Enhancement of DNA uptake into cell nucleus and methods of use

Abstract

Disclosed are methods of increasing transgene expression in a cell. The method can include providing a DNA vector comprising a Recognition Cassette (NUE-DNA vector), providing a nuclear uptake enhancer protein (NUE) for delivery into a cell and co-delivering the NUE-DNA vector and the NUE into the cell wherein co-delivering the NUE-DNA vector with the NUE increases transgene expression levels as compared to delivery of the NUE-DNA vector alone. The method can also include co-delivering a mRNA comprising a sequence encoding a NUE (NUE-mRNA) and a NUE-DNA vector into a cell wherein the co-delivery increases transgene expression levels as compared to delivery of the NUE-DNA vector alone. Constructs of NUEs are also provided.

Description

ENHANCEMENT OF DNA UPTAKE INTO CELL NUCLEUS AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Ser. No. 63 / 612,512, filed on December 20, 2023, and U.S. Ser. No. 63 / 735,536, filed on December 18, 2024, the disclosures of which are incorporated by reference herein in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 19, 2024, is named JBI6822WOPCT1_SL and is 161,000 bytes in size.BACKGROUND

[0003] Gene therapy is a potentially curative treatment approach for patients suffering from genetic diseases caused by inherited, disease-associated pathogenic mutations resulting in dysfunctional gene products and a pronounced disease phenotype. It involves the repair or modification of a gene to correct for a pathogenic mutation or to supplement a healthy copy of an otherwise diseased gene so that the expression of a functional protein can be achieved, which in turn leads to an improvement of disease symptoms or even a curative effect. Viruses have evolved effective mechanisms of introducing genetic material into cells and by using viral vectors these mechanisms can be harnessed for gene therapy. However, viral vectors have a number of disadvantages, such as immunogenicity, limited packaging capacity and high production costs. Thus, there is a need for new methods for delivering nucleic acids to subjects in need for gene therapy applications.SUMMARY

[0004] In a first aspect, a method of increasing transgene expression is provided. The method comprises providing at least one DNA vector comprising at least one Recognition Cassette (NUE-DNA vector); providing at least one nuclear uptake enhancer protein (NUE) for delivery into a cell, or providing at least one mRNA comprising a sequence encoding at least one NUE (NUE- mRNA); and co-delivering the at least one NUE-DNA vector and the at least one NUE into the cell or co-delivering the at least one NUE-DNA vector and the at least one NUE-mRNA into the cell, wherein co-delivering the at least one NUE-DNA vector with the at least one NUE or codelivering the at least one NUE-DNA vector with the at least one NUE-mRNA increases transgene expression levels as compared to delivery of the at least one NUE-DNA vector alone.

[0005] In some embodiments, the at least one Recognition Cassette comprises at least one Recognition Sequence (RS), wherein the at least one NUE binds the at least one Recognition sequence. In some embodiments, the at least one Recognition Sequence comprises about 5bp to about 45bp. In some embodiments, the at least one Recognition Sequence comprises about 9bp to about 40bp. In some embodiments, the at least one Recognition Cassette comprises between about 5 to about 30 Recognition Sequences.

[0006] In some embodiments, more than one NUE or more than one NUE-mRNA is provided, wherein each NUE comprises a different amino acid sequence from one another, wherein the Recognition Cassette comprises more than one Recognition Sequence, wherein the Recognition Sequences comprise different nucleic acid sequences from one another, and wherein each NUE comprises a specificity for a different Recognition Sequence. In some embodiments, more than one NUE or more than one NUE-mRNA is provided, wherein each NUE comprises a different amino acid sequence from one another, wherein the at least one NUE-DNA vector comprises more than one Recognition Cassette, wherein each Recognition Cassettes comprises different Recognition Sequences from each other, and wherein each NUE comprises a specificity for a different Recognition Sequence comprised within different Recognition Cassettes.

[0007] In some embodiments, the Recognition Cassette comprises at least one spacer, wherein the at least one spacer is located between the Recognition Sequences. In some embodiments, the at least one spacer prevents steric hindrance or electrostatic hindrance between 2 or more NUEs bound on the Recognition Sequences. In some embodiments, the at least one spacer comprises about 5 to about 45 bps. In some embodiments, the at least one spacer comprises 10 or 15 bp.

[0008] In some embodiments, the at least one NUE-DNA vector further comprises a gene encoding a cargo. In some embodiments, the cargo comprises a protein, an RNA or both. In someembodiments, the RNA is not translated. In some embodiments, the RNA comprises an interfering RNA, a gene silencing oligonucleotide, shRNA, miRNA, tRNAs or an RNA that mediates RNA editing. In some embodiments, the RNA comprises a hairpin RNA.

[0009] In some embodiments, the at least one NUE-DNA vector comprises a circular doublestranded plasmid DNA, a circular double-stranded DNA nanovector, a linear open-ended doublestranded DNA, a linear closed-ended double-stranded DNA, a linear single-stranded DNA containing double-stranded NUE recognition sequences or a circular single-stranded DNA containing double-stranded NUE recognition sequences. In some embodiments, the at least one NUE-DNA vector comprises a circular double-stranded DNA nanovector, and wherein the circular double-stranded DNA nanovector comprises a DNA nanoplasmid or DNA minicircle. In some embodiments, the at least one NUE-DNA vector comprises a DNA nanovector, and wherein the DNA nanovector comprises a linear closed ended double stranded DNA.

[0010] In some embodiments, 1, 2, 3, 4, or 5 NUEs are provided and co-delivered or 1, 2, 3, 4, or 5 NUE-mRNAs are provided and co -delivered.

[0011] In some embodiments, the at least one NUE comprises at least one DNA binding domain; at least one linker; and at least one effector domain. In some embodiments, more than one NUE is delivered or more than one mRNA comprising a sequence encoding at least one NUE (NUE- mRNA) is delivered, wherein each NUE comprises a different DNA binding domain from one another. In some embodiments, the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain.

[0012] In some embodiments, 2, 3, 4 or 5 NUEs are provided and co-delivered or 2, 3, 4, or 5 NUE-mRNAs are provided and co-delivered, wherein a first NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain and wherein a second NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

[0013] In some embodiments, 2, 3, 4 or 5 NUEs are provided and co-delivered or 2, 3, 4 or 5 NUE-mRNAs are provided and co-delivered, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain. In some embodiments, 2, 3, 4 or 5 NUEs are provided and co-delivered or 2, 3, 4 or 5 NUE-mRNAs are provided and co-delivered, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain. In some embodiments, 2, 3, 4 or 5 NUEs are provided and co-delivered or 2, 3, 4 or 5 NUE-mRNAs are provided and co-delivered, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain.

[0014] In some embodiments, at least one NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery. In some embodiments, the at least one NUE interacts with the nuclear import machinery. In some embodiments, the at least one NUE interacts with a cytoplasmic transport machinery. In some embodiments, the at least one NUE interacts with a nuclear import machinery and interacts with the cytoplasmic transport machinery. In some embodiments, a first NUE interacts with the nuclear import machinery and a second NUE interacts with the cytoplasmic transport machinery.

[0015] In some embodiments, the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids. In some embodiments, the at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids. In some embodiments, the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcription activator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof. In some embodiments, the at least one DNA binding domain comprises a C2H2-zinc finger DNA binding domain. In some embodiments, the at least one DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5.

[0016] In some embodiments, the nuclear import domain interacts with an endogenous nuclear import machinery. In some embodiments, the cytoplasmic transport domain interacts with the cytoplasmic transport machinery. In some embodiments, the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with thecytoplasmic transport machinery. In some embodiments, the nuclear import machinery comprises an importin dependent nuclear import machinery. In some embodiments, the nuclear import domain comprises a polypeptide that interact with proteins of a nucleoporin family, wherein the interaction or the binding of the nuclear import domain enables nuclear uptake of the NUE-DNA vector.

[0017] In some embodiments, the cytoplasmic transport domain comprises a polypeptide that binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that binds the Dynactin motor complex, wherein the interaction or the binding of the cytoplasmic transport domain enables nuclear uptake of the NUE-DNA vector. In some embodiments, the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p. In some embodiments, the nuclear import domain comprises an importin-P binding domain of importin-a. In some embodiments, the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9. In some embodiments, the nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11. In some embodiments, the nuclear import domain is mammalian or viral.

[0018] In some embodiments, the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery. In some embodiments, the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins. In some embodiments, the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35.

[0019] In some embodiments, the cargo comprises a therapeutic protein.

[0020] In some embodiments, the at least one NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162.

[0021] In some embodiments, the at least one NUE-mRNA and the at least one NUE-DNA vector are co-delivered in vivo. In some embodiments, the co-delivering is non-viral co-delivery of the at least one NUE-mRNA and at least one NUE-DNA vector.

[0022] In some embodiments, the method further comprises co-delivering a therapeutic cargo.

[0023] In some embodiments, the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18.

[0024] In some embodiments, the Recognition Cassette comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NO: 149-161.

[0025] In a second aspect, a nucleic acid encoding at least one nuclear uptake enhancer protein (NUE) for delivery into a cell is provided. In some embodiments, the NUE comprises at least one DNA binding domain; at least one linker; and at least one effector domain. In some embodiments, the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain. In some embodiments, the NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one at least one linker, a nuclear import domain and a cytoplasmic transport domain.

[0026] In some embodiments, the at least one NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery. In some embodiments, the at least one NUE interacts with the nuclear import machinery. In some embodiments, the at least one NUE interacts with the cytoplasmic transport machinery. In some embodiments, the NUE interacts with the nuclear import machinery and interacts with the cytoplasmic transport machinery.

[0027] In some embodiments of the nucleic acid, the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids. In some embodiments of the nucleic acid, the at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids. In some embodiments, the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcription activator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof. In some embodiments, the at least one DNA binding domain comprises a C2H2- zinc finger DNA binding domain.

[0028] In some embodiments of the nucleic acid, the at least one DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5. In some embodiments, the nuclear import domain interacts with an endogenous nuclear import machinery and / or the cytoplasmic transport domain interacts with the cytoplasmic transport machinery. In some embodiments, the nuclear import domain interacts with an endogenous nuclear import machinery. In some embodiments, the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

[0029] In some embodiments of the nucleic acid, the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with cytoplasmic transport machinery. In some embodiments, the nuclear import machinery comprises an importin dependent nuclear import machinery. In some embodiments, the nuclear import domain comprises a polypeptide that interacts or binds with proteins of a nucleoporin family, wherein interaction or binding enables nuclear uptake of the NUE-DNA vector.

