Repeat dosing of retrotransposons

Abstract

The present disclosure demonstrates that earlier editing of cells with ribosomal non-LTR retrotransposons (R elements) still allowed sufficient editing from subsequent dosing of R elements, without significant interference therebetween, whether they are the same R elements or different. Provided are compositions and methods for integrating one or more exogenous sequence(s) to the genome of a mammalian cell, comprising (a) introducing to the cell a first amount of one or more polynucleotide(s) comprising a first ribosomal non-long terminal repeat (LTR) retrotransposon (R element) which comprises a first exogenous sequence, and, (b) introducing to the cell a second amount of one or more polynucleotide(s) comprising a second R element.

Description

REPEAT DOSING OF RETROTRANSPOSONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of the United States Provisional Application Serial No. 63 / 631,911, filed April 9, 2024, the content of which is hereby incorporated by reference in its entirety.BACKGROUND

[0002] Gene therapy involves the use of nucleic acids (DNA or RNA) for treating and preventing a wide range of diseases, such as cancer, heart disease, diabetes, and viral infections. Genome editing technologies used in gene therapy mainly involve three strategies, namely gene correction, gene silencing, and gene addition. Gene correction modifies specific genes that cause diseases with healthy copies. Gene silencing (knock-out) inhibits the expression of an abnormal gene to eliminate or reduce the expression of the encoded protein. Gene addition (knock-in) adds new or functional genes to human cells using a vector.

[0003] The clustered regularly interspaced short palindromic repeat (CRISPR) / Cas9 mediated genome editing technology has seen wild use due to its simplicity and efficiency, leading to a new era of genetic engineering and gene therapy. The CRISPR / Cas9 system is composed of the Cas9 endonuclease and a single-guide RNA (sgRNA), which can guide Cas9 to the complementary sequence and induce double-stranded break (DSB) at the target site. The resulting DSB is repaired independently by two cellular DNA repair pathways, namely nonhom ologous end-joining (NHEJ) pathway and homology directed repair (HDR) pathway. Genome editing using the CRISPR-Cas9 system in human embryos has resulted in many unexpected mutations at or near its target site as well as in the neighboring areas. Furthermore, this system depends on HDR, which is regulated in host cells, indicating that the integration process of the exogenous gene itself cannot be regulated autonomously.

[0004] The safe use of gene therapy in medical applications requires strict sequence-specificity without generating any off-target effects. Targeted gene knock-in must center on transgene integration at harmless genomic sites and avoid unpredictable phenotypes caused by unexpected integration.

[0005] Transposons, also referred to as TE or mobile genetic elements, are able to move from one chromosomal location to another. The two major groups of transposons are DNA transposons and retrotransposons. DNA transposons, or class II elements, move via a “cut and paste” mechanism, by directly removing themselves from the original position and pasting at another position. Retrotransposons, or class I elements, move in a “copy and paste” manner, using RNA as an intermediate. Based on whether they are flanked by long terminal repeat (LTR) or not, retrotransposons are further classified as LTR type or non-LTR type. LTR retrotransposons are similar to retroviruses in structure and life cycle, and therefore their retrotransposition mechanisms are well-explained. However, the retrotransposition mechanisms of non-LTR type elements has not been well-clarified.

[0006] Non-LTR retrotransposons are sub-classified into two major groups, namely long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs). LINEs are autonomous elements that encode proteins to mediate their own mobility, whereas SINEs are nonautonomous elements that do not encode protein and consequently require LINEs for their propagation.

[0007] Most LINEs are randomly inserted throughout the host genome, such as LI and L2. Some elements are inserted into specific sites of repetitive genomic sequences, such as ribosomal DNA (rDNA), telomeric repeats, and microsatellites. Such sequence specific LINEs, therefore, can be suitable for integrating an exogenous nucleotide sequence into a target genome as specific locations.

