Peptide sequences for improved delivery of proteins into nucleus

Description

PEPTIDE SEQUENCES FOR IMPROVED DELIVERY OF PROTEINS INTO NUCLEUS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 650,085, filed on May 21, 2024, which is herein incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The sequence listing in the XML, named as 42796WO_SequenceListing.xml of 139,787 bytes, created on May 20, 2025, and submitted to the United States Patent and Trademark Office via Patent Center, is incorporated herein by reference. BACKGROUND

[0003] Clinical translation of biologics-based therapies, such as protein-based and gene-based therapies, has been a major challenge, because most biologics (e.g., gene-based and / or protein therapeutics) cannot efficiently penetrate the cell. Although significant efforts have focused on developing biologics for clinical use, significant obstacles exist in the development of effective systems for payload delivery. These include penetrating the cell membrane, release of payload, effective release such that the payload retains its biological activity, and / or release of payload in the absence of carrier molecules covalently or non-covalently attached to the payload.

[0004] In some instances, the payload comprises gene editing machinery. Once the payload penetrates the cell membrane, the payload is a gene editing machinery that must then enter the nucleus to be effective.

[0005] Therefore, there is a need for conjugates that confer improved intranuclear delivery of gene editing machinery into a cell. SUMMARY

[0006] The present disclosure is directed to fusion proteins comprising a nuclear localization sequence.

[0007] In some aspects, the present disclosure is directed to a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising an amino acid sequence that is at least 80% identical to a sequence selected fromSEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the NLS comprises a primary NLS peptide comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the NLS comprises a combination of the primary NLS peptide in tandem. In some embodiments, the combination comprises at least two copies of the primary NLS peptide. In some embodiments, the NLS comprises the sequence of any one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6. In some embodiments, the NLS comprises a combination of at least two different primary NLS peptides. In some embodiments, the NLS comprises a combination of at least two different primary NLS peptides, wherein each peptide of the combination comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the NLS comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the NLS comprises a sequence of either SEQ ID NO: 7 or SEQ ID NO: 8.

[0008] In some embodiments, the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide. In some embodiments, the secondary NLS peptide is selected from the group consisting of NLS of heterogeneous nuclear ribonucleoprotein D (hnRNP_D)-NLS (SEQ ID NO: 13), NLS of histone H3 (H3-NLS) (SEQ ID NO: 14), NLS of cold-inducible RNA-binding protein (CIRBP-RSY) (SEQ ID NO: 15), the NLS of SV40 large T antigen (SV40) (SEQ ID NO: 16), c-Myc-NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18). In some embodiments, the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and, the secondary NLS peptide is selected from the group consisting of NLS of heterogeneous nuclear ribonucleoprotein D (hnRNP_D)-NLS (SEQ ID NO: 13), NLS of histone H3 (H3-NLS) (SEQ ID NO: 14), NLS of cold-inducible RNA-binding protein (CIRBP- RSY) (SEQ ID NO: 15), the NLS of SV40 large T antigen (SV40) (SEQ ID NO: 16), c-Myc- NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18).

[0009] In some embodiments, the NLS comprises a sequence selected from the group consisting of NLS of Nipah virus W protein (NiV_W)-hnRNP_D (SEQ ID NO: 19), NLS of methyl-CpG binding protein 2 (MeCP2)-hnRNP_D (SEQ ID NO: 20), NiV_W-CIRBP-RSY (SEQ ID NO: 21), H3-NiV_W (SEQ ID NO: 22), MeCP2-H3 (SEQ ID NO: 23), H3-NiV_W-CIRBP-RSY(SEQ ID NO: 28), c-Myc-NiV_W-MeCP2 (SEQ ID NO: 62), SV40-NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W-MeCP2 (SEQ ID NO: 64).

[0010] In some embodiments, the NLS is linked to the polypeptide through a linker. In some embodiments, the primary NLS peptide and the secondary NLS peptide are linked through a linker.

[0011] In some embodiments, the polypeptide is an enzyme, a gene regulatory protein, a nucleic acid binding protein, or an antibody. In some embodiments, the enzyme is a Cas nuclease, zinc finger nuclease, transcription activator-like effector nuclease, a meganuclease, Cre recombinase, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, synthase, or recombinase. In some embodiments, the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa. In some embodiments, the nucleic acid binding protein is a natural or engineered single- or double- stranded DNA / RNA-binding protein. In some embodiments, the antibodies are natural or engineered antibodies and derivatives or fragments thereof. In some embodiments, the natural or engineered single- or double-stranded binding protein is an E. coli single stranded DNA binding protein, dCas protein, TAL effector, and zinc finger protein.

[0012] In some embodiments, the disclosure is directed to a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the fusion protein comprises a NLS operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the fusion protein further comprises a cell penetrating peptide (CPP). In some embodiments, the CPP is a transactivating transcriptional activator (TAT), an arginine 9 (R9) peptide, an 8-Lysine peptide, a polyhistidine KH27K peptide, a histidine-rich LAH4 peptide, an Antennapedia (Antp) peptide, a virus protein 22 (VP22) peptide, a Pep-1 peptide, a Tat-HA2, a human transcriptional factor Hph-1, a mHph1, a mHph2, an Azurin p18 peptide, a transportan, a SG3, a fibroblast growth factor (FGF)-12, or a proline rich peptide (PRO).

[0013] Some aspects of the present disclosure are directed to a nucleic acid encoding the fusion protein described herein. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid is a messenger ribonucleic acid (mRNA). In some embodiments, the nucleic acid is a viral genomic DNA.

[0014] In some embodiments, the disclosure is directed to an expression vector comprising the nucleic acid encoding the fusion protein described herein.

[0015] In some embodiments, the disclosure is directed to a virus with a genome comprising the nucleic acid encoding the fusion protein described herein.

[0016] In some embodiments, the disclosure is directed to a host cell comprising the expression vector comprising the nucleic acid encoding the fusion protein described herein.

[0017] Some aspects of the present disclosure are directed to a method of delivering a polypeptide into the nucleus of a cell, comprising introducing into the cell a fusion protein, wherein the fusion protein comprises a nuclear localization sequence (NLS) operably linked to the polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, thereby delivering the polypeptide into the nucleus of the cell. In some embodiments, the NLS comprises a primary NLS peptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the polypeptide is delivered into the nucleus of a mammalian cell, plant cell, or yeast cell. In some embodiments, the mammalian cell is a neuronal cell, stem cell, immune cell, glial cell, muscle cell, hepatocyte, or pulmonary epithelial cell. In some embodiments, the stem cell is an induced pluripotent stem cell, embryonic stem cell, neuronal stem cell and hematopoietic stem cell. In some embodiments, the cell is a cell of a mammalian subject. In some embodiments, the subject is in need of treatment. In some embodiments, the mammalian subject has a disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is an imprinting disorder, e.g. Angelman syndrome and Prader Willi syndrome. In some embodiments, the disease is an X-chromosome linked genetic disease, i.e., Rett Syndrome and CDKL5 deficiency disorder (CDD). In some embodiments, the disease is cancer, i.e. carcinomas, sarcoma, leukemias, lymphomas, melanomas, myelomas, germ cell cancers, gynecologic cancers, genitourinary, and neurological cancers.

[0018] In some embodiments, the fusion protein is introduced into the cell through the use of a transfection agent, electroporation, microinjection, nanoparticles, a cell penetrating peptide, STEP (Stimuli-responsive Traceless Engineering Platform) technology, a nucleic acid encoding the fusion protein, introducing mRNA into the cell, or through a virus. In some embodiments, the fusion protein is introduced into the cell without the use of a transfection agent, wherein the NLS comprises a primary NLS peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the fusion protein is introduced into the cell without the use of a transfection agent, wherein the NLS comprises a primary NLS peptide comprising the sequence of SEQ ID NO: 2. In some embodiments, the fusion protein is introduced into the cell without the use of a transfection agent, wherein the NLS comprises tandem repeats of SEQ ID NO: 2.

[0019] In some embodiments, the NLS comprises a combination of the primary NLS peptide in tandem. In some embodiments, the combination comprises at least two copies of the primary NLS peptide. In some embodiments, the NLS comprises the sequence of any one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO.6. In some embodiments, the NLS comprises a combination of at least two different primary NLS peptides. In some embodiments, the NLS comprises a combination of at least two different primary NLS peptides, each peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. In some embodiments, the NLS comprises a sequence that is at least 80% identical to the sequence of either SEQ ID NO: 7 or SEQ ID NO: 8. In some embodiments, the NLS comprises the sequence of either SEQ ID NO: 7 or SEQ ID NO: 8.

[0020] In some embodiments, the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide. In some embodiments, In some embodiments, the NLS comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide and the secondary NLS peptide is selected from the group consisting of NLS of heterogeneous nuclear ribonucleoprotein D (hnRNP_D)-NLS (SEQ ID NO: 13), NLS of histone H3 (H3-NLS) (SEQ ID NO: 14), NLS of cold-inducible RNA-binding protein (CIRBP-RSY) (SEQ ID NO: 15), the NLS of SV40 large T antigen (SV40) (SEQ ID NO: 16), NLS of c-Myc (SEQ ID NO: 17), or NLS of nucleoplasmin (SEQ ID NO: 18). In some embodiments, the NLS comprises a sequence selected from the group consisting of NLS of Nipah virus W protein (NiV_W)-hnRNP_D (SEQ ID NO:19), NLS of methyl-CpG binding protein 2 (MeCP2)-hnRNP_D (SEQ ID NO: 20), NiV_W- CIRBP-RSY (SEQ ID NO: 21), H3-NiV_W (SEQ ID NO: 22), MeCP2-H3 (SEQ ID NO: 23), H3-NiV_W-CIRBP-RSY (SEQ ID NO: 28), c-Myc-NiV_W-MeCP2 (SEQ ID NO: 62), SV40- NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W-MeCP2 (SEQ ID NO: 64).

[0021] In some embodiments, the NLS is linked to the polypeptide through a linker. In some embodiments, the primary NLS peptide and the secondary NLS peptide are linked through a linker.

[0022] In some embodiments, the polypeptide is an enzyme, gene regulatory protein, nucleic acid binding protein, or antibody. In some embodiments, the enzyme is a CAS nuclease, zinc finger nuclease, transcription activator-like effector nuclease, a meganuclease, Cre recombinase, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, synthase, or recombinase. In some embodiments, the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa. In some embodiments, the nucleic acid binding protein is a natural or engineered single- or double- stranded DNA / RNAbinding protein. In some embodiments,the antibodies are natural or engineered antibodies and derivatives or fragments thereof. In some embodiments, the natural or engineered single- or double-stranded binding protein is an E. coli single stranded DNA binding protein, dCas protein, TAL effector, zinc finger protein.

[0023] In some embodiments, the disclosure is directed to a method of delivering a polypeptide into the nucleus of a cell, comprising introducing into the cell a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the fusion protein further comprises a cell penetrating peptide (CPP). In some embodiments, the primary NLS peptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, wherein the fusion protein further comprises a CPP. In some embodiments, the CPP is a transactivating transcriptional activator (TAT), an arginine 9 (R9) peptide, an 8-Lysine peptide, a polyhistidine KH27K peptide, a histidine-rich LAH4 peptide, an Antennapedia (Antp) peptide, a virus protein 22 (VP22) peptide, a Pep-1 peptide, a Tat-HA2, a human transcriptional factor Hph-1, a mHph1, a mHph2, anAzurin p18 peptide, a transportan, a SG3, a fibroblast growth factor (FGF)-12, or a proline rich peptide (PRO).

[0024] Some aspects of the current disclosure are directed to a method of delivering a fusion protein into a cell, wherein the fusion protein comprises a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising a sequence that is at least 80% identical to SEQ ID NO: 2, thereby delivering the fusion protein into the cell. In some embodiments, the NLS comprises a primary NLS peptide comprising the sequence of SEQ ID NO: 2. In some embodiments, the cell is a mammalian cell, plant cell, or yeast cell. In some embodiments, the mammalian cell is a neuronal cell, stem cell, immune cell, glial cell, muscle cell, hepatocyte, or pulmonary epithelial cell. In some embodiments, the stem cell is an induced pluripotent stem cell, embryonic stem cell, neuronal stem cell and hematopoietic stem cell. In some embodiments, the cell is a cell of a mammalian subject. In some embodiments, the subject is in need of treatment. In some embodiments, the mammalian subject has a disease. In some embodiments, the disease is a genetic disease. In some embodiments, the disease is an imprinting disorder, e.g. Angelman syndrome and Prader Willi syndrome. In some embodiments, the disease is an X-chromosome linked genetic disease, i.e., Rett Syndrome and CDKL5 deficiency disorder (CDD). In some embodiments, the disease is cancer, e.g., carcinomas, sarcoma, leukemias, lymphomas, melanomas, myelomas, germ cell cancers, gynecologic cancers, genitourinary, and neurological cancers. In some embodiments, the NLS comprises a combination of the primary NLS peptide in tandem. In some embodiments, the combination comprises at least two copies of the primary NLS peptide. In some embodiments, the NLS comprises the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises a combination of two or more different primary NLS peptides, wherein one of the primary NLS peptides comprises the sequence of SEQ ID NO: 2 and the one or more additional primary NLS peptides comprises a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 3. In some embodiments, the NLS comprises a sequence that is at least 80% identical to the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 11. In some embodiments, the NLS comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 11. In some embodiments, the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide. In some embodiments, thesecondary NLS peptide is selected from the group consisting of hnRNP_D-NLS (SEQ ID NO: 13), H3-NLS (SEQ ID NO: 14), CIRBP-RSY-NLS (SEQ ID NO: 15), the NLS of SV40 large T antigen (SEQ ID NO: 16), c-Myc-NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18). In some embodiments, the NLS comprises a sequence selected from the group consisting of MeCP2-hnRNP_D (SEQ ID NO: 20), MeCP2-H3 (SEQ ID NO: 23), c-Myc-NiV_W-MeCP2 (SEQ ID NO: 62), SV40-NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W-MeCP2 (SEQ ID NO: 64). In some embodiments, the NLS is linked to the polypeptide through a linker. In some embodiments, the primary NLS peptide and the secondary NLS peptide are linked through a linker. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0026] FIGS.1A-C. FIG.1A shows a schematic representation of Ai9 loxP-flanked STOP reporter cassette. FIGS.1B and 1C present comparison of gene editing efficiency of various Cas9 / sgAB RNPs delivered by the cell penetrating peptide (CPP). Ai9 fibroblast cells were treated with indicated RNPs / CPP at Cas9 concentration of 1 µg / mL. 48 h after treatment, the cells were analyzed under fluorescence microscope, based on which the percentage of cells expressing tdTomato was quantified. Edited cells expressed tdTomato fluorescence (red).

[0027] FIG.2. A schematic of the amino acid sequences of NiV_W (SEQ ID NO: 1) and HeV_W (SEQ ID NO: 3). The non-conserved amino acids are underlined. The minor and major importin binding sites were boxed.