[0030] In some embodiments of the nucleic acid, the cytoplasmic transport domain comprises a polypeptide that interacts or binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that interacts or binds the Dynactin motor complex, wherein the interaction or the binding enables nuclear uptake of the NUE-DNA vector. In some embodiments, the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p. In some embodiments, the nuclear import domain comprises an importin-P binding domain of importin-a. In some embodiments, the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9. In some embodiments, the nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11. In some embodiments, the nuclear import domain is mammalian or viral. In some embodiments, the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery. In some embodiments, the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins. In some embodiments, the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35.

[0031] In some embodiments of the nucleic acid, the nucleic acid comprises DNA or RNA.

[0032] In some embodiments of the nucleic acid, the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162. In some embodiments, the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18.

[0033] In a third aspect, a NUE encoded by the nucleic acid of any one of the embodiments herein is provided.

[0034] In a fourth aspect, a nuclear uptake enhancer protein (NUE) is provided. The NUE comprises at least one DNA binding domain; at least one linker; and at least one effector domain. In some embodiments, the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain. In some embodiments, the NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain. In some embodiments, the NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain. In some embodiments, the NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain. In some embodiments, the NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery. In some embodiments, the NUE interacts with the nuclear import machinery. In some embodiments, the NUE interacts with the cytoplasmic transport machinery. In some embodiments, the NUE interacts with a nuclear import machinery and interacts with the cytoplasmic transport machinery.

[0035] In some embodiments of the NUE, the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids. In some embodiments, the at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids. In some embodiments, the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcription activator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof. In some embodiments, the at least one DNA binding domain comprises a C2H2-zinc finger DNA binding domain. In some embodiments, the at least one the DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequenceset forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5. In some embodiments, the nuclear import domain interacts with an endogenous nuclear import machinery. In some embodiments, the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

[0036] In some embodiments of the NUE, the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with the cytoplasmic transport machinery. In some embodiments, the nuclear import machinery comprises an importin dependent nuclear import machinery. In some embodiments, the nuclear import domain comprises a polypeptide that interacts or binds with proteins of a nucleoporin family, wherein the interaction or the binding enables nuclear uptake of a NUE-DNA vector. In some embodiments, the cytoplasmic transport domain comprises a polypeptide that binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that binds the Dynactin motor complex, wherein the interaction or the binding enables nuclear uptake of a NUE- DNA vector. In some embodiments, the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p. In some embodiments, the nuclear import domain comprises an importin-P binding domain of importin-a. In some embodiments, the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9.

[0037] In some embodiments of the NUE, the at least one nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11. In some embodiments, the at least one nuclear import domain is mammalian or viral. In some embodiments, the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery. In some embodiments, the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins. In some embodiments, the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35. In some embodiments, the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162. In some embodiments, the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs:

[0038] In a sixth aspect, a DNA vector comprising a Recognition Cassette (NUE-DNA vector) is provided. In some embodiments, the Recognition Cassette comprises at least Recognition Sequence (RS), and wherein a NUE binds the Recognition Sequence. In some embodiments, the at least one Recognition Sequence comprises about 5bp to about 45bp. In some embodiments, the Recognition Sequence comprises about 9bp to about 40bp. In some embodiments, the Recognition Cassette comprises between about 5 to about 30 Recognition Sequences.

[0039] In some embodiments of the NUE-DNA vector, the Recognition Cassette comprises more than one Recognition Sequence, wherein each Recognition Sequence comprises different nucleic acid sequences from one another and wherein each Recognition Sequence is specific for a different NUE. In some embodiments, the Recognition Cassette comprises at least one spacer, wherein the at least one spacer is located between the Recognition Sequences. In some embodiments, the at least one spacer prevents steric hindrance or electrostatic hindrance between 2 or more NUEs bound on the Recognition Sequences. In some embodiments, the at least one spacer comprises about 5 to about 45 bps. In some embodiments, the at least one spacer comprises 10 or 15 bp. In some embodiments, the NUE-DNA vector comprises more than one Recognition Cassette and wherein each Recognition Cassette have Recognition Sequences from each other and wherein each Recognition Cassette and Recognition Sequence is specific for a different NUE from one another.

[0040] In some embodiments of the NUE-DNA vector, the NUE-DNA vector further comprises a gene encoding a cargo. In some embodiments, the cargo comprises a protein, an RNA or both.

[0041] In some embodiments of the NUE-DNA vector, the Recognition Cassette comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 149-161.

[0042] In a seventh aspect, a cell is provided, wherein the cell comprises at least one NUE-DNA vector; and at least one nucleic acid encoding a NUE.

[0043] In an eighth aspect, a cell is provided, wherein the cell comprises at least one NUE-DNA vector; and at least one NUE.

[0044] In a ninth aspect, a composition is provided, wherein the composition comprises a cell of any one of the embodiments described herein.

[0045] In a tenth aspect, a composition comprising a cell is provided, wherein the cell comprises at least one NUE-DNA vector and at least one nucleic acid encoding an NUE.

[0046] In an eleventh aspect, a composition comprising a cell, wherein the cell comprises at least one NUE-DNA vector and at least one NUE, is provided.BRIEF DESCRIPTION OF THE FIGURES

[0047] Fig. 1 : depicts nuclear uptake of non-viral DNA gene therapy vectors with nuclear uptake enhancers. As shown, DNA with mRNA encoding at least one nuclear uptake enhancer (NUE) protein is co-delivered into a cell. The in-situ expression of the nuclear uptake enhancer mediates efficient interaction of the DNA payload with the nuclear import machinery and increased nuclear uptake.

[0048] Fig. 2: depicts a NUE protein binding to a NUE DNA vector by the DNA binding domain.

[0049] Fig. 3: Measurement of Luciferase expression from NUE-DNA vector encoding Luciferase in the presence of Aphidicolin. A549 Cells were transfected with 1) Luciferase NUE- DNA vector alone and a control mRNA in the presence of varying concentrations of Aphidicolin. As shown is a measurement of Luciferase expression in the presence and absence of different concentrations of Aphidicolin from a concentration of 0 pM to 10 pM.

[0050] Figs. 4A-4G: Measurement of Luciferase expression from NUE-DNA vector encoding Luciferase post co-transfection of various NUE mRNAs comprising DNA binding domain 1. As shown is the measurement of Luciferase expression upon transfection of 1 ) the Luciferase NUE- DNA vector comprising the Recognition Cassette V03 (SEQ ID NO: 151) with NUE mRNA. Plots shown in Figs. 4A-4G each shows a different NUE mRNA that was co-transfected with the Luciferase NUE-DNA vector comprising the Recognition Cassette V03 (SEQ ID NO: 151) 2) Luciferase NUE-DNA vector comprising the Recognition Cassette V03 with a mRNA encoding DNA binding domain 1 (SEQ ID NO: 1), 3) Luciferase NUE-DNA vector (V03) only. As shown in the X-axis are sequence identifiers for the NUEs for the NUE designs set forth in SEQ ID NOs: 56, 57, 68, 69, 70, 71 and 162.

[0051] Figs. 5A-5F: Measurement of Luciferase expression from NUE-DNA vector encodingLuciferase post co-transfection of various NUE mRNAs comprising DNA binding domain 2. As shown is the measurement of Luciferase expression upon transfection of 1 ) the Luciferase NUE- DNA vector comprising the Recognition Cassette V07 (SEQ ID NO: 155) with NUE mRNA. Plot A-F each shows a different NUE mRNA that was co-transfected with the Luciferase NUE-DNA vector comprising the Recognition Cassette V07 2) co-transfection of Luciferase NUE-DNA vector comprising the Recognition Cassette V07 with a mRNA encoding DNA binding domain 2 (SEQ ID NO: 2), 3) Luciferase NUE-DNA vector comprising the Recognition Cassette V07 only (SEQ ID NO: 155). As shown in the X-axis are sequence identifiers for the NUEs for the NUE design set forth in SEQ ID NOs: 72, 73, 76, 77, 86 and 87.

[0052] Figs. 6A- 6E: Measurement of Luciferase expression from NUE-DNA vector encoding Luciferase post co-transfection of various NUE mRNAs comprising DNA binding domain 4. As shown is the measurement of Luciferase expression upon transfection of 1 ) the Luciferase NUE- DNA vector comprising the Recognition Cassette VI 1 (SEQ ID NO: 159) with NUE mRNA. Plot A-E each shows a different NUE mRNA that was co-transfected with the Luciferase NUE-DNA vector comprising the Recognition Cassette VI 1 2) co-transfection of Luciferase NUE-DNA vector comprising the Recognition Cassette VI 1 with a mRNA encoding DNA binding domain 4 (SEQ ID NO: 4), 3) Luciferase NUE-DNA vector comprising the Recognition Cassette VI 1 only. (SEQ ID NO: 159). As shown in the X-axis are sequence identifiers for the NUEs which comprised the NUE sequences as set forth in SEQ ID NOs: 94, 98, 99, 105 and 108.

[0053] Figure 7 : Measurement of Luciferase expression from Luciferase encoding NUE-DNA vector containing different Recognition Cassettes in the presence of a NUE. As shown is the measurement of Luciferase expression upon co-transfection of different versions of the Luciferase NUE-DNA vector containing different Recognition Cassettes that each contain varying numbers of Recognition Sequences for DNA binding domain 1 in the presence of a NUE (SEQ ID NO: 71). Grey control NUE DNA, black Luciferase NUE DNA vector comprising Recognition Cassette V01 (SEQ ID NO: 149), black squared Luciferase NUE DNA vector comprising Recognition Cassette V02 (SEQ ID NO: 150), black dotted Luciferase NUE DNA vector comprising Recognition Cassette V03 (SEQ ID NO: 151), black striped Luciferase NUE DNA vector comprising Recognition Cassette V04 (SEQ ID NO: 152).

