produce mature FXN proteins. This experiment used L6 rat myoblasts and the same hFXN fusion proteins used in Example 5 and shown in Figure 8: TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN. Two additional hFXN fusion proteins, TAT-GG-hFXN-NES1 (SEQ ID NO: 60) and TAT-GG-SUMO-hFXN (SEQ ID NO: 81), were also used. The amino acid sequence of TAT-GG-SUMO-hFXN (SEQ ID NO: 81) is shown below. TIFF2026108757000025.tif40166

[0378] For this experiment, L6 cells were transfused with a DNA vector encoding an hFXN fusion protein, and the cells were incubated for 24 hours. The cells were collected and processed to isolate total protein, which was then separated on an SDS-PAGE gel. After SDS-PAGE separation, frataxin in the gel was analyzed using Western blotting with an anti-FXN antibody and a control anti-β-actin antibody.

[0379] Figure 11 is a Western blot image of total protein samples isolated from cells transfected with various hFXN fusion proteins and analyzed using anti-FXN antibodies. Lane 1 contains a sample of cells transfected with a construct encoding an hFXN control without TAT fusion. In this sample, a band of approximately 15 kDa corresponds to matured and processed hFXN resulting from double cleavage of the MTS sequence by endogenous mitochondrial α and β-MPP proteases.

[0380] Lane 2 corresponds to a sample of cells transfected with a construct encoding the TAT-GG-hFXN fusion protein. In this sample, a small amount of hFXN appears as processed and matured frataxin, but the majority of hFXN exists as an unprocessed, full-length fusion protein of approximately 25 kDa.

[0381] Lanes 3, 4, and 5 correspond to cell samples transfected with constructs encoding TAT-SUMO-hFXN, TAT-GG-ubiquitin-hFXN, and TAT-GG-EPLFAERK-hFXN, respectively. Under the same experimental conditions, these samples contain more mature hFXN than the samples in lanes 1 and 2. Intermediate cleavage products corresponding to incomplete hFXN processing and various SUMO cleavage are also present in lanes 3-5 (lane 3).

[0382] Lane 6 contains a sample of cells transfected with a construct encoding TAT-GG-hFXN-NES. This sample also contains a greater amount of mature hFXN than the samples in lanes 1 and 2. Intermediate cleavage products corresponding to incomplete hFXN processing are also present in Lane 6. Note that the molecular weights of the mature hFXN and intermediate cleavage products are greater than those of the mature hFXN and intermediate cleavage products in the other samples, as NES is not cleaved by processing and leaves a portion of the fusion protein after MTS cleavage.

[0383] The results shown in Figure 11 demonstrate that the novel hFXN fusion proteins of this disclosure, namely TAT-SUMO-hFXN, TAT-GG-ubiquitin-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-hFXN-NES, are processed intracellularly to produce mature hFXN. The results also demonstrate that the processing of the novel hFXN fusion proteins that produce mature hFXN is more efficient than the processing of the TAT-GG-hFXN fusion protein. The more efficient processing of the novel hFXN fusion proteins compared to the processing of TAT-GG-hFXN can be at least partially explained by the fact that the amount of novel hFXN fusion proteins localized in the nucleus is less compared to that of the TAT-GG-hFXN fusion protein, as shown in Figures 4 and 6.

[0384] [Example 8] Functional characterization analysis of hFXN fusion proteins The goal of this experiment was to characterize the function of a novel hFXN fusion protein. The applicant has previously shown that cells lacking LRPPRC exhibit high levels of cell medium acidification and secretion of SYR61 protein into the medium. The applicant has also previously shown that treatment of LRPPRC-deficient cells induces a reduction in both cell medium acidification and SYR61 protein secretion (see, e.g., WO2021 / 011929, the entirety of which disclosure is incorporated herein by reference). Thus, cell medium acidification and SYR61 protein secretion can be used as measures of intracellular FXN activity.

[0385] This experiment uses the cell line HEK293LRPPRC KD (clone 21C), which was previously described, for example, in WO2021 / 011929. Briefly, this cell line is an LRPPRC knockdown cell line created by transfecting HEK293 cells with LRPPRC shRNA to induce LRPPRC gene silencing. A control cell line transfected with a scrambled sequence (Scr-5) was also used in this experiment. This experiment also used the same hFXN fusion proteins used in Example 5 and shown in Figure 8, namely TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, and TAT-GG-ubiquitin-hFXN.

[0386] For this experiment, LRPPRC KD cells and Scr-5 cells were plated in complete DMEM (DMEM, 10% FBS, and 1% antibiotic: antifungal substance) at a seeding density of 50,000 cells per well in 24-well plates. On day 1, cells were treated with the medium, TAT-GG-hFXN, TAT-GG-EPLFAERK-hFXN, or TAT-GG-ubiquitin-hFXN at 37°C for 3 hours. Cells were treated with TAT-GG-hFXN fusion protein at fusion protein concentrations of 2.5 μM, 5 μM, and 10 μM. Cells were treated with TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN at fusion protein concentrations of 0.25 μM, 0.5 μM, and 1 μM. After 3 hours, complete medium was added to each well, and the cells were incubated overnight at 37°C.

[0387] The same procedure was repeated on the second day, and the cells were incubated overnight at 37°C. On the third day, the cells were incubated for a further 48 hours in complete culture medium.

[0388] After 48 hours, the culture medium was collected. CYR61 levels were analyzed by CYR61 ELISA using the Human Cyr61 / CCN1 Quantikine ELISA Kit (R&D Systems, catalog number DCYR10). Lactate levels in the culture medium were determined using the Lactate glo Kit (#J5021, Promega Corporation) according to the manufacturer's protocol.

[0389] Figure 12, Panel A, shows a photograph of a culture plate containing samples of LRPPRC KD cells and Scr-5 cells treated with the hFXN fusion protein of this disclosure. Acidification of the cell culture medium is reflected in the change in the phenol red color in the culture medium, from pink (weak acidification) to yellow (strong acidification). Figure 12, Panel A also shows a schematic diagram of the sample in the cell culture plate shown in the photograph.

[0390] Figure 12, the results in Panel A demonstrate that LRPPRC KD cells induce cell medium acidification (demonstrated by the yellow medium in wells 1-3 of row 4 of the plate) and that the degree of medium acidification is reduced in a dose-dependent manner after treatment with the tested hFXN fusion protein (demonstrated by the orange medium in wells 1-3 of row 3 of the plate, the pink medium in wells 1-3 of row 2, and the darker pink or red medium in wells 1-3 of row 1). The results also demonstrate that TAT-GG-hFXN can prevent medium acidification at a concentration of 10 μM, while TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN can prevent medium acidification at significantly lower concentrations of 1 μM each, compared to TAT-GG-hFXN.

[0391] Figure 12, Panel B, shows a series of photographs of LRPPRC KD and Scr-5 control cells treated with the highest concentrations of each hFXN fusion protein or medium tested. The results in Figure 12, Panel B, demonstrate that the acidification of the cell culture medium was not due to cell death.

[0392] Figure 12, Panel C is a graph showing the amount of CYR61 in the culture medium of LRPPRC KD cells treated with hFXN fusion protein. The results in Figure 12, Panel C demonstrate concentration-dependent inhibition of CYR61 secretion by LRPPRC KD cells after treatment with hFXN fusion protein. The results also demonstrate that the inhibitory effect on CYR61 secretion is observed at significantly lower concentrations of TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN compared to TAT-GG-hFXN. This effect is demonstrated by the IC of CYR61 inhibition of TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN. 50 The values ​​are for the IC TAT-GG-hFXN shown in the table on panel C in Figure 12. 50 This is also reflected in the fact that it is lower compared to the value.

[0393] Figure 12, Panel D is a graph showing the amount of lactate in the culture medium of LRPPRC KD cells treated with hFXN fusion protein. The results in Figure 12, Panel D demonstrate concentration-dependent inhibition of lactate production by LRPPRC KD cells after treatment with hFXN fusion protein. The results also demonstrate that the inhibitory effect on lactate production is observed at significantly lower concentrations of TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN compared to TAT-GG-hFXN. This effect is supported by the IC50 of the inhibitory effect of TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN on lactate production. 50 The values ​​are for the IC TAT-GG-hFXN shown in the table on panel D in Figure 12. 50 This is also reflected in the fact that it is lower compared to the value.

[0394] The results shown in Figure 12, panels A-D, demonstrate that TAT-GG-EPLFAERK-hFXN and TAT-GG-ubiquitin-hFXN are characterized by higher activity measured by inhibition of lactate production and CYR61 secretion compared to TAT-GG-hFXN. The results indicate that introducing TES into the fusion protein enhances its function.

[0395] [Example 9] Design and synthesis of novel PARKIN fusion constructs The goal of this experiment was to synthesize a novel fusion protein described herein, which includes the human PARKIN protein (SEQ ID NO: 7).

[0396] The design of the PARKIN fusion protein was similar to that of the hFXN fusion construct described in Example 2. Some PARKIN fusion proteins included those in which a cell-permeable peptide (CPP), such as the HIV-TAT (YGRKKRRQRRR; SEQ ID NO: 11) peptide, was fused to an amino acid sequence that could be cleaved by a TES, such as an endogenous intracellular protease, followed immediately by the target protein, PARKIN. The dipeptide GG was used as a linker between the CPP and the proteolytic cleavage domain. When TAT-GG is cleaved from the fusion protein in the cytosol, a PARKIN protein without TAT is produced. The proteolytic cleavage domains used in the PARKIN fusion proteins were ubiquitin (SEQ ID NO: 18) and EPLFAERK (SEQ ID NO: 45).

[0397] The alternative PARKIN fusion protein was designed to include a nuclear export signal (NES) to avoid PARKIN accumulation in the cell nucleus. This construct was designed to include a CPP linked to the target protein, PARKIN, linked to the NES.

[0398] A schematic diagram of the PARKIN fusion protein is shown in Figure 13, and an overview of the fusion protein construct is shown in Table 13.

[0399] [Table 13] The amino acid sequence of His6-SUMO-TAT-GG-PARKIN (SEQ ID NO: 82), used as a control, is shown below. Note that in the control fusion protein His6-SUMO-TAT-GG-PARKIN (TIFF2026108757000027.tif42168), the His6-SUMO portion is present solely for ease of purification. After purification using the His6 tag (SEQ ID NO: 85), the His6 tag (SEQ ID NO: 85) is removed by protease-mediated cleavage of the SUMO sequence. Therefore, the control fusion protein of SEQ ID NO: 82 does not contain the TES sequence.

[0400] [Example 10] Transduction ability of PARKIN fusion proteins The goal of this experiment was to transduce cells and determine the ability of PARKIN fusion proteins to localize in mitochondria.

[0401] This experiment used L6 rat myoblasts. This was because these cells have low endogenous PARKIN levels and therefore do not interfere with the signal generated by the TAT-GG-hFXN fusion protein. This experiment also used the PARKIN fusion protein described in Example 9.