[0028] FIG.3. The gene editing efficiency of various Cas9 / sgAB RNPs delivered by STEP technology. Ai9 fibroblast cells were treated with the indicated RNPs / STEP. After 48 h, the cells were imaged under fluorescence microscope and expressed as relative percentage of gene editing achieved through NiV_W-MeCP2, which was defined as 100%.

[0029] FIG.4. Representative images of brain slides prepared from adult Ai9 mice receiving one locoregional intrastriatal administration of Cas9 / sgAB delivered by STEP technology. Mice were sacrificed for histological analysis 14 days post injection. Edited cells expressed tdTomato fluorescence (red). Nuclear was stained with DAPI (blue).

[0030] FIG.5. Representative images of brain slides prepared from adult Ai9 mice receiving one locoregional intrastriatal administration of SpCas9-MeCP2-NiV_W RNPs delivered by STEP technology (40 ug Cas9 per injection). Mice were sacrificed at 28 days post injection. Co-staining of tdTomato with NeuN (a neuronal marker).

[0031] FIG.6. The gene editing efficiency of various Cas9 / sgAB RNPs with 28 different combinationa of NLSs delivered by STEP technology. The combinations represented in the figure are identified in Table 3. Ai9 fibroblast cells were treated with the indicated RNPs / STEP. After 48 h, the cells were imaged under fluorescence microscope and expressed as relative percentage of gene editing achieved through MeCP2-NiV_W, which was defined as 100%.

[0032] FIG.7. Gene editing efficiency of various Cas9 / sgAB RNPs delivered by Lipofectamine transfection agent. Ai9 fibroblast cells were treated with the indicated RNPs / Lipofectamine at Cas9 concentration of 2 µg / mL. 48 hours after treatment, cells were imaged under fluorescence microscope, based on which the percentage of cells expressing tdTomato was quantified.

[0033] FIG 8. Recombination efficiency of various Cre recombinase delivered by STEP technology. Ai9 fibroblast cells were treated with the indicated Cre / STEP at Cre concentration of 1 µg / mL. 48 hr after treatment, the cells were imaged under fluorescence microscope, based on which the percentage of cells expressing tdTomato was quantified.

[0034] FIG.9. Comparison of gene editing efficiency of free Cre recombinase protein and Cre- NiVW-MeCP2 fusion recombinase. Ai9 fibroblast cells were treated with Cre recombinase at indicated concentrations of 20 µg / mL or 40 µg / mL for free Cre recombinase and 20µg / mL or 40 µg / mL for Cre-NiVW-MeCP2 fusion recombinase. 48 h after treatment, the cells were analyzed under fluorescence microscope, based on which the percentage of cells expressing tdTomato was quantified. Edited cells expressed tdTomato fluorescence (red).

[0035] FIG.10. Comparison of gene editing efficiency of fusion Cre recombinases. All Cre recombinases used in the experiment have N-terminal NiVW-MeCP2 NLS and various C- terminal MeCP2 or NiVW repeats. The number of MeCP2 or NiVW repeats were indicated in the figure where MeCP2*1 includes one MeCP2 sequence, MeCP2*2 includes two MeCP2 sequences, MeCP2*3 includes three MeCP2 sequences, NiVW*1 includes one NiVW sequence, NiVW*2 includes two NiVW sequences, NiVW*3 includes three NiVW sequences. All sequence repeats are flanked by a GGS linker sequence as shown in Table 4. Ai9 fibroblast cells were treated with Cre recombinase at indicated concentrations of 20µg / mL or 40 µg / mL for Cre-NiVW-MeCP2 fusion recombinase. 48 hours after treatment, the cells were analyzed under fluorescence microscope, and the percentage of cells expressing tdTomato was quantified. Cells expressing tdTomato fluorescence (red) were penetrated by the Cre recombinase.

[0036] FIG.11. Ai9 fibroblasts were treated with GFP-Cas9-NiVW-MeCP2 fusion protein at 10 µg / mL for 1 h. After treatment, cells were washed, fixed, immunostained for EEA1 labeling endosome (CST 2411S), then co-immunostained for IgG and subjected to DAPI nuclear counterstain and visualized under fluorescence confocal microscopy (Leica TCS SP8). Cells showing GFP indicate penetration after an hour. INCORPORATION BY REFERENCE

[0037] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. DETAILED DESCRIPTION

[0038] The following description and examples illustrate embodiments of the present disclosure in detail. It is to be understood that this disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this disclosure, which are encompassed within its scope.

[0039] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0040] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0041] Although various features of the present disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination.Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0042] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0044] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0045] Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0046] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0047] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented withrespect to any method or composition of the present disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0048] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.

[0049] As used herein, the term “amino acid” refers to a molecule containing both an amino group and a carboxyl group. Amino acids include alpha-amino acids and beta-amino acids. In certain forms, an amino acid is an alpha-amino acid. Amino acids can be natural or synthetic. Amino acids include, but are not limited to, the twenty standard or canonical amino acids: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V). Common non-standard or non-canonical amino acids include, but are not limited to, selenocysteine, ornithine, pyrrolysine, and N-formylmethionine.

[0050] As used herein, the term “natural amino acid” refers to both the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letter abbreviations)).

[0051] The terms “synthetic amino acid”, “non-natural amino acid” and “unnatural amino acid,” are used interchangeably, and refer to an organic compound that has an amino group and a carboxyl group, and is not one of the D- and L-isomers of the 20 common naturally occurring amino acids found in peptides. Generally, it mimics the reactivity of a natural amino acid due to the presence of the amino and carboxyl groups. “Synthetic amino acid,” “non-natural amino acid,” or “unnatural amino acid” also refers to an amino acid that is not produced by an organism without genetic engineering. The synthetic amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., reactivity towards a desired molecule) when the synthetic amino acid is either substituted for a natural amino acid or incorporated into a peptide. “Synthetic amino acid,” “non-natural amino acid,” or “unnatural amino acid” can also refer to a natural amino acid whose side chain has been chemically modified to include a reactive group (e.g. alkyne; azide; alkene; triarylphosphine; aminooxy; carbonyl; hydrazide; sulfonyl chloride; maleimide; aziridine; -CN; acryloyl; acrylamide; sulfone; vinyl sulfone; cyanate; thiocyanate;isocyanate; isothiocyanate; alkoxysilane; dialkyl dialkoxysilane; diaryl dialkoxysilane; trialkyl monoalkoxysilane; vinyl silane; acetohydrazide; acyl azide; acyl halides; epoxide; glycidyl; carbodiimides; thiol; amine; phosphoramidate; vinyl ether; substituted hydrazine; an alkylene glycol bis(diester), e.g. ethylene glycol bis(succinate); thioester, e.g., alkyl thioester, α- thiophenylester, allyl thioester (e.g., allyl thioacetate, allyl thioproprionate); allyl ester (e.g., allyl acetate, allyl propionate); aryl acetate (e.g. phenacyl ester); orthoester; sulfonamide, e.g.2-N- acyl nitrobenzenesulfonamide; vinyl sulfide; or a combination thereof) such that the resulting amino acid is structurally different from any of the 20 canonical naturally occurring amino acids.

[0052] As used herein, “chemical moiety” describes a part of a molecule, such as an organic molecule.

[0053] “Conjugate,” “conjugation,” and related terms, refer to the covalent or non-covalent linkage of a molecule to another molecule, or one part of a molecule to a different part of the same molecule. The linkage can involve covalent or non-covalent linkage. Covalent linkages can be direct or indirect (i.e., mediated via a linker). “Covalent linkage”, refers to a bond or organic moiety that covalently links molecules or different parts of the same molecule. Non- covalent linkage includes electrostatic interactions, hydrogen bonding interactions, metal coordination, physical adsorption, host-guest interactions, hydrophobic interactions, π-stacking interactions, van der Waals interactions, magnetic interactions, and dipole-dipole interactions.

[0054] The terms “genome editing,” “genome engineering” or “genome mutagenesis” refer to selective and specific changes to one or more targeted genes or DNA sequences within a recipient cell, for example, via delivery of CRISPR-Cas system to the cell. The editing or changing of a targeted gene or genome can include one or more of a deletion, knock-in, point mutation, substitution mutation or any combination thereof in one or more genes of the recipient cell.

[0055] The terms “single guide RNA” or “sgRNA” refer to the polynucleotide sequence comprising the guide sequence, tracr sequence and the tracr mate sequence. “Guide sequence” refers to the around 20 base pair (bp) sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer.”

[0056] The terms “Cas9,” “Cas9 protein,” or “Cas9 nuclease” refer to an RNA-guided endonuclease that is a Cas9 protein that catalyzes the site-specific cleavage of double stranded DNA. Also, referred to as “Cas nuclease” or “CRISPR-associated nuclease.” In nature, theCRISPR system is an adaptive immune system found in bacteria that provides protection against mobile elements such as phage viruses and transposable elements. DNA binding and cleavage requires the Cas9 protein and two RNAs, a trans-encoded RNA (tracrRNA) and a CRISPR RNA (crRNA) in nature. Artificially, single-guided RNA or sgRNA can be engineered to incorporate aspects of both RNAs into a single species (Jinek, et al. Science, 337, 816-821, doi: 10.1126 / science.1225829 (2012)). The CRISPR system has two components: the Cas9 nuclease and a single guide RNA (sgRNA) that provides DNA sequence-targeting accuracy. The targeting of the Cas9-sgRNA complex is mediated by the protospacer adjacent motif (PAM) located at the DNA for Cas9 recognition and the homology between the ~20-nucleotide recognition sequence encoded in the sgRNA and the genomic DNA target. The targeted gene can be knocked out after the Cas9-sgRNA complex finds and cleaves the exonic region of the gene to generate frameshift mutations. Cas9 recognizes short motifs in CRISPR repeat sequences to help distinguish self from non-self. Cas9 nuclease sequences and structures are known to those of skill in the art (Ferretti, et al. Proc Natl Acad Sci U.S.A, 98, 4658-4863, doi: 10.1073 / pnas.071559398 (2001); Deltcheva, et al. Nature, 471, 602-607, doi: 10.1038 / nature09886 (2011)). Cas9 orthologs have been described in several species of bacteria, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus, Campylobacter jejuni and Neisseria meningitidis. (Slaymaker, et al. Science, 351, 84-88 doi: 10.1126 / science.aad5227 (2016); Kleinstiver, et al. Nature, 529, 490-495, doi: 10.1038 / nature16526 (2016); Chen, et al. Nature, 550, 407-410, doi: 10.1038 / nature24268 (2017); Casini, et al. Nat Biotechnol, 6, 265-271, doi: 10.1038 / nbt.4066 (2018); Lee, et al. Nat Commun,9, 3048, doi: 10.1038 / s41467-018-05477-x (2018); Vakulskas, et al. Nat Med, 24, 1216-1224, doi: 1.1038 / s41591-018-0137-0 (2018); Choi, et al. Nat Methods, 16, 722-730, doi: 10.1038 / s41592-019-0473-0 (2019); Kim, et al. Nat Commun, 8, 14500, doi: 10.1038 / ncomms14500 (2017); (Edraki, et al. Mol Cell, 73, 714-726, doi: (2019)).

[0057] “Peptide” refers of a chain of amino acids having a length of between 2 and 50 amino acids in length.

[0058] As used herein, “protein” refers to a chain of amino acids having a length of greater than 50 amino acids, such as greater than 50 amino acids and less than thirty-six thousand amino acids.

[0059] As used herein, “small molecule” refers to an organic molecule that is less than about 2500 g / mol in molecular weight, less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some forms, small molecules are non-polymeric and / or non-oligomeric.

[0060] The term “treating,” “preventing,” and a related term such as “treatment” mean to ameliorate, reduce or otherwise stop a disease, disorder, or condition from occurring or progressing in an animal which may be predisposed to the disease, disorder, and / or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder, or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and / or condition. Treating the disease or condition includes ameliorating at least one symptom of the particular disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with a genetic neuropathy, a genetic based musculopathy, a genetic eye disease or disorder, a genetic lung disease or disorder, a genetic liver disease or disorder, or cancer are mitigated or eliminated, including, but are not limited to, reducing and / or inhibiting rate of progress of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0061] As used herein, the term “edit,” “editing,” or “edited” refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., for example, a wild type naturally occurring nucleic acid sequence or a mutated naturally occurring sequence) by selective modification (e.g., deletion, insertion, or substitution) of one or more nucleotides in a specific genomic target. Such a specific genomic target includes, but is not limited to, a chromosomal region, a gene, a promoter, an open reading frame or any nucleic acid sequence.

[0062] As used herein, the term “target” or “target site” refers to a pre-identified nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, a chromosomal region, a gene, a promoter, an open reading frame or any nucleic acid sequence. Insome embodiments, the present invention interrogates these specific genomic target sequences with complementary sequences of gRNA.

[0063] The term “effective amount” as used herein, refers to a particular amount of a pharmaceutical composition comprising a therapeutic agent that achieves a clinically beneficial result (i.e., for example, a reduction of symptoms). Toxicity and therapeutic efficacy of such compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and additional animal studies can be used in formulating a range of dosage for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[0064] The term “pharmaceutically” or “pharmacologically acceptable”, as used herein, refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.

[0065] The term, “pharmaceutically acceptable carrier”, as used herein, includes any and all solvents, or a dispersion medium including, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils, coatings, isotonic and absorption delaying agents, liposome, commercially available cleansers, and the like. Supplementary bioactive ingredients also can be incorporated into such carriers.

[0066] The term “viral vector” encompasses any nucleic acid construct derived from a virus genome capable of incorporating heterologous nucleic acid sequences for expression in a host organism. For example, such viral vectors may include, but are not limited to, adeno-associated viral vectors, lentiviral vectors, SV40 viral vectors, retroviral vectors, or adenoviral vectors. Although viral vectors are occasionally created from pathogenic viruses, they may be modified in such a way as to minimize their overall health risk. In some embodiments, this involves the deletion of a part of the viral genome involved with viral replication. Such a virus can efficientlyinfect cells but, once the infection has taken place, the virus may require a helper virus to provide the missing proteins for production of new virions. Preferably, viral vectors should have a minimal effect on the physiology of the cell it infects and exhibit genetically stable properties (e.g., do not undergo spontaneous genome rearrangement). Most viral vectors are engineered to infect as wide a range of cell types as possible. Even so, a viral receptor can be modified to target the virus to a specific kind of cell. Viruses modified in this manner are said to be pseudotyped. Viral vectors are often engineered to incorporate certain genes that help identify which cells took up the viral genes. These genes are called marker genes. For example, a common marker gene confers antibiotic resistance to a certain antibiotic.

