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WO2025250877A1 - Mobile genetic elements from myotis myotis - Google Patents

Mobile genetic elements from myotis myotis

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Publication number
WO2025250877A1
WO2025250877A1 PCT/US2025/031562 US2025031562W WO2025250877A1 WO 2025250877 A1 WO2025250877 A1 WO 2025250877A1 US 2025031562 W US2025031562 W US 2025031562W WO 2025250877 A1 WO2025250877 A1 WO 2025250877A1
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Prior art keywords
seq
composition
enzyme
donor
nucleic acid
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French (fr)
Inventor
Joseph J. HIGGINS
Nancy Craig
David Ray
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Saliogen Therapeutics Inc
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Saliogen Therapeutics Inc
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Publication of WO2025250877A1 publication Critical patent/WO2025250877A1/en
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Definitions

  • the present disclosure relates to recombinant mobile element systems and uses thereof. Specifically, the recombinant mobile element systems of the present disclosure are derived from Myotis myotis. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63/654,350, filed on May 31, 2024, the entire contents which are incorporated herein in their entireties. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web.
  • a piggyBac transposon When combining a piggyBac transposon with a piggyBac helper enzyme, the DNA sequence from the transposon vector can be transferred to one of many specific nucleotide sequence (i.e., TTAA) sequences distributed throughout the genome.
  • TTAA specific nucleotide sequence
  • current transposition systems only find use in laboratory applications. Therapeutic uses have proven elusive. There is a need for novel and safer transposon systems for this technology to find use in medicine.
  • this disclosure describes, in part, a helper enzyme, optionally in the form of RNA, which is optionally engineered to target a single human genomic locus by introducing a DNA binding protein to yield a chimeric agent.
  • the present disclosure provides, inter alia, a composition comprising a recombinant mobile element enzyme and a DNA binder (e.g., without limitation, dCas9, TALEs, TnsD, and ZnF) that guide donor insertion to specific genomic sites.
  • a recombinant mobile element enzyme e.g., without limitation, dCas9, TALEs, TnsD, and ZnF
  • the helper enzyme is an engineered form of an enzyme reconstructed from Myotis myotis.
  • the enzyme includes but is not limited to an engineered version that is a monomer, dimer, tetramer (or DB1/ 159236086.1 SAL-049PC126933-5049 another multimer), hyperactive (Exc+), and/or has a reduced interaction with non-TTAA recognitions sites (Int-), of a helper enzyme reconstructed from Myotis myotis or a predecessor thereof.
  • the helper enzyme is a recombinant molecule which has at least about 90% identity to the nucleotide sequence of SEQ ID NO: 2 or the amino acid sequence SEQ ID NO: 1.
  • the helper enzyme has at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same.
  • the composition comprises a gene transfer construct.
  • the gene transfer construct comprises a donor (e.g., donor DNA) and can be or can comprise a vector comprising a mobile element comprising one or more end sequences recognized by the enzyme.
  • the end sequences are left and right end sequences that are recombinant or synthetic sequences.
  • the end sequences are from Myotis myotis, or end sequences with similarity to piggyBac-like mobile elements and exhibit duplications of their presumed TTAA target sites.
  • the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor.
  • the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor.
  • the end sequences which can be, e.g., from Myotis myotis, are optionally flanked by a TTAA sequence.
  • the enzyme is included in the gene transfer construct.
  • the composition comprises a nucleic acid binding component of a gene-editing system.
  • the gene-editing system is included in the gene transfer construct.
  • the gene-editing system comprises a CRISPR/Cas enzyme (class I, class II), or their six subtypes (type I–VI) (e.g., Cas9, Cas12a, Cas12j, Cas12k), or a variant thereof.
  • the gene-editing system comprises a nuclease-deficient a CRISPR/Cas enzyme (class I, class II), or their six subtypes (type I–VI) (e.g., dCas9, dCas12a, dCas12j, dCas12k).
  • the gene-editing system comprises Cas9, Cas12a, Cas12j, or Cas12k, or a variant thereof.
  • the gene-editing system comprises a nuclease-deficient dCas9, dCas12a, dCas12j, or dCas12k.
  • the gene-editing system comprises a TALE, ZnF, or TnsD.
  • the composition has the helper enzyme and the nucleic acid binding component of the gene-editing system.
  • the composition comprises a chimeric mobile element construct comprising the helper enzyme and the nucleic acid binding component of the gene-editing system fused or linked thereto.
  • the helper enzyme and the nucleic acid binding component of the gene-editing system can be fused or linked to one another via a linker, which can be a flexible linker.
  • the flexible linker can be substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is from about 1 to about 12.
  • the flexible linker is of or about 50, or about 100, or about 150, or about 200 amino acid residues.
  • the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt.
  • the helper enzyme is capable of inserting a donor at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule.
  • GSHS genomic safe harbor site
  • the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state.
  • a composition comprising (a) a nucleic acid binding component of a gene-editing system, and (b) a recombinant mammalian helper enzyme, the helper enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same.
  • the helper enzyme has at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same.
  • a mobile element construct comprises a helper enzyme (both herein called “helper”) constructed as a DNA vector or RNA vector (FIG.1A) fused or linked to a DNA binding domain (DBD), or TALE (FIG.1B), zinc finger (ZnF) (FIG.1C), inactive Cas protein (dCas9, dCas12a, dCas12j, or dCas12k) programmed by a guide RNA (gRNA) (FIG.1D), or a construct with an intein or dimerization enhancer such as SH3, biotin, avidin, or rapamycin binders (FIG. 1E).
  • helper both herein called “helper” constructed as a DNA vector or RNA vector (FIG.1A) fused or linked to a DNA binding domain (DBD), or TALE (FIG.1B), zinc finger (ZnF) (FIG.1C), inactive Cas protein (dCas9, dC
  • a composition comprising a recombinant mammalian helper enzyme in accordance with embodiments of the present disclosure can include one or more non-viral vectors.
  • the recombinant mammalian helper enzyme can be disposed on the same (cis) or different vector (trans) than a donor with a transgene. Accordingly, in embodiments, the recombinant mammalian helper enzyme and the donor encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the recombinant mammalian helper enzyme and the donor encompassing a transgene are in trans configuration such that they are included in different vectors.
  • the vector is any non-viral vector in accordance with the present disclosure.
  • the present disclosure provides a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure.
  • the donor comprises a gene encoding a complete polypeptide.
  • the donor comprises a gene which is defective or substantially absent in a disease state.
  • the present disclosure provides a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure and administering the cell to a subject in need thereof.
  • the present disclosure provides a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure or host cell of the present disclosure to a subject in need thereof.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof.
  • a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 2, or a functional variant thereof, operably linked to a heterologous promoter.
  • a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter.
  • FIG.1A - FIG.1E depict five illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid or miniplasmid) with a T7 promoter (cap dependent), beta-globin 5’-UTR, and a helper enzyme (SEQ ID NO: 1, SEQ ID NO: 2) from Myotis myotis followed by a beta-globin 3’-UTR, and a poly-alanine tail (FIG.1A).
  • a replication backbone e.g., plasmid or miniplasmid
  • T7 promoter cap dependent
  • beta-globin 5’-UTR e.g., beta-globin 5’-UTR
  • SEQ ID NO: 1 e.g., SEQ ID NO: 2
  • FIG.1A poly-alanine tail
  • TALEs (FIG.1B, TABLE 7 – TABLE 12), ZnF (FIG.1C, TABLE 13 – TABLE 17), or a dead Cas9 (dCas9) binding protein (FIG.1D, SEQ ID NO: 5, SEQ ID NO: 6) with guide RNAs (TABLE 1 – TABLE 6) were joined by a linker to the N-terminus to target the specific TTAA sites at hROSA 26, AAVS1, chromosome 4, chromosome 22, and chromosome X loci.
  • FIG.1E depicts a construct with a dimerization enhancer to assure activation of the two monomers.
  • FIG.2A depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter driving a gene of interest (GOI) with a polyA tail flanked by two insulators and ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting genomic safe harbor sites (GSHS) or other loci.
  • FIG.2B depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a splice acceptor site for exon 2 and other exons of a gene of interest (GOI) followed by a polyA tail and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting endogenous genes in the first intron (or other introns) to repair downstream mutations.
  • FIG.2C depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with tandem promoters to affect expression in different tissues (e.g., without limitation, liver specific promoter, cardiac specific promoter) and a gene(s) of interest (GOI) followed by a polyA tail and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used to differentially promote expression of genes in different organs, tissues or cell types.
  • FIG.2D depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with two or more genes of interest (GOI) linked by P2A “self-cleaving” peptides and followed by WPRE and a polyA tail.
  • the construct is flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for delivering multiple genes or genetic factors.
  • 2E depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter(s) driving the expression of two or more genes as in FIG.2D and linked to a sequence consisting of a 5’-miRNA, a sense and antisense miRNA pair, and completed with the 3’-miRNA.
  • the construct is followed by WPRE and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct combines protein replacement and miRNA to inhibit the expression of other related proteins.
  • FIG.3 depicts the TTAA site in hROSA26 (hg38 chr3:9,396,133-9,396,305) that is targeted by guideRNAs (TABLE 2), TALES (TABLE 8), and ZnF (TABLE 13).
  • FIG.4 depicts two TTAA sites in AAVS1 (hg38 chr19:55,112,851-55,113,324) that are targeted by guideRNAs (TABLE 3) or TALES (TABLE 9), and ZnF (TABLE 14).
  • FIG.5 depicts two TTAA sites in Chromosome 4 (hg38 chr4:30,793,534-30,875,476) that are targeted by guideRNAs (TABLE 4) or TALES (TABLE 10), and ZnF (TABLE 15).
  • FIG.6 depicts two TTAA sites in Chromosome 22 (hg38 chr22:35,370,000-35,380,000) that are targeted by guideRNAs (TABLE 5) or TALES (TABLE 11), and ZnF (TABLE 16).
  • FIG. 5 depicts two TTAA sites in Chromosome 4 (hg38 chr4:30,793,534-30,875,476) that are targeted by guideRNAs (TABLE 4) or TALES (TABLE 10), and ZnF (TABLE 15).
  • FIG.6 depicts two TTAA sites in Chromosome 22 (hg38 chr22:35,370,000-35,380,000) that are targeted by guideRNAs (TABLE 5) or
  • the present invention is based, in part, on the discovery of an engineered helper enzyme capable of gene insertion that finds uses in multiple applications, including, without limitation, in gene therapy.
  • an engineered enzyme from Myotis myotis e.g., having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, inclusive of variants generated (e.g., by random mutagenesis and/or site directed mutagenesis) (occasionally may be referred to as “engineered”, “the present MMT”, “hyperactive helper from Myotis myotis”, or “hyperactive helper”).
  • engineered refers to Myotis myotis helper, as engineered herein.
  • the present invention is based, in part, on the discovery that a helper enzyme, e.g., a recombinant helper enzyme derived from Myotis myotis, can be fused with a transcription activator-like effector proteins (TALE) DNA binding domain (DBD), a dCas9/gRNA, or a zinc finger sequence to thereby create a chimeric enzyme capable of a site- or locus-specific transposition.
  • TALE transcription activator-like effector proteins
  • DBD transcription activator-like effector proteins
  • DBD transcription activator-like effector proteins
  • dCas9/gRNA dCas9/gRNA
  • zinc finger sequence e.g., a chimeric helper
  • the enzyme e.g., without limitation, a chimeric helper
  • the chimeric helper in accordance with the present disclosure allows achieving targeted integration of a transgene.
  • the helper has one or more mutations that confer hyperactivity.
  • the helper is a mammal-derived helper, optionally a helper RNA helper.
  • the present compositions and methods for gene transfer utilize a dual donor/helper system.
  • Transposable elements are non-viral gene delivery vehicles found ubiquitously in nature. Donor-based vectors have the capacity of stable genomic integration and long-lasting expression of transgene constructs in cells.
  • dual donor and helper systems work via a cut-and-paste mechanism whereby donor DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a helper 6 DB1/ 159236086.1 SAL-049PC126933-5049 enzyme at a repetitive sequence site.
  • Dual donor/helper (or “donor/helper”) plasmid systems insert a transgene flanked by inverted terminal ends (“ends”), such as TTAA (SEQ ID NO: 440) tetranucleotide sites, without leaving a DNA footprint in the human genome.
  • the helper enzyme in embodiments, is transiently expressed (on the same or a different vector from a vector encoding the donor) and it catalyzes the insertion events from the donor plasmid to the host genome.
  • Genomic insertions primarily target introns but may target other TTAA (SEQ ID NO: 440) sites and integrate into approximately 50% of human genes.
  • This disclosure describes, in embodiments, a DNA integration system, which is highly active in mammals, and is derived from a mammalian mobile DNA element. In embodiments, this mammal-derived mobile genetic element is engineered to insert donor DNA at specific TTAA insertion “hotspots” that are frequently favored insertion sites for the un-engineered enzyme.
  • this technology exploits a helper RNA encoding enzyme with engineered DNA binding proteins and a donor DNA contained between the ends of a mobile element of the gene to be inserted into the genome.
  • the mammal-derived enzyme is fused to a protein domain at its N-terminus without loss of activity and “engineered” by fusing DNA binding domains (DBD) that can target almost any location in the genome.
  • DBD DNA binding domains
  • Excision competent/target binding defective enzymes (Exc+/Int) mutants are described, that when combined with programmable, synthetic DBDs only insert at a TTAAs at a single target site. This enzyme described in this disclosure displays several highly desirable features that are of great advantage for transgene integration.
  • no DNA double strand breaks are introduced into the target genome.
  • the flanking donor backbone ends are very efficiently rejoined, leaving no double strand break in the donor DNA to signal DNA damage.
  • the enzyme inserts the excised element at high frequency selectively into a TTAA target site.
  • excision from the donor site results in the covalent linkage of a TTAA segment to each 5’ donor end
  • the joining of the 3’ donor ends to staggered positions on the top and bottom strands of the DNA flanking the target TTAA, a simple ligation restores intact duplex DNA, and no DNA synthesis is required for repair.
  • the enzyme delivers a large cargo size as compared to other mobile genetic elements or integrating viral systems to date.
  • the enzyme is delivered as an RNA instead of as a DNA.
  • Other mobile genetic elements including helpers such as hyperactive PB and SB100X, when delivered as RNA, have significantly less activity when compared to DNA. See Bire, et al. (2013). Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition. BMC Biotechnol, 13, 75; Bire, et al. (2013). Optimization of the piggyBac donor using mRNA and insulators: toward a more reliable gene delivery system. PLoS One, 8(12), e82559; Wilber, et al. (2006).
  • RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther, 13(3), 625-630.
  • the enzyme described herein has the same or better activity when delivered as RNA.
  • helper RNA offers several advantages over delivery of a DNA molecule. Wilber, et al. (2006). RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther, 7 DB1/ 159236086.1 SAL-049PC126933-5049 13(3), 625-630.
  • the helper-encoding RNA sequence is incapable of integrating into the host genome, thereby eliminating concerns about long-term helper expression and destabilizing effects with respect to the gene of interest.
  • This safety feature prevents the integration of the enzyme gene into the human genome and circumvents potential oncogenic and mutagenic effects.
  • the present disclosure provides a dual DNA donor and RNA helper system.
  • the donor DNA plasmid contains helper-specific inverted terminal repeats (ITRs) flanking the transgene while the helper-RNA transiently expresses a synthetic enzyme that catalyzes the insertion events from the donor plasmid to the host genome.
  • ITRs helper-specific inverted terminal repeats
  • This two component DNA/RNA system is, in embodiments, co-encapsulated in a single lipid nanoparticle using microfluidic technology and the lipid nanoparticles protect the RNA from extracellular degradation by in vivo injection.
  • Helper Enzyme In embodiments, the present disclosure provides a composition comprising a helper enzyme or a nucleic acid encoding the enzyme, wherein the enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1.
  • SEQ ID NO: 1 amino acid sequence of a helper from Myotis myotis (634 amino acids) 1 MVKFSKDIES SDDEFYFENE DKSEKCNNDE IEFSEDASGD EQIAGPSGTT ERKTSLALPK 61 NLAESTDSDI EFIKAKRSRT IVYFESDVDI GDIIEKSGIR PSESYVSRGE NRKKKSWTST 121 SVNDKEPSRI PFSTGQLHVG PQVPSGCATP IDFFQLFFTE TLIKNITDET NEYARHKISQ 181 KELSQRSTNN WKDVTIEEMK AFLGVILNMG VLNHPNLQSY WSMDFESHIP FFRSVFKRER 241 FLQIFWMLHL KNDQKSSKDL RTRTEKVNCF LSYLEMKFRE RFCPGREIAV DEAVVGFKGK 301 IHFITYNPKK PTKWGIRLYV LSDSKCGYVH SF
  • the enzyme comprises an amino acid sequence of at least about 83% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 85% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 88% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 89% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 1.
  • the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 1. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions generated by by random mutagenesis and/or site directed mutagenesis. In embodiments, the nucleic acid that encodes the enzyme has a nucleotide sequence of SEQ ID NO: 2 or a codon- optimized form thereof.
  • SEQ ID NO: 2 nucleotide sequence encoding the helper from Myotis myotis (1905 nt) 1 ATGGTGAAAT TTTCTAAGGA CATTGAGTCT AGCGACGATG AGTTTTACTT CGAGAATGAG 61 GACAAGTCTG AGAAGTGCAA CAACGACGAG ATTGAGTTCA GCGAAGACGC CTCCGGGGAT 121 GAGCAGATCG CCGGACCTAG CGGAACCACC GAAAGGAAGA CCAGCCTGGC CCTCCCTAAA 181 AATCTGGCTG AGTCCACCGA TTCCGACATC GAATTCATCA AGGCCAAGCG CTCTCGGACA 241 ATCGTGTATT TCGAATCCGA CGTGGATATC GGCGACATCA TCGAGAAGTC CGGCATTAGG 301 CCTTCTGAAA GCTATGTGTC TCGAGGTGAG AACAGAAAGA AGAAGTCCTG GACAAGCACC 361 AGCGTGAACG ACAAGGAACC CTCTAGGATC
  • a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter.
  • the enzyme is excision positive.
  • the enzyme is integration deficient.
  • the enzyme has decreased integration activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof.
  • the enzyme has increased excision activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof.
  • a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 2, or a functional variant thereof, operably linked to a heterologous promoter.
  • a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter.
  • the enzyme comprises a targeting element.
  • the enzyme is capable of inserting a donor comprising a transgene in a genomic safe harbor site (GSHS).
  • the binding of a GSHS of a nucleic acid molecule in a mammalian cell is with high target specificity, relative to a control.
  • the control is a composition comprising an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 or a codon-optimized form thereof.
  • the targeting element is able to direct a transposition machinery to the GSHS of a nucleic acid molecule in a mammalian cell.
  • the GSHS is in an open chromatin location in a chromosome.
  • the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.
  • the GSHS is an adeno-associated virus site 1 (AAVS1).
  • the GSHS is a human Rosa26 locus.
  • the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
  • the GSHS is selected from TABLES 1-17.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TA-LER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6- 1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), catalytically inactive Zinc finger, catalytically inactive transcription factor, catalytically inactive 10 DB1/ 159236086.1 SAL-049PC126933-5049 nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof.
  • gRNA guide RNA
  • TALE transcription activator-like effector
  • DBD DNA binding domain
  • Zinc finger catalytically inactive Zinc finger
  • catalytically inactive transcription factor catalytically inactive
  • the targeting element comprises a TALE DBD.
  • the TALE DBD comprises one or more repeat sequences.
  • the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the repeat sequences each independently comprises about 33 or 34 amino acids.
  • the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively.
  • RVD recognizes one base pair in a target nucleic acid sequence.
  • the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI.
  • the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the target nucleic acid sequence and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG. In embodiments, the TALE DBD targets one or more of GSHS sites selected from TABLES 7-12.
  • the TALE DBD comprises one or more of RVD selected from TABLES 7-12, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations.
  • the targeting element comprises a Cas9 enzyme associated with a gRNA.
  • the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA.
  • the catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof.
  • SEQ ID NO: 5 nucleotide sequence of dead Cas9 DNA binding protein (5004 bp) 1 ATGGACAAGA AGTACTCCAT TGGGCTCGCT ATCGGCACAA ACAGCGTCGG CTGGGCCGTC 61 ATTACGGACG AGTACAAGGT GCCGAGCAAA AAATTCAAAG TTCTGGGCAA TACCGATCGC 121 CACAGCATAA AGAAGAACCT CATTGGCGCC CTCCTGTTCG ACTCCGGGGA GACGGCCGAA 181 GCCACGCGGC TCAAAAGAAC AGCACGGCGC AGATATACCC GCAGAAAGAA TCGGATCTGC 241 TACCTGCAGG AGATCTTTAG TAATGAGATG GCTAAGGTGG ATGACTCTTT CTTCCATAGG 301 CTGGAGGAGT CCTTTTTGGT GGAGGAGGAT AAAAAGCACG AGCGCCACCC AATCTTTGGC 361 AATATCGTGG ACGAGGTGGC GTACCATGAA AAGTACCCAA
  • the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a.
  • the targeting element comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA.
  • the guide RNA is selected from TABLES 1-6, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations.
  • the guide RNA targets one or more sites selected from TABLES 1-6.
  • the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence.
  • the zinc finger targets one or more sites selected from TABLES 13-17.
  • the targeting element comprises a nucleic acid binding component of a gene-editing system.
  • the enzyme or variant thereof and the targeting element are connected.
  • the enzyme and the targeting element are fused to one another or linked via a linker to one another.
  • the linker is a flexible linker.
  • the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is an integer from 1-12.
  • the flexible linker is 13 DB1/ 159236086.1 SAL-049PC126933-5049 of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues.
  • the enzyme is directly fused to the N-terminus of the targeting element and, optionally, wherein the targeting element is or comprises dCas9 enzyme.
  • the E is fusedCas9 enzyme.
  • TnsD comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 7.
  • the TnsD comprises a truncated TnsD.
  • the TnsD is truncated at its C-terminus.
  • the TnsD is truncated at its N-terminus.
  • the TnsD or variant thereof comprises a zinc finger motif.
  • the zinc finger motif comprises a C3H-type motif (e.g., CCCH).
  • the TnsD binds at or near a region downstream of the glmS gene.
  • GlmS L-glucosamine-fructose-6-phosphate aminotransferase
  • the TnsD binding region of glmS encodes the active site region of GlmS.
  • TnsD binds at or near the human homologs of glmS, e.g., gfpt-1 and gfpt-2.
  • TnsD binds the human glmS homologs gfpt-1 and gfpt-2.
  • the transgene is inserted into attTn7.
  • the TnsD comprises a nucleic acid binding component of a gene-editing system.
  • the enzyme or variant thereof (optionally, wherein the enzyme is a helper enzyme, optionally, wherein the helper enzyme is reconstructed from Myotis myotis) and the TnsD are connected.
  • the enzyme and the TnsD are fused to one another or linked via a linker to one another.
  • the linker is a flexible linker.
  • the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is an integer from 1-12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the enzyme is directly fused to the N- terminus of the TnsD.
  • the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 14 DB1/ 159236086.1 SAL-049PC126933-5049 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence.
  • the zinc finger targets one or more sites selected from TABLES 13-17.
  • the enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene.
  • the enzyme or variant thereof is able to directly or indirectly interact and/or form a complex with one or more proteins or nucleic acids.
  • the composition e.g., a helper of the present disclosure
  • system, or method further comprising a nucleic acid encoding a donor comprising a transgene to be integrated.
  • the transgene is defective or substantially absent in a disease state.
  • the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences.
  • the cargo nucleic acid sequence is flanked by the first and the second donor end sequences.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof.
  • the donor end sequences are selected from nucleotide sequences of SEQ ID NO: 3 and/or SEQ ID NO: 4, or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • SEQ ID NO: 3 Myotis myotis Left ITR (200 bp) 1 ccccctttgc actcggatgt cgagtgtgac tcgacgcggt tagcatcggt tgcagctcgt 61 atgttgagcc atactcgacc tgtagtttca ccgagggggg aagggggatt tttgtctatt 121 tttccagtat ttttcttgtttcattagca tgaaaggaca agtaaatgt aaatgccgtc 181 tcaactgatg ccaccaccta
  • the at least one repeat from the nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor.
  • the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 4.
  • the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor.
  • the present disclosure provides a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof.
  • the donor is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof.
  • the present disclosure provides a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the donor is suitable for transposition by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof.
  • the helper enzyme derived from Myotis myotis, the helper enzyme being suitable for transposition of a heterologous polynucleotide, the heterologous polynucleotide being flanked by two ends elements comprising the polynucleotide sequences of SEQ ID NO: 3, or a functional variant thereof and SEQ ID NO: 4, or a functional variant thereof.
  • the enzyme or variant thereof is incorporated into a vector or a vector-like particle.
  • the vector or a vector-like particle comprises one or more expression cassettes. In embodiments, the vector or a vector- like particle comprises one expression cassette. In embodiments, the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles.
  • the vector or vector-like particle is nonviral.
  • the composition comprises DNA, RNA, or both.
  • the enzyme or variant thereof is in the form of RNA. 16 DB1/ 159236086.1 SAL-049PC126933-5049
  • the donor is under the control of at least one tissue-specific promoter.
  • the at least one tissue-specific promoter is a single promoter.
  • the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter.
  • the transgene to be integrated comprises at least one gene of interest.
  • the transgene to be integrated comprises one gene of interest.
  • the transgene to be integrated comprises two genes of interest.
  • the transgene to be integrated comprises three genes of interest. In embodiments, the transgene to be integrated comprises four genes of interest. In embodiments, the transgene to be integrated comprises five genes of interest. In embodiments, the transgene to be integrated comprises six genes of interest. In embodiments, the at least one gene of interest comprises peptides for linking genes of interest. In embodiments, the peptides are 2A self-cleaving peptides, or functional variants thereof, wherein the 2A self-cleaving peptide is optionally selected from P2A, E2A, F2A, and T2A, or derivative thereof.
  • the at least one gene of interest is linked to polynucleotide comprising a sequence comprising a 5’- miRNA, a sense and antisense miRNA pair, and/or a 3’-miRNA.
  • Host Cell the present disclosure further provides a host cell comprising the composition in accordance with embodiments of the present disclosure.
  • Methods In certain embodiments, the present disclosure provides a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure. In embodiments, the method further comprises contacting the cell with a polynucleotide encoding a donor. In embodiments, the donor comprises a gene encoding a complete polypeptide.
  • the donor comprises a gene which is defective or substantially absent in a disease state.
  • the present disclosure provides a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure and administering the cell to a subject in need thereof.
  • the present disclosure provides a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure or host cell of the present disclosure to a subject in need thereof.
  • the transgene is an exogenous wild-type gene that, e.g., corrects a defective function of one or more mutations in a recipient.
  • the recipient may have a mutation that provides a disease phenotype (e.g., a defective or absent gene product).
  • the donor system or method of the present disclosure provides a correction that restores the gene product and diminishes the disease phenotype.
  • the transgene is a gene that replaces, inactivates, or provides suicide or helper functions.
  • the transgene and/or disease to be treated is one or more of: • beta-thalassemia: BCL11a or ⁇ -globin or ⁇ A-T87Q-globin, • LCA: RPE65, • LHON: ND4, • Achromatopsia: CNGA3 or CNGA3/CNGB3, • Choroideremia: REP1, • PKD: RPK (Red cell PK), • Hemophilia: F8, • ADA-SCID: ADA, • Fabry disease: GLA, • MPS type I: IDUA, • MPS type II: IDS, and • Cystic fibrosis: CFTR transgene.
  • the donor comprises a gene encoding a complete polypeptide.
  • the donor comprises a gene which is defective or substantially absent in a disease state.
  • the transfecting of the cell is carried out using electroporation or calcium phosphate precipitation.
  • the transfecting of the cell is carried out using a lipid vehicle, optionally N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2- dioleoyl-3-dimethylammonium-propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol, LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation), TRANSFECTAM (cationic liposome formulation), a lipid vehicle, optionally N
  • the transfecting of the cell is carried out using a lipid selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids.
  • a cationic lipid may be used to facilitate a charge-charge interaction with nucleic acids.
  • the lipid is a neutral lipid.
  • the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), 1,2-Dioleoyl- sn-glycero-3-phosphocholine (DOPC), or cholesterol.
  • DOPE dioleoylphosphatidylethanolamine
  • DOPC 1,2-Dioleoyl- sn-glycero-3-phosphocholine
  • cholesterol is derived from plant sources. In other embodiments, cholesterol is derived from animal, fungal, bacterial, or archaeal sources.
  • the lipid is a cationic lipid. In embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2-dioleoyl-3- dimethylammonium-propane (DODAP).
  • DOTMA N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane
  • DODAP 1,2-dioleoyl-3- dimethylammonium-propane
  • one or more of the phospholipids 18:0 PC, 18:1 PC, 18:2 PC, DMPC, DSPE, DOPE, 18:2 PE, DMPE, or a combination thereof are used as lipids.
  • the lipid is DOTMA and DOPE, optionally in a ratio of about 1:1.
  • the lipid is DHDOS and DOPE, optionally in a ratio of about 1:1.
  • the lipid is a commercially available product (e.g., LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (Life Technologies)).
  • the transfecting of the cell is carried out using a cationic vehicle, optionally LIPOFECTIN or TRANSFECTAM.
  • the transfecting of the cell is carried out using a lipid nanoparticle or a liposome.
  • the method is helper virus-free.
  • Epigenetic regulatory elements can be used to protect a transgene from unwanted epigenetic effects when placed near the transgene on a vector, including the transgene. See Ley et al., PloS One vol.8,4 e62784.30 Apr.2013.
  • MARs were shown to increase genomic integration and integration of a transgene while preventing heterochromatin silencing, as exemplified by the human MAR 1–68. See id.; see also Grandjean et al., Nucleic Acids Res.2011 Aug; 39(15):e104.
  • MARs can also act as insulators and thereby prevent the activation of neighboring cellular genes. Gaussin et al., Gene Ther.2012 Jan; 19(1):15-24.
  • the cell is further transfected with a third nucleic acid having at least one chromatin element, wherein the at least one chromatin element is optionally a Matrix Attachment Region (MAR) element.
  • MARs are expression- enhancing, epigenetic regulator elements which are used to enhance and/or facilitate transgene expression, as described, for example, in PCT/IB2010/002337 (WO2011033375), which is incorporated by reference herein in its entirety.
  • a MAR element can be located in cis or trans to the transgene.
  • the transgene has a size of 100,000 bases or less, e.g., about 100,000 bases, or about 50,000 bases, or about 30,000 bases, or about 10,000 bases, or about 5,000 bases, or about 10,000 to about 100,000 bases, or about 30,000 to about 100,000 bases, or about 50,000 to about 100,000 bases, or about 10,000 to about 50,000 bases, or about 10,000 to about 30,000 bases, or about 30,000 to about 50,000 bases.
  • the transgene has a size of about 200,000 bases or less, e.g., about 200,000 bases, or about 10,000 to about 200,000 bases, or about 30,000 to about 200,000 bases, or about 50,000 to about 200,000 bases, or about 100,000 to about 200,000 bases, or about 150,000 to about 200,000 bases.
  • the transgene has a size of about 300,000 bases or less, e.g., about 300,000 bases, or about 10,000 to about 300,000 bases, or about 30,000 to about 300,000 bases, or about 50,000 to about 300,000 bases, or about 100,000 to about 300,000 bases, or about 150,000 to about 300,000 bases.
  • the present disclosure provides for a donor system, e.g., in embodiments, a helper enzyme comprising a targeting element.
  • the helper enzyme associated with the targeting element is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS).
  • the helper enzyme associated with the targeting element has one or more mutations which confer hyperactivity.
  • the helper enzyme associated with the targeting element has gene cleavage (Exc) and/or gene integration (Int+) activity.
  • the helper enzyme associated with the targeting element has gene cleavage (Exc) and/or a lack of gene integration (Int-) activity.
  • the targeting element comprises one or more proteins or nucleic acids that are capable of binding to a nucleic acid.
  • the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, and paternally expressed gene 10 (PEG10).
  • TALE transcription activator-like effector
  • DBD DNA binding domain
  • the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule.
  • RVD repeat variable di-residue
  • the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome.
  • the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus.
  • AAVS1 adeno-associated virus site 1
  • CCR5 chemokine receptor 5
  • HIV-1 coreceptor HIV-1 coreceptor
  • Rosa26 locus human Rosa26 locus.
  • the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the targeting element comprises a Cas9 enzyme guide RNA complex.
  • the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex.
  • the targeting element comprises a Cas12 enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex.
  • the targeting element comprises a Cas12k enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex.
  • a targeting chimeric system or construct having a DBD fused to the helper enzyme directs binding of the helper to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di-residues (RVD) or gRNA) near an enzyme recognition site. The enzyme is thus prevented from binding to random recognition sites.
  • TALE transcription activator-like effector proteins
  • RVD repeat variable di-residues
  • gRNA binds to human GSHS.
  • dCas9 i.e., deficient for nuclease activity
  • gRNAs directed to bind at a desired sequence of DNA in GSHS.
  • TALEs described herein can physically sequester the enzyme to GSHS and promote transposition to nearby TTAA (SEQ ID NO: 440) sequences in close proximity to the RVD TALE nucleotide sequences.
