Engineered long interspersed repetitive element (LINE) transposons and methods for their use

Abstract

Engineered transposons and methods for their use are provided. Transposons typically contain an RNA component and a protein component. The RNA component can include, for example, a DNA targeting sequence, one or more protein binding motifs, and a nucleic acid sequence of interest to be integrated into the target DNA. The protein component is typically derived from an RLE LINE element protein and can include a DNA-binding domain, an RNA-binding domain, a reverse transcriptase, a linker domain, and an endonuclease. Pharmaceutical compositions and methods for introducing a nucleic acid sequence into the genome of a cell are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Patent Application No. 62 / 748,227, filed October 19, 2018, which is hereby incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY FUNDED RESEARCH This invention was made with government support under Grant 0950983 awarded by the National Science Foundation. The United States Government has certain rights in the invention.

[0003] Reference to sequence listing The sequence listing, submitted as a text file entitled "UTSB_18_47_PCT_ST25.txt" having a size of 17,183 bytes, is hereby incorporated by reference pursuant to 37 CFR 1.52(e)(5).

[0004] Technical Field The present invention relates generally to compositions and methods for genome modification. [Background technology]

[0005] 2. Background of the Invention Genome editing technology may be useful in the treatment of various diseases and disorders, including but not limited to cancer, genetic disorders, and HIV / AIDS. Somatic cell genome editing is a promising area for the development of therapeutics, and the complex enzyme editing tool CRISPR-Cas9 was used to remove the human beta globin (HBB) gene from the germline of human embryos (Otieno, (2015), J Clin Res Bioeth 6:253. doi: 10.4172 / 2155-9627.1000253). However, historically, the clinical application of gene editing technology has been limited by low frequency of editing events, high off-target events, or a combination of these, among other concerns.

[0006] It is therefore an object of the present invention to provide improved compositions and methods for gene delivery and gene editing. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Otieno, (2015), J Clin Res Bioeth 6:253. doi: 10.4172 / 2155-9627.1000253 Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention Provided are engineered transposons and their methods of use. Transposons typically include an RNA component and a protein component. The RNA component can include, for example, a DNA targeting sequence, one or more protein binding motifs, and a nucleic acid sequence of interest that is integrated into the DNA target site. The DNA targeting sequence, the protein binding motif, and the sequence of interest are typically operably linked to a protein component that is derived from a restriction enzyme-like endonuclease long interspersed repeat (RLE LINE) element protein, and are reverse transcribed, and the resulting cDNA is integrated into the DNA target site of DNA, for example, in a cell genome. The sequence of interest can, for example, code for a gene or a fragment thereof, or a functional nucleic acid.

[0009] A protein-binding motif (PMB), an RNA segment involved in binding to a protein, typically binds to the RNA-binding domain (domain-1), reverse transcriptase, linker domain, endonuclease, or a combination thereof, of a protein component.

[0010] The RNA component can include elements from or derived from the parent LINE or SINE backbone, and the nucleic acid sequence of interest of the RNA component is usually heterologous to the LINE or SINE. In a representative embodiment, the DNA targeting sequence is heterologous to the parent LINE or SINE. The RNA component can include, for example, a 3'PBM sequence from or derived from the parent LINE or SINE element, a CRISPR / Cas tracer sequence, a CRISPR / Cas guide sequence, or a combination thereof, a 5'PBM sequence from or derived from the parent LINE or SINE element, preferably a parent LINE or SINE element in which any IRES sequence is non-functional, a ribozyme, such as a hepatitis delta virus-like ribozyme, or any combination thereof.

[0011] The protein component is typically derived from an RLE LINE element protein and may include one or more DNA binding domains, one or more RNA binding domains, a reverse transcriptase, a linker domain, and an endonuclease. Typically, the DNA binding domain, the RNA binding domain, the reverse transcriptase, the linker domain, and the endonuclease are operably linked to bind the RNA component and DNA (e.g., cellular genomic DNA) at the DNA target site and facilitate reverse transcription of the RNA component into cDNA and integration of the cDNA into the DNA target site. Typically, the DNA binding domain is mutated compared to the parent LINE DNA binding domain, or the parent DNA binding domain is replaced with an alternative DNA binding domain. In some embodiments, the DNA binding domain is a DNA binding domain from another DNA binding protein, or a motif thereof, such as a helix-turn-helix, zinc finger, leucine zipper, winged helix, winged helix-turn-helix, helix-loop-helix, HMG box, Wor3 domain, OB-fold domain, immunoglobulin fold, B3 domain, TAL effector, or RNA guide domain. Typically, the sequences for one or more of the RNA binding domain, reverse transcriptase, linker domain, and endonuclease are the same as those of the LINE element protein, or are preferably mutated to improve binding and / or enzymatic activity to the RNA component or target DNA compared to the parent LINE element protein.

[0012] In some embodiments, the parent LINE or SINE backbone of the RNA component and the parent LINE backbone of the protein component are the same LINE and / or the SINE is derived from or is ancestral to a LINE. The RNA sequence of the RNA component, the amino acid sequence of the protein sequence, or combinations thereof, may be recombinant sequences and / or variants of the parent backbone.

[0013] Also provided are pharmaceutical compositions comprising vectors encoding the RNA and protein components, and the components, vectors, and / or engineered transposons formed therefrom. Preferably, the transposons are capable of forming productive four-way junctions during an integration reaction into a DNA target site.

[0014] Methods of use are also provided. For example, a method of introducing a nucleic acid sequence of interest into the genome of one or more cells may include contacting one or more cells with (i) an RNA component or a vector encoding an RNA component in combination with a protein component or a vector encoding a protein component, or (ii) an engineered transposon that includes both an RNA component and a protein component. The cells can be contacted in vitro or in vivo. In some embodiments, the ex vivo modified cells are then introduced into a subject in need. In some embodiments, the composition is administered directly to a subject in need.

[0015] Also provided is a method for treating disease and disorder.In such use, the expression of the nucleic acid sequence of interest in cells can improve one or more symptoms of disease or disorder, or the molecular pathway underlying disease or disorder.In a preferred embodiment, an effective number of cells are modified to treat the subject with disease or disorder. [Brief description of the drawings]

[0016] [Figure 1A]Figure 1A is a schematic diagram of the R2Bm structure. R2Bm RNA (wavy line) and open reading frame (ORF) structure (boxed). The ORF encodes conserved domains of known and unknown function: zinc finger (ZF), Myb (Myb), reverse transcriptase domain (RT), cysteine-histidine rich motif (CCHC), and PD-(D / E)XK-type restriction enzyme-like endonuclease (RLE). The RNA structures present in the 5' and 3' untranslated regions that bind the R2 protein are considered as 5' and 3' protein binding motifs (PBM), respectively. Square brackets indicate the individual segments of R2Bm RNA used in this document: 5'PBM RNA (320 nt), 3'PBM RNA (249 nt), RNA at the 5' end of the element (25 or 40 nt), and RNA 3' end (25 or 40 nt). [Figure 1B] FIG. 1B is a schematic of the R2Bm integration reaction. A four-step integration model is depicted for a segment of 28S rDNA (parallel lines). The R2 protein subunit (hexagon) is bound upstream of the insertion site (vertical bar) and the R2 protein subunit is bound downstream of the insertion site. The upstream subunit associates with the 3'PBM RNA, while the downstream subunit associates with the 5'PBM RNA. The footprints of the protein subunits on the target DNA are shown. The upstream footprint from -40bp to -20bp, but grows to just above the insertion site (vertical line) after first strand DNA cleavage. The downstream subunit footprint from just before the insertion site to +20bp (Christensen, et al., Nucleic Acids Res 33, 6461 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)). The four steps of integration are (1) bottom / first strand DNA cleavage of the target DNA, (2) TPRT, (3) top / second strand DNA cleavage of the target DNA, and (4) second strand DNA synthesis. The fourth step has never been observed directly in vitro before. The overlapping portions of the target sites used in Examples 1-8 are shown in brackets.

[0017] [Figure 2A] Figures 2A and 2B are schematic diagrams of the non-specific four-way junction (2A) and linear DNA (2B) DNA constructs. The design and sequence of the four-way junction was from (Middleton and Bond, Nucleic Acids Res 32, 5442 (2004)), which was formed by annealing b, x, h, and rDNA oligos. Each arm of the resulting junction was 25 bp. The linear DNA was generated by annealing oligo b to an oligo that was a combination of the x and h oligos. Thus, the junction DNA and linear DNA shared a common DNA oligo (oligo b). The 5' end of the shared DNA oligo was labeled with 32P (asterisk) prior to formation and purification of the linear and junction DNAs. [Figure 2B] Same as above.

[0018] [Diagram 3] FIG. 3 is a schematic diagram of several linear, 3-way, and 4-way branched DNA constructs. Straight lines represent DNA and wavy lines represent RNA. Thin lines represent non-specific DNA depicted in FIGS. 2A-2B. Bold lines represent sequences from the 28S rDNA as well as the R2 element. The R2 sequence is from the 5' and 3' ends of the element. The 28S sequence is the downstream DNA (28Sd) plus 7 bp of upstream DNA. The "arms" of each construct are 25 bp in length. Each construct is numbered for discussion. Asterisks indicate that the strands were end-labeled as in the previous figure. Two versions of construct v were tested, one with a DNA duplex in the R2 3' arm and the other with an RNA / DNA hybrid resulting from TPRT. No detectable second strand DNA cleavage was seen in constructs i-v. Second strand DNA cleavage could be detected in constructs vi-viii.

[0019] [Figure 4A]Figure 4A is a schematic diagram of several derivatives of the four-way junction from Figure 3 to test for cleavage versus partial junctions. Constructs are numbered. The 28S downstream (28Sd) DNA arm was increased by 47 bp to equal the amount of downstream DNA previously used in linear 28S target DNA (Christensen, et al., Nucleic Acids Res 33, 6461 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)). [Figure 4B] Figure 4B is a graph of the fraction cleaved (fCut) as a function of the fraction bound (fBind) for each set of constructs in Figure 4A. The diameter of the dots depicts the relative cleavability of the constructs by R2Bm. [Figure 4C] Figure 4C is a schematic diagram of a construct designed to test DNA cleavage at a four-way junction containing upstream 28S DNA. The 28S upstream (28Su) DNA arm is 73 bp, which corresponds to the amount of upstream DNA typically used in linear target DNA (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, J Mol Biol 336, 1035 (2004)). The black line is DNA, the thin line is non-specific DNA, and the thick line is either DNA from 28S or R2. [Figure 4D] Figure 4D is a graph of the fraction cleaved (fCut) as a function of the fraction bound (fBind) for each set of constructs in Figure 4C. The diameter of the dots depicts the relative cleavability of the constructs by R2Bm. Abbreviations and symbols are as in the previous figure.

[0020] [Diagram 5] FIG. 5 is a schematic diagram of a four-way junction for denaturing gel analysis of DNA cleavage (−dNTP) and cleavage and second strand synthesis (+dNTP) reactions.

[0021] [Figure 6A] Figure 6A is a schematic of a construct designed to hold an already cleaved product in close proximity and to test which arm would be used as a template. The lengths of the 5' and 3' arms were varied (40 bp vs. 25 bp). The 28S downstream arm was 47 bp and the 28S upstream arm was 73 bp. [Figure 6B] FIG. 6B is a schematic representation of constructs designed to test whether an upstream or downstream protein subunit is more likely to be involved in second strand synthesis. [Figure 6C] FIG. 6C is a graph of the fraction synthesized (f synthesized) as a function of the fraction bound (f bound).

[0022] [Figure 7] FIG. 7A is a schematic showing a new model of R2 integration. The R2 28S target site is labeled with the location of the first and second strand breaks that lead to the insertion of the new R2 element. The first steps (I, ii) of the integration reaction are similar to those in FIG. 1B, except that the target site is bent 90° near the second strand insertion site for illustration purposes. Step iii depicts a template jump / recombination event near the second strand break site that generates a four-way junction. Step iv depicts the second strand break. Finally, step v depicts second strand DNA synthesis. Abbreviations: up (target sequence upstream of insertion site), dwn (target sequence downstream of insertion site). FIG. 7B is a schematic showing a new model of L1 integration. The target site is labeled, and the first and second strand breaks are staggered to allow for target site duplication (tsd) upon element insertion. These steps are similar to those in R2, except that the template jump translocates / melts the targeted tsd region to generate a four-way junction.

[0023] [Figure 8A]FIG. 8A is a schematic diagram showing the R2 target site, 28S rDNA, and insertion model. The R2 protein associated with the 3'PBM RNA binds 20-40 bases upstream (28Su) of the insertion site (vertical line), and the protein associated with the 5'PBM RNA binds 20 bases downstream of the insertion site (Christensen, et al., Nucleic Acids Res. 33, 6461-6468 (2005), Christensen and Eickbush, J. Mol. Biol. 336, 1035-1045 (2004)). Insertion occurs in five steps: (1) first strand cleavage by the upstream protein subunit endonuclease; (2) first strand synthesis (TPRT) by the upstream protein subunit reverse transcriptase; and (3) template jump / recombination with the upstream target DNA (28Su) resulting in a four-way junction branched structure (expanded in the schematic). (4) Second strand cleavage by an endonuclease of the downstream protein subunit. (5) Second strand synthesis by the reverse transcriptase of the downstream protein subunit. [Figure 8B]FIG. 8B is a multiple sequence and secondary structure alignment of the linker region of RLE LINE (SEQ ID NOs: 31-44). Stars represent mutated residues and half triangles represent double point mutants generated in the putative α-finger and zinc knuckle regions. The double point mutants generated for this study were GR / AD / A, H / AIN / AALP, SR / AIR / A, SR / AGR / A, C / SC / SHC, CR / AAGCK / A, HILQ / AQ / A and RT / AH / A. As indicated by the square brackets at the top, the first four mutants are in the putative α-finger region and the last four mutants are in the zinc knuckle region. The secondary structure was predicted by Ali2D, with grey bars representing α-helices and arrows representing β-strands. Abbreviations: R2Bm=Bombyx mori, R2Dm=Drosophila melanogaster, R2Dana=Drosophila ananassae, R2Dwil=Drosophila willistoni, R2Dsim=Drosophila simulans, R2Dpse=Drosophila pseudoobscura, R2Fauric=Forficula auricularia, R2Amar=Anurida maritima, R2Nv-B=Nasonia vitripennis, R2Lp=Limulus polyphemus, R2Amel=Apis mellifera, R2Dr=Danio rerio, R8Hm-A=Hydra magnipapillata, R9Av-1=Adineta vaga.

[0024] [Figure 9] Figures 9A and 9B are bar graphs reporting the ability of mutants to bind target DNA in the presence of 3' (9A) and 5' PBM (9B) RNA. Wild-type (WT) protein activity was set to 1, so mutant protein activity is given as a percentage of WT activity (fWT activity). The bars in each graph represent, from left to right, R2:WT, H / AIN / AALP, C / SC, SHC.

[0025] [Figure 10-1]Figures 10A-10D are bar graphs showing DNA binding by α-finger mutant proteins. Figures 10A and 10B report the relative ability of mutants to bind linear target DNA. WT and KPD / A WT served as positive controls, while Pet28a and DNA only lanes served as negative controls. Standard deviations are presented at the top of the bars. Figure 10C reports binding to analogs of branched insertion intermediates. Stars in the substrate schematics indicate 5'-end labeled strands. Figure 10D reports linear target DNA binding activity of α-finger mutant proteins in the absence of RNA. Bars in each graph represent, from left to right, R2: KPD / A WT, GR / AD / A, SR / AIR / A, SR / AGR / A. [Figure 10-2] Same as above.

[0026] [Figure 11] Figure 11 is a scatter plot showing first strand DNA cleavage activity by alpha finger mutant proteins. The fraction of target DNA undergoing first strand cleavage (fCleavage) was quantified from denaturing gels. The scatter plot shows the fraction of target DNA cleaved (fCleavage) plotted as a function of the fraction of target DNA bound by the protein (fBinding) at each protein concentration. Data points for WT, GR / AD / A, SR / AIR / A and SR / AGR / A are represented by asterisks, white boxes, grey boxes and black boxes, respectively.

[0027] [Figure 12A] FIG. 12A is an illustration of the experimental setup for the first strand synthesis assay, in which pre-cleaved target DNA was incubated with R2 protein in the presence of 3′PBM RNA and dNTPs. [Figure 12B] Figure 12B is a scatter plot showing the fraction of DNA synthesized (f synthesis) as a function of the fraction of DNA bound by R2 protein (f binding) over a protein titration series. Symbols and abbreviations are as in the previous figure.

[0028] [Figure 13A] Figure 13A is a scatter plot of the second strand cleavage activity of α-finger mutant proteins against linear target DNA. EMSA gel was used to calculate the percentage of target DNA bound by R2 protein. Denaturing gel was used to calculate the percentage of target DNA cleaved by R2 protein. Symbols and abbreviations are as in the previous figure. [Figure 13B] Figure 13B is a scatter plot of the second strand DNA cleavage activity of α-finger mutant proteins against four-way junction DNA. EMSA gel was used to calculate the percentage of target DNA bound by R2 protein. Denaturing gel was used to calculate the percentage of target DNA cleaved by R2 protein. Symbols and abbreviations are as in the previous figure.

[0029] [Figure 14A] FIG. 14A is an illustration of the experimental setup for the second strand synthesis assay, in which pre-cleaved four-way junction DNA was incubated with R2 protein in the presence of dNTPs. [Figure 14B] Figure 14B is a scatter plot of second strand synthesis activity. Symbols and abbreviations are as in the previous figure.

[0030] [Figure 15A] 15A is a scatter plot showing first strand cleavage activity by zinc knuckle mutant proteins, where the fraction of target DNA cleaved (fcut) is plotted as a function of the fraction of target DNA bound by the protein (fbind) at each protein concentration. [Figure 15B] Figure 15B is a scatter plot showing first strand synthesis activity by zinc knuckle mutant proteins. The graph plots the fraction of target DNA that undergoes first strand synthesis by TPRT (f synthesis) as a function of the fraction of previously cleaved linear target DNA that is bound by the protein (f binding). [Figure 15C]Figure 15C is a scatter plot showing the second strand cleavage activity of zinc knuckle mutants against four-way junction target DNA. The graph plots the target DNA with second strand cleavage (fCleavage) as a function of the fraction of four-way junction DNA bound by the protein (fBinding). [Figure 15D] FIG. 15D is a scatter plot of second strand cleavage activity by zinc knuckle mutants against linear target DNA as a function of bound DNA.

[0031] [Figure 16] Figure 16 is a scatter plot of the second strand synthesis activity of zinc knuckle mutants. The experimental setup is the same as that of Figure 14A.

[0032] [Figure 17A] Figure 17A is a series of domain maps showing the ORF structures of R2Bm, human L1 (L1H), and Saccharomyces cerevisiae Prp8 (Mahbub, et al., Mob. DNA 8, 1-15 (2017), Wan, et al., Science (80-.)(2016) doi:10.1126 / science.aad6466; Bertram, et al., Cell (2017); doi:10.1016 / j.cell.2017.07.011; Qu, et al., Nat. Struct. Mol. Biol.(2016); doi:10.1038 / nsmb.3220; Nguyen, et al., Nature 530, 298-302 (2016); Galej, et al., Current Opinion in Structural Biology (2014). doi:10.1016 / j.sbi.2013.12.002; Blocker, et al., RNA 11, 14-28 (2005)). Within the linker region, the sequences of the α-helices marked with asterisks (rounded bars) are well aligned. The colored α-helices and the remainder of the β-strands (arrows) can form a structurally similar knuckle. [Figure 17B]Figure 17B is a model of RT and RLE of R2Bm (Mahbub, et al., Mob. DNA 8, 1-15 (2017)). [Figure 17C] Figure 17C is the cryo-Em structure of a large fragment of Prp8 (Wan, et al., Science (80-.). (2016). doi:10.1126 / science.aad6466). [Figure 17D] Figure 17D is a cryo-EM structure of Prp8 and RNA from the B spliceosome complex (Bertram, et al., Cell (2017). doi:10.1016 / j.cell.2017.07.011). Also shown is the branched structure formed by the RNA components of the spliceosome.