[0067] As used herein, a “spacer sequence,” sometimes also referred to herein and in the literature as a “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for cleavage by a Cas9. For clarity, the terms “spacer sequence”, “spacer,” “protospacer,” “guide sequence,” or “targeting sequence” as used herein, and unless specifically stated otherwise, may refer to an RNA molecule (comprising A, C, G, and U nucleotides) or to a DNA molecule encoding such an RNA molecule (comprising A, C, G, and T nucleotides) or complementary sequences thereof. A guide sequence can be 24, 23, 22, 21, 20 or fewer base pairs in length, e.g., in the case of Staphylococcus lugdunensis (i.e., SluCas9) or Staphylococcus aureus (i.e., SaCas9) and related Cas9 homologs / orthologs. In preferred embodiments, a guide / spacer sequence in the case of SluCas9 or SaCas9 is at least 20 base pairs in length, or more specifically, within 20-25 base pairs in length (see, e.g., Schmidt et al., 2021, Nature Communications, “Improved CRISPR genome editing using small highly active and specific engineered RNA-guided nucleases”). Shorter or longer sequences can also be used as guides, e.g., 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, or 25-nucleotides in length.

[0068] As used herein, the term “fluorescent protein” refers to a protein domain that comprises at least one organic compound moiety that emits fluorescent light in response to the appropriate wavelengths. For example, fluorescent proteins may emit red, blue and / or green light. Such proteins are readily commercially available including, but not limited to: i) mCherry (Clonetech Laboratories): excitation: 556 / 20 nm (wavelength / bandwidth); emission: 630 / 91 nm; ii) sfGFP (Invitrogen): excitation: 470 / 28 nm; emission: 512 / 23 nm; iii) TagBFP (Evrogen): excitation 387 / 11 nm; emission 464 / 23 nm.

[0069] As used herein, the term “orthogonal” refers to targets that are non-overlapping, uncorrelated, or independent. For example, if two orthogonal nuclease-deficient Cas9 genes fused to different effector domains were implemented, the sgRNAs coded for each would not cross-talk or overlap. Not all nuclease-deficient Cas9 genes operate the same. As such, the use of orthogonal nuclease-deficient Cas9 genes fused to different effector domains can be used provided the appropriate orthogonal sgRNAs are present.

[0070] As used herein, the term “phenotypic change” or “phenotype” refers to the composite of an organism's observable characteristics or traits, such as its morphology, development, biochemical or physiological properties, phenology, behavior, and products of behavior. Phenotypes result from the expression of an organism's genes as well as the influence of environmental factors and the interactions between the two.

[0071] “Nucleic acid sequence”, “polynucleotide sequence”, and “nucleotide sequence” as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

[0072] The term “an isolated nucleic acid”, as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and is, in a preferred embodiment, free of other genomic nucleic acid).

[0073] The terms “amino acid sequence” and “polypeptide sequence” as used herein, are interchangeable and refer to a sequence of amino acids.

[0074] As used herein the term “portion” when in reference to a protein (as in “a portion of a given protein”) refers to fragments of that protein. Unless specified otherwise, the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

[0075] The term “portion” when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. Unless specified otherwise, the fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

[0076] As used herein, the terms “complementary” or “complementarity” are used in reference to “polynucleotides” and “oligonucleotides” (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “C-A-G- T,” is complementary to the sequence “G-T-C-A.” Complementarity can be “partial” or “total.”“Partial” complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. “Total” or “complete” complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

[0077] The terms “homology” and “homologous” as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., “substantially homologous,” to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.

[0078] The terms “homology” and “homologous” as used herein in reference to amino acid sequences refer to the degree of identity of the primary structure between two amino acid sequences. Such a degree of identity may be directed to a portion of each amino acid sequence, or to the entire length of the amino acid sequence. Two or more amino acid sequences that are “substantially homologous” may have at least 75% identity, at least 85% identity, at least 95%, or 100% identity.

[0079] An oligonucleotide sequence which is a “homolog” is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity to a sequence, when sequences having a length of 100 bp or larger are compared.

[0080] As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the melting temperature of the formed hybrid, and the G:C ratio within the nucleic acids.

[0081] DNA and RNA molecules are said to have “5' ends” and “3' ends” because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the “5' end” if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the “3' end” if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3' ends. In either a linear or circular DNA or RNA molecule, discrete elements are referred to as being upstream” or 5' of the “downstream” or 3' elements. This terminology reflects the fact that transcription proceeds in a 5' to 3' fashion along the DNA or RNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5' or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region.

[0082] As used herein, “nuclear localization sequence” (NLS) or “nuclear localization signal” are used interchangeably to refer to an amino acid sequence that labels a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS.

[0083] The NLS allows entry into the nucleus through the nuclear envelope. The nuclear envelope consists of concentric membranes, the outer and the inner membrane. The inner and outer membranes connect at multiple sites, forming channels between the cytoplasm and the nucleoplasm. These channels are occupied by nuclear pore complexes (NPCs), complex multiprotein structures that mediate the transport across the nuclear membrane.

[0084] A protein translated with an NLS will bind strongly to importin (aka karyopherin), and, together, the complex will move through the nuclear pore. At this point, Ran-GTP will bind to the importin-protein complex, and its binding will cause the importin to lose affinity for the protein. The protein is released, and now the Ran-GTP / importin complex will move back out of the nucleus through the nuclear pore. A GTPase-activating protein (GAP) in the cytoplasm hydrolyzes the Ran-GTP to GDP, and this causes a conformational change in Ran, ultimately reducing its affinity for importin. Importin is released and Ran-GDP is recycled back to the nucleus where a Guanine nucleotide exchange factor (GEF) exchanges its GDP back for GTP.

[0085] The term “transfection” or “transfected” refers to the introduction of foreign DNA or RNA into a cell.

[0086] As used herein, the terms “nucleic acid molecule encoding”, “DNA (or RNA) sequence encoding,” and “DNA (or RNA) encoding” refer to the order or sequence of nucleotides along a strand of nucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence.

[0087] In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the RNA transcript. These sequences are referred to as “flanking” sequences or regions (these flanking sequences are located 5' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage, and polyadenylation. Fusion Protein

[0088] In one aspect, the present disclosure is directed to a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologousto the polypeptide. The NLS and the polypeptide being “heterologous” refers to the NLS and polypeptide originating from different native molecules.

[0089] In some embodiments, the NLS comprises a primary NLS peptide. In some embodiments, the primary NLS peptide comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of Nipah virus W protein, (designated herein as NiV_W): TCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: 1). In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 1. In some embodiments, the primary NLS peptide comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0090] In some embodiments, the primary NLS peptide comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of methyl-CpG binding protein 2 (designated herein as MeCP2): KRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 2). In some embodiments, theprimary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 2. In some embodiments, the primary NLS peptide comprises an amino acid sequence as shown in SEQ ID NO: 2.

[0091] In some embodiments, the primary NLS peptide comprises or consist of an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of Hendra virus W protein (designated herein as HeV_W): TCLGRRVVQPGMFADYPPTKKARVLLRRMS (SEQ ID NO: 3). In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequencethat is at least 92% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 3. In some embodiments, the primary NLS peptide comprises an amino acid sequence as shown in SEQ ID NO: 3. Nuclear Localization Sequence

[0092] In some embodiments, the NLS of the fusion protein comprises a primary NLS peptide which is a single NLS peptide. The single NLS peptide has a sequence selected from SEQ ID NOs: 1, 2, or 3. In some embodiments, the NLS comprises a combination of primary NLS peptides and the primary NLS peptides are arranged in tandem. In some embodiments, the NLS comprises a combination of primary NLS peptides wherein the combination comprises at least two (e.g., 2, 3, 4 or more) copies of the same primary NLS peptide. In embodiments where the NLS comprises a combination of primary NLS peptides, the primary NLS peptides can be connected to each other through a linker. In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of two copies of the primary NLS peptide NiV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising two copies of the primary NLS peptide NiV_W connected in tandem (e.g., via a linker) is (NiV_W- NiV_W): TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaTCLGRRVVQPGMFEDHPPTKKARVS MRRMS (SEQ ID NO: 4) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLScomprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 4. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 4.

[0093] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of two copies of the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising two copies of the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker) is (MeCP2-MeCP2): KRPGRKRKAEADPQAIPKKRGRKXaKRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 5) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 5. In someembodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 5. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 5.

[0094] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of two copies of the primary NLS peptide HeV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising two copies of the primary NLS peptide HeV_W connected in tandem (e.g., via a linker) is (HeV_W-HeV_W): TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaTCLGRRVVQPGMFADYPPTKKARVLL RRMS (SEQ ID NO: 6) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLScomprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 6. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 6.

[0095] In some embodiments, the NLS comprises a combination of primary NLS peptides wherein the combination comprises at least two (e.g., 2, 3, 4 or more) different primary NLS peptides in tandem connected, e.g., via a linker. In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide NiV_W and the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the primary NLS peptide NiV_W and the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker) is (NiV_W-MeCP2): TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 7) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 97%identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 7. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 7.

[0096] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide MeCP2 and the primary NLS peptide NiV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the sequence of the primary NLS peptide MeCP2 and the primary NLS peptide NiV_W connected in tandem (e.g., via a linker) is (MeCP2-NiV_W): KRPGRKRKAEADPQAIPKKRGRKXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: 8) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 8. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to thesequence of SEQ ID NO: 8. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 8.

[0097] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide HeV_W and the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the sequence of the primary NLS peptide HeV_W and the primary NLS peptide MeCP2 connected in tandem (e.g., via a linker) is (HeV_W-MeCP2): TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaKRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 9) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 9. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 9.

[0098] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof the primary NLS peptide HeV_W and the primary NLS peptide NiV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the sequence of the primary NLS peptide HeV_W and the primary NLS peptide NiV_W connected in tandem (e.g., via a linker) is (HeV_W-NiV_W): TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaTCLGRRVVQPGMFEDHPPTKKARVS MRRMS (SEQ ID NO: 10) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide MeCP2 and the primary NLS peptide HeV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the sequence of the primary NLS peptide MeCP2 and the primary NLS peptide HeV_W connected in tandem (e.g., via a linker) is (MeCP2-HeV_W): KRPGRKRKAEADPQAIPKKRGRKXaTCLGRRVVQPGMFADYPPTKKARVLLRRMS (SEQ ID NO: 11) where X is an amino acid selected from the group of A, S, G, or T, and a is 0- 12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 11. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to thesequence of SEQ ID NO: 11. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 11.

[0099] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide NiV_W and the primary NLS peptide HeV_W connected in tandem (e.g., via a linker). In some embodiments, the NLS comprising the sequence of the primary NLS peptide NiV_W and the primary NLS peptide HeV_W connected in tandem (e.g., via a linker) is (NiV_W-HeV_W): TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaTCLGRRVVQPGMFADYPPTKKARVL LRRMS (SEQ ID NO: 12) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 12. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 12.

[0100] In some embodiments, the NLS comprises a combination of primary NLS peptides comprising at least three copies of a primary NLS peptide, i.e. the NLS comprises an amino acidsequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of three copies of primary NLS peptide NiV_W connected in tandem, for example NiV_W-NiV_W-NiV_W. In some embodiments, the NLS comprises a combination of primary NLS peptides comprising at least three primary NLS peptides, i.e. the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the primary NLS peptide NiV_W, the primary NLS peptide HeV_W, and the primary NLS peptide MeCP2 connected in tandem, i.e. via a linker (NiV_W-HeV_W-MeCP2). In some embodiments, the NLS comprises a combination of primary NLS peptides comprising at least 4 copies of a primary NLS peptide. Secondary NLS peptide

[0101] In some embodiments, the NLS further comprises a secondary NLS peptide. The secondary NLS peptide is operably linked, e.g., via a linker, to either the N-terminus or C- terminus of the primary NLS peptide. In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of heterogeneous nuclear ribonucleoprotein D (designated herein as hnRNP_D): YSNQQSGYGKVSRRGGHQNSYKPY (SEQ ID NO: 13). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that isat least 95% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 13. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 13.

[0102] In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NLS of histone H3 (designated herein as H3): ARTKQTARKSTGGKAPRKQLATKAARKS (SEQ ID NO: 14). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO:14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 14. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 14.

[0103] In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of cold-inducible RNA-binding protein (designated herein as CIRBP-RSY): SRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNE (SEQ ID NO: 15). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 15. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO:15. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 15.

[0104] In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of SV40 large T antigen (designated herein as SV40): PKKKRKV (SEQ ID NO: 16). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 16. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 16.

[0105] In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of c-Myc (designated herein as c-Myc- NLS): PAAKKKKLD (SEQ ID NO: 17). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 17. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 17.

[0106] In some embodiments, the primary NLS peptide comprises a sequence as above, i.e. an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 and the secondary NLS peptide has an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of the NLS of nucleoplasmin: KRPAATKKAGQAKKKK (SEQ ID NO: 18). In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 80% identical to the sequence of SEQID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 18. In some embodiments, the secondary NLS peptide comprises the amino acid sequence of SEQ ID NO: 18.

[0107] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-hnRNP_D: TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaYSNQQSGYGKVSRRGGHQNSYKPY (SEQ ID NO: 19) where X is an amino acid selected from the group of A, S, G, or T, and a is 0- 12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 19. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 19. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 19. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO:19. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 19. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 19.

[0108] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of MeCP2-hnRNP_D: KRPGRKRKAEADPQAIPKKRGRKXaYSNQQSGYGKVSRRGGHQNSYKPY (SEQ ID NO: 20) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 20. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 20. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 20. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 20. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 20. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 20.

[0109] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-CIRBP-RSY: TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaSRDYYSSRSQSGGYSDRSSGGSYRDS YDSYATHNE (SEQ ID NO: 21) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 21. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 21. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 21. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 21. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 21. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 21.

[0110] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of H3-NiV_W: ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPGMFEDHPPTKKARVSMR RMS (SEQ ID NO: 22) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 22. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 22. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 22. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 22. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 22. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 22.

[0111] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of MeCP2-H3: KRPGRKRKAEADPQAIPKKRGRKXaARTKQTARKSTGGKAPRKQLATKAARKS (SEQ ID NO: 23) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 23. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 23. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 23. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 23. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 23. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 23.

[0112] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of hnRNP_D-NiV_W:YSNQQSGYGKVSRRGGHQNSYKPYXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: 24) where X is an amino acid selected from the group of A, S, G, or T, and a is 0- 12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 24. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 24. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 24. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 24. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 24. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 24.

[0113] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of CIRBP-RSY-NiV_W: SRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNEXaTCLGRRVVQPGMFEDHPPTKKA RVSMRRMS (SEQ ID NO: 25) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 25. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 25. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 25. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 25. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 25. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 25.

[0114] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of H3-MeCP2: ARTKQTARKSTGGKAPRKQLATKAARKSXaKRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 26) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acidsequence that is at least 95% identical to the sequence of SEQ ID NO: 26. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 26. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 26. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 26. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 26. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 26.