[0008] Ribosomal elements (R elements), which are sequence specific LINEs that target ribosomal DNA (28S, 18S, and 5.8S rDNAs), have a wide distribution among many species. Differences between insertion sites and RT domains have led to the identification of nine different R elements, including Rl, R2, R4, R5, R6, R7, R8, R9, and RT. Among them, Rl, R2, R4, R5, R6, R9, and RT elements insert into 28S rDNA, whereas R7 and R8 elements insert into 18S rDNA.SUMMARY

[0009] The present disclosure demonstrates that earlier editing of cells with ribosomal non- LTR retrotransposons (R elements) still allowed sufficient editing from subsequent dosing of R elements, without significant interference therebetween, whether they are the same R elements or different. It also successfully demonstrated that different payloads can be integrated concurrently or sequentially by the same R elements. Such a finding was unexpected at least because repeated dosing of other gene editing approaches, such as CRISPR / Cas, have failed due to cross-reactive immune responses (see, e.g., Ewaisha and Anderson “IMMUNOGENICITY OF CRISPR THERAPEUTICS-CRITICAL CONSIDERATIONS FOR CLINICAL TRANSLATION,” Front Bioeng Biotechnol. 2023; 11 : 1138596). This discovery, therefore, paves the way for increasing the editing efficiency of R elements by repeated dosing.

[0010] According to one embodiment of the present disclosure, therefore, provided is a method for integrating one or more exogenous sequence(s) to the genome of a mammalian cell, comprising (a) introducing to the cell a first amount of one or more polynucleotide(s) comprising a first ribosomal non-long terminal repeat (LTR) retrotransposon (R element) which comprises a first exogenous sequence, and, (b) introducing to the cell a second amount of one or more polynucleotide(s) comprising a second R element (which can be the same as the first R element or a new R element) which comprises a second exogenous sequence (which can be the same as the first exogenous sequence or a new exogenous sequence).

[0011] Also provided, in one embodiment, is a method for integrating one or more exogenous sequence(s) concurrently to the genome of a mammalian cell, comprising (a) introducing to the cell a first amount of one or more polynucleotide(s) comprising a first ribosomal non-long terminal repeat (LTR) retrotransposon (R element) which comprises a first exogenous sequence, and, (b) introducing to the cell a second amount of one or more polynucleotide(s) comprising a second R element which comprises a second exogenous sequence, wherein the first exogenous sequence is different from the second exogenous sequence.

[0012] In some embodiments, each R element is selected from the group of Rl, R2, R4, R5, R6, R7, R8, R9, and RT elements. In some embodiments, each R element is an R2 element or an R8 element. In some embodiments, each R element is an R2 element. In some embodiments, theR2 element comprises an open reading frame encoding a reverse transcriptase (RT) and an endonuclease (EN). In some embodiments, the R2 element further comprises a DNA-binding domain. In some embodiments, the DNA-binding domain comprises one or more zinc finger domains.

[0013] In some embodiments, the R2 element further comprises a 5’ untranslated region (UTR) and a 3 ’UTR. In some embodiments, the R2 element further comprises an upstream sequence and a downstream sequence that are complementary, respectively, to 28 S rDNA sequences upstream and downstream of a target site recognized by the R2 element.

[0014] In some embodiments, each exogenous sequence is flanked by the 5’UTR and 3 ’UTR, or by the upstream and downstream sequences.

[0015] In some embodiments, the one or more polynucleotide(s) are DNA molecule(s).

[0016] In some embodiments, the one or more polynucleotide(s) are RNA molecule(s). In some embodiments, the RNA molecule(s) are encapsulated in lipid nanoparticles (LNP).

[0017] In some embodiments, the second amount was introduced at least 48 hours after the first amount.

[0018] In some embodiments, the method further comprises at least 24 hours after introduction of the second amount, introducing to the cell a third amount of one or more polynucleotide(s) comprising a third R element which comprises a third exogenous sequence.

[0019] In some embodiments, the introducing is by admixing the cell and the one or more polynucleotide(s) in vitro. In some embodiments, the introducing is by administering the one or more polynucleotide(s) to a mammal subject comprising the cell. In some embodiments, the first exogenous sequence is the same as the second exogenous sequence.

[0020] In some embodiments, the first R element is the same as the second R element. In some embodiments, the first R element is different from the second R element.

[0021] In some embodiments, the first exogenous sequence and the second exogenous sequence are integrated into the genome of the cell.

[0022] In some embodiments, the second introduction is at least 24 hours after the first introduction.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the editing efficiencies following two rounds of transfection by R2- SpeciesA and R2-SpeciesB retrotransposon mRNA, respectively.

[0024] FIG. 2 shows the editing efficiencies following two rounds of transfection by R2- SpeciesA mRNA.