  • GSHS in open chromatin sites are specifically targeted based on the predilection for helpers to insert into open chromatin.
  • the helper enzyme is capable of targeted genomic integration by transposition is linked to or fused with a TALE DNA binding domain (DBD) or a Cas-based gene-editing system, such as, e.g., Cas9 or a variant thereof.
  • DBD TALE DNA binding domain
  • Cas-based gene-editing system such as, e.g., Cas9 or a variant thereof.
  • the targeting element targets the helper enzyme to a locus of interest.
  • the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeat
  • Cas9 CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816–821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091–6105.
  • Cas9 The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity.
  • Qi L. S. et al. (2013). Cell 152, 1173–1183.
  • CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol.2016;17:5–15; Wang et al., Annu Rev Biochem.2016;85:227–64.
  • dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome.
  • dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex
  • gRNA guide RNA
  • dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism’s genome.
  • gRNA guide RNA
  • dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism’s genome.
  • gRNA guide RNA
  • dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism’s genome.
  • dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription.
  • the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex.
  • the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex.
  • the dCas9/gRNA complex comprises a guide RNA selected from: GTTTAGCTCACCCGTGAGCC (SEQ ID NO: 91), CCCAATATTATTGTTCTCTG (SEQ ID NO: 92), GGGGTGGGATAGGGGATACG (SEQ ID NO: 93), GGATCCCCCTCTACATTTAA (SEQ ID NO: 94), GTGATCTTGTACAAATCATT (SEQ ID NO: 95), CTACACAGAATCTGTTAGAA (SEQ ID NO: 96), TAAGCTAGAGAATAGATCTC (SEQ ID NO: 97), and TCAATACACTTAATGATTTA (SEQ ID NO: 98), wherein the guide RNA directs the enzyme to a chemokine (C-C motif) receptor 5 (CCR5) gene.
  • C-C motif chemokine receptor 5
  • the dCas9/gRNA complex comprises a guide RNA selected from: CACCGGGAGCCACGAAAACAGATCC (SEQ ID NO: 99);CACCGCGAAAACAGATCCAGGGACA (SEQ ID NO: 100); CACCGAGATCCAGGGACACGGTGCT (SEQ ID NO: 101); CACCGGACACGGTGCTAGGACAGTG (SEQ ID NO: 102); CACCGGAAAATGACCCAACAGCCTC (SEQ ID NO: 103); CACCGGCCTGGCCGGCCTGACCACT (SEQ ID NO: 104); CACCGCTGAGCACTGAAGGCCTGGC (SEQ ID NO: 105); CACCGTGGTTTCCACTGAGCACTGA (SEQ ID NO: 106); CACCGGATAGCCAGGAGTCCTTTCG (SEQ ID NO: 107); CACCGGCGCTTCCAGTGCTCAGACT (SEQ ID NO: 108); CACCGCAGTGCTCAGACTAGGGAAG (SEQ ID NO: 109
  • the guide RNAs are: AATCGAGAAGCGACTCGACA (SEQ ID NO: 425), and tgccctgcaggggagtgagc (SEQ ID NO: 426).
  • the guide RNAs are gaagcgactcgacatggagg (SEQ ID NO: 427) and cctgcaggggagtgagcagc (SEQ ID NO: 428).
  • guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 1. TABLE 1.
  • RNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements in areas of open chromatin GSHS Identifier Sequence 26 DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 19F gcctggccggcctgaccact (SEQ ID NO: 34) DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 gAAVS12 taaggaatctgcctaacagg (SEQ ID NO: 72) DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 gAAVS34 gcccagggccaggaacgacg (SEQ ID NO: 94) DB1/ 159236086.1 SAL-049PC126933-5049 hROSA26 gHROSA26-3 gcagaacctgaggatatgga (SEQ ID NO: 116) hROSA26 gHROSA26-3 aat
  • DNA SEQUENCE DB1/ 159236086.1 SAL-049PC126933-5049 A AV GUIDE 20 CTGAGCACTGAAGGCCTGGCCGG (SEQ ID NO: 302) AAV GUIDE 21 TGGTTTCCACTGAGCACTGAAGG (SEQ ID NO: 303) ; 38 DB1/ 159236086.1 SAL-049PC126933-5049 G uide C4A2 ACAAGATGTGAACACGACGATGG (SEQ ID NO: 339) G uide C4A3 GTTGCACCGTTGATTCCTTCAGG (SEQ ID NO: 340) TABLE 5. Guide RNA seqences targeting chromosome 22 TTAA hotspot.
  • a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k).
  • the targeting element comprises a Cas12 enzyme guide RNA complex.
  • the targeting element is selected from a zinc finger (ZF), transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, any of which are, in embodiments, catalytically inactive.
  • CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof.
  • the CRISPR-associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof.
  • the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule.
  • the helper enzyme of the present disclosure is suitable for causing insertion of the donor DNA in a GSHS when contacted with a biological cell.
  • the targeting element is suitable for directing the helper enzyme of the present disclosure to the GSHS sequence.
  • the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD).
  • TALE DBD comprises one or more repeat sequences.
  • the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids. 41 DB1/ 159236086.1 SAL-049PC126933-5049
  • the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids.
  • the targeting element e.g., TALE or Cas (e.g., Cas9 or Cas12, or variants thereof) DBDs cause the the helper enzyme of the present disclosure to bind specifically to human GSHS.
  • the TALEs or Cas DBDs sequester the helper to GSHS and promote transposition to nearby TA dinucleotide or a TTAA tetranucleotide sites which can be located in proximity to the repeat variable di-residues (RVD) TALE or gRNA nucleotide sequences.
  • the GSHS regions are located in open chromatin sites that are susceptible to helper activity.
  • the helper enzyme of the present disclosure does not only operate based on its ability to recognize TA or TTAA sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Cas DBD.
  • the helper enzyme of the present disclosure in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies.
  • the helper enzyme of the present disclosure is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Cas DBD, for transposition.
  • a DBD fused thereto such as a TALE or Cas DBD
  • the described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk.
  • the described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies.
  • TALE or Cas DBDs are customizable, such as a TALE or Cas DBDs is selected for targeting a specific genomic location.
  • the genomic location is in proximity to a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site.
  • Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9/gRNA DBDs to target specific sites in a host genome.
  • the DNA targeting ability of a TALE or Cas DBD or dCas9/gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences.
  • TALE nucleases are a known tool for genome editing and introducing targeted double-stranded breaks.
  • TALENs comprise endonucleases, such as FokI nuclease domain, fused to a customizable DBD.
  • This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells.
  • the DBD includes a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition.
  • the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
  • the TALE DBD repeat sequences comprise 33 or 34 amino acids.
  • the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids.
  • the RVD can recognize certain base pair(s) or residue(s).
  • the RVD recognizes one base pair in the nucleic acid molecule.
  • the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI.
  • the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA.
  • the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG.
  • the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor; and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22 or X.
  • the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4.
  • the GSHS comprises one or more of TGGCCGGCCTGACCACTGG (SEQ ID NO: 418), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420), TCCACTGAGCACTGAAGGC (SEQ ID NO: 421), TGGTTTCCACTGAGCACTG (SEQ ID NO: 422), TGGGGAAAATGACCCAACA (SEQ ID NO: 423), TAGGACAGTGGGGAAAATG (SEQ ID NO: 424), TCCAGGGACACGGTGCTAG (SEQ ID NO: 425), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 426), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 427), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 428), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 429), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 418), T
  • the TALE DBD binds to one of TGGCCGGCCTGACCACTGG (SEQ ID NO: 418), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420), TCCACTGAGCACTGAAGGC (SEQ ID NO: 421), TGGTTTCCACTGAGCACTG (SEQ ID NO: 422), TGGGGAAAATGACCCAACA (SEQ ID NO: 423), TAGGACAGTGGGGAAAATG (SEQ ID NO: 424), TCCAGGGACACGGTGCTAG (SEQ ID NO: 425), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 426), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 427), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 428), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 429), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 418
  • the TALE DBD comprises one or more of NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG NH NH NI HD NI HD NG NH NH (SEQ ID NO: 486), NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD NG NH (SEQ ID NO: 487), NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD (SEQ ID NO: 488), HD HD NI HD NG NH NI NH HD NI HD NG NH NI NI NH NH HD (SEQ ID NO: 489), NH NH NG NG NG HD HD NI HD NG NH NI NI HD NG NH (SEQ ID NO: 490), NH NH NH NH NI NI NI NI NI HD NI HD NG NH (SEQ ID NO: 491), NI NH
  • the TALE DBD comprises one or more of the sequences outlined herein or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • the GSHS and the TALE DBD sequences are selected from: 48 DB1/ 159236086.1 SAL-049PC126933-5049 TGGCCGGCCTGACCACTGG (SEQ ID NO: 418) and NH NH HD HD NH NH HD NG NH NI HD HD NI HD NG NH NH (SEQ ID NO: 486); TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419) and NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH (SEQ ID NO: 487); TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420) and NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD (SEQ ID NO: 488); TCCACTGAGCACTGAAGGC (SEQ ID NO: 421) and HD HD NI HD NG NH NI NH HD NI HD NG NH NI NH NH
  • the GSHS is within about 25, or about 50, or about 100, or about 150, or about 200, or about 300, or about 500 nucleotides of the TA dinucleotide site or TTAA (SEQ ID NO: 2) tetranucleotide site.
  • Illustrative DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES encompassed by various embodiments are provided in TABLE 7. TABLE 7. DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES.
  • the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site. TABLE 8. TALE sequences targeting the genomic safe harbor site, hROSA26.
  • NAME DNA SEQUENCE RVD AMINO ACID CODE TCGCCCCTCAAATCTTACAG HD NH HD HD HD NG HD NI NI NI NI NG HD NG NG NI HD NI NH (SEQ ID Q 56 DB1/ 159236086.1 SAL-049PC126933-5049 TGCAGGGCAACGCCCAGGGA NH HD NI NH NH NH HD NI NI (SEQ ID R7 (SEQ ID NO: 560) NO: 574) TCTCGATTATGGGCGGGATT HD NG HD NH NI NG NG NI NG NH NH HD NH NH NH NI NI NG NG (SEQ Q Q Q 9.
  • TALE sequences targeting the chromosome 4 hotspot [hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)].
  • NAME DNA SEQUENCE RVD AMINO ACID CODE
  • T ALE4-R001 TCTTCCTAGTATTAAAGT (SEQ ID NO: HD NG NG HD HD NG NI NH NG NI NG NG NI NI NI I I I I D H 58 DB1/ 159236086.1 SAL-049PC126933-5049
  • T ALE4-F018 TAGCATGATGTTTCATGTTGTGACCT NI NH HD NI NG NH NI NG NH NG NG NG HD NI NG (SEQ ID NO: 633) NH NG NG NH NG NH NI HD HD NG (SEQ ID NO: 653) T ABLE 11.
  • TALE sequences targeting the chromosome 22 hotspot [hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)].
  • NAME DNA SEQUENCE RVD AMINO ACID CODE TALE22F- R 1 TCTTCCTAGTCTCTTCTCTACCCAGT (SEQ ID HD NG NG HD HD NG NI NH NG HD NG HD NG Q O: : ) I 59 DB1/ 159236086.1 SAL-049PC126933-5049 TALE22- TGCTATGTAAGGTAGCAAAAAGGTAACCT (SEQ NH HD NG NI NG NH NG NI NH NH NG NI NH F006A ID NO: 671) HD NI NI NI NI NI NH NH NG NI HD HD NG (SEQ ID NO: 691) H G NAME DNA SEQUENCE RVD A
  • the zinc finger targets one or more sites selected from TABLES 13-17.
  • Zinc finger sequences targeting the genomic safe harbor site hROSA26.
  • hROSA NAME TARGET SCO ZFP AMINO ACID CODE C T D C T D C T ) C T ) R C T ) 61 DB1/ 159236086.1 SAL-049PC126933-5049 5’
  • AHLERHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCP SEQ ID NO: 749)
  • ECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSDSGNLRVHQRTH S P H P H E E T Q C T C T ) R C T ) R C T ) TABLE 14.
  • Zinc finger sequences targeting the genomic safe harbor site AAVS1.
  • AAVS1 NAME TARGET SCO ZFP
  • Chr NAME TARGET SCO ZFP 22 RE TTA A Q K Q F S H K Q F T R C R S T C R ST K R T ST K Q S Y Q K Q F Y H O: T C R Q S K Q F P DB1/ 159236086.1 SAL-049PC126933-5049 YKCPECGKSFSQRANLRAHQRTHTGEKPYKCPECGKSFSQAGHLAS HQRTHTGEKPYKCPECGKSFSRNDALTEHQRTHTGEKPYKCPECGK SFSQRANLRAHQRTHTGEKPYKCPECGKSFSHRTTLTNHQRTHTGEK E K EK V S Y Q : S Y R K Q S T C RT QS Q K Q : SR C RT R K Q FS ST C R R S Y H O: 67 DB1/ 159236086.1 SAL-049PC126933-5049 3’ ZnF15 R GTCA
  • Zinc finger sequences targeting the chromosome X [hg38 chrX:134,476,304- 134,476,307 (85); chrX:134,476,337-134,476,340 (51)].
  • the present disclosure relates to a system having nucleic acids encoding the enzyme (e.g., without limitation, the helper enzyme) and the donor DNA, respectively.
  • the targeting element comprises a nucleic acid binding component of a gene-editing system.
  • the helper enzyme the targeting element are connected.
  • the targeting element may refer to a nucleic acid binding component of the gene-editing system.
  • the helper enzyme and the targeting element are connected.
  • the linker is a flexible linker.
  • the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly 4 Ser) n , where n is an integer from 1 to 12.
  • the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt.
  • Inteins are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Curr. Protein Pept. Sci., 20(5), 408–424, which is incorporated by reference herein in its entirety. Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein. Id. Inteins apply standard enzymatic strategies to excise themselves post-translationally from a 69 DB1/ 159236086.1 SAL-049PC126933-5049 precursor protein via protein splicing.
  • intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence.
  • split inteins have been used to control the delivery of heterologous genes into transgenic organisms. See Wood & Camarero (2014) J. Biol. Chem. 289(21):14512-14519. This approach relies on splitting the target protein into two segments, which are then post- translationally reconstituted in vivo by protein trans-splicing (PTS). See Aboye & Camarero (2012) J.
  • intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, TALEs, or ZnF, allows creation of a split-enzyme system such as, without limitation, split helper system, that permits reconstitution of the full-length enzyme, e.g., helper, from two smaller fragments.
  • a split-enzyme system such as, without limitation, split helper system
  • the full-length enzyme e.g., helper
  • the two portions of an enzyme, e.g., helper are fused to the intein and, after co-expression, the intein allows producing a full-length enzyme, e.g., helper, by post- translation modification.
  • a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein.
  • the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme.
  • an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Patent No.9,200,045; Mootz et al., J. Am. Chem. Soc.2002; 124, 9044-9045; Mootz et al., J. Am.
  • the intein is NpuN (Intein-N) (SEQ ID NO: 423) and/or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • SEQ ID NO: 868 nucleotide sequence of NpuN (Intein-N) GGCGGATCTGGCGGTAGTGCTGAGTATTGTCTGAGTTACGAAACGGAAATACTCACGGTTGAGTATGGGCTTCTTCC AATTGGCAAAATCGTTGAAAAGCGCATAGAGTGTACGGTGTATTCCGTCGATAACAACGGTAATATCTACACCCAGC CGGTAGCTCAGTGGCACGACCGAGGCGAACAGGAAGTGTTCGAGTATTGCTTGGAAGATGGCTCCCTTATCCGCGCC ACTAAAGACCATAAGTTTATGACGGTTGACGGGCAGATGCTGCCTATAGACGAAATATTTGAGAGAGCTGGACTT GATGAGAGTCGATAATCTGCCAAAT 70 DB1/ 159236086.1 SAL-049PC126933-5049 SEQ ID NO: 869: nucleotide sequence of NpuC (Intein-C) GGCGGATCTGGCGGTAGTGGGGGTTCCGGATCCATAAAGATAGCT
  • the nucleic acid encodes the enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and sec-ond portions are fused into a functional enzyme upon post-translational excision of the dimerization enhancer from the enzyme.
  • the dimerization enhancer is suitable for linking the helper enzyme and the targeting element.
  • the dimerization enhancer is selected from: a protein comprising a SRC Homology 3 Domain (or SH3 domain), biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 19, or a functional variant thereof and the right end comprises SEQ ID NO: 20, or a functional variant thereof.
  • a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 19, or a functional variant thereof and the right end comprises SEQ ID NO: 20, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 3, or a functional variant thereof.
  • a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 4, or a functional variant thereof, operably linked to a heterologous promoter.
  • a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 3, or a functional variant thereof, operably linked to a heterologous promoter.
  • a nucleic acid encoding the enzyme e.g., without limitation, the helper enzyme
  • a nucleic acid encoding the transgene is DNA.
  • the enzyme e.g., without limitation, the helper enzyme
  • the nucleic acid is RNA, optionally a helper RNA.
  • the nucleic acid is RNA that has a 5’-m7G cap (cap0, or cap1, or cap2), optionally with pseudouridine substitution (e.g., without limitation 71 DB1/ 159236086.1 SAL-049PC126933-5049 n-methyl-pseudouridine), and optionally a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length.
  • the poly-A tail is of about 30 nucleotides in length, optionally 34 nucleotides in length.
  • a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus.
  • the nucleic acid that is RNA has a 5’-m7G cap (cap 0, or cap 1, or cap 2).
  • the nucleic acid comprises a 5’ cap structure, a 5’-UTR comprising a Kozak consensus sequence, a 5’-UTR comprising a sequence that increases RNA stability in vivo, a 3’-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3’ poly(A) tail.
  • the enzyme e.g., without limitation, a helper
  • the vector is a non-viral vector.
  • a nucleic acid encoding the enzyme in accordance with embodiments of the present disclosure is DNA.
  • a construct comprising a donor is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector.
  • the construct is DNA, which is referred to herein as a donor DNA.
  • sequences of a nucleic acid encoding the donor is codon optimized to provide improved mRNA stability and protein expression in mammalian systems.
  • the enzyme and the donor are included in different vectors. In embodiments, the enzyme and the donor are included in the same vector.
  • a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition is RNA (e.g., helper RNA), and a nucleic acid encoding a donor is DNA.
  • a donor often includes an open reading frame that encodes a transgene at the middle of donor and terminal repeat sequences at the 5’ and 3’ end of the donor.
  • the translated helper binds to the 5’ and 3’ sequence of the donor and carries out the transposition function.
  • a donor is used interchangeably with transposable elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides.
  • the term donor is well known to those skilled in the art and includes classes of donors that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and/or directly repeated long terminal repeats (LTRs) at the ends.
  • the donor as described herein may be described as a piggyBac like element, e.g., a donor element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor.
  • TTAA SEQ ID NO: 440
  • the donor is flanked by one or more end sequences or terminal ends.
  • the donor is or comprises a gene encoding a complete polypeptide.
  • the donor is or comprises a gene which is defective or substantially absent in a disease state.
  • a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene.
  • a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes.
  • the insulators flank the donor (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences.
  • the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5’-HS4 chicken ⁇ -globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facioscapulohumeral muscular dystrophy (FSHD).
  • HS4 insulator 1.2-kb 5’-HS4 chicken ⁇ -globin (cHS4) insulator element
  • D4Z4 insulator tandem macrosatellite repeats linked to Facioscapulohumeral muscular dystrophy
  • the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety.
  • the transgene is inserted into a GSHS location in a host genome.
  • GSHSs is defined as loci well-suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto-oncogene activation, tumor suppressor inactivation, or insertional mutagenesis.
  • GSHSs can defined by the following criteria: (1) distance of at least 50 kb from the 5’ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat. Biotechnol.2011;29:73-8; Bejerano et al. Science 2004;304:1321-5.
  • CCR5 chemokine C-C motif receptor 5
  • a homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans.
  • Disrupted CCR5 expression naturally occurring in about 1% of the Caucasian population, does not appear to result in any reduction in immunity.
  • a clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases.
  • the donor is under control of a tissue-specific promoter.
  • the tissue-specific promoter is, e.g., without limitation, a liver-specific promoter.
  • the liver-specific promoter is an LP1 promoter that, in embodiments, is a human LP1 promoter.
  • the LP1 promoter is described, e.g., in Nathwani et al. Blood vol. 2006;107(7):2653-61, and it is constructed, without limitation, as described in Nathawani et al.
  • the present nucleic acids include polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs or derivatives thereof.
  • transcriptionally- activated polynucleotides such as methylated or capped polynucleotides are provided.
  • the present compositions are mRNA or DNA.
  • the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide.
  • the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences.
  • Such vectors may include, among others, chromosomal and episomal vectors, e.g., vectors bacterial plasmids, from donors, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors from combinations thereof.
  • the present constructs may contain control regions that regulate as well as engender expression.
  • the construct comprising the enzyme and/or transgene is codon optimized.
  • Transgene codon optimization is used to optimize therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in a host organism or cell. Codon optimization methods are known in the art and described in, for example, WO 2007/142954, which is incorporated by reference herein in its entirety. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases.
  • the construct comprising the enzyme and/or transgene includes several other regulatory elements that are selected to ensure stable expression of the construct.
  • the non-viral vector is a DNA plasmid that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes.
  • the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5′-HS4 chicken ⁇ - globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facioscapulohumeral muscular dystrophy (FSHD).
  • the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety.
  • the gene of the construct comprising the enzyme and/or transgene is capable of transposition in the presence of a helper.
  • the non-viral vector in accordance with embodiments of the present disclosure comprises a nucleic acid construct encoding a helper.
  • the helper e.g., without limitation, the helper enzyme of the present disclosure
  • the non-viral vector further comprises a nucleic 74 DB1/ 159236086.1 SAL-049PC126933-5049 acid construct encoding a DNA helper plasmid.
  • the helper is an in vitro-transcribed mRNA helper.
  • the helper e.g., without limitation, the helper enzyme of the present disclosure
  • the helper is capable of excising and/or transposing the gene from the construct comprising the enzyme and/or transgene to site- or locus-specific genomic regions.
  • the enzyme e.g., without limitation, the helper enzyme
  • the donor are included in the same vector.
  • the enzyme is disposed on the same (cis) or different vector (trans) than a donor with a transgene. Accordingly, in embodiments, the enzyme and the donor encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the enzyme and the donor encompassing a transgene are in trans configuration such that they are included in different vectors.
  • the vector is any non-viral vector in accordance with the present disclosure.
  • a nucleic acid encoding the donor system of the present disclosure capable of targeted genomic integration by transposition e.g., a helper
  • the nucleic acid is or comprises DNA or RNA.
  • the nucleic acid encoding the enzyme is DNA.
  • the nucleic acid encoding the enzyme capable of targeted genomic integration by transposition e.g., a helper of the present disclosure
  • RNA such as, e.g., helper RNA.
  • the helper is incorporated into a vector.
  • the vector is a non-viral vector.
  • a nucleic acid encoding the transgene in accordance with embodiments of the present disclosure is provided.
  • the nucleic acid is or comprises DNA or RNA.
  • the nucleic acid encoding the transgene is DNA.
  • the nucleic acid encoding the transgene is RNA such as, e.g., helper RNA.
  • the transgene is incorporated into a vector.
  • the vector is a non-viral vector.
  • the present enzyme can be in the form or an RNA or DNA and have one or two N-terminus nuclear localization signal (NLS) to shuttle the protein more efficiently into the nucleus.
  • NLS nuclear localization signal
  • the present enzyme further comprises one, two, three, four, five, or more NLSs. Examples of NLS are provided in Kosugi et al. (J. Biol. Chem. (2009) 284:478-485; incorporated by reference herein).
  • the NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 348). In an embodiment, the NLS comprises the consensus sequence (K/R)(K/R)X 10-12 (K/R) 3/5 (SEQ ID NO: 349), where (K/R) 3/5 represents at least three of the five amino acids is either lysine or arginine.
  • the NLS comprises the c-myc NLS. In a particular embodiment, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 70). In a particular embodiment, the NLS is the nucleoplasmin NLS.
  • the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 72).
  • the NLS comprises the SV40 Large T-antigen NLS.
  • the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 73).
  • the NLS comprises three SV40 Large T-antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ 75 DB1/ 159236086.1 SAL-049PC126933-5049 ID NO: 74).
  • the NLS may comprise mutations/variations in the above sequences such that they contain 1 or more substitutions, additions, or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions).
  • a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided.
  • Lipids and LNP Delivery In embodiments, a composition or a nucleic acid in accordance with embodiments of the present disclosure is provided wherein the composition is in the form of a lipid nanoparticle (LNP). In embodiments, the composition is encapsulated in an LNP.
  • LNP lipid nanoparticle
  • a nucleic acid encoding the enzyme and a nucleic acid encoding the transgene are contained within the same lipid nanoparticle (LNP).
  • the nucleic acid encoding the enzyme and the nucleic acid encoding the donor are a mixture incorporated into or associated with the same LNP.
  • the polynucleotide encoding the helper enzyme and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation.
  • the LNP is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol–2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol- 3phosphocholine (DSPC) and/or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc).
  • DOTAP 1,2-dio
  • an LNP is as described, e.g., in Patel et al., J Control Release 2019; 303:91-100.
  • the LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc).
  • a nanoparticle is a particle having a diameter of less than about 1000 nm.
  • nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm.
  • the cell in accordance with the present disclosure is prepared via an in vivo genetic modification method.
  • a genetic modification in accordance with the present disclosure is performed via an ex vivo method.
  • the cell in accordance with the present disclosure is prepared by contacting a cell with an enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) in vivo.
  • the cell is contacted with the enzyme ex vivo.
  • the present method provides high specific targeting as compared to a method that does not use the helper enzyme with a target selector.
  • the transgene of interest in accordance with embodiments of the present disclosure can encode various genes.
  • the helper enzyme and the donor are included in the same pharmaceutical composition.
  • the helper enzyme and the donor are included in different pharmaceutical compositions.
  • the helper enzyme and the donor are co-transfected.
  • the helper enzyme and the donor are transfected separately.
  • a transfected cell for gene therapy is provided, wherein the transfected cell is generated using the helper enzyme in accordance with embodiments of the present disclosure.
  • a method of delivering a cell therapy comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure.
  • a method of treating a disease or condition using a cell therapy comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure.
  • the disease or condition may comprise cancer.
  • the cancer is or comprises an adrenal cancer, a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, a cancer of the esophagus, a gastric cancer, a head/neck cancer, a hepatobiliary cancer, a kidney cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a pelvis cancer, a pleura cancer, a prostate cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a uterine cancer, a lymphoma, a melanoma, a multiple myeloma, or a leukemia.
  • an adrenal cancer a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer
  • the cancer is selected from one or more of the basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung 77 DB1/ 159236086.1 SAL-049PC126933-5049 cancer; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sar
  • the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
  • the disease or condition is or comprises an infectious disease.
  • the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof.
  • the infectious disease is or comprises a disease comprising a viral infection, a parasitic infection, or a bacterial infection.
  • the viral infection is caused by a virus of family Flaviviridae, a virus of family 78 DB1/ 159236086.1 SAL-049PC126933-5049 Picornaviridae, a virus of family Orthomyxoviridae, a virus of family Coronaviridae, a virus of family Retroviridae, a virus of family Paramyxoviridae, a virus of family Bunyaviridae, or a virus of family Reoviridae.
  • the virus of family Coronaviridae comprises a betacoronavirus or an alphacoronavirus, optionally wherein the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43, or the alphacoronavirus is selected from a HCoV-NL63 and HCoV-229E.
  • the infectious disease comprises a coronavirus infection 2019 (COVID-19).
  • the method requires a single administration. In embodiments, the method requires a plurality of administrations.
  • an isolated cell comprises the transfected cell in accordance with embodiments of the present disclosure, e.g., transfected with a helper and/or donor.
  • the present disclosure provides an ex vivo gene therapy approach. Accordingly, in embodiments, the method that is used to treat an inherited or acquired disease in a patient in need thereof comprises (a) contacting a cell obtained from a patient (autologous) or another individual (allogeneic) with a transfected cell in accordance with embodiments of the present disclosure; and (b) administering the cell to a patient in need thereof.
  • One of the advantages of ex vivo gene therapy is the ability to “sample” the transduced cells before patient administration.
  • a composition comprising transfected cells in accordance with the present disclosure comprises a pharmaceutically acceptable carrier, excipient, or diluent.
  • suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.).
  • compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile, and the fluid should be easy to draw up by a syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, 79 DB1/ 159236086.1 SAL-049PC126933-5049 for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer- sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid.
  • PCPP-SA poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid]
  • FAD-SA fatty acid dimer- sebacic acid
  • polyglycolic acid collagen
  • polyorthoesters polyethyleneglycol-coated liposomes
  • polylactic acid polylactic acid
  • the stable integration comprises an introduction of a polynucleotide into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome.
  • a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure.
  • the organism may be a mammal or an insect.
  • the organism may include, but is not limited to, a mouse, a rat, a chimpanzee, an elephant, a dog, a rabbit, and the like.
  • the organism may include, but is not limited to, a fruit fly, an ant, a mosquito, a bollworm, and the like. Definitions The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs. As used herein, “a,” “an,” or “the” can mean one or more than one.
  • an “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.
  • the term “in vivo” refers to an event that takes place in a subject’s body.
  • the term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject’s body.
  • the cell, tissue and/or organ may be returned to the subject’s body in a method of treatment or surgery.
  • the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions.
  • the variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.
  • Carrier or “vehicle” as used herein refer to carrier materials suitable for drug administration.
  • Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid, or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and 81 DB1/ 159236086.1 SAL-049PC126933-5049 animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients.
  • pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose.
  • the therapeutic agents are given at a pharmacologically effective dose.
  • a “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease.
  • An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease.
  • Therapeutic benefit also 82 DB1/ 159236086.1 SAL-049PC126933-5049 includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to about 50% of the population) and the ED 50 (the dose therapeutically effective in about 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD 50 /ED 50 .
  • compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 as determined in cell culture, or in an appropriate animal model.
  • Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.
  • the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation.
  • SEQ ID NO: 1 amino acid sequence of a helper from Myotis myotis (634 amino acids) 1 MVKFSKDIES SDDEFYFENE DKSEKCNNDE IEFSEDASGD EQIAGPSGTT ERKTSLALPK 61 NLAESTDSDI EFIKAKRSRT IVYFESDVDI GDIIEKSGIR PSESYVSRGE NRKKKSWTST 121 SVNDKEPSRI PFSTGQLHVG PQVPSGCATP IDFFQLFFTE TLIKNITDET NEYARHKISQ 181 KELSQRSTNN WKDVTIEEMK AFLGVILNMG VLNHPNLQSY WSMDFESHIP FFRSVFKRER 241 FLQIFWMLHL KNDQKSSKDL RTRTEKVNCF LSYLEMKFRE RFCPGREIAV DEAVVGFKGK 301 IHFITYNPKK PTKWGIRLYV LSDSKCGYVH SF
  • FIG.1A - FIG.1E depict five illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid or miniplasmid) with a T7 promoter (cap dependent), beta-globin 5’-UTR, and a helper enzyme (SEQ ID NO: 1, SEQ ID NO: 2) from Myotis myotis followed by a beta-globin 3’-UTR, and a poly-alanine tail (FIG.1A).
  • a replication backbone e.g., plasmid or miniplasmid
  • T7 promoter cap dependent
  • beta-globin 5’-UTR e.g., beta-globin 5’-UTR
  • SEQ ID NO: 1 e.g., SEQ ID NO: 2
  • FIG.1A depict five illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid or miniplasmid) with
  • FIG. 1E depicts a construct with a dimerization enhancer.
  • the dimerization enhancer may be selected from, without limitation, SH3, biotin, avidin, and rapamycin binders.
  • FIG.2A depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter driving a gene of interest (GOI) with a polyA tail flanked by two insulators and ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting genomic safe harbor sites (GSHS) or other loci.
  • FIG.2B depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a splice acceptor site for exon 2 and other exons of a gene of interest (GOI) followed by a polyA tail and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting endogenous genes in the first intron to repair downstream mutations.
  • FIG.2C depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with tandem promoters to affect expression in different tissues (e.g., without limitation, liver specific promoter, cardiac specific promoter) and a gene(s) of interest (GOI) followed by a polyA tail and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used to differentially promote expression of genes in different organs, tissues or cell types.
  • FIG.2D depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with two or more genes of interest (GOI) linked by 2A “self-cleaving” peptides and followed by WPRE and a polyA tail.
  • the construct is flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for delivering multiple genes or genetic factors.
  • 87 DB1/ 159236086.1 SAL-049PC126933-5049 FIG.
  • 2E depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter(s) driving the expression of two or more genes as in FIG.2D and linked to a sequence consisting of a 5’-miRNA, a sense and antisense miRNA pair, and completed with the 3’-miRNA.
  • the construct is followed by WPRE and flanked by ITRs.
  • the inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct combines protein replacement and miRNA to inhibit other related protein expression.