[0115] In some embodiments, where the NLS comprises a secondary NLS peptide, the NLS comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of H3-HeV_W: ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPGMFADYPPTKKARVLLRR MS (SEQ ID NO: 27) where X is an amino acid selected from the group of A, S, G, or T, and a is 0-12 and represents the number of amino acids. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 27. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 27. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 27. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 27. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 27. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 27.

[0116] In some embodiments, the NLS comprising a combination of primary and secondary NLS peptides comprises at least three NLS peptides. In some embodiments, the NLS comprises a combination of 2 primary NLS peptides and one secondary NLS peptide. In some embodiments, the NLS comprises a combination of one primary NLS peptide and two secondary NLS peptides.

[0117] In some embodiments, the NLS comprises two secondary NLS peptides. In some embodiments, the two secondary NLS peptides are on either side of the primary NLS peptide, i.e. one of the secondary NLS peptides attaches to the 5' end of the primary NLS peptide while another of the secondary NLS peptides attaches to the 3' end of the primary NLS peptide. Forexample, in some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of H3-NiV_W-CIRBP-RSY: ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPGMFEDHPPTKKARVSMR RMSYbSRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNE (SEQ ID NO: 28) where X is an amino acid selected from the group of A, S, G, or T, a is 0-12 and represents the number of amino acids, Y is an amino acid selected from the group of A, S, G, or T, and b is 0-12 and represents the number of amino acids, wherein X and Y are or are not the same and a and b are or are not the same. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 28.

[0118] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of c-Myc-NiV_W-MeCP2: PAAKKKKLDXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMSYbKRPGRKRKAEADPQ AIPKKRGRKASGG (SEQ ID NO: 62) where X is an amino acid selected from the group of A,S, G, or T, a is 0-12 and represents the number of amino acids, Y is an amino acid selected from the group of A, S, G, or T, and b is 0-12 and represents the number of amino acids, wherein X and Y are or are not the same and a and b are or are not the same. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 28. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 28.

[0119] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SV40-NiV_W-MeCP2: PKKKRKVXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMSYbKRPGRKRKAEADPQAIP KKRGRKASGG (SEQ ID NO: 63) where X is an amino acid selected from the group of A, S, G, or T, a is 0-12 and represents the number of amino acids, Y is an amino acid selected from the group of A, S, G, or T, and b is 0-12 and represents the number of amino acids, wherein X and Y are or are not the same and a and b are or are not the same. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 85%identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 63. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 63.

[0120] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of nucleoplasmin-NiV_W-MeCP2: KRPAATKKAGQAKKKKXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMSYbKRPGRKR KAEADPQAIPKKRGRKASGG (SEQ ID NO: 64) where X is an amino acid selected from the group of A, S, G, or T, a is 0-12 and represents the number of amino acids, Y is an amino acid selected from the group of A, S, G, or T, and b is 0-12 and represents the number of amino acids, wherein X and Y are or are not the same and a and b are or are not the same. In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 64. In someembodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 64. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 64.

[0121] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-c-Myc (SEQ ID NO: 29). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 29. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to thesequence of SEQ ID NO: 29. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 29.

[0122] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-SV40 (SEQ ID NO: 30). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 30. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 30.

[0123] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Nucleoplasmin (SEQ ID NO: 31). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to thesequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 31. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 31.

[0124] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-BDV-P1 (SEQ ID NO: 32). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 32. In someembodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 32. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 32.

[0125] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-HCVC2 (SEQ ID NO: 33). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 33. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 33.

[0126] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-QKI-5 (SEQ ID NO: 34). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLScomprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 34. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 34.

[0127] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Amida (SEQ ID NO:35). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises anamino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 35. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 35.

[0128] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-HIV-1R (SEQ ID NO: 36). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 36. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 36.

[0129] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-RanBP3 (SEQ ID NO: 37). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequencethat is at least 90% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 37. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 37.

[0130] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-HCVC4 (SEQ ID NO: 38). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 97%identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 38. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 38.

[0131] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-BDV-P2 (SEQ ID NO: 39). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 39. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 39.

[0132] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-EBNA1 (SEQ ID NO: 40). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to thesequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 40.

[0133] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-FluA (SEQ ID NO: 41). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO:41. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 41. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 41.

[0134] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-MyoD (SEQ ID NO: 42). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 42. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 42.

[0135] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-polyoma1 (SEQ ID NO: 43). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 43. In someembodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 43. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 43.

[0136] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-E1a (SEQ ID NO: 44). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLScomprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 44. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 44.

[0137] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-VirD2C (SEQ ID NO: 45). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 45. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 45.

[0138] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-cFOS (SEQ ID NO: 46). In some embodiments, the NLS comprises anamino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 46. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 46.

[0139] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-NS5A (SEQ ID NO: 47). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 40. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequencethat is at least 95% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 47. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 47.

[0140] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Max (SEQ ID NO: 48). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 48. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 48.

[0141] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequenceof NiV_W-MeCP2-polyoma2 (SEQ ID NO: 49). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 49. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 49.

[0142] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-L29 (SEQ ID NO: 50). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to thesequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 50. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 50.

[0143] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Pax-PD (SEQ ID NO: 51). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 51. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 51.

[0144] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Mat-a (SEQ ID NO: 52). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 52. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 52.

[0145] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-Rex (SEQ ID NO: 53). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises anamino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 53. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 53.

[0146] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of NiV_W-MeCP2-M9 (SEQ ID NO: 54). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 54. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to thesequence of SEQ ID NO: 54. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 54.

[0147] In some embodiments, the NLS comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of or NiV_W-MeCP2-hARNT (SEQ ID NO: 55). In some embodiments, the NLS comprises an amino acid sequence that is at least 80% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 85% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 91% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 92% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 93% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 94% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 96% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 97% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 98% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises an amino acid sequence that is at least 99% identical to the sequence of SEQ ID NO: 55. In some embodiments, the NLS comprises the amino acid sequence of SEQ ID NO: 55. Polypeptide of the Fusion Protein

[0148] In some aspects of the disclosure, the fusion protein comprises a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide.

[0149] In some embodiments, the polypeptide operably linked to the NLS is an enzyme, a gene regulatory protein, a nucleic acid binding protein, or an antibody.

[0150] In some embodiments, the polypeptide is an enzyme. In some embodiments, the enzyme is a Cas nuclease, zinc finger nuclease, transcription activator-like effector nuclease, a meganuclease, Cre recombinase, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, a synthase, or a recombinase.

[0151] In some embodiments, the polypeptide is a gene regulatory protein, wherein the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa.

[0152] In some embodiments, the polypeptide is a nucleic acid binding protein. In some embodiments, the nucleic acid binding protein is a natural binding protein. In some embodiments, the nucleic acid binding protein is an engineered binding protein. In some embodiments, the nucleic acid binding protein is a single- stranded binding protein. In some embodiments, the nucleic acid binding protein is a double-stranded binding protein. In some embodiments, the nucleic acid binding protein is an E. coli single stranded DNA binding protein. In some embodiments, the nucleic acid binding protein is a dCas protein. In some embodiments, the nucleic acid binding protein is a TAL effector. In some embodiments, the nucleic acid binding protein is a zinc finger protein.

[0153] In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a natural antibody. In some embodiments, the antibody is an engineered antibody. In some embodiments, the antibody is a derivative of a natural or engineered antibody. In some embodiments, the antibody is a fragment of a natural or engineered antibody.

[0154] In some embodiments of the disclosure, the fusion protein further comprises a cell penetrating peptide (CPP). CPPs are a well-studied group of compounds widely used as carriers for the delivery of therapeutic agents. CPPs are relatively short peptides, 4–40 aa, with the ability to gain access to the cell interior by means of different mechanisms, mainly including endocytosis, and / or with the capacity to promote the intracellular effects by these peptides themselves, or by the delivered covalently or non-covalently conjugated bioactive cargoes. In some embodiments, the CPP is a transactivating transcriptional activator (TAT). In some embodiments, the CPP is an arginine 9 (R9) peptide. In some embodiments, the CPP is an 8- Lysine peptide. In some embodiments, the CPP is a polyhistidine KH27K peptide. In some embodiments, the CPP is a histidine-rich LAH4 peptide. In some embodiments, the CPP is anAntennapedia (Antp) peptide. In some embodiments, the CPP is a virus protein 22 (VP22) peptide. In some embodiments, the CPP is a Pep-1 peptide. In some embodiments, the CPP is a Tat-HA2. In some embodiments, the CPP is a human transcriptional factor Hph-1. In some embodiments, the CPP is a mHph1. In some embodiments, the CPP is a mHph2. In some embodiments, the CPP is an Azurin p18 peptide. In some embodiments, the CPP is a transportan. In some embodiments, the CPP is a SG3. In some embodiments, the CPP is a fibroblast growth factor (FGF)-12. In some embodiments, the CPP is a proline rich peptide. Linkers

[0155] In some embodiments, the fusion protein comprises a linker between the NLS peptides. In embodiments where the NLS comprises two primary NLS peptides, the linker is located between the two primary NLS peptides and functions to “link” the two NLS peptides together. In embodiments wherein the NLS comprises a primary NLS peptide and a secondary NLS peptide, the linker is located between the primary NLS peptide and the secondary NLS peptide and functions to “link” the two NLS peptides together. The linker of such embodiments facilitates the binding of the NLS peptides to each other. In some embodiments, the fusion protein comprises a linker between the primary NLS peptide and the secondary NLS peptide and a linker between the secondary NLS peptide and tertiary NLS peptide. The two linkers function to “link” the three NLS peptides together.

[0156] In some embodiments, the linker between the NLS peptides is 1-12 amino acids in length. In some embodiments, the linker between the NLS peptides is 2 amino acids in length. In some embodiments, the linker between the NLS peptides is 3 amino acids in length. In some embodiments, the linker between the NLS peptides is 4 amino acids in length. In some embodiments, the linker between the NLS peptides is 5 amino acids in length. In some embodiments, the linker between the NLS peptides is 6 amino acids in length. In some embodiments, the linker between the NLS peptides is 7 amino acids in length. In some embodiments, the linker between the NLS peptides is 8 amino acids in length. In some embodiments, the linker between the NLS peptides is 9 amino acids in length. In some embodiments, the linker between the NLS peptides is 10 amino acids in length. In some embodiments, the linker between the NLS peptides is 11 amino acids in length. In some embodiments, the linker between the NLS peptides is at least 12 amino acids in length.

[0157] In some embodiments, the linker between the NLS peptides comprises alanine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 alanine residues. In some embodiments, the linker comprises 1 alanine residue. In some embodiments, the linker comprises 2 alanine residues. In some embodiments, the linker comprises 3 alanine residues. In some embodiments, the linker comprises 4 alanine residues. In some embodiments, the linker comprises 5 alanine residues. In some embodiments, the linker comprises 6 alanine residues. In some embodiments, the linker comprises 7 alanine residues. In some embodiments, the linker comprises 8 alanine residues. In some embodiments, the linker comprises 9 alanine residues.

[0158] In some embodiments, the linker between the NLS peptides comprises serine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 serine residues. In some embodiments, the linker comprises 1 serine residue. In some embodiments, the linker comprises 2 serine residues. In some embodiments, the linker comprises 3 serine residues. In some embodiments, the linker comprises 4 serine residues. In some embodiments, the linker comprises 5 serine residues. In some embodiments, the linker comprises 6 serine residues. In some embodiments, the linker comprises 7 serine residues. In some embodiments, the linker comprises 8 serine residues. In some embodiments, the linker comprises 9 serine residues.

[0159] In some embodiments, the linker between the NLS peptides comprises glycine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 glycine residues. In some embodiments, the linker comprises 1 glycine residue. In some embodiments, the linker comprises 2 glycine residues. In some embodiments, the linker comprises 3 glycine residues. In some embodiments, the linker comprises 4 glycine residues. In some embodiments, the linker comprises 5 glycine residues. In some embodiments, the linker comprises 6 glycine residues. In some embodiments, the linker comprises 7 glycine residues. In some embodiments, the linker comprises 8 glycine residues. In some embodiments, the linker comprises 9 glycine residues.

[0160] In some embodiments, the linker between the NLS peptides comprises one or more threonine residues. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 threonine residues. In some embodiments, the linker comprises 1 threonine residue. In some embodiments, the linker comprises 2 threonine residues. In some embodiments, the linker comprises 3 threonine residues. In some embodiments, the linker comprises 4 threonine residues. In some embodiments, the linker comprises 5 threonine residues. In some embodiments, the linker comprises 6 threonine residues. In some embodiments, the linkercomprises 7 threonine residues. In some embodiments, the linker comprises 8 threonine residues. In some embodiments, the linker comprises 9 threonine residues.

[0161] In some embodiments, the linker between the NLS peptides comprises a mixture of alanine, serine, glycine, and / or threonine residues.

[0162] In some embodiments, the linker between the NLS peptides has an amino acid sequence of ASGGGGS (SEQ ID NO: 56). For example, in some embodiments, the combination of NiV_W-NiV_W above includes the ASGGGGS linker between the two copies of NiV_W and therefore would have an amino acid sequence of TCLGRRVVQPGMFEDHPPTKKARVSMRRMSASGGGGSTCLGRRVVQPGMFEDHPPTK KARVSMRRMS (SEQ ID NO: 58) where the linker is ASGGGGS (SEQ ID NO: 56) in bold.

[0163] In some embodiments, the fusion protein comprises more than one linker in the NLS, wherein the first linker is located as described above, i.e. between two of the NLS peptides. However, in embodiments where the NLS comprises more than two NLS peptides in tandem, i.e., three or more NLS peptides, there can be a subsequent linker between each NLS peptide. In some embodiments, the linker between the NLS peptides is 1-12 amino acids in length. In some embodiments, the linker between the NLS peptides is 2 amino acids in length. In some embodiments, the linker between the NLS peptides is 3 amino acids in length. In some embodiments, the linker between the NLS peptides is 4 amino acids in length. In some embodiments, the linker between the NLS peptides is 5 amino acids in length. In some embodiments, the linker between the NLS peptides is 6 amino acids in length. In some embodiments, the linker between the NLS peptides is 7 amino acids in length. In some embodiments, the linker between the NLS peptides is 8 amino acids in length. In some embodiments, the linker between the NLS peptides is 9 amino acids in length. In some embodiments, the linker between the NLS peptides is 10 amino acids in length. In some embodiments, the linker between the NLS peptides is 11 amino acids in length. In some embodiments, the linker between the NLS peptides is at least 12 amino acids in length.

[0164] In some embodiments, the linker between the NLS peptides comprises alanine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 alanine residues. In some embodiments, the linker comprises 1 alanine residue. In some embodiments, the linker comprises 2 alanine residues. In some embodiments, the linker comprises 3 alanine residues. In some embodiments, the linker comprises 4 alanine residues. In some embodiments, the linkercomprises 5 alanine residues. In some embodiments, the linker comprises 6 alanine residues. In some embodiments, the linker comprises 7 alanine residues. In some embodiments, the linker comprises 8 alanine residues. In some embodiments, the linker comprises 9 alanine residues.