[0054] Figure 8: Measurement of Luciferase expression from NUE-DNA vector encodingLuciferase post co-transfection of various NUE-mRNAs encoding a nuclear import domain and NUE-mRNAs encoding a cytoplasmic transport domain. As shown is the measurement of Luciferase expression upon co-transfection of 1) The Luciferase NUE-DNA vector comprising the recognition Cassette V13 (SEQ ID NO: 161) with two different NUE mRNAs. The plot shows different NUE mRNAs encoding cytoplasmic transport domains (SEQ ID NOs: 134-138) in combination with NUE-mRNA (SEQ ID NO: 108). Depicted is the fold increase in Luciferase reporter gene expression over NUE (SEQ ID NO: 108) alone. Relative Luciferase expression of co-transfection of mRNAs encoding the two DNA binding domains (SEQ ID NO: 4 and SEQ ID NO: 1) not encoding any effector domains were used as negative control.Definitions

[0055] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0056] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like. The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of up to and including ±10% (e.g., 1%, 5%, or 10%) from the specified value, as such variations are appropriate to perform the disclosed methods. Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0057] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0058] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0059] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. NatT. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0060] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389- 3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0061] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an Expectation Value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0062] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. NatT. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0063] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0064] The meaning of “substantially the same” can differ depending on the context in which the term is used. Because of the natural sequence variation likely to exist among heavy and light chains and the genes encoding them, one would expect to find some level of variation within the amino acid sequences or the genes encoding the NUEs or NUE-DNA vectors described herein, with little or no impact on their unique binding properties (e.g., specificity and affinity). Such anexpectation is due in part to the degeneracy of the genetic code, as well as to the evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, in the context of nucleic acid sequences, “substantially the same” means at least 65% identity between two or more sequences. Preferably, the term refers to at least 70% identity between two or more sequences, more preferably at least 75% identity, more preferably at least 80% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 91% identity, more preferably at least 92% identity, more preferably at least 93% identity, more preferably at least 94% identity, more preferably at least 95% identity, more preferably at least 96% identity, more preferably at least 97% identity, more preferably at least 98% identity, and more preferably at least 99% or greater identity. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two nucleotide or amino acid sequences may e.g., be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci 4, 11-17 (1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences may be determined using the Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970) algorithm.

[0065] The degree of variation that may occur within the amino acid sequence of a protein without having a substantial effect on protein function is much lower than that of a nucleic acid sequence, since the same degeneracy principles do not apply to amino acid sequences. Accordingly, in the context of a protein, “substantially the same” means proteins having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the proteins described.

[0066] The terms “express” and “produce” (or “expression” and “production”) are used synonymously herein, and refer to the biosynthesis of a gene product. These terms encompass the transcription of a gene into RNA. These terms also encompass translation of RNA into one or more polypeptides, and further encompass all naturally occurring post-transcriptional and post- translational modifications. The expression or production of protein may be within the cytoplasm of the cell.

[0067] “Polynucleotide,” synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be singlestranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to double-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications may be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides .

[0068] The term “transgene” as described herein refers to a sequence that encodes an exogenous protein or RNA to be expressed in a transduced cell.

[0069] The term “vector,” and “DNA vector” refers to a nucleic acid sequence for delivery of a nucleic acid that includes at least one modification compared to a naturally occurring sequence and may contain a transgene that encodes an exogenous protein, an RNA, cargo or therapeutic cargo. In some embodiments described herein, the transgene comprises nucleic acid sequences encoding a Crispr endonuclease and / or a Crispr guide RNA. In some embodiments herein, a vector is provided, wherein the vector comprises a plasmid DNA, nanoplasmid DNA, linear closed end DNA or any other type of DNA transgene expression vector. The DNA vector can be described as a plasmid DNA, nanoplasmid DNA, linear closed end DNA.

[0070] The term “nuclear uptake enhancer protein (NUE)” refers to a protein, that upon expression, increases the uptake of a DNA vector into the cell nucleus by a) sequence specific binding to a DNA vector, b) binding to cellular proteins that are involved in the transport of molecules through the nuclear pore and into the nucleus (the nuclear import machinery) and / or c) the ability to interact with / bind to components of the cytoplasmic transport machinery. NUEs aredesigned as bifunctional or multifunctional proteins that are modular in nature. The NUE comprises at least one DNA binding domain, at least one linker and at least one effector domain. The at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain. NUEs may be obtained by rational design, domain shuffling and protein evolution.

[0071] For example, in a designed NUE, the two (or more) functions can be comprised within two (or more) distinct protein domains, that are separated by a linker sequence in such a way that enables optimal activity of each domain.

[0072] A “NUE-DNA vector” refers to a DNA vector comprising a Recognition Sequence. The NUE specifically binds the Recognition Sequence within the NUE-DNA vector. In some embodiments described herein, the NUE-DNA vector comprises a circular double-stranded plasmid DNA, a circular double-stranded DNA nanovector, a linear open-ended double-stranded DNA, a linear closed-ended double-stranded DNA, a linear single-stranded DNA containing double-stranded NUE recognition sequences or circular single-stranded DNA containing doublestranded NUE recognition sequences. In some embodiments, the NUE-DNA vector comprises a closed ended linear DNA. In some embodiments, the NUE-DNA vector further comprises at least one chromatin interacting sequence.

[0073] “Recognition Sequence (RS)” as described herein is a DNA sequence that can be bound specifically by the DNA binding domain of the NUE. The Recognition Sequence is a NUE binding site and a NUE recognition site. A DNA vector that contains one or more Recognition Sequences that can be bound by a NUE is referred to as a “NUE-DNA vector.” The DNA binding domain of a NUE is specific for a Recognition Sequence on the DNA vector (NUE-DNA vector) and is selected from the zinc finger, transcription activator-like effector or CRISPR / Cas family, or any other kind of DNA binding protein. DNA binding proteins are known to those of skill in the art. The binding affinity of the NUE to the NUE-DNA vector is determined by the type and number of DNA binding domains within each NUE as well as the number Recognition Sequences encoded within the NUE-DNA vector. In the embodiments herein, the NUE-DNA vector comprises DNA Recognition Sequence(s), that can be bound specifically by a DNA binding domain of the NUE. In some embodiments, the NUE is delivered by an mRNA encoding the NUE, referred to as the “NUE-mRNA.” In some embodiments, the NUE-mRNA encodes at least one NUE.

[0074] “Recognition Cassette” as described herein, comprises an array of varying number of functionally spaced Recognition Sequences. In some embodiments described herein, a DNA vectoris provided, wherein the vector comprises at least one Recognition Cassette (NUE-DNA vector). As described herein, a DNA vector comprising at least one Recognition Cassette is referred to as a “NUE-DNA vector.” The Recognition Cassette comprises at least one Recognition Sequence. In some embodiments of any one of the embodiment herein, a single NUE-DNA vector has more than one Recognition Cassette. In some embodiments, the multiple Recognition Cassettes have different sequences and different Recognition Sequences from one another. In some embodiments, the different Recognition Cassettes have specificity for different NUEs from each other.

[0075] “Spacers” or “spacer sequence” as described herein, refers to DNA sequences of varying length that separate the Recognition Sequences in a Recognition Cassette to facilitate optimal binding of one or multiple NUEs. In some embodiments, the Recognition Cassette comprises at least one spacer. In some embodiments described herein, an NUE-DNA vector is provided, wherein the NUE-DNA vector comprises a Recognition Cassette, wherein the Recognition Cassette comprises Recognition Sequences and spacers. In some embodiments, the spacer comprises about 5 to about 45 bps. In some embodiments, the spacer comprises about 10 to about 40 bps. In some embodiments, the spacer comprises about 10 to about 15 bps. In some embodiments, the spacer comprises 10 or 15 bp. In some embodiments, the spacer comprises 10 bp. In some embodiments, the spacer comprises 15 bp.

[0076] “Cytoplasmic transport machinery,” as described herein refers to a network of cellular proteins and co-factors that mediate transport of molecules through the cellular cytoplasm, e.g., along the microtubule network. Without being limiting, this may include members of dynein / dynactin complexes, for example.

[0077] “Nuclear import machinery,” as described herein, refers to a network of cellular proteins and co-factors that mediate and control nuclear import within a cell. Without being limiting, this may include the importin family of proteins, for example.

[0078] “DNA binding domain(s),” as described herein, refers to a protein domain that mediates specific binding to a Recognition Sequence or Recognition Sequences. Without being limiting, the DNA binding domain can be a Zinc finger, a TALE protein, a Cas protein, a Leucine Zipper protein, or any other specific DNA binding protein, for example. The DNA binding domain of a NUE, mediates sequence specific binding of the NUE to the Recognition Sequence. DNA binding properties can be controlled by engineering the DNA binding domain. Different binding properties, such as binding affinity, can influence the assembly (and disassembly) of a functionalNUE-DNA vector / NUE complex and thus the efficiency of nuclear import and subsequent transgene expression.

[0079] “Effector domains,” as described herein, refer to a protein domain that directs nuclear import or cytoplasmic transport. In some embodiments, the effector domain comprises a nuclear import domain. In some embodiments, the effector domain comprises a cytoplasmic transport domain. In some embodiments, the effector domain comprises a nuclear transport domain and a cytoplasmic transport domain. In some embodiments, the effector domain has a binding specificity for components of the cytoplasmic transport machinery. In some embodiments, the effector domain has a binding specificity for components of the nuclear import machinery.

[0080] “Nuclear import domain (NID),” as described herein, refers to a protein domain that binds to one or more components of the nuclear import machinery. The nuclear import domain mediates interaction of the NUE-DNA vector bound to the NUE with the cellular nuclear import machinery or directly with the nuclear pore complex (NPC). In a principle NUE design, the nuclear import domain is a nuclear localization sequence (NLS), that is bound by importin alpha or other members of the importin family of proteins. A variety of NLS sequences have been described in the literature. Well known examples include the SV40 large T antigen NLS or the c-Myc NLS. Some NLS can interact directly with e.g., importin beta, without requiring importin-alpha as an adapter. Further, Importin-beta binding domains of importin-alpha, or other members of the importin family of proteins can serve as a nuclear import domain. Besides incorporating peptide sequences (NLS) or endogenous protein domains that bind to Importin beta, binding of the NUE to the nuclear import machinery can also be mediated by the incorporation of engineered binding domains like single chain antibody fragments, or so-called alternative scaffold binding proteins (e.g., DARPINs, Anticalins, Fynomers, Centyrins or any other protein that has been engineered to bind components of the nuclear import machinery).

[0081] “Cytoplasmic transport domain (CTD)” as described herein, refers to a protein domain that binds to one or more components of the cytoplasmic transport machinery. The high viscosity of the cytoplasm limits free diffusion of large molecules in the cell and movement and distribution of vector DNA introduced into a cell is very limited.