[0033] [Figure 18A] Figure 18A is a schematic diagram of the RNA components of engineered LINEs. HDV = Hepatitis delta virus ribozyme (optional); PBM = protein binding motif (can be from one element or from two elements when forming a hetero-RNP); Prom = pol II promoter and associated transcription factor binding sites for ORF expression; ORF = ORF of gene brought into genome by TPRT; tracr = tracer RNA; tracr / guide = canonical cas9 targeting RNA; TS = target sequence. Tracer, guide, or tracer / guide can be provided in cis (as above) or in trans. [Figure 18B] Figure 18B is a schematic diagram of the RLE ORF with engineered DNA binding domain. R2 or other RLE protein expression constructs can be expressed in bacterial (to be purified for use) or eukaryotic expression systems for direct production in the cells of interest. Engineered DB = ZF from ZF library, or talen, or cas9 (EN-). Note: DB in R2 is ZF and Myb. αF = α finger. [Figure 18C]FIG. 18C is a schematic representation of two different models of RLE LINE binding at the target site. [Figure 18D] FIG. 18D is a schematic diagram of two different models of RLE LINE integration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description of the Invention I. Definition As used herein, the term "carrier" or "excipient" refers to a natural or synthetic inert ingredient in a formulation, an organic or inorganic ingredient with which one or more active ingredients are combined.

[0035] As used herein, the term "pharmacologically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.

[0036] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a dosage sufficient to alleviate one or more symptoms of the disorder, disease or condition being treated, or otherwise provide the desired pharmacological and / or physiological effect. The exact dosage will vary depending on a variety of factors, such as subject-dependent variables (e.g., age, immune system health, etc.), the disease or disorder being treated, and the route of administration and pharmacokinetics of the agent being administered.

[0037] As used herein, the term "prevention" or "preventing" means administering a composition to a subject or system at risk of or predisposed to one or more symptoms caused by a disease or disorder, to cause the arrest of a particular symptom of the disease or disorder, the reduction or prevention of one or more symptoms of the disease or disorder, the lessening of the severity of the disease or disorder, the complete eradication of the disease or disorder, the stabilization of the disease or disorder or the delay in its onset or progression.

[0038] As used herein, the term "construct" refers to a recombinant genetic molecule having one or more isolated polynucleotide sequences.

[0039] As used herein, the term "regulatory sequence" refers to a nucleic acid sequence that controls and regulates the function, for example, transcription and / or translation, of another nucleic acid sequence. Control sequences suitable for prokaryotes may include a promoter, optionally an operator sequence and / or a ribosome binding site. It is known that eukaryotic cells utilize sequences such as promoters, terminators, polyadenylation signals and enhancers. Regulatory sequences include viral protein recognition elements that control the transcription and replication of viral genes.

[0040] As used herein, the term "gene" refers to a DNA sequence that, via its template or messenger RNA, codes for a sequence of amino acids specific to a particular peptide, polypeptide or protein. The term "gene" also refers to a DNA sequence that codes for an RNA product. The term gene, as used herein with respect to genomic DNA, includes not only regulatory sequences but also intervening non-coding regions and may include the 5' and 3' ends.

[0041] As used herein, the term polypeptide includes proteins and fragments thereof. Polypeptides may be "endogenous" or "exogenous", meaning that they are "heterologous", i.e., foreign to the host cell utilized, e.g., human polypeptides produced by bacterial cells. Polypeptides are disclosed herein as amino acid residue sequences.

[0042] As used herein, the term "vector" refers to a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted to bring about the replication of the inserted segment. The vector can be an expression vector.

[0043] As used herein, the term "expression vector" refers to a vector that contains one or more expression control sequences.

[0044] As used herein, the term "transfected" or "transduced" refers to a host cell or organism into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule may be stably integrated into the genome of the host, or the nucleic acid molecule may exist as a stable or unstable extrachromosomal structure. Such extrachromosomal structures may be self-replicating. A transformed cell or organism may include not only the end product of a transformation process, but also its transgenic progeny. A "non-transformed" or "non-transduced" host refers to a cell or organism that does not contain a heterologous nucleic acid molecule.

[0045] As used herein, the term "exogenous" with respect to nucleic acids refers to nucleic acids that are normally present within the host body.

[0046] As used herein, the term "heterologous" refers to elements that are present in a place where they are not normally found. For example, an endogenous promoter may be linked to a heterologous nucleic acid sequence, such as a sequence that is not normally found operably linked to a promoter. When used herein to describe a promoter element, heterologous means a promoter element that is different in either sequence, species, or number from the one normally found in the native promoter. For example, a heterologous control element in a promoter sequence may be a control / regulatory element of a different promoter that is added to enhance promoter control, or may be an additional control element of the same promoter. Thus, the term "heterologous" may also encompass "foreign" and "non-native" elements. II. Engineered Transposons

[0047] Long interspersed repetitive elements (LINEs) are a prolific and diverse group of autonomous transposable elements (TEs) found in eukaryotic genomes across the tree of life. LINEs also recruit non-autonomous short interspersed repetitive elements (SINEs). SINEs utilize the LINE protein machinery to replicate. LINE and SINE migration has been implicated in cancer progression, in genome evolution, including modulation of gene expression, genome rearrangements, DNA repair, and as a source of new genes. LINEs replicate by a process called target primed reverse transcription (TPRT), in which element RNA is reverse transcribed into the DNA insertion site using a nick in the target DNA as a primer for reverse transcription (Luan, et al., Cell 72, 595 (1993);Cost, et al., EMBO J 21, 5899 (2002);Moran, et al., Eds.(ASM Press, Washington, DC, 2002), pp. 836-869). LINEs encode proteins that are used to carry out the key steps of the insertion reaction: LINE proteins bind their own mRNA, recognize target DNA, perform first-strand target DNA cleavage, and carry out TPRT.These proteins are also thought to perform second strand target DNA cleavage and second strand element DNA synthesis, although there is little evidence for this (Luan, et al., Cell 72, 595 (1993);Cost, et al., EMBO J 21, 5899 (2002);Moran, et al., Eds.(ASM Press, Washington, DC, 2002), pp. 836-869;Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005);Kulpa and Moran, Nat Struct Mol Biol 13, 655 (2006);Dewannieux and Heidmann, Cytogenet Genome Res 110, 35 (2005);Doucet, et al. Mol Cell 60, 728 (2015);Christensen, et al., Nucleic Acids Res 33, 6461 (2005);Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016);Martin, RNA Biol 7, 67 (2010);Martin, J Biomed Biotechnol 2006, 45621 (2006);Matsumoto, et al., Mol Cell Biol 26, 5168 (2006);Zingler et al., Genome Res 15, 780 (2005);Kurzynska-Kokorniak, et al., J Mol Biol 374, 322 (2007);Ichiyanagi, et al. N. Okada, Genome Res 17, 33 (2007);Gasior, et al., J Mol Biol 357, 1383 (2006);Suzuki et al., PLoS Genet 5, e1000461 (2009); Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)).

[0048] Early divergent lineages of LINEs encode restriction enzyme-like endonucleases (RLEs), whereas later divergent LINEs encode apurinic-apyrimidinic DNA endonucleases (APEs) (Eickbush and Malik, in Origins and Evolution of Retrotransposons, Craig, NL, Craigie, R, Gellert, M, AM Lambowitz, Eds. (ASM Press, Washington, DC, 2002), pp. 1111-1146; Yang, et al., Proc Natl Acad Sci USA 96, 7847 (1999); Feng, et al., Cell 87, 905 (1996); Weichenrieder, et al., Structure 12, 975 (2004)). Both types of elements appear to be integrated by functionally equivalent integration processes (Moran, et al., Eds. (ASM Press, Washington, DC, 2002), pp. 836-869; Han, Mob DNA 1, 15 (2010); Fujiwara, Microbiol Spectr 3, MDNA3 (2015); Eickbush and Eickbush, Microbiol Spectr 3, MDNA3 (2015)).

[0049] Replication occurs through a series of ordered DNA cleavage and polymerization events using the encoded nucleic acid binding, endonuclease, and polymerase functions (Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006); Shivram, et al., Mobile Genetic Elements, 1:3, 169-178 (2011); see also Examples below). When translated, the proteins encoded by the elements, along with the transcripts they were translated from, form ribonucleoprotein (RNP) particles, a process called cis-type preference. The RNP binds to the target DNA, cleaves one of the DNA strands, and reverse transcribes the element RNA into cDNA (cDNA) using the exposed 3'-OH of the target site as a primer - a process called target primed reverse transcription (TPRT). The opposite target DNA strand is then cleaved. The cDNA becomes double-stranded DNA and the integration event is completed. Successful integration of the newly reverse transcribed DNA into the target site depends on interactions between the DNA, RNA, and protein components of the transposon and the target site DNA.

[0050] Engineered RNA and protein components utilizing sequences and mechanisms from or derived from LINE and SINE retrotransposons, and engineered transposons formed therefrom, are provided. As used herein, "derived from" a LINE or SINE means that the RNA and / or protein component traces the origin of one or more of its domains back to the corresponding RNA or protein component of the parent LINE or SINE. In some embodiments, the engineered RNA or protein component has one or more domains deleted, replaced, added or mutated compared to the corresponding RNA or protein component of the parent LINE or SINE. In some embodiments, the engineered RNA and / or protein component has at least 50, 60, 70, 75, 80, 85, 80, 95 or more percent sequence identity to the nucleic acid or amino acid sequence of the corresponding RNA or protein component of the parent LINE or SINE. The engineered RNA and / or protein component can include sequences that are heterologous to the corresponding RNA or protein component of the parent LINE or SINE, such as entire domains. The engineered RNA and / or protein component can also be recombinant sequences.

[0051] Typically, an RNA component containing the gene of interest to be inserted / delivered into the genome can be linked to an engineered protein component. The RNA is converted to DNA and inserted into the genome by target primed reverse transcription mediated by the protein component (first strand DNA cleavage, priming of cDNA from the released target site 3-OH, second strand cleavage, second strand synthesis).

[0052] To change the insertion site, the existing DNA binding regions of the RLE LINE, including the amino-terminal ZF / myb, the alpha finger of the linker (see examples below), and the RLE (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)), can be modified or replaced to bind and cleave the new site of interest. The ZF / myb is a candidate to be replaced with a DNA binding domain that targets the new site of interest. In some embodiments, the linker, RT, and RLE can generally be modified in place. Different RLE LINE backbones can be used and exchanged in whole and in part. Potential sources of DNA binding modules for use in the amino-terminal domain include zinc fingers from zinc finger libraries, Talen, CRISPR / cas, and others as discussed in more detail below.

[0053] When altering the coding and non-coding nucleic acid sequences of a transposon to engineer a retargeted gene delivery system, steps must be taken to ensure that each of the component parts of the system maintains structural and functional compatibility while also specifically targeting the desired site (e.g., gene location). Design considerations for key structural elements are discussed in detail below. Regardless of the component parts chosen by the practitioner, care should be taken to ensure that the engineered transposon is capable of the essential activities of RNA binding activity, DNA binding activity, DNA endonuclease activity, reverse transcriptase (RT) activity, and completion of integration by second strand synthesis. A. Engineered transposon structures

[0054] An exemplary engineered transposon-based RLE LINE backbone is outlined in Figures 18A-18D. The engineered transposon comprises an RNA component and a protein component. 1.RNA components

[0055] In general, the RNA component comprises elements that allow or facilitate binding of the protein component to the RNA component; elements that allow or facilitate targeting, preferably binding (e.g., priming), of the engineered transposon to the DNA target site; and elements that allow or facilitate one or more of the endonuclease activity, reverse transcription activity, and integration activity of the protein component or other endonuclease, reverse transcriptase, or auxiliary elements provided in trans. At a minimum, the design of the RNA component, including both its primary and secondary structure, must not interfere with, and preferably aid in, proper integration of the open reading frame of interest into the DNA target site.

[0056] Exemplary RNA components of engineered transposons are illustrated in Figure 18A. Thus, for example, the RNA components of engineered transposons can include one or more of the following: a target sequence (TS), a ribozyme (e.g., Hepatitis Delta Virus Ribozyme) (HDV), a tracr sequence (e.g., tracr, guide, or tracr / guide sequence, e.g., Cas9 targeting RNA), a sequence encoding an IRES / PBM protein binding motif domain, a promoter (e.g., a pol II promoter or a transcription factor binding site to ensure ORF expression) (Prom), an open reading frame (ORF) encoding a transgene of interest for insertion into the target site, and a PBM protein binding motif. The tracer, guide, or tracer / guide can be provided in cis or in trans. The RNA component need not, and preferably does not, include a sequence encoding an open reading from a LINE transposon.

[0057] Short Interspersed Repetitive Elements (SINEs) are parasites of APE LINEs. SINEs recruit the protein components of LINEs to integrate into the genome. Such SINEs represent, or at least approximately represent, the minimal RNA requirements for binding LINE proteins and for insertion into the genome. SINEs of RLE LINEs are called SIDEs, which represent Short Internally Deleted Elements. RLE LINE R2 has a SIDE present in various Drosophila species that contains a hepatitis delta virus-like ribozyme and the 3'PBM RNA component of the parent LINE element (DG Eickbush, TH Eickbush, Mob DNA 3, 10 (2012)).

[0058] Ribozymes are used to cleave element RNA from rRNA / R2 cotranscripts, and are present in SIDE as well as parent R2 (Eickbush, et al., Mol Cell Biol (2010); Eickbush, et al., Mob DNA 3, 10 (2012)). Many of the HDV ribozymes encoded by R2 elements cleave rDNA / R2 element cotranscripts, for example, leaving some ribosomal sequence at the 5' end of the element RNA. As explained in the experiments presented below, target sequences, if present, are used to anneal to upstream target sequences after TPRT to form a four-way junction integration intermediate. The four-way junction integration intermediate is the entry point to the second half of the integration reaction. For R2 elements where HDV removes all target sequences, template jumping occurs to form a four-way junction. Ribozymes can be optional in engineered RNAs, since RNAs are not made as cotranscripts. However, the presence of a ribozyme (e.g., the HDV ribozyme) may help protect the element RNA from degradation by cellular RNAses. In addition, the R2 protein may interact with the HDV ribozyme and / or aid in the integration reaction.

[0059] The presence of a target sequence on the engineered RNA may aid in the formation of a four-way junction, especially when using protein and RNA components from the R2 element that are known to leave a target sequence on that mRNA.

[0060] When CRISPR / Cas is used to help drive engineered RNA-protein particles (RNPs) as either a DNA-binding domain or a DNA-binding + DNA-cleavage domain, the RNA component of the engineered CRISPR / Cas-9 system can be included in the engineered R2 "SIDE" RNA.

[0061] The 3'PBM is a critical RNA element. It is the only structural component of the RNA that binds to the R2 protein that can undergo TPRT, and therefore it will be a critical component for the engineered RNA to be integrated into the genome. The sequence and structure of the 3'PBM RNA used for the engineered RNA must match the parent LINE RNA and the parent protein that binds it.

[0062] Although the 5'PBM RNA is not required for SIDE integration, it is generally a critical component of the integrated full-length R2 element. Its presence helps to form integratable RNA-protein particles (RNPs), helps to protect the RNA from degradation, and serves as a timing mechanism for entering the later stages of the integration reaction (Christensen, et al., Proc Natl Acad Sci USA 103, 17602 (2006); see also Examples below). Contained within the 5'PBM is a potential internal ribosome entry site (IRES) used by the R2 LINE to translate its mRNA. If the 5'PBM RNA is used in an engineered RNA, it may be necessary to render the IRES non-functional (e.g., mutated, deleted, eliminated, etc.).

[0063] The LINE ORF sequence within the engineered RNA component can be replaced with a gene or regulatory sequence of interest that is to be integrated into the genome. 2. Protein Ingredients

[0064] Engineered RLE LINE proteins are designed to bind the RNA component and, alone or in conjunction with other endonucleases, reverse transcriptases or auxiliary elements provided in trans, facilitate reverse transcription and integration of the gene of interest into the DNA target site. LINE-based proteins can contain many or all of the protein domains of the open reading frame of the LINE transposon. In general, engineered LINE proteins are designed to bind the RNA component, bind genomic DNA, cleave the first strand of the target DNA, perform TPRT, bind to the four-way junction intermediate, cleave the four-way junction, and facilitate second strand synthesis.

[0065] The protein components are illustrated in Figure 18B using the generic RLE ORF backbone as an example. The illustrated protein includes an N-terminal DNA binding domain (DB), an RNA binding domain (RB), a reverse transcriptase (RT), a putative α-finger (αF) and a linker containing a zinc knuckle-like CCHC motif, and a restriction enzyme-like DNA endonuclease (RLE).

[0066] The DB in R2Bm has a ZF and a myb. In R2Lp, R8Hm and R9Av, it has three ZFs and one myb. In NeSL-1, it has two ZFs. In R2Bm, myb is known to position protein subunits downstream of the insertion site and does so in the presence of 5'PBM RNA (Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)). In R2Lp, which targets the same site, myb binds upstream of the target site. The sequence where myb binds upstream of the insertion site is a degenerate palindrome of the downstream site (Thompson and Christensen, Mobile Genetic Elements 1, 29 (2011)). In NeSL, the ZF is thought to bind upstream of the insertion site and aid in targeting to the first strand break (Shivram, et al., Mob Genet Elements 1, 169 (2011)). The zinc finger in R2Bm is thought to be involved in targeting to the first strand DNA break, as in NeSL (Shivram, et al., Mob Genet Elements 1, 169 (2011)). R2 phylogenetic elements, including R8 and R9, also use ZF and myb to assist in binding of protein subunits to upstream and possibly downstream sequences (Shivram, et al., Mob Genet Elements 1, 169 (2011)). As mentioned above, the R2 SIDE lacks the 5'PBM RNA and does not itself preposition protein subunits downstream as the parental LINE does. The DB from the backbone LINE transposon can be mutated in place or replaced with a different DNA binding domain, such as a ZF from the library or an otherwise known ZF, or talen, or cas9, to target to the new site. The DB is believed to contact both upstream and downstream of the insertion site in the case of the R2 element, but only upstream of the target sequence in the case of NeSL-1.In some cases, the engineered protein can be designed to bind to an upstream sequence, and in other cases to bind to both upstream and downstream sequences.

[0067] The linker domain contains αF and CCHC zinc knuckle-like domains, as depicted in FIG. 18B (Mahbub, et al., Mob DNA 8, 16 (2017)). As described in the Examples below, αF and CCHC zinc knuckles position the target DNA in the proper position for cleavage and synthesis at all stages of the integration reaction. In particular, αF is important for binding and recognition of the four-way junction. The four-way junction is the entry point for second strand DNA cleavage and second strand DNA synthesis. In R2Bm, the sequence downstream of the insertion site (i.e., the north arm of the four-way junction) is important for DNA cleavage and is recognized by the DB. In the R2 LINE RNP, the protein subunit is already bound to the downstream DNA sequence by association with the 5'PBM RNA. The structure and sequence of the south, west and east arms are also recognized by the protein. The R2 SIDE RNP prepositions the protein subunit only at the upstream site, not downstream of the insertion site. NeSL-like elements are unlikely to bind sequences downstream of the insertion site by DB. Instead, the recognition of the four-way junction and the positioning of the endonuclease are performed by the linker, especially αF. The recognition of the four-way junction is both sequence-specific and structure-specific. αF is thought to contact the center of the four-way junction, which is similar to αF of Prp8, which binds to the multibranched RNA of the 5' splice site in the splicosome (Mahbub, et al., Mob DNA 8, 16 (2017)). See also experiments below. Thus, engineering of RLE LINE proteins to target new sites may involve modification of not only the amino-terminal DNA binding domain but also the linker, especially αF.