  • the sense and anti-sense miRNA pair regulate the sense miRNAs, probably via modulating the chromatin architectures of the resided genomic loci. See Brown, T., Howe, F. S., Murray, S. C., Wouters, M., Lorenz, P., Seward, E., ... Mellor, J. (2016). Antisense transcription-dependent chromatin signature modulates sense transcript dynamics. Mol Syst Biol, 14(2), e8007; Murray, S. C., Haenni, S., Howe, F. S., Fischl, H., Chocian, K., Nair, A., & Mellor, J. (2015). Sense and antisense transcription are associated with distinct chromatin architectures across genes.
  • Example 2 Identification of Excision Positive and Integration Negative Mutants Random mutagenesis and/or site directed mutagenesis will be performed on the Myotis myotis helper enzyme of SEQ ID NO: 1. The variants will be screened using integration and excision assays.
  • the excision assay is a PCR-based assay to test for excision of the donor DNA.
  • a HEK293 cell line that expresses GFP at a known genomic site would be transfected with helper plasmid alone to excise the donor GFP DNA at the genomic locus by recognizing the end sequences.
  • HEK293 cells will be plated in 12-well size plates the day before transfection.
  • the day of the transfection the media will be exchanged 1 hour and 30 min before the transfection is performed.
  • a 3:1 ratio of X-tremeGENETM 9 DNA Transfection Reagent protocol reagent will be used to co-transfect a donor plasmid containing GFP and a helper plasmid in duplicate using 600ng of DNA each.
  • Forty-eight (48) hrs after transfection the cells will be analyzed by flow cytometry to count the percentage of GFP expressing cells to measure transient transfection efficiency. The cells will be gated to distinguish them from debris and 20,000 cells will be counted.
  • the cultures will be grown for 15-20 days without antibiotic. Cells will be passaged 2 to 3 times per week.
  • Flow cytometry will be used to count the percentage of GFP expressing cells to measure integration efficiency at 2 weeks.
  • the final integration efficiency will be calculated by dividing the 2-week percentage of GFP cells by the percentage of GFP cell at 48hr.
  • the excision assay will be performed by measuring the percentage of GFP cells in a cell line with a known GFP donor integration. The cells will be grown to 80% confluency and analyzed by flow cytometry to count the percentage of GFP expressing cells as a baseline measurement. This percentage will be used as the standard (i.e., 100%).
  • X- tremeGENETM 9 DNA Transfection Reagent protocol reagent will be used to transfect helper plasmid in duplicate using 600ng of DNA.
  • the cells will be gated to distinguish them from debris and 20,000 cells will be counted. Forty-eight (48) 88 DB1/ 159236086.1 SAL-049PC126933-5049 hrs after transfection, the cells will be analyzed by flow cytometry to count the percentage of GFP expressing cells. The cells will be gated to distinguish them from debris and 20,000 cells will be counted. The final integration efficiency will be calculated by the baseline percentage of GFP cells by the percentage of GFP cells at 48hr. Excision positive (EXC+) and integration deficient (INT-) mutants will be identified using the method described above. Example 3 – Identification of Deletion Mutants and Fusion Protein Mutants Multiple deletion mutations will be generated using known methods.
  • Some deletion mutants will be deleted at the N- terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted at the C- terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted in between the N-terminus and the C-terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted at the N-terminus and at the C-terminus. Some deletion mutants will be deleted at the N-terminus, at the C- terminus, and in between the N-terminus and the C-terminus relative to SEQ ID NO: 1. Integration and excision activity will be tested on the mutants.
  • Mutants with high excision activity and low integration activity will be selected as lead candidates for further optimization (e.g., without limitation, additional rounds of screening and/or addition of fusion proteins as described below). It is contemplated that an additional of a tag, e.g., an HA-tag, would not alter the excision and integration activity of the mutants.
  • the helper enzyme from Myotis myotis will also be subjected to fusion with protein binding domains (e.g., without limitation, TALEs, TnsD, dCas9, and dCas12j) as described throughout the present application. Fusion proteins mutants will be generated using known method and the mutants will be screened for integration and excision activity.
  • FIG.6 depicts the TTAA site in hROSA26 (hg38 chr3:9,396,133-9,396,305) that is targeted by guideRNAs (TABLE 2), TALES (TABLE 8), and ZnF (TABLE 13).
  • FIG.7 depicts two TTAA sites in AAVS1 (hg38 chr19:55,112,851-55,113,324) that are targeted by guideRNAs (TABLE 3) or TALES (TABLE 9), and ZnF (TABLE 14).
  • FIG.8 depicts two TTAA sites in Chromosome 4 (hg38 chr4:30,793,534-30,875,476) that are targeted by guideRNAs (TABLE 4) or TALES (TABLE 10), and ZnF (TABLE 15).
  • FIG.9 depicts two TTAA sites in Chromosome 22 (hg38 chr22:35,370,000-35,380,000) that are targeted by guideRNAs (TABLE 5) or TALES (TABLE 11), and ZnF (TABLE 16).
  • FIG. 10 depicts two TTAA sites in Chromosome X (hg38 chrX:134,419,661-134,541,172) that are targeted by guideRNAs (TABLE 6) or TALES (TABLE 12), and ZnF (TABLE 17).

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Abstract

Gene therapy compositions and methods related to transposition are provided, e.g., those engineered from Myotis myotis.

Description

SAL-049PC/126933-5049 MOBILE GENETIC ELEMENTS FROM MYOTIS MYOTIS FIELD The present disclosure relates to recombinant mobile element systems and uses thereof. Specifically, the recombinant mobile element systems of the present disclosure are derived from Myotis myotis. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No.63/654,350, filed on May 31, 2024, the entire contents which are incorporated herein in their entireties. DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY The instant application contains a sequence listing, which has been submitted in XML format via EFS-Web. The contents of the XML copy named “SAL-049PC_126933-5049_Sequence_Listing,” which was created on May 30, 2025 and is 810,833 bytes in size, the contents of which are incorporated herein by reference in their entirety. BACKGROUND Mobile elements are genetic sequences that are found, with small exceptions, in all living organisms. These elements have deep evolutionary origins and diversification and have an astonishing variety of forms and shapes. The most widely used transposon system is that of the piggyBac system, which was originally identified in moths in 1983. When combining a piggyBac transposon with a piggyBac helper enzyme, the DNA sequence from the transposon vector can be transferred to one of many specific nucleotide sequence (i.e., TTAA) sequences distributed throughout the genome. However, current transposition systems only find use in laboratory applications. Therapeutic uses have proven elusive. There is a need for novel and safer transposon systems for this technology to find use in medicine. SUMMARY Accordingly, this disclosure describes, in part, a helper enzyme, optionally in the form of RNA, which is optionally engineered to target a single human genomic locus by introducing a DNA binding protein to yield a chimeric agent. The present disclosure provides, inter alia, a composition comprising a recombinant mobile element enzyme and a DNA binder (e.g., without limitation, dCas9, TALEs, TnsD, and ZnF) that guide donor insertion to specific genomic sites. In embodiments, the helper enzyme is an engineered form of an enzyme reconstructed from Myotis myotis. In embodiments, the enzyme includes but is not limited to an engineered version that is a monomer, dimer, tetramer (or DB1/ 159236086.1 SAL-049PC126933-5049 another multimer), hyperactive (Exc+), and/or has a reduced interaction with non-TTAA recognitions sites (Int-), of a helper enzyme reconstructed from Myotis myotis or a predecessor thereof. In embodiments, the helper enzyme is a recombinant molecule which has at least about 90% identity to the nucleotide sequence of SEQ ID NO: 2 or the amino acid sequence SEQ ID NO: 1. In embodiments, the helper enzyme has at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same. In embodiments, the composition comprises a gene transfer construct. In embodiments, the gene transfer construct comprises a donor (e.g., donor DNA) and can be or can comprise a vector comprising a mobile element comprising one or more end sequences recognized by the enzyme. In embodiments, the end sequences are left and right end sequences that are recombinant or synthetic sequences. In embodiments, the end sequences are from Myotis myotis, or end sequences with similarity to piggyBac-like mobile elements and exhibit duplications of their presumed TTAA target sites. In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor. In embodiments, the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4, and wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor. In embodiments, the end sequences, which can be, e.g., from Myotis myotis, are optionally flanked by a TTAA sequence. In embodiments, the enzyme is included in the gene transfer construct. In embodiments, the composition comprises a nucleic acid binding component of a gene-editing system. In embodiments, the gene-editing system is included in the gene transfer construct. In embodiments, the gene-editing system comprises a CRISPR/Cas enzyme (class I, class II), or their six subtypes (type I–VI) (e.g., Cas9, Cas12a, Cas12j, Cas12k), or a variant thereof. In embodiments, the gene-editing system comprises a nuclease-deficient a CRISPR/Cas enzyme (class I, class II), or their six subtypes (type I–VI) (e.g., dCas9, dCas12a, dCas12j, dCas12k). In embodiments, the gene-editing system comprises Cas9, Cas12a, Cas12j, or Cas12k, or a variant thereof. For example, the gene-editing system comprises a nuclease-deficient dCas9, dCas12a, dCas12j, or dCas12k. In embodiments, the gene-editing system comprises a TALE, ZnF, or TnsD. In embodiments, the composition has the helper enzyme and the nucleic acid binding component of the gene-editing system. 2 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the composition comprises a chimeric mobile element construct comprising the helper enzyme and the nucleic acid binding component of the gene-editing system fused or linked thereto. In embodiments, the helper enzyme and the nucleic acid binding component of the gene-editing system can be fused or linked to one another via a linker, which can be a flexible linker. In embodiments, the flexible linker can be substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is from about 1 to about 12. In embodiments, the flexible linker is of or about 50, or about 100, or about 150, or about 200 amino acid residues. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt. In embodiments, the helper enzyme is capable of inserting a donor at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule. In embodiments, the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state. In aspects, a composition is provided comprising (a) a nucleic acid binding component of a gene-editing system, and (b) a recombinant mammalian helper enzyme, the helper enzyme having at least about 90% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same. In embodiments, the helper enzyme has at least about 95%, or at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding the same. In embodiments, a mobile element construct comprises a helper enzyme (both herein called “helper”) constructed as a DNA vector or RNA vector (FIG.1A) fused or linked to a DNA binding domain (DBD), or TALE (FIG.1B), zinc finger (ZnF) (FIG.1C), inactive Cas protein (dCas9, dCas12a, dCas12j, or dCas12k) programmed by a guide RNA (gRNA) (FIG.1D), or a construct with an intein or dimerization enhancer such as SH3, biotin, avidin, or rapamycin binders (FIG. 1E). In embodiments, a composition comprising a recombinant mammalian helper enzyme in accordance with embodiments of the present disclosure can include one or more non-viral vectors. Also, in embodiments, the recombinant mammalian helper enzyme can be disposed on the same (cis) or different vector (trans) than a donor with a transgene. Accordingly, in embodiments, the recombinant mammalian helper enzyme and the donor encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the recombinant mammalian helper enzyme and the donor encompassing a transgene are in trans configuration such that they are included in different vectors. In embodiments, the vector is any non-viral vector in accordance with the present disclosure. In embodiments, the present disclosure provides a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure. In embodiments, 3 DB1/ 159236086.1 SAL-049PC126933-5049 the method of the present disclosure further comprising contacting the cell with a polynucleotide encoding a donor DNA. In embodiments, the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state. In embodiments, the present disclosure provides a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure and administering the cell to a subject in need thereof. In embodiments, the present disclosure provides a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure or host cell of the present disclosure to a subject in need thereof. In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof. In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 2, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter. The details of the invention are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, illustrative methods and materials are now described. Other features, objects, and advantages of the invention will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. BRIEF DESCRIPTION OF DRAWINGS 4 DB1/ 159236086.1 SAL-049PC126933-5049 FIG.1A - FIG.1E depict five illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid or miniplasmid) with a T7 promoter (cap dependent), beta-globin 5’-UTR, and a helper enzyme (SEQ ID NO: 1, SEQ ID NO: 2) from Myotis myotis followed by a beta-globin 3’-UTR, and a poly-alanine tail (FIG.1A). TALEs (FIG.1B, TABLE 7 – TABLE 12), ZnF (FIG.1C, TABLE 13 – TABLE 17), or a dead Cas9 (dCas9) binding protein (FIG.1D, SEQ ID NO: 5, SEQ ID NO: 6) with guide RNAs (TABLE 1 – TABLE 6) were joined by a linker to the N-terminus to target the specific TTAA sites at hROSA 26, AAVS1, chromosome 4, chromosome 22, and chromosome X loci. FIG.1E depicts a construct with a dimerization enhancer to assure activation of the two monomers. FIG.2A depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter driving a gene of interest (GOI) with a polyA tail flanked by two insulators and ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting genomic safe harbor sites (GSHS) or other loci. FIG.2B depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a splice acceptor site for exon 2 and other exons of a gene of interest (GOI) followed by a polyA tail and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting endogenous genes in the first intron (or other introns) to repair downstream mutations. FIG.2C depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with tandem promoters to affect expression in different tissues (e.g., without limitation, liver specific promoter, cardiac specific promoter) and a gene(s) of interest (GOI) followed by a polyA tail and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used to differentially promote expression of genes in different organs, tissues or cell types. FIG.2D depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with two or more genes of interest (GOI) linked by P2A “self-cleaving” peptides and followed by WPRE and a polyA tail. The construct is flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for delivering multiple genes or genetic factors. FIG. 2E depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter(s) driving the expression of two or more genes as in FIG.2D and linked to a sequence consisting of a 5’-miRNA, a sense and antisense miRNA pair, and completed with the 3’-miRNA. The construct is followed by WPRE and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct combines protein replacement and miRNA to inhibit the expression of other related proteins. 5 DB1/ 159236086.1 SAL-049PC126933-5049 FIG.3 depicts the TTAA site in hROSA26 (hg38 chr3:9,396,133-9,396,305) that is targeted by guideRNAs (TABLE 2), TALES (TABLE 8), and ZnF (TABLE 13). FIG.4 depicts two TTAA sites in AAVS1 (hg38 chr19:55,112,851-55,113,324) that are targeted by guideRNAs (TABLE 3) or TALES (TABLE 9), and ZnF (TABLE 14). FIG.5 depicts two TTAA sites in Chromosome 4 (hg38 chr4:30,793,534-30,875,476) that are targeted by guideRNAs (TABLE 4) or TALES (TABLE 10), and ZnF (TABLE 15). FIG.6 depicts two TTAA sites in Chromosome 22 (hg38 chr22:35,370,000-35,380,000) that are targeted by guideRNAs (TABLE 5) or TALES (TABLE 11), and ZnF (TABLE 16). FIG. 7 depicts two TTAA sites in Chromosome X (hg38 chrX:134,419,661-134,541,172) that are targeted by guideRNAs (TABLE 6) or TALES (TABLE 12), and ZnF (TABLE 17). DETAILED DESCRIPTION The present invention is based, in part, on the discovery of an engineered helper enzyme capable of gene insertion that finds uses in multiple applications, including, without limitation, in gene therapy. In aspects, there is provided an engineered enzyme from Myotis myotis, e.g., having an amino acid sequence of SEQ ID NO: 1 or a variant thereof, inclusive of variants generated (e.g., by random mutagenesis and/or site directed mutagenesis) (occasionally may be referred to as “engineered”, “the present MMT”, “hyperactive helper from Myotis myotis”, or “hyperactive helper”). “MMT”, as used herein, refers to Myotis myotis helper, as engineered herein. The present invention is based, in part, on the discovery that a helper enzyme, e.g., a recombinant helper enzyme derived from Myotis myotis, can be fused with a transcription activator-like effector proteins (TALE) DNA binding domain (DBD), a dCas9/gRNA, or a zinc finger sequence to thereby create a chimeric enzyme capable of a site- or locus-specific transposition. For instance, in the case of a fusion to a TALD DBD, the enzyme (e.g., without limitation, a chimeric helper) utilizes the specificity of TALE DBD to certain sites within a host genome, which allows using DBDs to target any desired location in the genome. In this way, the chimeric helper in accordance with the present disclosure allows achieving targeted integration of a transgene. In embodiments, the helper has one or more mutations that confer hyperactivity. In embodiments, the helper is a mammal-derived helper, optionally a helper RNA helper. Thus, in embodiments, the present compositions and methods for gene transfer utilize a dual donor/helper system. Transposable elements are non-viral gene delivery vehicles found ubiquitously in nature. Donor-based vectors have the capacity of stable genomic integration and long-lasting expression of transgene constructs in cells. Generally, dual donor and helper systems work via a cut-and-paste mechanism whereby donor DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a helper 6 DB1/ 159236086.1 SAL-049PC126933-5049 enzyme at a repetitive sequence site. Dual donor/helper (or “donor/helper”) plasmid systems insert a transgene flanked by inverted terminal ends (“ends”), such as TTAA (SEQ ID NO: 440) tetranucleotide sites, without leaving a DNA footprint in the human genome. The helper enzyme, in embodiments, is transiently expressed (on the same or a different vector from a vector encoding the donor) and it catalyzes the insertion events from the donor plasmid to the host genome. Genomic insertions primarily target introns but may target other TTAA (SEQ ID NO: 440) sites and integrate into approximately 50% of human genes. This disclosure describes, in embodiments, a DNA integration system, which is highly active in mammals, and is derived from a mammalian mobile DNA element. In embodiments, this mammal-derived mobile genetic element is engineered to insert donor DNA at specific TTAA insertion “hotspots” that are frequently favored insertion sites for the un-engineered enzyme. In embodiments, this technology exploits a helper RNA encoding enzyme with engineered DNA binding proteins and a donor DNA contained between the ends of a mobile element of the gene to be inserted into the genome. In embodiments, the mammal-derived enzyme is fused to a protein domain at its N-terminus without loss of activity and “engineered” by fusing DNA binding domains (DBD) that can target almost any location in the genome. Excision competent/target binding defective enzymes (Exc+/Int) mutants are described, that when combined with programmable, synthetic DBDs only insert at a TTAAs at a single target site. This enzyme described in this disclosure displays several highly desirable features that are of great advantage for transgene integration. In embodiments, no DNA double strand breaks are introduced into the target genome. Furthermore, upon enzyme- mediated excision containing a gene of interest from its donor DNA, the flanking donor backbone ends are very efficiently rejoined, leaving no double strand break in the donor DNA to signal DNA damage. In embodiments, the enzyme inserts the excised element at high frequency selectively into a TTAA target site. Notably, because excision from the donor site results in the covalent linkage of a TTAA segment to each 5’ donor end, the joining of the 3’ donor ends to staggered positions on the top and bottom strands of the DNA flanking the target TTAA, a simple ligation restores intact duplex DNA, and no DNA synthesis is required for repair. In embodiments, the enzyme delivers a large cargo size as compared to other mobile genetic elements or integrating viral systems to date. In embodiments, the enzyme is delivered as an RNA instead of as a DNA. Other mobile genetic elements including helpers such as hyperactive PB and SB100X, when delivered as RNA, have significantly less activity when compared to DNA. See Bire, et al. (2013). Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition. BMC Biotechnol, 13, 75; Bire, et al. (2013). Optimization of the piggyBac donor using mRNA and insulators: toward a more reliable gene delivery system. PLoS One, 8(12), e82559; Wilber, et al. (2006). RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther, 13(3), 625-630. In embodiments, the enzyme described herein has the same or better activity when delivered as RNA. The use of helper RNA offers several advantages over delivery of a DNA molecule. Wilber, et al. (2006). RNA as a source of helper for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. Mol Ther, 7 DB1/ 159236086.1 SAL-049PC126933-5049 13(3), 625-630. For instance, without wishing to be bound by theory, there is improved control with respect to the duration of enzyme expression, minimizing persistence in the tissue, and there is potential for transgene re-mobilization and re-insertion following the initial transposition event. Furthermore, in embodiments, the helper-encoding RNA sequence is incapable of integrating into the host genome, thereby eliminating concerns about long-term helper expression and destabilizing effects with respect to the gene of interest. This safety feature, in embodiments, prevents the integration of the enzyme gene into the human genome and circumvents potential oncogenic and mutagenic effects. In embodiments, the present disclosure provides a dual DNA donor and RNA helper system. The donor DNA plasmid contains helper-specific inverted terminal repeats (ITRs) flanking the transgene while the helper-RNA transiently expresses a synthetic enzyme that catalyzes the insertion events from the donor plasmid to the host genome. This two component DNA/RNA system is, in embodiments, co-encapsulated in a single lipid nanoparticle using microfluidic technology and the lipid nanoparticles protect the RNA from extracellular degradation by in vivo injection. Helper Enzyme In embodiments, the present disclosure provides a composition comprising a helper enzyme or a nucleic acid encoding the enzyme, wherein the enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1. SEQ ID NO: 1: amino acid sequence of a helper from Myotis myotis (634 amino acids) 1 MVKFSKDIES SDDEFYFENE DKSEKCNNDE IEFSEDASGD EQIAGPSGTT ERKTSLALPK 61 NLAESTDSDI EFIKAKRSRT IVYFESDVDI GDIIEKSGIR PSESYVSRGE NRKKKSWTST 121 SVNDKEPSRI PFSTGQLHVG PQVPSGCATP IDFFQLFFTE TLIKNITDET NEYARHKISQ 181 KELSQRSTNN WKDVTIEEMK AFLGVILNMG VLNHPNLQSY WSMDFESHIP FFRSVFKRER 241 FLQIFWMLHL KNDQKSSKDL RTRTEKVNCF LSYLEMKFRE RFCPGREIAV DEAVVGFKGK 301 IHFITYNPKK PTKWGIRLYV LSDSKCGYVH SFVPYYGGIT SETLVRPDLP FTSRIVLELH 361 ERLKNSVPGS QGYHFFTDRY YTSVTLAKEL FKEKTHLTGT IMPNRKDNPP VIKHQKLKKG 421 EIVAFRDENV MLLAWKDKRI VTMLSTWDPS ETESVERRVP GGGKEIVLKP KVVTNYTKFM 481 GGVDIADHYT STYCFMRKTL KWWRTLFFWG LEVSVVNSYI LYKECQKRKN EKPITHVKFI 541 RKLVHDLVGE FRDGTLTSRG RLLSTNLEQR LDGKLHIITP HPNKKHKDCV VCSNRKIKGG 601 RRETIYICET CECKPGLHVG ECFKKYHTMK NYRD In embodiments, the enzyme comprises an amino acid sequence of at least about 80% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 83% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 85% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 88% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 89% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 1. In 8 DB1/ 159236086.1 SAL-049PC126933-5049 embodiments, the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 1. In embodiments, the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 1. In embodiments, the enzyme has one or more mutations which confer hyperactivity. In embodiments, the enzyme has one or more amino acid substitutions generated by by random mutagenesis and/or site directed mutagenesis. In embodiments, the nucleic acid that encodes the enzyme has a nucleotide sequence of SEQ ID NO: 2 or a codon- optimized form thereof. SEQ ID NO: 2: nucleotide sequence encoding the helper from Myotis myotis (1905 nt) 1 ATGGTGAAAT TTTCTAAGGA CATTGAGTCT AGCGACGATG AGTTTTACTT CGAGAATGAG 61 GACAAGTCTG AGAAGTGCAA CAACGACGAG ATTGAGTTCA GCGAAGACGC CTCCGGGGAT 121 GAGCAGATCG CCGGACCTAG CGGAACCACC GAAAGGAAGA CCAGCCTGGC CCTCCCTAAA 181 AATCTGGCTG AGTCCACCGA TTCCGACATC GAATTCATCA AGGCCAAGCG CTCTCGGACA 241 ATCGTGTATT TCGAATCCGA CGTGGATATC GGCGACATCA TCGAGAAGTC CGGCATTAGG 301 CCTTCTGAAA GCTATGTGTC TCGAGGTGAG AACAGAAAGA AGAAGTCCTG GACAAGCACC 361 AGCGTGAACG ACAAGGAACC CTCTAGGATC CCTTTCAGCA CCGGCCAGCT GCACGTGGGC 421 CCACAGGTGC CTAGCGGCTG CGCCACCCCT ATTGATTTCT TTCAGCTGTT CTTTACCGAG 481 ACCCTGATTA AGAATATCAC CGACGAGACA AACGAGTACG CCCGCCACAA GATCAGCCAG 541 AAGGAGCTGA GCCAGAGAAG CACTAACAAC TGGAAGGACG TGACCATCGA GGAAATGAAG 601 GCCTTCCTCG GGGTGATCCT GAACATGGGC GTGCTGAATC ACCCAAATCT GCAGAGCTAC 661 TGGAGTATGG ATTTCGAGTC TCACATTCCC TTTTTTAGAT CAGTGTTTAA GCGGGAGAGA 721 TTTCTGCAGA TCTTCTGGAT GCTGCACCTG AAGAATGATC AGAAGAGCAG CAAAGACCTG 781 CGCACTCGGA CCGAGAAAGT CAACTGCTTT CTGTCCTACC TCGAGATGAA ATTCAGGGAG 841 CGGTTTTGCC CTGGCAGGGA GATCGCTGTG GATGAAGCCG TGGTGGGCTT CAAGGGTAAG 901 ATCCACTTCA TTACATACAA CCCCAAGAAG CCCACCAAGT GGGGCATTCG CCTCTACGTG 961 CTGTCAGACT CCAAATGCGG CTACGTGCAC TCTTTCGTGC CTTACTATGG AGGGATCACC 1021 TCCGAAACTC TGGTGAGACC CGACCTGCCC TTTACTTCTA GAATTGTGCT GGAGCTGCAC 1081 GAGCGGCTGA AGAACAGCGT GCCAGGGTCC CAGGGCTATC ACTTTTTCAC CGACCGGTAC 1141 TATACCTCCG TGACCCTGGC CAAGGAGCTG TTTAAGGAGA AGACACATCT TACGGGCACC 1201 ATCATGCCAA ATAGGAAAGA CAATCCTCCC GTGATCAAGC ACCAGAAGCT GAAAAAGGGG 1261 GAGATTGTGG CTTTTCGGGA CGAAAATGTG ATGCTGCTGG CTTGGAAGGA TAAGAGAATC 1321 GTGACTATGC TGTCCACCTG GGACCCCTCT GAAACCGAGA GTGTGGAGCG CAGAGTGCCA 1381 GGCGGAGGCA AGGAGATTGT GCTGAAGCCA AAAGTGGTGA CAAACTACAC TAAGTTTATG 1441 GGCGGCGTGG ACATCGCCGA CCACTACACC AGCACCTACT GTTTCATGCG CAAAACCCTG 1501 AAGTGGTGGC GTACCCTCTT TTTCTGGGGC CTGGAGGTGA GTGTGGTGAA CTCTTACATC 1561 CTGTACAAGG AGTGCCAGAA GCGGAAGAAC GAGAAGCCCA TCACCCACGT GAAGTTCATC 1621 CGCAAGCTGG TGCACGACCT GGTTGGCGAG TTCAGAGATG GAACCCTGAC ATCCAGAGGG 1681 CGCCTGCTGA GCACCAATCT GGAGCAGAGG CTGGATGGCA AACTCCACAT TATCACTCCC 1741 CACCCTAACA AGAAGCACAA GGATTGTGTG GTGTGTAGCA ACAGGAAGAT CAAGGGAGGG 1801 CGGCGCGAGA CCATCTACAT CTGTGAGACC TGCGAATGCA AGCCCGGCCT GCACGTGGGC 1861 GAATGTTTCA AGAAATACCA TACTATGAAG AATTACAGGG ATTGA In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 2, or a functional variant thereof, operably linked to a heterologous promoter. 9 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, the enzyme is excision positive. In embodiments, the enzyme is integration deficient. In embodiments, the enzyme has decreased integration activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. In embodiments, the enzyme has increased excision activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 2, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, the enzyme comprises a targeting element. In embodiments, the enzyme is capable of inserting a donor comprising a transgene in a genomic safe harbor site (GSHS). In embodiments, the binding of a GSHS of a nucleic acid molecule in a mammalian cell is with high target specificity, relative to a control. In embodiments, the control is a composition comprising an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 or a codon-optimized form thereof. In embodiments, the targeting element is able to direct a transposition machinery to the GSHS of a nucleic acid molecule in a mammalian cell. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. In embodiments, the GSHS is an adeno-associated virus site 1 (AAVS1). In embodiments, the GSHS is a human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X. In embodiments, the GSHS is selected from TABLES 1-17. In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TA-LER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6- 1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4. In embodiments, the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), catalytically inactive Zinc finger, catalytically inactive transcription factor, catalytically inactive 10 DB1/ 159236086.1 SAL-049PC126933-5049 nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof. In embodiments, the targeting element comprises a TALE DBD. In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the repeat sequences each independently comprises about 33 or 34 amino acids. In embodiments, the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively. In embodiments, the RVD recognizes one base pair in a target nucleic acid sequence. In embodiments, the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the target nucleic acid sequence and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG. In embodiments, the TALE DBD targets one or more of GSHS sites selected from TABLES 7-12. In embodiments, the TALE DBD comprises one or more of RVD selected from TABLES 7-12, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. In embodiments, the targeting element comprises a Cas9 enzyme associated with a gRNA. In embodiments, the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA. In embodiments, the catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof. SEQ ID NO: 5: nucleotide sequence of dead Cas9 DNA binding protein (5004 bp) 1 ATGGACAAGA AGTACTCCAT TGGGCTCGCT ATCGGCACAA ACAGCGTCGG CTGGGCCGTC 61 ATTACGGACG AGTACAAGGT GCCGAGCAAA AAATTCAAAG TTCTGGGCAA TACCGATCGC 121 CACAGCATAA AGAAGAACCT CATTGGCGCC CTCCTGTTCG ACTCCGGGGA GACGGCCGAA 181 GCCACGCGGC TCAAAAGAAC AGCACGGCGC AGATATACCC GCAGAAAGAA TCGGATCTGC 241 TACCTGCAGG AGATCTTTAG TAATGAGATG GCTAAGGTGG ATGACTCTTT CTTCCATAGG 301 CTGGAGGAGT CCTTTTTGGT GGAGGAGGAT AAAAAGCACG AGCGCCACCC AATCTTTGGC 361 AATATCGTGG ACGAGGTGGC GTACCATGAA AAGTACCCAA CCATATATCA TCTGAGGAAG 421 AAGCTTGTAG ACAGTACTGA TAAGGCTGAC TTGCGGTTGA TCTATCTCGC GCTGGCGCAT 481 ATGATCAAAT TTCGGGGACA CTTCCTCATC GAGGGGGACC TGAACCCAGA CAACAGCGAT 541 GTCGACAAAC TCTTTATCCA ACTGGTTCAG ACTTACAATC AGCTTTTCGA AGAGAACCCG 601 ATCAACGCAT CCGGAGTTGA CGCCAAAGCA ATCCTGAGCG CTAGGCTGTC CAAATCCCGG 661 CGGCTCGAAA ACCTCATCGC ACAGCTCCCT GGGGAGAAGA AGAACGGCCT GTTTGGTAAT 721 CTTATCGCCC TGTCACTCGG GCTGACCCCC AACTTTAAAT CTAACTTCGA CCTGGCCGAA 781 GATGCCAAGC TTCAACTGAG CAAAGACACC TACGATGATG ATCTCGACAA TCTGCTGGCC 11 DB1/ 159236086.1 SAL-049PC126933-5049 841 CAGATCGGCG ACCAGTACGC AGACCTTTTT TTGGCGGCAA AGAACCTGTC AGACGCCATT 901 CTGCTGAGTG ATATTCTGCG AGTGAACACG GAGATCACCA AAGCTCCGCT GAGCGCTAGT 961 ATGATCAAGC GCTATGATGA GCACCACCAA GACTTGACTT TGCTGAAGGC CCTTGTCAGA 1021 CAGCAACTGC CTGAGAAGTA CAAGGAAATT TTCTTCGATC AGTCTAAAAA TGGCTACGCC 1081 GGATACATTG ACGGCGGAGC AAGCCAGGAG GAATTTTACA AATTTATTAA GCCCATCTTG 1141 GAAAAAATGG ACGGCACCGA GGAGCTGCTG GTAAAGCTTA ACAGAGAAGA TCTGTTGCGC 1201 AAACAGCGCA CTTTCGACAA TGGAAGCATC CCCCACCAGA TTCACCTGGG CGAACTGCAC 1261 GCTATCCTCA GGCGGCAAGA GGATTTCTAC CCCTTTTTGA AAGATAACAG GGAAAAGATT 1321 GAGAAAATCC TCACATTTCG GATACCCTAC TATGTAGGCC CCCTCGCCCG GGGAAATTCC 1381 AGATTCGCGT GGATGACTCG CAAATCAGAA GAGACCATCA CTCCCTGGAA CTTCGAGGAA 1441 GTCGTGGATA AGGGGGCCTC TGCCCAGTCC TTCATCGAAA GGATGACTAA CTTTGATAAA 1501 AATCTGCCTA ACGAAAAGGT GCTTCCTAAA CACTCTCTGC TGTACGAGTA CTTCACAGTT 1561 TATAACGAGC TCACCAAGGT CAAATACGTC ACAGAAGGGA TGAGAAAGCC AGCATTCCTG 1621 TCTGGAGAGC AGAAGAAAGC TATCGTGGAC CTCCTCTTCA AGACGAACCG GAAAGTTACC 1681 GTGAAACAGC TCAAAGAAGA CTATTTCAAA AAGATTGAAT GTTTCGACTC TGTTGAAATC 1741 AGCGGAGTGG AGGATCGCTT CAACGCATCC CTGGGAACGT ATCACGATCT CCTGAAAATC 1801 ATTAAAGACA AGGACTTCCT GGACAATGAG GAGAACGAGG ACATTCTTGA GGACATTGTC 1861 CTCACCCTTA CGTTGTTTGA AGATAGGGAG ATGATTGAAG AACGCTTGAA AACTTACGCT 1921 CATCTCTTCG ACGACAAAGT CATGAAACAG CTCAAGAGGC GCCGATATAC AGGATGGGGG 1981 CGGCTGTCAA GAAAACTGAT CAATGGGATC CGAGACAAGC AGAGTGGAAA GACAATCCTG 2041 GATTTTCTTA AGTCCGATGG ATTTGCCAAC CGGAACTTCA TGCAGTTGAT CCATGATGAC 2101 TCTCTCACCT TTAAGGAGGA CATCCAGAAA GCACAAGTTT CTGGCCAGGG GGACAGTCTT 2161 CACGAGCACA TCGCTAATCT TGCAGGTAGC CCAGCTATCA AAAAGGGAAT ACTGCAGACC 2221 GTTAAGGTCG TGGATGAACT CGTCAAAGTA ATGGGAAGGC ATAAGCCCGA GAATATCGTT 2281 ATCGAGATGG CCCGAGAGAA CCAAACTACC CAGAAGGGAC AGAAGAACAG TAGGGAAAGG 2341 ATGAAGAGGA TTGAAGAGGG TATAAAAGAA CTGGGGTCCC AAATCCTTAA GGAACACCCA 2401 GTTGAAAACA CCCAGCTTCA GAATGAGAAG CTCTACCTGT ACTACCTGCA GAACGGCAGG 2461 GACATGTACG TGGATCAGGA ACTGGACATC AATCGGCTCT CCGACTACGA CGTGGCTGCT 2521 ATCGTGCCCC AGTCTTTTCT CAAAGATGAT TCTATTGATA ATAAAGTGTT GACAAGATCC 2581 GATAAAGCTA GAGGGAAGAG TGATAACGTC CCCTCAGAAG AAGTTGTCAA GAAAATGAAA 2641 AATTATTGGC GGCAGCTGCT GAACGCCAAA CTGATCACAC AACGGAAGTT CGATAATCTG 2701 ACTAAGGCTG AACGAGGTGG CCTGTCTGAG TTGGATAAAG CCGGCTTCAT CAAAAGGCAG 2761 CTTGTTGAGA CACGCCAGAT CACCAAGCAC GTGGCCCAAA TTCTCGATTC ACGCATGAAC 2821 ACCAAGTACG ATGAAAATGA CAAACTGATT CGAGAGGTGA AAGTTATTAC TCTGAAGTCT 2881 AAGCTGGTCT CAGATTTCAG AAAGGACTTT CAGTTTTATA AGGTGAGAGA GATCAACAAT 2941 TACCACCATG CGCATGATGC CTACCTGAAT GCAGTGGTAG GCACTGCACT TATCAAAAAA 3001 TATCCCAAGC TTGAATCTGA ATTTGTTTAC GGAGACTATA AAGTGTACGA TGTTAGGAAA 3061 ATGATCGCAA AGTCTGAGCA GGAAATAGGC AAGGCCACCG CTAAGTACTT CTTTTACAGC 3121 AATATTATGA ATTTTTTCAA GACCGAGATT ACACTGGCCA ATGGAGAGAT TCGGAAGCGA 3181 CCACTTATCG AAACAAACGG AGAAACAGGA GAAATCGTGT GGGACAAGGG TAGGGATTTC 3241 GCGACAGTCC GGAAGGTCCT GTCCATGCCG CAGGTGAACA TCGTTAAAAA GACCGAAGTA 3301 CAGACCGGAG GCTTCTCCAA GGAAAGTATC CTCCCGAAAA GGAACAGCGA CAAGCTGATC 3361 GCACGCAAAA AAGATTGGGA CCCCAAGAAA TACGGCGGAT TCGATTCTCC TACAGTCGCT 3421 TACAGTGTAC TGGTTGTGGC CAAAGTGGAG AAAGGGAAGT CTAAAAAACT CAAAAGCGTC 3481 AAGGAACTGC TGGGCATCAC AATCATGGAG CGATCAAGCT TCGAAAAAAA CCCCATCGAC 3541 TTTCTGGAGG CGAAAGGATA TAAAGAGGTC AAAAAAGACC TCATCATTAA GCTTCCCAAG 3601 TACTCTCTCT TTGAGCTTGA AAACGGCCGG AAACGAATGC TCGCTAGTGC GGGCGAGCTG 3661 CAGAAAGGTA ACGAGCTGGC ACTGCCCTCT AAATACGTTA ATTTCTTGTA TCTGGCCAGC 3721 CACTATGAAA AGCTCAAAGG GTCTCCCGAA GATAATGAGC AGAAGCAGCT GTTCGTGGAA 3781 CAACACAAAC ACTACCTTGA TGAGATCATC GAGCAAATAA GCGAATTCTC CAAAAGAGTG 3841 ATCCTCGCCG ACGCTAACCT CGATAAGGTG CTTTCTGCTT ACAATAAGCA CAGGGATAAG 3901 CCCATCAGGG AGCAGGCAGA AAACATTATC CACTTGTTTA CTCTGACCAA CTTGGGCGCG 3961 CCTGCAGCCT TCAAGTACTT CGACACCACC ATAGACAGAA AGCGGTACAC CTCTACAAAG 4021 GAGGTCCTGG ACGCCACACT GATTCATCAG TCAATTACGG GGCTCTATGA AACAAGAATC 4081 GACCTCTCTC AGCTCGGTGG AGAC 12 DB1/ 159236086.1 SAL-049PC126933-5049 SEQ ID NO: 6: amino acid sequence of dead Cas9 DNA binding protein (1368 amino acids) 1 MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVAA 841 IVPQSFLKDD SIDNKVLTRS DKARGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA 1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD In embodiments, the targeting element comprises a Cas12 enzyme associated with a gRNA. In embodiments, the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a. In embodiments, the targeting element comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA. In embodiments, the guide RNA is selected from TABLES 1-6, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. In embodiments, the guide RNA targets one or more sites selected from TABLES 1-6. In embodiments, the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLES 13-17. In embodiments, the targeting element comprises a nucleic acid binding component of a gene-editing system. In embodiments, the enzyme or variant thereof and the targeting element are connected. In embodiments, the enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. In embodiments, the flexible linker is 13 DB1/ 159236086.1 SAL-049PC126933-5049 of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the enzyme is directly fused to the N-terminus of the targeting element and, optionally, wherein the targeting element is or comprises dCas9 enzyme. In embodiments, the E. coli TnsD comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 7. In embodiments, the TnsD comprises a truncated TnsD. In embodiments, the TnsD is truncated at its C-terminus. In embodiments, the TnsD is truncated at its N-terminus. In embodiments, the TnsD or variant thereof comprises a zinc finger motif. In embodiments, the zinc finger motif comprises a C3H-type motif (e.g., CCCH). SEQ ID NO: 7: amino acid sequence of E. coli TnsD (508 amino acids) 1 MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH 61 QTGRYAVQQL IYEHTLFPLY APFVGKERRD EAIRLMEYQA QGAVHLMLGV AASRVKSDNR 121 FRYCPDCVAL QLNRYGEAFW QRDWYLPALP YCPKHGALVF FDRAVDDHRH QFWALGHTEL 181 LSDYPKDSLS QLTALAAYIA PLLDAPRAQE LSPSLEQWTL FYQRLAQDLG LTKSKHIRHD 241 LVAERVRQTF SDEALEKLDL KLAENKDTCW LKSIFRKHRK AFSYLQHSIV WQALLPKLTV 301 IEALQQASAL TEHSITTRPV SQSVQPNSED LSVKHKDWQQ LVHKYQGIKA ARQSLEGGVL 361 YAWLYRHDRD WLVHWNQQHQ QERLAPAPRV DWNQRDRIAV RQLLRIIKRL DSSLDHPRAT 421 SSWLLKQTPN GTSLAKNLQK LPLVALCLKR YSESVEDYQI RRISQAFIKL KQEDVELRRW 481 RLLRSATLSK ERITEEAQRF LEMVYGEE In embodiments, the TnsD binds at or near an attTn7 attachment site. In embodiments, the TnsD binds at or near a region downstream of the glmS gene. GlmS (L-glucosamine-fructose-6-phosphate aminotransferase) is highly conserved and found in a wide variety of organisms from bacteria to humans. In embodiments, the TnsD binding region of glmS encodes the active site region of GlmS. In embodiments, TnsD binds at or near the human homologs of glmS, e.g., gfpt-1 and gfpt-2. In embodiments, TnsD binds the human glmS homologs gfpt-1 and gfpt-2. In embodiments, the transgene is inserted into attTn7. In embodiments, the TnsD comprises a nucleic acid binding component of a gene-editing system. In embodiments, the enzyme or variant thereof (optionally, wherein the enzyme is a helper enzyme, optionally, wherein the helper enzyme is reconstructed from Myotis myotis) and the TnsD are connected. In embodiments, the enzyme and the TnsD are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the enzyme is directly fused to the N- terminus of the TnsD. In embodiments, the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 14 DB1/ 159236086.1 SAL-049PC126933-5049 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLES 13-17. In embodiments, the enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene. In embodiments, the enzyme or variant thereof is able to directly or indirectly interact and/or form a complex with one or more proteins or nucleic acids. Construct In embodiments, the composition (e.g., a helper of the present disclosure), system, or method further comprising a nucleic acid encoding a donor comprising a transgene to be integrated. In embodiments, the transgene is defective or substantially absent in a disease state. In embodiments, the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. In embodiments, the cargo nucleic acid sequence is flanked by the first and the second donor end sequences. In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof. In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. In embodiments, the donor end sequences are selected from nucleotide sequences of SEQ ID NO: 3 and/or SEQ ID NO: 4, or a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. SEQ ID NO: 3: Myotis myotis Left ITR (200 bp) 1 ccccctttgc actcggatgt cgagtgtgac tcgacgcggt tagcatcggt tgcagctcgt 61 atgttgagcc atactcgacc tgtagtttca ccgagggggg aagggggatt tttgtctatt 121 tttccagtat ttttcttgtt ttcattagca tgaaaggaca agtaaaatgt aaatgccgtc 181 tcaactgatg ccaccaccta SEQ ID NO: 4: Myotis myotis Right ITR (200 bp) 1 tgaaaaatta tagagattaa aattactctt tgaatgtatc aataatttga aatataaaaa 61 aatccaaata aataagtttg tatgaaaaga aactccagtt ttttattcta ctgccgcact 121 ttgtaaaatc tggggtattt aaaaaattaa atcccgagta gaataaagga atcgagaaaa 181 aagcaagcga gtgcaaaggg 15 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor. In embodiments, the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity to the nucleotide sequence of SEQ ID NO: 4. In embodiments, the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor. In embodiments, the present disclosure provides a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof. In embodiments, the donor is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. In embodiments, the present disclosure provides a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the donor is suitable for transposition by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. In embodiments, the helper enzyme derived from Myotis myotis, the helper enzyme being suitable for transposition of a heterologous polynucleotide, the heterologous polynucleotide being flanked by two ends elements comprising the polynucleotide sequences of SEQ ID NO: 3, or a functional variant thereof and SEQ ID NO: 4, or a functional variant thereof. In embodiments, the enzyme or variant thereof is incorporated into a vector or a vector-like particle. In embodiments, the vector or a vector-like particle comprises one or more expression cassettes. In embodiments, the vector or a vector- like particle comprises one expression cassette. In embodiments, the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. In embodiments, the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles. In embodiments, the vector or vector-like particle is nonviral. In embodiments, the composition comprises DNA, RNA, or both. In embodiments, the enzyme or variant thereof is in the form of RNA. 16 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the donor is under the control of at least one tissue-specific promoter. In embodiments, the at least one tissue-specific promoter is a single promoter. In embodiments, the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter. In embodiments, the transgene to be integrated comprises at least one gene of interest. In embodiments, the transgene to be integrated comprises one gene of interest. In embodiments, the transgene to be integrated comprises two genes of interest. In embodiments, the transgene to be integrated comprises three genes of interest. In embodiments, the transgene to be integrated comprises four genes of interest. In embodiments, the transgene to be integrated comprises five genes of interest. In embodiments, the transgene to be integrated comprises six genes of interest. In embodiments, the at least one gene of interest comprises peptides for linking genes of interest. In embodiments, the peptides are 2A self-cleaving peptides, or functional variants thereof, wherein the 2A self-cleaving peptide is optionally selected from P2A, E2A, F2A, and T2A, or derivative thereof. In embodiments, the at least one gene of interest is linked to polynucleotide comprising a sequence comprising a 5’- miRNA, a sense and antisense miRNA pair, and/or a 3’-miRNA. Host Cell In aspects, the present disclosure further provides a host cell comprising the composition in accordance with embodiments of the present disclosure. Methods In certain embodiments, the present disclosure provides a method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure. In embodiments, the method further comprises contacting the cell with a polynucleotide encoding a donor. In embodiments, the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state. In certain embodiments, the present disclosure provides a method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of the present disclosure or host cell of the present disclosure and administering the cell to a subject in need thereof. In certain embodiments, the present disclosure provides a method for treating a disease or disorder in vivo, comprising administering the composition of the present disclosure or host cell of the present disclosure to a subject in need thereof. Transgene 17 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the transgene is an exogenous wild-type gene that, e.g., corrects a defective function of one or more mutations in a recipient. For instance, in embodiments, the recipient may have a mutation that provides a disease phenotype (e.g., a defective or absent gene product). In embodiments, the donor system or method of the present disclosure provides a correction that restores the gene product and diminishes the disease phenotype. In embodiments, the transgene is a gene that replaces, inactivates, or provides suicide or helper functions. In embodiments, the transgene and/or disease to be treated is one or more of: • beta-thalassemia: BCL11a or β-globin or βA-T87Q-globin, • LCA: RPE65, • LHON: ND4, • Achromatopsia: CNGA3 or CNGA3/CNGB3, • Choroideremia: REP1, • PKD: RPK (Red cell PK), • Hemophilia: F8, • ADA-SCID: ADA, • Fabry disease: GLA, • MPS type I: IDUA, • MPS type II: IDS, and • Cystic fibrosis: CFTR transgene. In embodiments, the donor comprises a gene encoding a complete polypeptide. In embodiments, the donor comprises a gene which is defective or substantially absent in a disease state. In embodiments, the transfecting of the cell is carried out using electroporation or calcium phosphate precipitation. In embodiments, the transfecting of the cell is carried out using a lipid vehicle, optionally N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2- dioleoyl-3-dimethylammonium-propane (DODAP), dioleoylphosphatidylethanolamine (DOPE), cholesterol, LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation), TRANSFECTAM (cationic liposome formulation), a lipid nanoparticle, or a liposome and combinations thereof. 18 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the transfecting of the cell is carried out using a lipid selected from one or more of the following categories: cationic lipids; anionic lipids; neutral lipids; multi-valent charged lipids; and zwitterionic lipids. In embodiments, a cationic lipid may be used to facilitate a charge-charge interaction with nucleic acids. In embodiments, the lipid is a neutral lipid. In embodiments, the neutral lipid is dioleoylphosphatidylethanolamine (DOPE), 1,2-Dioleoyl- sn-glycero-3-phosphocholine (DOPC), or cholesterol. In embodiments, cholesterol is derived from plant sources. In other embodiments, cholesterol is derived from animal, fungal, bacterial, or archaeal sources. In embodiments, the lipid is a cationic lipid. In embodiments, the cationic lipid is N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonia) propane (DOTAP), or 1,2-dioleoyl-3- dimethylammonium-propane (DODAP). In embodiments, one or more of the phospholipids 18:0 PC, 18:1 PC, 18:2 PC, DMPC, DSPE, DOPE, 18:2 PE, DMPE, or a combination thereof are used as lipids. In embodiments, the lipid is DOTMA and DOPE, optionally in a ratio of about 1:1. In embodiments, the lipid is DHDOS and DOPE, optionally in a ratio of about 1:1. In embodiments, the lipid is a commercially available product (e.g., LIPOFECTIN (cationic liposome formulation), LIPOFECTAMINE (cationic liposome formulation), LIPOFECTAMINE 2000 (cationic liposome formulation), LIPOFECTAMINE 3000 (cationic liposome formulation) (Life Technologies)). In embodiments, the transfecting of the cell is carried out using a cationic vehicle, optionally LIPOFECTIN or TRANSFECTAM. In embodiments, the transfecting of the cell is carried out using a lipid nanoparticle or a liposome. In embodiments, the method is helper virus-free. Epigenetic regulatory elements can be used to protect a transgene from unwanted epigenetic effects when placed near the transgene on a vector, including the transgene. See Ley et al., PloS One vol.8,4 e62784.30 Apr.2013. For example, MARs were shown to increase genomic integration and integration of a transgene while preventing heterochromatin silencing, as exemplified by the human MAR 1–68. See id.; see also Grandjean et al., Nucleic Acids Res.2011 Aug; 39(15):e104. MARs can also act as insulators and thereby prevent the activation of neighboring cellular genes. Gaussin et al., Gene Ther.2012 Jan; 19(1):15-24. It has been shown that a piggyBac donor containing human MARs in CHO cells mediated efficient and sustained expression from a few transgene copies, using cell populations generated without an antibiotic selection procedure. See Ley et al. (2013). In embodiments, the cell is further transfected with a third nucleic acid having at least one chromatin element, wherein the at least one chromatin element is optionally a Matrix Attachment Region (MAR) element. MARs are expression- enhancing, epigenetic regulator elements which are used to enhance and/or facilitate transgene expression, as described, for example, in PCT/IB2010/002337 (WO2011033375), which is incorporated by reference herein in its entirety. A MAR element can be located in cis or trans to the transgene. 19 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the transgene has a size of 100,000 bases or less, e.g., about 100,000 bases, or about 50,000 bases, or about 30,000 bases, or about 10,000 bases, or about 5,000 bases, or about 10,000 to about 100,000 bases, or about 30,000 to about 100,000 bases, or about 50,000 to about 100,000 bases, or about 10,000 to about 50,000 bases, or about 10,000 to about 30,000 bases, or about 30,000 to about 50,000 bases. In embodiments, the transgene has a size of about 200,000 bases or less, e.g., about 200,000 bases, or about 10,000 to about 200,000 bases, or about 30,000 to about 200,000 bases, or about 50,000 to about 200,000 bases, or about 100,000 to about 200,000 bases, or about 150,000 to about 200,000 bases. In embodiments, the transgene has a size of about 300,000 bases or less, e.g., about 300,000 bases, or about 10,000 to about 300,000 bases, or about 30,000 to about 300,000 bases, or about 50,000 to about 300,000 bases, or about 100,000 to about 300,000 bases, or about 150,000 to about 300,000 bases. Targeting Chimeric Constructs In aspects, the present disclosure provides for a donor system, e.g., in embodiments, a helper enzyme comprising a targeting element. In embodiments, the helper enzyme associated with the targeting element, is capable of inserting the donor comprising a transgene, optionally at a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site in a genomic safe harbor site (GSHS). In embodiments, the helper enzyme associated with the targeting element has one or more mutations which confer hyperactivity. In embodiments, the helper enzyme associated with the targeting element has gene cleavage (Exc) and/or gene integration (Int+) activity. In embodiments, the helper enzyme associated with the targeting element has gene cleavage (Exc) and/or a lack of gene integration (Int-) activity. In embodiments, the targeting element comprises one or more proteins or nucleic acids that are capable of binding to a nucleic acid. In embodiments, the targeting element comprises one or more of a gRNA, optionally associated with a Cas enzyme, which is optionally catalytically inactive, transcription activator-like effector (TALE), catalytically inactive Zinc finger, catalytically inactive transcription factor, nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, and paternally expressed gene 10 (PEG10). In embodiments, the targeting element comprises a transcription activator-like effector (TALE) DNA binding domain (DBD). 20 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X. In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4. In embodiments, the targeting element comprises a Cas9 enzyme guide RNA complex. In embodiments, the Cas9 enzyme guide RNA complex comprises a nuclease-deficient dCas9 guide RNA complex. In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element comprises a Cas12k enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12k guide RNA complex. In embodiments, a targeting chimeric system or construct, having a DBD fused to the helper enzyme directs binding of the helper to a specific sequence (e.g., transcription activator-like effector proteins (TALE) repeat variable di-residues (RVD) or gRNA) near an enzyme recognition site. The enzyme is thus prevented from binding to random recognition sites. In embodiments, the targeting chimeric construct binds to human GSHS. In embodiments, dCas9 (i.e., deficient for nuclease activity) is programmed with gRNAs directed to bind at a desired sequence of DNA in GSHS. In embodiments, TALEs described herein can physically sequester the enzyme to GSHS and promote transposition to nearby TTAA (SEQ ID NO: 440) sequences in close proximity to the RVD TALE nucleotide sequences. GSHS in open chromatin sites are specifically targeted based on the predilection for helpers to insert into open chromatin. 