[0082] The “linker” or “linker sequence(s)” as described within, is comprised within a NUE and enables proper positioning of the DNA-binding domains, nuclear import domains orcytoplasmic transport domains and NUE-DNA vector to enable functional DNA import into the nucleus. The linkers are polypeptide sequences of various lengths and of either flexible, structured, or flexible and structured nature. The linkers may serve as a spacer between functional domains for the NUE that enables optimal folding, spacing and function of the domains within the NUE. Within the NUE, the functional domains are linked by peptide sequences (linkers).

[0083] The linkers are designed to provide the right orientation of and the right distance between the functional domains. Linkers can be designed from described structured or unstructured amino acid sequences. The linker sequence can vary in length and usually ranges from 1-40 amino acids but can also be significantly longer in order to separate the functional domains of a NUE even further by the introduction linker sequences that fold into stable protein structures themselves.DETAILED DESCRIPTION

[0084] Advances in gene therapy and therapeutic protein expression in cells have been reported, such as engineering viral vectors to improve cellular transduction and subsequent protein expression; however, the use of viral vectors for therapeutic transgene delivery and production has unmet challenges, such as immunogenicity and high production costs. A major advantage of providing non-viral DNA vectors is biosafety, however, it has been noted to have poor efficiency due to low transient expression. The present invention, against this backdrop, meets these various challenges, which has the advantage of reduced immunogenicity, re-dosability, scalable manufacturing and increased transgene capacity with the delivery of the NUE with the NUE-DNA vector, allowing access to safe and efficacious gene therapy products. Additionally, such methods can be used for manufacturing cells for use in cell therapy. Consequently, there is significant need for novel technologies that enable in vivo non-viral delivery of DNA into the nucleus to broaden the applicability and access to the potentially curative approach of gene therapy as well as cell therapies. The present invention is directed to such improved methods and materials.

[0085] Until today, most non-viral gene therapies have not delivered on their promise mainly due to a lack of efficacy and / or a lack of efficient transgene expression. One of the main limitations of DNA based non-viral vectors is the inefficient nuclear uptake of DNA, particularly in nondividing cells. Molecular import into the cell nucleus is a tightly regulated process and large molecular cargo designated for the cell nucleus needs to be actively transported through the nuclearpore complex. In general, this process is orchestrated by a family of importin proteins that bind to peptidic nuclear localization signals that are typically present within proteins but not DNA. Efficient nuclear access for larger DNA molecules such as plasmid DNA can mostly only occur in dividing cells where the nucleus disassembles during mitosis. However, the majority of cells that are to be targeted by gene therapies are non- or only very slowly dividing cells where the absence of nuclear localization signals and the hydrodynamic diameter of larger DNA molecules hinders efficient nuclear import.

[0086] DNA viruses can efficiently deliver their DNA cargo into the host cell nucleus by hijacking the host cell cytoplasmic transport and nuclear import machinery. The present disclosure provides, in part, methods for efficient nuclear import of DNA by co-delivery of NUE-mRNA together with a cognate NUE-DNA vector or the co-delivery of the NUE with the cognate NUE- DNA vector. These NUE-DNA vectors may be used as non-viral DNA gene therapy vectors with NUEs. (Fig. 1). Once delivered into the cytoplasm, the NUE binds to the NUE-DNA vector or the NUE-mRNA is translated into a NUE transiently, which binds the NUE-DNA vector (Fig. 2), facilitating its transport to the perinuclear space and / or interacting with the nuclear import machinery to actively drive translocation of the DNA cargo vector into the nucleus.

[0087] NUE-mRNA and cognate NUE-DNA vector or NUE and cognate NUE-DNA vector are co-delivered into the target cell. They may be also delivered sequentially in any order (in vivo), wherein the NUE will bind the NUE-DNA vector. Non-viral delivery vehicles for in vivo codelivery include but are not restricted to lipid nanoparticles, polymeric nanoparticles or exosomes. Targeting ligands e.g. directed towards cell surface molecules may be included to achieve targeted payload delivery. Co-formulated NUE-mRNA and NUE-DNA vector or NUE-mRNA, NUE-DNA vector are then being administered to the patient. Respective route of administration depends on the indication and target organ and includes but is not restricted to intravenous administration. In some embodiments, the NUE-mRNA and NUE-DNA vector are co-delivered using in vivo administration methodologies. In some embodiments, the NUE-mRNA and NUE-DNA vector are co-delivered within carrier particles in vivo. Formulations for in vivo delivery of the NUE-DNA vector, NUE and NUE-mRNA are also contemplated. Formulations for non-viral gene delivery systems have been described in Sung et al. (Cancer Med J. 2019 June ; 2(1): 6-13), Mohammad et al. (Drug Discov Today. 2022 Jan;27(l):292-303) and Sung et al. (Biomaterials Research (2019) 23:8)), incorporated by reference in their entirety.

[0088] In addition, physical methods of delivery are provided. Without being limiting, this includes electroporation, cell squeezing, ultrasound, injection and methods using mechanical stress that can be applied to co-deliver nuclear uptake enhancer and a cognate NUE-DNA vector. In some embodiments described herein, a DNA vector is provided, wherein the vector comprises at least one Recognition Cassette (NUE-DNA vector), wherein the at least one Recognition Cassette comprises at least one Recognition Sequence and wherein the at least one Recognition Sequence can be recognized and bound by at least one NUE. In some embodiments, the Recognition Cassette comprises between about 5 to about 30 Recognition Sequences. In some embodiments, the length of the Recognition Sequence comprises between about 9bp to about 40bp to provide a sufficient level of DNA sequence selectivity over genomic DNA sequences. In some embodiments, multiple copies of a Recognition Sequence are comprised into a NUE-DNA vector, within a Recognition Cassette. In some embodiments, the NUE-DNA vector further comprises at least one gene encoding a cargo. In some embodiments, the cargo comprises a protein, an RNA or both. In some embodiments, the cargo comprises at least one protein. In some embodiments, the NUE-DNA vector comprises multiple genes encoding multiple proteins. In some embodiments, theRecognition Cassette comprises 10 Recognition Sequences. In some embodiments, theRecognition Cassette comprises 15 Recognition Sequences. In some embodiments, theRecognition Cassette comprises 20 Recognition Sequences. In some embodiments, theRecognition Cassette comprises 25 Recognition Sequences. In some embodiments, theRecognition Cassette comprises 30 Recognition Sequences. In some embodiments, theRecognition Cassette comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 149- 161. In some embodiments, the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 151. In some embodiments, the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 155. In some embodiments, the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 159.

[0089] The NUE is a complex protein comprising domains that interact with the NUE-DNA vector as well as domains that may interact with the nuclear import machinery and / or interacts with the cytoplasmic transport machinery. In some embodiments, the NUE comprises at least one DNA binding domain, at least one linker, and at least one effector domain. In some embodiments, at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transportdomain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker and at least one nuclear import domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker and at least one cytoplasmic transport domain. In some embodiments, the at least one NUE comprises at least one DNA binding domain, at least one linker, at least one nuclear import domain and at least one cytoplasmic transport domain.

[0090] In some embodiments, the methods include at least one NUE or at least one NUE- mRNA. In some embodiments, a first NUE comprises at least one DNA binding domain, at least one linker and at least one nuclear import domain and a second NUE comprises at least one DNA binding domain, at least one linker and at least one cytoplasmic transport domain. In some embodiments, one NUE comprises at least one DNA binding domain, at least one linker, at least one nuclear import domain and at least one cytoplasmic transport domain. In some embodiments, the at least one NUE interacts with a nuclear import machinery and / or interacts with the cytoplasmic transport machinery. In some embodiments, the at least one NUE interacts with the nuclear import machinery. In some embodiments, the at least one NUE interacts with the cytoplasmic transport machinery. In some embodiments, the at least one NUE interacts with a nuclear import machinery and interacts with the cytoplasmic transport machinery. In some embodiments, a first NUE interacts with the nuclear import machinery and a second NUE interacts with the cytoplasmic transport machinery. In some embodiments, a first NUE interacts with the nuclear import machinery and the cytoplasmic transport machinery.

[0091] In some embodiments, an NUE comprising a nuclear import domain is provided, wherein the NUE comprises a sequence set forth in SEQ ID NO: 108. In some embodiments, the NUE comprising a nuclear import domain is provided concurrently with an NUE comprising a cytoplasmic transport domain. In some embodiments, NUE-mRNA encoding an NUE comprising a nuclear import domain is provided, wherein the NUE comprises a sequence set forth in SEQ ID NO: 108. In some embodiments, wherein the NUE-mRNA encoding an NUE comprising a nuclear import domain is provided, an NUE-mRNA encoding an NUE comprising a cytoplasmic transport domain is also provided concurrently. In some embodiments, the NUE comprising the cytoplasmic transport domain comprises a sequence set forth in SEQ ID NO: 134, 135, 136, 137 or 138.

[0092] The NUE comprises a DNA binding domain that interacts with the NUE-DNA vector. The DNA binding domain of the NUE is comprised of sequences from DNA binding proteins. Avariety of DNA binding proteins that can be engineered to adjust their sequence specificity has been described, including, but not limited to designed zinc-finger proteins, Transcription activatorlike effector (TALE) proteins, Meganucleases and the Cas family of ribonucleoproteins or any other protein that can bind a DNA sequence specifically. A DNA binding domain suitable as a component of a NUE does not confer additional functionality beyond DNA recognition and can be engineered to recognize and bind to a specific DNA recognition element. In some embodiments, the DNA binding domain of the NUE comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-5, or a sequence having at least about 80%, about 85%, about 90% about 95% or about 100% identity thereto. In some embodiments, the DNA binding domain(s) comprise a sequence as shown in Table 1 below.

[0093] Table 1. DNA binding domains.

[0094] In some embodiments of the methods described herein, at least one NUE is provided, wherein the NUE comprises at least one DNA binding domain, at least one linker, and at least one effector domain. In some embodiments, the at least one effector domain comprises a nuclear import domain. In some embodiments, the effector domain is a nuclear import domain comprising a sequence set forth in any one of SEQ ID NOs: 6-11. In some embodiments, the effector domain is a nuclear import domain comprising a sequence that is at least about 80%, about 85%, about 90%about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11.Sequences of example natural nuclear import domains are provided in Table 2, below.