[0068] Although much of the target cleavage specificity can be attributed to the RLE tethered to the DB and linker, the endonuclease appears to make some key contact points with the target DNA and have some specificity (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016) and experiments below). Thus, targeting of the transposon to a new site may involve modification of the RLE.

[0069] The RNA binding domain (RB) of R2Bm binds both the 3' and 5' PBM RNAs (Jamburuthugoda and Eickbush, Nucleic Acids Res 42, 8405 (2014)). The RNA binding domain must be able to bind the RNA of the engineered transposon in a manner that leads to reverse transcription and integration at the target site. Typically, this can be accomplished by using a parental protein and a PBM RNA from the same parental LINE. However, it may be advantageous to use one parental LINE for the upstream 3' PBM binding subunit and another parental LINE for the downstream 5' PBM binding subunit. The RNA binding domain can be mutated as necessary to correct for perturbations introduced by engineering the protein and RNA components.

[0070] Figures 18C and 18D illustrate two models of engineered transposons binding to RNA moieties (18C) and reverse transcription and integration into DNA target sites (18D). Protein subunits are engineered to bind to desired gene locations. Since different RLE lineages appear to use various configurations of amino-terminal DBs to bind upstream and downstream of the insertion site, the protein subunits can be of the same parent RLE origin or different parent RLE origins. The design can take into account two insertion models: (1) R2 LINE-like integration and (2) R2 SIDE-like integration (Figure 18D).

[0071] Mutations (eg, point mutations) in the DB, linker and RLE would likely be required during retargeting to the element, as DNA binding and recognition involves each of these domains. B. Sequence Sources of RNA and Protein Components 1. Parent retrotransposon

[0072] Engineered retrotransposons are typically constructed from pre-existing LINEs or SINEs / SIDEs, or LINE or SINE / SIDE backbones, also referred to as parental LINEs or SINEs / SIDEs. Thus, the appropriate nucleic acid and amino acid sequences of the LINEs and SINEs can be adjusted, mutated, or otherwise modified as necessary to effect integration of a gene of interest at a desired target site.

[0073] For example, the RNA component sequence may be derived from a known RLE LINE or SIDE, including, but not limited to, a 3' PBM. The protein component sequence is typically derived from an RLE LINE. As discussed above, the RNA and protein components must be compatible to ensure proper reverse transcription and integration of the gene of interest.

[0074] There are two major LINE families. The two families share a common RT and linker (αF and IAP / gag-like CCHC zinc knuckles). The two families differ in their open reading frame (ORF) structure, RNA-binding domain, DNA-binding domain, and DNA endonuclease domain used for element RNP formation and integration into host DNA.

[0075] The earlier clade has a single ORF. The ORF encodes a multifunctional protein with an N-terminal zinc finger and Myb motif, an RT, a gag knuckle-like motif, and a type II restriction enzyme-like endonuclease (RLE) with a restriction enzyme-like endonuclease-like fold (RLE) (Eickbush, et al., Microbiol Spectr. 2015;3:MDNA3-0011. doi: 10.1128 / microbiolspec.MDNA3-0011-2014; and Eickbush, "R2 and related site-specific non-long terminal repeat Retrotransposons." In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington, DC: ASM Press; 2002. p. 813-35). This group of LINEs is generally site-specific during integration.

[0076] Insect R2 elements are a well-studied example of this early-diverging LINE group. Muhbub, et al., Mobile DNA (2017) 8:16 DOI 10.1186 / s13100-017-0097-9n present an updated model of the R2 RT together with an analysis of the linker region between the RT and the endonuclease. The R2 proteolysis data, together with a sequence-structure alignment of the RT, linker and RLE, show that the RLE LINE shares many commonalities with a large fragment of Prp8, a highly conserved eukaryotic splicing factor that contains RT and RLE domains.

[0077] RLE LINEs and their SIDEs can be used as parental scaffolds or as a basis for deriving the RNA and protein components of engineered transposons. 2. Source of DNA-binding domain

[0078] In some embodiments, one or more DNA binding domains or their motifs of LINE or SINE can be modified or replaced with alternative DNA binding domains. For example, N-terminal ZF (and Myb motif, if present) can represent the majority of targeting modules for all site-specific RLE-bearing non-LTR retrotransposons that contain these motifs. Myb and ZF can undergo modification that allows targeting to new sites. During modification, individual ZF and Myb motifs can be gained or lost. In addition, the configuration of physical / temporal associations between various nucleic acid binding activities (5'UTR RNA binding, 3'UTR RNA binding, upstream DNA binding, and downstream DNA binding) and catalytic activities (first strand cleavage, TPRT, second strand cleavage, and second strand synthesis) can be reconfigured to target new sites in the genome as the element translocates. Specific considerations regarding integration and linker regions are also discussed above.

[0079] In some embodiments, the surrogate DNA binding domain is derived from the DNA binding domain or motif of DNA binding protein.Examples of DNA binding domain include, but are not limited to, helix-turn-helix, zinc finger, leucine zipper, winged helix, winged helix-turn-helix, helix-loop-helix, HMG box, Wor3 domain, OB-fold domain, immunoglobulin fold, B3 domain, TAL effector, RNA guide domain, such as those in Cas protein. 3. Transgene Source

[0080] As introduced above, the RNA component typically encodes a gene of interest, also referred to herein as a transgene, and an open reading frame of interest. In some embodiments, the transgene sequence encodes one or more proteins or functional nucleic acids. The transgene may be monocistronic or polycistronic. In some embodiments, the transgene is polygenic. The transgenes may be of similar size, since LINEs range from 3-7 KB and their SINEs / SIDEs are several hundred bases. Larger transgenes may also be possible.

[0081] The disclosed engineered transposons can be used to induce gene correction, gene replacement, gene transfer, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc. For example, transposons can be used to add, i.e., insert or supplement, nucleic acid material to a target DNA sequence (e.g., to "knock in" a nucleic acid encoding a protein, siRNA, miRNA, etc.), to add tags (e.g., 6xHis, fluorescent proteins (e.g., green fluorescent protein, yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add regulatory sequences to genes (e.g., promoters, polyadenylation signals, internal ribosome entry sites (IRES), 2A peptides, start codons, stop codons, splice signals, localization signals, etc.), to modify nucleic acid sequences (e.g., to introduce mutations), etc. Thus, the compositions can be used, for example, in gene therapy, e.g., to treat disease or as anti-viral, anti-pathogen or anti-cancer therapeutics, to modify DNA in a site-specific or "targeted" manner, e.g., for gene knock-out, gene knock-in, gene editing, gene tagging, etc.

[0082] Thus, the sequence of the RNA component to be integrated at the target site is typically referred to herein as a gene of interest, a transgene, or an open reading frame of interest, although it will be understood that in some embodiments the gene of interest is not a full-length gene or a full-length transgene, but rather a fragment of a gene, a regulatory element, or another untranslated element. A polypeptide of interest

[0083] The transgene can code for one or more polypeptides of interest. The polypeptide can be any polypeptide. For example, the polypeptide of interest encoded by the transgene can be a polypeptide that provides a therapeutic or preventive effect to an organism, or can be used to diagnose a disease or disorder of an organism. The transgene can compensate for or otherwise correct a genetic disease or disorder. The transgene can function in the treatment of cancer, autoimmune disorders, parasitic infections, viral infections, bacterial infections, fungal infections, or other infectious diseases. The expressed transgene can code for a polypeptide that functions as a ligand or receptor for cells of the immune system, or can code for a polypeptide that can function to stimulate or inhibit the immune system of an organism.

[0084] In some embodiments, the transgene comprises a selectable marker, e.g., a selectable marker that is effective in eukaryotic cells, e.g., a drug resistance selection marker. The selectable marker gene can encode a factor necessary for the survival or growth of transduced host cells grown in a selective culture medium. Host cells that are not transduced with the selection gene will not survive in the culture medium. Exemplary selection genes encode proteins that confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, kanamycin, gentamicin, zeocin, or tetracycline, proteins that complement auxotrophic deficiencies, or proteins that supply vital nutrients that are subtracted from the medium.

[0085] In some embodiments, the transgene comprises a reporter gene. A reporter gene is typically a gene that is not present or expressed in the host cell. A reporter gene typically encodes a protein that provides some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the glucuronidase (GUS) gene and the GFP gene.

[0086] Additional genes, including those that produce iPCs, interleukins, receptors, transcription factors and pro- and anti-apoptotic proteins. b.Functional nucleic acid

[0087] The transgene can code for a functional nucleic acid. A functional nucleic acid is a nucleic acid molecule that has a specific function, such as binding to a target molecule or catalyzing a specific reaction. Functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNAs, miRNAs, aptamers, ribozymes, triplex-forming molecules, RNAi, and external guide sequences. Functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of the specific activity of target molecules, or functional nucleic acid molecules can have novel activities that are independent of any other molecule.

[0088] Functional nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide or sugar chain. Thus, functional nucleic acid can interact with the mRNA or genomic DNA of target polypeptide, or can interact with the polypeptide itself. In many cases, functional nucleic acid is designed to interact with other nucleic acid based on the sequence homology between target molecule and functional nucleic acid molecule. In other situations, specific recognition between functional nucleic acid molecule and target molecule is not based on the sequence homology between functional nucleic acid molecule and target molecule, but rather based on the formation of tertiary structure that allows specific recognition to occur. C. expression element

[0089] As introduced above, the transgene can contain or be operably linked to an expression control sequence that allows transgene expression when integrated into the target DNA site. Operable linkage means that the disclosed sequence is incorporated into a genetic construct such that the expression control sequence effectively controls the expression of the sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. A promoter is an expression control sequence that is comprised of a region of a nucleic acid sequence molecule, typically within 100 nucleotides upstream of the point where transcription starts (generally near the start site of RNA polymerase II).

[0090] Some promoters are "constitutive" and direct transcription in the absence of regulatory influences. Some promoters are "tissue specific" and initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are "inducible" and achieve gene transcription under the influence of an inducer. Induction can occur, for example, as a result of a physiological response, a response to an external signal, or as a result of artificial manipulation. Some promoters respond to the presence of tetracycline, and "rtTA" is a reverse tetracycline-controlled transactivator. Such promoters are well known to those skilled in the art. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome can be used to provide other genetic elements, such as SV40 origin, early and late promoters, enhancers, splice and polyadenylation sites, for expression of structural gene sequences in mammalian host cells. Viral early and late promoters are particularly useful because both are easily obtained from the viral genome as a fragment that may also contain the viral origin of replication. Exemplary expression vectors for use in mammalian host cells are well known in the art.

[0091] To place a coding sequence under the control of a promoter, it is preferred to place the translation start site of the translation reading frame of the polypeptide between 1 nucleotide and about 50 nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. Enhancers can also be located downstream from the transcription start site. A coding sequence is "operably linked" to and "under the control" of an expression control sequence in a cell if RNA polymerase is capable of transcribing the coding sequence into mRNA, which can then be translated into the protein encoded by the coding sequence. C. Design Considerations

[0092] An important consideration when designing an engineered transposon is how it will integrate into the target site. Modifications to the RNA and protein components must be made in a way that ensures integration of the gene of interest into the target site. 1. Four-way branched DNA intermediate

[0093] Second strand DNA cleavage remains puzzling because cleavage sites are generally not palindromic: the sequence surrounding the second cleavage site is often unrelated to the sequence surrounding the first strand site. In addition, cleavage can produce blunt-ended or staggered states, the latter leading to either target site duplication or target site deletion depending on the staggering of the cleavage event for that element. This staggered cleavage can be a few bases apart (e.g., 2 bp for R2Bm) or much farther apart, e.g., 126 bp for R9 (Gladyshev and Arkhipova, Gene 448, 145 (2009), Christensen and Eickbush, J Mol Biol 336, 1035 (2004)). In APE LINEs, cuts are generally staggered to produce, for example, small 10-20 target site overlaps upon insertion (Zingler, et al., Cytogenet Genome Res 110, 250 (2005); Christensen, et al. Genetica 110, 245 (2001); Ostertag, et al., Annu Rev Genet 35, 501 (2001)). Endonucleases from APE bearing LINEs (APE LINEs) appear to have some specificity for the first DNA cleavage site on the linear target DNA but much less specificity for the second (Feng, et al., Cell 87, 905 (1996), Zingler, et al., Cytogenet Genome Res 110, 250 (2005), Christensen, et al. Genetica 110, 245 (2001), Feng, et al., Proc Natl Acad Sci USA 95, 2083 (1998), Maita, et al., Nucleic Acids Res 35, 3918 (2007)).Endonucleases from RLEs with LINEs (RLE LINEs) are similarly involved in target site recognition (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)). However, in both cases, further specifiers of cleavage were evoked to explain the different specificities of first and second strand cleavage, involving endonucleases tethered to DNA by unidentified DNA binding domains in the protein. Another complicating factor is that the first cleavage event should occur in the presence of element RNA, while the second cleavage event, by a priori inference, should occur in the absence of element RNA, which has been difficult to demonstrate in vitro (Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)).

[0094] Second strand DNA synthesis has remained unsolved for more than 20 years and has never been directly observed in vitro (Cost, et al., EMBO J 21, 5899 (2002), Zingler et al., Genome Res 15, 780 (2005), Han, Mob DNA 1, 15 (2010), Eickbush, et al., PLoS One 8, e66441 (2013), Kajikawa, et al., Gene 505, 345 (2012)). Second strand synthesis is thought to be mediated by element-encoded reverse transcriptase, which is activated by the loss of priming activity of the free 3'-OH generated by the second strand cleavage event. It is unclear how the proposed primer-template association occurs when the target (ds)DNA ends are separated from each other after second-strand DNA cleavage in in vitro reactions (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)).

[0095] The R2 element from Bombyx mori, R2Bm, is one of many model systems that have been used to study LINE insertion reactions (Eickbush and Eickbush, Microbiol Spectr 3, MDNA3 (2015)). R2 elements are site-specific and target the "R2 site" of the 28S rRNA gene (Eickbush and Eickbush, Microbiol Spectr 3, MDNA3 (2015)). R2 elements encode a single open reading frame with an N-terminal zinc finger (ZF) and myb domain (Myb), a central reverse transcriptase (RT), a restriction enzyme-like endonuclease (RLE), and a C-terminal gag knuckle-like CCHC motif (Figure 1A). The R2Bm protein was expressed in E. coli and purified for use in in vitro reactions.

[0096] In vitro studies of R2Bm protein and RNA have led to a model for R2Bm integration (Figure 1B) (Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)). Two subunits of the R2 protein are thought to be involved in the integration reaction, one binding to the 3' protein binding motif (PBM) of the R2 RNA and the other to the 5' PBM. The 5' and 3' PBM RNAs direct the roles of the two subunits, coordinating a series of DNA cleavage and polymerization steps that result in element integration by TPRT (Figure 1A). The protein subunit bound to the 3' PBM of the element interacts with 28S rDNA sequences upstream of the R2 interaction site. The RLE of the upstream subunit cleaves the first (bottom / antisense) DNA strand. After first-strand target DNA cleavage, the RT of the subunit performs TPRT to synthesize first-strand cDNA using the 3'-OH generated by the cleavage event as a primer. The protein subunit bound to the 5'PBM RNA interacts with the 28S rDNA sequence downstream of the R2 insertion site by its ZF and Myb domains. The RLE of the downstream subunit cleaves the second (top / sense) DNA strand. However, second-strand DNA cleavage does not occur until the 5'PBM RNA has been extracted from the subunit, presumably by a process of TPRT that places the protein in an "RNA-free" conformation. Second-strand DNA cleavage does not occur in the absence of RNA in in vitro reactions. Second-strand cleavage required a narrow range of R2 protein, 5'PBM RNA, and target DNA ratios to be observed, until this report (Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)).In addition, because second strand breaks separate the upstream target-DNA from the downstream target-DNA, initiation of second strand DNA synthesis from the upstream target-DNA to the TPRT product binding to the downstream-DNA is problematic (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)).

[0097] DNA endonucleases play a central role in the LINE integration reaction. The RLEs found in early-diverging LINEs are variants of the PD-(D / E)XK superfamily of endonucleases (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016), Yang, et al., Proc Natl Acad Sci USA 96, 7847 (1999)). LINE RLEs share sequence and structural homology to archaeal Holliday junction resolvases (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)). However, previous studies left open the question of whether the R2 protein can function as a Holliday junction resolvase and what, if any, relevance this putative function may have in the insertion mechanism. The ability of the R2 protein to perform its transfer function on branched DNA is investigated in detail in the following examples. The results indicate that the integration-specific four-way junction is a key intermediate in the integration event and the entry point to the later stages of the integration event. This four-way junction is recognized by the RLE protein by both structure and sequence. The structural and sequence requirements can be used to aid in the design of functional engineered transposons. a. The R2 protein is not a general Holliday junction resolvase, but cleaves its own integration intermediate in a resolvase-like reaction.

[0098] It was found that the R2 protein preferentially binds to Holliday junctions, which are nonspecific four-way DNA junctions, over nonspecific linear DNA. When the R2 protein is in a minus-RNA conformation, it appears to have a larger surface for binding to junction DNA. This makes mechanistic sense for R2 incorporation, since the minus-RNA conformation of the R2 protein is more likely to perform second-strand DNA cleavage. The presence of the 5' RNA abolishes binding to nonspecific junction DNA (and nonspecific DNA in general). It is unclear which part of the R2 protein binds to the four-way DNA junction, and it may not be an endonuclease. Indeed, the experiments described below implicate the linker, and in particular the α-finger of the linker, as the primary determinant of four-way junction DNA recognition and binding. It is also unclear whether the 5' PBM binding site overlaps the junction binding surface, or whether the lack of RNA promotes a protein conformational change that in turn exposes the junction binding surface. The binding surfaces for 5' and 3' PBM RNA are thought to be distributed over most of the R2 protein, but currently the only identified RNA binding areas are domain-1 and domain-0 (Jamburuthugoda and Eickbush, Nucleic Acids Res 42, 8405 (2014)). The CCHC zinc knuckle is also thought to bind element RNA, but its true function remains unclear. It is also possible that the 5' PBM RNA forms a four-way junction-like mimic. The DNA binding surface of Holliday junction resolvase is large and highly positively charged, so it makes sense that the R2 protein would use a portion of this positive surface to bind and aid in binding to the R2 RNA (Wyatt and West, Cold Spring Harb Perspect Biol 6, a023192 (2014)).

[0099] R2 binds to non-specific DNA junctions in the absence of RNA, but then fails to resolve these junctions, and no DNA cleavage, especially no symmetric DNA cleavage, occurs. Therefore, the R2 protein is not strictly speaking a Holliday junction resolvase. However, at the more specific four-way junctions containing 28S rDNA and R2 sequences, the second / top strand 28S rDNA cleavage events were nearly symmetric with the bottom / first strand cleavage engineered into the four-way junction. This DNA cleavage activity is very similar to a Holliday junction resolvase.

[0100] Template jumping and the presence of a double-stranded 5' (south) arm appeared to be the most important junction determinants for cleavability, beyond the presence of a target sequence in the downstream 28S rDNA (north) arm. A single-stranded yeast arm was more stimulatory.

[0101] Interestingly, unless the R2 protein exists as a dimer in solution (for which there is no convincing evidence), the binding versus DNA activity graph is linear and thus consistent with the endonuclease being monomeric (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006)). The DNA sequence in the center of the junction may also be important, but the constructs tested do not accommodate this possibility, since all of the R2-specific junctions contained 5-7 bases of 28S sequence on either side of the insertion site. In addition, each junction contained at least 25 bp of R2 5'-end sequence and 25 bp of R2 3'-end sequence. The R2 3'-arm appeared to be less important. Having the R2 3'-arm formed a duplex was also inhibitory. In all DNA versions, removal of the R2 3' arm was still cleavable, albeit marginally. All DNA versions circularized by a covalent four-way junction were also difficult to cleave by the R2 protein, but the presence of a first strand cleavage event also appeared to contribute to cleavability, as the lack of an RNA-DNA hybrid, especially in the 5' arm, could contribute to reduced cleavability.