[0402] For this experiment, L6 cells were plated in complete DMEM medium (DMEM, 10% FBS, and 1% antibiotic: antifungal substance) at a seeding density of 8,000 cells per well in a 96-well plate (corning 3904) and incubated overnight at 37°C. On day 1, cells were treated with 0.0625 μM, 0.125 μM, 0.25 μM, 0.5 μM, and 1 μM of PARKIN fusion protein for 2 hours at 37°C. After 2 hours, the same amount of complete medium was added to each well and incubated overnight at 37°C. The same treatment was repeated on day 2.

[0403] On day 3, the cells were washed with PBS, triedpsinized, resuspended in complete cell culture medium, transferred to a fibronectin-coated glass-bottom plate, and deposited overnight at 37°C. On day 4, the cells were washed with PBS and treated with freshly prepared 4% paraformaldehyde solution at room temperature for 10 minutes. After 10 minutes, the cells were washed again with PBS, and then 50 μL of blocking buffer (PBS containing 0.3% Triton-X 100 and 5% normal goat serum) was added per well, and the cells were incubated at room temperature for 1 hour. After 1 hour, the blocking buffer was aspirated, and primary antibodies diluted in blocking buffer (anti-Parkin antibody 12235-1-AP (1:100) and anti-TOMM20 antibody EMD Millipore MABT166 (1:500)) were added per well, and the cells were incubated overnight at 4°C.

[0404] On day 5, the cells were washed twice with PBS, and 50 μL of secondary antibodies diluted in blocking buffer (anti-mouse IgG AlexaFluor594, ab150116 abcam(1:1000), and anti-rabbit IgG AlexaFluor488, ab150077 abcam(1:1000)) were added to each well. The cells were incubated at room temperature for 1 hour, then gently washed with PBS, mixed with 50 μL of 300 nM Hoescht 33342 stain, and allowed to stand at room temperature for 3 minutes. The cells were then washed twice with PBS and imaged. The ratio of PARKIN protein co-localized with mitochondria was evaluated using Harmony software on an Operetta (Perkin Elmer high-content imager), and the ratio of PARKIN localized to mitochondria to protein concentration (μM) was plotted. Specifically, the ratio of the amount of AlexaFluor488 stain (corresponding to PARKIN) to the amount of AlexaFluor594 stain (corresponding to mitochondria) was calculated and plotted on a graph to compare the transduction efficiency of various PARKIN fusion proteins.

[0405] The experimental results are shown in Figure 14. Specifically, Figure 14 is a graph showing the ratio of PARKIN staining to mitochondrial staining as a function of the PARKIN fusion protein concentration in cells treated with various concentrations of His6-SUMO-TAT-GG-PARKIN, TAT-GG-EPLFAERK-PARKIN, and TAT-GG-ubiquitin-PARKIN. The results shown in Figure 14 demonstrate that cell transduction by PARKIN fusion proteins occurs in a dose-dependent manner, with higher ratios indicating greater mitochondrial localization. The results show that PARKIN fusion proteins containing TES, namely TAT-GG-EPLFAERK-PARKIN and TAT-GG-ubiquitin-PARKIN, have greater mitochondrial localization at a concentration of 1 μM than the control PARKIN fusion protein His6-SUMO-TAT-GG-PARKIN, which does not contain TES. The results of this experiment indicate that the PARKIN fusion protein containing TES is transduced into cells and correctly localizes to mitochondria in greater quantities than the control His6-SUMO-TAT-PARKIN fusion protein.

[0406] [Example 11] Activity of PARKIN fusion protein The goal of this experiment was to test the activity of PARKIN in cells treated with a novel PARKIN fusion protein. Specifically, the goal was to determine whether PARKIN localizes to mitochondria and induces mitophagy in the presence of the mitochondrial uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) after treating cells with the novel PARKIN fusion protein.

[0407] For this experiment, L6 rat myoblasts were treated with 0 μM, 0.5 μM, and 1 μM His6-SUMO-TAT-GG-PARKIN, TAT-GG-EPLFAERK-PARKIN, or TAT-GG-ubiquitin-PARKIN in the presence and absence of 10 μM CCCP. Immunostaining and microscopy were then performed essentially as described in Example 10.

[0408] The experimental results for TAT-GG-EPLFAERK-PARKIN are shown in Figure 15. Specifically, Figure 15 is a series of photographs of L6 rat myoblasts treated with 0 μM or 0.5 μM TAT-GG-EPLFAERK-PARKIN in the absence or presence of 10 μM CCCP. The results shown in Figure 15 demonstrate that control L6 rat myoblasts show no morphological differences in the absence or presence of CCCP. Figure 15 also shows that endogenous PARKIN remains undetectable in L6 cells in the absence of treatment with the PARKIN fusion protein. The results shown in Figure 15 also show that localization of PARKIN to mitochondria is observed in L6 rat myoblasts treated with TAT-GG-EPLFAERK-PARKIN in the presence of CCCP. This observation is consistent with the known role of endogenous PARKIN in mitophagy, which involves mitochondrial relocalization of PARKIN to mitochondria during mitochondrial membrane depolarization. This disclosure includes the following embodiments. [1] The protein to be delivered to the cell, Cell-permeable peptides (CPPs), and Target-enhancing sequences (TES) A fusion protein containing [the specified ingredient]. [2] The fusion protein according to Embodiment 1, wherein CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP. [3] Starting from the N-terminus, CPP, TES, and Target protein A fusion protein according to Embodiment 1 or 2, comprising or comprising the above. [4] The fusion protein according to Embodiment 1, wherein CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP. [5] Starting from the C-terminus, The target protein, TES, and CPP A fusion protein according to Embodiment 1 or 4, comprising or comprising the above. [6] A fusion protein according to any one of embodiments 1 to 5, wherein the target protein lacks OTS. [7] The fusion protein according to Embodiment 6, further comprising an exogenous organelle transition sequence (OTS) for the target protein. [8] A fusion protein according to any one of Embodiments 1 to 5, wherein the target protein includes OTS. [9] The fusion protein according to Embodiment 8, wherein the OTS is heterogeneous to the target protein.

[10] The fusion protein according to Embodiment 8, wherein the OTS is endogenous to the target protein.

[11] A fusion protein according to any one of Embodiments 1 to 5, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and its variants or derivatives.

[12] The fusion protein of Embodiment 11, wherein the target protein comprises frataxin (FXN), PARK2 protein, or a variant or derivative thereof.

[13] A fusion protein according to any one of embodiments 1 to 12, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeability peptide database CPPsite2.0.

[14] A fusion protein according to any one of Embodiments 1 to 13, wherein the CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

[15] The fusion protein according to Embodiment 14, wherein CPP comprises HIV-TAT or a variant or derivative thereof.

[16] A fusion protein according to any one of embodiments 1 to 15, wherein TES is a nuclear export signal peptide.

[17] The fusion protein according to Embodiment 16, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs. 36-43.

[18] The fusion protein according to Embodiment 17, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives.

[19] The fusion protein according to Embodiment 18, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1 and NES2, or variants or derivatives thereof.

[20] A fusion protein according to any one of embodiments 1 to 19, wherein TES is a protease-sensitive peptide.

[21] The fusion protein according to Embodiment 20, wherein the protease-sensitive peptide contains a ubiquitin-like modifying factor.

[22] The fusion protein according to Embodiment 20, wherein the protease-sensitive peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18-31.

[23] The fusion protein according to Embodiment 20, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

[24] The fusion protein according to Embodiment 1, wherein the fusion protein contains an amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

[25] Proteins to be delivered to non-nuclear organelles, Organelle transition sequences (OTS), Cell-permeable peptides (CPPs), and Target-enhancing sequences (TES) Including CPP, CPP can interfere with the delivery of target proteins to non-nuclear organelles. TES prevents the aforementioned interference caused by CPP. Fusion protein.

[26] The fusion protein according to Embodiment 25, wherein CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP.

[27] Starting from the N-terminus, CPP, TES, OTS, and Target protein The fusion protein according to Embodiment 25 or 26, including

[28] The fusion protein according to Embodiment 25, wherein CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP.

[29] A fusion protein according to any one of embodiments 25 to 28, wherein the non-nuclear organelle is selected from the group consisting of mitochondria, cytosol, lysosome, endoplasmic reticulum (ER), peroxisome, and Golgi apparatus.

[30] The fusion protein according to Embodiment 25 or 26, wherein the target protein is localized in the mitochondria, cytosol, lysosome, endoplasmic reticulum (ER), peroxisome, or Golgi apparatus in its natural form.

[31] A fusion protein according to any one of Embodiments 25 to 30, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and its variants or derivatives.

[32] The fusion protein according to Embodiment 31, wherein the target protein is frataxin (FXN) or a variant or derivative thereof.

[33] The fusion protein according to Embodiment 31, wherein the target protein is pyruvate dehydrogenase (PDH) or a variant or derivative thereof.

[34] A fusion protein according to any of embodiments 25 to 33, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeability peptide database CPPsite2.0.

[35] A fusion protein according to any one of Embodiments 25 to 34, wherein the CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

[36] The fusion protein according to Embodiment 35, wherein CPP comprises HIV-TAT or a variant or derivative thereof.

[37] A fusion protein according to any one of embodiments 25 to 36, wherein TES is a nuclear export signaling peptide.

[38] The fusion protein according to Embodiment 37, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs. 36-43.

[39] The fusion protein according to Embodiment 38, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives.

[40] The fusion protein according to Embodiment 39, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, and their variants or derivatives.

[41] A fusion protein according to any one of embodiments 25 to 36, wherein TES is a protease-sensitive peptide.

[42] The fusion protein according to Embodiment 41, wherein the protease-sensitive peptide contains a ubiquitin-like modifying factor.

[43] The fusion protein according to Embodiment 41, wherein the protease-sensitive peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18-31.

[44] The fusion protein according to Embodiment 41, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

[45] A fusion protein according to any of embodiments 25 to 44, further comprising a secretory signal (SS).

[46] A fusion protein according to any one of embodiments 25 to 45, further comprising an extracellular proteolytic site (EPS).

[47] A fusion protein according to any one of embodiments 25 to 46, wherein the fusion protein delivers the target protein to a non-nuclear organelle.

[48] ​​A fusion protein according to any one of embodiments 25 to 47, wherein the fusion protein delivers the target protein to the mitochondria.

[49] The fusion protein according to Embodiment 25, wherein the fusion protein contains an amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

[50] A nucleic acid encoding a fusion protein as described in any of Embodiments 1 to 49.

[51] An expression vector for introducing a fusion protein into a cell, comprising the nucleic acid described in Embodiment 50.

[52] The expression vector according to Embodiment 51, wherein the expression vector is selected from the group consisting of retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, phagemids, baculoviruses, and combinations thereof.

[53] A conjugate for intracellular delivery of a protein to a nonnuclear organelle, comprising a fusion protein described in any of Embodiments 1 to 49 and a portion linked to the fusion protein, wherein the portion is selected from the group consisting of radiolabeled, fluorescently labeled, small molecule, and polymer molecule.