21 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the helper enzyme is capable of targeted genomic integration by transposition is linked to or fused with a TALE DNA binding domain (DBD) or a Cas-based gene-editing system, such as, e.g., Cas9 or a variant thereof. In embodiments, the targeting element targets the helper enzyme to a locus of interest. In embodiments, the targeting element comprises CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) associated protein 9 (Cas9), or a variant thereof. A CRISPR/Cas9 tool only requires Cas9 nuclease for DNA cleavage and a single-guide RNA (sgRNA) for target specificity. See Jinek et al. (2012) Science 337, 816–821; Chylinski et al. (2014) Nucleic Acids Res 42, 6091–6105. The inactivated form of Cas9, which is a nuclease-deficient (or inactive, or “catalytically dead” Cas9, is typically denoted as “dCas9,” has no substantial nuclease activity. Qi, L. S. et al. (2013). Cell 152, 1173–1183. CRISPR/dCas9 binds precisely to specific genomic sequences through targeting of guide RNA (gRNA) sequences. See Dominguez et al., Nat Rev Mol Cell Biol.2016;17:5–15; Wang et al., Annu Rev Biochem.2016;85:227–64. dCas9 is utilized to edit gene expression when applied to the transcription binding site of a desired site and/or locus in a genome. When the dCas9 protein is coupled to guide RNA (gRNA) to create dCas9 guide RNA complex, dCas9 prevents the proliferation of repeating codons and DNA sequences that might be harmful to an organism’s genome. Essentially, when multiple repeat codons are produced, it elicits a response, or recruits an abundance of dCas9 to combat the overproduction of those codons and results in the shut-down of transcription. Thus, dCas9 works synergistically with gRNA and directly affects the DNA polymerase II from continuing transcription. In embodiments, the targeting element comprises a nuclease-deficient Cas enzyme guide RNA complex. In embodiments, the targeting element comprises a nuclease-deficient (or inactive, or “catalytically dead” Cas, e.g., Cas9, typically denoted as “dCas” or “dCas9”) guide RNA complex. In embodiments, the dCas9/gRNA complex comprises a guide RNA selected from: GTTTAGCTCACCCGTGAGCC (SEQ ID NO: 91), CCCAATATTATTGTTCTCTG (SEQ ID NO: 92), GGGGTGGGATAGGGGATACG (SEQ ID NO: 93), GGATCCCCCTCTACATTTAA (SEQ ID NO: 94), GTGATCTTGTACAAATCATT (SEQ ID NO: 95), CTACACAGAATCTGTTAGAA (SEQ ID NO: 96), TAAGCTAGAGAATAGATCTC (SEQ ID NO: 97), and TCAATACACTTAATGATTTA (SEQ ID NO: 98), wherein the guide RNA directs the enzyme to a chemokine (C-C motif) receptor 5 (CCR5) gene. In embodiments, the dCas9/gRNA complex comprises a guide RNA selected from: CACCGGGAGCCACGAAAACAGATCC (SEQ ID NO: 99);CACCGCGAAAACAGATCCAGGGACA (SEQ ID NO: 100); CACCGAGATCCAGGGACACGGTGCT (SEQ ID NO: 101); CACCGGACACGGTGCTAGGACAGTG (SEQ ID NO: 102); CACCGGAAAATGACCCAACAGCCTC (SEQ ID NO: 103); CACCGGCCTGGCCGGCCTGACCACT (SEQ ID NO: 104); CACCGCTGAGCACTGAAGGCCTGGC (SEQ ID NO: 105); CACCGTGGTTTCCACTGAGCACTGA (SEQ ID NO: 106); CACCGGATAGCCAGGAGTCCTTTCG (SEQ ID NO: 107); CACCGGCGCTTCCAGTGCTCAGACT (SEQ ID NO: 108); CACCGCAGTGCTCAGACTAGGGAAG (SEQ ID NO: 109); 22 DB1/ 159236086.1 SAL-049PC126933-5049 CACCGGCCCCTCCTCCTTCAGAGCC (SEQ ID NO: 110); CACCGTCCTTCAGAGCCAGGAGTCC (SEQ ID NO: 111); CACCGTGGTTTCCGAGCTTGACCCT (SEQ ID NO: 112); CACCGCTGCAGAGTATCTGCTGGGG (SEQ ID NO: 113); CACCGCGTTCCTGCAGAGTATCTGC (SEQ ID NO: 114); AAACGGATCTGTTTTCGTGGCTCCC (SEQ ID NO: 115); AAACTGTCCCTGGATCTGTTTTCGC (SEQ ID NO: 116); AAACAGCACCGTGTCCCTGGATCTC (SEQ ID NO: 117); AAACCACTGTCCTAGCACCGTGTCC (SEQ ID NO: 118); AAACGAGGCTGTTGGGTCATTTTCC (SEQ ID NO: 119); AAACAGTGGTCAGGCCGGCCAGGCC (SEQ ID NO: 120); AAACGCCAGGCCTTCAGTGCTCAGC (SEQ ID NO: 121); AAACTCAGTGCTCAGTGGAAACCAC (SEQ ID NO: 122); AAACCGAAAGGACTCCTGGCTATCC (SEQ ID NO: 123); AAACAGTCTGAGCACTGGAAGCGCC (SEQ ID NO: 124); AAACCTTCCCTAGTCTGAGCACTGC (SEQ ID NO: 125); AAACGGCTCTGAAGGAGGAGGGGCC (SEQ ID NO: 126); AAACGGACTCCTGGCTCTGAAGGAC (SEQ ID NO: 127); AAACAGGGTCAAGCTCGGAAACCAC (SEQ ID NO: 128); AAACCCCCAGCAGATACTCTGCAGC (SEQ ID NO: 129); AAACGCAGATACTCTGCAGGAACGC (SEQ ID NO: 130); TCCCCTCCCAGAAAGACCTG (SEQ ID NO: 131); TGGGCTCCAAGCAATCCTGG (SEQ ID NO: 132); GTGGCTCAGGAGGTACCTGG (SEQ ID NO: 133); GAGCCACGAAAACAGATCCA (SEQ ID NO: 134); AAGTGAACGGGGAAGGGAGG (SEQ ID NO: 135); GACAAAAGCCGAAGTCCAGG (SEQ ID NO: 136); GTGGTTGATAAACCCACGTG (SEQ ID NO: 137); TGGGAACAGCCACAGCAGGG (SEQ ID NO: 138); GCAGGGGAACGGGGATGCAG (SEQ ID NO: 139); GAGATGGTGGACGAGGAAGG (SEQ ID NO: 140); GAGATGGCTCCAGGAAATGG (SEQ ID NO: 141); TAAGGAATCTGCCTAACAGG (SEQ ID NO: 142); TCAGGAGACTAGGAAGGAGG (SEQ ID NO: 143); TATAAGGTGGTCCCAGCTCG (SEQ ID NO: 144); CTGGAAGATGCCATGACAGG (SEQ ID NO: 145); GCACAGACTAGAGAGGTAAG (SEQ ID NO: 146); ACAGACTAGAGAGGTAAGGG (SEQ ID NO: 147); GAGAGGTGACCCGAATCCAC (SEQ ID NO: 148); GCACAGGCCCCAGAAGGAGA (SEQ ID NO: 149); CCGGAGAGGACCCAGACACG (SEQ ID NO: 150); GAGAGGACCCAGACACGGGG (SEQ ID NO: 151); GCAACACAGCAGAGAGCAAG (SEQ ID NO: 152); GAAGAGGGAGTGGAGGAAGA (SEQ ID NO: 153); AAGACGGAACCTGAAGGAGG (SEQ ID NO: 154); AGAAAGCGGCACAGGCCCAG (SEQ ID NO: 155); GGGAAACAGTGGGCCAGAGG (SEQ ID NO: 156); GTCCGGACTCAGGAGAGAGA (SEQ ID NO: 157); GGCACAGCAAGGGCACTCGG (SEQ ID NO: 158); GAAGAGGGGAAGTCGAGGGA (SEQ ID NO: 159); GGGAATGGTAAGGAGGCCTG (SEQ ID NO: 160); GCAGAGTGGTCAGCACAGAG (SEQ ID NO: 161); GCACAGAGTGGCTAAGCCCA (SEQ ID NO: 162); GACGGGGTGTCAGCATAGGG (SEQ ID NO: 163); GCCCAGGGCCAGGAACGACG (SEQ ID NO: 164); GGTGGAGTCCAGCACGGCGC (SEQ ID NO: 165); ACAGGCCGCCAGGAACTCGG (SEQ ID NO: 166); ACTAGGAAGTGTGTAGCACC (SEQ ID NO: 167); ATGAATAGCAGACTGCCCCG (SEQ ID NO: 168); ACACCCCTAAAAGCACAGTG (SEQ ID NO: 169); CAAGGAGTTCCAGCAGGTGG (SEQ ID NO: 170); AAGGAGTTCCAGCAGGTGGG (SEQ ID NO: 171); TGGAAAGAGGAGGGAAGAGG (SEQ ID NO: 172); TCGAATTCCTAACTGCCCCG (SEQ ID NO: 173); GACCTGCCCAGCACACCCTG (SEQ ID NO: 174); 23 DB1/ 159236086.1 SAL-049PC126933-5049 GGAGCAGCTGCGGCAGTGGG (SEQ ID NO: 175); GGGAGGGAGAGCTTGGCAGG (SEQ ID NO: 176); GTTACGTGGCCAAGAAGCAG (SEQ ID NO: 177); GCTGAACAGAGAAGAGCTGG (SEQ ID NO: 178); TCTGAGGGTGGAGGGACTGG (SEQ ID NO: 179); GGAGAGGTGAGGGACTTGGG (SEQ ID NO: 180); GTGAACCAGGCAGACAACGA (SEQ ID NO: 181); CAGGTACCTCCTGAGCCACG (SEQ ID NO: 182); GGGGGAGTAGGGGCATGCAG (SEQ ID NO: 183); GCAAATGGCCAGCAAGGGTG (SEQ ID NO: 184); CAAATGGCCAGCAAGGGTGG (SEQ ID NO: 309); GCAGAACCTGAGGATATGGA (SEQ ID NO: 310); AATACACAGAATGAAAATAG (SEQ ID NO: 311); CTGGTGACTAGAATAGGCAG (SEQ ID NO: 312); TGGTGACTAGAATAGGCAGT (SEQ ID NO: 313); TAAAAGAATGTGAAAAGATG (SEQ ID NO: 314); TCAGGAGTTCAAGACCACCC (SEQ ID NO: 315); TGTAGTCCCAGTTATGCAGG (SEQ ID NO: 316); GGGTTCACACCACAAATGCA (SEQ ID NO: 317); GGCAAATGGCCAGCAAGGGT (SEQ ID NO: 318); AGAAACCAATCCCAAAGCAA (SEQ ID NO: 319); GCCAAGGACACCAAAACCCA (SEQ ID NO: 320); AGTGGTGATAAGGCAACAGT (SEQ ID NO: 321); CCTGAGACAGAAGTATTAAG (SEQ ID NO: 322); AAGGTCACACAATGAATAGG (SEQ ID NO: 323); CACCATACTAGGGAAGAAGA (SEQ ID NO: 324); CAATACCCTGCCCTTAGTGG (SEQ ID NO: 327); AATACCCTGCCCTTAGTGGG (SEQ ID NO: 325); TTAGTGGGGGGTGGAGTGGG (SEQ ID NO: 326); GTGGGGGGTGGAGTGGGGGG (SEQ ID NO: 328); GGGGGGTGGAGTGGGGGGTG (SEQ ID NO: 329); GGGGTGGAGTGGGGGGTGGG (SEQ ID NO: 330); GGGTGGAGTGGGGGGTGGGG (SEQ ID NO: 331); GGGGGTGGGGAAAGACATCG (SEQ ID NO: 332); GCAGCTGTGAATTCTGATAG (SEQ ID NO: 333); GAGATCAGAGAAACCAGATG (SEQ ID NO: 334); TCTATACTGATTGCAGCCAG (SEQ ID NO: 335); CACCGAATCGAGAAGCGACTCGACA (SEQ ID NO: 185); CACCGGTCCCTGGGCGTTGCCCTGC (SEQ ID NO: 186); CACCGCCCTGGGCGTTGCCCTGCAG (SEQ ID NO: 187); CACCGCCGTGGGAAGATAAACTAAT (SEQ ID NO: 188); CACCGTCCCCTGCAGGGCAACGCCC (SEQ ID NO: 189); CACCGGTCGAGTCGCTTCTCGATTA (SEQ ID NO: 190); CACCGCTGCTGCCTCCCGTCTTGTA (SEQ ID NO: 191); CACCGGAGTGCCGCAATACCTTTAT (SEQ ID NO: 192); CACCGACACTTTGGTGGTGCAGCAA (SEQ ID NO: 193); CACCGTCTCAAATGGTATAAAACTC (SEQ ID NO: 194); CACCGAATCCCGCCCATAATCGAGA (SEQ ID NO: 195); CACCGTCCCGCCCATAATCGAGAAG (SEQ ID NO: 196); CACCGCCCATAATCGAGAAGCGACT (SEQ ID NO: 197); CACCGGAGAAGCGACTCGACATGGA (SEQ ID NO: 198); CACCGGAAGCGACTCGACATGGAGG (SEQ ID NO: 199); CACCGGCGACTCGACATGGAGGCGA (SEQ ID NO: 200); AAACTGTCGAGTCGCTTCTCGATTC (SEQ ID NO: 201); AAACGCAGGGCAACGCCCAGGGACC (SEQ ID NO: 202); AAACCTGCAGGGCAACGCCCAGGGC (SEQ ID NO: 203); AAACATTAGTTTATCTTCCCACGGC (SEQ ID NO: 204); AAACGGGCGTTGCCCTGCAGGGGAC (SEQ ID NO: 205); AAACTAATCGAGAAGCGACTCGACC (SEQ ID NO: 206); AAACTACAAGACGGGAGGCAGCAGC (SEQ ID NO: 207); AAACATAAAGGTATTGCGGCACTCC (SEQ ID NO: 208); AAACTTGCTGCACCACCAAAGTGTC (SEQ ID NO: 209); AAACGAGTTTTATACCATTTGAGAC (SEQ ID NO: 210); AAACTCTCGATTATGGGCGGGATTC (SEQ ID NO: 211); 24 DB1/ 159236086.1 SAL-049PC126933-5049 AAACCTTCTCGATTATGGGCGGGAC (SEQ ID NO: 212); AAACAGTCGCTTCTCGATTATGGGC (SEQ ID NO: 213); AAACTCCATGTCGAGTCGCTTCTCC (SEQ ID NO: 214); AAACCCTCCATGTCGAGTCGCTTCC (SEQ ID NO: 215); AAACTCGCCTCCATGTCGAGTCGCC (SEQ ID NO: 216); CACCGACAGGGTTAATGTGAAGTCC (SEQ ID NO: 217); CACCGTCCCCCTCTACATTTAAAGT (SEQ ID NO: 218); CACCGCATTTAAAGTTGGTTTAAGT (SEQ ID NO: 219); CACCGTTAGAAAATATAAAGAATAA (SEQ ID NO: 220); CACCGTAAATGCTTACTGGTTTGAA (SEQ ID NO: 221); CACCGTCCTGGGTCCAGAAAAAGAT (SEQ ID NO: 222); CACCGTTGGGTGGTGAGCATCTGTG (SEQ ID NO: 223); CACCGCGGGGAGAGTGGAGAAAAAG (SEQ ID NO: 224); CACCGGTTAAAACTCTTTAGACAAC (SEQ ID NO: 225); CACCGGAAAATCCCCACTAAGATCC (SEQ ID NO: 226); AAACGGACTTCACATTAACCCTGTC (SEQ ID NO: 227); AAACACTTTAAATGTAGAGGGGGAC (SEQ ID NO: 228); AAACACTTAAACCAACTTTAAATGC (SEQ ID NO: 229); AAACTTATTCTTTATATTTTCTAAC (SEQ ID NO: 230); AAACTTCAAACCAGTAAGCATTTAC (SEQ ID NO: 231); AAACATCTTTTTCTGGACCCAGGAC (SEQ ID NO: 232); AAACCACAGATGCTCACCACCCAAC (SEQ ID NO: 233); AAACCTTTTTCTCCACTCTCCCCGC (SEQ ID NO: 234); AAACGTTGTCTAAAGAGTTTTAACC (SEQ ID NO: 235); AAACGGATCTTAGTGGGGATTTTCC (SEQ ID NO: 236); AGTAGCAGTAATGAAGCTGG (SEQ ID NO: 237); ATACCCAGACGAGAAAGCTG (SEQ ID NO: 238); TACCCAGACGAGAAAGCTGA (SEQ ID NO: 239); GGTGGTGAGCATCTGTGTGG (SEQ ID NO: 240); AAATGAGAAGAAGAGGCACA (SEQ ID NO: 241); CTTGTGGCCTGGGAGAGCTG (SEQ ID NO: 242); GCTGTAGAAGGAGACAGAGC (SEQ ID NO: 243); GAGCTGGTTGGGAAGACATG (SEQ ID NO: 244); CTGGTTGGGAAGACATGGGG (SEQ ID NO: 245); CGTGAGGATGGGAAGGAGGG (SEQ ID NO: 246); ATGCAGAGTCAGCAGAACTG (SEQ ID NO: 247); AAGACATCAAGCACAGAAGG (SEQ ID NO: 248); TCAAGCACAGAAGGAGGAGG (SEQ ID NO: 249); AACCGTCAATAGGCAAAGGG (SEQ ID NO: 250); CCGTATTTCAGACTGAATGG (SEQ ID NO: 251); GAGAGGACAGGTGCTACAGG (SEQ ID NO: 252); AACCAAGGAAGGGCAGGAGG (SEQ ID NO: 253); GACCTCTGGGTGGAGACAGA (SEQ ID NO: 254); CAGATGACCATGACAAGCAG (SEQ ID NO: 255); AACACCAGTGAGTAGAGCGG (SEQ ID NO: 256); AGGACCTTGAAGCACAGAGA (SEQ ID NO: 257); TACAGAGGCAGACTAACCCA (SEQ ID NO: 258); ACAGAGGCAGACTAACCCAG (SEQ ID NO: 259); TAAATGACGTGCTAGACCTG (SEQ ID NO: 260); AGTAACCACTCAGGACAGGG (SEQ ID NO: 261); ACCACAAAACAGAAACACCA (SEQ ID NO: 262); GTTTGAAGACAAGCCTGAGG (SEQ ID NO: 263); GCTGAACCCCAAAAGACAGG (SEQ ID NO: 264); GCAGCTGAGACACACACCAG (SEQ ID NO: 265); AGGACACCCCAAAGAAGCTG (SEQ ID NO: 266); GGACACCCCAAAGAAGCTGA (SEQ ID NO: 267); CCAGTGCAATGGACAGAAGA (SEQ ID NO: 268); AGAAGAGGGAGCCTGCAAGT (SEQ ID NO: 269); GTGTTTGGGCCCTAGAGCGA (SEQ ID NO: 270); CATGTGCCTGGTGCAATGCA (SEQ ID NO: 271); TACAAAGAGGAAGATAAGTG (SEQ ID NO: 272); GTCACAGAATACACCACTAG (SEQ ID NO: 273); GGGTTACCCTGGACATGGAA (SEQ ID NO: 274); CATGGAAGGGTATTCACTCG (SEQ ID NO: 275); AGAGTGGCCTAGACAGGCTG (SEQ ID NO: 276); CATGCTGGACAGCTCGGCAG (SEQ ID NO: 277); 25 DB1/ 159236086.1 SAL-049PC126933-5049 AGTGAAAGAAGAGAAAATTC (SEQ ID NO: 278); TGGTAAGTCTAAGAAACCTA (SEQ ID NO: 279); CCCACAGCCTAACCACCCTA (SEQ ID NO: 280); AATATTTCAAAGCCCTAGGG (SEQ ID NO: 281); GCACTCGGAACAGGGTCTGG (SEQ ID NO: 282); AGATAGGAGCTCCAACAGTG (SEQ ID NO: 283); AAGTTAGAGCAGCCAGGAAA (SEQ ID NO: 284); TAGAGCAGCCAGGAAAGGGA (SEQ ID NO: 285); TGAATACCCTTCCATGTCCA (SEQ ID NO: 286); CCTGCATTGCACCAGGCACA (SEQ ID NO: 287); TCTAGGGCCCAAACACACCT (SEQ ID NO: 288); TCCCTCCATCTATCAAAAGG (SEQ ID NO: 289); AGCCCTGAGACAGAAGCAGG (SEQ ID NO: 290); GCCCTGAGACAGAAGCAGGT (SEQ ID NO: 291); AGGAGATGCAGTGATACGCA (SEQ ID NO: 292); ACAATACCAAGGGTATCCGG (SEQ ID NO: 293); TGATAAAGAAAACAAAGTGA (SEQ ID NO: 294); AAAGAAAACAAAGTGAGGGA (SEQ ID NO: 295); GTGGCAAGTGGAGAAATTGA (SEQ ID NO: 296); CAAGTGGAGAAATTGAGGGA (SEQ ID NO: 297); GTGGTGATGATTGCAGCTGG (SEQ ID NO: 298); CTATGTGCCTGACACACAGG (SEQ ID NO: 299); GGGTTGGACCAGGAAAGAGG (SEQ ID NO: 300); GATGCCTGGAAAAGGAAAGA (SEQ ID NO: 301); TAGTATGCACCTGCAAGAGG (SEQ ID NO: 302); TATGCACCTGCAAGAGGCGG (SEQ ID NO: 303); AGGGGAAGAAGAGAAGCAGA (SEQ ID NO: 304); GCTGAATCAAGAGACAAGCG (SEQ ID NO: 305); AAGCAAATAAATCTCCTGGG (SEQ ID NO: 306); AGATGAGTGCTAGAGACTGG (SEQ ID NO: 307); and CTGATGGTTGAGCACAGCAG (SEQ ID NO: 308). In embodiments, the guide RNAs are: AATCGAGAAGCGACTCGACA (SEQ ID NO: 425), and tgccctgcaggggagtgagc (SEQ ID NO: 426). In embodiments, the guide RNAs are gaagcgactcgacatggagg (SEQ ID NO: 427) and cctgcaggggagtgagcagc (SEQ ID NO: 428). In embodiments, guide RNAs (gRNAs) for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation dCas, in areas of open chromatin are as shown in TABLE 1. TABLE 1. Guide RNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements in areas of open chromatin. GSHS Identifier Sequence 26 DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 19F gcctggccggcctgaccact (SEQ ID NO: 34) DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 gAAVS12 taaggaatctgcctaacagg (SEQ ID NO: 72) DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 gAAVS34 gcccagggccaggaacgacg (SEQ ID NO: 94) DB1/ 159236086.1 SAL-049PC126933-5049 hROSA26 gHROSA26-3 gcagaacctgaggatatgga (SEQ ID NO: 116) hROSA26 gHROSA26-3 aatacacagaatgaaaatag (SEQ ID NO: 117) hROSA26 gHROSA26-4 ctggtgactagaataggcag (SEQ ID NO: 118) hROSA26 gHROSA26-5 tggtgactagaataggcagt (SEQ ID NO: hROSA26 gHROSA26-6 taaaagaatgtgaaaagatg (SEQ ID NO: hROSA26 gHROSA26-7 tcaggagttcaagaccaccc (SEQ ID NO: hROSA26 gHROSA26-8 tgtagtcccagttatgcagg (SEQ ID NO: hROSA26 gHROSA26-9 gggttcacaccacaaatgca (SEQ ID NO: hROSA26 gHROSA26-10 ggcaaatggccagcaagggt (SEQ ID NO: hROSA26 gHROSA26-11 agaaaccaatcccaaagcaa (SEQ ID NO: 125) hROSA26 gHROSA26-12 gccaaggacaccaaaaccca (SEQ ID NO: 126) hROSA26 gHROSA26-13 agtggtgataaggcaacagt (SEQ ID NO: 127) hROSA26 gHROSA26-14 cctgagacagaagtattaag (SEQ ID NO: 128) hROSA26 gHROSA26-15 aaggtcacacaatgaatagg (SEQ ID NO: 129) hROSA26 gHROSA26-16 caccatactagggaagaaga (SEQ ID NO: 130) SAL-049PC126933-5049 hROSA26 gHROSA26-24 gggggtggggaaagacatcg (SEQ ID NO: 138) hROSA26 hROSA26 hROSA26 hROSA26 hROSA26 hROSA26 hROSA26 hROSA26 gHROSA26-32 tcaggagttcaagaccaccc (SEQ ID NO: 121) hROSA26 hROSA26 hROSA26 gHROSA26-35 ggcaaatggccagcaagggt (SEQ ID NO: hROSA26 gHROSA26-36 agaaaccaatcccaaagcaa (SEQ ID NO: 125) hROSA26 gHROSA26-37 gccaaggacaccaaaaccca (SEQ ID NO: 126) hROSA26 gHROSA26-38 agtggtgataaggcaacagt (SEQ ID NO: 127) hROSA26 gHROSA26-39 cctgagacagaagtattaag (SEQ ID NO: 128) hROSA26 gHROSA26-40 aaggtcacacaatgaatagg (SEQ ID NO: 129) hROSA26 gHROSA26-41 caccatactagggaagaaga (SEQ ID NO: 130) SAL-049PC126933-5049 hROSA26 gHROSA26-46 ggggggtggagtggggggtg (SEQ ID NO: 135) DB1/ 159236086.1 SAL-049PC126933-5049 hROSA26 12F gagaagcgactcgacatgga (SEQ ID NO: 155) DB1/ 159236086.1 SAL-049PC126933-5049 CCR5 gCCR5-7 gctgtagaaggagacagagc (SEQ ID NO: 200) DB1/ 159236086.1 SAL-049PC126933-5049 chr4 gchr4-2 gcagctgagacacacaccag (SEQ ID NO: 222) DB1/ 159236086.1 SAL-049PC126933-5049 chr6 gchr6-20 cctgcattgcaccaggcaca (SEQ ID NO: 244) DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, gRNAs for targeting human genomic safe harbor sites using any of the gRNA-based targeting elements, e.g., without limitation, dCas, in areas of open chromatin are shown in TABLES 2-6. TABLE 2. Guide RNA sequences targeting the genomic safe harbor site, hROSA26. HROSA26 GUIDE NO. DNA SEQUENCE GUIDE 44 AATCGAGAAGCGACTCGACA (SEQ ID NO: 270) TABLE 3. Guide RNA sequences targeting the genomic safe harbor site, AAVS1. AAVS1 GUIDE NO. DNA SEQUENCE DB1/ 159236086.1 SAL-049PC126933-5049 AAV GUIDE 20 CTGAGCACTGAAGGCCTGGCCGG (SEQ ID NO: 302) AAV GUIDE 21 TGGTTTCCACTGAGCACTGAAGG (SEQ ID NO: 303) ; 38 DB1/ 159236086.1 SAL-049PC126933-5049 Guide C4A2 ACAAGATGTGAACACGACGATGG (SEQ ID NO: 339) Guide C4A3 GTTGCACCGTTGATTCCTTCAGG (SEQ ID NO: 340) TABLE 5. Guide RNA seqences targeting chromosome 22 TTAA hotspot. [hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)]. CHR22 GUIDE NO. DNA SEQUENCE Guide C22-1 ATAACACGTGAGCCGTCCTAAGG (SEQ ID NO: 358) DB1/ 159236086.1 SAL-049PC126933-5049 Guide C22-19 CAGGTGCAAAAAGGTTGCTGTGG (SEQ ID NO: 376) Guide C22-20 GCAAGAGATGAAATTCCATATGG (SEQ ID NO: 377) TABLE 6. Guide RNA sequences targeting chromosome X (HPRT) TTAA hotspot. [hg38 chrX:134,476,304- 134,476,307 (85); chrX:134,476,337-134,476,340 (51)]. CHRX GUIDE NO. DNA SEQUENCE Guide CX-1 GTTACGTTATGACTAATCTTTGG (SEQ ID NO: 398) i X 2 TA TTAT A TAAT TTT E ID N 40 DB1/ 159236086.1 SAL-049PC126933-5049 Guide CX-16 CATCAATCCTGTAGGTGATTGGG (SEQ ID NO: 413) Guide CX-17 GTTATAAGATCAATTCTGAGTGG (SEQ ID NO: 414) , g p q q g at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, a Cas-based targeting element comprises Cas12 or a variant thereof, e.g., without limitation, Cas12a (e.g., dCas12a), or Cas12j (e.g., dCas12j), or Cas12k (e.g., dCas12k). In embodiments, the targeting element comprises a Cas12 enzyme guide RNA complex. In embodiments, comprises a nuclease-deficient dCas12 guide RNA complex, optionally dCas12j guide RNA complex or dCas12a guide RNA complex. In embodiments, the targeting element is selected from a zinc finger (ZF), transcription activator-like effector (TALE), meganuclease, and clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, any of which are, in embodiments, catalytically inactive. In embodiments, the CRISPR-associated protein is selected from Cas9, CasX, CasY, Cas12a (Cpf1), and gRNA complexes thereof. In embodiments, the CRISPR-associated protein is selected from Cas9, xCas9, Cas 6, Cas7, Cas8, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, MG1 nuclease, MG2 nuclease, MG3 nuclease, or catalytically inactive forms thereof, and gRNA complexes thereof. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site or a TTAA tetranucleotide site in a genomic safe harbor site (GSHS) of a nucleic acid molecule. The helper enzyme of the present disclosure is suitable for causing insertion of the donor DNA in a GSHS when contacted with a biological cell. In embodiments, the targeting element is suitable for directing the helper enzyme of the present disclosure to the GSHS sequence. In embodiments, the targeting element comprises transcription activator-like effector (TALE) DNA binding domain (DBD). The TALE DBD comprises one or more repeat sequences. For example, in embodiments, the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. 41 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the one or more of the TALE DBD repeat sequences comprise a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids. In embodiments, the targeting element (e.g., TALE or Cas (e.g., Cas9 or Cas12, or variants thereof) DBDs cause the the helper enzyme of the present disclosure to bind specifically to human GSHS. In embodiments, the TALEs or Cas DBDs sequester the helper to GSHS and promote transposition to nearby TA dinucleotide or a TTAA tetranucleotide sites which can be located in proximity to the repeat variable di-residues (RVD) TALE or gRNA nucleotide sequences. The GSHS regions are located in open chromatin sites that are susceptible to helper activity. Accordingly, the helper enzyme of the present disclosure does not only operate based on its ability to recognize TA or TTAA sites, but it also directs a donor DNA (having a transgene) to specific locations in proximity to a TALE or Cas DBD. The helper enzyme of the present disclosure in accordance with embodiments of the present disclosure has negligible risk of genotoxicity and exhibits superior features as compared to existing gene therapies. In embodiments, the helper enzyme of the present disclosure is mutated to be characterized by reduced or inhibited binding of off-target sequences and consequently reliant on a DBD fused thereto, such as a TALE or Cas DBD, for transposition. The described cells, compositions, and methods allow reducing vector and transgene insertions that increase a mutagenic risk. The described cells and methods make use of a gene transfer system that reduces genotoxicity compared to viral- and nuclease-mediated gene therapies. In embodiments, TALE or Cas DBDs are customizable, such as a TALE or Cas DBDs is selected for targeting a specific genomic location. In embodiments, the genomic location is in proximity to a TA dinucleotide site or a TTAA (SEQ ID NO: 440) tetranucleotide site. Embodiments of the present disclosure make use of the ability of TALE or Cas or dCas9/gRNA DBDs to target specific sites in a host genome. The DNA targeting ability of a TALE or Cas DBD or dCas9/gRNA DBD is provided by TALE repeat sequences (e.g., modular arrays) or gRNA which are linked together to recognize flanking DNA sequences. Each TALE or gRNA can recognize certain base pair(s) or residue(s). TALE nucleases (TALENs) are a known tool for genome editing and introducing targeted double-stranded breaks. TALENs comprise endonucleases, such as FokI nuclease domain, fused to a customizable DBD. This DBD is composed of highly conserved repeats from TALEs, which are proteins secreted by Xanthomonas bacteria to alter transcription of genes in host plant cells. The DBD includes a repeated highly conserved 33–34 amino acid sequence with divergent 12th and 13th amino acids. These two positions, referred to as the RVD, are highly variable and show a strong correlation with specific base pair or nucleotide recognition. This straightforward relationship between amino 42 DB1/ 159236086.1 SAL-049PC126933-5049 acid sequence and DNA recognition has allowed for the engineering of specific DBDs by selecting a combination of repeat segments containing the appropriate RVDs. Boch et al. Nat. Biotechnol.2011; 29 (2):135–6. Accordingly, TALENs can be readily designed using a “protein-DNA code” that relates modular DNA-binding TALE repeat domains to individual bases in a target-binding site. See Joung et al. Nat Rev Mol Cell Biol.2013;14(1):49-55. The following table, for example, shows such code: RVD Nucleotide RVD Nucleotide It has been demonstrated that TALENs can be used to target essentially any DNA sequence of interest in human cell. Miller et al. Nat. Biotechnol.2011;29:143–148. Guidelines for selection of potential target sites and for use of particular TALE repeat domains (harboring NH residues at the hypervariable positions) for recognition of G bases have been proposed. See Streubel et al. Nat. Biotechnol.2012;30:593–595. Accordingly, in embodiments, the TALE DBD comprises one or more repeat sequences. In embodiments, the TALE DBD comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. In embodiments, the TALE DBD repeat sequences comprise 33 or 34 amino acids. In embodiments, the one or more of the TALE DBD repeat sequences comprise an RVD at residue 12 or 13 of the 33 or 34 amino acids. The RVD can recognize certain base pair(s) or residue(s). In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(gap), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(gap), and IG. In embodiments, the GSHS is in an open chromatin location in a chromosome. In embodiments, the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor; and human Rosa26 locus. In embodiments, the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22 or X. 43 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11-1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4. In embodiments, the GSHS comprises one or more of TGGCCGGCCTGACCACTGG (SEQ ID NO: 418), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420), TCCACTGAGCACTGAAGGC (SEQ ID NO: 421), TGGTTTCCACTGAGCACTG (SEQ ID NO: 422), TGGGGAAAATGACCCAACA (SEQ ID NO: 423), TAGGACAGTGGGGAAAATG (SEQ ID NO: 424), TCCAGGGACACGGTGCTAG (SEQ ID NO: 425), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 426), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 427), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 428), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 429), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 430), TGGTTTCCGAGCTTGACCC (SEQ ID NO: 431), TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 432), TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 433), TGCAGAGTATCTGCTGGGG (SEQ ID NO: 434), CCAATCCCCTCAGT (SEQ ID NO: 435), CAGTGCTCAGTGGAA (SEQ ID NO: 436), GAAACATCCGGCGACTCA (SEQ ID NO: 437), TCGCCCCTCAAATCTTACA (SEQ ID NO: 438), TCAAATCTTACAGCTGCTC (SEQ ID NO: 439), TCTTACAGCTGCTCACTCC (SEQ ID NO: 440), TACAGCTGCTCACTCCCCT (SEQ ID NO: 441), TGCTCACTCCCCTGCAGGG (SEQ ID NO: 442), TCCCCTGCAGGGCAACGCC (SEQ ID NO: 443), TGCAGGGCAACGCCCAGGG (SEQ ID NO: 444), TCTCGATTATGGGCGGGAT (SEQ ID NO: 445), TCGCTTCTCGATTATGGGC (SEQ ID NO: 446), TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 447), TCCATGTCGAGTCGCTTCT (SEQ ID NO: 448), TCGCCTCCATGTCGAGTCG (SEQ ID NO: 449), TCGTCATCGCCTCCATGTC (SEQ ID NO: 450), TGATCTCGTCATCGCCTCC (SEQ ID NO: 451), GCTTCAGCTTCCTA (SEQ ID NO: 452), CTGTGATCATGCCA (SEQ ID NO: 453), ACAGTGGTACACACCT (SEQ ID NO: 454), CCACCCCCCACTAAG (SEQ ID NO: 455), CATTGGCCGGGCAC (SEQ ID NO: 456), GCTTGAACCCAGGAGA (SEQ ID NO: 457), ACACCCGATCCACTGGG (SEQ ID NO: 458), GCTGCATCAACCCC (SEQ ID NO: 459), GCCACAAACAGAAATA (SEQ ID NO: 460), GGTGGCTCATGCCTG (SEQ ID NO: 461), GATTTGCACAGCTCAT (SEQ ID NO: 462), AAGCTCTGAGGAGCA (SEQ ID NO: 463), CCCTAGCTGTCCC (SEQ ID NO: 464), GCCTAGCATGCTAG (SEQ ID NO: 465), ATGGGCTTCACGGAT (SEQ ID NO: 466), GAAACTATGCCTGC (SEQ ID NO: 467), GCACCATTGCTCCC (SEQ ID NO: 468), GACATGCAACTCAG (SEQ ID NO: 469), ACACCACTAGGGGT (SEQ ID NO: 470), GTCTGCTAGACAGG (SEQ ID NO: 471), GGCCTAGACAGGCTG (SEQ ID NO: 472), GAGGCATTCTTATCG (SEQ ID NO: 473), GCCTGGAAACGTTCC (SEQ ID NO: 474), GTGCTCTGACAATA (SEQ ID NO: 475), GTTTTGCAGCCTCC (SEQ ID NO: 476), ACAGCTGTGGAACGT (SEQ ID NO: 477), GGCTCTCTTCCTCCT (SEQ ID NO: 478), CTATCCCAAAACTCT (SEQ 44 DB1/ 159236086.1 SAL-049PC126933-5049 ID NO: 479), GAAAAACTATGTAT (SEQ ID NO: 480), AGGCAGGCTGGTTGA (SEQ ID NO: 481), CAATACAACCACGC (SEQ ID NO: 482), ATGACGGACTCAACT (SEQ ID NO: 483), CACAACATTTGTAA (SEQ ID NO: 484), and ATTTCCAGTGCACA (SEQ ID NO: 485). In embodiments, the TALE DBD binds to one of TGGCCGGCCTGACCACTGG (SEQ ID NO: 418), TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419), TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420), TCCACTGAGCACTGAAGGC (SEQ ID NO: 421), TGGTTTCCACTGAGCACTG (SEQ ID NO: 422), TGGGGAAAATGACCCAACA (SEQ ID NO: 423), TAGGACAGTGGGGAAAATG (SEQ ID NO: 424), TCCAGGGACACGGTGCTAG (SEQ ID NO: 425), TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 426), TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 427), TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 428), TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 429), TCCGAGCTTGACCCTTGGA (SEQ ID NO: 430), TGGTTTCCGAGCTTGACCC (SEQ ID NO: 431), TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 432), TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 433), TGCAGAGTATCTGCTGGGG (SEQ ID NO: 434), CCAATCCCCTCAGT (SEQ ID NO: 435), CAGTGCTCAGTGGAA (SEQ ID NO: 436), GAAACATCCGGCGACTCA (SEQ ID NO: 437), TCGCCCCTCAAATCTTACA (SEQ ID NO: 438), TCAAATCTTACAGCTGCTC (SEQ ID NO: 439), TCTTACAGCTGCTCACTCC (SEQ ID NO: 440), TACAGCTGCTCACTCCCCT (SEQ ID NO: 441), TGCTCACTCCCCTGCAGGG (SEQ ID NO: 442), TCCCCTGCAGGGCAACGCC (SEQ ID NO: 443), TGCAGGGCAACGCCCAGGG (SEQ ID NO: 444), TCTCGATTATGGGCGGGAT (SEQ ID NO: 445), TCGCTTCTCGATTATGGGC (SEQ ID NO: 446), TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 447), TCCATGTCGAGTCGCTTCT (SEQ ID NO: 448), TCGCCTCCATGTCGAGTCG (SEQ ID NO: 449), TCGTCATCGCCTCCATGTC (SEQ ID NO: 450), TGATCTCGTCATCGCCTCC (SEQ ID NO: 451), GCTTCAGCTTCCTA (SEQ ID NO: 452), CTGTGATCATGCCA (SEQ ID NO: 453), ACAGTGGTACACACCT (SEQ ID NO: 454), CCACCCCCCACTAAG (SEQ ID NO: 455), CATTGGCCGGGCAC (SEQ ID NO: 456), GCTTGAACCCAGGAGA (SEQ ID NO: 457), ACACCCGATCCACTGGG (SEQ ID NO: 458), GCTGCATCAACCCC (SEQ ID NO: 459), GCCACAAACAGAAATA (SEQ ID NO: 460), GGTGGCTCATGCCTG (SEQ ID NO: 461), GATTTGCACAGCTCAT (SEQ ID NO: 462), AAGCTCTGAGGAGCA (SEQ ID NO: 463), CCCTAGCTGTCCC (SEQ ID NO: 464), GCCTAGCATGCTAG (SEQ ID NO: 465), ATGGGCTTCACGGAT (SEQ ID NO: 466), GAAACTATGCCTGC (SEQ ID NO: 467), GCACCATTGCTCCC (SEQ ID NO: 468), GACATGCAACTCAG (SEQ ID NO: 469), ACACCACTAGGGGT (SEQ ID NO: 470), GTCTGCTAGACAGG (SEQ ID NO: 471), GGCCTAGACAGGCTG (SEQ ID NO: 472), GAGGCATTCTTATCG (SEQ ID NO: 473), GCCTGGAAACGTTCC (SEQ ID NO: 474), GTGCTCTGACAATA (SEQ ID NO: 475), GTTTTGCAGCCTCC (SEQ ID NO: 476), ACAGCTGTGGAACGT (SEQ ID NO: 477), GGCTCTCTTCCTCCT (SEQ ID NO: 478), CTATCCCAAAACTCT (SEQ ID NO: 479), GAAAAACTATGTAT (SEQ ID NO: 480), AGGCAGGCTGGTTGA (SEQ ID NO: 481), 45 DB1/ 159236086.1 SAL-049PC126933-5049 CAATACAACCACGC (SEQ ID NO: 482), ATGACGGACTCAACT (SEQ ID NO: 483), CACAACATTTGTAA (SEQ ID NO: 484), and ATTTCCAGTGCACA (SEQ ID NO: 485). In embodiments, the TALE DBD comprises one or more of NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG NH NH (SEQ ID NO: 486), NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH (SEQ ID NO: 487), NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD (SEQ ID NO: 488), HD HD NI HD NG NH NI NH HD NI HD NG NH NI NI NH NH HD (SEQ ID NO: 489), NH NH NG NG NG HD HD NI HD NG NH NI NH HD NI HD NG NH (SEQ ID NO: 490), NH NH NH NH NI NI NI NI NG NH NI HD HD HD NI NI HD NI (SEQ ID NO: 491), NI NH NH NI HD NI NH NG NH NH NH NH NI NI NI NI NG NH (SEQ ID NO: 492), HD HD NI NH NH NH NI HD NI HD NH NH NG NH HD NG NI NH (SEQ ID NO: 493), HD NI NH NI NH HD HD NI NH NH NI NH NG HD HD NG NH NH (SEQ ID NO: 494), HD HD NG NG HD NI NH NI NH HD HD NI NH NH NI NH NG HD (SEQ ID NO: 495), HD HD NG HD HD NG NG HD NI NH NI NH HD HD NI NH NH NI (SEQ ID NO: 496), HD HD NI NH HD HD HD HD NG HD HD NG HD HD NG NG HD NI (SEQ ID NO: 497), HD HD NH NI NH HD NG NG NH NI HD HD HD NG NG NH NH NI (SEQ ID NO: 498), NH NH NG NG NG HD HD NH NI NH HD NG NG NH NI HD HD HD (SEQ ID NO: 499), NH NH NH NH NG NH NH NG NG NG HD HD NH NI NH HD NG NG (SEQ ID NO: 500), HD NG NH HD NG NH NH NH NH NG NH NH NG NG NG HD HD NH (SEQ ID NO: 501), NH HD NI NH NI NH NG NI NG HD NG NH HD NG NH NH NH NH (SEQ ID NO: 502), HD HD NI NI NG HD HD HD HD NG HD NI NH NG (SEQ ID NO: 503), HD NI NH NG NH HD NG HD NI NH NG NH NH NI NI (SEQ ID NO: 504), NH NI NI NI HD NI NG HD HD NH NH HD NH NI HD NG HD NI (SEQ ID NO: 505), HD NH HD HD HD HD NG HD NI NI NI NG HD NG NG NI HD NI (SEQ ID NO: 506), HD NI NI NI NG HD NG NG NI HD NI NH HD NG NH HD NG HD (SEQ ID NO: 507), HD NG NG NI HD NI NH HD NG NH HD NG HD NI HD NG HD HD (SEQ ID NO: 508), 46 DB1/ 159236086.1 SAL-049PC126933-5049 NI HD NI NH HD NG NH HD NG HD NI HD NG HD HD HD HD NG (SEQ ID NO: 509), NH HD NG HD NI HD NG HD HD HD HD NG NH HD NI NH NH NH (SEQ ID NO: 510), HD HD HD HD NG NH HD NI NH NH NH HD NI NI HD NH HD HD (SEQ ID NO: 511), NH HD NI NH NH NH HD NI NI HD NH HD HD HD NI NH NH NH (SEQ ID NO: 512), HD NG HD NH NI NG NG NI NG NH NH NH HD NH NH NH NI NG (SEQ ID NO: 513), HD NH HD NG NG HD NG HD NH NI NG NG NI NG NH NH NH HD (SEQ ID NO: 514), NH NG HD NH NI NH NG HD NH HD NG NG HD NG HD NH NI NG (SEQ ID NO: 515), HD HD NI NG NH NG HD NH NI NH NG HD NH HD NG NG HD NG (SEQ ID NO: 516), HD NH HD HD NG HD HD NI NG NH NG HD NH NI NH NG HD NH (SEQ ID NO: 517), HD NH NG HD NI NG HD NH HD HD NG HD HD NI NG NH NG HD (SEQ ID NO: 518), NH NI NG HD NG HD NH NG HD NI NG HD NH HD HD NG HD HD (SEQ ID NO: 519), NH HD NG NG HD NI NH HD NG NG HD HD NG NI (SEQ ID NO: 520), HD NG NK NG NH NI NG HD NI NG NH HD HD NI (SEQ ID NO: 521), NI HD NI NN NG NN NN NG NI HD NI HD NI HD HD NG (SEQ ID NO: 522), HD HD NI HD HD HD HD HD HD NI HD NG NI NI NN (SEQ ID NO: 523), HD NI NG NG NN NN HD HD NN NN NN HD NI HD (SEQ ID NO: 524), NN HD NG NG NN NI NI HD HD HD NI NN NN NI NN NI (SEQ ID NO: 525), NI HD NI HD HD HD NN NI NG HD HD NI HD NG NN NN NN (SEQ ID NO: 526), NN HD NG NN HD NI NG HD NI NI HD HD HD HD (SEQ ID NO: 527), NN NN HD NI HD NN NI NI NI HD NI HD HD HD NG HD HD (SEQ ID NO: 528), NN NN NG NN NN HD NG HD NI NG NN HD HD NG NN (SEQ ID NO: 529), NN NI NG NG NG NN HD NI HD NI NN HD NG HD NI NG (SEQ ID NO: 530), NI NI NH HD NG HD NG NH NI NH NH NI NH HD (SEQ ID NO: 531), HD HD HD NG NI NK HD NG NH NG HD HD HD HD (SEQ ID NO: 532), NH HD HD NG NI NH HD NI NG NH HD NG NI NH (SEQ ID NO: 533), 47 DB1/ 159236086.1 SAL-049PC126933-5049 NI NG NH NH NH HD NG NG HD NI HD NH NH NI NG (SEQ ID NO: 534), NH NI NI NI HD NG NI NG NH HD HD NG NH HD (SEQ ID NO: 535), NH HD NI HD HD NI NG NG NH HD NG HD HD HD (SEQ ID NO: 536), NH NI HD NI NG NH HD NI NI HD NG HD NI NH (SEQ ID NO: 537), NI HD NI HD HD NI HD NG NI NH NH NH NH NG (SEQ ID NO: 538), NH NG HD NG NH HD NG NI NH NI HD NI NH NH (SEQ ID NO: 539), NH NH HD HD NG NI NH NI HD NI NH NH HD NG NH (SEQ ID NO: 540), NH NI NH NH HD NI NG NG HD NG NG NI NG HD NH (SEQ ID NO: 541), NN HD HD NG NN NN NI NI NI HD NN NG NG HD HD (SEQ ID NO: 542), NN NG NN HD NG HD NG NN NI HD NI NI NG NI (SEQ ID NO: 543), NN NG NG NG NG NN HD NI NN HD HD NG HD HD (SEQ ID NO: 544), NI HD NI NN HD NG NN NG NN NN NI NI HD NN NG (SEQ ID NO: 545), HD NI NI NN NI HD HD NN NI NN HD NI HD NG NN HD NG NN (SEQ ID NO: 546), HD NG NI NG HD HD HD NI NI NI NI HD NG HD NG (SEQ ID NO: 547), NH NI NI NI NI NI HD NG NI NG NH NG NI NG (SEQ ID NO: 548), NI NH NH HD NI NH NH HD NG NH NH NG NG NH NI (SEQ ID NO: 549), HD NI NI NG NI HD NI NI HD HD NI HD NN HD (SEQ ID NO: 550), NI NG NN NI HD NN NN NI HD NG HD NI NI HD NG (SEQ ID NO: 551), HD NI HD NI NI HD NI NG NG NG NN NG NI NI (SEQ ID NO: 552), and NI NG NG NG HD HD NI NN NG NN HD NI HD NI (SEQ ID NO: 553). In embodiments, the TALE DBD comprises one or more of the sequences outlined herein or a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. In embodiments, the GSHS and the TALE DBD sequences are