[0102] The presence of all target sites within a four-way junction was inhibitory to DNA cleavage, except when the West arm (i.e., the 28S upstream DNA arm) contained a template jump structure ("gap with flap"). This data further indicates that the West arm derived from the template jump must be within a very narrow stability range and is inhibitory if it is too stable or too rigid. A melting temperature that is too low results in the dissociation and / or formation of a large amount of single-stranded mobile region and a concomitant decrease in cleavage fidelity. b. New model with R2Bm built-in

[0103] A deeper understanding of the second half of the R2Bm insertion reaction allowed us to present an improved R2Bm integration model (Figure 7A). The first half of the integration reaction is identical to steps 1 and 2 in Figure 1B. However, after TPRT, the new model proposes a template jump or recombination event (step 3) from the 5' end of the R2 RNA to the top strand of the 28S rDNA upstream of the R2 insertion site, which results in the formation of a four-way junction. This step does not currently occur in vitro, but even if it did, it is a step that could be formed with the aid of host factors. However, the association of the cDNA with the upstream target DNA is consistent with much previous data, and the four-way junction presents a simple unified mechanism of 5' junction formation, second strand DNA cleavage and second strand DNA synthesis, leading to full-length element insertion.

[0104] This model makes sense of previous in vivo experiments that stated that an "upstream" ribosomal RNA sequence attached to the 5' end of the R2Bm element RNA is required for full-length element insertion (Fujimoto et al., Nucleic Acids Res 32, 1555 (2004), Eickbush, et al., Mol Cell Biol 20, 213 (2000)). More recently, bioinformatics and in vitro studies of the R2 RNA transcript have determined that R2 RNA and ribosomal RNA are cotranscribed as part of the same larger transcript (Eickbush, et al., PLoS One 8, e66441 (2013), Eickbush and Eickbush, Mol Cell Biol (2010)). The R2 RNA is then processed from the bulk ribosomal RNA by an HDV-like ribozyme found near the 5' end of the R2 RNA (Eickbush, et al., PLoS One 8, e66441 (2013), Eickbush and Eickbush, Mol Cell Biol (2010)). However, for many R2 elements, the final processed R2 RNA retains some ribosomal RNA on the 5' end 27 nt of the ribosomal RNA, in the case of R2Bm (Eickbush, et al., PLoS One 8, e66441 (2013)). For elements that harbor this abundance of ribosomal RNA, template jumping may not be template jumping but rather a strand invasion or recombination event (Fujimoto et al., Nucleic Acids Res 32, 1555 (2004); Eickbush, et al., Mol Cell Biol 20, 213 (2000)).However, for other R2 elements, the ribozyme does not leave a ribosomal sequence on the processed R2 RNA (e.g., Drosophila simulans R2) and template jumping is predicted to occur as illustrated in Figure 7A (Kurzynska-Kokorniak, et al., J Mol Biol 374, 322 (2007), Eickbush, et al., PLoS One 8, e66441 (2013), Stage and Eickbush, Genome Biol 10, R49 (2009), Bibillo and Eickbush, J Mol Biol 316, 459 (2002)). Both APE and RLE LINE RTs have been shown to be capable of jumping from one end of the template to the start of the other without using any homology (Bibillo and Eickbush, J Mol Biol 316, 459 (2002)). Template jumping has long been thought to be involved in 5' junction formation for both types of elements (Kurzynska-Kokorniak, et al., J Mol Biol 374, 322 (2007), Eickbush, et al., PLoS One 8, e66441 (2013), Stage and Eickbush, Genome Biol 10, R49 (2009), Bibillo and Eickbush, J Mol Biol 316, 459 (2002)). In addition to template jumping, LINE reverse transcriptases can use both DNA and RNA as templates during DNA synthesis and can shift the duplex during polymerization (Kurzynska-Kokorniak, et al., J Mol Biol 374, 322 (2007)).

[0105] The recently reported similarity of the R2 RLE to archaeal Holliday junction resolvase raised the question of whether R2 could bind and cleave branched DNA (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016); Mukha, et al., Front Genet 4, 63 (2013)). As it turns out, the R2 protein can indeed bind and cleave four-way junctions in the absence of RNA. Second-strand DNA cleavage is step 4 in Figure 7A. Second-strand cleavage occurs directly opposite the first-strand cleavage at the R2-specific four-way junction, a reaction reminiscent of Holliday junction resolvase. Second-strand cleavage is both structure- and sequence-dependent, since sequences from the adjacent insertion site area and downstream of the insertion site helped drive cleavage.

[0106] The south arm, or R2 5' arm, was the critical cleavage determinant. The presence of 5'PBM RNA prevents binding to nonspecific four-way junctions and prevents DNA cleavage at specific junctions. The R2 protein cleaves only in the absence of RNA. Three-way TPRT junctions were not good substrates for DNA cleavage.

[0107] For elements with rRNA sequences at the 5' end, such as R2Bm, it is unclear what happens to the RNA strand displaced from the heteroduplex while the cDNA strand forms the junction depicted in Figure 2-8A step 3 or to the displaced "bottom strand" target DNA flap, and what role, if any, the displaced strand plays in DNA cleavage. The displaced RNA was not included in the R2Bm-integrated four-way junction construct, and the flap was non-specific DNA. In addition, it has also not been investigated whether the upstream protein subunit is removed by jumping / recombination if the 27 nt ribosomal sequence invades the minimal DNase footprint observed when that subunit binds to linear 28S rDNA (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, J Mol Biol 336, 1035 (2004)). The constructs in Figures 4A and 4C, which contain the entire target sequence along with the transferred target DNA strand, behave much more like a junction lacking the upstream target sequence than a junction with the entire target sequence and no transferred target DNA. The recombined cDNA / target DNA duplex was 27 bp in these constructs that matched that expected for R2Bm (Eickbush, et al., PLoS One 8, e66441 (2013)).

[0108] The fifth and final piece of evidence supporting this model is that cleavage of the four-way junction generates a natural primer-template for second-strand DNA synthesis. The "downstream-binding" subunit appears to prime second-strand DNA synthesis (Figure 7A, step 5).

[0109] In vivo, host factors may help hold the junction halves together long enough to prime second strand synthesis. In vitro, the primer template is released, at least if the upstream target DNA arm consists of nonspecific DNA. c. Extrapolation of the R2 model to LINEs with different cutoff offsets

[0110] The location of the second strand DNA cleavage site relative to the first strand cleavage site varies considerably among species and even more among R2 phylogenetic groups. The misalignment of the first and second DNA cleavage events in R2Bm is a small 5' overhang of 2 bp, which results in a 2 bp target site deletion upon element insertion. In Drosophila, the R2 endonuclease produces a blunt-end type cleavage (Stage and Eickbush, Genome Biol 10, R49 (2009)). Other R2 elements produce small 3' overhangs. The model presented in Figure 7A is equally effective for elements with any of these small misalignments. The model can be adapted to elements with moderate 3' overhang misalignments by assuming local melting or movement of the TSD region to generate a four-way junction, followed by template crossover. APE LINEs tend to produce modest 3' overhang position shifts ranging from 10 to 20. Whether APE LINEs use a four-way junction structure to drive second-strand DNA cleavage and synthesis remains to be determined. Bioinformatics analysis of the 5' junctions of full-length L1 and Alu elements shows template jumping to upstream target sequences, indicating that DNA repair processes may be an alternative route to 5' junction formation for failed insertion events (Zingler et al., Genome Res 15, 780 (2005), Ichiyanagi, et al. N. Okada, Genome Res 17, 33 (2007), Gasior and Deininger, DNA Repair (Amst) 7, 983 (2008), Coufal, et al., Proc Natl Acad Sci USA 108, 20382 (2011), Richardson, et al., Microbiol Spectr 3, MDNA3 (2015)).

[0111] Twin priming of L1 is a related, but unusual, phenomenon for second strand synthesis (Ostertag and Kazazian, Genome Res 11, 2059 (2001)). Association between cDNA and upstream target DNA has been suggested for some R1 events (Stage and Eickbush, Genome Biol 10, R49 (2009)). Ribosomal sequences are important for element-RNA / target-DNA interactions during first strand synthesis of R1Bm as well as several other site-specific LINEs, but appear to be dispensable for R2Bm (Fujiwara, Microbiol Spectr 3, MDNA3 (2015), Anzai, et al., Nucleic Acids Res 33, 1993 (2005), Luan, et al., Mol Cell Biol 16, 4726 (1996)). A minority of LINEs have a much more staggered arrangement. The R9 Av element, a member of the R2 complex, produces a misalignment of 126 bp (Arkhipova, et al., Mob DNA 3, 19 (2012)). At larger misalignments, the opening of the D-loop allows template jumping and the formation of a four-way junction. d. Design considerations for maintaining embeddedness

[0112] Care must be taken in the design of the genomic DNA target site and the engineered RNA that will be inserted into the genome by the engineered LINE protein to ensure that a productive four-way junction is formed during the integration reaction. The presence or absence of a target sequence at the 5' end of the engineered RNA will depend on whether the HDV of the parent LINE leaves a target sequence behind when it is cleaved. Most ribozymes leave 10-25 nt of RNA derived from the target DNA. The R2Bm ribozyme leaves the target sequence behind. The R2Dm ribozyme does not. The survival of the target sequence determines how the four-way junction is formed, the stability of the junction's waist arm, and the location and fidelity of the second strand cleavage event. The stability of the waist arm (the size of the template jump area) appears to be determined in part by how far upstream of the insertion site of the upstream subunit it is designed to bind. For R2 elements and NeSLs, this distance is about 10-20 bases upstream of the insertion site leaving room for the formation of about two turns of the waist arm helix. Since R2BM is the parental LINE (on which most of the supporting biochemistry has been carried out), R2Bm is the preferred parental LINE protein and parental RNA.

[0113] The misalignment of the DNA cleavage event determines whether the East arm of the four-way junction becomes single-stranded or double-stranded. A misalignment that results in a 3' overhang creates a four-way junction with a single-stranded East arm. A single-stranded East arm stimulates second-strand DNA cleavage. In R2Bm, the misalignment is such that the East arm is an RNA / DNA duplex until such time that cellular RNAses remove RNA from the RNA / DNA duplex of the East arm.

[0114] Because the South arm is also the primary determinant for four-way junction recognition and cleavage, the engineered RNA will need to maintain the sequence and structural elements of that arm by ensuring that the sequence at the 5' end of the engineered RNA that will become the South arm has the appropriate sequence and properties relative to the parent LINE protein / RNA. 2. Linker region

[0115] LINEs are integrated into new sites by a process called target primed reverse transcription (TPRT). An element-encoded DNA endonuclease nicks the host chromatin to expose a free 3'-OH group, which is used by the element-encoded reverse transcriptase as a primer for reverse transcription of the element RNA at the insertion site. LINEs encode an invariant gag-like zinc knuckle cysteine / histidine-rich motif (CX2-3CX7-8HX4C) downstream of the reverse transcriptase (Jakubczak, et al., J. Mol. Biol.(1990). doi:10.1016 / 0022-2836(90)90303-4, Matsumoto, et al., Mol. Cell. Biol. 26, 5168-5179 (2006)). The spacing of the cysteines and histidines within the knuckle is typical of knuckles found in LINEs. Immediately upstream of the zinc knuckle are a series of predicted helices (Mahbub, et al., Mob. DNA 8, 1-15 (2017)).

[0116] The R2 LINE from Bombyx mori (R2Bm) is a site-specific LINE that serves as a model system that can be purified in active form and used in in vitro assays to dissect the LINE integration reaction at the biochemical level as a protein (Jakubczak, et al., J. Mol. Biol.(1990). doi:10.1016 / 0022-2836(90)90303-4, Kojima, et al., Mol. Biol. Evol.(2006). doi:10.1093 / molbev / msl067; Gladyshev, et al., Gene (2009). doi:10.1016 / j.gene.2009.08.016). The R2 ORF encodes a multifunctional protein with N-terminal zinc-finger (ZF) and myb domains involved in DNA binding, an RNA-binding (RB) domain, a central reverse transcriptase (RT), a linker region containing several conserved predicted helices (HINALP motif) and a gag-like zinc knuckle (CCHC motif), and a PD-(D / E)XK type II restriction enzyme-like endonuclease (RLE) domain (Figure 1A) (Jakubczak, et al., J. Mol. Biol.(1990). doi:10.1016 / 0022-2836(90)90303-4, Mahbub, et al., Mob. DNA 8, 1-15 (2017), Burke, et al., Mol. Cell. Biol.(1987). doi:10.1128 / MCB.7.6.2221.Updated, Yang, et al. al., Proc. Natl. Acad. Sci. USA 96, 7847-52 (1999), Christensen, et al., Nucleic Acids Res.33, 6461-6468 (2005), Jamburuthugoda, et al., Nucleic Acids Res.42, 8405-8415 (2014), Christensen, et al., Mol. Cell. Biol. 25, 6617-6628 (2005)).The R2 RNA sequences corresponding to the 5' and 3' untranslated regions (UTRs) fold into distinct structures that are known to bind the R2 protein and are therefore referred to as the 5'PBM and 3'PBM, respectively (Figure 1A) (Kierzek, et al., Nucleic Acids Res. (2008), doi:10.1093 / nar / gkm1085, Kierzek, et al., J. Mol. Biol. 390, 428-442 (2009), Christensen, et al., Proc. Natl. Acad. Sci. USA 103, 17602-17607 (2006)). Binding of the 5'PBM and 3'PBM to RNA controls protein conformation and its role in the integration reaction (Figure 8B) (Christensen, et al., Mol. Cell. Biol. 25, 6617-6628 (2005)). Selective addition of RNA, DNA and protein components allows for assaying separate steps of the integration reaction.

[0117] The R2 protein bound to the 3'PBM adopts a conformation that allows the protein to bind to the upstream 28S DNA sequence (28Su) relative to the insertion site. The domain of the R2 protein that contacts 28Su to form the upstream protein subunit remains largely unidentified (Govindaraju, et al., Nucleic Acids Res.44, 3276-3287 (2016), Thompson, et al., Elements 1, 29-37 (2011), Shivram, et al., Mob. Genet. Elements 1, 169-178 (2011)). The R2 protein bound to the 5'PBM adopts a conformation that allows the protein to bind to the downstream 28S DNA sequence (28Sd). The ZF and Myb motifs of the R2 protein contain key residues known to interact with 28Sd to form the downstream protein subunit (Christensen, et al., Nucleic Acids Res.33, 6461-6468 (2005)). The upstream and downstream protein subunits catalyze the incorporation of the R2 element in two half-reactions, each involving DNA cleavage followed by DNA synthesis (Christensen, et al., Mol. Cell. Biol. 25, 6617-6628 (2005)). The five steps of integration include: (1) an endonuclease from the upstream subunit nicks the target DNA to expose a 3'-OH at the insertion site; (2) the exposed 3'-OH is used as a primer for TPRT by the reverse transcriptase of the upstream subunit; (3) a template jump or recombination event occurs in which cDNA from the 5' end of the reverse transcribed fragment associates with the upstream target DNA sequence to form a four-way junction; (4) the downstream subunit cleaves the four-way DNA junction; and (5) the 3'-OH generated by the cleavage event is used as a primer for second strand DNA synthesis of the element.

[0118] The role of the linker region located after the RT in all LINEs has long been elusive (Mahbub, et al., Mob. DNA 8, 1-15 (2017)). Point mutations were introduced into the gag-like zinc knuckle and putative α-finger of the linker (Figure 8B). The spacing of the CCHC motif is unique to LINEs (Malik, et al., Mol. Biol. Evol. 16, 793-805 (1999), Fanning and Singer, Nucleic Acids Res.(1987). doi:10.1093 / nar / 15.5.2251). In a previous in vivo study using APE-containing human LINE-1 elements, mutation of the first two cysteines within the CCHC motif of the linker region significantly reduced LINE-1 retrotransposition (Moran, et al., Cell 87, 917-927 (1996)). Another in vivo study with human LINE-1 observed reduced levels of RNP complexes when the first two cysteines were mutated, indicating their possible role in nucleic acid binding (Doucet, et al., PLoS Genet.6, 1-19 (2010)). No reduction in RNA-binding activity was reported in vitro for human LINE-1 elements when the zinc knuckle structure was altered by substitution of the first three cysteines with serines (Piskareva, et al., FEBS Open Bio 3, 433-437 (2013)). However, in the same study, sequences C-terminal to the RT were found to be involved in RNA binding. Mutation of residues upstream of the putative α-finger in LINE-1 elements reduced retrotransposition activity in vivo (Moran, et al., Cell 87, 917-927 (1996)).The zinc knuckle itself as well as the upstream helix of the zinc knuckle have been reported to align with the α-finger and non-zinc knuckles of the eukaryotic splicing factor Prp8 (Mahbub, et al., Mob. DNA 8, 1-15 (2017), Wan, et al., Science (80-.). (2016). doi:10.1126 / science.aad6466, Bertram, et al., Cell (2017). doi:10.1016 / j.cell.2017.07.011).

[0119] The examples below test the effect on in vitro function of a series of double mutations generated throughout the putative alpha finger and zinc knuckle of R2Bm under conditions testing for DNA binding, first-strand DNA cleavage, first-strand DNA synthesis, second-strand DNA cleavage, and second-strand DNA synthesis. The results yield conclusions that can be used to aid in the design of functional engineered transposons. a. The primary role of the linker does not appear to be to join the element RNAs.

[0120] CCHC mutations reduced accumulation of ORF2 protein in ribonucleoprotein (RNP) complexes, implicating its possible involvement in binding to element RNA (Doucet, et al., PLoS Genet.6, 1-19 (2010)). Similarly, sequences upstream of the putative α-finger were found to reduce retrotransposition activity in vivo (Moran, et al., Cell 87, 917-927 (1996)). Domain swapping experiments between human and mouse L1 elements also indicate that sequences immediately upstream of the zinc knuckle are important for retrotransposition in vivo (Wagstaff, et al., PLoS One 6, (2011)). The upstream sequence functionally links the zinc knuckle to the rest of the protein in a complex but modular manner that is not well understood. A number of these domain swaps are present in the middle finger of the putative α-finger. In addition, a polypeptide containing the C-terminal 180 amino acids of ORF2 of L1H, which contains most of the α-finger and zinc knuckle, was found to bind nonspecifically to RNA in vitro, but mutation of the cysteines did not affect nucleic acid binding (Piskareva, et al., FEBS Open Bio 3, 433-437 (2013)).

[0121] In vitro studies have found that mutations in the zinc knuckle and alpha finger of R2Bm do not appreciably reduce binding to the elements 5'PBM RNA or 3'PBM RNA. However, it is noteworthy that RNA binding is inferred by the formation of distinct DNA-RNA-protein complexes in EMSA gels (Jamburuthugoda, et al., Nucleic Acids Res. 42, 8405-8415 (2014), Christensen, et al., Proc. Natl. Acad. Sci. USA 103, 17602-17607 (2006)). Protein-DNA and protein DNA-RNA complexes with either 5'PBM RNA or 3'PBM RNA have unique, well-defined migration patterns in EMSA gels (Christensen, et al., Mol. Cell. Biol. 25, 6617-6628 (2005)). Thus, amino acids that affect RNA incorporation into protein-nucleic acid complexes can be detected as changes in the ratio of protein-DNA complexes to protein-DNA-RNA complexes in a general protein titration series. Using the same assay system, the RT -1 and RT 0 domains were determined to be RNA-binding domains (Jamburuthugoda, et al., Nucleic Acids Res. 42, 8405-8415 (2014)). RNA titrations, rather than protein titrations, were also performed with some of the mutations because the changes to RNA binding were unclear. However, a role for RNA binding cannot be discounted. The RNA-binding surface is too large and point mutations are too widely distributed across the surface of the R2 protein to produce observable differences in the assay. This is one of the reasons why double point mutations were used rather than single point mutations (Jamburuthugoda, et al., Nucleic Acids Res. 42, 8405-8415 (2014)).