[54] The conjugate according to Embodiment 53, wherein the polymer molecule is polyethylene glycol (PEG).

[55] Cells comprising a fusion protein according to any of Embodiments 1 to 49, a nucleic acid according to Embodiment 50, an expression vector according to any of Embodiments 51 to 52, a conjugate according to any of Embodiments 53 to 54, or a combination thereof.

[56] The cell according to Embodiment 55, which is a stem cell or an iPS cell.

[57] The cell according to Embodiment 55, selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cell lines, or yeast cell lines.

[58] A pharmaceutical composition comprising a fusion protein according to any of Embodiments 1 to 49, a conjugate according to any of Embodiments 53 to 54, or a combination thereof, and a pharmaceutically acceptable diluent, carrier, additive, or excipient.

[59] A method for delivering a target protein to a cell, comprising contacting the cell with a fusion protein according to any of Embodiments 1 to 49, a nucleic acid according to Embodiment 50, a vector according to any of Embodiments 51 to 52, or a conjugate according to any of Embodiments 53 to 54.

[60] A method for intracellular delivery of a target protein to a non-nuclear organelle within a cell, comprising contacting the cell with a fusion protein according to any of Embodiments 1 to 49, a nucleic acid according to Embodiment 50, a vector according to any of Embodiments 51 to 52, or a conjugate according to any of Embodiments 53 to 54.

[61] The method according to embodiment 59, wherein the non-nuclear organelle is a mitochondria.

[62] A therapeutic compound for treating nonnuclear organelle-related disorders comprising a fusion protein according to any of Embodiments 1 to 49, a nucleic acid according to Embodiment 50, a vector according to any of Embodiments 51 to 52, or a conjugate according to any of Embodiments 53 to 54.

[63] The therapeutic compound according to Embodiment 62, wherein the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, fibrosis, and PLA2G6-associated neurodegenerative disease (PLAN).

[64] The therapeutic compound according to Embodiment 63, wherein the nonnuclear organelle-associated disorder is FDRA.

[65] The therapeutic compound according to Embodiment 63, wherein the nonnuclear organelle-associated disorder is Parkinson's disease.

[66] A method for treating a nonnuclear organelle-related disorder, comprising administering to a subject in need of such treatment a fusion protein according to any of Embodiments 1 to 49, a nucleic acid according to Embodiment 50, a vector according to any of Embodiments 51 to 52, a conjugate according to any of Embodiments 53 to 54, or a pharmaceutical composition according to Embodiment 58, such treatment of the nonnuclear organelle-related disorder.

[67] The method according to Embodiment 66, wherein the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, and fibrosis.

[68] The method according to embodiment 67, wherein the nonnuclear organelle-related disorder is FDRA.

[69] The method according to embodiment 67, wherein the nonnuclear organelle-associated disorder is Parkinson's disease.

[70] Any of the methods of embodiments 66 to 69, wherein the subject is a human.

[71] A method for increasing the amount of a target protein delivered to a cell, The steps include: modifying the sequence of a fusion protein containing a target protein and a cell-permeable peptide (CPP) by introducing a target-promoting sequence (TES) into the fusion protein, thereby generating a modified fusion protein; and Steps to bring cells into contact with the modified fusion protein. A method comprising, thereby increasing the amount of target protein delivered to cells compared to the amount of target protein delivered to cells by a fusion protein without TES.

[72] The method according to Embodiment 71, wherein in the modified fusion protein, CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP.

[73] The modified fusion protein starts from the N terminus, CPP, TES, and Target protein The method according to embodiment 71 or 72, including the method described in embodiment 71 or 72.

[74] The modified fusion protein starts from the N terminus, CPP, TES, OTS, and Target protein The method according to embodiment 71 or 72, including the method described in embodiment 71 or 72.

[75] The method according to Embodiment 71, wherein in the modified fusion protein, CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP.

[76] The method according to Embodiment 74, wherein the amount of target protein delivered to the organelle by OTS is increased compared to the amount of target protein delivered to the organelle by the fusion protein without TES.

[77] Any method according to Embodiments 71 to 76, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, and type VI phospholipase A2, or variants or derivatives thereof.

[78] The method of Embodiment 77, wherein the target protein is frataxin (FXN) or a variant or derivative thereof.

[79] The method of Embodiment 77, wherein the target protein is the PARK2 protein or a variant or derivative thereof.

[80] The method according to any one of embodiments 71 to 79, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.0.

[81] The method according to any one of embodiments 71 to 79, wherein the CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

[82] The method according to Embodiment 81, wherein the CPP comprises HIV-TAT or a variant or derivative thereof.

[83] Any method of Embodiments 71 to 82, wherein TES is a nuclear export signal peptide.

[84] The method according to Embodiment 83, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 36-43.

[85] The method according to Embodiment 84, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives.

[86] The method of Embodiment 85, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, and variants or derivatives thereof.

[87] The method according to any one of embodiments 71 to 82, wherein TES is a protease-sensitive peptide.

[88] The method of Embodiment 87, wherein the protease-sensitive peptide comprises a ubiquitin-like modifying factor.

[89] The method according to Embodiment 87, wherein the protease-sensitive peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18-31.

[90] The method according to Embodiment 87, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