[0096] In some embodiments, the NUE protein concept includes the use of cytoplasmic transport domains that enable the interaction of NUE-DNA vectors with the cytoplasmic transport machinery, i.e. the microtubule network and dynein / dynactin motor proteins or associated proteins. Without being limiting, dynein includes three families of cytoskeletal motor proteins and includes nine major classes of dynein heavy chain (Wickstead et al. Traffic. 2007;8:1708-1721). Active transport of the NUE-DNA vector along the microtubule network will increase the local concentration of NUE-DNA vector in the perinuclear space and improve its uptake into the nucleus. In some embodiments, the NUE comprises a polypeptide that binds to the cytoplasmic transport machinery. In some embodiments, the cytoplasmic transport domain comprises a polypeptide, wherein the polypeptide binds to proteins of the dynein multiprotein complex, dynactin proteins or associated proteins. In a NUE construct, a nuclear transport domain is linked to a DNA binding domain, thus enabling the interaction of NUE-bound NUE-DNA vector complex with the cytoplasmic transport machinery. In some embodiments herein, the effector domain of the NUE comprises a cytoplasmic transport domain. In some embodiments, a NUE comprises a DNA binding domain linked to a cytoplasmic transport domain and be used in combination with a second NUE comprising a DNA binding domain and a nuclear import domain, or the NUE can be designed to contain both, a cytoplasmic transport domain and a nuclear import domain functionally linked to a DNA binding domain.

[0097] Without being limiting, the dynein / dynactin motor proteins or associated proteins include Dynein heavy chain, Dynein light intermediate chain, Dynein intermediate chain and theDynein light chain proteins DYNLRB (RobI), DYNLT (Tctexl / rp3), DYNLL (LC8), dynein activators and adaptors, cytoplasmic dynein- 1, BICD1, BICD2, Hookl, Hook3, Spindly, dynactin, ninein and ninein-like proteins. Dyenein / dynactin motor proteins or associated proteins have been described in Olenick et al. (Olenick et al. J. Cell. Sci. 2019; 132(6)), which is incorporated by reference in its entirety. In some embodiments, the NUE-bound NUE-vector complex interacts with any one of or a combination of Dynein heavy chain, Dynein light intermediate chain, Dynein intermediate chain and the Dynein light chain proteins DYNLRB (RobI), DYNLT (Tctexl / rp3), DYNLL (LC8), dynein activators and adaptors, cytoplasmic dynein- 1, BICD1, BICD2, Hookl, Hook3, Spindly, dynactin, ninein and ninein-like proteins.

[0098] Without being limiting, the cytoplasmic transport machinery may comprise the cytoplasmic dynein transport machinery and associated proteins. In some embodiments, the NUE- bound NUE-DNA vector complex interacts with any one of or a combination of cytoplasmic dynein transport machinery and associated proteins.

[0099] Several viral capsid proteins have been shown to engage the cytoplasmic transport machinery, resulting in transport of the viral capsid to the perinuclear space.

[0100] A cytoplasmic transport domain comprising of a viral protein or a domain of such a protein whose function mediates cytoplasmic transport has been described. Eurther, a cytoplasmic transport domain can comprise at least one or a combination of multiple peptide sequence(s) derived from such proteins or consensus motives of several such peptides, known to bind to one or multiple members of the dynein motor complex. In some embodiments, an NUE comprises a cytoplasmic transport domain, wherein the cytoplasmic transport domains comprises a viral protein that functions in cytoplasmic transport.

[0101] The cytoplasmic transport domain can further comprise of designed proteins that are engineered to bind to members of the cytoplasmic transport machinery. Without being limiting, such engineered binders can be selected from peptide libraries, antibody fragment based-libraries or alternative scaffold binder libraries (such as designed ankyrin repeat proteins, DARPins, Anticalins, Centyrins or Fynomers), for example. In some embodiments, the cytoplasmic transport domain comprises a sequence set forth in any one of SEQ ID Nos: 19-35, or a sequence having at least about 80%, about 85%, about 90% about 95% or about 100% identity thereto. The sequences can be found in Table 3, below.

[0102] Table 3: Cytoplasmic transport domains.

[0103] In some embodiments, the linkers comprise a sequence set forth in any one of SEQ ID NOs: 12-18. In some embodiments, the linkers comprise a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18. Linkers are shown below in Table 4.

[0104] Table 4. Linker amino acid sequences

[0105] In some embodiments, the methods comprise providing a NUE-DNA vector comprising a gene that is transcribed into an RNA. In some embodiments, such RNA comprises an interfering RNA, a gene silencing oligonucleotide, shRNA, miRNA or tRNAs.

[0001] In some embodiments, genetically modifying a target gene comprises reducing mRNA of the target gene through RNA interference (RNAi) system. RNA interference (RNAi) is the biological process of mRNA degradation induced by complementary sequences doublestranded (ds) small interfering RNAs (siRNA) and suppression of target gene expression. Any suitable RNAi system known in the art can be used for reducing mRNA of a target gene. See, for example, Xu et al., Comprehensive Biotechnology. 2019 : 560-575 for a review of RNAi technology.

[0106] In some embodiments, the RNAi system comprises short hairpin RNAs (shRNAs), dicer-produced siRNAs, endoribonuclease-prepared short interfering RNAs (esiRNAs), microRNAs and mimics, pro-siRNAs, miR-adapted shRNAs, or a combination thereof.

[0107] In some embodiments, the NUE-DNA vector comprises elements for long term nuclear retention and stable episome formation. The NUE-DNA vector is selected from various DNA vector modalities including circular double-stranded plasmid DNA, circular double-stranded DNA nanovectors (e.g., DNA nanoplasmid, DNA minicircle), linear open-ended double-stranded DNA, linear closed-ended double-stranded DNA, linear single-stranded DNA containing doublestranded NUE binding elements and circular single-stranded DNA containing double-stranded NUE binding elements.

[0108] In some embodiments, a method of increasing transgene expression is provided, wherein the method comprises providing a NUE-DNA vector, providing at least one NUE or NUE-mRNA for delivery into a cell, and delivering the NUE-DNA vector and the at least one NUE into the cell, wherein delivering the NUE-DNA vector with the NUE increases transgene expression levels as compared to delivery of the NUE-DNA vector alone or a DNA vector without a Recognition Sequence. In some embodiments, the method comprises a first delivery of the NUE-DNA vectorinto the cell and a second delivery of the NUE into the cell. In some embodiments, the method comprises a first delivery of the NUE into the cell and a second delivery of the NUE-DNA vector into the cell. In some embodiments, the method comprises a first delivery of the NUE-DNA vector into the cell and a second delivery of the NUE-mRNA into the cell. In some embodiments, the method comprises a first delivery of the NUE-mRNA into the cell and a second delivery of the NUE-DNA vector into the cell. In some embodiments wherein the NUE and the NUE-DNA vector are delivered sequentially, the NUE-DNA vector and the NUE are present in the cell at the same time and the NUE-DNA vector and the NUE interact, forming a NUE / NUE-DNA vector complex.

[0109] In some embodiments, the method of increasing transgene expression is provided, wherein the method comprises providing a NUE-DNA vector, providing at least one NUE-mRNA, and delivering the NUE-DNA vector and the at least one NUE-mRNA into the cell, wherein delivering the NUE-DNA vector with the at least one NUE-mRNA increases transgene expression levels as compared to delivery of the NUE-DNA vector alone or a DNA vector without a Recognition Sequence. In some embodiments, the method comprises a first delivery of the NUE- DNA vector into the cell and a second delivery of the NUE-mRNA into the cell. In some embodiments, the method comprises a first delivery of the NUE-mRNA into the cell and a second delivery of the NUE-DNA vector into the cell. In some embodiments wherein the NUE-mRNA and the NUE-DNA vector are delivered sequentially, the NUE-DNA vector and the NUE-mRNA are present in the cell at the same time and the NUE-DNA vector and the translated NUE interact, forming a NUE / NUE-DNA vector complex.

[0110] In some embodiments of the methods described herein, the NUE-mRNA and cognate NUE-DNA vector are co-delivered in vivo into the target cell. Non-viral delivery vehicles for in vivo co-delivery include but are not restricted to lipid nanoparticles, polymeric nanoparticles or exosomes. Additional targeting surface molecules may be included to achieve targeted payload delivery. In some embodiments, co-formulated NUE-mRNA and NUE-DNA vector or coformulated NUE- mRNA and NUE-DNA vector are administered to the patient. In some embodiments, the method further comprises co-delivering mRNA-encoded gene editing tools. Respective route of administration may depend on the indication and target organ and includes but is not restricted to intravenous administration. In some embodiments, the NUE-mRNA and NUE- DNA vector are co-delivered within carrier particles in vivo. In some embodiments, the carrier particles comprise neutral or cationic carrier particles.

[0111] In some embodiments herein, the NUE comprises a sequence set forth in any one of SEQ ID NOs: 36-148 or 162 or a sequence having at least about 80%, about 85%, about 90%, about 95% or about 100% identity thereto. A selection of NUE designs is shown in Table 5, below.

[0112] In some embodiments, the NUE comprises a sequence set forth in any one of SEQ ID NOs: 36-148 or 162. In some embodiments, the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162. In some embodiments, the NUE comprises a sequence set forth in any one of SEQ ID NOs: 56, 57, and 68-71, or 162. In some embodiments, the NUE binds to a Recognition Cassette, wherein the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 151.

[0113] In some embodiments, the NUE comprises a sequence set forth in any one of SEQ ID NOs: 72, 73, 76, 77, 86 or 87. In some embodiments, the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 72, 73, 76, 77, 86 or 87. In some embodiments, the NUE binds to a Recognition Cassette, wherein the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 155.

[0114] In some embodiments, the NUE comprises a sequence set forth in any one of SEQ ID NOs: 94, 98, 99, 105 or 108. In some embodiments, the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 94, 98, 99, 105 or 108. In some embodiments, the NUE binds to aRecognition Cassette, wherein the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 159.

[0115] In some embodiments, the NUE comprises a sequence set forth in SEQ ID NO: 71. In some embodiments, the NUE binds to a Recognition Cassette, wherein the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 151.

[0116] In some embodiments, the NUE comprises a sequence set forth in SEQ ID NO: 108. In some embodiments, the NUE binds to a Recognition Cassette, wherein the Recognition Cassette comprises a sequence set forth in SEQ ID NO: 159.EXAMPLES

[0117] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Design and testing of NUE-mRNA for nuclear uptake of a NUE-DNA vector.

[0118] As described below are embodiments of the DNA delivery system, including design and testing of a NUE-mRNA that increased the nuclear uptake of a NUE-DNA vector which led to increased reporter transgene expression which serves as the primary read out in this assay.