[0122] Mutations to the core CCHC motif (C / SC / SHC) in the zinc knuckle and to the HINALP motif (H / AIN / AALP) in the putative α-finger are consistent with localized disruption of protein structure that results in instability to form stable gel-migrating protein-nucleic acid complexes in EMSA gels. EMSA with these two mutants did not allow us to discern whether RNA was bound or not, since no distinct protein-DNA or protein-DNA-RNA bands were observed. All other mutations in the zinc knuckle and α-finger regions retained the ability to efficiently form the appropriate protein-RNA-DNA complexes in a pattern similar to that of the WT protein. b. The linker presents the nucleic acid to the RLE and RT during the first half of the integration reaction.

[0123] A comparative summary of DNA binding, cleavage, and synthesis results for each of the mutants tested in this study is presented in Table 2 below. Mutations to the core of the CCHC motif (C / SC / SHC) and to the core of the HINALP motif (H / AIN / AALP) result in an unconstrained DNA endonuclease and an inability to form stable upstream binding protein-nucleic acid complexes. All other mutants are able to form normal upstream protein-RNA-DNA complexes. Two of the alpha finger mutations (SR / AIR / A and SR / AGR / A) resulted in an overly constrained endonuclease that did not cleave. The inability to perform first strand cleavage was unrelated to the ability of the mutants to bind to upstream DNA sequences, as one of the mutants did not compromise DNA binding in the presence of 3'PBM RNA, and the other mutation actually increased the ability of the protein to bind to the target DNA in the presence of 3'PBM RNA. Rather, residues R849, R851, R854 and R856 are used to position the target DNA and / or the DNA endonuclease for first strand DNA cleavage.

[0124] Upon cleavage, the α-finger GR / AD / A and SR / AIR / A mutants were unable to perform first-strand cDNA synthesis (TPRT) on already nicked target DNA, indicating a role for the mutated residues in positioning the RT and / or nucleic acid components relative to each other. Indeed, the GR / AD / A mutant lacked any other major phenotype other than the inability to perform TPRT and slightly reduced binding to upstream DNA sequences. The zinc knuckle mutants CR / AAGCK / A, HILQ / AQ / A and RT / AH / A had slightly reduced first-strand DNA cleavage and retained near wild-type first-strand DNA synthesis activity. Upstream DNA binding was not examined carefully and appeared normal. c. The linker region is the key to the second half of the integration reaction.

[0125] The second half of the integration reaction begins with the R2 protein associating with the 5'PBM RNA and thus binding to the DNA sequence downstream of the insertion site on the linear target DNA. Mutations to the core of the CCHC motif (C / SC / SHC) and to the core of the HINALP motif (H / AIN / AALP) result in unconstrained DNA endonucleases and an inability to form stable downstream binding protein-nucleic acid complexes. All other mutants were able to form normal downstream protein-RNA-DNA complexes with linear target DNA and appeared to have minimal effects on binding to linear DNA. However, the SR / AIR / A mutations showed a slight reduction in binding to downstream sequences of linear DNA, and the zinc knuckle mutants were not quantitatively tested.

[0126] The second half of integration proceeds only when the downstream subunit is in an "RNA-free" state (Christensen, et al., Proc. Natl. Acad. Sci. USA 103, 17602-17607 (2006)). Second-strand DNA cleavage can occur on linear DNA, but this requires a complex set of 5' RNA, DNA and protein ratios, and second-strand DNA cleavage is non-productive in the sense that no second-strand synthesis occurs (Christensen, et al., Mol. Cell. Biol. 25, 6617-6628 (2005), Christensen, et al., Proc. Natl. Acad. Sci. USA 103, 17602-17607 (2006)). For this reason, it is now believed that the formation of a four-way junction is mechanistically required for the later stages of the integration reaction, specifically second strand DNA cleavage and second strand synthesis (see Examples 1-8), which properly cleaves the junction in the absence of RNA, and the cleavage products are substrates for second strand synthesis (see Examples 1-8).

[0127] All zinc knuckle and α-finger mutants tested, except for the CR / AAGCK / A mutant, were unable to perform second-strand cleavage on linear DNA (Table 2), but importantly, the zinc knuckle mutants did not impair second-strand cleavage on the more critical four-way junction. The α-finger mutations closest to the zinc knuckle, SR / AIR / A and SR / AGR / A, greatly reduce binding to four-way junctions and abolish second-strand DNA cleavage. Second-strand synthesis was similarly affected by these two sets of mutations. The results indicate that the α-finger is important for recognition of the four-way junction as well as presenting the bound DNA to endonucleases and reverse transcriptase. The zinc knuckle mutants HILQ / AQ / A and RT / AH / A significantly reduced second-strand synthesis. This indicates that the zinc knuckle residues are involved in positioning the junction to be cleaved and / or reverse transcriptase for primer extension. d. Structural and functional connections with APE LINE and with Prp8

[0128] The protein encoded by R2Bm was determined to consist of two globular domains. The larger of the two domains (colored in Figures 17A-17D) contains the RT, RLE, and a region between the two called the linker (Mahbub, et al., Mob. DNA 8, 1-15 (2017)). The end of the linker region contains an invariant zinc knuckle and several conserved helices upstream of the zinc knuckle. The upstream helix is ​​referred to herein as the "putative α-finger" and its HINALP motif is in the center of the α-finger in R2Bm. The APE LINE also contains a "linker" with a putative α-finger and a zinc knuckle located beyond the RT (Figures 17A-17D).

[0129] The large globular domain of R2Bm, the RLE LINE, shares not only sequence similarity but also structural similarity with a large fragment of the eukaryotic splicing factor Prp8 (see Figures 17A-17D). Prp8 has an RT, an RLE, and a linker region between the RT and the RLE. Towards the end of the linker region in Prp8, there is a non-zinc knuckle structure. Upstream of the non-zinc knuckle are a series of helices that align with the helices found upstream of the zinc knuckle in the LINE. The helices upstream of the non-zinc knuckle in Prp8 form a highly promising and important α-finger. The α-finger protrudes beyond the reverse transcriptase (see Figure 17C) (Bertram, et al., Cell (2017). doi:10.1016 / j.cell.2017.07.011). Due to similarity with the α-finger in Prp8, the corresponding region of the RLE LINE is called the "putative α-finger" (Mahbub, et al., Mob. DNA 8, 1-15 (2017)). In Prp8, the non-zinc knuckle, α-finger and RT thumb act together to bind splice sites and spliceosomal RNAs. The non-zinc knuckle and α-finger are dynamic in Prp8, undergoing / facilitating protein and protein-RNA conformational changes across all aspects of the splicing region. The fact that the α-finger and non-zinc knuckle bind key branched RNA structures in the U4 / U6.U5 tri-snRNP and in the B complex is particularly interesting.

[0130] The data reported here indicate that whatever the actual structure of the R2Bm linker, it is central to the recognition of the four-way junction integration intermediate and also acts as a protein-DNA conformational switch or hub to correctly position the EN, RT, and substrate DNA relative to each other. e. Design considerations for maintaining embeddedness

[0131] The linker region is an important DNA binding region and protein-nucleic acid conformation control region. The linker region makes specific or non-specific contact points. Both α-fingers and IAP / Gag-like zinc knuckles modulate DNA cleavage and DNA synthesis events. In particular, α-fingers are involved in binding to four-way junctions. They are thought to contact the center of the four-way junction, like the α-fingers of Prp8, a multi-branched RNA structure, which is located at the center of the 5' splice site. Transposon α-fingers are also likely to make base-specific contact points in addition to non-specific contact points. The linker is also thought to be involved in binding to LINE RNA. When designing engineered LINE proteins, engineered RNAs, and target DNAs, care must be taken to either maintain parental protein contacts between a particular target DNA sequence and RNA sequence, or to mutate the linker to make new desired DNA / RNA contact points. III.How to use

[0132] The disclosed compositions can be used ex vivo or in vivo to introduce a gene of interest into a DNA target site of interest. For example, in a preferred embodiment, the RNA and protein components of an engineered transposon are delivered to or otherwise expressed in a cell, and the gene of interest is integrated into the cell's genome at the DNA target site of interest. The RNA component can be delivered as RNA, or can be delivered as DNA (e.g., an expression vector) that codes for the RNA component. The protein component can be delivered as a protein, or can be delivered as RNA or DNA (e.g., an expression vector) that codes for the protein component. In some embodiments, a vector that codes for the protein is expressed in a bacterial or eukaryotic expression system, and the protein is recovered and delivered to the target cell. In some embodiments, the RNA is prepared by in vitro transcription, and / or the protein is prepared by in vitro transcription / translation. The RNA and protein components can be expressed from the same or different vectors. A. Vectors and Host Cells

[0133] Also provided are vectors and host cells for preparing engineered transposons.Suitable expression vectors include, but are not limited to, plasmids and viral vectors derived from, for example, bacteriophage, baculovirus, tobacco mosaic virus, herpes virus, cytomegalovirus, retrovirus, vaccinia virus, adenovirus and adeno-associated virus.A large number of vectors and expression systems are commercially available from companies such as Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen Life Technologies (Carlsbad, CA).

[0134] The expression vector can include a tag sequence. The tag sequence is typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere in the polypeptide, such as at either the carboxyl or amino terminus. Useful tags include, but are not limited to, green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, Flag™ (Kodak, New Haven, CT), maltose E binding protein and protein A.

[0135] A vector containing a nucleic acid to be expressed can be transferred into a host cell. The term "host cell" is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced. As used herein, "transformed" and "transfected" encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques. Without being limited to a particular technique, many of these techniques are well established in the art. Prokaryotic cells can be transformed with a nucleic acid, for example, by electroporation or calcium chloride-mediated transformation. Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection.

[0136] Useful prokaryotic and eukaryotic cell systems for expressing and producing polypeptides are well known in the art, for example, Escherichia coli strains, such as BL-21, and cultured mammalian cells, such as CHO cells. B. Methods for editing a cell's genome

[0137] The method typically includes contacting a cell with an effective amount of engineered transposon to modify the genome of the cell. As discussed herein, contacting a cell with an engineered retrotransposon means that both the RNA component and the protein component are present in the same cell at the same time. In some embodiments, the RNA and protein components are mixed before contacting the cell. In some embodiments, they are contacted with the cell separately, and they form a complex only in the cell. In some embodiments, one or both of the components are delivered when the DNA is expressed in the cell. Any of these embodiments may include the use of electroporation, lipofection, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran or other suitable transfection methods to facilitate delivery of nucleic acid or protein to the cell.

[0138] As discussed in more detail below, the contacting can be performed ex vivo or in vivo. In a preferred embodiment, the method comprises contacting a population of target cells with an effective amount of an engineered retrotransposon to achieve a therapeutic result.

[0139] For example, an effective amount or therapeutically effective amount can be a dosage sufficient to treat, inhibit or alleviate one or more symptoms of a disease or disorder, or otherwise provide a desired physiological effect, such as the alleviation, inhibition or reversal of one or more of the underlying pathophysiological mechanisms underlying a disease or disorder.

[0140] The formulation is made to suit the method of administration. Pharmaceutically acceptable carriers depend in part on the specific composition to be administered, and also on the specific method used to administer the composition.Therefore, there are a wide variety of suitable formulations of pharmaceutical compositions containing nucleic acids and proteins.The exact dosage will vary according to various factors, such as subject-dependent variables (e.g., age, immune health, clinical condition, etc.). 1. Ex vivo gene therapy

[0141] In some embodiments, ex vivo gene therapy of cells is used to treat a disease or disorder, including but not limited to a genetic disorder, in a subject. For ex vivo gene therapy, cells can be isolated from a subject and contacted with a composition ex vivo to generate cells containing an inserted transgene. In a preferred embodiment, the cells are isolated from the subject to be treated or from a syngenic host. The target cells are removed from the subject prior to contact with the engineered retrotransposon. In some embodiments, the cells are hematopoietic progenitor or stem cells. In a preferred embodiment, the target cells are CD34 +Hematopoietic stem cells.Hematopoietic stem cells (HSCs), such as CD34+ cells, are pluripotent stem cells that give rise to all blood cell types, including red blood cells.Therefore, CD34+ cells can be isolated from patients with, for example, thalassemia, sickle cell disease or lysosomal storage disease, and the mutant gene can be modified or repaired ex vivo using the disclosed compositions and methods, and the cells can be introduced back into the patient as treatment or therapy.

[0142] Stem cells may be isolated and enriched by one skilled in the art. + Such methods of isolating and enriching for hematopoietic and other cells are known in the art and are disclosed, for example, in U.S. Patent Nos. 4,965,204, 4,714,680, 5,061,620, 5,643,741, 5,677,136, 5,716,827, 5,750,397 and 5,759,793. As used herein with reference to compositions enriched for hematopoietic progenitor and stem cells, "enriched" refers to a population of desired elements (e.g., hematopoietic progenitor and stem cells) that are present to a higher degree than that found in the natural source of the cells. The composition of cells may be enriched by at least one order of magnitude, preferably two or three orders of magnitude, more preferably 10, 100, 200 or 1000 orders of magnitude, than the natural source of the cells.

[0143] Once progenitor or stem cells are isolated, they can be propagated by growing in any suitable medium.For example, progenitor or stem cells can be grown in conditioned medium from stromal cells, such as those that can be obtained from bone marrow or liver in relation to secretion of factors, or in medium that contains cell surface factors that support the proliferation of stem cells.Stromal cells can be removed from hematopoietic cells using monoclonal antibodies suitable for removing unwanted cells.

[0144] The modified cells can also be maintained or propagated in culture prior to administration to a subject. Culture conditions are generally known in the art depending on the cell type.

[0145] In other embodiments, the technology is used as part of a CAR T-based therapy. Immune cells are harvested (e.g., T cells) and taken from the patient's blood. A chimeric antigen receptor (CAR) is introduced into the cell's genome at a targeted site using an engineered transposon. Large numbers of CAR T cells can be grown in the laboratory and administered to the patient by infusion. CAR T cell therapy is used to treat several types of cancer. 2. In vivo gene therapy

[0146] The disclosed compositions can be administered directly to a subject for in vivo gene therapy. A. Pharmaceutical preparations

[0147] The disclosed compositions can be preferably utilized in combination with a pharmaceutical carrier suitable for therapeutic use. Such compositions include an effective amount of the composition and a pharma- ceutically acceptable carrier or excipient.

[0148] Those skilled in the art will understand that nucleotides administered in vivo are taken up and distributed in cells and tissues (Huang, et al., FEBS Lett., 558(1-3):69-73 (2004)). For example, Nyce et al. have shown that antisense oligodeoxyribonucleotides (ODNs) bind to endogenous surfactant (lipid produced by lung cells) when inhaled and are taken up by lung cells without the need for additional carrier lipids (Nyce, et al., Nature, 385:721-725 (1997)). Small nucleic acids are easily taken up by T24 bladder cancer tissue culture cells (Ma, et al., Antisense Nucleic Acid Drug Dev., 8:415-426 (1998)).

[0149] The disclosed compositions may be in a formulation for local, topical or systemic administration in a suitable pharmaceutical carrier.Remington's Pharmaceutical Sciences, 15th Edition by EW Martin (Mark Publishing Company, 1975) discloses representative carriers and preparation methods.The compound may be encapsulated in suitable biocompatible microcapsules, microparticles, nanoparticles or microspheres formed of biodegradable or non-biodegradable polymers or proteins, or in liposomes for cell targeting.Such systems are well known to those skilled in the art and can be optimized for use with suitable nucleic acids.

[0150] Various methods for nucleic acid delivery are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1994). Such nucleic acid delivery systems include the desired nucleic acid, for example, but not limited to, either in "naked" form as a "naked" nucleic acid, or in a suitable vehicle for delivery, for example, in a complex with a cationic molecule or a liposome-forming lipid, or as a component of a vector or a pharmaceutical composition. The nucleic acid delivery system can be provided to a cell directly, for example, by contacting it with the cell, or can be provided to a cell indirectly, for example, by the action of any biological process. Nucleic acid delivery system can be provided to cells by physical means such as endocytosis, receptor targeting, coupling with native or synthetic cell membrane fragments, electroporation, combining nucleic acid delivery system with polymer carriers, such as controlled release films or nanoparticles or microparticles, using vectors, injecting nucleic acid delivery system into tissue or fluid surrounding cells, simple diffusion of nucleic acid delivery system across cell membrane, or any active or passive transport mechanism across cell membrane.In addition, nucleic acid delivery system can be provided to cells using techniques such as antibody-associated targeting and antibody-mediated immobilization of viral vectors.

[0151] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, or thickeners may be used as desired.

[0152] For example, suitable formulations for parenteral administration, such as administration by intra-articular (into a joint), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, solutions or emulsions, which may contain suspending agents, solubilizing agents, thickening agents, dispersing agents, stabilizers, and preservatives. Preparations for injection can be provided in unit dosage form, for example in ampoules or in multi-dose containers, with preservatives added as necessary. Compositions can take the form of sterile aqueous or non-aqueous solutions, suspensions and emulsions, which in certain embodiments may be isotonic with the blood of the subject. Examples of non-aqueous solvents are polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or fixed oils such as synthetic mono- or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, such as saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers (such as those based on Ringer's dextrose). Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases. In addition, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injections. Carrier formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. Those skilled in the art can easily determine various parameters for preparing and formulating compositions without resorting to undue experimentation.

[0153] The disclosed compositions, alone or in combination with other suitable components, can also be made into aerosol formulations (i.e., they can be "nebulized") to be administered by inhalation.Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen and air.For administration by inhalation, the compound is delivered in the form of aerosol spray extrusion from pressurized packs or nebulizers by using suitable propellants.

[0154] In some embodiments, the composition comprises a pharma- ceutically acceptable carrier together with formulation components, such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, fillers, desiccants, antioxidants, antimicrobial agents, preservatives, binders, bulking agents, silica, solubilizers or stabilizers.In one embodiment, nucleic acid is conjugated to lipophilic groups, such as cholesterol derivatives, lauric acid derivatives and lithocholic acid derivatives, with C32 functional groups, to improve cellular uptake.For example, cholesterol has been demonstrated to enhance the uptake and serum stability of siRNA in vitro (Lorenz, et al., Bioorg. Med. Chem. Lett., 14(19):4975-4977 (2004)) and in vivo (Soutschek, et al., Nature, 432(7014):173-178 (2004)). In addition, it has been shown that the binding of steroid-conjugated oligonucleotides to various lipoproteins in the bloodstream, such as LDL, protects their integrity and promotes biodistribution (Rump, et al., Biochem. Pharmacol., 59(11):1407-1416 (2000)).Other groups that can be attached or conjugated to the above compounds to increase cellular uptake include acridine derivatives; crosslinkers, such as psoralen derivatives, azidophenacyl, proflavine, and azidoproflavine; artificial endonucleases; metal complexes, such as EDTA-Fe(II) and porphyrin-Fe(II); alkylating moieties; nucleases, such as alkaline phosphatase; terminal transferase; antibody enzymes; cholesteryl moieties; lipophilic carriers; peptide conjugates; long-chain alcohols; phosphate esters; radioactive markers; non-radioactive markers; carbohydrates; and polylysine or other polyamines. Enhanced delivery methods are also described in U.S. Patent No. 6,919,208 to Levy et al. These pharmaceutical preparations can be manufactured in a manner known per se, for example, by conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. b. Method of administration

[0155] In general, the method of administering nucleic acid and protein compositions is well known in the art.In particular, the administration route already used for nucleic acid therapeutics, together with the currently used formulations, provides the preferred administration and formulation route of the above engineered transposon.Preferably, the composition is injected into the organism that undergoes genetic engineering, such as the animal that needs gene therapy.