[91] The method according to Embodiment 71, wherein the modified fusion protein comprises an amino acid sequence having at least 85% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81. SEQUENCE LISTING <110> LARIMAR THERAPEUTICS, INC. <120> MOLECULES FOR ORGANELLE-SPECIFIC PROTEIN DELIVERY <130> PA26-138 <150> 63 / 000,138 <151> 2020-03-26 <160> 88 <170> PatentIn version 3.5 <210> 1 <211> 210 <212> PRT <213> Homo sapiens <400> 1 Met Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro 1 5 10 15 Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu 20 25 30 Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala 35 40 45 Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln 50 55 60 Ile Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys 65 70 75 80 Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu 85 90 95 Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp 100 105 110 Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly 115 120 125 Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val 130 135 140 Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser 145 150 155 160 Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser 165 170 175 His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys 180 185 190 Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys 195 200 205 Asp Ala 210 <210> 2 <211> 130 <212> PRT <213> Homo sapiens <400> 2 Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu 1 5 10 15 Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp 20 25 30 Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly 35 40 45 Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val 50 55 60 Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser 65 70 75 80 Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser 85 90 95 His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys 100 105 110 Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys 115 120 125 Asp Ala 130 <210> 3 <211> 292 <212> PRT <213> Homo sapiens <400> 3 Met Pro Leu His Val Lys Trp Pro Phe Pro Ala Val Pro Pro Leu Thr 1 5 10 15 Trp Thr Leu Ala Ser Ser Val Val Met Gly Leu Val Gly Thr Tyr Ser 20 25 30 Cys Phe Trp Thr Lys Tyr Met Asn His Leu Thr Val His Asn Arg Glu 35 40 45 Val Leu Tyr Glu Leu Ile Glu Lys Arg Gly Pro Ala Thr Pro Leu Ile 50 55 60 Thr Val Ser Asn His Gln Ser Cys Met Asp Asp Pro His Leu Trp Gly 65 70 75 80 Ile Leu Lys Leu Arg His Ile Trp Asn Leu Lys Leu Met Arg Trp Thr 85 90 95 Pro Ala Ala Ala Asp Ile Cys Phe Thr Lys Glu Leu His Ser His Phe 100 105 110 Phe Ser Leu Gly Lys Cys Val Pro Val Cys Arg Gly Ala Glu Phe Phe 115 120 125 Gln Ala Glu Asn Glu Gly Lys Gly Val Leu Asp Thr Gly Arg His Met 130 135 140 Pro Gly Ala Gly Lys Arg Arg Glu Lys Gly Asp Gly Val Tyr Gln Lys 145 150 155 160 Gly Met Asp Phe Ile Leu Glu Lys Leu Asn His Gly Asp Trp Val His 165 170 175 Ile Phe Pro Glu Gly Lys Val Asn Met Ser Ser Glu Phe Leu Arg Phe 180 185 190 Lys Trp Gly Ile Gly Arg Leu Ile Ala Glu Cys His Leu Asn Pro Ile 195 200 205 Ile Leu Pro Leu Trp His Val Gly Met Asn Asp Val Leu Pro Asn Ser 210 215 220 Pro Pro Tyr Phe Pro Arg Phe Gly Gln Lys Ile Thr Val Leu Ile Gly 225 230 235 240 Lys Pro Phe Ser Ala Leu Pro Val Leu Glu Arg Leu Arg Ala Glu Asn 245 250 255 Lys Ser Ala Val Glu Met Arg Lys Ala Leu Thr Asp Phe Ile Gln Glu 260 265 270 Glu Phe Gln His Leu Lys Thr Gln Ala Glu Gln Leu His Asn His Leu 275 280 285 Gln Pro Gly Arg 290 <210> 4 <211> 359 <212> PRT <213> Homo sapiens <400> 4 Met Ala Ala Val Ser Gly Leu Val Arg Arg Pro Leu Arg Glu Val Ser 1 5 10 15 Gly Leu Leu Lys Arg Arg Phe His Trp Thr Ala Pro Ala Ala Leu Gln 20 25 30 Val Thr Val Arg Asp Ala Ile Asn Gln Gly Met Asp Glu Glu Leu Glu 35 40 45 Arg Asp Glu Lys Val Phe Leu Leu Gly Glu Glu Val Ala Gln Tyr Asp 50 55 60 Gly Ala Tyr Lys Val Ser Arg Gly Leu Trp Lys Lys Tyr Gly Asp Lys 65 70 75 80 Arg Ile Ile Asp Thr Pro Ile Ser Glu Met Gly Phe Ala Gly Ile Ala 85 90 95 Val Gly Ala Ala Met Ala Gly Leu Arg Pro Ile Cys Glu Phe Met Thr 100 105 110 Phe Asn Phe Ser Met Gln Ala Ile Asp Gln Val Ile Asn Ser Ala Ala 115 120 125 Lys Thr Tyr Tyr Met Ser Gly Gly Leu Gln Pro Val Pro Ile Val Phe 130 135 140 Arg Gly Pro Asn Gly Ala Ser Ala Gly Val Ala Ala Gln His Ser Gln 145 150 155 160 Cys Phe Ala Ala Trp Tyr Gly His Cys Pro Gly Leu Lys Val Val Ser 165 170 175 Pro Trp Asn Ser Glu Asp Ala Lys Gly Leu Ile Lys Ser Ala Ile Arg 180 185 190 Asp Asn Asn Pro Val Val Val Leu Glu Asn Glu Leu Met Tyr Gly Val 195 200 205 Pro Phe Glu Phe Pro Pro Glu Ala Gln Ser Lys Asp Phe Leu Ile Pro 210 215 220 Ile Gly Lys Ala Lys Ile Glu Arg Gln Gly Thr His Ile Thr Val Val 225 230 235 240 Ser His Ser Arg Pro Val Gly His Cys Leu Glu Ala Ala Ala Val Leu 245 250 255 Ser Lys Glu Gly Val Glu Cys Glu Val Ile Asn Met Arg Thr Ile Arg 260 265 270 Pro Met Asp Met Glu Thr Ile Glu Ala Ser Val Met Lys Thr Asn His 275 280 285 Leu Val Thr Val Glu Gly Gly Trp Pro Gln Phe Gly Val Gly Ala Glu 290 295 300 Ile Cys Ala Arg Ile Met Glu Gly Pro Ala Phe Asn Phe Leu Asp Ala 305 310 315 320 Pro Ala Val Arg Val Thr Gly Ala Asp Val Pro Met Pro Tyr Ala Lys 325 330 335 Ile Leu Glu Asp Asn Ser Ile Pro Gln Val Lys Asp Ile Ile Phe Ala 340 345 350 Ile Lys Lys Thr Leu Asn Ile 355 <210> 5 <211> 1394 <212> PRT <213> Homo sapiens <400> 5 Met Ala Ala Leu Leu Arg Ser Ala Arg Trp Leu Leu Arg Ala Gly Ala 1 5 10 15 Ala Pro Arg Leu Pro Leu Ser Leu Arg Leu Leu Pro Gly Gly Pro Gly 20 25 30 Arg Leu His Ala Ala Ser Tyr Leu Pro Ala Ala Arg Ala Gly Pro Val 35 40 45 Ala Gly Gly Leu Leu Ser Pro Ala Arg Leu Tyr Ala Ile Ala Ala Lys 50 55 60 Glu Lys Asp Ile Gln Glu Glu Ser Thr Phe Ser Ser Arg Lys Ile Ser 65 70 75 80 Asn Gln Phe Asp Trp Ala Leu Met Arg Leu Asp Leu Ser Val Arg Arg 85 90 95 Thr Gly Arg Ile Pro Lys Lys Leu Leu Gln Lys Val Phe Asn Asp Thr 100 105 110 Cys Arg Ser Gly Gly Leu Gly Gly Ser His Ala Leu Leu Leu Leu Arg 115 120 125 Ser Cys Gly Ser Leu Leu Pro Glu Leu Lys Leu Glu Glu Arg Thr Glu 130 135 140 Phe Ala His Arg Ile Trp Asp Thr Leu Gln Lys Leu Gly Ala Val Tyr 145 150 155 160 Asp Val Ser His Tyr Asn Ala Leu Leu Lys Val Tyr Leu Gln Asn Glu 165 170 175 Tyr Lys Phe Ser Pro Thr Asp Phe Leu Ala Lys Met Glu Glu Ala Asn 180 185 190 Ile Gln Pro Asn Arg Val Thr Tyr Gln Arg Leu Ile Ala Ser Tyr Cys 195 200 205 Asn Val Gly Asp Ile Glu Gly Ala Ser Lys Ile Leu Gly Phe Met Lys 210 215 220 Thr Lys Asp Leu Pro Val Thr Glu Ala Val Phe Ser Ala Leu Val Thr 225 230 235 240 Gly His Ala Arg Ala Gly Asp Met Glu Asn Ala Glu Asn Ile Leu Thr 245 250 255 Val Met Arg Asp Ala Gly Ile Glu Pro Gly Pro Asp Thr Tyr Leu Ala 260 265 270 Leu Leu Asn Ala Tyr Ala Glu Lys Gly Asp Ile Asp His Val Lys Gln 275 280 285 Thr Leu Glu Lys Val Glu Lys Ser Glu Leu His Leu Met Asp Arg Asp 290 295 300 Leu Leu Gln Ile Ile Phe Ser Phe Ser Lys Ala Gly Tyr Pro Gln Tyr 305 310 315 320 Val Ser Glu Ile Leu Glu Lys Val Thr Cys Glu Arg Arg Tyr Ile Pro 325 330 335 Asp Ala Met Asn Leu Ile Leu Leu Leu Val Thr Glu Lys Leu Glu Asp 340 345 350 Val Ala Leu Gln Ile Leu Leu Ala Cys Pro Val Ser Lys Glu Asp Gly 355 360 365 Pro Ser Val Phe Gly Ser Phe Phe Leu Gln His Cys Val Thr Met Asn 370 375 380 Thr Pro Val Glu Lys Leu Thr Asp Tyr Cys Lys Lys Leu Lys Glu Val 385 390 395 400 Gln Met His Ser Phe Pro Leu Gln Phe Thr Leu His Cys Ala Leu Leu 405 410 415 Ala Asn Lys Thr Asp Leu Ala Lys Ala Leu Met Lys Ala Val Lys Glu 420 425 430 Glu Gly Phe Pro Ile Arg Pro His Tyr Phe Trp Pro Leu Leu Val Gly 435 440 445 Arg Arg Lys Glu Lys Asn Val Gln Gly Ile Ile Glu Ile Leu Lys Gly 450 455 460 Met Gln Glu Leu Gly Val His Pro Asp Gln Glu Thr Tyr Thr Asp Tyr 465 470 475 480 Val Ile Pro Cys Phe Asp Ser Val Asn Ser Ala Arg Ala Ile Leu Gln 485 490 495 Glu Asn Gly Cys Leu Ser Asp Ser Asp Met Phe Ser Gln Ala Gly Leu 500 505 510 Arg Ser Glu Ala Ala Asn Gly Asn Leu Asp Phe Val Leu Ser Phe Leu 515 520 525 Lys Ser Asn Thr Leu Pro Ile Ser Leu Gln Ser Ile Arg Ser Ser Leu 530 535 540 Leu Leu Gly Phe Arg Arg Ser Met Asn Ile Asn Leu Trp Ser Glu Ile 545 550 555 560 Thr Glu Leu Leu Tyr Lys Asp Gly Arg Tyr Cys Gln Glu Pro Arg Gly 565 570 575 Pro Thr Glu Ala Val Gly Tyr Phe Leu Tyr Asn Leu Ile Asp Ser Met 580 585 590 Ser Asp Ser Glu Val Gln Ala Lys Glu Glu His Leu Arg Gln Tyr Phe 595 600 605 His Gln Leu Glu Lys Met Asn Val Lys Ile Pro Glu Asn Ile Tyr Arg 610 615 620 Gly Ile Arg Asn Leu Leu Glu Ser Tyr His Val Pro Glu Leu Ile Lys 625 630 635 640 Asp Ala His Leu Leu Val Glu Ser Lys Asn Leu Asp Phe Gln Lys Thr 645 650 655 Val Gln Leu Thr Ser Ser Glu Leu Glu Ser Thr Leu Glu Thr Leu Lys 660 665 670 Ala Glu Asn Gln Pro Ile Arg Asp Val Leu Lys Gln Leu Ile Leu Val 675 680 685 Leu Cys Ser Glu Glu Asn Met Gln Lys Ala Leu Glu Leu Lys Ala Lys 690 695 700 Tyr Glu Ser Asp Met Val Thr Gly Gly Tyr Ala Ala Leu Ile Asn Leu 705 710 715 720 Cys Cys Arg His Asp Lys Val Glu Asp Ala Leu Asn Leu Lys Glu Glu 725 730 735 Phe Asp Arg Leu Asp Ser Ser Ala Val Leu Asp Thr Gly Lys Tyr Val 740 745 750 Gly Leu Val Arg Val Leu Ala Lys His Gly Lys Leu Gln Asp Ala Ile 755 760 765 Asn Ile Leu Lys Glu Met Lys Glu Lys Asp Val Leu Ile Lys Asp Thr 770 775 780 Thr Ala Leu Ser Phe Phe His Met Leu Asn Gly Ala Ala Leu Arg Gly 785 790 795 800 Glu Ile Glu Thr Val Lys Gln Leu His Glu Ala Ile Val Thr Leu Gly 805 810 815 Leu Ala Glu Pro Ser Thr Asn Ile Ser Phe Pro Leu Val Thr Val His 820 825 830 Leu Glu Lys Gly Asp Leu Ser Thr Ala Leu Glu Val Ala Ile Asp Cys 835 840 845 Tyr Glu Lys Tyr Lys Val Leu Pro Arg Ile His Asp Val Leu Cys Lys 850 855 860 Leu Val Glu Lys Gly Glu Thr Asp Leu Ile Gln Lys Ala Met Asp Phe 865 870 875 880 Val Ser Gln Glu Gln Gly Glu Met Val Met Leu Tyr Asp Leu Phe Phe 885 890 895 Ala Phe Leu Gln Thr Gly Asn Tyr Lys Glu Ala Lys Lys Ile Ile Glu 900 905 910 Thr Pro Gly Ile Arg Ala Arg Ser Ala Arg Leu Gln Trp Phe Cys Asp 915 920 925 Arg Cys Val Ala Asn Asn Gln Val Glu Thr Leu Glu Lys Leu Val Glu 930 935 940 Leu Thr Gln Lys Leu Phe Glu Cys Asp Arg Asp Gln Met Tyr Tyr Asn 945 950 955 960 Leu Leu Lys Leu Tyr Lys Ile Asn Gly Asp Trp Gln Arg Ala Asp Ala 965 970 975 Val Trp Asn Lys Ile Gln Glu Glu Asn Val Ile Pro Arg Glu Lys Thr 980 985 990 Leu Arg Leu Leu Ala Glu Ile Leu Arg Glu Gly Asn Gln Glu Val Pro 995 1000 1005 Phe Asp Val Pro Glu Leu Trp Tyr Glu Asp Glu Lys His Ser Leu 1010 1015 1020 Asn Ser Ser Ser Ala Ser Thr Thr Glu Pro Asp Phe Gln Lys Asp 1025 