[0165] In some embodiments, the linker between the NLS peptides comprises serine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 serine residues. In some embodiments, the linker comprises 1 serine residue. In some embodiments, the linker comprises 2 serine residues. In some embodiments, the linker comprises 3 serine residues. In some embodiments, the linker comprises 4 serine residues. In some embodiments, the linker comprises 5 serine residues. In some embodiments, the linker comprises 6 serine residues. In some embodiments, the linker comprises 7 serine residues. In some embodiments, the linker comprises 8 serine residues. In some embodiments, the linker comprises 9 serine residues.

[0166] In some embodiments, the linker between the NLS peptides comprises glycine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 glycine residues. In some embodiments, the linker comprises 1 glycine residue. In some embodiments, the linker comprises 2 glycine residues. In some embodiments, the linker comprises 3 glycine residues. In some embodiments, the linker comprises 4 glycine residues. In some embodiments, the linker comprises 5 glycine residues. In some embodiments, the linker comprises 6 glycine residues. In some embodiments, the linker comprises 7 glycine residues. In some embodiments, the linker comprises 8 glycine residues. In some embodiments, the linker comprises 9 glycine residues.

[0167] In some embodiments, the linker between the NLS peptides comprises one or more threonine residues. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 threonine residues. In some embodiments, the linker comprises 1 threonine residue. In some embodiments, the linker comprises 2 threonine residues. In some embodiments, the linker comprises 3 threonine residues. In some embodiments, the linker comprises 4 threonine residues. In some embodiments, the linker comprises 5 threonine residues. In some embodiments, the linker comprises 6 threonine residues. In some embodiments, the linker comprises 7 threonine residues. In some embodiments, the linker comprises 8 threonine residues. In some embodiments, the linker comprises 9 threonine residues.

[0168] In some embodiments, the linker between the NLS peptides comprises a mixture of alanine, serine, glycine, and / or threonine residues.

[0169] The desired properties of the linkers will be known to one having skill in the art. In some embodiments, the linker is a multiple of (GGGS)m followed by (G)n where m= the number of multiples of (GGGS) and n= the number of repeated (G). The GS linker is known in the art to be the most common flexible linker used in the art. You can use other linkers or omit the linker as well. In some embodiments, the linker can be as short as GS to multiples of G's and S's. In some embodiments, the linker includes multiples of G’s and S’s along with alanine (A) and / or threonine (T) residues. In some embodiments, the linker can serve as a recognition site for a restriction enzyme. In a non-limiting example, ATGGGS (SEQ ID NO: 89) is a flexible linker that also introduces an AgeI restriction site, which can help with subsequent cloning.

[0170] In some embodiments, the linker between the NLS peptides has an amino acid sequence of ASGGGGS (SEQ ID NO: 56). In some embodiments, the linker between the NLS peptides has an amino acid sequence of ASGG (SEQ ID NO: 57).

[0171] In some embodiments, the one or more linkers do not have to comprise the same amino acid sequence. In some embodiments, the one or more linkers do comprise the same amino acid sequence. For example, in some embodiments, the combination of H3-NiV_W-CIRBP-RSY above includes an ASGGGGS (SEQ ID NO: 56) linker between the H3 and NiV_W and also between the NiV_W and CIRBP-RSY. Therefore, such a combination would have an amino acid sequence of ARTKQTARKSTGGKAPRKQLATKAARKSASGGGGSTCLGRRVVQPGMFEDHPPTKKA RVSMRRMSASGGGGSSRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNE (SEQ ID NO: 59) where the linkers are in bold. In some embodiments, the one or more linkers do not comprise the same amino acid sequence. For example, in some embodiments, the tandem NLS peptides of NiV_W-MeCP2-c-Myc comprise an amino acid sequence of TCLGRRVVQPGMFEDHPPTKKARVSMRRMSASGGGGSKRPGRKRKAEADPQAIPKKR GRKASGGPAAKKKKLD (SEQ ID NO: 60) where the linkers are in bold.

[0172] In some embodiments, the fusion protein comprises a linker between the NLS and the operably linked polypeptide. In some embodiments, the linker between the NLS and polypeptide is 1-12 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 2 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 3 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 4 amino acids in length. In some embodiments, the linker between the NLS andpolypeptide is 5 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 6 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 7 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 8 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 9 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 10 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is 11 amino acids in length. In some embodiments, the linker between the NLS and polypeptide is at least 12 amino acids in length.

[0173] In some embodiments, the linker between the NLS and polypeptide comprises alanine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 alanine residues. In some embodiments, the linker comprises 1 alanine residue. In some embodiments, the linker comprises 2 alanine residues. In some embodiments, the linker comprises 3 alanine residues. In some embodiments, the linker comprises 4 alanine residues. In some embodiments, the linker comprises 5 alanine residues. In some embodiments, the linker comprises 6 alanine residues. In some embodiments, the linker comprises 7 alanine residues. In some embodiments, the linker comprises 8 alanine residues. In some embodiments, the linker comprises 9 alanine residues.

[0174] In some embodiments, the linker between the NLS and polypeptide comprises serine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 serine residues. In some embodiments, the linker comprises 1 serine residue. In some embodiments, the linker comprises 2 serine residues. In some embodiments, the linker comprises 3 serine residues. In some embodiments, the linker comprises 4 serine residues. In some embodiments, the linker comprises 5 serine residues. In some embodiments, the linker comprises 6 serine residues. In some embodiments, the linker comprises 7 serine residues. In some embodiments, the linker comprises 8 serine residues. In some embodiments, the linker comprises 9 serine residues.

[0175] In some embodiments, the linker between the NLS and polypeptide comprises glycine. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 glycine residues. In some embodiments, the linker comprises 1 glycine residue. In some embodiments, the linker comprises 2 glycine residues. In some embodiments, the linker comprises 3 glycine residues. In some embodiments, the linker comprises 4 glycine residues. In some embodiments, the linker comprises 5 glycine residues. In some embodiments, the linker comprises 6 glycine residues. Insome embodiments, the linker comprises 7 glycine residues. In some embodiments, the linker comprises 8 glycine residues. In some embodiments, the linker comprises 9 glycine residues.

[0176] In some embodiments, the linker between the NLS and polypeptide comprises one or more threonine residues. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 threonine residues. In some embodiments, the linker comprises 1 threonine residue. In some embodiments, the linker comprises 2 threonine residues. In some embodiments, the linker comprises 3 threonine residues. In some embodiments, the linker comprises 4 threonine residues. In some embodiments, the linker comprises 5 threonine residues. In some embodiments, the linker comprises 6 threonine residues. In some embodiments, the linker comprises 7 threonine residues. In some embodiments, the linker comprises 8 threonine residues. In some embodiments, the linker comprises 9 threonine residues.

[0177] In some embodiments, the linker between the NLS and polypeptide comprises a mixture of alanine, serine, glycine, and / or threonine residues.

[0178] In some embodiments, the linker between the NLS and polypeptide has an amino acid sequence of ASTGGGS (SEQ ID NO: 61). Methods of Delivering a Polypeptide into the Nucleus of a Cell

[0179] In some aspects, the disclosure provides methods of delivering a polypeptide into the nucleus of a cell, comprising introducing into the cell a fusion protein, wherein the fusion protein comprises a nuclear localization sequence (NLS) operably linked to the polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, thereby delivering the polypeptide into the nucleus of the cell.

[0180] In some embodiments, the polypeptide is delivered into a mammalian cell. In some embodiments, the mammalian cell is a neuronal cell. In some embodiments, the mammalian cell is a stem cell. In some embodiments, the mammalian cell is an immune cell. In some embodiments, the mammalian cell is a glial cell. In some embodiments, the mammalian cell is a muscle cell. In some embodiments, the mammalian cell is a hepatocyte. In some embodiments, the mammalian cell is a germ cell. In some embodiments, the mammalian cell is a pulmonary epithelial cell. In some embodiments, the stem cell is an induced pluripotent stem cell. In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a neuronal stem cell. In some embodiments, the stem cell is a hematopoietic stem cell.

[0181] In some embodiments of the disclosure, the polypeptide is delivered into a plant cell. In some embodiments, the polypeptide is delivered into a yeast cell.

[0182] In some embodiments, where the fusion protein comprises an NLS, the fusion protein confers cell penetrating ability.

[0183] In some embodiments of the disclosure, the fusion protein is introduced into the cell through methods known in the art.

[0184] In some embodiments, the fusion protein is introduced into the cell through the use of a transfection agent. Transfection agents are known in the art. A non-limiting example of a transfection agent is lipofectamine.

[0185] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of electroporation.

[0186] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of microinjection.

[0187] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of nanoparticles. Nanoparticles (NP) are known in the art. NP are found in 3 main classes, lipid-based NPs, polymeric NPs, and inorganic NPs. Lipid-based NPs offer many advantages as a delivery system including formulation simplicity, self-assembly, biocompatibility, high bioavailability, ability to carry large payloads and a range of physicochemical properties that can be controlled to modulate their biological characteristics. Some non-limiting examples of lipid-based NPs are liposomes, lipid nanoparticles, and oil emulsions. Liposome NPs are typically composed of phospholipids, which can form unilamellar and multilamellar vesicular structures, allowing the liposome to carry and deliver hydrophilic, hydrophobic and lipophilic drugs. Liposomes can entrap hydrophilic and lipophilic compounds in the same system. Lipid nanoparticles (LNPs) are liposome-like structures widely used for the delivery of nucleic acids. LNPs are typically composed of four major components: cationic or ionizable lipids that complex with negatively charged genetic material and aid endosomal escape, phospholipids for particle structure, cholesterol for stability and membrane fusion, and PEGylated lipids to improve stability and circulation. Polymeric NPs can be synthesized from natural or synthetic materials, as well as monomers or preformed polymers, which allows for a wide variety of possible structures and characteristics. Polymeric NPs can be formulated to enable precise control of multiple NP features and are generally good delivery vehicles becausethey are biocompatible and have simple formulation parameters. Polymeric NPs are synthesized using various techniques such as emulsification (solvent displacement or diffusion), nanoprecipitation, ionic gelation and microfluidics, which all result in different final products. Polymeric NPs also have variable drug delivery capabilities; therapeutics can be encapsulated within the NP core, entrapped in the polymer matrix, chemically conjugated to the polymer or bound to the NP surface. This enables delivery of various payloads including hydrophobic and hydrophilic compounds, as well as cargos with different molecular weights such as small molecules, biological macromolecules, proteins and vaccines. Some of the most common forms of polymeric NPs are nanocapsules (cavities surrounded by a polymeric membrane or shell) and nanospheres (solid matrix systems), which can be further divided into shapes such as polymersomes, micelles and dendrimers. Polymersomes are artificial vesicles, with membranes made using amphiphilic block copolymers. Dendrimers are a type of hyperbranched polymeric NPs with complex three-dimensional architectures for which the mass, size, shape and surface chemistry can be highly controlled. Functional groups present on the exterior of dendrimers enable conjugation of biomolecules or contrast agents to the surface while drugs can be loaded in the interior. Inorganic NPs made of materials such as gold, iron and silica have been used for various drug delivery and imaging applications. Magnetic iron oxide NPs, which are composed of magnetite (Fe3O4) or maghemite (Fe2O3), possess superparamagnetic properties at certain sizes and have shown success as contrast agents, drug delivery vehicles and thermal-based therapeutics. Additional non-limiting examples of inorganic NPs include calcium phosphate and mesoporous silica NPs, which have both been used successfully for gene and drug delivery.

[0188] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of a cell penetrating peptide. CPPs are relatively short peptides, 4–40 aa, with the ability to gain access to the cell interior by means of different mechanisms, mainly including endocytosis, and / or with the capacity to promote the intracellular effects by these peptides themselves, or by the delivered covalently or non-covalently conjugated bioactive cargoes. Some non-limiting examples of CPPs are: virus-derived or mimicking polymers such as TAT, influenza fusion peptide, rabies virus glycoprotein fragment (RVG), neuropilin, penetratin, polyarginines, R9 peptide, 8-Lysine peptide, KH27K, LAH4, Antp, VP22, Pep-1, Tat-HA2, Hph-1, mHph1, Azurin p18, transportan, SG3, FGF-12, and proline rich peptide.

[0189] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of a nucleic acid encoding the fusion protein. Delivery systems that transport genes of interest typically consist of two components, 1) nucleic acids with the genetic information encoding for the desired protein and 2) a gene delivery material that will transport the nucleic acid across the cell membrane. Plasmid DNA (pDNA) was the first nucleic acid to be pursued as a therapeutic molecule and remains popular in gene therapy. In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of introducing mRNA into the cell. Mesenger RNA (mRNA) does not hold risks associated with genome integration. mRNA does not require transport across the nuclear membrane, it typically acts effectively upon release in the cytosol and is functional in dividing and non-dividing cells. However, the use of mRNA molecules for expressing a desired protein has been hindered due to technological challenges, such as the ability to successfully transcribe in vitro mRNA in large amounts, instability in vivo, and immunogenicity. Chemically modified mRNAs (cmRNAs) remove or replace structural elements and modified nucleosides are used to circumvent such hinderances. Modifications such as pseudouridine (Ψ), 5-methylcytidine (m5C), N6- methyladenosine (m6A), 5-methyluridine (m5U), and or 2-thiouridine (s2U) have been shown to stabilize mRNA in that such modifications avoid stimulating specific Toll-like receptors in the cells innate immune system.

[0190] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of a virus. Viral delivery systems are known in the art. The three main vector strategies are based on adenoviruses, adeno-associated viruses (AAVs), and lentiviruses. Adenoviruses are non-enveloped double-stranded DNA viruses containing genomes of approximately 34-44 kilobase pairs. AAVs are replication-defective, nonenveloped viruses that have linear single-stranded DNA genome of approximately 4.8 kilobases. AAV vectors can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. AAVs can be engineered to deliver DNA to target cells. Lentiviruses are retroviruses that are enveloped RNA viruses containing two copies of a positive-sense single-stranded RNA genome. Lentiviral vectors are viral vectors with an ability to infect both dividing and non-dividing cells, integrate transgenes into the host cell genome, and efficiently transduce non-proliferating or slowly proliferating cells, such as CD34 + stem cells.

[0191] In some embodiments of the disclosure, the fusion protein is introduced into the cell through the use of STEP technology. STEP stands for stimuli-responsive traceless engineering platform which can be used for efficient delivery of biologics, such as proteins, into cells without compromising the biological activities the biologics. STEP technology is explained in detail in International Patent Application No. PCT / US2023 / 082123 filed December 1, 2023, the disclosure of which is incorporated by reference herein in its entirety. Briefly, conjugates contain the payload, a chemical linker, and a cell membrane fusogenic molecule. The cell membrane fusogenic molecule facilitates intracellular uptake of the conjugates. In some forms, the conjugate enters the cell through a non-endocytic pathway. The chemical linker can be a traceless chemical linker or an untraceless chemical linker. When the chemical linker is a traceless chemical linker, it contains a stimuli-responsive chemical moiety and / or a self- immolative chemical moiety. In some embodiments, the traceless chemical linker contains a stimuli-responsive chemical moiety and a self-immolative chemical moiety. Upon entry into a cell, the conjugate is exposed to one or more stimuli, such as a reducing and / or an acidic environment, that cleave the stimuli-responsive chemical moiety. This cleavage event activates self-immolation of the chemical linker via an electronic cascade and / or cyclization elimination.