[0119] The NUEs that were tested comprised a DNA binding domain, a DNA binding domain as set forth in the sequences comprising SEQ ID NOs: 1-5, that specifically bound to a 9-18 base pair long Recognition Sequence present within the Recognition Cassette of the NUE-DNA vector as set forth in any one of SEQ ID NOs: 149-156, and an NUE that comprised a nuclear import domain that efficiently interacted with the endogenous nuclear transport machinery.

[0120] The nuclear import domains that were tested belong to a family of nuclear localization sequences as set forth in the sequences comprising SEQ ID NOs: 6-9, that are known to efficiently interact with the nuclear import machinery. In addition, interaction with the nuclear import machinery was found to also be achieved by using importin beta binding domains as fusion partners (as set forth in any one of SEQ ID NOs: 10-11).

[0121] By combining these two domains (nuclear import domain and importin binding domain) into one bi-functional protein, the resulting NUE specifically bound the NUE-DNA vector andsubsequently mediated nuclear uptake of the resulting NUE / NUE-DNA vector complex by interacting with the nuclear uptake machinery (e.g. Importin a and Importin [3 ).

[0122] To ensure that the bi-functional NUEs can efficiently bind the NUE-DNA vector and simultaneously provide optimal interaction with the nuclear uptake machinery, several linker sequences were designed and tested. The linkers comprised the sequences set forth in any one of SEQ ID NOs: 12-18.

[0123] For the designed NUE-DNA vector, a circular plasmid backbone of approximately 4.1 kb (pVax) was chosen in which multiple DNA recognition sequences were incorporated into the “Recognition Cassette”. The Recognition Cassette was introduced by molecular cloning between the Kanamycin resistance gene and the origin of replication. Each NUE-DNA vector is specific for a given NUE since the DNA binding domain of the NUE must recognize the Recognition Sequence within the Recognition Cassette of the NUE-DNA vector. For all NUE-DNA vectors, a secreted Metridia Luciferase gene was cloned into the multiple cloning site and expressed under the control of a CMV promoter, these are designated as Luciferase NUE-DNA vector below.

[0124] In the examples presented here, the embodiments described show improved nuclear uptake measured by the increase in reporter transgene expression upon co-transfection of the Luciferase NUE-DNA vector and a NUE encoding mRNA (NUE-mRNA). As a reference, transgene expression was measured upon co-transfection of an irrelevant mRNA (coding for DNA binding domain with no additional functionalities e.g. SEQ ID NO:1 for example 2 and example 4 and SEQ ID NO: 2 for example 3).

[0125] Example 1: Expression of Luciferase NUE-DNA vector in the presence of Aphidicolin

[0126] mRNA encoding NUE protein (NUE-mRNA) was produced by in vitro transcription from linearized plasmid DNA using modified nucleotides, and transcripts were purified by HPLC.

[0127] When cultured in vitro, most cell lines that proliferate can be transfected by DNA- reporter vectors because during the cell division process the nuclear envelop is dissolved and reformed. When the nuclear envelope is re-formed in the presence of a DNA-vector it can enter the nucleus by passive diffusion. It has been shown that the amount of plasmid DNA that can enter the nucleus positively correlates with increased transgene expression, which will serve as a surrogate read out to prove the concept. Terminally differentiated or resting cells do not undergo cell division and nuclear envelope remodeling which is the reason that these cells cannot be transfected efficiently by standard lipofection based protocols. Proliferating cells can bechemically growth arrested in S-Phase by the addition of the DNA polymerase oc / 8 inhibitor Aphidicolin. This chemical growth arrest blocks cell proliferation while cells remain metabolically viable.

[0128] The effect of Aphidicolin treatment on the transfection efficiency of a DNA vector comprising a Recognition Sequence comprising a sequence set forth in SEQ ID NO: 151 (V03) and a sequence encoding Luciferase (Luciferase NUE-DNA vector) was tested for its ability to express Luciferase in the presence of increasing concentrations of Aphidicolin. A549 cells were co-transfected with 25 ng of Luciferase NUE-DNA vector comprising the V03 Recognition Sequence (SEQ ID NO: 151) and 50 ng of an irrelevant control mRNA (SEQ ID NO: 1) according to standard protocols and in the presence of Aphidicolin (OpM to lOpM). Transgene expression was then measured 24h post transfection by quantifying the amount of secreted Luciferase according to standard protocols. In the absence of Aphidicolin significant transgene expression was observed. However, in the presence of 1 pM Aphidicolin, almost no Luciferase signal was obtained. This result demonstrates that in the absence of cell division DNA-vectors cannot enter the nucleus and thus transgene expression is inhibited (Eig. 3). 1 pM Aphidicolin was then used in the assays of Examples 2-4.Example 2: Expression of Luciferase from the Luciferase NUE-DNA vector in the presence of NUEs comprising DNA binding domain 1

[0129] The bioactivity of designed NUEs on Luciferase NUE-DNA vector containing the Recognition Sequence as set forth in SEQ ID NO: 151 (V03) was tested in the presence of 1 pM Aphidicolin in A549 cells (ATCC: CCL-185™). The designed NUEs comprised amino acid sequences from the DNA binding domain as set forth in SEQ ID NO: 1. The cells were seeded in a 96 well plate in the presence of 1 pM Aphidicolin. Cells were then incubated at 37 °C and 5% CO2 for 24h. Prior to transfection, the Luciferase NUE-DNA vector transfection-mix and the NUE-mRNA transfection mix were prepared separately according to standard protocols and commonly used reagents (Lipofectamine and Messenger Max). The DNA transfection mix contained approximately 25 ng of Luciferase NUE-DNA vector and the RNA transfection mix contained varying amounts of NUE-mRNA for expression of the designed NUEs. Luciferase NUE-DNA vector and NUE-mRNA transfections were then conducted following standard transfection protocols. A reference control was prepared by combining 25 ng Luciferase NUE- DNA vector with a control mRNA, wherein the control mRNA encoded a DNA binding domainthat lacked the nuclear uptake enhancer functionality (SEQ ID NO: 1). Reporter gene expression was then analyzed 24 hours post transfection. Several NUEs were designed and tested containing different nuclear localization sequences, different linkers and different protein geometries. It was surprisingly found that co-transfection of mRNAs encoding for different NUEs (SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70 and SEQ ID NO: 71) led to a significant increase in transgene expression from the Luciferase NUE-DNA vector. The overall bioactivity of the tested NUEs depends both on the NUE composition and its geometry (Figs. 4A- 4F).

[0130] Example 3: Expression of Luciferase from the Luciferase NUE-DNA vector in the presence of NUEs comprising DNA binding domain 2

[0131] The expression of Luciferase from the Luciferase NUE-DNA vector containing the Recognition Sequence as set forth in SEQ ID NO: 155 (V07) was again assayed in the presence of control mRNAs or designed NUE mRNAs comprising a DNA binding domain as set forth in SEQ ID NO: 2 and a nuclear import domain as set forth in any one of SEQ ID NOs: 6-11. The A549 cells (ATCC: CCL-185™) were prepared for co-transfection as described above in Example 2. Cells were then co-transfected and analyzed as described previously. Several NUEs based on DNA binding domain 2 also showed significantly increased reporter gene expression as shown in Figs. 5A-5F. Of all DNA binding domain 2-based NUEs tested, several showed strong increases in reporter gene expression, including SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 86 and SEQ ID NO: 87. Increase in reporter gene expression was again dependent on the geometry of the Nuclear uptake enhancer protein and its overall composition. Together these results demonstrate that the activity of a designed NUE protein needs to be verified and tested experimentally.

[0132] Example 4: Expression of Luciferase from the Luciferase NUE-DNA vector in the presence of NUEs comprising DNA binding domain 4

[0133] The expression of Luciferase from the Luciferase NUE-DNA vector containing the Recognition Sequence as set forth in SEQ ID NO: 159 (VI 1) was finally assayed in the presence of control mRNAs or designed NUE mRNAs comprising a DNA binding domain as set forth in SEQ ID NO: 4 and a nuclear import domain as set forth in any one of SEQ ID NOs: 6-11. The A549 cells (ATCC: CCL-185™) were prepared for co-transfection as described above in Example 2. Cells were then co-transfected and analyzed as described previously. Several NUEs based onDNA binding domain 4 showed significantly increased reporter gene expression as shown in Figs. 6A-6E. Of all DNA binding domain 4-based NUEs tested, several showed strong increases in reporter gene expression, including SEQ ID NO: 94, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 105 and SEQ ID NO: 108. Increase in reporter gene expression was again dependent on the geometry of the Nuclear uptake enhancer protein and its overall composition. Together these results demonstrate that the activity of a designed NUE protein needs to be verified and tested experimentally

[0134] Example 5: The activity on designed NUE proteins depend on the number of recognition elements present in the Luciferase NUE-DNA vector.

[0135] To further demonstrate that the concept of improving DNA nuclear uptake by cotransfection of designed Luciferase NUE-DNA vectors and NUE encoding mRNAs depend on the careful design of corresponding NUE / NUE-DNA vector pairs, the activity of different Luciferase NUE-DNA vectors in combination with a NUE (SEQ ID NO: 71) was evaluated. Each Luciferase NUE-DNA vector under evaluation contained different Recognition Cassettes that were each based on the recognition sequence of DNA binding domain 1 (SEQ ID NO: 1) and differed in the number of recognition sequences varying from 5 recognition sequence motives to 30. Luciferase NUE-DNA vector comprising the Recognition Cassette set forth in SEQ ID NO: 149 (V01) comprises 5 RS, Luciferase NUE-DNA vector comprising the Recognition Cassette set forth in SEQ ID NO: 150 (V02) comprises 10 RS, Luciferase NUE-DNA vector comprising the Recognition Cassette set forth in SEQ ID: 151 (V03) comprises 20 RS and Luciferase NUE-DNA vector comprising the Recognition Cassette set forth in SEQ ID NO: 152 (V04) comprises 30 RS. Luciferase NUE-DNA vector and a NUE mRNA (encoding NUE comprising SEQ ID NO: 71) was transfected into Aphidicolin treated A594 cells as described under Example 2 and Luciferase expression was analyzed accordingly. It was surprisingly found that enhanced nuclear uptake and transgene expression strength was significantly influenced by the number of recognition sequences that were incorporated into the Recognition Cassette of the Luciferase NUE-DNA vector (Fig. 7). When only 5 recognition sequences were incorporated no increase in nuclear uptake was observed. The highest NUE activity could be achieved by Luciferase NUE-DNA vector V03 (SEQ ID NO: 151) which contained 20 recognition sequences while the activity of NUE-DNA vector V04 (SEQ ID NO: 152) containing 30 recognitions sequences was significantly lower. These results demonstrate that the design of the Recognition Cassette of NUE-DNA vectors is crucial for theoverall bioactivity of a NUE-mRNA NUE-DNA vector pair. The sequences comprised in the Recognition Cassettes are shown below in Table 6.