1030 1035 Ile Leu Ile Ala Cys Arg Leu Asn Gln Lys Lys Gly Ala Tyr Asp 1040 1045 1050 Ile Phe Leu Asn Ala Lys Glu Gln Asn Ile Val Phe Asn Ala Glu 1055 1060 1065 Thr Tyr Ser Asn Leu Ile Lys Leu Leu Met Ser Glu Asp Tyr Phe 1070 1075 1080 Thr Gln Ala Met Glu Val Lys Ala Phe Ala Glu Thr His Ile Lys 1085 1090 1095 Gly Phe Thr Leu Asn Asp Ala Ala Asn Ser Arg Leu Ile Ile Thr 1100 1105 1110 Gln Val Arg Arg Asp Tyr Leu Lys Glu Ala Val Thr Thr Leu Lys 1115 1120 1125 Thr Val Leu Asp Gln Gln Gln Thr Pro Ser Arg Leu Ala Val Thr 1130 1135 1140 Arg Val Ile Gln Ala Leu Ala Met Lys Gly Asp Val Glu Asn Ile 1145 1150 1155 Glu Val Val Gln Lys Met Leu Asn Gly Leu Glu Asp Ser Ile Gly 1160 1165 1170 Leu Ser Lys Met Val Phe Ile Asn Asn Ile Ala Leu Ala Gln Ile 1175 1180 1185 Lys Asn Asn Asn Ile Asp Ala Ala Ile Glu Asn Ile Glu Asn Met 1190 1195 1200 Leu Thr Ser Glu Asn Lys Val Ile Glu Pro Gln Tyr Phe Gly Leu 1205 1210 1215 Ala Tyr Leu Phe Arg Lys Val Ile Glu Glu Gln Leu Glu Pro Ala 1220 1225 1230 Val Glu Lys Ile Ser Ile Met Ala Glu Arg Leu Ala Asn Gln Phe 1235 1240 1245 Ala Ile Tyr Lys Pro Val Thr Asp Phe Phe Leu Gln Leu Val Asp 1250 1255 1260 Ala Gly Lys Val Asp Asp Ala Arg Ala Leu Leu Gln Arg Cys Gly 1265 1270 1275 Ala Ile Ala Glu Gln Thr Pro Ile Leu Leu Leu Phe Leu Leu Arg 1280 1285 1290 Asn Ser Arg Lys Gln Gly Lys Ala Ser Thr Val Lys Ser Val Leu 1295 1300 1305 Glu Leu Ile Pro Glu Leu Asn Glu Lys Glu Glu Ala Tyr Asn Ser 1310 1315 1320 Leu Met Lys Ser Tyr Val Ser Glu Lys Asp Val Thr Ser Ala Lys 1325 1330 1335 Ala Leu Tyr Glu His Leu Thr Ala Lys Asn Thr Lys Leu Asp Asp 1340 1345 1350 Leu Phe Leu Lys Arg Tyr Ala Ser Leu Leu Lys Tyr Ala Gly Glu 1355 1360 1365 Pro Val Pro Phe Ile Glu Pro Pro Glu Ser Phe Glu Phe Tyr Ala 1370 1375 1380 Gln Gln Leu Arg Lys Leu Arg Glu Asn Ser Ser 1385 1390 <210> 6 <211> 109 <212> PRT <213> Homo sapiens <400> 6 Met Ala Ala Ser Ala Ala Arg Gly Ala Ala Ala Leu Arg Arg Ser Ile 1 5 10 15 Asn Gln Pro Val Ala Phe Val Arg Arg Ile Pro Trp Thr Ala Ala Ser 20 25 30 Ser Gln Leu Lys Glu His Phe Ala Gln Phe Gly His Val Arg Arg Cys 35 40 45 Ile Leu Pro Phe Asp Lys Glu Thr Gly Phe His Arg Gly Leu Gly Trp 50 55 60 Val Gln Phe Ser Ser Glu Glu Gly Leu Arg Asn Ala Leu Gln Gln Glu 65 70 75 80 Asn His Ile Ile Asp Gly Val Lys Val Gln Val His Thr Arg Arg Pro 85 90 95 Lys Leu Pro Gln Thr Ser Asp Asp Glu Lys Lys Asp Phe 100 105 <210> 7 <211> 465 <212> PRT <213> Homo sapiens <400> 7 Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu 1 5 10 15 Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys 20 25 30 Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys 35 40 45 Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln 50 55 60 Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met 65 70 75 80 Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu 85 90 95 Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu 100 105 110 Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg 115 120 125 Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn 130 135 140 Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly 145 150 155 160 Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu 165 170 175 Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met 180 185 190 Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe 195 200 205 Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val 210 215 220 Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr 225 230 235 240 Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg 245 250 255 His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu 260 265 270 Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro 275 280 285 Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe 290 295 300 Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala 305 310 315 320 Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly 325 330 335 Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys 340 345 350 Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys 355 360 365 Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser 370 375 380 Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala Glu Gln 385 390 395 400 Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys 405 410 415 Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met 420 425 430 His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn 435 440 445 Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp 450 455 460 Val 465 <210> 8 <211> 581 <212> PRT <213> Homo sapiens <400> 8 Met Ala Val Arg Gln Ala Leu Gly Arg Gly Leu Gln Leu Gly Arg Ala 1 5 10 15 Leu Leu Leu Arg Phe Thr Gly Lys Pro Gly Arg Ala Tyr Gly Leu Gly 20 25 30 Arg Pro Gly Pro Ala Ala Gly Cys Val Arg Gly Glu Arg Pro Gly Trp 35 40 45 Ala Ala Gly Pro Gly Ala Glu Pro Arg Arg Val Gly Leu Gly Leu Pro 50 55 60 Asn Arg Leu Arg Phe Phe Arg Gln Ser Val Ala Gly Leu Ala Ala Arg 65 70 75 80 Leu Gln Arg Gln Phe Val Val Arg Ala Trp Gly Cys Ala Gly Pro Cys 85 90 95 Gly Arg Ala Val Phe Leu Ala Phe Gly Leu Gly Leu Gly Leu Ile Glu 100 105 110 Glu Lys Gln Ala Glu Ser Arg Arg Ala Val Ser Ala Cys Gln Glu Ile 115 120 125 Gln Ala Ile Phe Thr Gln Lys Ser Lys Pro Gly Pro Asp Pro Leu Asp 130 135 140 Thr Arg Arg Leu Gln Gly Phe Arg Leu Glu Glu Tyr Leu Ile Gly Gln 145 150 155 160 Ser Ile Gly Lys Gly Cys Ser Ala Ala Val Tyr Glu Ala Thr Met Pro 165 170 175 Thr Leu Pro Gln Asn Leu Glu Val Thr Lys Ser Thr Gly Leu Leu Pro 180 185 190 Gly Arg Gly Pro Gly Thr Ser Ala Pro Gly Glu Gly Gln Glu Arg Ala 195 200 205 Pro Gly Ala Pro Ala Phe Pro Leu Ala Ile Lys Met Met Trp Asn Ile 210 215 220 Ser Ala Gly Ser Ser Ser Glu Ala Ile Leu Asn Thr Met Ser Gln Glu 225 230 235 240 Leu Val Pro Ala Ser Arg Val Ala Leu Ala Gly Glu Tyr Gly Ala Val 245 250 255 Thr Tyr Arg Lys Ser Lys Arg Gly Pro Lys Gln Leu Ala Pro His Pro 260 265 270 Asn Ile Ile Arg Val Leu Arg Ala Phe Thr Ser Ser Val Pro Leu Leu 275 280 285 Pro Gly Ala Leu Val Asp Tyr Pro Asp Val Leu Pro Ser Arg Leu His 290 295 300 Pro Glu Gly Leu Gly His Gly Arg Thr Leu Phe Leu Val Met Lys Asn 305 310 315 320 Tyr Pro Cys Thr Leu Arg Gln Tyr Leu Cys Val Asn Thr Pro Ser Pro 325 330 335 Arg Leu Ala Ala Met Met Leu Leu Gln Leu Leu Glu Gly Val Asp His 340 345 350 Leu Val Gln Gln Gly Ile Ala His Arg Asp Leu Lys Ser Asp Asn Ile 355 360 365 Leu Val Glu Leu Asp Pro Asp Gly Cys Pro Trp Leu Val Ile Ala Asp 370 375 380 Phe Gly Cys Cys Leu Ala Asp Glu Ser Ile Gly Leu Gln Leu Pro Phe 385 390 395 400 Ser Ser Trp Tyr Val Asp Arg Gly Gly Asn Gly Cys Leu Met Ala Pro 405 410 415 Glu Val Ser Thr Ala Arg Pro Gly Pro Arg Ala Val Ile Asp Tyr Ser 420 425 430 Lys Ala Asp Ala Trp Ala Val Gly Ala Ile Ala Tyr Glu Ile Phe Gly 435 440 445 Leu Val Asn Pro Phe Tyr Gly Gln Gly Lys Ala His Leu Glu Ser Arg 450 455 460 Ser Tyr Gln Glu Ala Gln Leu Pro Ala Leu Pro Glu Ser Val Pro Pro 465 470 475 480 Asp Val Arg Gln Leu Val Arg Ala Leu Leu Gln Arg Glu Ala Ser Lys 485 490 495 Arg Pro Ser Ala Arg Val Ala Ala Asn Val Leu His Leu Ser Leu Trp 500 505 510 Gly Glu His Ile Leu Ala Leu Lys Asn Leu Lys Leu Asp Lys Met Val 515 520 525 Gly Trp Leu Leu Gln Gln Ser Ala Ala Thr Leu Leu Ala Asn Arg Leu 530 535 540 Thr Glu Lys Cys Cys Val Glu Thr Lys Met Lys Met Leu Phe Leu Ala 545 550 555 560 Asn Leu Glu Cys Glu Thr Leu Cys Gln Ala Ala Leu Leu Leu Cys Ser 565 570 575 Trp Arg Ala Ala Leo 580 <210> 9 <211> 189 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Lys Arg Ala Leu Val Ile Leu Ala Lys Gly Ala Glu Glu 1 5 10 15 Met Glu Thr Val Ile Pro Val Asp Val Met Arg Arg Ala Gly Ile Lys 20 25 30 Val Thr Val Ala Gly Leu Ala Gly Lys Asp Pro Val Gln Cys Ser Arg 35 40 45 Asp Val Val Ile Cys Pro Asp Ala Ser Leu Glu Asp Ala Lys Lys Glu 50 55 60 Gly Pro Tyr Asp Val Val Val Leu Pro Gly Gly Asn Leu Gly Ala Gln 65 70 75 80 Asn Leu Ser Glu Ser Ala Ala Val Lys Glu Ile Leu Lys Glu Gln Glu 85 90 95 Asn Arg Lys Gly Leu Ile Ala Ala Ile Cys Ala Gly Pro Thr Ala Leu 100 105 110 Leu Ala His Glu Ile Gly Phe Gly Ser Lys Val Thr Thr His Pro Leu 115 120 125 Ala Lys Asp Lys Met Met Asn Gly Gly His Tyr Thr Tyr Ser Glu Asn 130 135 140 Arg Val Glu Lys Asp Gly Leu Ile Leu Thr Ser Arg Gly Pro Gly Thr 145 150 155 160 Ser Phe Glu Phe Ala Leu Ala Ile Val Glu Ala Leu Asn Gly Lys Glu 165 170 175 Val Ala Ala Gln Val Lys Ala Pro Leu Val Leu Lys Asp 180 185 <210> 10 <211> 80 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 10 Met Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro 1 5 10 15 Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu 20 25 30 Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala 35 40 45 Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln 50 55 60 Ile Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys 65 70 75 80 <210> 11 <211> 11 <212> PRT <213> Human immunodeficiency virus <400> 11 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10 <210> 12 <211> 30 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 12 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Pro His Ala Val 1 5 10 15 Gly Asn His Arg Ser Phe Ser Asp Lys Asn Gly Leu Thr Ser 20 25 30 <210> 13 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 13 Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 1 5 10 <210> 14 <211> 27 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 14 Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15 Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25 <210> 15 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 15 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 16 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 17 <211> 34 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 17 Asp Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1 5 10 15 Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg Pro 20 25 30 Val Glu <210> 18 <211> 76 <212> PRT <213> Homo sapiens <400> 18 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 <210> 19 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: caspase cleavage domain sequence" <400> 19 Asp Glu Val Asp 1 <210> 20 <211> 8 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: calpain cleavage domain sequence" <400> 20 Glu Pro Leu Phe Ala Glu Arg Lys 1 5 <210> 21 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: calpain cleavage domain sequence" <400> 21 Leu Leu Val Tyr 1 <210> 22 <211> 5 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Enterokinase protease cleavage site sequence" <400> 22 Asp Asp Asp Asp Lys 1 5 <210> 23 <211> 7 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: human Rhinovirus protease cleavage site sequence" <400> 23 Leu Glu Val Leu Phe Gln Pro 1 5 <210> 24 <211> 7 <212> PRT <213> Tobacco Etch Virus <400> 24 Leu Glu Val Leu Phe Gly Pro 1 5 <210> 25 <211> 7 <212> PRT <213> Tobacco Etch Virus <400> 25 Glu Asn Leu Tyr Phe Gln Ser 1 5 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <220> <221> MOD_RES <222> (2)..