[0156] The disclosed compositions can be administered by a number of routes, including but not limited to oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, rectal, intranasal, pulmonary, and other suitable means.The compositions can also be administered by liposomes.Such administration routes and suitable formulations are generally known to those skilled in the art.

[0157] Administration of the formulations can be accomplished by any acceptable method that allows the gene editing compositions to reach their targets.

[0158] Any acceptable method known to those of skill in the art can be used to administer the formulation to a subject. Administration can be localized (i.e., to a particular area, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated.

[0159] Injection can be, for example, intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal injection. In some embodiments, injection can be administered at multiple locations. Implantation includes inserting an implantable drug delivery system, for example, a microsphere, a hydrogel, a polymer reservoir, a cholesterol matrix, a polymer system, for example, a matrix erosion and / or diffusion system, and a non-polymer system, for example, a compressed, fused or partially fused pellet. Inhalation includes administering the composition alone or bound to a carrier that can be absorbed with an aerosol in an inhaler. For systemic administration, it may be preferable that the composition is encapsulated in a liposome.

[0160] The composition can be delivered in a manner that allows tissue-specific uptake of the drug and / or nucleotide delivery system. Techniques include the use of tissue or organ localization devices, such as wound dressings or transdermal delivery systems, the use of invasive devices, such as vascular or urinary catheters, and the use of interventional devices, such as stents capable of drug delivery and configured as expansion devices or stent grafts.

[0161] The formulation can be delivered by diffusion or by degradation of the polymer matrix using biodegradable implants.In certain embodiments, the administration of the formulation can be designed to result in continuous exposure to the composition over a certain period of time, for example, hours, days, weeks, months or years.This can be achieved, for example, by repeated administration of the formulation, or by a sustained or controlled release delivery system in which the composition is delivered over an extended period of time without repeated administration.The administration of the formulation using such a delivery system can be, for example, by oral dosage form, bolus injection, transdermal patch or subcutaneous implant.In some cases, it may be preferable to maintain a substantially constant concentration of the composition.

[0162] Other suitable delivery systems include sustained, delayed, sustained or controlled release delivery systems. Such systems can often avoid repeated administration, thus increasing the convenience for subjects and physicians. Many types of release delivery systems are available and known to those skilled in the art. They include polymer-based systems, such as, for example, polylactic acid and / or polyglycolic acid, polyanhydrides, polycaprolactones, copolyoxalates, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and / or combinations thereof. Microcapsules of the aforementioned polymers containing nucleic acids are described, for example, in U.S. Pat. No. 5,075,109. Other examples include lipid-based non-polymeric systems, such as sterols, such as cholesterol, cholesterol esters, and fatty acids or meso-fatty acids, such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid-based systems; silastic systems; peptide-based systems; wax coatings; compressed tablets using conventional binders and excipients; or partial fusion implants. Specific examples include erosion systems in which oligonucleotides are contained in the formulation within a matrix (e.g., those described in U.S. Pat. Nos. 4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and 5,239,660), or diffusion systems in which the active ingredient controls the release rate (e.g., those described in U.S. Pat. Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The formulation may be present, for example, as a microsphere, hydrogel, polymer reservoir, cholesterol matrix, or polymer system. In some embodiments, the system may allow for sustained or controlled release of the composition, for example, by controlling the diffusion or erosion / degradation rate of the formulation containing engineered transposon. Additionally, one or more embodiments can be delivered using a delivery system that is a pump-based device.

[0163] Examples of the system that releases in a burst include the system that the composition is entrapped in liposomes that are encapsulated in a polymer matrix; the liposomes that are sensitive to certain stimuli, such as temperature, pH, light, or degrading enzymes; and the system that the composition is encapsulated in ion-coated microcapsules with microcapsule core degrading enzymes.Examples of the system that releases inhibitors gradually and continuously include, for example, the erosion system that contains the composition in a form that is in a matrix, and the leaching system that the composition permeates, for example, through a polymer, at a controlled rate.Such a sustained release system can be in the form of pellets or capsules.

[0164] In some embodiments, the use of long-term release implants may be particularly suitable.As used herein, "long-term release" means that the implant containing the composition is constructed and arranged to deliver therapeutically effective levels of the composition for at least 30 or 45 days, preferably at least 60 or 90 days, or in some cases even longer.Long-term release implants are well known to those skilled in the art and include some of the release systems described above. c. Preferred Formulations for Mucosal and Pulmonary Administration

[0165] The active agents and compositions thereof can be formulated for pulmonary or mucosal administration. Administration can include delivery of the compositions to the pulmonary, nasal, oral (sublingual, buccal), vaginal or rectal mucosa.

[0166] In one embodiment, the compound is formulated for intrapulmonary delivery, such as intranasal administration or oral inhalation. The airways are structures involved in gas exchange between the outside air and the bloodstream. The lungs are branched structures with alveoli at the end, where gas exchange occurs. The surface area of ​​the alveoli is the largest in the respiratory system, where drug absorption takes place. The alveoli are covered by a thin, non-ciliated epithelium, or mucus layer, and secrete phospholipids, which are surfactants. The airways include the upper airways, which include the oropharynx and larynx, followed by the lower airways, which include the trachea, which then bifurcates into the bronchi and bronchioles. The upper and lower airways are called conducting airways. The terminal bronchioles then divide into respiratory bronchioles, which then lead to the alveoli, or deep lungs, which are the final respiratory zone. The deep lungs, or alveoli, are the primary target of therapeutic aerosols inhaled for systemic drug delivery.

[0167] Pulmonary administration of therapeutic compositions composed of low molecular weight drugs is seen, for example, in beta-androgen antagonists for treating asthma. Other therapeutic agents that are active in the lungs are administered systemically and targeted by pulmonary absorption. Nasal delivery is considered a promising technique for administration of therapeutic agents for the following reasons: the nose has a large surface area available for drug absorption due to the numerous microvilli that cover the epithelium; the subepithelial layer is rich in blood vessels; venous blood passes directly from the nose into the systemic circulation, thus avoiding loss of drug through first-pass metabolism in the liver; nasal delivery allows for lower doses, more rapid attainment of therapeutic agent blood levels, more rapid onset of pharmacological activity, fewer side effects, and fewer clots. 3 It provides a high total blood flow per septum (porous epithelial basement membrane); and intranasal delivery is easy to access.

[0168] The term aerosol as used herein refers to any preparation of a mist of particles, which may be in solution or suspension, whether or not it is generated using a propellant. Aerosols can be generated using standard techniques such as ultrasound or high pressure processing.

[0169] Carriers for pulmonary formulations can be divided into those for dry powder formulations and those for administration as a solution. Aerosols for delivering therapeutic agents to the respiratory tract are known in the art. Formulations for administration via the upper respiratory tract can be formulated in buffered or unbuffered solutions, such as water or isotonic saline, or as suspensions for intranasal administration as drops or as a spray. Preferably, such solutions or suspensions are isotonic with respect to nasal secretions and have about the same pH, for example, a pH range of about pH 4.0 to about pH 7.4, or pH 6.0 to pH 7.0. Buffers should be physiologically compatible and include, by way of example only, phosphate buffers. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2. Those skilled in the art can easily determine the appropriate salinity and pH for a non-invasive aqueous solution for nasal and / or upper respiratory tract administration.

[0170] Preferably, the aqueous solution is water, a physiologically acceptable aqueous solution containing salts and / or buffers, such as phosphate buffered saline (PBS), or any other aqueous solution acceptable for administration to animals or humans. Such solutions are well known to those skilled in the art and include, but are not limited to, distilled water, deionized water, pure or ultrapure water, saline, phosphate buffered saline (PBS). Other suitable aqueous vehicles include, but are not limited to, Ringer's solution and isotonic sodium chloride. Aqueous suspensions may contain suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and tragacanth gum, and wetting agents, such as lecithin. Suitable preservatives for aqueous suspensions include ethyl p-hydroxybenzoate and n-propyl p-hydroxybenzoate.

[0171] In another embodiment, solvents that are low toxicity organic (i.e., non-aqueous) Class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol, may be used in the formulation. The solvent is selected based on its ability to easily aerosolize the formulation. The solvent must not adversely react with the compound. A suitable solvent should be used that dissolves the compound or forms a suspension of the compound. The solvent should be sufficiently volatile to allow the formation of an aerosol of the solution or suspension. Additional solvents or aerosolizing agents, such as Freon, may be added as desired to increase the volatility of the solution or suspension.

[0172] In one embodiment, the composition may contain small amounts of polymers, surfactants or other excipients known to those skilled in the art. "Small amounts" in this context means the absence of excipients that may affect or mediate uptake of the compound into the lungs, and that any excipients that are present are present in amounts that do not adversely affect uptake of the compound into the lungs.

[0173] Dry lipid powders can be dispersed directly in ethanol due to their hydrophobicity. For lipids stored in organic solvents such as chloroform, the desired amount of solution is placed in a vial and the chloroform is evaporated under a nitrogen stream to form a dry thin film on the surface of the glass vial. This thin film swells easily upon reconstitution in ethanol. The suspension is sonicated to completely disperse the lipid molecules in the organic solvent. A non-aqueous suspension of lipids can also be prepared in absolute ethanol using a reusable PARI LC Jet+ nebulizer (PARI Respiratory Equipment, Monterey, CA). C. The disease being treated

[0174] The disclosed engineered transposons are particularly useful for the treatment of genetic defects, genetic disorders and diseases caused by single gene mutations, for example, for the correction of genetic defects, genetic disorders and diseases caused by point mutations. When the target gene contains a mutation that is the cause of a genetic disorder, the disclosed compositions can be used for mutation repair, which can restore the DNA sequence of the target gene to normal. The target sequence can be within the coding DNA sequence of the gene, or within an intron. The target sequence can also be within the DNA sequence that regulates the expression of the target gene, such as a promoter or enhancer sequence. The disclosed transposons can additionally or alternatively deliver wild-type or equivalent and enhanced versions of the gene of interest to cells, or deliver new (e.g., heterologous) genes to cells. Thus, this technology can repair or replace genes, complement genes, or add new genes.

[0175] When the target gene is an oncogene that causes unregulated proliferation, for example in cancer cells, engineered transposons are useful for inactivating the gene and causing mutations that terminate or reduce the uncontrolled proliferation of cells. Engineered transposons are also useful anti-cancer drugs for activating repressor genes that have lost their ability to suppress proliferation. The target gene may also be a gene that codes for an immune regulator, such as PD-1, to enhance the host's immune response to cancer. Thus, engineered transposons can be designed to reduce or prevent the expression of PD-1, and can be administered in an amount effective to reduce or prevent it.

[0176] Engineered transposons can be used as antiviral agents, for example, when designed to modify specific portions of the viral genome necessary for proper growth or function of the virus. EXAMPLES

[0177] Muhbub, et al., Mobile DNA (2017) 8:16 DOI 10.1186 / s13100-017-0097-9 is specifically incorporated by reference in its entirety. Example 1 The R2 protein preferentially binds nonspecific four-way junction DNA over nonspecific linear DNA. material and method Protein purification

[0178] R2Bm protein expression and purification was performed as previously published (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)). Briefly, BL21 cells containing the R2 expression plasmid were grown in LB broth and induced with IPTG. Induced cells were pelleted by centrifugation, resuspended, and gently lysed in HEPES buffer containing lysozyme and Triton X-100. Cellular DNA and debris were spun down, and the supernatant containing the R2Bm protein was purified with Talon resin (Clontech #635501). R2Bm protein was eluted from the Talon resin column and stored in protein storage buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 50% glycerol, 0.1% Triton X-100, 0.1 mg / ml bovine serum albumin (BSA), and 2 mM dithiothreitol (DTT) and stored at -20°C. R2 protein was quantified by SYPRO Orange (Sigma #S5692) staining of samples run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which BSA was added for storage. All quantification was performed using FIJI software analysis of digital photographs (Schindelin et al., Nat Methods 9, 676 (2012)). Nucleic acid preparation

[0179] Oligos containing the 28S R2 target DNA sequence, non-target (non-specific) DNA sequence and the R2 sequence were ordered from Sigma-Aldrich. The designations upstream (28Su) and downstream (28Sd) target DNA refer to the R2 insertion dyad in the 28S rRNA gene. The oligo sequences are reported in Table 1.

[0180] All linear DNAs were 50 bp in length. Each arm for the majority of the 3-way and 4-way junctions was 25 bp in length, except for the junctions tested for cDNA synthesis where the 28S DNA arm length was strategically varied to observe second strand synthesis products. Schematic diagrams of the constructs are provided in the main figure. Oligos with 28Sd sequences contained either 25 bp or 47 bp after the R2 insertion site of the 28S rDNA. Seven base pairs of upstream sequence were also included in these "downstream" oligos to span the insertion site. Oligos with 28Su sequences also contained 72 bp before the insertion site and 5 bp after the R2 insertion site of the 28S rDNA. The largest oligo contained 72 bp upstream and 47 bp downstream of the 28S rDNA. Some oligos incorporated 25 bp of sequence complementary to either the 3' or 5' RNA. Shorter oligos (25 bp) of sequence corresponding to the first and last 25 bp of R2Bm were also used in many of the constructs. Sequences for the x, h, b and r strands of the non-specific 4-way junction were obtained from Middleton et al. (Middleton and Bond, Nucleic Acids Res 32, 5442 (2004)). Constructs were formed by a component oligo annealing procedure: 20 pmol of the labeled oligo was mixed with 66 pmol of each cold oligo. Primers were annealed in SSC buffer (15 mM sodium citrate and 0.15 M sodium chloride) for 2 min at 95°C, followed by 10 min at 65°C, 10 min at 37°C, and finally 10 min at room temperature. One of the component oligos was 5'32P end-labeled before the other component oligo was annealed. Annealed junctions were purified by polyacrylamide gel electrophoresis, eluted in gel elution buffer (0.3 M sodium acetate, 0.05% SDS and 0.5 mM EDTA pH 8.0), extracted with chloroform, precipitated with ethanol and resuspended in Tris-EDTA. Junctions sharing a common labeled oligo were matched to equal DNA numbers; otherwise equal volumes of purified constructs were generally used for the R2 reaction.R2 3'PBM RNA (249 nt), 5'PBM RNA (320 nt), and nonspecific RNA (180 nt) were generated by in vitro transcription as previously published (Gasior, et al., J Mol Biol 357, 1383 (2006)). R2Bm reaction and analysis

[0181] R2 protein and target DNA binding and cleavage reactions were performed primarily as previously reported (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)). Briefly, each DNA construct was tested for its ability to bind R2 protein and undergo DNA cleavage in the presence and absence of 5'PBM RNA, 3'PBM RNA and non-specific RNA. All reactions contained excess low-temperature competitor DNA, dIdC. Reactions were loaded onto electrophoretic mobility shift assay (EMSA) gels and comparative denaturing gels for analysis. Ability to bind branched and linear DNA was obtained from the EMSA gel, and ability to cleave DNA and cleavage location was obtained from the denaturing urea gel. An A+G ladder was run in parallel with the reactions on the denaturing gel to aid in mapping the cleavage. Second strand synthesis assays were performed by adding dNTPs to the DNA cleavage reactions in the absence of RNA. All gels were dried, exposed to a phosphorimager screen, and scanned using a phosphorimager (Molecular dynamics STORM 840). The resulting 16-bit TIFF images were linearly corrected so that the most intense bands were dark gray. Corrected TIFF files were quantified using FIJI (Schindelin et al., Nat Methods 9, 676 (2012)).

[0182] Table 1. A table presenting the DNA and RNA oligonucleotides used to construct the linear and junction DNA. "Comp" stands for the complementary strand. [Table 1-1] [Table 1-2] result

[0183] Holliday junction resolvase binds to and symmetrically cleaves four-way DNA junctions (Holliday junctions), resulting in the resolution of the junctions into linear DNA. Holliday junction resolvase recognizes DNA structure rather than DNA sequence. R2 RLE, which shares structural and amino acid sequence homology with archaeal Holliday junction resolvase, may exhibit similar DNA binding and cleavage activity.

[0184] The potential of R2 protein to recognize and bind to four-way DNA branched structures was tested by comparing the relative binding abilities of R2 protein to nonspecific linear and nonspecific four-way junction DNA - individually and competitively (Figure 2A-2B). Linear and junction DNA were formed by annealing complementary oligos. Linear and junction DNA shared a common DNA oligo that was radioactively labeled before annealing. Sharing a common labeled DNA strand allowed radioactive decay counts to serve as a proxy for equalizing DNA concentrations between linear and junction DNA and to probe for similar DNA sequences. DNA binding was analyzed by electrophoretic mobility shift assay (EMSA). In the absence of RNA (Figure 2A-2B), R2 protein bound with nearly equal efficiency to both nonspecific linear and nonspecific four-way junction DNA when examined individually across a protein concentration series. However, in the competitive binding reactions, the R2 protein had a clear preference for binding to the four-way junction over the linear DNA. Note that the junction DNA contained a greater number of total base pairs (100 bp; each arm is 25 bp), whereas the linear DNA contained fewer (50 bp). However, because the R2 protein did not bind to the linear DNA until most of the junction DNA had finished binding, it is unlikely that the difference in DNA "length" (greater than a two-fold difference) had a significant effect on the binding affinity observed in the competitive reactions.

[0185] The migration patterns for both linear and junctional DNA were similar. A portion of the signal was packed into the well, with a smear extending down from the well to the faint protein-DNA complex in the gel. The gel-migrating protein-DNA complexes for linear and junctional DNA migrated to approximately the same position in the gel. In the case of linear DNA, the smear continued all the way from the well to the free DNA. The migration patterns, especially that of the R2 protein bound to junctional DNA, were similar to that of the R2 protein bound to its own target DNA in the absence of RNA before DNA cleavage (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005); Christensen and Eickbush, J Mol Biol 336, 1035 (2004)).

[0186] In the presence of non-specific RNA (nsRNA for short), R2 protein again bound preferentially to junction DNA, as did the absence of RNA. Again, there was a smear extending from the well to the major complex in the gel. Junction and linear protein-RNA-DNA complexes migrated to similar, but distinctly different, positions in the gel. In the presence of R2 3'PBM RNA, R2 protein bound to junction DNA, as was nearly the same for non-specific RNA, and again 4-way junction DNA was preferred over non-specific linear DNA. Interestingly, in the presence of 5'PBM RNA, the behavior was different (see next section). Example 2 5'PBM RNA, but not 3'PBM RNA, inhibits binding to nonspecific four-way DNA junctions.

[0187] An assay was designed to directly compare R2 protein binding to four-way junction DNA across a range of RNA concentrations for nonspecific RNA, 3'PBM RNA, and 5'PBM RNA. For each RNA titration set, the amount of protein used was sufficient to bind to the majority of the junction DNA in reactions lacking RNA. In general, addition of any of the three RNAs pulled material out of the well and into the gel. R2 RNA was more efficient at pulling material out of the well and into the gel. A similar phenomenon is observed when R2 protein is bound to its normal (linear) 28S target DNA in the presence of R2 RNA (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005), Christensen and Eickbush, Proc Natl Acad Sci USA 103, 17602 (2006), Christensen and Eickbush, J Mol Biol 336, 1035 (2004)). Unlike binding to linear 28S target DNA, the presence of 5'PBM RNA strongly inhibited the binding of R2 protein to four-way junction DNA. Only the presence of 5'PBM RNA had a significant effect on the binding of R2 protein to junction DNA, and the inhibition scaled with the 5'PBM RNA concentration. Binding to nonspecific linear DNA and three-way junctions was less affected by the presence of 5'RNA, but was still reduced in its presence. This inhibition is not observed when downstream 28S rDNA sequences are present in either of the DNA constructs (Christensen, et al., Nucleic Acids Res 33, 6461 (2005), Zingler, et al., Cytogenet Genome Res 110, 250 (2005)). Example 3 The R2 protein does not degrade nonspecific four-way junction DNA.