[0136] Example 7: Expression of Luciferase from the Luciferase NUE-DNA vector can be further increased in the presence of NUEs that comprise a nuclear import domain and NUEs that comprise a cytoplasmic transport domain

[0137] The expression of Luciferase from the Luciferase NUE-DNA vector containing the Recognition Sequence as set forth in SEQ ID NO: 161 (V13) was assayed in the presence of a NUE protein of known activity (SEQ ID NO: 108) and mRNAs or designed NUE mRNAs comprising a DNA binding domain as set forth in SEQ ID NO: 1 and a cytoplasmic transport domain as set forth in SEQ ID NO: 19-35. The A549 cells (ATCC: CCL-185™) were prepared for co-transfection as described above in Example 2. Cells were then co-transfected with two NUE mRNAs and the NUE-DNA vector and analyzed as described previously. It was surprisingly found that in the presence of a NUE-mRNA encoding for a cytoplasmic transport domain the activity of a NUE-mRNA containing a nuclear import domain (SEQ ID NO: 108) could be even further increased. As shown in Fig. 8 the activity of NUE SEQ ID NO: 108 was increased by up to 4-fold when NUE (SEQ ID NO: 134; 135; 136; 137 or 138) were co-transfected. Demonstrating the synergistic effect of combining NUEs with nuclear import domains with NUEs containing cytoplasmic transport domains.

[0138] Table 6: Recognition Cassette Sequences* * *

[0139] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein willbecome apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0140] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entireties as if physically present in this specification and each were individually and specifically mentioned to be incorporated by reference in their entireties.

Claims

CLAIMS1. A method of increasing transgene expression, the method comprising: a. providing at least one DNA vector comprising at least one Recognition Cassette (NUE-DNA vector); b. providing at least one nuclear uptake enhancer protein (NUE) for delivery into a cell, or providing at least one mRNA comprising a sequence encoding at least one NUE (NUE-mRNA); and c. co-delivering the at least one NUE-DNA vector and the at least one NUE into the cell or co-delivering the at least one NUE-DNA vector and the at least one NUE- mRNA into the cell, wherein co-delivering the at least one NUE-DNA vector with the at least one NUE or co-delivering the at least one NUE-DNA vector with the at least one NUE-mRNA increases transgene expression levels as compared to delivery of the at least one NUE-DNA vector alone.

2. The method of claim 1 , wherein the at least one Recognition Cassette comprises at least one Recognition Sequence (RS), wherein the at least one NUE binds the at least one Recognition sequence.

3. The method of claim 2, wherein the at least one Recognition Sequence comprises about 5bp to about 45bp.

4. The method of claim 2 or 3, wherein the at least one Recognition Sequence comprises about 9bp to about 40bp.

5. The method of any one of claims 2-4, wherein the at least one Recognition Cassette comprises between about 5 to about 30 Recognition Sequences.

6. The method of any one of claims 2-5, wherein more than one NUE or more than one NUE-mRNA is provided, wherein each NUE comprises a different amino acid sequencefrom one another, wherein the at least one NUE-DNA vector comprises more than one Recognition Cassette, wherein each Recognition Cassettes comprises different Recognition Sequences from each other, and wherein each NUE comprises a specificity for a different Recognition Sequence comprised within different Recognition Cassettes.

7. The method of any one of claims 2-6, wherein more than one NUE or more than one NUE-mRNA is provided, wherein each NUE comprises a different amino acid sequence from one another, wherein the Recognition Cassette comprises more than one Recognition Sequence, wherein the Recognition Sequences comprise different nucleic acid sequences from one another, and wherein each NUE comprises a specificity for a different Recognition Sequence.

8. The method of any one of claims 2-7, wherein the Recognition Cassette comprises at least one spacer, wherein the at least one spacer is located between the Recognition Sequences.

9. The method of claim 8, wherein the at least one spacer prevents steric hindrance or electrostatic hindrance between 2 or more NUEs bound on the Recognition Sequences.

10. The method of claim 8 or 9, wherein the at least one spacer comprises about 5 to about 45 bps.

11. The method of any one of claims 8-10, wherein the at least one spacer comprises 10 or 15 bp.

12. The method of any one of claims 1-11, wherein the at least one NUE-DNA vector further comprises a gene encoding a cargo.

13. The method of claim 12, wherein the cargo comprises a protein, an RNA or both.

14. The method of claim 13, wherein the RNA is not translated.

15. The method of claim 13 or 14, wherein the RNA comprises an interfering RNA, a gene silencing oligonucleotide, shRNA, miRNA, tRNAs or an RNA that mediates RNA editing.

16. The method of any one of claims 13-15, wherein the RNA comprises a hairpin RNA.

17. The method of any one of claims 1-16, wherein the at least one NUE-DNA vector comprises a circular double-stranded plasmid DNA, a circular double-stranded DNA nanovector, a linear open-ended double-stranded DNA, a linear closed-ended doublestranded DNA, a linear single-stranded DNA containing double-stranded NUE recognition sequences or a circular single-stranded DNA containing double-stranded NUE recognition sequences.

18. The method of any one of claims 1-17, wherein the at least one NUE-DNA vector comprises a circular double-stranded DNA nanovector, and wherein the circular doublestranded DNA nanovector comprises a DNA nanoplasmid or DNA minicircle.

19. The method of any one of claims 1-17, wherein the at least one NUE-DNA vector comprises a DNA nanovector, and wherein the DNA nanovector comprises a linear closed ended double stranded DNA.

20. The method of any one of claims 1-19, wherein 1, 2, 3, 4, or 5 NUEs are provided and codelivered or wherein 1, 2, 3, 4, or 5 NUE-mRNAs are provided and co -delivered.

21. The method of any one of claims 1-20, wherein the at least one NUE comprises a. at least one DNA binding domain; b. at least one linker; and c. at least one effector domain.

22. The method of claim 21, wherein more than one NUE is delivered or wherein more than one mRNA comprising a sequence encoding at least one NUE (NUE-mRNA) is delivered, wherein each NUE comprises a different DNA binding domain from one another.

23. The method of claim 21 or 22, wherein the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain.

24. The method of claim 23, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain.

25. The method of claim 23, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

26. The method of any one of claims 23-25, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain.

27. The method of any one of claims 23-26, wherein 2, 3, 4 or 5 NUEs are provided and codelivered or wherein 2, 3, 4, or 5 NUE-mRNAs are provided and co-delivered, wherein a first NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain and wherein a second NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

28. The method of any one of claims 23-27, wherein 2, 3, 4 or 5 NUEs are provided and codelivered or 2, 3, 4 or 5 NUE-mRNAs are provided and co-delivered, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain.

29. The method of any one of claims 23-28, wherein 2, 3, 4 or 5 NUEs are provided and codelivered or 2, 3, 4 or 5 NUE-mRNAs are provided and co-delivered, wherein the at leastone NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

30. The method of any one of claims 23-29, wherein 2, 3, 4 or 5 NUEs or 2, 3, 4 or 5 NUE- mRNAs are provided and co-delivered, and wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain.

31. The method of any one of claims 1-30, wherein the at least one NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery.

32. The method of claim 31, wherein the at least one NUE interacts with the nuclear import machinery.

33. The method of any one of claim 31 or 32, wherein the at least one NUE interacts with the cytoplasmic transport machinery.

34. The method of any one of claims 31-33, wherein the at least one NUE interacts with the nuclear import machinery and interacts with the cytoplasmic transport machinery.

35. The method of any one of claims 31-34, wherein a first NUE interacts with the nuclear import machinery and a second NUE interacts with the cytoplasmic transport machinery.

36. The method of any one of claims 21-35, wherein the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids.

37. The method of claim 21-36, wherein the at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids.

38. The method of any one of claims 21-37, wherein the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcriptionactivator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof.

39. The method of any one of claims 21-38, wherein the at least one DNA binding domain comprises a C2H2-zinc finger DNA binding domain.

40. The method of any one of claims claim 21-39, wherein the at least one DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5.

41. The method of any one of claims 23-40, wherein the nuclear import domain interacts with an endogenous nuclear import machinery.

42. The method of any one of claims 31-41 wherein the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

43. The method of claim 41 or 42, wherein the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

44. The method of any one of claims 31-43, wherein the nuclear import machinery comprises an importin dependent nuclear import machinery.

45. The method of any one of claims 23-44, wherein the nuclear import domain comprises a polypeptide that interact with proteins of a nucleoporin family, wherein the interaction or the binding of the nuclear import domain enables nuclear uptake of the NUE-DNA vector.

46. The method of any one of claims 23-45, wherein the cytoplasmic transport domain comprises a polypeptide that binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that binds the Dynactin motor complex, wherein the interaction or the binding of the cytoplasmic transport domain enables nuclear uptake of the NUE-DNA vector.

47. The method of any one of claims 23-46, wherein the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p.

48. The method of any one of claims 23-46, wherein the nuclear import domain comprises an importin-P binding domain of importin-a.

49. The method of any one of claims 23-48, wherein the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9.

50. The method of any one of claims 23-49, wherein the nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11.

51. The method of any one of claims 23-50, wherein the nuclear import domain is mammalian or viral.

52. The method of any one of claims 31-51, wherein the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery.

53. The method of any one of claims 23-52, wherein the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins.

54. The method of any one of claims 23-53, wherein the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35.

55. The method of any one of claims 12-54, wherein the cargo comprises a therapeutic protein.

56. The method of any one of claims 1-55, wherein the at least one NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162.

57. The method of any one of claims 1-56, wherein the at least one NUE-mRNA and the at least one NUE-DNA vector are co-delivered in vivo.

58. The method of any one of claims 1-57, wherein the co-delivering is non- viral co-delivery of the at least one NUE-mRNA and at least one NUE-DNA vector.

59. The method of any one of claims 1-58, wherein the method further comprises codelivering a therapeutic cargo.

60. The method of any one of claims 21-59, wherein the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18.

61. The method of any one of claims 1-60, wherein the Recognition Cassette comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NO: 149-161.