(3) <223> Any amino acid <220> <221> MOD_RES <222> (5)..(5) <223> Any amino acid <400> 26 Glu Xaa Xaa Tyr Xaa Gln Gly 1 5 <210> 27 <211> 7 <212> PRT <213> Tobacco vein mottling virus <400> 27 Glu Thr Val Arg Phe Gln Ser 1 5 <210> 28 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Factor Xa protease cleavage site sequence" <400> 28 Ile Glu Gly Arg 1 <210> 29 <211> 4 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Factor Xa protease cleavage site sequence" <400> 29 Ile Asp Gly Arg 1 <210> 30 <211> 5 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Thrombin cleavage site sequence" <400> 30 Leu Val Pro Arg Ser 1 5 <210> 31 <211> 5 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: Thrombin cleavage site sequence" <400> 31 Leu Val Pro Gly Ser 1 5 <210> 32 <211> 97 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: SUMO1 sequence" <400> 32 Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys 1 5 10 15 Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser 20 25 30 Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys 35 40 45 Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe 50 55 60 Leu Phe Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro Lys Glu Leu 65 70 75 80 Gly Met Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu Gln Thr Gly 85 90 95 Gly <210> 33 <211> 95 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: SUMO2 sequence" <400> 33 Met Ala Asp Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asn Asp 1 5 10 15 His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe 20 25 30 Lys Ile Lys Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys 35 40 45 Glu Arg Gln Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly 50 55 60 Gln Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp 65 70 75 80 Glu Asp Thr Ile Asp Val Phe Gln Gln Gln Thr Gly Gly Val Tyr 85 90 95 <210> 34 <211> 103 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: SUMO3 sequence" <400> 34 Met Ser Glu Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asp His 1 5 10 15 Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe Lys 20 25 30 Ile Lys Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys Glu 35 40 45 Arg Gln Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly Gln 50 55 60 Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp Glu 65 70 75 80 Asp Thr Ile Asp Val Phe Gln Gln Gln Thr Gly Gly Val Pro Glu Ser 85 90 95 Ser Leu Ala Gly His Ser Phe 100 <210> 35 <211> 95 <212> PRT <213> Unknown <220> <221> source <223> / note="Description of Unknown: SUMO4 sequence" <400> 35 Met Ala Asn Glu Lys Pro Thr Glu Glu Val Lys Thr Glu Asn Asn Asn 1 5 10 15 His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe 20 25 30 Lys Ile Lys Arg Gln Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys 35 40 45 Glu Pro Arg Gly Leu Ser Met Lys Gln Ile Arg Phe Arg Phe Gly Gly 50 55 60 Gln Pro Ile Ser Gly Thr Asp Lys Pro Ala Gln Leu Glu Met Glu Asp 65 70 75 80 Glu Asp Thr Ile Asp Val Phe Gln Gln Pro Thr Gly Gly Val Tyr 85 90 95 <210> 36 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 36 Leu Ala Leu Lys Leu Ala Gly Leu Asp Leu 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 37 Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu 1 5 10 <210> 38 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 38 Met Gln Glu Leu Ser Asn Ile Leu Asn Leu 1 5 10 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 39 Leu Pro Pro Leu Glu Arg Leu Thr Leu 1 5 <210> 40 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 40 Leu Cys Gln Ala Phe Ser Asp Val Ile Leu 1 5 10 <210> 41 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 41 Arg Thr Phe Asp Met His Ser Leu Glu Ser Ser Leu Ile Asp Ile Met 1 5 10 15 Arg <210> 42 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 42 Thr Asn Leu Glu Ala Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp 1 5 10 15 Glu <210> 43 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 43 Arg Ser Phe Glu Met Thr Glu Phe Asn Gln Ala Leu Glu Glu Ile Lys 1 5 10 15 Gly <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 44 Gly Gly Gly Gly Ser Leu Val Pro Arg Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 45 <211> 6 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 45 Gly Ser Gly Ser Gly Ser 1 5 <210> 46 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 46 Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser 1 5 10 15 <210> 47 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 47 Gly Gly Ser Gly Gly His Met Gly Ser Gly Gly 1 5 10 <210> 48 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 48 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 1 5 10 <210> 49 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 49 Gly Gly Ser Gly Gly 1 5 <210> 50 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 50 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 1 5 10 15 Gly Gly <210> 51 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 51 Ala Ala Gly Ala Ala Thr Ala Ala 1 5 <210> 52 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 52 Gly Gly Gly Gly Gly 1 5 <210> 53 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 53 Gly Gly Ser Ser Gly 1 5 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic peptide" <400> 54 Gly Ser Gly Gly Gly Thr Gly Gly Gly Ser Gly 1 5 10 <210> 55 <211> 321 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 55 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Met Ser 1 5 10 15 Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys Lys Glu 20 25 30 Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser Glu Ile 35 40 45 His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys Glu Ser 50 55 60 Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe Leu Phe 65 70 75 80 Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro Lys Glu Leu Gly Met 85 90 95 Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu Gln Thr Gly Gly Met 100 105 110 Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro Ser 115 120 125 Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu Leu 130 135 140 Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr 145 150 155 160 Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile 165 170 175 Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys Ser 180 185 190 Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu Arg 195 200 205 Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp Leu 210 215 220 Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly Ser 225 230 235 240 Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val Ile 245 250 255 Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser 260 265 270 Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His 275 280 285 Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala 290 295 300 Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp 305 310 315 320 Ala <210> 56 <211> 300 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 56 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Met Gln 1 5 10 15 Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu 20 25 30 Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu 35 40 45 Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu 50 55 60 Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr 65 70 75 80 Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Trp Thr Leu Gly Arg 85 90 95 Arg Ala Val Ala Gly Leu Leu Ala Ser Pro Ser Pro Ala Gln Ala Gln 100 105 110 Thr Leu Thr Arg Val Pro Arg Pro Ala Glu Leu Ala Pro Leu Cys Gly 115 120 125 Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr Cys Thr Pro Arg Arg 130 135 140 Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile Trp Asn Val Lys Lys 145 150 155 160 Gln Ser Val Tyr Leu Met Asn Leu Arg Lys Ser Gly Thr Leu Gly His 165 170 175 Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu Arg Leu Ala Glu Glu Thr 180 185 190 Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp Leu Ala Asp Lys Pro Tyr 195 200 205 Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly Ser Gly Val Leu Thr Val 210 215 220 Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro 225 230 235 240 Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr 245 250 255 Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp Gly Val Ser Leu 260 265 270 His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu Lys Thr Lys Leu 275 280 285 Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala 290 295 300 <210> 57 <211> 228 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 57 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Asp Glu 1 5 10 15 Val Asp Met Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala 20 25 30 Ser Pro Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro 35 40 45 Ala Glu Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile 50 55 60 Asp Ala Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu 65 70 75 80 Asn Gln Ile Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu 85 90 95 Arg Lys Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr 100 105 110 Tyr Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe 115 120 125 Glu Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser 130 135 140 Phe Gly Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr 145 150 155 160 Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser 165 170 175 Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val 180 185 190 Tyr Ser His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu 195 200 205 Thr Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser 210 215 220 Gly Lys Asp Ala 225 <210> 58 <211> 232 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 58 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Glu Pro 1 