[0192] STEP is achieved through chemical conjugation of membrane fusogenic molecules to amino- or thiol- groups on the surface of protein payloads via stimuli-responsive linkers that can be cleaved and completely removed by intracellular stimuli, such as a reducing and / or an acidic environment. The membrane fusogenic molecules are cell membrane fusogenic molecules. These cell membrane fusogenic molecules contain proteins, peptides, lipids, and / or small molecules. These cell membrane fusogenic molecules enhance fusion between the conjugate and a cell membrane and / or facilitate intracellular uptake of the conjugate. Cell membrane fusogenic molecules include peptides, lipids, small molecule moieties such as pentacyclic or tetracyclic moieties of cholesterol, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens, or progestogens, phytosterols (e.g., β-sitosterol). In some embodiments, the cell membrane fusogenic molecule contains small molecule moieties such as pentacyclic or tetracyclic moieties of cholesterol.

[0193] Examples of peptides that can be utilized as cell membrane fusogenic molecules include cell-penetrating peptides, also known as cell permeable peptides, protein transduction domains (PTDs), membrane translocating sequences (MTSs) and Trojan peptides. Cell penetratingpeptides (CPPs) include, but are not limited to, virus-derived or mimicking polymers such as TAT, influenza fusion peptide, rabies virus glycoprotein fragment (RVG), neuropilin, penetratin, and polyarginines. Examples of lipids that can be utilized as cell membrane fusogenic molecules include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; phosphatidylethanolamine; 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (16-PC); 1-palmitoyl-2-oleoyl-glycero-3- phosphocholine (18-PC); 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), or other related phosphatidylethanolamine with two attached fatty acyl chains, preferably unsaturated fatty acyl chains; lysolipids, etc. Examples of small molecule moieties that can be utilized as cell membrane fusogenic molecules include pentacyclic or tetracyclic moieties of cholesterol, steroid hormones, glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, phytosterols (e.g., β-sitosterol); or hydrophobic amino acid residues (e.g., tyrosine, phenylamine, tryptophan, etc.).

[0194] As a result of STEP technology, the payloads (e.g., protein) are released without any trace molecules and thus fully recover their biological functions after cell penetration.

[0195] In some embodiments of the disclosure, the fusion protein is introduced into the cell by way of the NLS sequence. This occurs as the NLS sequence functions as a CPP, i.e. the NLS not only penetrates the nucleus of the cell, but the NLS is also able to penetrate the cell membrane and reach the cytoplasm. In some embodiments where the NLS functions as a CPP, NLS comprises a MeCP2 NLS peptide. In some embodiments, the NLS peptide comprises the sequence of SEQ ID NO: 2. In some embodiments, the NLS peptide comprises a tandem repeat of the sequence of SEQ ID NO: 2. In some embodiments, the NLS peptide comprises tandem repeats of the sequence of SEQ ID NO: 2. In some embodiments, the linker between the repeats of SEQ ID NO: 2 of the NLS peptide is GGS.

[0196] The methods disclosed herein are suitable for delivery of any of the fusion proteins described herein into a cell and then into the nucleus of the cell. Nucleic Acids

[0197] Some aspects of the disclosure are directed to a nucleic acid encoding the fusion proteins described herein. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA). In some embodiments, the nucleic acid is a messenger ribonucleic acid (mRNA). In some embodiments, the nucleic acid is a viral genomic DNA.Vectors

[0198] Some aspects of the current disclosure are directed to a vector comprising a nucleic acid encoding a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the vector is selected from the group of adeno- associated virus, adenovirus, lentivirus, bacteriophage, and virus-like particles. In some embodiments, the vector is an adeno-associated virus.

[0199] Some aspects of the current disclosure are directed to a virus with a genome comprising the nucleic acids described herein, i.e. a nucleic acid encoding a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. Isolated Cells

[0200] Certain aspects of the current disclosure are directed to an isolated cell comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0201] Some aspects of the current disclosure are directed to a host cell comprising an expression vector described herein, i.e. a vector comprising a nucleic acid encoding a fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0202] The term “host cell” refers to a cell into which an expression vector has been introduced. The term encompasses not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in successive generations due to either environmental influences or mutation, such progeny may not be identical to the parent cell, but are still included within the scope of the term “host cell.” In some embodiments, eukaryotic and prokaryotic host cells, including mammalian cells, may be used as host cells. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These hostcells include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, Expi 293 cells, HEK-293 cells, other transient 293 expression systems known in the art, and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindnen), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Methods of Treatment

[0203] The term “treatment” or “treating” refers to preventing or delaying the onset, slowing down the progression, and / or or ameliorating the symptoms of the disorder. In some embodiments, any of the proteins disclosed herein (e.g., any of the fusion proteins comprising a NLS operably linked to a polypeptide disclosed herein) may be administered to a subject. In some embodiments, the subject has a disease. In some embodiments, the subject is a mammal. The term “mammal” refers to any animal species of the Mammalian class. Examples of mammals include humans; laboratory animals such as rats, mice, simians and guinea pigs; domestic animals such as rabbits, cattle, sheep, goats, cats, dogs, horses, and pigs and the like.

[0204] In some embodiments, the disease is genetic disease. In some embodiments, the disease is an imprinting disorder. In some embodiments, the disease is Angelman syndrome. In some embodiments, the disease is Prader Willi syndrome. In some embodiments, the disease is an X- chromosome linked genetic disease. In some embodiments, the disease is Rett Syndrome. In some embodiments, the disease is CDKL5 deficiency disorder (CDD). In some embodiments, the disease is cancer. Some non-limiting examples of cancer include carcinomas, sarcoma,leukemias, lymphomas, melanomas, myelomas, germ cell cancers, gynecologic cancers, genitourinary, and neurological cancers.

[0205] In some embodiments, any of the proteins disclosed herein are administered to the subject in need of treatment. In some embodiments, the proteins administered to the subject are a fusion protein comprising a NLS operably linked to a polypeptide. In some embodiments, the polypeptide is an enzyme, gene regulatory protein, nucleic acid binding protein, or antibody. In some embodiments, the enzyme is a CAS nuclease, zinc finger nuclease, transcription activator- like effector nuclease, a meganuclease, CRE, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, synthase, or recombinase. In some embodiments, the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa. In some embodiments, the nucleic acid binding protein is a natural or engineered single- or double-stranded binding protein. In some embodiments, the antibodies are natural or engineered antibodies and derivatives or fragments thereof. In some embodiments, the natural or engineered single- or double-stranded binding protein is an E. coli single stranded DNA binding protein, dCas protein, TAL effector, and zinc finger protein. In some embodiments, the fusion protein further comprises a cell penetrating peptide (CPP). In some embodiments, the CPP is a TAT, R9, 8-Lysine, LAH4, Antp, VP22, Pep-1, Tat-HA2, Hph- 1, mHph1, mHph2, Azurin p18, Transportan, SG3, FGF, or Pro.

[0206] Depending on the severity of the subject’s disease, the frequency and the duration of the treatment can be adjusted. In certain embodiments, the conjugates that confer improved intranuclear delivery of gene editing machinery described in the present disclosure can be administered at an initial dose. In some embodiments, the conjugates that confer improved intranuclear delivery of gene editing machinery described in the present disclosure can be administered at an initial dose, followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of conjugates that confer improved intranuclear delivery of gene editing machinery described herein in an amount that can be approximately the same, less than that of the initial dose, or more than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10weeks; at least 12 weeks; at least 14 weeks; at least 16 weeks; at least 18 weeks; at least 20 weeks; at least 22 weeks; at least 24 weeks; at least 26 weeks; at least 28 weeks; at least 30 weeks; at least 32 weeks; at least 34 weeks; at least 36 weeks; at least 38 weeks; at least 40 weeks; at least 42 weeks; at least 44 weeks; at least 46 weeks; at least 48 weeks; at least 50 weeks; or at least 52 weeks.

[0207] The mode of administration of the intranuclear gene editor delivery disclosed herein can vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal or intra-arterial. EXAMPLES

[0208] The following examples are presented to illustrate the present disclosure. The examples are not intended to be limiting in any manner. Example 1. Construction of Cas9 plasmids carrying various types of NLSs

[0209] To study the impact of NLSs on transporting proteins into the nucleus of a cell, a collection of Cas9 plasmids were created to encode well-defined NLSs with various amino acid sequence features and different importin binding specificities. cDNA encoding the various single NLSs or different combinations of NLSs were cloned into the 3’-end of Cas9 cDNA. Efficiency of the resulting Cas9 in editing cells were evaluated in vitro with cultured cell line or in vivo by injecting into a mouse brain. Example 2. Gene editing activity of disclosed Cas9-NLSs (NiV_W, HeV_W, and MeCP2)

[0210] A collection of cDNA encoding NLSs with various amino acid sequence features and different importin binding specificities were cloned individually into the 3’ end of Cas9 cDNA (Table 1). Experiments were performed to determine whether the disclosed Cas9-NLSs provide unique benefits in gene editing than Cas9 fused with common classical NLSs. RNPs were assembled through incubation of Cas9 nuclease with sgRNAs 276 (sgA) and 280 (sgB) that target the loxP-flanked STOP cassette in Ai9 cells (Table 1 and FIG.1A). The Cas9 / sgAB RNPs were then complexed with cell penetrating peptide (CPP) for intracellular delivery. The resulting RNPs / CPP were evaluated in primary fibroblasts isolated from Ai9 mice for their capacity forgenome editing, which was determined based on the expression of tdTomato. The results showed that the disclosed SpCas9-NiV_W, SpCa9-HeV_W, and SpCas9-MeCP2 all showed higher cell editing efficiency than the classical SpCas9-SV40 (Table 1 and FIG.1B and C). For example, SpCas9-NiV_W, SpCa9-HeV_W, and SpCas9-MeCP2 achieved 41.8%, 39.8 and 43.6% editing efficiency at Cas9 concentration of 1 µg / mL. However, SpCas9-SV40 edited only 23.4% cells. These findings showed that the disclosed Cas9-NLSs provided unique benefits in gene editing than Cas9 fused with common classical NLS and usage of these NLSs enhances the delivery and / or editing efficiencies. TABLE 1 Gene Binding Editing NLS Amino Acid Sequence NLS Type Importins Efficiency TCLGRRVVQPGMFEDHPPTKKARVSMRRMS bipartite KPNA4, NiV_W (SEQ ID NO: 1) NLS KPNA2 High TCLGRRVVQPGMFADYPPTKKARVLLRRMS bipartite KPNA4, HeV_W (SEQ ID NO: 3) NLS KPNA2 High KRPGRKRKAEADPQAIPKKRGRK bipartite KPNA4, MeCP2 (SEQ ID NO: 2) NLS KPNA3 High hnRNP_ YSNQQSGYGKVSRRGGHQNSYKPY D (SEQ ID NO: 13) PY NLS TNPO1 Low ARTKQTARKSTGGKAPRKQLATKAARKS nonclassical TNPO1 / IPO H3 (SEQ ID NO: 14) NLS 5 Low SRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATH CIRBP- NE RSY (SEQ ID NO: 15) RS NLS TNPO3 Low Example 3. NiV_W and HeV_W NLSs are identical in essential sequence and almost interchangeable functionally.

[0211] NiV_W and HeV_W NLSs have almost identical amino acid sequences, differing by only four amino acids (FIG.2). The two NLSs share the same minor and major importin binding sites, and the four amino acid mutations don’t impact their function significantly (FIG.1B). Similar gene editing efficiencies were observed when NiV_W was replaced by HeV_W. Only NiV_W was used as examples in the subsequent studies.Example 4. Better gene editing activities in disclosed NLS combination-Cas9 than single Cas9 in Ai9 reporter fibroblasts

[0212] Further experiments were performed to determine whether Cas9 proteins carrying different combinations of disclosed NLSs, especially the ones containing NiV_W, HeV_W, and / or MeCP2 provide additional benefits in gene editing than common NLS Cas9 in Ai9 reporter fibroblasts (Table 2). The Cas9 / sgAB RNPs were chemically engineered with STEP delivery technology for gene editing activity analysis in vitro Ai9 reporter fibroblasts. The results showed that Cas9 carrying any combinations of NiV_W, HeV_W, or MeCP2, such as SpCas9-H3-NiV_W, SpCas9-MeCP2-hnRNP, and SpCas9-NiV_W-MeCP2 showed higher cell editing efficiency than the classical SpCas9-SV40 in Ai9 fibroblasts. SpCas9-H3-NiV_W and SpCas9-NiV_W-MeCP2 also showed higher editing efficacy than single NLS -Cas9 including SpCas9-NiV_W and SpCas9-MeCP2. Notably Cas9 protein NiV_W-MeCP2 NLS combination showed the best editing efficiency in the study (FIG.3). TABLE 2 NLS combination Resulting gene editing efficiency NiV_W-NiV_W +++++ MeCP2-MeCP2 +++++ NiV_W-MeCP2 +++++ NiV_W-hnRNP_D  ++++ MeCP2-hnRNP_D  ++++ NiV_W-CIRBP-RSY  +++ H3-NiV_W ++++ MeCP2-H3 ++++ H3-NiV_W-CIRBP-RSY  ++++ HeV_W-MeCP2 ++++ H3-HeV_W ++++ Example 5. Better gene editing activity of disclosed NLS combinations than common NLS- Cas9 in the brain

[0213] Further experiments were performed to determine whether Cas9 proteins carrying the disclosed NLS combinations provide unique benefits in gene editing than common NLS Cas9 in the brain. The Cas9 / sgAB RNPs were chemically engineered with STEP for in vivo brain localdelivery. The resulting RNPs / STEP were evaluated in adult Ai9 mice for their capacity for genome editing of brain neuronal cells, which was determined based on the expression of tdTomato (red). The results showed that the disclosed SpCa9-H3-NiV_W (G5) and SpCas9- MeCP2-hnRNP (G6) both showed higher cell editing efficiency in the brain tissue than the classical SpCas9-SV40 with larger editing areas and more edited tdTomato+ cells (FIG.4). These findings showed that the disclosed NLSs Cas9 provided unique benefit in neuronal cells gene editing than classical NLS Cas9 and the use of disclosed NLSs enhances in vivo delivery and / or editing efficiencies in the brain. Example 6. Increased Cas9 gene editing activity of disclosed MeCP2-NiV_W NLS in the brain