62. A nucleic acid encoding at least one nuclear uptake enhancer protein (NUE) for delivery into a cell, wherein the NUE comprisesa. at least one DNA binding domain; b. at least one linker; and c. at least one effector domain.

63. The nucleic acid of claim 62, wherein the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain.

64. The nucleic acid of claim 62 or 63, wherein the at least one NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain.

65. The nucleic acid of any one of claims 62-64, wherein the NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

66. The nucleic acid of any one of claims 62-65, wherein the at least one NUE comprises at least one DNA binding domain, at least one at least one linker, a nuclear import domain and a cytoplasmic transport domain.

67. The nucleic acid of any one of claims 62-66, wherein the at least one NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery.

68. The nucleic acid of claim 67, wherein the at least one NUE interacts with the nuclear import machinery.

69. The nucleic acid of claim 67 or 68, wherein the at least one NUE interacts with the cytoplasmic transport machinery.

70. The nucleic acid of any one of claims 67-69, wherein the NUE interacts with the nuclear import machinery and interacts with the cytoplasmic transport machinery.

71. The nucleic acid of any one of claims 62-70, wherein the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids.

72. The nucleic acid of any one of claims 62-71, wherein the at least one at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids.

73. The nucleic acid of any one of claims 62-72, wherein the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcription activator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof.

74. The nucleic acid of any one of claims 62-73, wherein the at least one DNA binding domain comprises a C2H2-zinc finger DNA binding domain.

75. The nucleic acid of any one of claims claim 62-74, wherein the at least one DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5.

76. The nucleic acid of any one of claims 67-75, wherein the nuclear import domain interacts with an endogenous nuclear import machinery and / or the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

77. The nucleic acid of anyone of claims 67-76, wherein the nuclear import domain interacts with an endogenous nuclear import machinery.

78. The nucleic acid of any one of claims 67-77, wherein the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

79. The nucleic acid of any one of claims 76-78, wherein the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with cytoplasmic transport machinery.

80. The nucleic acid of any one of claims 67-79, wherein the nuclear import machinery comprises an importin dependent nuclear import machinery.

81. The nucleic acid of any one of claims 63-80, wherein the nuclear import domain comprises a polypeptide that interacts or binds with proteins of a nucleoporin family, wherein interaction or binding enables nuclear uptake of the NUE-DNA vector.

82. The nucleic acid of any one of claims 63-81, wherein the cytoplasmic transport domain comprises a polypeptide that interacts or binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that interacts or binds the Dynactin motor complex, wherein the interaction or the binding enables nuclear uptake of the NUE- DNA vector.

83. The nucleic acid of any one of claims 63-82, wherein the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p.

84. The nucleic acid of any one of claims 63-83, wherein the nuclear import domain comprises an importin-P binding domain of importin-a.

85. The nucleic acid of any one of claims 63-84, wherein the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9.

86. The nucleic acid of any one of claims 63-85, wherein the nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11.

87. The nucleic acid of any one of claims 63-86, wherein the nuclear import domain is mammalian or viral.

88. The nucleic acid of any one of claims 67-87, wherein the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery.

89. The nucleic acid of any one of claims 63-88, wherein the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins.

90. The nucleic acid of any one of claims 63-89, wherein the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35.

91. The nucleic acid of any one of claims 62-90, wherein the nucleic acid comprises DNA or RNA.

92. The nucleic acid of any one of claims 62-91, wherein the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 16293. The nucleic acid of any one of claims 62-92, wherein the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18.

94. A NUE encoded by the nucleic acid of any one of claims 62-93.

95. A nuclear uptake enhancer protein (NUE) comprising: a. at least one DNA binding domain; b. at least one linker; and c. at least one effector domain.

96. The NUE of claim 95 wherein the at least one effector domain comprises a nuclear import domain and / or a cytoplasmic transport domain.

97. The NUE of claim 96, wherein the NUE comprises at least one DNA binding domain, at least one linker and a nuclear import domain.

98. The NUE of claim 96 or 97, wherein the NUE comprises at least one DNA binding domain, at least one linker and a cytoplasmic transport domain.

99. The NUE of any one of claims 96-98, wherein the NUE comprises at least one DNA binding domain, at least one linker, a nuclear import domain and a cytoplasmic transport domain.

100. The NUE any one of claims 95-99, wherein the NUE interacts with a nuclear import machinery and / or interacts with a cytoplasmic transport machinery.

101. The NUE of claim 100, wherein the NUE interacts with the nuclear import machinery.

102. The NUE of claim 100 or 101, wherein the NUE interacts with the cytoplasmic transport machinery.

103. The NUE any one of claims 100-102, wherein the NUE interacts with a nuclear import machinery and interacts with the cytoplasmic transport machinery.

104. The NUE any one of claims 95-103, wherein the at least one DNA binding domain comprises about 70 amino acids to about 1600 amino acids.

105. The NUE of any one of claims 95-104, wherein the at least one DNA binding domain comprises about 80 amino acids to about 140 amino acids.

106. The NUE of any one of claims 95-105, wherein the at least one DNA binding domain comprises a zinc finger protein, a meganuclease, a leucine zipper protein, a transcription activator-like effector (TALE) protein, or a DNA binding domain that belongs to the family of CRISPR / Cas DNA binding proteins, or a portion thereof.

107. The NUE of any one of claims 95- 106, wherein the at least one DNA binding domain comprises a C2H2-zinc finger DNA binding domain.

108. The NUE of any one of claims claim 95-107, wherein the at least one DNA binding domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4 or 5.

109. The NUE of any one of claims 96-108, wherein the nuclear import domain interacts with an endogenous nuclear import machinery.

110. The NUE of any one of claims 100-109 wherein the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.11 l.The NUE of claim 109 or 110, wherein the nuclear import domain interacts with the endogenous nuclear import machinery and the cytoplasmic transport domain interacts with the cytoplasmic transport machinery.

112. The NUE of any one of claims 100-111, wherein the nuclear import machinery comprises an importin dependent nuclear import machinery.

113. The NUE of any one of claims 96-112, wherein the nuclear import domain comprises a polypeptide that interacts or binds with proteins of a nucleoporin family, wherein the interaction or the binding enables nuclear uptake of a NUE-DNA vector.

114. The NUE of any one of claims 96-113, wherein the cytoplasmic transport domain comprises a polypeptide that binds a Dynein motor complex and / or the cytoplasmic transport domain comprises a polypeptide that binds the Dynactin motor complex, wherein the interaction or the binding enables nuclear uptake of a NUE-DNA vector.

115. The NUE of any one of claims 96-114, wherein the nuclear import domain comprises a polypeptide that binds to importin-a or importin-p.

116. The NUE of any one of claims 96-114, wherein the nuclear import domain comprises an importin-P binding domain of importin-a.

117. The NUE of any one of claims 96-114, wherein the nuclear import domain comprises a nuclear localization signal (NLS) derived from a SV40 large T antigen, or a nuclear localization sequence derived from c-Myc, or a nuclear localization peptide sequence M9.

118. The NUE of any one of claims 96-117, wherein the at least one nuclear import domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 6-11.

119. The NUE of any one of claims 96-118, wherein the at least one nuclear import domain is mammalian or viral.

120. The NUE any one of claims 100-119, wherein the NUE further comprises a polypeptide that binds the cytoplasmic transport machinery.

121. The NUE of any one of claims 96-120, wherein the cytoplasmic transport domain comprises a polypeptide that binds to Dynein / dynactin motor proteins and / or associated proteins.

122. The NUE of any one of claims 96-121, wherein the cytoplasmic transport domain comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 19-35.

123. The NUE of any one of claims 95-122, wherein the NUE comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 36-148 or 162.

124. The NUE of any one of claims 95-123, wherein the at least one linker comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 12-18.

125. A DNA vector comprising a Recognition Cassette (NUE-DNA vector), wherein the Recognition Cassette comprises at least one Recognition Sequence (RS), and wherein a NUE binds the at least Recognition Sequence.

126. The NUE-DNA vector of claim 125, wherein the at least one Recognition Sequence comprises about 5bp to about 45bp.

127. The NUE-DNA vector of claim 125 or 126, wherein the Recognition Sequence comprises about 9bp to about 40bp.

128. The NUE-DNA vector of any one of claims 125-127, wherein the Recognition Cassette comprises between about 5 to about 30 Recognition Sequences.

129. The NUE-DNA vector of any one of claims 125-128, wherein the NUE-DNA vector comprises more than one Recognition Cassette and wherein each Recognition Cassette have Recognition Sequences from each other and wherein each Recognition Cassette and Recognition Sequence is specific for a different NUE from one another.

130. The NUE-DNA vector of any one of claims 125-129, wherein the Recognition Cassette comprises more than one Recognition Sequence, wherein each Recognition Sequence comprises different nucleic acid sequences from one another and wherein each Recognition Sequence is specific for a different NUE.

131. The NUE-DNA vector of any one of claims 125-130, wherein the Recognition Cassette comprises at least one spacer, wherein the at least one spacer is located between the Recognition Sequences.

132. The NUE-DNA vector of claim 131, wherein the at least one spacer prevents steric hindrance or electrostatic hindrance between 2 or more NUEs bound on the Recognition Sequences.

133. The NUE-DNA vector of claim 131 or 132, wherein the at least one spacer comprises about 5 to about 45 bps.

134. The NUE-DNA vector of any one of claims 131-133, wherein the at least one spacer comprises 10 or 15 bp.

135. The NUE DNA vector of any one of claims 125-134, wherein the at least one NUE-DNA vector further comprises a gene encoding a cargo.

136. The NUE-DNA vector of claim 135, wherein the cargo comprises a protein, an RNA or both.

137. The NUE-DNA vector of any one of claims 125-135, wherein the Recognition Cassette comprises a sequence that is at least about 80%, about 85%, about 90% about 95% or about 100% identical to a sequence set forth in any one of SEQ ID NOs: 149-161.

138. A cell comprising a. at least one NUE-DNA vector; and b. at least one nucleic acid encoding a NUE.

139. A cell comprising a. at least one NUE-DNA vector; and b. at least one NUE.

140. A composition comprising the cell of claim 138 or 139.

141. A composition comprising a cell, wherein the cell comprises at least one NUE-DNA vector and at least one nucleic acid encoding an NUE.

142. A composition comprising a cell, wherein the cell comprises at least one NUE-DNA vector and at least one NUE.

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