5 10 15 Leu Phe Ala Glu Arg Lys Met Trp Thr Leu Gly Arg Arg Ala Val Ala 20 25 30 Gly Leu Leu Ala Ser Pro Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg 35 40 45 Val Pro Arg Pro Ala Glu Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu 50 55 60 Arg Thr Asp Ile Asp Ala Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn 65 70 75 80 Gln Arg Gly Leu Asn Gln Ile Trp Asn Val Lys Lys Gln Ser Val Tyr 85 90 95 Leu Met Asn Leu Arg Lys Ser Gly Thr Leu Gly His Pro Gly Ser Leu 100 105 110 Asp Glu Thr Thr Tyr Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu 115 120 125 Ala Glu Phe Phe Glu Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp 130 135 140 Tyr Asp Val Ser Phe Gly Ser Gly Val Leu Thr Val Lys Leu Gly Gly 145 150 155 160 Asp Leu Gly Thr Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile 165 170 175 Trp Leu Ser Ser Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly 180 185 190 Lys Asn Trp Val Tyr Ser His Asp Gly Val Ser Leu His Glu Leu Leu 195 200 205 Ala Ala Glu Leu Thr Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser 210 215 220 Leu Ala Tyr Ser Gly Lys Asp Ala 225 230 <210> 59 <211> 228 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 59 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Leu Leu 1 5 10 15 Val Tyr Met Trp Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala 20 25 30 Ser Pro Ser Pro Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro 35 40 45 Ala Glu Leu Ala Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile 50 55 60 Asp Ala Thr Cys Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu 65 70 75 80 Asn Gln Ile Trp Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu 85 90 95 Arg Lys Ser Gly Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr 100 105 110 Tyr Glu Arg Leu Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe 115 120 125 Glu Asp Leu Ala Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser 130 135 140 Phe Gly Ser Gly Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr 145 150 155 160 Tyr Val Ile Asn Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser 165 170 175 Pro Ser Ser Gly Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val 180 185 190 Tyr Ser His Asp Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu 195 200 205 Thr Lys Ala Leu Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser 210 215 220 Gly Lys Asp Ala 225 <210> 60 <211> 234 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 60 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Met Trp 1 5 10 15 Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro Ser Pro 20 25 30 Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu Leu Ala 35 40 45 Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr Cys 50 55 60 Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile Trp 65 70 75 80 Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys Ser Gly 85 90 95 Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu Arg Leu 100 105 110 Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp Leu Ala 115 120 125 Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly Ser Gly 130 135 140 Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val Ile Asn 145 150 155 160 Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly 165 170 175 Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp 180 185 190 Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu 195 200 205 Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala 210 215 220 Leu Ala Leu Lys Leu Ala Gly Leu Asp Leu 225 230 <210> 61 <211> 234 <212> PRT <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polypeptide" <400> 61 Met Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Met Trp 1 5 10 15 Thr Leu Gly Arg Arg Ala Val Ala Gly Leu Leu Ala Ser Pro Ser Pro 20 25 30 Ala Gln Ala Gln Thr Leu Thr Arg Val Pro Arg Pro Ala Glu Leu Ala 35 40 45 Pro Leu Cys Gly Arg Arg Gly Leu Arg Thr Asp Ile Asp Ala Thr Cys 50 55 60 Thr Pro Arg Arg Ala Ser Ser Asn Gln Arg Gly Leu Asn Gln Ile Trp 65 70 75 80 Asn Val Lys Lys Gln Ser Val Tyr Leu Met Asn Leu Arg Lys Ser Gly 85 90 95 Thr Leu Gly His Pro Gly Ser Leu Asp Glu Thr Thr Tyr Glu Arg Leu 100 105 110 Ala Glu Glu Thr Leu Asp Ser Leu Ala Glu Phe Phe Glu Asp Leu Ala 115 120 125 Asp Lys Pro Tyr Thr Phe Glu Asp Tyr Asp Val Ser Phe Gly Ser Gly 130 135 140 Val Leu Thr Val Lys Leu Gly Gly Asp Leu Gly Thr Tyr Val Ile Asn 145 150 155 160 Lys Gln Thr Pro Asn Lys Gln Ile Trp Leu Ser Ser Pro Ser Ser Gly 165 170 175 Pro Lys Arg Tyr Asp Trp Thr Gly Lys Asn Trp Val Tyr Ser His Asp 180 185 190 Gly Val Ser Leu His Glu Leu Leu Ala Ala Glu Leu Thr Lys Ala Leu 195 200 205 Lys Thr Lys Leu Asp Leu Ser Ser Leu Ala Tyr Ser Gly Lys Asp Ala 210 215 220 Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu 225 230 <210> 62 <211> 966 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 62 atgtacggcc gcaagaagcg gagacagagg cgccggggag gaatgtccga ccaggaggcc 60 aagccctcta ccgaggacct gggcgataag aagggcg agtacatcaa gctgaaagtg 120 atcggccagg atagctccga gatcccttc aaggtgaaga tgaccacaca cctgaagaag 180 ctgaaggagt cttattgcca gcggcagggc gtgccaatga acagcctgag attcctgttt 240 gagggccaga ggatcgccga caatcacacc cccaaggagc tgggcatgga ggaggaggat 300 gtgatcgagg tgtatcagga gcagaccggc ggcatgtgga cactgggcag aagggcagtg 360 gcaggcctgc tggcatcccc atctcctgca caggcacaga ccctgacacg cgtgccacgg 420 cccgcagagc tggcaccact gtgcggccgg agaggcctga ggacagacat cgatgccacc 480 tgtacaccta gaagggcctc tagcaaccag cggggcctga accagatctg gaatgtgaag 540 aagcagtccg tgtacctgat gaatctgaga aagagcggca ccctgggaca ccctggctcc 600 ctggacgaga aacatatga gaggctggcc gaggagacac tggattctct ggccgagttc 660 tttgaggacc tggccgataa gccatacacc ttcgaggact atgatgtgag ctttggctcc 720 ggcgtgctga cagtgaagct gggaggcgac ctgggcacct acgtgatcaa caagcagaca 780 cctaataagc agatctggct gtcctctcct agctccggcc caaagcggta cgactggacc 840 ggcaagaact gggtgtattc tcacgatggc gtgagcctgc acgagctgct ggcagcagag 900 ctgaccaagg ccctgaagac aaagctggac ctgtctagcc tggcctatag cggcaaggat 960 gcctga 966 <210> 63 <211> 903 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 63 atgtacggcc ggaagaagcg gagacagagg cgccggggag gaatgcagat cttcgtgaag 60 accctgacag gcaagaccat cacactggag gtggagccct ctgacaccat cgagaacgtg 120 aaggccaaga tccaggacaa ggagggcatc ccccctgatc agcagcgcct gatctttgca 180 ggcaagcagc tggaggacgg acggaccctg tctgattata atatccagaa ggagagcaca 240 ctgcacctgg tgctgaggct gaggggagga atgtggaccc tgggcagaag ggcagtggca 300 ggcctgctgg cctctccaag cccagcacag gcacagaccc tgacaagagt gcctaggcca 360 gcagagctgg caccactgtg cggccggaga ggcctgagaa cagacatcga tgccacctgt 420 acacccagaa gggccagctc caaccagagg ggcctgaacc agatctggaa tgtgaagaag 480 cagagcgtgt acctgatgaa tctgaggaag tccggcaccc tgggacaccc tggctctctg 540 gacgagacaa catatgagcg gctggccgag gagacactgg attccctggc cgagttcttt 600 gaggacctgg ccgataagcc atacaccttc gaggactatg acgtgagctt cggctctggc 660 gtgctgacag tgaagctggg cggcgatctg ggcacctacg tgatcaacaa gcagacacct 720 aataagcaga tctggctgtc tagcccctcc tctggcccta agagatacga ctggaccggc 780 aagaactggg tgtatagcca cgatggcgtg tccctgcacg agctgctggc agcagagctg 840 accaaggccc tgaagacaaa gctggacctg agctccctgg cctattccgg caaggatgcc 900 tga 903 <210> 64 <211> 687 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 64 atgtacggca gaaagaagag gcggcagaga cgcaggggag gcgacgaggt ggatatgtgg 60 accctgggcc ggagagcagt ggcaggactg ctggcctctc ccagccctgc ccaggcccag 120 accctgacac gcgtgccaag gccagcagag ctggcaccac tgtgcggccg caggggcctg 180 cggacagaca tcgatgccac ctgtacacct cggagagcca gctccaacca gagaggcctg 240 aaccagatct ggaatgtgaa gaagcagtcc gtgtacctga tgaatctgag gaagtctggc 300 accctgggac acccaggcag cctggacgag accacatacg agaggctggc cgaggagaca 360 ctggattctc tggccgagtt ctttgaggac ctggccgata agccctacac cttcgaggac 420 tacgacgtga gcttcggctc tggcgtgctg acagtgaagc tgggcggcga cctgggcacc 480 tacgtgatca acaagcagac acctaataag cagatctggc tgtctagccc ttcctctggc 540 ccaaagaggt acgactggac cggcaagaac tgggtgtaca gccacgatgg cgtgtccctg 600 cacgagctgc tggcagcaga gctgaccaag gccctgaaga caaagctgga cctgagctcc 660 ctggcctaca gcggcaagga tgcctga 687 <210> 65 <211> 699 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 65 atgtatggaa ggaagaagag acggcagaga cggagaggag gcgagcccct gtttgctgag 60 cggaagatgt ggaccctggg aaggcgggca gtggcaggcc tgctggcaag cccatcccct 120 gcacaggcac agaccctgac aagggtgcca cggcccgcag agctggcacc actgtgcggc 180 aggcggggcc tgagaaccga catcgatgcc acctgtacac ctagaagggc cagctccaac 240 cagaggggcc tgaaccagat ctggaatgtg aagaagcaga gcgtgtacct gatgaatctg 300 aggaagagcg gcaccctggg acacccaggc tccctggacg agacaacata cgagaggctg 360 gccgaggaga cactggattc cctggccgag ttctttgagg acctggccga taagccctac 420 accttcgagg actacgacgt gagcttcggc agcggcgtgc tgacagtgaa gctgggaggc 480 gacctgggca cctacgtgat caacaagcag acacctaata agcagatctg gctgtctagc 540 ccttcctctg gcccaaagcg gtacgactgg accggcaaga actgggtgta ctcccacgat 600 ggcgtgtctc tgcacgagct gctggcagca gagctgacaa aagcactgaa aacaaaactg 660 gacctgtcat cactggcata ctctggaaag gacgcataa 699 <210> 66 <211> 687 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 66 atgtacggca gaaagaagag gcggcagaga cgcaggggag gactgctggt gtacatgtgg 60 accctgggcc ggagagcagt ggcaggactg ctggcctctc ccagccctgc ccaggcccag 120 accctgacac gcgtgccaag gccagcagag ctggcaccac tgtgcggccg caggggcctg 180 cggacagaca tcgatgccac ctgtacacct cggagagcca gctccaacca gagaggcctg 240 aaccagatct ggaatgtgaa gaagcagtcc gtgtacctga tgaatctgag gaagtctggc 300 accctgggac acccaggcag cctggacgag accacatacg agaggctggc cgaggagaca 360 ctggattctc tggccgagtt ctttgaggac ctggccgata agccctacac cttcgaggac 420 tacgacgtga gcttcggctc tggcgtgctg acagtgaagc tgggcggcga cctgggcacc 480 tacgtgatca acaagcagac acctaataag cagatctggc tgtctagccc ttcctctggc 540 ccaaagaggt acgactggac cggcaagaac tgggtgtaca gccacgatgg cgtgtccctg 600 cacgagctgc tggcagcaga gctgaccaag gccctgaaga caaagctgga cctgagctcc 660 ctggcctaca gcggcaagga tgcctga 687 <210> 67 <211> 699 <212> DNA <213> Artificial Sequence <220> <221> source <223> / note="Description of Artificial Sequence: Synthetic polynucleotide" <400> 67 atgtatggaa ggaagaagag acggcagaga cggagaggag gcgagcccct gtttgctgag 60 cggaagatgt ggaccctggg aaggcgggca gtggcaggcc tgctggcaag cccatcccct 120 gcacaggcac agaccctgac aagggtgcca cggcccgcag agctggcacc actgtgcggc 180 aggcggggcc tgagaaccga catcgatgcc acctgtacac ctagaagggc cagctccaac 240 cagaggggc...