selected from: 48 DB1/ 159236086.1 SAL-049PC126933-5049 TGGCCGGCCTGACCACTGG (SEQ ID NO: 418) and NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG NH NH (SEQ ID NO: 486); TGAAGGCCTGGCCGGCCTG (SEQ ID NO: 419) and NH NI NI NH NH HD HD NG NH NH HD HD NH NH HD HD NG NH (SEQ ID NO: 487); TGAGCACTGAAGGCCTGGC (SEQ ID NO: 420) and NH NI NH HD NI HD NG NH NI NI NH NH HD HD NG NH NH HD (SEQ ID NO: 488); TCCACTGAGCACTGAAGGC (SEQ ID NO: 421) and HD HD NI HD NG NH NI NH HD NI HD NG NH NI NI NH NH HD (SEQ ID NO: 489); TGGTTTCCACTGAGCACTG (SEQ ID NO: 422) and NH NH NG NG NG HD HD NI HD NG NH NI NH HD NI HD NG NH (SEQ ID NO: 490); TGGGGAAAATGACCCAACA (SEQ ID NO: 423) and NH NH NH NH NI NI NI NI NG NH NI HD HD HD NI NI HD NI (SEQ ID NO: 491); TAGGACAGTGGGGAAAATG (SEQ ID NO: 424) and NI NH NH NI HD NI NH NG NH NH NH NH NI NI NI NI NG NH (SEQ ID NO: 492); TCCAGGGACACGGTGCTAG (SEQ ID NO: 425) and HD HD NI NH NH NH NI HD NI HD NH NH NG NH HD NG NI NH (SEQ ID NO: 493); TCAGAGCCAGGAGTCCTGG (SEQ ID NO: 426) and HD NI NH NI NH HD HD NI NH NH NI NH NG HD HD NG NH NH (SEQ ID NO: 494); TCCTTCAGAGCCAGGAGTC (SEQ ID NO: 427) and HD HD NG NG HD NI NH NI NH HD HD NI NH NH NI NH NG HD (SEQ ID NO: 495); TCCTCCTTCAGAGCCAGGA (SEQ ID NO: 428) and HD HD NG HD HD NG NG HD NI NH NI NH HD HD NI NH NH NI (SEQ ID NO: 496); TCCAGCCCCTCCTCCTTCA (SEQ ID NO: 429) and HD HD NI NH HD HD HD HD NG HD HD NG HD HD NG NG HD NI (SEQ ID NO: 497); TCCGAGCTTGACCCTTGGA (SEQ ID NO: 430) and HD HD NH NI NH HD NG NG NH NI HD HD HD NG NG NH NH NI (SEQ ID NO: 498); TGGTTTCCGAGCTTGACCC (SEQ ID NO: 431) and NH NH NG NG NG HD HD NH NI NH HD NG NG NH NI HD HD HD (SEQ ID NO: 499); 49 DB1/ 159236086.1 SAL-049PC126933-5049 TGGGGTGGTTTCCGAGCTT (SEQ ID NO: 432) and NH NH NH NH NG NH NH NG NG NG HD HD NH NI NH HD NG NG (SEQ ID NO: 500); TCTGCTGGGGTGGTTTCCG (SEQ ID NO: 433) and HD NG NH HD NG NH NH NH NH NG NH NH NG NG NG HD HD NH (SEQ ID NO: 501); TGCAGAGTATCTGCTGGGG (SEQ ID NO: 434) and NH HD NI NH NI NH NG NI NG HD NG NH HD NG NH NH NH NH (SEQ ID NO: 502); CCAATCCCCTCAGT (SEQ ID NO: 435) and HD HD NI NI NG HD HD HD HD NG HD NI NH NG (SEQ ID NO: 503); CAGTGCTCAGTGGAA (SEQ ID NO: 436) and HD NI NH NG NH HD NG HD NI NH NG NH NH NI NI (SEQ ID NO: 504); GAAACATCCGGCGACTCA (SEQ ID NO: 437) and NH NI NI NI HD NI NG HD HD NH NH HD NH NI HD NG HD NI (SEQ ID NO: 505); TCGCCCCTCAAATCTTACA (SEQ ID NO: 438) and HD NH HD HD HD HD NG HD NI NI NI NG HD NG NG NI HD NI (SEQ ID NO: 506); TCAAATCTTACAGCTGCTC (SEQ ID NO: 439) and HD NI NI NI NG HD NG NG NI HD NI NH HD NG NH HD NG HD (SEQ ID NO: 507); TCTTACAGCTGCTCACTCC (SEQ ID NO: 440) and HD NG NG NI HD NI NH HD NG NH HD NG HD NI HD NG HD HD (SEQ ID NO: 508); TACAGCTGCTCACTCCCCT (SEQ ID NO: 441) and NI HD NI NH HD NG NH HD NG HD NI HD NG HD HD HD HD NG (SEQ ID NO: 509); TGCTCACTCCCCTGCAGGG (SEQ ID NO: 442) and NH HD NG HD NI HD NG HD HD HD HD NG NH HD NI NH NH NH (SEQ ID NO: 510); TCCCCTGCAGGGCAACGCC (SEQ ID NO: 443) and HD HD HD HD NG NH HD NI NH NH NH HD NI NI HD NH HD HD (SEQ ID NO: 511); TGCAGGGCAACGCCCAGGG (SEQ ID NO: 444) and NH HD NI NH NH NH HD NI NI HD NH HD HD HD NI NH NH NH (SEQ ID NO: 512); TCTCGATTATGGGCGGGAT (SEQ ID NO: 445) and HD NG HD NH NI NG NG NI NG NH NH NH HD NH NH NH NI NG (SEQ ID NO: 513); TCGCTTCTCGATTATGGGC (SEQ ID NO: 446) and HD NH HD NG NG HD NG HD NH NI NG NG NI NG NH NH NH HD (SEQ ID NO: 514); 50 DB1/ 159236086.1 SAL-049PC126933-5049 TGTCGAGTCGCTTCTCGAT (SEQ ID NO: 447) and NH NG HD NH NI NH NG HD NH HD NG NG HD NG HD NH NI NG (SEQ ID NO: 515); TCCATGTCGAGTCGCTTCT (SEQ ID NO: 448) and HD HD NI NG NH NG HD NH NI NH NG HD NH HD NG NG HD NG (SEQ ID NO: 516); TCGCCTCCATGTCGAGTCG (SEQ ID NO: 449) and HD NH HD HD NG HD HD NI NG NH NG HD NH NI NH NG HD NH (SEQ ID NO: 517); TCGTCATCGCCTCCATGTC (SEQ ID NO: 450) and HD NH NG HD NI NG HD NH HD HD NG HD HD NI NG NH NG HD (SEQ ID NO: 518); TGATCTCGTCATCGCCTCC (SEQ ID NO: 451) and NH NI NG HD NG HD NH NG HD NI NG HD NH HD HD NG HD HD (SEQ ID NO: 519); GCTTCAGCTTCCTA (SEQ ID NO: 452) and NH HD NG NG HD NI NH HD NG NG HD HD NG NI (SEQ ID NO: 520); CTGTGATCATGCCA (SEQ ID NO: 453) and HD NG NK NG NH NI NG HD NI NG NH HD HD NI (SEQ ID NO: 521); ACAGTGGTACACACCT (SEQ ID NO: 454) and NI HD NI NN NG NN NN NG NI HD NI HD NI HD HD NG (SEQ ID NO: 522); CCACCCCCCACTAAG (SEQ ID NO: 455) and HD HD NI HD HD HD HD HD HD NI HD NG NI NI NN (SEQ ID NO: 523); CATTGGCCGGGCAC (SEQ ID NO: 456) and HD NI NG NG NN NN HD HD NN NN NN HD NI HD (SEQ ID NO: 524); GCTTGAACCCAGGAGA (SEQ ID NO: 457) and NN HD NG NG NN NI NI HD HD HD NI NN NN NI NN NI (SEQ ID NO: 525); ACACCCGATCCACTGGG (SEQ ID NO: 458) and NI HD NI HD HD HD NN NI NG HD HD NI HD NG NN NN NN (SEQ ID NO: 526); GCTGCATCAACCCC (SEQ ID NO: 459) and NN HD NG NN HD NI NG HD NI NI HD HD HD HD (SEQ ID NO: 527); GCCACAAACAGAAATA (SEQ ID NO: 460) and NN NN HD NI HD NN NI NI NI HD NI HD HD HD NG HD HD (SEQ ID NO: 528); GGTGGCTCATGCCTG (SEQ ID NO: 461) and NN NN NG NN NN HD NG HD NI NG NN HD HD NG NN (SEQ ID NO: 529); GATTTGCACAGCTCAT (SEQ ID NO: 462) and NN NI NG NG NG NN HD NI HD NI NN HD NG HD NI NG (SEQ ID NO: 530); 51 DB1/ 159236086.1 SAL-049PC126933-5049 AAGCTCTGAGGAGCA (SEQ ID NO: 463) and NI NI NH HD NG HD NG NH NI NH NH NI NH HD (SEQ ID NO: 531); CCCTAGCTGTCCC (SEQ ID NO: 464) and HD HD HD NG NI NK HD NG NH NG HD HD HD HD (SEQ ID NO: 532); GCCTAGCATGCTAG (SEQ ID NO: 465) and NH HD HD NG NI NH HD NI NG NH HD NG NI NH (SEQ ID NO: 533); ATGGGCTTCACGGAT (SEQ ID NO: 466) and NI NG NH NH NH HD NG NG HD NI HD NH NH NI NG (SEQ ID NO: 534); GAAACTATGCCTGC (SEQ ID NO: 467) and NH NI NI NI HD NG NI NG NH HD HD NG NH HD (SEQ ID NO: 535); GCACCATTGCTCCC (SEQ ID NO: 468) and NH HD NI HD HD NI NG NG NH HD NG HD HD HD (SEQ ID NO: 536); GACATGCAACTCAG (SEQ ID NO: 469) and NH NI HD NI NG NH HD NI NI HD NG HD NI NH (SEQ ID NO: 537); ACACCACTAGGGGT (SEQ ID NO: 470) and NI HD NI HD HD NI HD NG NI NH NH NH NH NG (SEQ ID NO: 538); GTCTGCTAGACAGG (SEQ ID NO: 471) and NH NG HD NG NH HD NG NI NH NI HD NI NH NH (SEQ ID NO: 539); GGCCTAGACAGGCTG (SEQ ID NO: 472) and NH NH HD HD NG NI NH NI HD NI NH NH HD NG NH (SEQ ID NO: 540); GAGGCATTCTTATCG (SEQ ID NO: 473) and NH NI NH NH HD NI NG NG HD NG NG NI NG HD NH (SEQ ID NO: 541); GCCTGGAAACGTTCC (SEQ ID NO: 474) and NN HD HD NG NN NN NI NI NI HD NN NG NG HD HD (SEQ ID NO: 542); GTGCTCTGACAATA (SEQ ID NO: 475) and NN NG NN HD NG HD NG NN NI HD NI NI NG NI (SEQ ID NO: 543); GTTTTGCAGCCTCC (SEQ ID NO: 476) and NN NG NG NG NG NN HD NI NN HD HD NG HD HD (SEQ ID NO: 544); ACAGCTGTGGAACGT (SEQ ID NO: 477) and NI HD NI NN HD NG NN NG NN NN NI NI HD NN NG (SEQ ID NO: 545); GGCTCTCTTCCTCCT (SEQ ID NO: 478) and HD NI NI NN NI HD HD NN NI NN HD NI HD NG NN HD NG NN (SEQ ID NO: 546); CTATCCCAAAACTCT (SEQ ID NO: 479) and HD NG NI NG HD HD HD NI NI NI NI HD NG HD NG (SEQ ID NO: 547); GAAAAACTATGTAT (SEQ ID NO: 480) and NH NI NI NI NI NI HD NG NI NG NH NG NI NG (SEQ ID NO: 548); AGGCAGGCTGGTTGA (SEQ ID NO: 481) and NI NH NH HD NI NH NH HD NG NH NH NG NG NH NI (SEQ ID NO: 549); 52 DB1/ 159236086.1 SAL-049PC126933-5049 CAATACAACCACGC (SEQ ID NO: 482) and HD NI NI NG NI HD NI NI HD HD NI HD NN HD (SEQ ID NO: 550); ATGACGGACTCAACT (SEQ ID NO: 483) and NI NG NN NI HD NN NN NI HD NG HD NI NI HD NG (SEQ ID NO: 551); and CACAACATTTGTAA (SEQ ID NO: 484) and HD NI HD NI NI HD NI NG NG NG NN NG NI NI (SEQ ID NO: 552). In embodiments, the GSHS is within about 25, or about 50, or about 100, or about 150, or about 200, or about 300, or about 500 nucleotides of the TA dinucleotide site or TTAA (SEQ ID NO: 2) tetranucleotide site. Illustrative DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES, encompassed by various embodiments are provided in TABLE 7. TABLE 7. DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES. GSHS ID Sequence TALE (DNA binding code) D D H G I D H H H I H G D G 53 DB1/ 159236086.1 SAL-049PC126933-5049 AAVS1 15 tggggtggtttccgagctt (SEQ ID NH NH NH NH NG NH NH NG NG NG HD NO: 432) HD NH NI NH HD NG NG (SEQ ID NO: 500) H : H I H D G D G D G D D hROSA26 8R tctcgattatgggcgggat (SEQ ID HD NG HD NH NI NG NG NI NG NH NH NH NO: 445) HD NH NH NH NI NG (SEQ ID NO: 513) hROSA26 9R tcgcttctcgattatgggc (SEQ ID HD NH HD NG NG HD NG HD NH NI NG NO: 446) NG NI NG NH NH NH HD (SEQ ID NO: 514) hROSA26 10R tgtcgagtcgcttctcgat (SEQ ID NH NG HD NH NI NH NG HD NH HD NG NG NO: 447) HD NG HD NH NI NG (SEQ ID NO: 515) tccatgtcgagtcgcttct (SEQ ID HD HD NI NG NH NG HD NH NI NH NG HD hROSA26 11R NO: 448) NH HD NG NG HD NG (SEQ ID NO: 516) hROSA26 12R tcgcctccatgtcgagtcg (SEQ ID HD NH HD HD NG HD HD NI NG NH NG HD NO: 449) NH NI NH NG HD NH (SEQ ID NO: 517) hROSA26 13R tcgtcatcgcctccatgtc (SEQ ID HD NH NG HD NI NG HD NH HD HD NG HD NO: 450) HD NI NG NH NG HD (SEQ ID NO: 518) hROSA26 14R tgatctcgtcatcgcctcc (SEQ ID NH NI NG HD NG HD NH NG HD NI NG HD NO: 451) NH HD HD NG HD HD (SEQ ID NO: 519) hROSA26 ROSA1 GCTTCAGCTTCCTA (SEQ NH HD NG NG HD NI NH HD NG NG HD HD ID NO: 452) NG NI (SEQ ID NO: 520) 54 DB1/ 159236086.1 SAL-049PC126933-5049 hROSA26 ROSA2 CTGTGATCATGCCA (SEQ HD NG NK NG NH NI NG HD NI NG NH HD ID NO: 453) HD NI (SEQ ID NO: 521) I G D N I D D D D I D G H G D I H I H I G DB1/ 159236086.1 SAL-049PC126933-5049 Chr 10 SHCHR10-2 GTGCTCTGACAATA (SEQ NN NG NN HD NG HD NG NN NI HD NI NI ID NO: 475) NG NI (SEQ ID NO: 543) D I D G I G N I G I Further illustrative DNA binding codes for targeting human genomic safe harbor in areas of open chromatin via TALES, encompassed by embodiments are provided in TABLES 8-12. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TA dinucleotide site. In embodiments, the helper enzyme of the present disclosure is capable of inserting a donor DNA at a TTAA (SEQ ID NO: 440) tetranucleotide site. TABLE 8. TALE sequences targeting the genomic safe harbor site, hROSA26. NAME DNA SEQUENCE RVD AMINO ACID CODE TCGCCCCTCAAATCTTACAG HD NH HD HD HD HD NG HD NI NI NI NG HD NG NG NI HD NI NH (SEQ ID Q 56 DB1/ 159236086.1 SAL-049PC126933-5049 TGCAGGGCAACGCCCAGGGA NH HD NI NH NH NH HD NI NI HD NH HD HD HD NI NH NH NH NI (SEQ ID R7 (SEQ ID NO: 560) NO: 574) TCTCGATTATGGGCGGGATT HD NG HD NH NI NG NG NI NG NH NH NH HD NH NH NH NI NG NG (SEQ Q Q Q Q 9. sequencesargeng e genomc sae aror se, S. NAME DNA SEQUENCE RVD AMINO ACID CODE TGGCCGGCCTGACCACTGGG (SEQ ID NH NH HD HD NH NH HD HD NG NH NI HD HD NI HD NG G H H DB1/ 159236086.1 SAL-049PC126933-5049 TGGGGTGGTTTCCGAGCTTG (SEQ ID NH NH NH NH NG NH NH NG NG NG HD HD NH NI NH HD AAV15c NO: 596) NG NG NH (SEQ ID NO: 613) TCTGCTGGGGTGGTTTCCGA (SEQ ID HD NG NH HD NG NH NH NH NH NG NH NH NG NG NG HD HD NH NI (SEQ ID NO: 614) TGCAGAGTATCTGCTGGGGT (SEQ ID NH HD NI NH NI NH NG NI NG HD NG NH HD NG NH NH NH AAV17c NO: 598) NH NG (SEQ ID NO: 615) T ABLE 10. TALE sequences targeting the chromosome 4 hotspot. [hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)]. NAME DNA SEQUENCE RVD AMINO ACID CODE TALE4-R001 TCTTCCTAGTATTAAAGT (SEQ ID NO: HD NG NG HD HD NG NI NH NG NI NG NG NI NI NI I I I D H 58 DB1/ 159236086.1 SAL-049PC126933-5049 TALE4-F018 TAGCATGATGTTTCATGTTGTGACCT NI NH HD NI NG NH NI NG NH NG NG NG HD NI NG (SEQ ID NO: 633) NH NG NG NH NG NH NI HD HD NG (SEQ ID NO: 653) T ABLE 11. TALE sequences targeting the chromosome 22 hotspot. [hg38 chr22:35,373,912-35,373,916 (861); chr22:35,377,843-35,377,847 (1153)]. NAME DNA SEQUENCE RVD AMINO ACID CODE TALE22F- R 1 TCTTCCTAGTCTCTTCTCTACCCAGT (SEQ ID HD NG NG HD HD NG NI NH NG HD NG HD NG Q O: : ) I 59 DB1/ 159236086.1 SAL-049PC126933-5049 TALE22- TGCTATGTAAGGTAGCAAAAAGGTAACCT (SEQ NH HD NG NI NG NH NG NI NI NH NH NG NI NH F006A ID NO: 671) HD NI NI NI NI NI NH NH NG NI NI HD HD NG (SEQ ID NO: 691) H G NAME DNA SEQUENCE RVD AMINO ACID CODE TALE F002 TTTAGCAGATGCATCAGC (SEQ ID NO: NG NG NI NH HD NI NH NI NG NH HD NI NG HD NI NH HD H G D D G D 60 DB1/ 159236086.1 SAL-049PC126933-5049 TALE F036* TAGAGTCAAAGCATGTACT (SEQ ID NO: NI NH NI NH NG HD NI NI NI NH HD NI NG NH NG NI HD NG 712) (SEQ ID NO: 736) TALE F037* TCCTACCCATAAGCTCCT (SEQ ID NO: HD HD NG NI HD HD HD NI NG NI NI NH HD NG HD HD NG D Q n em o mens, e znc nger comprses one o e sequences seece rom S 3- , or varans ereo comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence. In embodiments, the zinc finger targets one or more sites selected from TABLES 13-17. TABLE 13. Zinc finger sequences targeting the genomic safe harbor site, hROSA26. hROSA NAME TARGET SCO ZFP AMINO ACID CODE C T D C T D C T ) C T ) R C T ) 61 DB1/ 159236086.1 SAL-049PC126933-5049 5’ ZnF5 e GGC AAC GCC 57.3 LEPGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSQR CAG GGA CCA 2 AHLERHQRTHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCP (SEQ ID NO: 749) ECGKSFSDCRDLARHQRTHTGEKPYKCPECGKSFSDSGNLRVHQRTH S P H P H E E T Q C T C T ) R C T ) R C T ) TABLE 14. Zinc finger sequences targeting the genomic safe harbor site, AAVS1. AAVS1 NAME TARGET SCO ZFP AMINO ACID CODE Y S G E R R S Y S TE E C 62 DB1/ 159236086.1 SAL-049PC126933-5049 3’ ZnF1 2b GCA GAT AGC CAG GAG (SEQ ID 59.9 LEPGEKPYKCPECGKSFSRSDNLVRHQRTHTGEKPY NO: 772) 7 KCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKS FSERSHLREHQRTHTGEKPYKCPECGKSFSTSGNLV K F E E Y S A G E K Y S T G Q Y S TE E C G T Y S G E 2) Y S V G E C R Y S V G E S 63 DB1/ 159236086.1 SAL-049PC126933-5049 3’ ZnF2 0b ATA GCC CTG GGC CCA CGG 59.5 LEPGEKPYKCPECGKSFSSRRTCRAHQRTHTGEKPY CTT CGT (SEQ ID NO: 780) 3 KCPECGKSFSTTGALTEHQRTHTGEKPYKCPECGKS FSRSDKLTEHQRTHTGEKPYKCPECGKSFSTSHSLTE E C S Y S G Y S T G E R R Y S E E K F E E C G T K : TABLE 15. Zinc finger sequences targeting the chromosome 4 hotspot. [hg38 chr4:30,793,533-30,793,537 (9677); chr4:30,875,472-30,875,476 (8948)]. Chr4 NAME TARGET SCO ZFP AMINO ACID CODE TTAA RE Y S A G Y S G 64 DB1/ 159236086.1 SAL-049PC126933-5049 EKPYKCPECGKSFSTTGALTEHQRTHTGKKTS (SEQ ID NO: 810) 5’ ZnF3 ATACTAGGAAGAAATACAATA 57.2 LEPGEKPYKCPECGKSFSQKSSLIAHQRTHTGEKPYK F E C Y S V G Q Y S V G E T R Y O: K F E G Y S E E C G K F R E C Y S R G E S R 65 DB1/ 159236086.1 SAL-049PC126933-5049 TABLE 16. Zinc finger sequences targeting the chromosome 22 hotspot. Chr NAME TARGET SCO ZFP 22 RE TTA A Q K Q F S H K Q F T R C R S T C R ST K R T ST K Q S Y Q K Q F Y H O: T C R Q S K Q F P DB1/ 159236086.1 SAL-049PC126933-5049 YKCPECGKSFSQRANLRAHQRTHTGEKPYKCPECGKSFSQAGHLAS HQRTHTGEKPYKCPECGKSFSRNDALTEHQRTHTGEKPYKCPECGK SFSQRANLRAHQRTHTGEKPYKCPECGKSFSHRTTLTNHQRTHTGEK E K EK V S Y Q : S Y R K Q S T C RT QS Q K Q : SR C RT R K Q FS ST C R R S Y H O: 67 DB1/ 159236086.1 SAL-049PC126933-5049 3’ ZnF15 R GTCAAGCAACAGGATG 59.2 LEPGEKPYKCPECGKSFSHKNALQNHQRTHTGEKPYKCPECGKSFST ATCCAAATGCTATT 2 SGELVRHQRTHTGEKPYKCPECGKSFSTTGNLTVHQRTHTGEKPYKC (SEQ ID NO: 834) PECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSGNLVRHQRT R K Q TABLE 17. Zinc finger sequences targeting the chromosome X (HPRT) hotspot. [hg38 chrX:134,476,304- 134,476,307 (85); chrX:134,476,337-134,476,340 (51)]. ChrX NAME TARGET SCO ZFP AMINO ACID CODE TTAA RE 5’ ZnF4 GTAGAAACTCGCCTTATG (SEQ 54.0 LEPGEKPYKCPECGKSFSRRDELNVHQRTHTGEKPY S R E K F E E C Y S R E C Y S R G Y S T G Y S A K G L T Y S DB1/ 159236086.1 SAL-049PC126933-5049 FSQRANLRAHQRTHTGEKPYKCPECGKSFSQSGNLT EHQRTHTGEKPYKCPECGKSFSTSGHLVRHQRTHTG EKPYKCPECGKSFSQRANLRAHQRTHTGEKPYKCPE S R Y S V G E T In embodiments, the present disclosure relates to a system having nucleic acids encoding the enzyme (e.g., without limitation, the helper enzyme) and the donor DNA, respectively. Linkers In embodiments, the targeting element comprises a nucleic acid binding component of a gene-editing system. In embodiments, the helper enzyme the targeting element are connected. Without wishing to be bound by a particular theory, the targeting element may refer to a nucleic acid binding component of the gene-editing system. In embodiments, the helper enzyme and the targeting element are connected. For example, in embodiments, the the helper enzyme and the targeting element are fused to one another or linked via a linker to one another. In embodiments, the linker is a flexible linker. In embodiments, the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1 to 12. In embodiments, the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. In embodiments, the flexible linker is about 50, or about 100, or about 150, or about 200 amino acid residues in length. In embodiments, the flexible linker comprises at least about 150 nucleotides (nt), or at least about 200 nt, or at least about 250 nt, or at least about 300 nt, or at least about 350 nt, or at least about 400 nt, or at least about 450 nt, or at least about 500 nt, or at least about 500 nt, or at least about 600 nt. In embodiments, the flexible linker comprises from about 450 nt to about 500 nt. Inteins Inteins (INTervening protEINS) are mobile genetic elements that are protein domains, found in nature, with the capability to carry out the process of protein splicing. See Sarmiento & Camarero (2019) Curr. Protein Pept. Sci., 20(5), 408–424, which is incorporated by reference herein in its entirety. Protein spicing is a post-translation biochemical modification which results in the cleavage and formation of peptide bonds between precursor polypeptide segments flanking the intein. Id. Inteins apply standard enzymatic strategies to excise themselves post-translationally from a 69 DB1/ 159236086.1 SAL-049PC126933-5049 precursor protein via protein splicing. Nanda A, Nasker SS, Mehra A, Panda S, Nayak S. Inteins in Science: Evolution to Application. Microorganisms. 2020; 8(12):2004. An intein can splice its flanking N- and C-terminal domains to become a mature protein and excise itself from a sequence. For example, split inteins have been used to control the delivery of heterologous genes into transgenic organisms. See Wood & Camarero (2014) J. Biol. Chem. 289(21):14512-14519. This approach relies on splitting the target protein into two segments, which are then post- translationally reconstituted in vivo by protein trans-splicing (PTS). See Aboye & Camarero (2012) J. Biol. Chem.287, 27026–27032. More recently, an intein-mediated split-Cas9 system has been developed to incorporate Cas9 into cells and reconstitute nuclease activity efficiently. Truong et al., Nucleic Acids Res.2015, 43(13), 6450–6458. The protein splicing excises the internal region of the precursor protein, which is then followed by the ligation of the N-extein and C-extein fragments, resulting in two polypeptides--the excised intein and the new polypeptide produced by joining the C- and N-exteins. Sarmiento & Camarero (2019) Curr. Protein Pept. Sci., 20(5), 408–424. In embodiments, intein-mediated incorporation of DNA binders such as, without limitation, dCas9, dCas12j, TALEs, or ZnF, allows creation of a split-enzyme system such as, without limitation, split helper system, that permits reconstitution of the full-length enzyme, e.g., helper, from two smaller fragments. This allows avoiding the need to express DNA binders at the N- or C-terminus of an enzyme, e.g., helper. In this approach, the two portions of an enzyme, e.g., helper, are fused to the intein and, after co-expression, the intein allows producing a full-length enzyme, e.g., helper, by post- translation modification. Thus, in embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme. In embodiments, an intein is a suitable ligand-dependent intein, for example, an intein selected from those described in U.S. Patent No.9,200,045; Mootz et al., J. Am. Chem. Soc.2002; 124, 9044-9045; Mootz et al., J. Am. Chem. Soc. 2003; 125, 10561-10569; Buskirk et al., PNAS 2004; 101, 10505-10510; Skretas & Wood. Protein Sci.2005; 14, 523- 532; Schwartz, et al., Nat. Chem. Biol.2007; 3, 50-54; Peck et al., Chem. Biol.2011; 18(5), 619-630; the entire contents of each of which are hereby incorporated by reference herein. In embodiments the intein is NpuN (Intein-N) (SEQ ID NO: 423) and/or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof, e.g., a sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. SEQ ID NO: 868: nucleotide sequence of NpuN (Intein-N) GGCGGATCTGGCGGTAGTGCTGAGTATTGTCTGAGTTACGAAACGGAAATACTCACGGTTGAGTATGGGCTTCTTCC AATTGGCAAAATCGTTGAAAAGCGCATAGAGTGTACGGTGTATTCCGTCGATAACAACGGTAATATCTACACCCAGC CGGTAGCTCAGTGGCACGACCGAGGCGAACAGGAAGTGTTCGAGTATTGCTTGGAAGATGGCTCCCTTATCCGCGCC ACTAAAGACCATAAGTTTATGACGGTTGACGGGCAGATGCTGCCTATAGACGAAATATTTGAGAGAGAGCTGGACTT GATGAGAGTCGATAATCTGCCAAAT 70 DB1/ 159236086.1 SAL-049PC126933-5049 SEQ ID NO: 869: nucleotide sequence of NpuC (Intein-C) GGCGGATCTGGCGGTAGTGGGGGTTCCGGATCCATAAAGATAGCTACTAGGAAATATCTTGGCAAACAAAACGTCTA TGACATAGGAGTTGAGCGAGATCACAATTTTGCTTTGAAGAATGGGTTCATCGCGTCTAATTGCTTCAACGCTAGCG GCGGGTCAGGAGGCTCTGGTGGAAGC Dimerization Enhancers In embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises a dimerization enhancer. In embodiments, the nucleic acid encodes the enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and sec-ond portions are fused into a functional enzyme upon post-translational excision of the dimerization enhancer from the enzyme. In embodiments, the dimerization enhancer is suitable for linking the helper enzyme and the targeting element. In embodiments, the dimerization enhancer is selected from: a protein comprising a SRC Homology 3 Domain (or SH3 domain), biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof. Nucleic Acids of the Disclosure In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 19, or a functional variant thereof and the right end comprises SEQ ID NO: 20, or a functional variant thereof. In embodiments, there is provided a donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 19, or a functional variant thereof and the right end comprises SEQ ID NO: 20, or a functional variant thereof, wherein the heterologous polynucleotide is transposable by a helper enzyme having the sequence of SEQ ID NO: 3, or a functional variant thereof. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a helper enzyme which is at least 90% identical to SEQ ID NO: 4, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, there is provided a polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 3, or a functional variant thereof, operably linked to a heterologous promoter. In embodiments, a nucleic acid encoding the enzyme (e.g., without limitation, the helper enzyme) is RNA. In embodiments, a nucleic acid encoding the transgene is DNA. In embodiments, the enzyme (e.g., without limitation, the helper enzyme) is encoded by a recombinant or synthetic nucleic acid. In embodiments, the nucleic acid is RNA, optionally a helper RNA. In embodiments, the nucleic acid is RNA that has a 5’-m7G cap (cap0, or cap1, or cap2), optionally with pseudouridine substitution (e.g., without limitation 71 DB1/ 159236086.1 SAL-049PC126933-5049 n-methyl-pseudouridine), and optionally a poly-A tail of about 30, or about 50, or about 100, of about 150 nucleotides in length. In embodiments, the poly-A tail is of about 30 nucleotides in length, optionally 34 nucleotides in length. In embodiments, a nuclear localization signal is placed before the enzyme start codon at the N-terminus, optionally at the C-terminus. In embodiments, the nucleic acid that is RNA has a 5’-m7G cap (cap 0, or cap 1, or cap 2). In embodiments, the nucleic acid comprises a 5’ cap structure, a 5’-UTR comprising a Kozak consensus sequence, a 5’-UTR comprising a sequence that increases RNA stability in vivo, a 3’-UTR comprising a sequence that increases RNA stability in vivo, and/or a 3’ poly(A) tail. In embodiments, the enzyme (e.g., without limitation, a helper) is incorporated into a vector or a vector-like particle. In embodiments, the vector is a non-viral vector. In embodiments, a nucleic acid encoding the enzyme in accordance with embodiments of the present disclosure, is DNA. In various embodiments, a construct comprising a donor is any suitable genetic construct, such as a nucleic acid construct, a plasmid, or a vector. In various embodiments, the construct is DNA, which is referred to herein as a donor DNA. In embodiments, sequences of a nucleic acid encoding the donor is codon optimized to provide improved mRNA stability and protein expression in mammalian systems. In embodiments, the enzyme and the donor are included in different vectors. In embodiments, the enzyme and the donor are included in the same vector. In various embodiments, a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) is RNA (e.g., helper RNA), and a nucleic acid encoding a donor is DNA. As would be appreciated in the art, a donor often includes an open reading frame that encodes a transgene at the middle of donor and terminal repeat sequences at the 5’ and 3’ end of the donor. The translated helper (e.g., without limitation, the helper enzyme) binds to the 5’ and 3’ sequence of the donor and carries out the transposition function. In embodiments, a donor is used interchangeably with transposable elements, which are used to refer to polynucleotides capable of inserting copies of themselves into other polynucleotides. The term donor is well known to those skilled in the art and includes classes of donors that can be distinguished on the basis of sequence organization, for example inverted terminal sequences at each end, and/or directly repeated long terminal repeats (LTRs) at the ends. In embodiments, the donor as described herein may be described as a piggyBac like element, e.g., a donor element that is characterized by its traceless excision, which recognizes TTAA (SEQ ID NO: 440) sequence and restores the sequence at the insert site back to the original TTAA (SEQ ID NO: 440) sequence after removal of the donor. 72 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, the donor is flanked by one or more end sequences or terminal ends. In embodiments, the donor is or comprises a gene encoding a complete polypeptide. In embodiments, the donor is or comprises a gene which is defective or substantially absent in a disease state. In embodiments, a transgene is associated with various regulatory elements that are selected to ensure stable expression of a construct with the transgene. Thus, in embodiments, a transgene is encoded by a non-viral vector (e.g., without limitation, a DNA plasmid) that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. The insulators flank the donor (transgene cassette) to reduce transcriptional silencing and position effects imparted by chromosomal sequences. As an additional effect, the insulators can eliminate functional interactions of the transgene enhancer and promoter sequences with neighboring chromosomal sequences. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5’-HS4 chicken β-globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facioscapulohumeral muscular dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety. In embodiments, the transgene is inserted into a GSHS location in a host genome. GSHSs is defined as loci well-suited for gene transfer, as integrations within these sites are not associated with adverse effects such as proto-oncogene activation, tumor suppressor inactivation, or insertional mutagenesis. GSHSs can defined by the following criteria: (1) distance of at least 50 kb from the 5’ end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any microRNA (miRNA), (4) location outside a transcription unit, and (5) location outside ultra-conserved regions (UCRs) of the human genome. See Papapetrou et al. Nat. Biotechnol.2011;29:73-8; Bejerano et al. Science 2004;304:1321-5. Furthermore, the use of GSHS locations can allow stable transgene expression across multiple cell types. One such site, chemokine C-C motif receptor 5 (CCR5) has been identified and used for integrative gene transfer. CCR5 is a member of the beta chemokine receptor family and is required for the entry of R5 tropic viral strains involved in primary infections. A homozygous 32 bp deletion in the CCR5 gene confers resistance to HIV-1 virus infections in humans. Disrupted CCR5 expression, naturally occurring in about 1% of the Caucasian population, does not appear to result in any reduction in immunity. Lobritz et al., Viruses 2010;2:1069-105. A clinical trial has demonstrated safety and efficacy of disrupting CCR5 via targetable nucleases. Tebas et al., HIV. N Engl J Med 2014;370:901-10. In embodiments, the donor is under control of a tissue-specific promoter. The tissue-specific promoter is, e.g., without limitation, a liver-specific promoter. In embodiments, the liver-specific promoter is an LP1 promoter that, in embodiments, is a human LP1 promoter. The LP1 promoter is described, e.g., in Nathwani et al. Blood vol. 2006;107(7):2653-61, and it is constructed, without limitation, as described in Nathawani et al. 73 DB1/ 159236086.1 SAL-049PC126933-5049 It should be appreciated however that a variety of promoters can be used, including other tissue-specific promoters, inducible promoters, constitutive promoters, etc. In embodiments, the present nucleic acids include polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs or derivatives thereof. In embodiments, there is provided double- and single- stranded DNA, as well as double- and single-stranded RNA, and RNA-DNA hybrids. In embodiments, transcriptionally- activated polynucleotides such as methylated or capped polynucleotides are provided. In embodiments, the present compositions are mRNA or DNA. In embodiments, the present non-viral vectors are linear or circular DNA molecules that comprise a polynucleotide encoding a polypeptide and is operably linked to control sequences, wherein the control sequences provide for expression of the polynucleotide encoding the polypeptide. In embodiments, the non-viral vector comprises a promoter sequence, and transcriptional and translational stop signal sequences. Such vectors may include, among others, chromosomal and episomal vectors, e.g., vectors bacterial plasmids, from donors, from yeast episomes, from insertion elements, from yeast chromosomal elements, and vectors from combinations thereof. The present constructs may contain control regions that regulate as well as engender expression. In embodiments, the construct comprising the enzyme and/or transgene is codon optimized. Transgene codon optimization is used to optimize therapeutic potential of the transgene and its expression in the host organism. Codon optimization is performed to match the codon usage in the transgene with the abundance of transfer RNA (tRNA) for each codon in a host organism or cell. Codon optimization methods are known in the art and described in, for example, WO 2007/142954, which is incorporated by reference herein in its entirety. Optimization strategies can include, for example, the modification of translation initiation regions, alteration of mRNA structural elements, and the use of different codon biases. In embodiments, the construct comprising the enzyme and/or transgene includes several other regulatory elements that are selected to ensure stable expression of the construct. Thus, in embodiments, the non-viral vector is a DNA plasmid that can comprise one or more insulator sequences that prevent or mitigate activation or inactivation of nearby genes. In embodiments, the one or more insulator sequences comprise an HS4 insulator (1.2-kb 5′-HS4 chicken β- globin (cHS4) insulator element) and an D4Z4 insulator (tandem macrosatellite repeats linked to Facioscapulohumeral muscular dystrophy (FSHD). In embodiments, the sequences of the HS4 insulator and the D4Z4 insulator are as described in Rival-Gervier et al. Mol Ther.2013 Aug; 21(8):1536-50, which is incorporated herein by reference in its entirety. In embodiments, the gene of the construct comprising the enzyme and/or transgene is capable of transposition in the presence of a helper. In embodiments, the non-viral vector in accordance with embodiments of the present disclosure comprises a nucleic acid construct encoding a helper. The helper (e.g., without limitation, the helper enzyme of the present disclosure) is an RNA helper plasmid. In embodiments, the non-viral vector further comprises a nucleic 74 DB1/ 159236086.1 SAL-049PC126933-5049 acid construct encoding a DNA helper plasmid. In embodiments, the helper is an in vitro-transcribed mRNA helper. The helper (e.g., without limitation, the helper enzyme of the present disclosure) is capable of excising and/or transposing the gene from the construct comprising the enzyme and/or transgene to site- or locus-specific genomic regions. In embodiments, the enzyme (e.g., without limitation, the helper enzyme) and the donor are included in the same vector. In embodiments, the enzyme is disposed on the same (cis) or different vector (trans) than a donor with a transgene. Accordingly, in embodiments, the enzyme and the donor encompassing a transgene are in cis configuration such that they are included in the same vector. In embodiments, the enzyme and the donor encompassing a transgene are in trans configuration such that they are included in different vectors. The vector is any non-viral vector in accordance with the present disclosure. In aspects, a nucleic acid encoding the donor system of the present disclosure capable of targeted genomic integration by transposition (e.g., a helper) in accordance with embodiments of the present disclosure is provided. The nucleic acid is or comprises DNA or RNA. In embodiments, the nucleic acid encoding the enzyme is DNA. In embodiments, the nucleic acid encoding the enzyme capable of targeted genomic integration by transposition (e.g., a helper of the present disclosure) is RNA such as, e.g., helper RNA. In embodiments, the helper is incorporated into a vector. In embodiments, the vector is a non-viral vector. In embodiments, a nucleic acid encoding the transgene in accordance with embodiments of the present disclosure is provided. The nucleic acid is or comprises DNA or RNA. In embodiments, the nucleic acid encoding the transgene is DNA. In embodiments, the nucleic acid encoding the transgene is RNA such as, e.g., helper RNA. In embodiments, the transgene is incorporated into a vector. In embodiments, the vector is a non-viral vector. In embodiments, the present enzyme can be in the form or an RNA or DNA and have one or two N-terminus nuclear localization signal (NLS) to shuttle the protein more efficiently into the nucleus. For example, in embodiments, the present enzyme further comprises one, two, three, four, five, or more NLSs. Examples of NLS are provided in Kosugi et al. (J. Biol. Chem. (2009) 284:478-485; incorporated by reference herein). In a particular embodiment, the NLS comprises the consensus sequence K(K/R)X(K/R) (SEQ ID NO: 348). In an embodiment, the NLS comprises the consensus sequence (K/R)(K/R)X10-12(K/R)3/5 (SEQ ID NO: 349), where (K/R)3/5 represents at least three of the five amino acids is either lysine or arginine. In an embodiment, the NLS comprises the c-myc NLS. In a particular embodiment, the c-myc NLS comprises the sequence PAAKRVKLD (SEQ ID NO: 70). In a particular embodiment, the NLS is the nucleoplasmin NLS. In embodiments, the nucleoplasmin NLS comprises the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 72). In embodiments, the NLS comprises the SV40 Large T-antigen NLS. In embodiments, the SV40 Large T-antigen NLS comprises the sequence PKKKRKV (SEQ ID NO: 73). In a particular embodiment, the NLS comprises three SV40 Large T-antigen NLSs (e.g., DPKKKRKVDPKKKRKVDPKKKRKV (SEQ 75 DB1/ 159236086.1 SAL-049PC126933-5049 ID NO: 74). In embodiments, the NLS may comprise mutations/variations in the above sequences such that they contain 1 or more substitutions, additions, or deletions (e.g., about 1, or about 2, or about 3, or about 4, or about 5, or about 10 substitutions, additions, or deletions). In aspects, a host cell comprising the nucleic acid in accordance with embodiments of the present disclosure is provided. Lipids and LNP Delivery In embodiments, a composition or a nucleic acid in accordance with embodiments of the present disclosure is provided wherein the composition is in the form of a lipid nanoparticle (LNP). In embodiments, the composition is encapsulated in an LNP. In embodiments, a nucleic acid encoding the enzyme and a nucleic acid encoding the transgene are contained within the same lipid nanoparticle (LNP). In embodiments, the nucleic acid encoding the enzyme and the nucleic acid encoding the donor are a mixture incorporated into or associated with the same LNP. In embodiments, the polynucleotide encoding the helper enzyme and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation. In embodiments, the LNP is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2- dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol–2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol- 3phosphocholine (DSPC) and/or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc). In embodiments, an LNP is as described, e.g., in Patel et al., J Control Release 2019; 303:91-100. The LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc). In embodiments, a nanoparticle is a particle having a diameter of less than about 1000 nm. In embodiments, nanoparticles of the present disclosure have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In embodiments, nanoparticles of the present disclosure have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In embodiments, the nanoparticles of the present disclosure have a greatest dimension (e.g., a diameter) of about 100 nm. In aspects, the cell in accordance with the present disclosure is prepared via an in vivo genetic modification method. In embodiments, a genetic modification in accordance with the present disclosure is performed via an ex vivo method. 76 DB1/ 159236086.1 SAL-049PC126933-5049 In aspects, the cell in accordance with the present disclosure is prepared by contacting a cell with an enzyme capable of targeted genomic integration by transposition (e.g., without limitation, the helper enzyme) in vivo. In embodiments, the cell is contacted with the enzyme ex vivo. In embodiments, the present method provides high specific targeting as compared to a method that does not use the helper enzyme with a target selector. Therapeutic Applications In embodiments, the transgene of interest in accordance with embodiments of the present disclosure can encode various genes. In embodiments, the helper enzyme and the donor are included in the same pharmaceutical composition. In embodiments, the helper enzyme and the donor are included in different pharmaceutical compositions. In embodiments, the helper enzyme and the donor are co-transfected. In embodiments the helper enzyme and the donor are transfected separately. In embodiments, a transfected cell for gene therapy is provided, wherein the transfected cell is generated using the helper enzyme in accordance with embodiments of the present disclosure. In embodiments, a method of delivering a cell therapy is provided, comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure. In embodiments, a method of treating a disease or condition using a cell therapy, comprising administering to a patient in need thereof the transfected cell generated using the helper enzyme in accordance with embodiments of the present disclosure. In embodiments, the disease or condition may comprise cancer. In embodiments, the cancer is or comprises an adrenal cancer, a biliary track cancer, a bladder cancer, a bone/bone marrow cancer, a brain cancer, a breast cancer, a cervical cancer, a colorectal cancer, a cancer of the esophagus, a gastric cancer, a head/neck cancer, a hepatobiliary cancer, a kidney cancer, a liver cancer, a lung cancer, an ovarian cancer, a pancreatic cancer, a pelvis cancer, a pleura cancer, a prostate cancer, a renal cancer, a skin cancer, a stomach cancer, a testis cancer, a thymus cancer, a thyroid cancer, a uterine cancer, a lymphoma, a melanoma, a multiple myeloma, or a leukemia. In embodiments, the cancer is selected from one or more of the basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer; glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung 77 DB1/ 159236086.1 SAL-049PC126933-5049 cancer; melanoma; myeloma; neuroblastoma; oral cavity cancer; ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; Hodgkin's lymphoma; non-Hodgkin's lymphoma; B-cell lymphoma; small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); and Hairy cell leukemia. In embodiments, the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphoma including Hodgkin's and non-Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs syndrome. In embodiments, the disease or condition is or comprises an infectious disease. In embodiments, the infectious disease is a coronavirus infection, optionally selected from infection with SAR-CoV, MERS-CoV, and SARS-CoV-2, or variants thereof. In embodiments, the infectious disease is or comprises a disease comprising a viral infection, a parasitic infection, or a bacterial infection. In embodiments, the viral infection is caused by a virus of family Flaviviridae, a virus of family 78 DB1/ 159236086.1 SAL-049PC126933-5049 Picornaviridae, a virus of family Orthomyxoviridae, a virus of family Coronaviridae, a virus of family Retroviridae, a virus of family Paramyxoviridae, a virus of family Bunyaviridae, or a virus of family Reoviridae. In embodiments, the virus of family Coronaviridae comprises a betacoronavirus or an alphacoronavirus, optionally wherein the betacoronavirus is selected from SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-HKU1, and HCoV-OC43, or the alphacoronavirus is selected from a HCoV-NL63 and HCoV-229E. In embodiments, the infectious disease comprises a coronavirus infection 2019 (COVID-19). In embodiments, the method requires a single administration. In embodiments, the method requires a plurality of administrations. Isolated Cell In aspects of the present disclosure, an isolated cell is provided that comprises the transfected cell in accordance with embodiments of the present disclosure, e.g., transfected with a helper and/or donor. In aspects, the present disclosure provides an ex vivo gene therapy approach. Accordingly, in embodiments, the method that is used to treat an inherited or acquired disease in a patient in need thereof comprises (a) contacting a cell obtained from a patient (autologous) or another individual (allogeneic) with a transfected cell in accordance with embodiments of the present disclosure; and (b) administering the cell to a patient in need thereof. One of the advantages of ex vivo gene therapy is the ability to “sample” the transduced cells before patient administration. This facilitates efficacy and allows performing safety checks before introducing the cell(s) to the patient. For example, the transduction efficiency and/or the clonality of integration can be assessed before infusion of the product. The present disclosure provides transfected cells and methods that can be effectively used for ex vivo gene modification. In embodiments, a composition comprising transfected cells in accordance with the present disclosure comprises a pharmaceutically acceptable carrier, excipient, or diluent. Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, N.Y.). For example, pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile, and the fluid should be easy to draw up by a syringe. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, 79 DB1/ 159236086.1 SAL-049PC126933-5049 for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Therapeutic compounds can be prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as collagen, ethylene vinyl acetate, polyanhydrides (e.g., poly[1,3-bis(carboxyphenoxy)propane-co-sebacic-acid] (PCPP-SA) matrix, fatty acid dimer- sebacic acid (FAD-SA) copolymer, poly(lactide-co-glycolide)), polyglycolic acid, collagen, polyorthoesters, polyethyleneglycol-coated liposomes, and polylactic acid. Such formulations can be prepared using standard techniques, or obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. Semisolid, gelling, soft-gel, or other formulations (including controlled release) can be used, e.g., when administration to a surgical site is desired. Methods of making such formulations are known in the art and can include the use of biodegradable, biocompatible polymers. See, e.g., Sawyer et al., Yale J Biol Med.2006; 79(3-4): 141-152. In embodiments, there is provided a method of transforming a cell using the construct comprising the ends and/or transgene described herein in the presence of a helper (e.g., without limitation, the helper enzyme) to produce a stably transfected cell which results from the stable integration of a gene of interest into the cell. In embodiments, the stable integration comprises an introduction of a polynucleotide into a chromosome or mini-chromosome of the cell and, therefore, becomes a relatively permanent part of the cellular genome. 80 DB1/ 159236086.1 SAL-049PC126933-5049 In embodiments, there is provided a transgenic organism that may comprise cells which have been transformed by the methods of the present disclosure. In embodiments, the organism may be a mammal or an insect. When the organism is a mammal, the organism may include, but is not limited to, a mouse, a rat, a chimpanzee, an elephant, a dog, a rabbit, and the like. When the organism is an insect, the organism may include, but is not limited to, a fruit fly, an ant, a mosquito, a bollworm, and the like. Definitions The following definitions are used in connection with the disclosure disclosed herein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of skill in the art to which this invention belongs. As used herein, “a,” “an,” or “the” can mean one or more than one. Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55. An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest. The term “in vivo” refers to an event that takes place in a subject’s body. The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and/or organ which has been removed from a subject’s body. Aptly, the cell, tissue and/or organ may be returned to the subject’s body in a method of treatment or surgery. As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and/or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids. “Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid, or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner. The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and 81 DB1/ 159236086.1 SAL-049PC126933-5049 animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods. As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of” or “consisting essentially of.” As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology. The amount of compositions described herein needed for achieving a therapeutic effect may be determined empirically in accordance with conventional procedures for the particular purpose. Generally, for administering therapeutic agents for therapeutic purposes, the therapeutic agents are given at a pharmacologically effective dose. A “pharmacologically effective amount,” “pharmacologically effective dose,” “therapeutically effective amount,” or “effective amount” refers to an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., slow the progression of a symptom of the disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease, and reverse a symptom of a disorder or disease. Therapeutic benefit also 82 DB1/ 159236086.1 SAL-049PC126933-5049 includes halting or slowing the progression of the underlying disease or disorder, regardless of whether improvement is realized. Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to about 50% of the population) and the ED50 (the dose therapeutically effective in about 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. In embodiments, compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays, including, for example, cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture, or in an appropriate animal model. Levels of the described compositions in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of a medicaments for treating the diseases or disorders described herein. SELECTED SEQUENCES In embodiments, the present disclosure provides for any of the sequence provided herein, including the below, and a variant sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto, or at least about 10 mutations, or at least about 9 mutations, or at least about 8 mutations, or at least about 7 mutations, or at least about 6 mutations, or at least about 5 mutations, or at least about 4 mutations, or at least about 3 mutations, or at least about 2 mutations, or at least about 1 mutation. SEQ ID NO: 1: amino acid sequence of a helper from Myotis myotis (634 amino acids) 1 MVKFSKDIES SDDEFYFENE DKSEKCNNDE IEFSEDASGD EQIAGPSGTT ERKTSLALPK 61 NLAESTDSDI EFIKAKRSRT IVYFESDVDI GDIIEKSGIR PSESYVSRGE NRKKKSWTST 121 SVNDKEPSRI PFSTGQLHVG PQVPSGCATP IDFFQLFFTE TLIKNITDET NEYARHKISQ 181 KELSQRSTNN WKDVTIEEMK AFLGVILNMG VLNHPNLQSY WSMDFESHIP FFRSVFKRER 241 FLQIFWMLHL KNDQKSSKDL RTRTEKVNCF LSYLEMKFRE RFCPGREIAV DEAVVGFKGK 301 IHFITYNPKK PTKWGIRLYV LSDSKCGYVH SFVPYYGGIT SETLVRPDLP FTSRIVLELH 361 ERLKNSVPGS QGYHFFTDRY YTSVTLAKEL FKEKTHLTGT IMPNRKDNPP VIKHQKLKKG 421 EIVAFRDENV MLLAWKDKRI VTMLSTWDPS ETESVERRVP GGGKEIVLKP KVVTNYTKFM 481 GGVDIADHYT STYCFMRKTL KWWRTLFFWG LEVSVVNSYI LYKECQKRKN EKPITHVKFI 541 RKLVHDLVGE FRDGTLTSRG RLLSTNLEQR LDGKLHIITP HPNKKHKDCV VCSNRKIKGG 601 RRETIYICET CECKPGLHVG ECFKKYHTMK NYRD 83 DB1/ 159236086.1 SAL-049PC126933-5049 SEQ ID NO: 2: nucleotide sequence encoding the helper from Myotis myotis (1905 nt) 1 ATGGTGAAAT TTTCTAAGGA CATTGAGTCT AGCGACGATG AGTTTTACTT CGAGAATGAG 61 GACAAGTCTG AGAAGTGCAA CAACGACGAG ATTGAGTTCA GCGAAGACGC CTCCGGGGAT 121 GAGCAGATCG CCGGACCTAG CGGAACCACC GAAAGGAAGA CCAGCCTGGC CCTCCCTAAA 181 AATCTGGCTG AGTCCACCGA TTCCGACATC GAATTCATCA AGGCCAAGCG CTCTCGGACA 241 ATCGTGTATT TCGAATCCGA CGTGGATATC GGCGACATCA TCGAGAAGTC CGGCATTAGG 301 CCTTCTGAAA GCTATGTGTC TCGAGGTGAG AACAGAAAGA AGAAGTCCTG GACAAGCACC 361 AGCGTGAACG ACAAGGAACC CTCTAGGATC CCTTTCAGCA CCGGCCAGCT GCACGTGGGC 421 CCACAGGTGC CTAGCGGCTG CGCCACCCCT ATTGATTTCT TTCAGCTGTT CTTTACCGAG 481 ACCCTGATTA AGAATATCAC CGACGAGACA AACGAGTACG CCCGCCACAA GATCAGCCAG 541 AAGGAGCTGA GCCAGAGAAG CACTAACAAC TGGAAGGACG TGACCATCGA GGAAATGAAG 601 GCCTTCCTCG GGGTGATCCT GAACATGGGC GTGCTGAATC ACCCAAATCT GCAGAGCTAC 661 TGGAGTATGG ATTTCGAGTC TCACATTCCC TTTTTTAGAT CAGTGTTTAA GCGGGAGAGA 721 TTTCTGCAGA TCTTCTGGAT GCTGCACCTG AAGAATGATC AGAAGAGCAG CAAAGACCTG 781 CGCACTCGGA CCGAGAAAGT CAACTGCTTT CTGTCCTACC TCGAGATGAA ATTCAGGGAG 841 CGGTTTTGCC CTGGCAGGGA GATCGCTGTG GATGAAGCCG TGGTGGGCTT CAAGGGTAAG 901 ATCCACTTCA TTACATACAA CCCCAAGAAG CCCACCAAGT GGGGCATTCG CCTCTACGTG 961 CTGTCAGACT CCAAATGCGG CTACGTGCAC TCTTTCGTGC CTTACTATGG AGGGATCACC 1021 TCCGAAACTC TGGTGAGACC CGACCTGCCC TTTACTTCTA GAATTGTGCT GGAGCTGCAC 1081 GAGCGGCTGA AGAACAGCGT GCCAGGGTCC CAGGGCTATC ACTTTTTCAC CGACCGGTAC 1141 TATACCTCCG TGACCCTGGC CAAGGAGCTG TTTAAGGAGA AGACACATCT TACGGGCACC 1201 ATCATGCCAA ATAGGAAAGA CAATCCTCCC GTGATCAAGC ACCAGAAGCT GAAAAAGGGG 1261 GAGATTGTGG CTTTTCGGGA CGAAAATGTG ATGCTGCTGG CTTGGAAGGA TAAGAGAATC 1321 GTGACTATGC TGTCCACCTG GGACCCCTCT GAAACCGAGA GTGTGGAGCG CAGAGTGCCA 1381 GGCGGAGGCA AGGAGATTGT GCTGAAGCCA AAAGTGGTGA CAAACTACAC TAAGTTTATG 1441 GGCGGCGTGG ACATCGCCGA CCACTACACC AGCACCTACT GTTTCATGCG CAAAACCCTG 1501 AAGTGGTGGC GTACCCTCTT TTTCTGGGGC CTGGAGGTGA GTGTGGTGAA CTCTTACATC 1561 CTGTACAAGG AGTGCCAGAA GCGGAAGAAC GAGAAGCCCA TCACCCACGT GAAGTTCATC 1621 CGCAAGCTGG TGCACGACCT GGTTGGCGAG TTCAGAGATG GAACCCTGAC ATCCAGAGGG 1681 CGCCTGCTGA GCACCAATCT GGAGCAGAGG CTGGATGGCA AACTCCACAT TATCACTCCC 1741 CACCCTAACA AGAAGCACAA GGATTGTGTG GTGTGTAGCA ACAGGAAGAT CAAGGGAGGG 1801 CGGCGCGAGA CCATCTACAT CTGTGAGACC TGCGAATGCA AGCCCGGCCT GCACGTGGGC 1861 GAATGTTTCA AGAAATACCA TACTATGAAG AATTACAGGG ATTGA SEQ ID NO: 3: Myotis myotis Left ITR (200 bp) 1 ccccctttgc actcggatgt cgagtgtgac tcgacgcggt tagcatcggt tgcagctcgt 61 atgttgagcc atactcgacc tgtagtttca ccgagggggg aagggggatt tttgtctatt 121 tttccagtat ttttcttgtt ttcattagca tgaaaggaca agtaaaatgt aaatgccgtc 181 tcaactgatg ccaccaccta SEQ ID NO: 4: Myotis myotis Right ITR (200 bp) 1 tgaaaaatta tagagattaa aattactctt tgaatgtatc aataatttga aatataaaaa 61 aatccaaata aataagtttg tatgaaaaga aactccagtt ttttattcta ctgccgcact 121 ttgtaaaatc tggggtattt aaaaaattaa atcccgagta gaataaagga atcgagaaaa 181 aagcaagcga gtgcaaaggg SEQ ID NO: 5: nucleotide sequence of dead Cas9 DNA binding protein (5004 bp) 1 ATGGACAAGA AGTACTCCAT TGGGCTCGCT ATCGGCACAA ACAGCGTCGG CTGGGCCGTC 61 ATTACGGACG AGTACAAGGT GCCGAGCAAA AAATTCAAAG TTCTGGGCAA TACCGATCGC 121 CACAGCATAA AGAAGAACCT CATTGGCGCC CTCCTGTTCG ACTCCGGGGA GACGGCCGAA 181 GCCACGCGGC TCAAAAGAAC AGCACGGCGC AGATATACCC GCAGAAAGAA TCGGATCTGC 84 DB1/ 159236086.1 SAL-049PC126933-5049 241 TACCTGCAGG AGATCTTTAG TAATGAGATG GCTAAGGTGG ATGACTCTTT CTTCCATAGG 301 CTGGAGGAGT CCTTTTTGGT GGAGGAGGAT AAAAAGCACG AGCGCCACCC AATCTTTGGC 361 AATATCGTGG ACGAGGTGGC GTACCATGAA AAGTACCCAA CCATATATCA TCTGAGGAAG 421 AAGCTTGTAG ACAGTACTGA TAAGGCTGAC TTGCGGTTGA TCTATCTCGC GCTGGCGCAT 481 ATGATCAAAT TTCGGGGACA CTTCCTCATC GAGGGGGACC TGAACCCAGA CAACAGCGAT 541 GTCGACAAAC TCTTTATCCA ACTGGTTCAG ACTTACAATC AGCTTTTCGA AGAGAACCCG 601 ATCAACGCAT CCGGAGTTGA CGCCAAAGCA ATCCTGAGCG CTAGGCTGTC CAAATCCCGG 661 CGGCTCGAAA ACCTCATCGC ACAGCTCCCT GGGGAGAAGA AGAACGGCCT GTTTGGTAAT 721 CTTATCGCCC TGTCACTCGG GCTGACCCCC AACTTTAAAT CTAACTTCGA CCTGGCCGAA 781 GATGCCAAGC TTCAACTGAG CAAAGACACC TACGATGATG ATCTCGACAA TCTGCTGGCC 841 CAGATCGGCG ACCAGTACGC AGACCTTTTT TTGGCGGCAA AGAACCTGTC AGACGCCATT 901 CTGCTGAGTG ATATTCTGCG AGTGAACACG GAGATCACCA AAGCTCCGCT GAGCGCTAGT 961 ATGATCAAGC GCTATGATGA GCACCACCAA GACTTGACTT TGCTGAAGGC CCTTGTCAGA 1021 CAGCAACTGC CTGAGAAGTA CAAGGAAATT TTCTTCGATC AGTCTAAAAA TGGCTACGCC 1081 GGATACATTG ACGGCGGAGC AAGCCAGGAG GAATTTTACA AATTTATTAA GCCCATCTTG 1141 GAAAAAATGG ACGGCACCGA GGAGCTGCTG GTAAAGCTTA ACAGAGAAGA TCTGTTGCGC 1201 AAACAGCGCA CTTTCGACAA TGGAAGCATC CCCCACCAGA TTCACCTGGG CGAACTGCAC 1261 GCTATCCTCA GGCGGCAAGA GGATTTCTAC CCCTTTTTGA AAGATAACAG GGAAAAGATT 1321 GAGAAAATCC TCACATTTCG GATACCCTAC TATGTAGGCC CCCTCGCCCG GGGAAATTCC 1381 AGATTCGCGT GGATGACTCG CAAATCAGAA GAGACCATCA CTCCCTGGAA CTTCGAGGAA 1441 GTCGTGGATA AGGGGGCCTC TGCCCAGTCC TTCATCGAAA GGATGACTAA CTTTGATAAA 1501 AATCTGCCTA ACGAAAAGGT GCTTCCTAAA CACTCTCTGC TGTACGAGTA CTTCACAGTT 1561 TATAACGAGC TCACCAAGGT CAAATACGTC ACAGAAGGGA TGAGAAAGCC AGCATTCCTG 1621 TCTGGAGAGC AGAAGAAAGC TATCGTGGAC CTCCTCTTCA AGACGAACCG GAAAGTTACC 1681 GTGAAACAGC TCAAAGAAGA CTATTTCAAA AAGATTGAAT GTTTCGACTC TGTTGAAATC 1741 AGCGGAGTGG AGGATCGCTT CAACGCATCC CTGGGAACGT ATCACGATCT CCTGAAAATC 1801 ATTAAAGACA AGGACTTCCT GGACAATGAG GAGAACGAGG ACATTCTTGA GGACATTGTC 1861 CTCACCCTTA CGTTGTTTGA AGATAGGGAG ATGATTGAAG AACGCTTGAA AACTTACGCT 1921 CATCTCTTCG ACGACAAAGT CATGAAACAG CTCAAGAGGC GCCGATATAC AGGATGGGGG 1981 CGGCTGTCAA GAAAACTGAT CAATGGGATC CGAGACAAGC AGAGTGGAAA GACAATCCTG 2041 GATTTTCTTA AGTCCGATGG ATTTGCCAAC CGGAACTTCA TGCAGTTGAT CCATGATGAC 2101 TCTCTCACCT TTAAGGAGGA CATCCAGAAA GCACAAGTTT CTGGCCAGGG GGACAGTCTT 2161 CACGAGCACA TCGCTAATCT TGCAGGTAGC CCAGCTATCA AAAAGGGAAT ACTGCAGACC 2221 GTTAAGGTCG TGGATGAACT CGTCAAAGTA ATGGGAAGGC ATAAGCCCGA GAATATCGTT 2281 ATCGAGATGG CCCGAGAGAA CCAAACTACC CAGAAGGGAC AGAAGAACAG TAGGGAAAGG 2341 ATGAAGAGGA TTGAAGAGGG TATAAAAGAA CTGGGGTCCC AAATCCTTAA GGAACACCCA 2401 GTTGAAAACA CCCAGCTTCA GAATGAGAAG CTCTACCTGT ACTACCTGCA GAACGGCAGG 2461 GACATGTACG TGGATCAGGA ACTGGACATC AATCGGCTCT CCGACTACGA CGTGGCTGCT 2521 ATCGTGCCCC AGTCTTTTCT CAAAGATGAT TCTATTGATA ATAAAGTGTT GACAAGATCC 2581 GATAAAGCTA GAGGGAAGAG TGATAACGTC CCCTCAGAAG AAGTTGTCAA GAAAATGAAA 2641 AATTATTGGC GGCAGCTGCT GAACGCCAAA CTGATCACAC AACGGAAGTT CGATAATCTG 2701 ACTAAGGCTG AACGAGGTGG CCTGTCTGAG TTGGATAAAG CCGGCTTCAT CAAAAGGCAG 2761 CTTGTTGAGA CACGCCAGAT CACCAAGCAC GTGGCCCAAA TTCTCGATTC ACGCATGAAC 2821 ACCAAGTACG ATGAAAATGA CAAACTGATT CGAGAGGTGA AAGTTATTAC TCTGAAGTCT 2881 AAGCTGGTCT CAGATTTCAG AAAGGACTTT CAGTTTTATA AGGTGAGAGA GATCAACAAT 2941 TACCACCATG CGCATGATGC CTACCTGAAT GCAGTGGTAG GCACTGCACT TATCAAAAAA 3001 TATCCCAAGC TTGAATCTGA ATTTGTTTAC GGAGACTATA AAGTGTACGA TGTTAGGAAA 3061 ATGATCGCAA AGTCTGAGCA GGAAATAGGC AAGGCCACCG CTAAGTACTT CTTTTACAGC 3121 AATATTATGA ATTTTTTCAA GACCGAGATT ACACTGGCCA ATGGAGAGAT TCGGAAGCGA 3181 CCACTTATCG AAACAAACGG AGAAACAGGA GAAATCGTGT GGGACAAGGG TAGGGATTTC 3241 GCGACAGTCC GGAAGGTCCT GTCCATGCCG CAGGTGAACA TCGTTAAAAA GACCGAAGTA 3301 CAGACCGGAG GCTTCTCCAA GGAAAGTATC CTCCCGAAAA GGAACAGCGA CAAGCTGATC 3361 GCACGCAAAA AAGATTGGGA CCCCAAGAAA TACGGCGGAT TCGATTCTCC TACAGTCGCT 3421 TACAGTGTAC TGGTTGTGGC CAAAGTGGAG AAAGGGAAGT CTAAAAAACT CAAAAGCGTC 3481 AAGGAACTGC TGGGCATCAC AATCATGGAG CGATCAAGCT TCGAAAAAAA CCCCATCGAC 3541 TTTCTGGAGG CGAAAGGATA TAAAGAGGTC AAAAAAGACC TCATCATTAA GCTTCCCAAG 85 DB1/ 159236086.1 SAL-049PC126933-5049 3601 TACTCTCTCT TTGAGCTTGA AAACGGCCGG AAACGAATGC TCGCTAGTGC GGGCGAGCTG 3661 CAGAAAGGTA ACGAGCTGGC ACTGCCCTCT AAATACGTTA ATTTCTTGTA TCTGGCCAGC 3721 CACTATGAAA AGCTCAAAGG GTCTCCCGAA GATAATGAGC AGAAGCAGCT GTTCGTGGAA 3781 CAACACAAAC ACTACCTTGA TGAGATCATC GAGCAAATAA GCGAATTCTC CAAAAGAGTG 3841 ATCCTCGCCG ACGCTAACCT CGATAAGGTG CTTTCTGCTT ACAATAAGCA CAGGGATAAG 3901 CCCATCAGGG AGCAGGCAGA AAACATTATC CACTTGTTTA CTCTGACCAA CTTGGGCGCG 3961 CCTGCAGCCT TCAAGTACTT CGACACCACC ATAGACAGAA AGCGGTACAC CTCTACAAAG 4021 GAGGTCCTGG ACGCCACACT GATTCATCAG TCAATTACGG GGCTCTATGA AACAAGAATC 4081 GACCTCTCTC AGCTCGGTGG AGAC SEQ ID NO: 6: amino acid sequence of dead Cas9 DNA binding protein (1368 amino acids) 1 MDKKYSIGLA IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 61 ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 121 NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 181 VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 241 LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 301 LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 361 GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 421 AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 481 VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 541 SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 601 IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 661 RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 721 HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 781 MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVAA 841 IVPQSFLKDD SIDNKVLTRS DKARGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 901 TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 961 KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK 1021 MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1081 ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1141 YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1201 YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1261 QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA 1321 PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGD SEQ ID NO: 7: amino acid sequence of E. coli TnsD (508 amino acids) 1 MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH 61 QTGRYAVQQL IYEHTLFPLY APFVGKERRD EAIRLMEYQA QGAVHLMLGV AASRVKSDNR 121 FRYCPDCVAL QLNRYGEAFW QRDWYLPALP YCPKHGALVF FDRAVDDHRH QFWALGHTEL 181 LSDYPKDSLS QLTALAAYIA PLLDAPRAQE LSPSLEQWTL FYQRLAQDLG LTKSKHIRHD 241 LVAERVRQTF SDEALEKLDL KLAENKDTCW LKSIFRKHRK AFSYLQHSIV WQALLPKLTV 301 IEALQQASAL TEHSITTRPV SQSVQPNSED LSVKHKDWQQ LVHKYQGIKA ARQSLEGGVL 361 YAWLYRHDRD WLVHWNQQHQ QERLAPAPRV DWNQRDRIAV RQLLRIIKRL DSSLDHPRAT 421 SSWLLKQTPN GTSLAKNLQK LPLVALCLKR YSESVEDYQI RRISQAFIKL KQEDVELRRW 481 RLLRSATLSK ERITEEAQRF LEMVYGEE This invention is further illustrated by the following non-limiting examples. EXAMPLES 86 DB1/ 159236086.1 SAL-049PC126933-5049 Hereinafter, the present disclosure will be described in further detail with reference to examples. These examples are illustrative purposes only and are not to be construed to limit the scope of the present invention. In addition, various modifications and variations can be made without departing from the technical scope of the present invention. Example 1 – Design of Transposon System FIG.1A - FIG.1E depict five illustrative bioengineered RNA helper constructs that are contained in a replication backbone (e.g., plasmid or miniplasmid) with a T7 promoter (cap dependent), beta-globin 5’-UTR, and a helper enzyme (SEQ ID NO: 1, SEQ ID NO: 2) from Myotis myotis followed by a beta-globin 3’-UTR, and a poly-alanine tail (FIG.1A). TALEs (FIG.1B, TABLE 7 – TABLE 12), ZnF (FIG.1C, TABLE 13 – TABLE 17), or a dead Cas9 (dCas9) binding protein (FIG.1D, SEQ ID NO: 5, SEQ ID NO: 6) with guide RNAs (TABLE 1 – TABLE 6) were linked to the N-terminus to target the specific TTAA sites at hROSA 26, AAVS1, chromosome 4, chromosome 22, and chromosome X loci. FIG. 1E depicts a construct with a dimerization enhancer. The dimerization enhancer may be selected from, without limitation, SH3, biotin, avidin, and rapamycin binders. The dimerization enhancer can be replaced with an intein. FIG.2A depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter driving a gene of interest (GOI) with a polyA tail flanked by two insulators and ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting genomic safe harbor sites (GSHS) or other loci. FIG.2B depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a splice acceptor site for exon 2 and other exons of a gene of interest (GOI) followed by a polyA tail and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for targeting endogenous genes in the first intron to repair downstream mutations. FIG.2C depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with tandem promoters to affect expression in different tissues (e.g., without limitation, liver specific promoter, cardiac specific promoter) and a gene(s) of interest (GOI) followed by a polyA tail and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used to differentially promote expression of genes in different organs, tissues or cell types. FIG.2D depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with two or more genes of interest (GOI) linked by 2A “self-cleaving” peptides and followed by WPRE and a polyA tail. The construct is flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct is used for delivering multiple genes or genetic factors. 