[0214] The neuronal editing efficiency of the optimized second generation SpCas9-MeCP2- NiV_W was further studied in vivo. SpCas9-MeCP2-NiV_W / sgAB RNP was chemically engineered with STEP technology and followed by intrastriatal administration in adult Ai9 mouse. As shown in FIG.5, SpCas9-MeCP2-NiV_W RNPs are highly efficient in editing neurons in adult mouse brain with wide editing area, which is larger than the editing area by other Cas9s shown in FIG.4. The results confirm the highly efficient activity of disclosed combinational NLSs SpCas9-MeCP2-NiV_W for neuronal gene editing (FIG.5). Example 7. Triple NLSs showed enhanced gene editing activity

[0215] A further NLS was added to the C terminus of SpCas9-MeCP2-NiV_W and a new library of SpCas9 with triple NLSs was created (Table 3). These SpCas9 RNP were delivered by STEP technology and tested in Ai9 fibroblasts. Among the SpCas9 library, as shown in FIG.6, 2 candidates, SpCas9-MeCP2-NiV_W-Max and SpCas9-MeCP2-NiV_W-HCVC2, were found with higher gene editing efficiency than SpCas9-MeCP2-NiV_W. Other candidates did not show significantly increased editing activity compared to SpCas9-NiV_W-MeCP2. The results further confirm that the disclosed Cas9 proteins carrying an NLS such as NiV_W, HeV_W, or MeCP2, as well as any NLS combinations containing NiV_W, HeV_W, and / or MeCP2 show enhanced gene editing efficiency compared to common classical NLS.Example 8. NLSs enhance Cas9 gene editing activity

[0216] After confirming the increased editing activity of the disclosed Cas9-NLSs over common classical NLSs-Cas9 using STEP technology as the delivery tool both in vitro and in vivo, editing efficiency of an additional delivery system was tested. Commercially available Lipofectamine transfection agent was used to deliver the disclosed Cas9-NLSs RNP and their editing activity assessed in Ai9 reporter cells. As depicted in FIG.7, both SpCas9-MeCP2-NiV_W and SpCas9- H3-NiV_W demonstrated significantly higher editing efficiency compared to the classical SpCas9-SV40. Additionally, SpCas9-MeCP2-NiV_W exhibited superior editing activity over SpCas9-H3-NiV_W. These results support the notion that the disclosed NLSs could enhance the editing efficacy of Cas9 RNP regardless of the delivery method. TABLE 3 NLS added to NLS sequence added to NiVW-MeCP2SEQ ID Figure NiVW-MeCP2NO: Label Control G221 c-Myc PAAKKKKLD 62 G304 SV40 PKKKRKV 63 G305 Nucleoplasmin KRPAATKKAGQAKKKK 64 G306 BDV-P1 PPRIYPQLPSAPT 65 G307 HCVC2 PRRGPR 66 G308 QKI-5 RVHPYQR 67 G309 Amida RGRRRRQR 68 G310 HIV-1R RQARRNRRRRWR 69 G311 RanBP3 SDREDGNYCPPVKRERTS 70 G312 HCVC4 PRGRRQPIPKARRP 71 G313 BDV-P2 PRPRKIPR 72 G314 EBNA1 LKRPRSPSS 73 G315 FluA MASQGTKRSYEQM 74 G316 MyoD VNEAFETLKRC 75 G317 polyoma1 VSRKRPRPA 76 G318 E1a KRPRP 77 G319 VirD2C PKRPRDRHDGELGGRKRARG 78 G320 cFOS RRERNKMAAAKCRNRRR 79 G321 NS5A PPRKKRTVV 80 G322 Max PQSRKKLR 81 G323polyoma2 PKKARED 82 G324 L29 KTRKHRG 83 G325 Pax-PD VSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPE 84 G326 Mat-a MNKIPIKDLLNPG 85 G327 Rex MPKTRRRPRRSQRKRPPT 86 G328 M9 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY 87 G329 hARNT RAIKRRPGLDFDDDGEGNSKFLR 88 G330 Example 9. Disclosed NLSs enhance Cre recombinase activity in Ai9 reporter fibroblasts

[0217] To assess whether the disclosed NLSs could improve the delivery of non-Cas9 protein payloads into the nucleus, Cre recombinase was fused with the disclosed NLSs or SV40 NLSs. The recombination activity was evaluated in Ai9 reporter cells. As illustrated in FIG.8, both Cre-MeCP2-NiV_W and Cre-H3-NiV_W exhibited significantly higher recombination efficiency compared to the classical Cre-SV40. Furthermore, Cre-MeCP2-NiV_W demonstrated superior activity over Cre-H3-NiV_W. These results parallel those observed with Cas9-NLS RNP. In summary, the disclosed NLSs hold promise for delivering other types of biologics targeting the nucleus. Example 10. NLS Comprising MeCP2 Show Cell Penetration Activity

[0218] Experiments were performed to determine the cell penetration activity of the NiVW- MeCP2 NLS (FIG.9). It was shown that Free Cre recombinase cannot edit Ai9 reporter fibroblasts due to the lack of cell penetration vehicle (FIG.9). However, when NiVW-MeCP2 NLS was fused to the C-terminus of Cre recombinase, the fusion recombinase showed gene editing activity at high concentrations (FIG.9), indicating NiVW-MeCP2 has cell-penetration activity. In an attempt to optimize the NLS and study the impact of individual NLS to the cell- penetration activity the NiVW-MeCP2 NLS combo was moved to the N-terminus of Cre recombinase and tandem repeats of MeCP2 or NiVW were fused to the C-terminus (Table 4 and FIG.10). With the increase of C-terminal MeCP2 repeat from 1 to 3, we observed increasing gene editing activities, while increasing C-terminal NiVW repeats didn’t improve gene editing activities. The results imply that the C-terminal MeCP2 is the reason for the cell penetration activities of the C-terminal NiVW- MeCP2 NLS.

[0219] Table 4 includes information related to the optimization study shown in FIG.10. The MeCP2 and NiVW repeats were flanked with a GGS linker which is italicized in the table.

[0220] Further experiments were performed to visualize the cell penetration of GFP-Cas9- NiVW-MeCP2 fusion protein in live Ai9 fibroblast cells (FIG.11). After 1-hr treatment of GFP-Cas9-NiVW-MeCP2 fusion protein, green fluorescence from GFP was observed in the cytoplasm, indicating the GFP-Cas9-NiVW-MeCP2 fusion protein has entered the cell; however, little has entered the nucleus after such a time period. TABLE 4 N-Terminal C-Terminal Name NLS NLS Amino Acid Sequence MeCP2*1 MeCP2 KRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 2) MeCP2- KRPGRKRKAEADPQAIPKKRGRK GGS MeCP2*2 MeCP2 KRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 90) MeCP2- KRPGRKRKAEADPQAIPKKRGRK MeCP2- GGSKRPGRKRKAEADPQAIPKKRGRK GGS MeCP2*3 MeCP2 KRPGRKRKAEADPQAIPKKRGRK (SEQ ID NO: 91) TCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: NiVW- NiV_W*1 NiVW 1) MeCP2 TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS TCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: NiV_W*2 NiVW-NiVW 92) TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS NiVW-NiVW- TCLGRRVVQPGMFEDHPPTKKARVSMRRMS (SEQ ID NO: NiV_W*3 NiVW 93) Description of Sequences

[0221] Table 5 is a description of the sequences included in the electronic sequence listing. The table provides the SEQ ID NO and a description of the sequence. These sequences are included in the aforementioned electronic sequence listing (42796P_SequenceListing; Size: 132 KB; and Date of Creation: May 20, 2024) incorporated by reference in its entirety.TABLE 5 SEQ ID Description Sequence 1 NiV_W TCLGRRVVQPGMFEDHPPTKKARVSMRRMS 2 MeCP2 KRPGRKRKAEADPQAIPKKRGRK 3 HeV_W TCLGRRVVQPGMFADYPPTKKARVLLRRMS TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaTCLGRRVVQ 4 NiV_W-NiV_W PGMFEDHPPTKKARVSMRRMS KRPGRKRKAEADPQAIPKKRGRKXaKRPGRKRKAEADPQAIP 5 MeCP2- MeCP2 KKRGRK HeV_W - TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaTCLGRRVVQP 6 HeV_W GMFADYPPTKKARVLLRRMS NiV_W-MeCP2 TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA 7 EADPQAIPKKRGRK MeCP2-NiV_W KRPGRKRKAEADPQAIPKKRGRKXaTCLGRRVVQPGMFEDHP 8 PTKKARVSMRRMS HeV_W-MeCP2 TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaKRPGRKRKA 9 EADPQAIPKKRGRK HeV_W-NiV_W TCLGRRVVQPGMFADYPPTKKARVLLRRMSXaTCLGRRVVQP 10 GMFEDHPPTKKARVSMRRMS MeCP2-HeV_W KRPGRKRKAEADPQAIPKKRGRKXaTCLGRRVVQPGMFADYP 11 PTKKARVLLRRMS NiV_W-HeV_W TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaTCLGRRVVQ 12 PGMFADYPPTKKARVLLRRMS 13 hnRNP_D YSNQQSGYGKVSRRGGHQNSYKPY 14 H3 ARTKQTARKSTGGKAPRKQLATKAARKS 15 CIRBP-RSY SRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNE 16 SV40 PKKKRKV 17 c-Myc PAAKKKKLD 18 nucleoplasmin KRPAATKKAGQAKKKK NiV_W- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaYSNQQSGYG 19 hnRNP_D KVSRRGGHQNSYKPY MeCP2- KRPGRKRKAEADPQAIPKKRGRKXaYSNQQSGYGKVSRRGGH 20 hnRNP_D QNSYKPY NiV_W-CIRBP- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaSRDYYSSRSQ 21 RSY SGGYSDRSSGGSYRDSYDSYATHNEH3-NiV_W ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPG MFEDHPPTKKARVSMRRMS MeCP2-H3 KRPGRKRKAEADPQAIPKKRGRKXaARTKQTARKSTGGKAPR KQLATKAARKS hnRNP_D- YSNQQSGYGKVSRRGGHQNSYKPYXaTCLGRRVVQPGMFED NiV_W HPPTKKARVSMRRMS CIRBP-RSY- SRDYYSSRSQSGGYSDRSSGGSYRDSYDSYATHNEXaTCLGRR NiV_W VVQPGMFEDHPPTKKARVSMRRMS H3-MeCP2 ARTKQTARKSTGGKAPRKQLATKAARKSXaKRPGRKRKAEA DPQAIPKKRGRK H3-HeV_W ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPG MFADYPPTKKARVLLRRMS H3-NiV_W- ARTKQTARKSTGGKAPRKQLATKAARKSXaTCLGRRVVQPG CIRBP-RSY MFEDHPPTKKARVSMRRMSYbSRDYYSSRSQSGGYSDRSSGG SYRDSYDSYATHNE NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA c-Myc EADPQAIPKKRGRK YbPAAKKKKLD NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA SV40 EADPQAIPKKRGRK YbPKKKRKV NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Nucleoplasmin EADPQAIPKKRGRK YbKRPAATKKAGQAKKKK NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA BDV-P1  EADPQAIPKKRGRKYbPPRIYPQLPSAPT NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA HCVC2  EADPQAIPKKRGRKYbPRRGPR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA QKI-5  EADPQAIPKKRGRKYbRVHPYQR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Amida  EADPQAIPKKRGRKYbRGRRRRQR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA HIV-1R  EADPQAIPKKRGRKYbRQARRNRRRRWR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA RanBP3 EADPQAIPKKRGRKYbSDREDGNYCPPVKRERTS NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA HCVC4  EADPQAIPKKRGRKYbPRGRRQPIPKARRPNiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA BDV-P2  EADPQAIPKKRGRKYbPRPRKIPR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA EBNA1  EADPQAIPKKRGRKYbLKRPRSPSS NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA FluA  EADPQAIPKKRGRKYbMASQGTKRSYEQM NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA MyoD  EADPQAIPKKRGRK YbVNEAFETLKRC NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA polyoma1  EADPQAIPKKRGRKYbVSRKRPRPA NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA E1a  EADPQAIPKKRGRKYbKRPRP NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA VirD2C  EADPQAIPKKRGRKYbPKRPRDRHDGELGGRKRARG NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA cFOS  EADPQAIPKKRGRKYbRRERNKMAAAKCRNRRR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA NS5A  EADPQAIPKKRGRKYbPPRKKRTVV NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Max  EADPQAIPKKRGRKYbPQSRKKLR NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaSKRPGRKRK polyoma2 AEADPQAIPKKRGRKYbPKKARED NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA L29  EADPQAIPKKRGRKYbKTRKHRG NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Pax-PD  EADPQAIPKKRGRK YbVSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPE NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Mat-a  EADPQAIPKKRGRKYbMNKIPIKDLLNPG NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA Rex  EADPQAIPKKRGRKYbMPKTRRRPRRSQRKRPPT NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA M9  EADPQAIPKKRGRKYbNQSSNFGPMKGGNFGGRSSGPYGGGG QYFAKPRNQGGY NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSXaKRPGRKRKA hARNT  EADPQAIPKKRGRKYbRAIKRRPGLDFDDDGEGNSKFLRASGGGGS ASGGGGS linker  ASGG linker ASGG NiV_W-NiV_W TCLGRRVVQPGMFEDHPPTKKARVSMRRMSASGGGGSTCLG + linker RRVVQPGMFEDHPPTKKARVSMRRMS H3-NiV_W- ARTKQTARKSTGGKAPRKQLATKAARKSASGGGGSTCLGRR CIRBP-RSY + VVQPGMFEDHPPTKKARVSMRRMSASGGGGSSRDYYSSRSQ linkers SGGYSDRSSGGSYRDSYDSYATHNE NiV_W-MeCP2- TCLGRRVVQPGMFEDHPPTKKARVSMRRMSASGGGGSKRPG c-Myc + linkers RKRKAEADPQAIPKKRGRKASGGPAAKKKKLD ASTGGGS ASTGGGS linker c-Myc-NiV_W- PAAKKKKLDXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMS MeCP2 YbKRPGRKRKAEADPQAIPKKRGRKASGG SV40-NiV_W- PKKKRKVXaTCLGRRVVQPGMFEDHPPTKKARVSMRRMSYb MeCP2 KRPGRKRKAEADPQAIPKKRGRKASGG nucleoplasmin- KRPAATKKAGQAKKKKXaTCLGRRVVQPGMFEDHPPTKKAR NiV_W-MeCP2 VSMRRMSYbKRPGRKRKAEADPQAIPKKRGRKASGG BDV-P1 PPRIYPQLPSAPT HCVC2 PRRGPR QKI-5 RVHPYQR Amida RGRRRRQR HIV-1R RQARRNRRRRWR RanBP3 SDREDGNYCPPVKRERTS HCVC4 PRGRRQPIPKARRP BDV-P2 PRPRKIPR EBNA1 LKRPRSPSS FluA MASQGTKRSYEQM MyoD VNEAFETLKRC polyoma1 VSRKRPRPA E1a KRPRP VirD2C PKRPRDRHDGELGGRKRARG cFOS RRERNKMAAAKCRNRRR NS5A PPRKKRTVV Max PQSRKKLR polyoma2 PKKAREDL29 KTRKHRG Pax-PD VSNGCVSKILGRYYETGSIRPRAIGGSKPRVATPE Mat-a MNKIPIKDLLNPG Rex MPKTRRRPRRSQRKRPPT M9 NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY hARNT RAIKRRPGLDFDDDGEGNSKFLR ATGGGS Linker ATGGGS MeCP2*2 KRPGRKRKAEADPQAIPKKRGRK GGS KRPGRKRKAEADPQAIPKKRGRK MeCP2*3 KRPGRKRKAEADPQAIPKKRGRK GGS KRPGRKRKAEADPQAIPKKRGRK GGS KRPGRKRKAEADPQAIPKKRGRK NiV_W*2 TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS TCLGRRVVQPGMFEDHPPTKKARVSMRRMS NiV_W*3 TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS TCLGRRVVQPGMFEDHPPTKKARVSMRRMS GGS TCLGRRVVQPGMFEDHPPTKKARVSMRRMS

Claims

WHAT IS CLAIMED IS:

1. A fusion protein comprising a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising an amino acid sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

2. The fusion protein of claim 1, wherein the NLS comprises a primary NLS peptide comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

3. The fusion protein of claim 1 or 2, wherein the NLS comprises a combination of the primary NLS peptide in tandem.

4. The fusion protein of claim 3, wherein the combination comprises at least two copies of the primary NLS peptide.

5. The fusion protein of claim 4, wherein the NLS comprises the amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

6. The fusion protein of claim 1, wherein the NLS comprises a combination of at least two different primary NLS peptides.