Claims

1. The protein to be delivered to the cell, Cell-permeable peptides (CPPs), and Target-enhancing sequences (TES) A fusion protein containing [the specified ingredient].

2. The fusion protein according to claim 1, wherein CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP.

3. Starting from the N-terminus, CPP, TES, and Target protein A fusion protein according to claim 1 or 2, comprising or consisting of the following.

4. The fusion protein according to claim 1, wherein CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP.

5. Starting from the C-terminus, The target protein, TES, and CPP A fusion protein according to claim 1 or 4, comprising or consisting of the following.

6. A fusion protein according to any one of claims 1 to 5, wherein the target protein lacks OTS.

7. The fusion protein according to claim 6, further comprising an exogenous organelle transition sequence (OTS) for the target protein.

8. A fusion protein according to any one of claims 1 to 5, wherein the target protein includes OTS.

9. The fusion protein according to claim 8, wherein the OTS is heterogeneous to the target protein.

10. The fusion protein according to claim 8, wherein the OTS is endogenous to the target protein.

11. The fusion protein according to any one of claims 1 to 5, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and its variants or derivatives.

12. The fusion protein according to claim 11, wherein the target protein comprises frataxin (FXN), PARK2 protein, or a variant or derivative thereof.

13. The fusion protein according to any one of claims 1 to 12, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.

0.

14. The fusion protein according to any one of claims 1 to 13, wherein CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

15. The fusion protein according to claim 14, wherein CPP comprises HIV-TAT or a variant or derivative thereof.

16. The fusion protein according to any one of claims 1 to 15, wherein TES is a nuclear export signal peptide.

17. The fusion protein according to claim 16, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs. 36 to 43.

18. The fusion protein according to claim 17, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8, and their variants or derivatives.

19. The fusion protein according to claim 18, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1 and NES2, or variants or derivatives thereof.

20. The fusion protein according to any one of claims 1 to 19, wherein TES is a protease-sensitive peptide.

21. The fusion protein according to claim 20, wherein the protease-sensitive peptide contains a ubiquitin-like modifying factor.

22. The fusion protein according to claim 20, wherein the protease-sensitive peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18 to 31.

23. The fusion protein according to claim 20, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

24. The fusion protein according to claim 1, wherein the fusion protein contains an amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

25. Proteins to be delivered to non-nuclear organelles, Organelle transition sequences (OTS), Cell-permeable peptides (CPPs), and Target-enhancing sequences (TES) Including CPP, CPP can interfere with the delivery of target proteins to non-nuclear organelles. TES prevents the aforementioned interference caused by CPP. Fusion protein.

26. The fusion protein according to claim 25, wherein CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP.

27. Starting from the N-terminus, CPP, TES, OTS, and Target protein The fusion protein according to claim 25 or 26, comprising

28. The fusion protein according to claim 25, wherein CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP.

29. The fusion protein according to any one of claims 25 to 28, wherein the non-nuclear organelle is selected from the group consisting of mitochondria, cytosol, lysosome, endoplasmic reticulum (ER), peroxisome, and Golgi apparatus.

30. The fusion protein according to claim 25 or 26, wherein the target protein is localized in the mitochondria, cytosol, lysosome, endoplasmic reticulum (ER), peroxisome, or Golgi apparatus in its naturally occurring form.

31. The fusion protein according to any one of claims 25 to 30, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, phospholipase type VI A2, and its variants or derivatives.

32. The fusion protein according to claim 31, wherein the target protein is frataxin (FXN) or a variant or derivative thereof.

33. The fusion protein according to claim 31, wherein the target protein is pyruvate dehydrogenase (PDH) or a variant or derivative thereof.

34. The fusion protein according to any one of claims 25 to 33, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.

0.

35. The fusion protein according to any one of claims 25 to 34, wherein CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

36. The fusion protein according to claim 35, wherein CPP comprises HIV-TAT or a variant or derivative thereof.

37. The fusion protein according to any one of claims 25 to 36, wherein TES is a nuclear export signal peptide.

38. The fusion protein according to claim 37, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs. 36 to 43.

39. The fusion protein according to claim 38, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8, and their variants or derivatives.

40. The fusion protein according to claim 39, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, and their variants or derivatives.

41. The fusion protein according to any one of claims 25 to 36, wherein TES is a protease-sensitive peptide.

42. The fusion protein according to claim 41, wherein the protease-sensitive peptide contains a ubiquitin-like modifying factor.

43. The fusion protein according to claim 41, wherein the protease-sensitive peptide contains a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18 to 31.

44. The fusion protein according to claim 41, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

45. A fusion protein according to any one of claims 25 to 44, further comprising a secretory signal (SS).

46. A fusion protein according to any one of claims 25 to 45, further comprising an extracellular protein degradation site (EPS).

47. The fusion protein according to any one of claims 25 to 46, wherein the fusion protein delivers the target protein to a non-nuclear organelle.

48. The fusion protein according to any one of claims 25 to 47, wherein the fusion protein delivers the target protein to the mitochondria.

49. The fusion protein according to claim 25, wherein the fusion protein contains an amino acid sequence having at least 85%, 90%, or 95% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

50. A nucleic acid encoding a fusion protein according to any one of claims 1 to 49.

51. An expression vector for introducing a fusion protein into a cell, comprising the nucleic acid described in claim 50.

52. The expression vector according to claim 51, wherein the expression vector is selected from the group consisting of retroviral vectors, DNA vectors, plasmids, RNA vectors, adenovirus vectors, adenovirus-associated vectors, lentiviral vectors, phagemids, baculoviruses, and combinations thereof.

53. A conjugate for intracellular delivery of a protein to a non-nuclear organelle, comprising a fusion protein according to any one of claims 1 to 49 and a portion linked to the fusion protein, wherein the portion is selected from the group consisting of radiolabeled, fluorescently labeled, small molecule, and polymer molecule.

54. The conjugate according to claim 53, wherein the polymer molecule is polyethylene glycol (PEG).

55. A cell comprising a fusion protein according to any one of claims 1 to 49, a nucleic acid according to claim 50, an expression vector according to any one of claims 51 to 52, a conjugate according to any one of claims 53 to 54, or a combination thereof.

56. The cell according to claim 55, which is a stem cell or an iPS cell.

57. The cell according to claim 55, selected from the group consisting of muscle progenitor cells, neuronal progenitor cells, bone marrow stem cells, bacterial cell lines, or yeast cell lines.

58. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 49, a conjugate according to any one of claims 53 to 54, or a combination thereof, and a pharmaceutically acceptable diluent, carrier, additive, or excipient.

59. A method for delivering a target protein to a cell, comprising contacting the cell with a fusion protein according to any one of claims 1 to 49, a nucleic acid according to claim 50, a vector according to any one of claims 51 to 52, or a conjugate according to any one of claims 53 to 54.

60. A method for intracellular delivery of a target protein to a non-nuclear organelle within a cell, comprising contacting the cell with a fusion protein according to any one of claims 1 to 49, a nucleic acid according to claim 50, a vector according to any one of claims 51 to 52, or a conjugate according to any one of claims 53 to 54.

61. The method according to claim 59, wherein the non-nuclear organelle is a mitochondria.

62. A therapeutic compound for treating nonnuclear organelle-related disorders, comprising a fusion protein according to any one of claims 1 to 49, a nucleic acid according to claim 50, a vector according to any one of claims 51 to 52, or a conjugate according to any one of claims 53 to 54.

63. The therapeutic compound according to claim 62, wherein the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth's syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, fibrosis, and PLA2G6-associated neurodegeneration (PLAN).

64. The therapeutic compound according to claim 63, wherein the nonnuclear organelle-associated disorder is FDRA.

65. The therapeutic compound according to claim 63, wherein the non-nuclear organelle-related disorder is Parkinson's disease.

66. A method for treating a nonnuclear organelle-related disorder, comprising administering to a subject in need of such treatment a fusion protein according to any one of claims 1 to 49, a nucleic acid according to claim 50, a vector according to any one of claims 51 to 52, a conjugate according to any one of claims 53 to 54, or a pharmaceutical composition according to claim 58, such that the nonnuclear organelle-related disorder is treated.

67. The method according to claim 66, wherein the nonnuclear organelle-related disorder is selected from the group consisting of Friedreich's ataxia (FDRA), Barth syndrome, Parkinson's disease, Wilson's disease, Leigh syndrome, and fibrosis.

68. The method according to claim 67, wherein the non-nuclear organelle-related disorder is FDRA.

69. The method according to claim 67, wherein the nonnuclear organelle-related disorder is Parkinson's disease.

70. The method according to any one of claims 66 to 69, wherein the subject is a human.

71. A method for increasing the amount of a target protein delivered to a cell, The steps include: modifying the sequence of a fusion protein containing a target protein and a cell-permeable peptide (CPP) by introducing a target-promoting sequence (TES) into the fusion protein, thereby generating a modified fusion protein; and Steps to bring cells into contact with the modified fusion protein. A method comprising, thereby increasing the amount of target protein delivered to cells compared to the amount of target protein delivered to cells by a fusion protein without TES.

72. The method according to claim 71, wherein in the modified fusion protein, CPP is located at the N-terminus of the fusion protein and TES is fused to the C-terminus of CPP.

73. The modified fusion protein starts from the N-terminus, CPP, TES, and Target protein The method according to claim 71 or 72, including the method described in claim 71 or 72.

74. The modified fusion protein starts from the N-terminus, CPP, TES, OTS, and Target protein The method according to claim 71 or 72, including the method described in claim 71 or 72.

75. The method according to claim 71, wherein in the modified fusion protein, CPP is located at the C-terminus of the fusion protein and TES is fused to the N-terminus of CPP.

76. The method according to claim 74, wherein the amount of target protein delivered to the organelle by OTS is increased compared to the amount of target protein delivered to the organelle by the fusion protein without TES.

77. The method according to any one of claims 71 to 76, wherein the target protein is selected from the group consisting of frataxin (FXN), tafadin, pyruvate dehydrogenase (PDH), SLIRP, LRPPC, PARK2 protein, PARK6 protein, PARK7 protein, and type VI phospholipase A2, or variants or derivatives thereof.

78. The method according to claim 77, wherein the target protein is frataxin (FXN) or a variant or derivative thereof.

79. The method according to claim 77, wherein the target protein is the PARK2 protein or a variant or derivative thereof.

80. The method according to any one of claims 71 to 79, wherein the CPP comprises a peptide selected from the group of CPPs listed in the cell permeable peptide database CPPsite2.

0.

81. The method according to any one of claims 71 to 79, wherein CPP comprises a peptide selected from the group consisting of HIV-TAT, galanin, mastoparan, transportan, penetratin, polyarginine, VP22, and variants or derivatives thereof.

82. The method according to claim 81, wherein CPP comprises HIV-TAT or a variant or derivative thereof.

83. The method according to any one of claims 71 to 82, wherein TES is a nuclear export signal peptide.

84. The method according to claim 83, wherein the nuclear export signal peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 36 to 43.

85. The method according to claim 84, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, NES3, NES4, NES5, NES6, NES7, NES8 and their variants or derivatives.

86. The method according to claim 85, wherein the nuclear export signal peptide comprises a peptide selected from the group consisting of NES1, NES2, and variants or derivatives thereof.

87. The method according to any one of claims 71 to 82, wherein TES is a protease-sensitive peptide.

88. The method according to claim 87, wherein the protease-sensitive peptide comprises a ubiquitin-like modifying factor.

89. The method according to claim 87, wherein the protease-sensitive peptide comprises a sequence having at least 85% sequence identity with any one of SEQ ID NOs: 18 to 31.

90. The method according to claim 87, wherein the protease-sensitive peptide comprises a peptide selected from the group consisting of ubiquitin, a caspase cleavage domain, a calpain cleavage domain, SUMO1, SUMO2, SUMO3, SUMO4, ISG15, Atg8, Atg12, NEDD8, and variants or derivatives thereof.

91. The method according to claim 71, wherein the modified fusion protein contains an amino acid sequence having at least 85% sequence identity with any of SEQ ID NOs. 55-61, 69-71, and 81.

Images