[0188] DNA from reactions of R2 protein bound to nonspecific linear and nonspecific four-way junctions across a range of protein concentrations in the absence of RNA was analyzed for DNA cleavage events by denaturing polyacrylamide gel electrophoresis. Each strand of junction and linear DNA was followed independently for DNA cleavage events by sequentially radiolabeling the 5' ends of different DNA strands. Complex patterns of random low intensity background cleavage occurred, especially in the case of protein excess. Similar background cleavage events occurred for R2 protein bound to its normal 28S target DNA in the absence of RNA when R2 protein was present in excess. Background cleavage for nonspecific junctions was not structure driven, as cleavage occurred at identical positions within linear DNA of the same sequence. The presence of any of the three RNAs (5'PBM RNA > 3'PBM RNA > nonspecific RNA) abolished random background DNA cleavage. Example 4 Linear target DNA and TPRT products are poor substrates for second strand cleavage.

[0189] R2Bm is inserted at a specific site in the 28S rDNA. It was found that a protein subunit bound to a target sequence downstream of the insertion site provides the endonuclease responsible for second-strand (i.e., top-strand) DNA cleavage. However, second-strand cleavage, while not novel, has been difficult to achieve and study. Until now, second-strand cleavage required a narrow range of 5'PBM RNA, R2 protein, and DNA ratios. Previous data showed that first-strand DNA cleavage is almost certainly required before the second strand can be cleaved, that the downstream subunit must be bound to DNA (which requires 5'PBM RNA), and that the 5'PBM RNA must then dissociate from the downstream subunit for second-strand cleavage to occur. In vivo, with respect to the full-length R2 RNA, the TPRT process would be thought to remove the 5'PBM RNA from the downstream subunit, thereby rendering the downstream subunit "RNA-unbound" and thus initiating second-strand DNA cleavage.

[0190] Given that the R2 protein can bind branched DNA in the absence of RNA, the role of DNA structure in the ability of the downstream subunit to cleave DNA in the absence of RNA was investigated. To isolate the activity associated with the downstream subunit, the DNA construct contained the binding site of the downstream R2 protein subunit rather than that of the upstream bound R2 protein subunit. The upstream DNA sequence was replaced with non-specific DNA derived from the four-way junction used in the previous figure. Linear DNA containing the downstream 28S DNA was not a substrate for second-strand cleavage, regardless of the presence or absence of a first-strand DNA cleavage event (Figure 2, constructs iii and iv). A post-TPRT analog (construct v) that can be cleaved by the R2 protein was also not a substrate. The TPRT analog was a three-way junction containing the downstream 28S DNA that had already been cleaved at the first (bottom) strand cleavage site in terms of the TPRT reaction and was covalently linked to a cDNA sequence corresponding to the 3' end of the R2 element. Annealed to the cDNA portion of the construct was either the 25 bp R2 RNA or a DNA version of the same 25 bp. The R2Bm protein was unable to cleave the top strand of these three-way junctions, whether the R2 3′ sequence-containing arm was in the form of an RNA-DNA duplex or a DNA duplex. Example 5 A specific four-way junction is cleaved by the R2 protein.

[0191] Unlike linear and TPRT-junction (Figure 3, constructs iii-v) DNA, a four-way junction containing the target sequence and the R2 sequence was found to be cleaved by the R2 protein (Figure 3, construct viii). Construct viii was similar to the TPRT-junction (construct v) but had an additional arm, the 5'R2 arm. Both the R2 5' arm and the R2 3' arm were 25 bp in length and consisted of an RNA-DNA duplex. Construct viii mimics the proposed association between the cDNA and the target DNA. The 5' end of R2Bm mRNA is believed to contain rRNA sequences corresponding to the upstream target DNA (Eickbush, et al., PLoS One 8, e66441 (2013), Stage and Eickbush, Genome Biol 10, R49 (2009), Fujimoto et al., Nucleic Acids Res 32, 1555 (2004), Eickbush, et al., Mol Cell Biol 20, 213 (2000)). Thus, the reverse transcribed cDNA could hybridize with the top strand of the target to form a four-way junction. We were also able to cleave, albeit to a lesser extent, a fully covalently closed all-DNA version of the same junction (see Figure 3, construct vi), as well as a construct lacking the R2 3' arm (construct vii). Example 6 Further exploration of second-strand DNA breaks.

[0192] To further explore the structural requirements for second strand cleavage, a number of structural variants (i.e., partial junctions) of Figure 3 construct viii were tested for cleavability (Figure 4A-4B, constructs i-viii). Figure 3 construct viii is identical to Figure 4A construct i, except that the length of the 28S downstream arm was increased to 47 bp instead of the original 25 bp used in Figure 3 construct viii. This adjustment was made to match the downstream DNA in Figure 4A-4B constructs with the amount of downstream DNA included in previous linear DNA constructs used in a previous publication (Govindaraju, et al., Nucleic Acids Res 44, 3276 (2016)). The reason for testing the cleavability of partial junctions (Figure 4A-4B, junctions ii-viii) was to determine how much, if any, of the DNA cleavage signal observed in Figure 3 could have been due to contaminating partial junctions present in the binding and cleavage reaction, even though they may have been present in small amounts. It was also to determine whether constructs that mimicked cellular removal of the RNA component (e.g., by cellular RNases; constructs vi-viii) were better or worse at being cleaved by the R2 protein than constructs with an intact RNA-DNA duplex. It appeared that some of the partial junctions (complexes ii and iii) could be cleaved and thus potentially contributed to some degree to the total cleavage in reactions containing the complete junction (complex i). The four-way junction that lacked both RNA components (complex vi) was nearly uncleavable, indicating the requirement for the double-stranded R2 arm. The four-way junction that lacked the 5'-terminal RNA but contained a 3'-terminal RNA (construct vii) also could not be appreciably cleaved, indicating the importance of the presence of the RNA-DNA duplex in the R2 5'-arm. The four-way junction that lacked the 3'-terminal RNA but contained a 5'-terminal RNA (construct vii) was cleaved well; in fact, it was cleaved more efficiently than construct i. This indicates that the presence of a duplex in the R2 3' arm is somewhat inhibitory, but that the presence of a duplex in the 5' arm is stimulatory.

[0193] To investigate the relative importance of the upstream target sequence for second strand DNA cleavage, 73 bp of upstream 28S DNA was incorporated into the four-way junction (Figure 4C-4D; constructs ii-iv). In construct ii, 47 bp of downstream 28S DNA was replaced with non-specific DNA, and construct iii contained the entire target DNA sequence (73 bp of upstream 28S DNA and 47 bp of downstream 28S DNA). Construct ii could be cleaved, albeit with much lower efficiency than construct i, which contained the downstream target DNA as in the previous figure but not the upstream one. The fact that construct ii could be cleaved indicates that perhaps the 12 bp common to both constructs i and ii (7 bp upstream and 5 bp downstream of the DNA) may be involved in helping direct DNA cleavage. Paradoxically, construct iii, which contained the entire target sequence, was cleaved less efficiently than construct ii. The addition of a flap, or strand shift (construct iv), which is thought to occur during template jumping, significantly increased the scissionability of the junction. Example 7 Second strand cleavage leads to second strand synthesis in the presence of dNTPs.

[0194] To test whether the second strand cleavage can proceed to second strand synthesis, dNTPs were added to the DNA cleavage reaction. The construct used to test for second strand synthesis was construct i in Figures 4A-4B. This cleaved relatively well. Various R2 protein concentrations were used and the reactions were analyzed by denaturing (Figure 5) and non-denaturing polyacrylamide gel electrophoresis. The labeled strand of the four-way junction was 72 nt uncleaved and 24 nt in length at the time of second strand DNA cleavage (marked SSC on the denaturing gel). Second strand synthesis (SSS), i.e., extension of the labeled strand after DNA cleavage, will generate a 50 nt product when analyzed on a denaturing gel. Second strand DNA synthesis was only observed at the top of the protein titration series in the denaturing gel. The reason for this becomes clear in the non-denaturing (EMSA) gel. Upon cleavage, the four-way junction is resolved into two linear DNAs, one DNA containing the downstream arm and the R2 3' arm, and one DNA containing the "upstream" arm and the R2 5' arm. The R2 protein appeared to remain bound to DNA containing the downstream 28S DNA after DNA cleavage, while DNA containing nonspecific "upstream" DNA was released. The free DNA primer-template was extended by R2 RT only when the protein was present in excess. The migration positions of the products of second strand cleavage and second strand synthesis are shown next to the EMSA gel.

[0195] In the presence of dNTPs, the higher signal on a denaturing gel than the full-length oligo results from the original full-length oligo being extended by R2. Given a template in cis or in trans, R2 can capture and extend almost any 3' end (Bibillo, et al., J Biol Chem 279, 14945 (2004), Bibillo and Eickbush, J Mol Biol 316, 459 (2002)). Example 8 Second strand synthesis on previously cleaved DNA constructs.

[0196] The primer-template is released from the protein-DNA complex when the upstream DNA is not present at the four-way junction, but one would think that this would not occur in vivo at junctions that contain the entire target sequence. In part, this is because the downstream subunit is thought to carry out second-strand synthesis (Christensen and Eickbush, Mol Cell Biol 25, 6617 (2005)). Unfortunately, junctions with the entire target sequence are poorly cleaved (Figures 4C and 4D), and second-strand synthesis is below detection levels when tested in vitro. For this reason, a post-second-strand cleavage analog was generated. To keep the second-strand cleavage products tethered together, the R2 3' and 5' terminal "RNA" were covalently linked, but DNA was used instead of RNA for convenience. The upstream 28S DNA containing the second-strand cleavage products can undergo primer extension (i.e., second-strand synthesis) in a tethered configuration. The 5' terminal cDNA strand was used as the template (Figure 6A).

[0197] To determine which R2 protein subunits are used for second-strand synthesis, linear (Figure 6B, complexes iv and v) and tethered (Figure 6B, complexes i and iii) post-second-strand cleavage products were tested for their relative ability to undergo second-strand synthesis (Figure 6C). Results are consistent with the subunits bound to the four-way junction involved in second-strand cleavage. Complex iii was the most efficient substrate for second-strand synthesis, and complex iv was the least efficient substrate. Example 9 Mutation of core residues in the HINALP and CCHC motifs affects target DNA binding and results in loss of DNA cleavage specificity. material and method mutation

[0198] To investigate the role of the putative α-finger (HINALP motif region) and zinc knuckle (CCHC motif region) in the linker region, a number of double point mutants were generated (Figure 8B). Mutations in the putative α-finger region included GR / AD / A, VH / ATH / A, H / AIN / ALP, SR / AIR / A and SR / AGR / A. The H / AIN / AALP and SR / AIR / A mutations resulted in reduced soluble protein recovered compared to the wild-type (WT) protein. The VH / ATH / A mutation did not result in soluble protein and was therefore excluded from this study. Mutations in the zinc knuckle region were C / SC / SHC, CR / AAGCK / A, E / AT / AT, HILQ / AQ / A and RT / AH / A (Figure 8B). The C / SC / SHC mutation resulted in a significant reduction in soluble protein recovered compared to the wild-type (WT) protein. The E / AT / AT mutation did not yield usable amounts of protein and was therefore excluded from this study. Protein and nucleic acid preparation

[0199] Proteins were expressed and purified as previously published (Govindaraju, et al., Nucleic Acids Res.44, 3276-3287 (2016)). GR / AD / A, SR / AIR / A, SR / AGR / A, H / AIN / ALP, C / SC / SHC, CR / AAGCK / A, HILQ / AQ / A and RT / AH / A mutants were generated using QuikChange site-directed mutagenesis kit (Stratagene #200523-5). 5'PBM (320nt), 3'PBM (249nt), linear target DNA, and four-way junctions were prepared as previously published (Govindaraju, et al., Nucleic Acids Res.44, 3276-3287 (2016)). R2Bm reaction and analysis

[0200] DNA binding, first- and second-strand cleavage, and first- and second-strand synthesis reactions were performed as previously reported (Govindaraju, et al., Nucleic Acids Res.44, 3276-3287 (2016)).

[0201] For DNA binding assays, a master mix containing all components except protein was made and aliquoted. Binding reactions were initiated by adding 3ul of protein at known, equalized concentrations across all proteins tested in the data set. Duplicate reactions were set up for each data set, and two different data sets were generated, each with a different protein concentration. WT and WT KPD / A proteins served as binding activity reference and positive controls for endonuclease activity and endonuclease-deficient mutations, respectively.

[0202] For the DNA cleavage assay, a master mix containing all components except protein and DNA was made and aliquoted. Proteins from the protein dilution series were left for 5 minutes at 37°C to bind to RNA, after which target DNA was added to initiate the cleavage reaction. Reactions were incubated for 30 minutes at 37°C. Reactions were kept on ice and then run on a 5% native (1x Tris-borate-EDTA) polyacrylamide gel and a denaturing (8M urea) 7% polyacrylamide gel.

[0203] First and second strand synthesis reactions contained labeled target DNA in a master mix with all other components except protein. Linear DNA that had already been cleaved was used so that mutants without DNA cleavage could be tested along with mutants with normal cleavage ability. The target DNA substrate for the second strand synthesis assay was a four-way junction DNA in which the second strand had already been cleaved. This substrate is described in Chapter 2. As with the cleavage assay, the reactions were analyzed by both native and denaturing polyacrylamide gels.

[0204] All gels were dried and quantified using a phosphorimager (Molecular dynamics STORM 840) and FIJI (Schindelin, et al., Nat. Methods (2012). doi:10.1038 / nmeth.2019.Fiji). result

[0205] Four double point mutants were generated in the HINALP region and four in the zinc knuckle region. The H / AIN / AALP and C / SC / SHC mutants appear to have nearly identical phenotypes. Both sets of mutations severely impair DNA binding to linear DNA and also the ability of linear DNA to form correct DNA-RNA-protein complexes in EMSA gels (Figures 9A-9B). Only well complexes and a diffuse smear down from the well to free DNA were observed (Figures 9A-9B). This observation applies to both upstream binding conditions (i.e., in the presence of 3'PBM RNA) and downstream binding conditions (i.e., in the presence of 5'PBM RNA). The cysteine ​​and histidine residues of the zinc knuckle motif are putative zinc coordinating residues. The C / SC / SHC mutations may promote local misfolding of the linker. The H / AIN / AALP mutations may also affect linker folding.

[0206] In the presence of 3'PBM RNA, the H / AIN / AALP and C / SC / SHC mutants showed little or no first-strand breaks at the insertion site. Second-strand DNA breaks were similarly abolished in the presence of 5'PBM RNA. Abundant, indiscriminate breaks, rather than site-specific DNA breaks, were observed at aberrant sites on both strands of the target DNA. Example 10 Mutations in the putative α-finger affect DNA binding, particularly to specific branched integration intermediate analogs.

[0207] To better determine whether the putative α-finger is involved in anchoring the protein to upstream and / or downstream target DNA sequences, mutations around the core HINALP motif were tested. GR / AD / A, SR / AIR / A and SR / AGR / A mutants were tested for their ability to bind linear targets in the presence of 3'PBM RNA and in the presence of 5'PBM RNA. Two positive controls, WT R2 protein and an R2 protein with the catalytic residue of RLE (KPD / A) mutated to alanine, were used to knock out DNA cleavage but not DNA binding, to appropriately control for α-finger mutations that may or may not affect DNA cleavage (see next section). Electrophoretic mobility shift assays (EMSA) were used to assay the DNA binding ability of the mutants relative to the control R2 protein (Figures 10A-10B). Duplicate lanes were loaded and duplicate binding reactions were performed. A vector control extract lane and a no protein lane served as negative control lanes.

[0208] Upstream target DNA binding was moderately reduced (24%) by the GR / AD / A mutation and only mildly reduced (13%) by the SR / AIR / A mutation. However, upstream target DNA binding activity was significantly increased by 32% by the SR / AGR / A mutant (Figures 10A-10B). Downstream target DNA binding activity for the GR / AD / A and SR / AGR / A mutants was similar to WT activity, with only a mild reduction of about 13%. The SR / AIR / A mutation reduced binding in the range of 19-28%. All three mutants did not appear to have a large, if any, effect on the migration pattern of the protein-RNA-DNA complex, although more well complex formation was observed for the SR / AIR / A mutant (Figures 10A-10B). The ability of the mutants to bind linear target DNA in the absence of RNA is presented in Figure 10D.

[0209] The ability of the mutants to bind to the four-way junction integration intermediate was also tested. The four-way junction mimics the branched structure that the 28S rDNA adopts after the template jump step and contains the 28Sd rDNA sequence (north arm), a non-specific sequence (west arm), the R2 5'-terminal RNA-DNA duplex (south arm), and the R2 3'-terminal RNA-DNA duplex (east arm) (Figure 10C) (see also Examples 1-8). The four-way junction DNA was labeled with a radioisotope in the top strand at the 5' end of the west arm. The junction DNA was incubated with R2 protein in the absence of RNA, and aliquots were run on an EMSA gel (Figure 10C). After quantification as described above, two mutants were shown to significantly reduce the ability of R2 protein to bind to four-way junctions, by 63% for SR / AIR / A and 48% for SR / AGR / A, whereas the binding activity of the GR / AD / A mutant was comparable to that of the WT activity, showing only a mild reduction of 12%. Example 11 Mutations in the putative α-finger reduce first-strand DNA cleavage

[0210] The ability of the GR / AD / A, SR / AIR / A and SR / AGR / A mutants to carry out first strand DNA cleavage was assayed. R2 protein was pre-bound to the 3'PBM and then incubated with the target DNA. A protein titration series was used (seven 1:3 protein dilutions). Aliquots of each reaction were run on an EMSA gel and on a denaturing (8M urea) polyacrylamide gel. The target DNA was pre-bound at the 5' end of the bottom strand (i.e., the 28S antisense strand). 32 The strand was labeled with P so that cleavage of this strand could be followed in denaturing gels.

[0211] In the higher protein concentration lanes of the EMSA gel (first two), when the RNA concentration was held constant, protein-DNA complexes corresponding to those seen in the absence of RNA were observed for the WT, GR / AD / A and SR / AGR / A mutants, and as the protein approached equivalence with the RNA concentration, the DNA-complex appeared along with the protein-RNA-DNA complex, and then all packed into the well. The mutations did not appear to significantly affect the migration pattern of the protein-RNA-DNA complex compared to the WT. The cleavage activity for each of the mutants is reported as a scatter plot of the fraction of cleaved DNA (fCut) calculated from the urea denaturing gel as a function of the fraction of bound DNA (fBound) calculated from the EMSA gel. The GR / AD / A mutant did not affect the first-strand cleavage activity of the R2 protein, whereas the SR / AIR / A and SR / AGR / A mutants significantly reduced the ability of the bound protein to undergo first-strand DNA cleavage (Figure 11). Cleavage beyond the R2 cleavage site was not observed for either the WT or the mutants. Example 12 Mutations in the putative α-finger reduce first-strand cDNA synthesis

[0212] To investigate whether the HINALP region affects TPRT (first strand DNA synthesis), previously cleaved target DNA with a nick at the insertion site on the first / bottom strand was incubated with R2 protein in the presence of 3'PBM RNA and dNTPs (Figure 12A). Target DNA was radioisotope-labeled at the 5' end of the bottom strand to follow the formation of TPRT products. Aliquots of reactions over a protein titration series were assayed by EMSA and denaturing polyacrylamide gel. A graph of the fraction of target DNA that underwent TPRT (f synthesis) as a function of the fraction of target DNA bound by R2 protein (f binding) is reported in Figure 12B. GR / AD / A and SR / AIR / A mutants completely abolished TPRT activity, while the SR / AGR / A mutant reduced first strand synthesis activity by approximately 50% (Figure 12B). Example 13 Mutations in the putative α-finger affect second-strand DNA cleavage

[0213] To determine the role, if any, that the GR / AD / A, SR / AIR / A and SR / AGR / A mutants play on second strand cleavage, two different cleavage assays were undertaken: (1) cleavage on linear target DNA in the presence of 5'PBM RNA, and (2) cleavage on four-way junction DNA in the absence of RNA. On linear DNA, the R2 protein bound downstream of the insertion site in the presence of 5'PBM RNA, but cleaved only upon dissociation of the RNA from the complex. This dissociation occurred as the ratio of RNA to protein was decreased (holding RNA constant) over a protein titration series (Christensen, et al., Proc. Natl. Acad. Sci. USA 103, 17602-17607 (2006)). In EMSA gels, the migration patterns of the mutant protein-RNA-DNA complexes were similar to that of the WT, except for the bands corresponding to the second strand cleavage products located just below the major protein-RNA-DNA complex, which were absent for the SR / AIR / A and SR / AGR / A mutants. In denaturing gels, the signals of the second strand cleavage products were not visible for the SR / AIR / A and SR / AGR / A mutants. Nonspecific cleavage was not observed for any of the mutants. GR / AD / A showed WT activity, whereas the SR / AIR / A and SR / AGR / A mutants knocked out the endonuclease activity of the R2 protein to cause second strand cleavage of linear target DNA (Figure 13A).