87 DB1/ 159236086.1 SAL-049PC126933-5049 FIG. 2E depicts an illustrative core donor construct that is contained in a replication backbone (e.g., plasmid or miniplasmid) with a promoter(s) driving the expression of two or more genes as in FIG.2D and linked to a sequence consisting of a 5’-miRNA, a sense and antisense miRNA pair, and completed with the 3’-miRNA. The construct is followed by WPRE and flanked by ITRs. The inverted terminal repeat (ITR) recognition sequences are included at the 5’- (SEQ ID NO: 3) and 3’-ends (SEQ ID NO: 4). This construct combines protein replacement and miRNA to inhibit other related protein expression. The sense and anti-sense miRNA pair regulate the sense miRNAs, probably via modulating the chromatin architectures of the resided genomic loci. See Brown, T., Howe, F. S., Murray, S. C., Wouters, M., Lorenz, P., Seward, E., ... Mellor, J. (2018). Antisense transcription-dependent chromatin signature modulates sense transcript dynamics. Mol Syst Biol, 14(2), e8007; Murray, S. C., Haenni, S., Howe, F. S., Fischl, H., Chocian, K., Nair, A., & Mellor, J. (2015). Sense and antisense transcription are associated with distinct chromatin architectures across genes. Nucleic Acids Res, 43(16), 7823-7837. Example 2 – Identification of Excision Positive and Integration Negative Mutants Random mutagenesis and/or site directed mutagenesis will be performed on the Myotis myotis helper enzyme of SEQ ID NO: 1. The variants will be screened using integration and excision assays. The excision assay is a PCR-based assay to test for excision of the donor DNA. A HEK293 cell line that expresses GFP at a known genomic site would be transfected with helper plasmid alone to excise the donor GFP DNA at the genomic locus by recognizing the end sequences. For the integration assay, HEK293 cells will be plated in 12-well size plates the day before transfection. The day of the transfection the media will be exchanged 1 hour and 30 min before the transfection is performed. A 3:1 ratio of X-tremeGENE™ 9 DNA Transfection Reagent protocol reagent will be used to co-transfect a donor plasmid containing GFP and a helper plasmid in duplicate using 600ng of DNA each. Forty-eight (48) hrs after transfection, the cells will be analyzed by flow cytometry to count the percentage of GFP expressing cells to measure transient transfection efficiency. The cells will be gated to distinguish them from debris and 20,000 cells will be counted. The cultures will be grown for 15-20 days without antibiotic. Cells will be passaged 2 to 3 times per week. Flow cytometry will be used to count the percentage of GFP expressing cells to measure integration efficiency at 2 weeks. The final integration efficiency will be calculated by dividing the 2-week percentage of GFP cells by the percentage of GFP cell at 48hr. The excision assay will be performed by measuring the percentage of GFP cells in a cell line with a known GFP donor integration. The cells will be grown to 80% confluency and analyzed by flow cytometry to count the percentage of GFP expressing cells as a baseline measurement. This percentage will be used as the standard (i.e., 100%). X- tremeGENE™ 9 DNA Transfection Reagent protocol reagent will be used to transfect helper plasmid in duplicate using 600ng of DNA. The cells will be gated to distinguish them from debris and 20,000 cells will be counted. Forty-eight (48) 88 DB1/ 159236086.1 SAL-049PC126933-5049 hrs after transfection, the cells will be analyzed by flow cytometry to count the percentage of GFP expressing cells. The cells will be gated to distinguish them from debris and 20,000 cells will be counted. The final integration efficiency will be calculated by the baseline percentage of GFP cells by the percentage of GFP cells at 48hr. Excision positive (EXC+) and integration deficient (INT-) mutants will be identified using the method described above. Example 3 – Identification of Deletion Mutants and Fusion Protein Mutants Multiple deletion mutations will be generated using known methods. Some deletion mutants will be deleted at the N- terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted at the C- terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted in between the N-terminus and the C-terminus at varying number of residues relative to SEQ ID NO: 1. Some deletion mutants will be deleted at the N-terminus and at the C-terminus. Some deletion mutants will be deleted at the N-terminus, at the C- terminus, and in between the N-terminus and the C-terminus relative to SEQ ID NO: 1. Integration and excision activity will be tested on the mutants. Mutants with high excision activity and low integration activity will be selected as lead candidates for further optimization (e.g., without limitation, additional rounds of screening and/or addition of fusion proteins as described below). It is contemplated that an additional of a tag, e.g., an HA-tag, would not alter the excision and integration activity of the mutants. The helper enzyme from Myotis myotis will also be subjected to fusion with protein binding domains (e.g., without limitation, TALEs, TnsD, dCas9, and dCas12j) as described throughout the present application. Fusion proteins mutants will be generated using known method and the mutants will be screened for integration and excision activity. Mutants that show optimized activity will be selected as candidates for additional rounds of optimization (e.g., without limitation, additional rounds of screening and/or addition of fusion proteins as described herein). Example 4 – Construction of targeting elements directed to TTAA sites in hROSA26, AAVS1, chromosome 4, chromosome 22, and chromosome X targeted by guideRNAs, TALES, and ZnF FIG.6 depicts the TTAA site in hROSA26 (hg38 chr3:9,396,133-9,396,305) that is targeted by guideRNAs (TABLE 2), TALES (TABLE 8), and ZnF (TABLE 13). FIG.7 depicts two TTAA sites in AAVS1 (hg38 chr19:55,112,851-55,113,324) that are targeted by guideRNAs (TABLE 3) or TALES (TABLE 9), and ZnF (TABLE 14). FIG.8 depicts two TTAA sites in Chromosome 4 (hg38 chr4:30,793,534-30,875,476) that are targeted by guideRNAs (TABLE 4) or TALES (TABLE 10), and ZnF (TABLE 15). 89 DB1/ 159236086.1 SAL-049PC126933-5049 FIG.9 depicts two TTAA sites in Chromosome 22 (hg38 chr22:35,370,000-35,380,000) that are targeted by guideRNAs (TABLE 5) or TALES (TABLE 11), and ZnF (TABLE 16). FIG. 10 depicts two TTAA sites in Chromosome X (hg38 chrX:134,419,661-134,541,172) that are targeted by guideRNAs (TABLE 6) or TALES (TABLE 12), and ZnF (TABLE 17). 90 DB1/ 159236086.1 SAL-049PC126933-5049 EQUIVALENTS While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein set forth and as follows in the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. 91 DB1/ 159236086.1

Claims

SAL-049PC126933-5049 CLAIMS What is claimed is: 1. A composition comprising a helper enzyme or a nucleic acid encoding the enzyme, wherein the enzyme comprises an amino acid sequence having at least about 80% sequence identity to SEQ ID NO: 1. 2. The composition of claim 1, wherein the enzyme comprises an amino acid sequence of at least about 90% identity to SEQ ID NO: 1. 3. The composition of claim 1, wherein the enzyme comprises an amino acid sequence of at least about 93% identity to SEQ ID NO: 1. 4. The composition of claim 1, wherein the enzyme comprises an amino acid sequence of at least about 95% identity to SEQ ID NO: 1. 5. The composition of claim 1, wherein the enzyme comprises an amino acid sequence of at least about 98% identity to SEQ ID NO: 1. 6. The composition of claim 1, wherein the enzyme comprises an amino acid sequence of at least about 99% identity to SEQ ID NO: 1. 7. The composition of any one of claims 1-6, wherein the enzyme has one or more mutations which confer hyperactivity. 8. The composition of any one of claims 1-7, wherein the enzyme has one or more amino acid substitutions. 9. The composition of any one of claims 1-8, wherein the enzyme is a hyperactive variant of SEQ ID NO: 1. 10. The composition of any one of claims 1-6, wherein the nucleic acid that encodes the enzyme has a nucleotide sequence of at least about 80% identical to SEQ ID NO: 2 or a codon-optimized form thereof. 11. The composition of claim 10, wherein the nucleic acid that encodes the enzyme has a nucleotide sequence of at least about 90% identical to SEQ ID NO: 2 or a codon-optimized form thereof. 12. The composition of claim 10, wherein the nucleic acid that encodes the enzyme has a nucleotide sequence of at least about 93% identical to SEQ ID NO: 2 or a codon-optimized form thereof. 13. The composition of any one of claims 1-12, wherein the enzyme has increased activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. 14. The composition of any one of claims 1-13, wherein the enzyme is excision positive. 15. The composition of any one of claims 1-14, wherein the enzyme is integration deficient. 92 DB1/ 159236086.1 SAL-049PC126933-5049 16. The composition of any one of claims 14-15, wherein the enzyme has decreased integration activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. 17. The composition of any one of claims 14-16, wherein the enzyme has increased excision activity relative to an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or functional equivalent thereof. 18. The composition of any one of claims 1-17, wherein the enzyme comprises a targeting element. 19. The composition of any one of claims 1-18, wherein the enzyme is capable of inserting a donor comprising a transgene in a genomic safe harbor site (GSHS). 20. The composition of claim 19, wherein the binding of a GSHS of a nucleic acid molecule in a mammalian cell is with high target specificity, relative to a control. 21. The composition of claim 20, wherein the control is a composition comprising an enzyme comprising an amino acid sequence of SEQ ID NO: 1 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 2 or a codon- optimized form thereof. 22. The composition of any one of claims 18-21, wherein the targeting element is able to direct a transposition machinery to the GSHS of a nucleic acid molecule in a mammalian cell. 23. The composition of any one of claims 18-22, wherein the GSHS is in an open chromatin location in a chromosome. 24. The composition of any one of claims 18-23, wherein the GSHS is selected from adeno-associated virus site 1 (AAVS1), chemokine (C-C motif) receptor 5 (CCR5) gene, HIV-1 coreceptor, and human Rosa26 locus. 25. The composition of any one of claims 18-24, wherein the GSHS is an adeno-associated virus site 1 (AAVS1). 26. The composition of any one of claims 18-25, wherein the GSHS is a human Rosa26 locus. 27. The composition of any one of claims 18-26, wherein the GSHS is located on human chromosome 2, 4, 6, 10, 11, 17, 22, or X. 28. The composition of any one of claims 18-27, wherein the GSHS is selected from TABLES 1-17. 29. The composition of any one of claims 18-28, wherein the GSHS is selected from TALC1, TALC2, TALC3, TALC4, TALC5, TALC7, TALC8, AVS1, AVS2, AVS3, ROSA1, ROSA2, TALER1, TALER2, TALER3, TALER4, TALER5, SHCHR2-1, SHCHR2-2, SHCHR2-3, SHCHR2-4, SHCHR4-1, SHCHR4-2, SHCHR4-3, SHCHR6-1, SHCHR6-2, SHCHR6-3, SHCHR6-4, SHCHR10-1, SHCHR10-2, SHCHR10-3, SHCHR10-4, SHCHR10-5, SHCHR11- 1, SHCHR11-2, SHCHR11-3, SHCHR17-1, SHCHR17-2, SHCHR17-3, and SHCHR17-4. 93 DB1/ 159236086.1 SAL-049PC126933-5049 30. The composition of any one of claims 18-29, wherein the targeting element is or comprises one or more of a Cas enzyme, which is optionally catalytically inactive and which is optionally associated with a guide RNA (gRNA), transcription activator-like effector (TALE) DNA binding domain (DBD), catalytically inactive Zinc finger, catalytically inactive transcription factor, catalytically inactive nickase, a transcriptional activator, a transcriptional repressor, a recombinase, a DNA methyltransferase, a histone methyltransferase, a paternally expressed gene 10 (PEG10), and a transposon-encoded polypeptide D (TnsD) or a variant thereof. 31. The composition of claim 30, wherein the targeting element comprises a TALE DBD. 32. The composition of claim 31, wherein the TALE DBD comprises one or more repeat sequences. 33. The composition of claim 32, wherein the TALE DBD comprises about 14, or about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences. 34. The composition of claim 32 or claim 33, wherein the repeat sequences each independently comprises about 33 or 34 amino acids. 35. The composition of claim 34, wherein the repeat sequences each independently comprises a repeat variable di-residue (RVD) at residue 12 or 13 of the 33 or 34 amino acids, respectively. 36. The composition of claim 35, wherein the RVD recognizes one base pair in a target nucleic acid sequence. 37. The composition of claim 34 or claim 35, wherein the RVD recognizes a C residue in the target nucleic acid sequence and is selected from HD, N(gap), HA, ND, and HI. 38. The composition of claim 34 or claim 35, wherein the RVD recognizes a G residue in the target nucleic acid sequence and is selected from NN, NH, NK, HN, and NA. 39. The composition of claim 34 or claim 35, wherein the RVD recognizes an A residue in the target nucleic acid sequence and is selected from NI and NS. 40. The composition of claim 34 or claim 35, wherein the RVD recognizes a T residue in the target nucleic acid sequence and is selected from NG, HG, H(gap), and IG. 41. The composition of claim 30-40, wherein the TALE DBD targets one or more of GSHS sites selected from TABLES 7-12. 42. The composition of any one of claims 30-41, wherein the TALE DBD comprises one or more of RVD selected from TABLES 7-12, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. 94 DB1/ 159236086.1 SAL-049PC126933-5049 43. The composition of claim 30, wherein the targeting element comprises a Cas9 enzyme associated with a gRNA. 44. The composition of claim 43, wherein the Cas9 enzyme associated with a gRNA comprises a catalytically inactive dCas9 associated with a gRNA. 45. The composition of claim 44, wherein catalytically inactive dCas9 comprises at least about 90%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% identity to an amino acid sequence of SEQ ID NO: 6 or a nucleic acid comprising a nucleotide sequence of SEQ ID NO: 5 or a codon-optimized form thereof. 46. The composition of any one of claims 30 or 43-45, wherein the targeting element comprises a Cas12 enzyme associated with a gRNA. 47. The composition of claim 46, wherein the targeting element comprises a catalytically inactive Cas12 associated with a gRNA, optionally wherein the catalytically inactive Cas12 is dCas12j or dCas12a. 48. The composition of any one of claims 30 or 43-45, wherein the targeting element comprises a TnsC, TnsB, TnsA, TniQ, Cas6, Cas7, Cas8 enzyme associated with a gRNA. 49. The composition of any one of claims 30 or 43-45, wherein the targeting element comprises a TnsD. 50. The composition of claims 30 or 43-47, wherein the guide RNA is selected from TABLES 1-6, or variants thereof comprising about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 mutations. 51. The composition of claims 30 or 43-47, wherein the guide RNA targets one or more sites selected from TABLES 1-6. 52. The composition of claim 30, wherein the zinc finger comprises one of the sequences selected from TABLES 13-17, or variants thereof comprising about 99, about 98, about 97, about 95, about 94, about 93, about 92, about 91, about 90, about 89, about 88, about 87, about 86, about 85, about 84, about 83, about 82, about 81, about 80 percent identity to the sequence. 53. The composition of claim 30, wherein the zinc finger targets one or more sites selected from TABLES 13-17. 54. The composition of any one of claims 30-53, wherein the targeting element comprises a nucleic acid binding component of a gene-editing system. 55. The composition of any one of claims 30-54, wherein the enzyme or variant thereof and the targeting element are connected. 95 DB1/ 159236086.1 SAL-049PC126933-5049 56. The composition of claim 55, wherein the enzyme and the targeting element are fused to one another or linked via a linker to one another. 57. The composition of claim 56, wherein the linker is a flexible linker. 58. The composition of claim 57, wherein the flexible linker is substantially comprised of glycine and serine residues, optionally wherein the flexible linker comprises (Gly4Ser)n, where n is an integer from 1-12. 59. The composition of claim 58, wherein the flexible linker is of about 20, or about 30, or about 40, or about 50, or about 60 amino acid residues. 60. The composition of claim 59, wherein the enzyme is directly fused to the N-terminus of the targeting element and, optionally, wherein the targeting element is or comprises dCas9 enzyme. 61. The composition of any one of claims 1-60, wherein the enzyme or variant thereof is able to directly or indirectly cause transposition of a target gene. 62. The composition of any one of claims 1-61, wherein the enzyme or variant thereof is able to directly or indirectly interact and/or form a complex with one or more proteins or nucleic acids. 63. The composition of any one of the preceding claims, wherein a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises an intein, optionally NpuN (Intein-N) (SEQ ID NO: 423) and/or NpuC (Intein-C) (SEQ ID NO: 424), or a variant thereof. 64. The composition of claim 63, wherein the nucleic acid encodes the enzyme in the form of first and second portions with the intein encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the intein from the enzyme. 65. The composition of claim 63 or claim 64, wherein the intein is suitable for linking the helper enzyme and the targeting element. 66. The composition of any one of the preceding claims, wherein a nucleic acid encoding the enzyme capable of targeted genomic integration by transposition comprises a dimerization enhancer. 67. The composition of claim 66, wherein the nucleic acid encodes the enzyme in the form of first and second portions with the dimerization enhancer encoded between the first and second portions, such that the first and second portions are fused into a functional enzyme upon post-translational excision of the dimerization enhancer from the enzyme. 68. The composition of claim 66 or claim 67, wherein the dimerization enhancer is suitable for linking the helper enzyme and the targeting element. 96 DB1/ 159236086.1 SAL-049PC126933-5049 69. The composition of any one of claims 66-68, wherein the dimerization enhancer is selected from: a protein comprising a SH3 domain, biotin, avidin, or a rapamycin binder, optionally, wherein the rapamycin binder is FKBP12 or mTOR, or a variant thereof. 70. The composition of any one of claims 1-69, further comprising a nucleic acid encoding a donor comprising a transgene to be integrated, optionally wherein the transgene is defective or substantially absent in a disease state. 71. The composition of claim 70, wherein the transgene comprises a cargo nucleic acid sequence and a first and a second donor end sequences. 72. The composition of claim 71, wherein the cargo nucleic acid sequence is flanked by the first and the second donor end sequences. 73. The composition of claim 71 or claim 72, wherein the donor end sequences are selected from nucleotide sequences of SEQ ID NO: 3 and/or SEQ ID NO: 4, or a nucleotide sequence having at least about 90% identity thereto. 74. The composition of any one of claims 71-73, wherein the end sequences include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3. 75. The composition of claim 74, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 3 is positioned at the 5’ end of the donor. 76. The composition of any one of claims 71-75, wherein the end sequences can further include at least one repeat from a nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4. 77. The composition of any one of claims 72-76, wherein the at least one repeat from the nucleotide sequence having at least about 90% identity to the nucleotide sequence of SEQ ID NO: 4 is positioned at the 3’ end of the donor. 78. The composition of any one of claims 1-77, wherein the donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof. 79. The composition of any one of claims 1-78, wherein the the polynucleotide comprising an open reading frame encoding a transposase, the amino acid sequence of which is at least 90% identical to SEQ ID NO: 1, or a functional variant thereof, operably linked to a heterologous promoter. 80. The composition of claim 79, wherein the enzyme or variant thereof is incorporated into a vector or a vector- like particle, wherein the vector or a vector-like particle comprises one or more expression cassettes, and/or wherein the vector or a vector-like particle comprises one expression cassette. 81. The composition of claim 80, wherein the expression cassette further comprises the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof. 97 DB1/ 159236086.1 SAL-049PC126933-5049 82. The composition of claim 81, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or a combination thereof are incorporated into one or more vectors or vector-like particles. 83. The composition of claim 81, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof are incorporated into a same vector or vector-like particle. 84. The composition of claim 81, wherein the enzyme or variant thereof, the transgene, the donor end sequences, or combination thereof is incorporated into different vectors or vector-like particles. 85. The composition of any one of claims 78-84, wherein the vector or vector-like particle is nonviral. 86. The composition of any one of claims 70-85, wherein the donor is under the control of at least one tissue- specific promoter. 87. The composition of claim 86, wherein the at least one tissue-specific promoter is a single promoter. 88. The composition of claim 86, wherein the at least one tissue-specific promoter is under the control of a dual promoter or a tandem promoter. 89. The composition of any one of claims 70-88, wherein the transgene to be integrated comprises at least one gene of interest. 90. The composition of any one of claims 70-89, wherein the transgene to be integrated comprises one gene of interest. 91. The composition of any one of claims 70-89, wherein the transgene to be integrated comprises two or more genes of interest. 92. The composition of any one of claims 70-91, wherein the at least one gene of interest comprises peptides for linking genes of interest. 93. The composition of claim 92, wherein the peptides are 2A self-cleaving peptides, or functional variants thereof, wherein the 2A self-cleaving peptide is optionally selected from P2A, E2A, F2A, and T2A, or derivative thereof. 94. The composition of any one of claims 70-93, wherein the at least one gene of interest is linked to polynucleotide comprising a sequence comprising a 5’-miRNA, a sense and antisense miRNA pair, and/or a 3’-miRNA. 95. The composition of any one of claims 1-94, wherein the composition comprises DNA, RNA, or both. 96. The composition of any one of claims 1-95, wherein the enzyme or variant thereof is in the form of RNA. 97. A host cell comprising the composition any one of claims 1-96. 98 DB1/ 159236086.1 SAL-049PC126933-5049 98. The composition of any one of claims 1-96, wherein the composition is encapsulated in a lipid nanoparticle (LNP). 99. The composition of any one of claims 1-96, wherein the polynucleotide encoding the enzyme or variant thereof and the polynucleotide encoding the donor are in the form of the same LNP, optionally in a co-formulation. 100. The composition of claim 98 or claim 99, wherein the LNP comprises one or more lipids selected from 1,2- dioleoyl-3-trimethylammonium propane (DOTAP), a cationic cholesterol derivative mixed with dimethylaminoethane- carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3- methoxypolyethyleneglycol–2000 (DMG-PEG 2K), and 1,2 distearol -sn-glycerol-3phosphocholine (DSPC) and/or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc). 101. A method for inserting a gene into the genome of a cell, comprising contacting a cell with the composition of any one of claims 1-96 or 98-100 or host cell of claim 97. 102. The method of claim 101, further comprising contacting the cell with a polynucleotide encoding a donor DNA. 103. The method of claim 101 or claim 102, wherein the donor comprises a gene encoding a complete polypeptide. 104. The method of any one of claims 101-103, wherein the donor comprises a gene which is defective or substantially absent in a disease state. 105. A method for treating a disease or disorder ex vivo, comprising contacting a cell with the composition of any one of claims 1-96 or 98-100 or host cell of claim 97 and administering the cell to a subject in need thereof. 106. A method for treating a disease or disorder in vivo, comprising administering the composition of any one of claims 1-96 or 98-100 or host cell of claim 97 to a subject in need thereof. 107. A donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the left end comprises SEQ ID NO: 3, or a functional variant thereof and the right end comprises SEQ ID NO: 4, or a functional variant thereof. 108. The donor construct of claim 107, wherein the donor is transposable by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. 109. A donor construct comprising a heterologous polynucleotide between left and right transposon ends, wherein the donor is suitable for transposition by a helper enzyme having the sequence of SEQ ID NO: 1, or a functional variant thereof. 99 DB1/ 159236086.1 SAL-049PC126933-5049 110. A helper enzyme derived from Myotis myotis, the helper enzyme being suitable for transposition of a heterologous polynucleotide, the heterologous polynucleotide being flanked by two ends elements comprising the polynucleotide sequences of SEQ ID NO: 3, or a functional variant thereof and SEQ ID NO: 4, or a functional variant thereof. 100 DB1/ 159236086.1
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