7. The fusion protein of claim 6, wherein each peptide of the combination comprises a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

8. The fusion protein of claim 7, wherein the NLS comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 7 or SEQ ID NO:

8.

9. The fusion protein of claim 7, wherein the NLS comprises a sequence of either SEQ ID NO: 7 or SEQ ID NO:

8.

10. The fusion protein of any one of claims 1-9, wherein the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide.

11. The fusion protein of claim 10, wherein the secondary NLS peptide is selected from the group consisting of NLS of heterogeneous nuclear ribonucleoprotein D (hnRNP_D)-NLS (SEQID NO: 13), NLS of histone H3 (H3-NLS) (SEQ ID NO: 14), NLS of cold-inducible RNA-binding protein (CIRBP-RSY) (SEQ ID NO: 15), the NLS of SV40 large T antigen (SV40) (SEQ ID NO: 16), c-Myc-NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18).

12. The fusion protein of claim 11, wherein the NLS comprises a sequence selected from the group consisting of NLS of Nipah virus W protein (NiV_W)-hnRNP_D (SEQ ID NO: 19), NLS of methyl-CpG binding protein 2 (MeCP2)-hnRNP_D (SEQ ID NO: 20), NiV_W-CIRBP-RSY (SEQ ID NO: 21), H3-NiV_W (SEQ ID NO: 22), MeCP2-H3 (SEQ ID NO: 23), H3-NiV_W- CIRBP-RSY (SEQ ID NO: 28), c-Myc-NiV_W-MeCP2 (SEQ ID NO: 62), SV40-NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W-MeCP2 (SEQ ID NO: 64).

13. The fusion protein of any one of claims 1-12, wherein the NLS is linked to the polypeptide through a linker.

14. The fusion protein of claim 10, wherein the primary NLS peptide and the secondary NLS peptide are linked through a linker.

15. The fusion protein of any one of claims 1-14, wherein the polypeptide is an enzyme, a gene regulatory protein, a nucleic acid binding protein, or an antibody.

16. The fusion protein of claim 15, wherein the enzyme is a CAS nuclease, zinc finger nuclease, transcription activator-like effector nuclease, a meganuclease, CRE, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, synthase, or recombinase.

17. The fusion protein of claim 15, wherein the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa.

18. The fusion protein of claim 15, wherein the nucleic acid binding protein is a natural or engineered single- or double-stranded binding protein.

19. The fusion protein of claim 15, wherein the antibodies are natural or engineered antibodies and derivatives or fragments thereof.

20. The fusion protein of claim 18, wherein the natural or engineered single- or double-stranded binding protein is an E. coli single stranded DNA binding protein, dCas protein, TAL effector, zinc finger protein.

21. The fusion protein of any one of claims 1-20, further comprising a cell penetrating peptide (CPP).

22. The fusion protein of claim 21, wherein the CPP is a transactivating transcriptional activator (TAT), an arginine 9 (R9) peptide, an 8-Lysine peptide, a polyhistidine KH27K peptide, a histidine-rich LAH4 peptide, an Antennapedia (Antp) peptide, a virus protein 22 (VP22) peptide, a Pep-1 peptide, a Tat-HA2, a human transcriptional factor Hph-1, a mHph1, a mHph2, an Azurin p18 peptide, a transportan, a SG3, a fibroblast growth factor (FGF)-12, or a proline rich peptide (PRO).

23. A nucleic acid encoding the fusion protein according to any one of claims 1-22.

24. The nucleic acid of claim 23, wherein the nucleic acid is a deoxyribonucleic acid (DNA).

25. The nucleic acid of claim 23, wherein the nucleic acid is a messenger ribonucleic acid (mRNA).

26. The nucleic acid of claim 23, wherein the nucleic acid is a viral genomic DNA.

27. An expression vector comprising the nucleic acid of claim 23.

28. A virus with a genome comprising the nucleic acid of claim 23.

29. A host cell comprising the expression vector of claim 27.

30. A method of delivering a polypeptide into the nucleus of a cell, comprising introducing into the cell a fusion protein, wherein the fusion protein comprises a nuclear localization sequence (NLS) operably linked to the polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide having a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, thereby delivering the polypeptide into the nucleus of the cell.

31. The method of claim 30, wherein the NLS comprises a primary NLS peptide comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

32. The method of claim 30 or 31, wherein the cell is a mammalian cell, plant cell, or yeast cell.

33. The method of claim 32, wherein the mammalian cell is a neuronal cell, stem cell, immune cell, glial cell, muscle cell, hepatocyte, germ cell, or pulmonary epithelial cell.

34. The method of claim 33, wherein the stem cell is an induced pluripotent stem cell, embryonic stem cell, neuronal stem cell, and hematopoietic stem cell.

35. The method of any one of claims 30-34, wherein the cell is a cell of a mammalian subject.

36. The method of claim 35, wherein the subject is in need of treatment.

37. The method of claim 36, wherein the mammalian subject has a disease.

38. The method of claim 37, wherein the disease is a genetic disease.

39. The method of claim 38, wherein the disease is an imprinting disorder, e.g., Angelman syndrome and Prader Willi syndrome.

40. The method of claim 38, wherein the disease is an X-chromosome linked genetic disease, e.g., Rett Syndrome and CDKL5 deficiency disorder (CDD).

41. The method of claim 37, wherein the disease is cancer, e.g., carcinomas, sarcoma, leukemias, lymphomas, melanomas, myelomas, germ cell cancers, gynecologic cancers, genitourinary, and neurological cancers.

42. The method of any one of claims 30 to 41, wherein the fusion protein is introduced into the cell through the use of a transfection agent, electroporation, microinjection, nanoparticles, a cell penetrating peptide, STEP technology, a nucleic acid encoding the fusion protein, introducing mRNA into the cell, or through a virus.

43. The method of any one of claims 27 to 37, wherein the fusion protein is introduced into the cell without the use of a transfection agent, wherein the NLS comprises an NLS peptide comprising a sequence having at least 80% sequence identity to SEQ ID NO:

2.

44. The method of claim 43, wherein the fusion protein is introduced into the cell without the use of a transfection agent, wherein the NLS comprises an NLS peptide comprising the sequence of SEQ ID NO:

2.

45. The method of any one of claims 30-44, wherein the NLS comprises a combination of the primary NLS peptide in tandem.

46. The method of claim 45, wherein the combination comprises at least two copies of the primary NLS peptide.

47. The method of claim 46, wherein the NLS comprises the sequence of any one of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

48. The method of claim 30, wherein the NLS comprises a combination of at least two different primary NLS peptides.

49. The method of claim 48, wherein the NLS comprises a combination of at least two different primary NLS peptides, each peptide having a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

50. The method of claim 48, wherein the NLS comprises a sequence that is at least 80% identical to the sequence of either SEQ ID NO: 7 or SEQ ID NO:

8.

51. The method of claim 50, wherein the NLS comprises the sequence of either SEQ ID NO: 7 or SEQ ID NO:

8.

52. The method of any one of claims 30-50, wherein the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide.

53. The method of claim 52, wherein the secondary NLS peptide is selected from the group consisting of hnRNP_D-NLS (SEQ ID NO: 13), H3-NLS (SEQ ID NO: 14), CIRBP-RSY-NLS(SEQ ID NO: 15), the NLS of SV40 large T antigen (SEQ ID NO: 16), c-Myc-NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18).

54. The method of claim 30, wherein the NLS comprises a sequence selected from the group consisting of NiV_W-hnRNP_D (SEQ ID NO: 19), MeCP2-hnRNP_D (SEQ ID NO: 20), NiV_W-CIRBP-RSY (SEQ ID NO: 21), H3-NiV_W (SEQ ID NO: 22), MeCP2-H3 (SEQ ID NO: 23), H3-NiV_W-CIRBP-RSY (SEQ ID NO: 28), c-Myc-NiV_W-MeCP2 (SEQ ID NO: 62), SV40-NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W-MeCP2 (SEQ ID NO: 64).

55. The method of any one of claims 30-54, wherein the NLS is linked to the polypeptide through a linker.

56. The method of claim 52, wherein the primary NLS peptide and the secondary NLS peptide are linked through a linker.

57. The method of any one of claims 30-56, wherein the polypeptide is an enzyme, gene regulatory protein, nucleic acid binding protein, or antibody.

58. The method of claim 57, wherein the enzyme is a CAS nuclease, zinc finger nuclease, transcription activator-like effector nuclease, a meganuclease, CRE, partially or fully deactivated nuclease, nucleases fused with nucleic acid binding protein, demethylases, deaminases, polymerases, synthase, or recombinase.

59. The method of claim 57, wherein the gene regulatory protein is a transcription factor or proteins associated with transcription factors, gene regulatory proteins fused to a nucleic acid binding domain, CRISPRi, or CRISPRa.

60. The method of claim 57, wherein the nucleic acid binding protein is a natural or engineered single- or double-stranded binding protein.

61. The method of claim 57, wherein the antibodies are natural or engineered antibodies and derivatives or fragments thereof.

62. The method of claim 60, wherein the natural or engineered single- or double-stranded binding protein is an E. coli single stranded DNA binding protein, dCas protein, TAL effector, zinc finger protein 63. The method of any one of claims 30-62, further comprising a cell penetrating peptide (CPP).

64. The method of claim 63, wherein the CPP is a TAT, R9, 8-Lysine, LAH4, Antp, VP22, Pep- 1, Tat-HA2, Hph-1, mHph1, mHph2, Azurin p18, Transportan, SG3, FGF, or Pro.

65. A method of delivering a fusion protein into a cell, wherein the fusion protein comprises a nuclear localization sequence (NLS) operably linked to a polypeptide, wherein the NLS is heterologous to the polypeptide, and wherein the NLS comprises a primary NLS peptide comprising a sequence that is at least 80% identical to SEQ ID NO: 2, thereby delivering the fusion protein into the cell.

66. The method of claim 65, wherein the NLS comprises a primary NLS peptide comprising the sequence of SEQ ID NO:

2.

67. The method of claim 65 or 66, wherein the cell the cell is a mammalian cell, plant cell, or yeast cell.

68. The method of claim 67, wherein the mammalian cell is a neuronal cell, stem cell, immune cell, glial cell, muscle cell, hepatocyte, or pulmonary epithelial cell.

69. The method of claim 68, wherein the stem cell is an induced pluripotent stem cell, embryonic stem cell, neuronal stem cell and hematopoietic stem cell.

70. The method of any one of claims 65-69, wherein the cell is a cell of a mammalian subject.

71. The method of claim 70, wherein the subject is in need of treatment.

72. The method of claim 71, wherein the mammalian subject has a disease.

73. The method of claim 72, wherein the disease is a genetic disease.

74. The method of claim 73, wherein the disease is an imprinting disorder, i.e., Angelman syndrome and Prader Willi syndrome.

75. The method of claim 73, wherein the disease is an X-chromosome linked genetic disease, i.e., Rett Syndrome and CDKL5 deficiency disorder.

76. The method of claim 72, wherein the disease is cancer, e.g., carcinomas, sarcoma, leukemias, lymphomas, melanomas, myelomas, germ cell cancers, gynecologic cancers, genitourinary, and neurological cancers.

77. The method of any one of claims 65-76, wherein the NLS comprises a combination of the primary NLS peptide in tandem.

78. The method of claim 77, wherein the combination comprises at least two copies of the primary NLS peptide.

79. The method of claim 77, wherein the NLS comprises the sequence of SEQ ID NO:

5.

80. The method of claim 77, wherein the NLS comprises a combination of two or more different primary NLS peptides, wherein one primary NLS peptide comprises SEQ ID NO: 2 and the one or more additional primary NLS peptides comprises a sequence selected from SEQ ID NO: 1 or SEQ ID NO:

3.

81. The method of claim 80, wherein the NLS comprises a sequence that is at least 80% identical to a sequence selected from SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

11.

82. The method of claim 80, wherein the NLS comprises the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

11.

83. The method of any one of claims 65-82, wherein the NLS further comprises a secondary NLS peptide that is operably linked to either the N- or C-terminus of the primary NLS peptide.

84. The method of claim 83, wherein the secondary NLS peptide is selected from the group consisting of hnRNP_D-NLS (SEQ ID NO: 13), H3-NLS (SEQ ID NO: 14), CIRBP-RSY-NLS(SEQ ID NO: 15), the NLS of SV40 large T antigen (SEQ ID NO: 16), c-Myc-NLS (SEQ ID NO: 17), or nucleoplasmin-NLS (SEQ ID NO: 18).

85. The method of claim 65, wherein the NLS comprises a sequence selected from the group consisting of MeCP2-hnRNP_D (SEQ ID NO: 20), MeCP2-H3 (SEQ ID NO: 23), c-Myc-NiV_W- MeCP2 (SEQ ID NO: 62), SV40-NiV_W-MeCP2 (SEQ ID NO: 63), and nucleoplasmin-NiV_W- MeCP2 (SEQ ID NO: 64).

86. The method of any one of claims 65-85, wherein the NLS is linked to the polypeptide through a linker.

87. The method of claim 83, wherein the primary NLS peptide and the secondary NLS peptide are linked through a linker.

Images