[0214] As described above, second-strand cleavage activity was also tested using the four-way junction integration intermediate (Figure 13B). Second-strand DNA cleavage is thought to occur when the protein is in an "RNA-free" state. 16Therefore, the appropriate substrate for DNA cleavage is the four-way junction intermediate formed by template jumping. A schematic diagram of the junction DNA used is shown in Figure 10C. The junction DNA was radioisotope-labeled at the 5' end of the waist arm to follow cleavage against the top strand of 28S DNA. The cleavage activity of the mutants was tested against the WT as shown for the previous targeted DNA cleavage assay, but in the absence of RNA. As shown in the scatter plot (Figure 13B), the endonuclease activity cleaving the second strand of the four-way junction DNA was completely knocked out by the SR / AIR / A and SR / AGR / A mutants, while the GR / AD / A mutant showed WT cleavage activity or better. Example 14 Mutations in the putative α-finger affect second-strand synthesis

[0215] In addition to testing the second strand cleavage activity of HINALP mutants, the same mutants were subjected to experiments designed to test second strand DNA synthesis activity. Since DNA cleavage is less efficient, already cleaved DNA was used, and since the upstream and downstream ends separate after DNA cleavage in vitro, the two ends were held together by a covalent linkage between the East and South arms (i.e., between the R2 5'-end sequence and the R2 3'-end sequence) (see schematic diagrams in Figures 14A-14B) (see also Examples 1-8). This post-second strand cleavage analog was developed and reported in previous studies. HINALP mutants were tested for second strand DNA synthesis activity using this construct (Figure 14C). The 5'-end of the West arm was radioactively labeled to visualize the newly synthesized second strand on a denaturing gel (indicated by a black star in Figures 14A-14B). The graphs shown in Figure 14C were obtained from EMSA and denaturing gels as previously described for the first strand synthesis assay. The GR / AD / A mutant appears to behave more like the WT, except that at the highest protein concentrations, the amount of second strand synthesis is reduced. The SR / AIR / A mutant appears more like the WT until approximately 40% of the target DNA is protein bound, at which point second strand synthesis is significantly reduced with increasing protein concentration. The SR / AGR / A mutant greatly reduces the ability of the R2 protein to synthesize second strands as shown in the Figure 14C graph. Example 15 Mutations in residues in the zinc knuckle region affect targeted DNA cleavage and second-strand synthesis

[0216] Although the C / SC / SHC mutants were shown to affect target DNA binding and cleavage, the role of the CCHC region was further investigated using three additional double point mutants in this region: CR / AAGCK / A, HILQ / AQ / A, and RT / AH / A (Figure 8B). These mutants were assayed for DNA cleavage and new strand synthesis activity as previously described.

[0217] All three mutants only slightly reduced the ability of the R2 protein to cleave the first strand at the insertion site (Figure 15A), and they did not appear to have any effect on first strand synthesis activity by TPRT (Figure 15B). The CR / AAGCK / A, HILQ / AQ / A, and RT / AH / A mutants were nearly WT for first strand cleavage and synthesis, but at least two of these mutants, HILQ / AQ / A and RT / AH / A, significantly abolished second strand cleavage activity on linear DNA (Figure 15D). In addition to reduced second strand cleavage activity at the insertion site, the endonuclease of the RT / AH / A mutant was also found to cleave the top strand of a linear target at an adjacent site. The second strand cleavage activity of the mutants was also tested using a four-way junction target DNA, and all three mutants showed WT activity (Figure 15C). Furthermore, again, the RT / AH / A mutant endonuclease showed additional cleavage at non-R2 specific sites.

[0218] Second strand synthesis assays using pre-nicked four-way junction DNA as shown in Figure 14 were performed for the three CCHC domain mutants as previously described for the HINALP domain mutants. As shown in Figure 16, second strand synthesis product formation per binding unit of target DNA for CR / AAGCK / A was very similar to that of WT, whereas for HILQ / AQ / A and RT / AH / A there was a dramatic reduction in second strand synthesis product formation.

[0219] Table 2: Summary of DNA binding, cleavage and synthesis results. [Table 2] Not Applicable (NA), Not Tested (NT) "++": +30% and above 「+」:+15%~30% "WT": 15%-15% of WT activity: functionally WT "-": -15%~-30%: slight reduction "--": -30% to -50%: Significant reduction "---": -50% to 75%: Significant reduction "φ": 75% or higher: functionally ineffective

[0220] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Publications cited herein, and the material for which they are cited, are specifically incorporated by reference.

[0221] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the scope of the following claims. The present invention provides, for example, the following items. (Item 1) An RNA component comprising a DNA targeting sequence, one or more protein binding motifs (PBMs), and a nucleic acid sequence of interest to be integrated into a DNA target site, wherein said DNA targeting sequence, said protein binding motifs, and said sequence of interest are operably linked such that said RNA component can bind to a protein component derived from a parent long interspersed repeat (LINE) element protein, be reverse transcribed into cDNA, and said cDNA can be integrated into said DNA target site in DNA. (Item 2) 2. The RNA component according to claim 1, wherein the protein component comprises one or more of an RNA binding domain, a linker domain, a reverse transcriptase, a DNA endonuclease, and the one or more protein binding motifs bind the RNA component to the RNA binding domain, the linker domain, the reverse transcriptase, the DNA endonuclease, or a combination thereof of the protein component. (Item 3) contains elements from or derived from a parent LINE or SINE backbone, and said nucleic acid sequence of interest in the RNA component is heterologous to said LINE or SINE; the protein component contains elements from or derived from a parent LINE; or It is a combination of these, 3. The RNA component according to item 1 or 2. (Item 4) 4. The RNA moiety according to any one of items 1 to 3, wherein the DNA targeting sequence is heterologous to the parent LINE or SINE. (Item 5) 5. The RNA component according to any one of items 1 to 4, wherein the sequence of interest encodes a gene, a fragment of a gene, or a functional nucleic acid. (Item 6) 6. The RNA moiety according to any one of items 1 to 5, comprising a 3' PBM sequence from or derived from a parent LINE or SINE element. (Item 7) 7. The RNA component according to any one of items 1 to 6, comprising a CRISPR / Cas tracer sequence, a CRISPR / Cas guide sequence, or a combination thereof. (Item 8) 8. The RNA moiety according to any one of items 1 to 7, comprising a 5' PBM sequence from or derived from the parent LINE or SINE element. (Item 9) 9. The RNA moiety of item 8, wherein the 5' PBM comprises a non-functional IRES sequence. (Item 10) 10. The RNA moiety according to any one of items 1 to 9, further comprising a ribozyme. (Item 11) 11. The RNA moiety according to item 10, wherein the ribozyme is a hepatitis delta virus-like ribozyme. (Item 12) 11. The RNA moiety according to any one of items 2 to 10, wherein the parent LINE or SINE is a restriction enzyme-like endonuclease (RLE) LINE. (Item 13) 11. The RNA component according to item 10, wherein the RLE LINE is an R2 LINE. (Item 14) 14. The RNA component according to any one of items 3 to 13, wherein the parent LINE or SINE backbone of the RNA component and the parent LINE backbone of the protein component are the same LINE and / or the SINE is derived from or is ancestral to the LINE. (Item 15) A protein component comprising a DNA-binding domain, an RNA-binding domain, a reverse transcriptase, a linker domain and an endonuclease, wherein the DNA-binding domain, the RNA-binding domain, the reverse transcriptase, the linker domain and the endonuclease are operably linked such that they are capable of binding to an RNA component and DNA at a DNA target site and facilitating reverse transcription of the RNA component into cDNA and integration of the cDNA into the DNA at the DNA target site. (Item 16) 16. The protein component according to item 15, wherein the RNA component comprises a DNA targeting sequence, one or more protein binding motifs, and a nucleic acid sequence of interest that is integrated into the DNA target site. (Item 17) the RNA component comprises elements from or derived from a parent LINE or SINE backbone, and the nucleic acid sequence of interest of the RNA component is heterologous to the LINE or SINE; the protein component contains elements from or derived from a parent LINE; or It is a combination of these, 17. The protein component according to item 15 or 16. (Item 18) 18. The protein component according to any one of items 15 to 17, wherein the DNA-binding domain is mutated compared to the parent LINE DNA-binding domain. (Item 19) 18. The protein component according to any one of items 15 to 17, wherein the DNA-binding domain has been replaced with an alternative DNA-binding domain compared to the parent LINE DNA-binding domain. (Item 20) 20. The protein component according to item 19, wherein the DNA-binding domain is a DNA-binding domain from another DNA-binding protein. (Item 21) 21. The protein component according to item 19 or 20, wherein the DNA-binding domain comprises one or more of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix-turn-helix, a helix-loop-helix, an HMG box, a Wor3 domain, an OB-fold domain, an immunoglobulin fold, a B3 domain, a TAL effector, or an RNA guide domain. (Item 22) 23. The protein component according to any one of items 15 to 22, wherein the sequences of one or more of the RNA binding domain, reverse transcriptase, linker domain and endonuclease are identical to those of the parent LINE element protein or are mutated to improve binding or enzymatic activity towards the RNA component compared to the parent LINE element protein. (Item 23) 23. The protein moiety according to any one of items 17 to 22, wherein the parent LINE or SINE is a restriction enzyme-like endonuclease (RLE) LINE. (Item 24) 24. The protein component according to item 23, wherein the RLE LINE is an R2 LINE. (Item 25) 25. The protein component according to any one of items 17 to 24, wherein the parent LINE or SINE backbone of the RNA component and the parent LINE backbone of the protein component are the same LINE and / or the SINE is derived from or is ancestral to the LINE. (Item 26) 15. A vector encoding the RNA component according to any one of items 1 to 14. (Item 27) 26. A vector encoding the protein component according to any one of items 15 to 25. (Item 28) 26. An engineered transposon comprising an RNA component according to any one of items 1 to 14 and a protein component according to any one of items 15 to 25. (Item 29) 29. The transposon of item 28, wherein a productive four-way junction is formed during an integration reaction at the DNA target site. (Item 30) 27. A pharmaceutical composition comprising the RNA component according to any one of items 1 to 14, the protein component according to any one of items 15 to 25, the vector according to item 26, the vector according to item 27, the engineered transposon according to item 28 or 29, or any combination thereof. (Item 31) A method for introducing a nucleic acid sequence of interest into the genome of one or more cells, comprising the step of contacting said one or more cells with (i) the RNA component according to any one of items 1 to 14 or the vector according to item 26 in combination with the protein component according to any one of items 15 to 25 or the vector according to item 17, or (ii) the engineered transposon according to item 28 or 29. (Item 32) 32. The method of claim 31, wherein the cell is contacted in vitro. (Item 33) The method of claim 32, wherein the cells are then introduced into a subject. (Item 34) 32. The method of claim 31, wherein the cell is contacted in vivo. (Item 35) 35. The method of any one of items 31 to 34, wherein expression of the nucleic acid sequence of interest in the cell ameliorates one or more symptoms of a disease or disorder, or a molecular pathway underlying a disease or disorder. (Item 36) The method of item 35, wherein an effective number of cells are modified to treat a subject in need thereof.

Claims

1. 1. An engineered RNA moiety comprising a heterologous DNA targeting sequence, a nucleic acid sequence of interest to be integrated into a DNA target site, and a scaffold from a parent long interspersed nuclear element (LINE) or short interspersed nuclear element (SINE) or a variant thereof comprising at least 90% sequence identity thereto, said scaffold comprising a 3′ protein binding motif (PBM); The DNA targeting sequence, the 3' protein binding motif, and the sequence of interest are operably linked to an RNA component that can bind to a protein component comprising a DNA binding domain and elements from the LINE, the LINE from which the SINE is derived, or a variant thereof comprising at least 90% sequence identity thereto, and be reverse transcribed into cDNA, and the cDNA can be integrated into DNA at the DNA target site, wherein the DNA binding domain is a DNA binding domain that is distinct from the parent LINE DNA binding domain, and wherein the elements comprise an RNA binding domain, a reverse transcriptase, a linker domain, and an endonuclease.

2. The RNA moiety of claim 1 , wherein the backbone further comprises a target sequence and a 5′ PBM.

3. The RNA moiety of claim 2 , wherein the target sequence aids in the formation of a four-way junction integration intermediate and double-stranded breaks in the target DNA.

4. The RNA moiety of claim 2 , wherein the 5′ PBM comprises a non-functional IRES sequence.

5. The RNA component according to any one of claims 1 to 4, wherein the 3' and / or 5' protein binding motif binds the RNA component to the RNA binding domain.

6. The RNA component according to any one of claims 1 to 5, wherein the nucleic acid sequence of interest of the RNA component is heterologous to the LINE or SINE.

7. The RNA component according to any one of claims 1 to 6, wherein the sequence of interest codes for a gene, a fragment of a gene, or a functional nucleic acid.

8. The RNA moiety of any one of claims 1 to 7, wherein the DNA targeting sequence comprises a CRISPR / Cas tracer sequence, a CRISPR / Cas guide sequence, or a combination thereof.

9. The RNA moiety of any one of claims 1 to 8, further comprising a ribozyme.

10. The RNA moiety of claim 9, wherein the ribozyme is a Hepatitis Delta virus-like ribozyme.

11. The RNA moiety according to any one of claims 1 to 10, wherein the parent LINE or SINE is a restriction enzyme-like endonuclease (RLE) LINE.

12. The RNA moiety of claim 11 , wherein the restriction enzyme-like endonuclease (RLE) LINE is an R2 LINE.

13. a protein component comprising a DNA binding domain and an element from a parent long interspersed nuclear element (LINE) or a variant derived therefrom comprising at least 90% sequence identity thereto, said element comprising an RNA binding domain, a reverse transcriptase, a linker domain and an endonuclease, said DNA binding domain, RNA binding domain, reverse transcriptase, linker domain and endonuclease being operably linked such that they can bind to an RNA component and DNA at a DNA target site and facilitate reverse transcription of said RNA component into cDNA and integration of said cDNA into said DNA at said DNA target site; the DNA-binding domain is a different DNA-binding domain from a parent LINE DNA-binding domain, A protein component, wherein the RNA component comprises a DNA targeting sequence, a nucleic acid sequence of interest to be integrated into the DNA target site, and a scaffold from a parent LINE or short interspersed repeat nuclear element (SINE) or a variant thereof comprising at least 90% sequence identity thereto, the scaffold comprising a 3' protein binding motif (PBM) and, optionally, a 5' protein binding motif, and the DNA targeting sequence is heterologous to the parent LINE or SINE.

14. The protein component of claim 13 , wherein the nucleic acid sequence of interest of the RNA component is heterologous to the LINE or SINE.

15. 15. A protein component according to claim 13 or 14, wherein the DNA binding domain is heterologous to the parent LINE DNA binding domain.

16. 16. The protein moiety of any one of claims 13 to 15, wherein the DNA binding domain comprises one or more of a helix-turn-helix, a zinc finger, a leucine zipper, a winged helix, a winged helix-turn-helix, a helix-loop-helix, an HMG box, a Wor3 domain, an OB-fold domain, an immunoglobulin fold, a B3 domain, a TAL effector, or an RNA guide domain.

17. The protein component according to any one of claims 13 to 16, wherein the sequences of one or more of the RNA binding domain, reverse transcriptase, linker domain and endonuclease are the same as the sequence of the parent LINE.

18. The protein moiety according to any one of claims 13 to 17, wherein the parent LINE or SINE is a restriction enzyme-like endonuclease (RLE) LINE.

19. The protein component according to claim 18 , wherein the restriction enzyme-like endonuclease (RLE) LINE is an R2 LINE.

20. A vector encoding an RNA component according to any one of claims 1 to 12.

21. A vector encoding a protein component according to any one of claims 13 to 19.

22. 20. An engineered transposon comprising an RNA component according to any one of claims 1 to 12 and a protein component according to any one of claims 13 to 19.

23. 23. The engineered transposon of claim 22, wherein a productive four-way junction is formed during an integration reaction at the DNA target site.

24. 20. A pharmaceutical composition comprising an RNA component according to any one of claims 1 to 12, a protein component according to any one of claims 13 to 19, a vector according to claim 20, a vector according to claim 21, an engineered transposon according to claim 22 or 23, or any combination thereof, and a pharma- ceutically acceptable carrier.

25. A combination for use in a method for introducing a nucleic acid sequence of interest into the genome of one or more cells, comprising (i) an RNA component according to any one of claims 1 to 12 or a vector according to claim 20, and (ii) a protein component according to any one of claims 13 to 19 or a vector according to claim 21, A combination, wherein the method comprises the step of contacting the one or more cells with an RNA component according to any one of claims 1 to 12 or a vector according to claim 20 in combination with a protein component according to any one of claims 13 to 19 or a vector according to claim 21.

26. 24. A composition for use in a method for introducing a nucleic acid sequence of interest into the genome of one or more cells, comprising the engineered transposon of claim 22 or 23, 24. A composition, wherein the method comprises contacting the one or more cells with the engineered transposon of claim 22 or 23.

27. 27. The combination of claim 25 or the composition of claim 26, wherein the cells are contacted in vitro.

28. 28. The combination or composition of claim 27, wherein the cells are then introduced into a subject.

29. 27. The combination of claim 25 or the composition of claim 26, wherein the cells are contacted in vivo.

30. A combination according to any one of claims 25 and 27 to 29 or a composition according to any one of claims 26 to 29, wherein expression of said nucleic acid sequence of interest in said cell ameliorates one or more symptoms of a disease or disorder, or a molecular pathway underlying a disease or disorder.

31. 31. The combination or composition of claim 30, wherein an effective number of cells are modified to treat a subject in need thereof.

32. A composition for use in a method for introducing a nucleic acid sequence of interest into the genome of one or more cells, comprising an RNA component according to any one of claims 1 to 12 or a vector according to claim 20, A composition, wherein the method comprises the step of contacting the one or more cells with an RNA component according to any one of claims 1 to 12 or a vector according to claim 20 in combination with a protein component according to any one of claims 13 to 19 or a vector according to claim 21.

33. A composition for use in a method for introducing a nucleic acid sequence of interest into the genome of one or more cells, comprising a protein component according to any one of claims 13 to 19 or a vector according to claim 21, A composition, wherein the method comprises the step of contacting the one or more cells with a protein component according to any one of claims 13 to 19 or a vector according to claim 21 in combination with an RNA component according to any one of claims 1 to 12 or a vector according to claim 20.

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