[0025] FIG. 3A-C demonstrate successful sequential dosing of same or different payloads by R2 constructs.

[0026] FIG. 4A-B demonstrate successful multiple concurrent dosing of different payloads by R2 constructs.

[0027] It will be recognized that some or all of the figures are schematic representations for purpose of illustration.DETAILED DESCRIPTION

[0028] The following description sets forth exemplary embodiments of the present technology. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.Repeat Dosing of Ribosomal Non-LTR Retrotransposons

[0029] Repeated dosing of gene addition templates has met with mixed success in the gene therapy field. Randomly-integrating gene technologies, such as lentiviral vectors, have been sequentially administered in an ex vivo setting (see, for example, Scaramuzza et al., Mol Ther, 2013 Jan;21(l): 175-84) with an evident increase in overall gene delivery. However, increases in overall gene delivery after sequential administration of site-specific gene addition technologieshave remained a challenge. In the case of CRISPR / Cas based genome editing technologies, it has been widely acknowledged that gene insertion (often via homology directed repair) competes with other endogenous DNA repair mechanisms, including non-homologous end joining (NHEJ). DNA cleavage and re-cleavage via CRISPR-Cas technologies typically occurs at unedited / WT loci. As loci are cleaved and edited via HDR, the locus becomes unavailable for subsequent gene addition. Similarly, as loci are cleaved and edited through NHEJ pathways to result in disruption of the endogenous site through various insertion / deletion patterns (InDeis), the edited loci become unavailable for subsequent editing or gene addition. As most loci are available in the cell in two copies (one on each chromosome), genome editing with CRISPR-Cas technologies can limit or potentially exhaust the number of target sites available for subsequent re-dosing applications. So, while it has been published in the field that gene disruption can occur via exposure of cells or animals to sequential rounds of CRISPR / Cas-based technology (see, for example Finn et al., Cell Rep, 2018 Feb 27;22(9):2227-2235), it was not expected that sequential rounds of gene addition would be successful to increase overall gene addition efficiency, particularly since the pathway leading to gene disruption competes with the pathway of gene addition.

[0030] Ribosomal Non-LTR Retrotransposons (R elements) have shown promises in gene editing. In particular, R2 elements insert into 28S rDNA, and R8 elements insert into 18S rDNA. It was reported that R2 elements are widely distributed in many eukaryotic genomes but not in mammals. Two notable examples are R2Bm, isolated from the silkworm Bombyx mori, and R2O1, isolated from the medaka fish, Oryzias latipes.

[0031] Given that these ribosomal elements do not naturally exist in humans and there exists limited prior application of ribosomal elements to gene therapy or gene addition, many features of use of ribosomal elements remain unclear. For example, it has been published that there is widespread heterogeneity of human ribosomal RNA genes (Fan, et al., RNA, 2022 Apr;28(4):478-492). R2-based gene insertion relies upon certain integration site-specific interactions (Wilkinson et al., Science, 2023 Apr 21;380(6642):301-308) but may also rely on less-well characterized distal secondary structure characteristics at the rDNA locus (Ye et al., Cytogenet Genome Res, 2005; 110(l-4):299-306). Moreover, these ribosomal elements insert sequences into a target site via a target-primed reverse transcription (TPRT) mechanism, whichis initiated by an encoded endonuclease (EN) domain that nicks one strand of DNA at the target site. Such nicking, even without subsequent successful insertion, may inhibit further editing by the same ribosomal element at the same target site, following a subsequent dosing. Consequently, at the level of the anticipated R2 integration target site at the rDNA locus, it has not been clear whether a first administration of an R2-mediated gene delivery system would access, saturate and / or mediate an InDel-like response on all available loci in a cell, which individually or in aggregate could prevent subsequent access to a naive intact target locus of a later administration of R2-mediated gene delivery system.

[0032] Furthermore, it is unclear whether further innate immune responses within a target cell following a first round of R2-mediated gene insertion may limit the success of a second round of R2 -mediated gene insertion. For example, the presence of R2 donor or helper mRNA or protein upon a first administration could trigger intracellular consequences, including pattern recognition receptors and an associated interferon-like response, a senescence response, or even the activation of other mobile genetic elements such as Alu elements similar to other non-LTR retrotransposons like the human LINE-1 (for example, Freeman et al., Nucleic Acids Res. 2022 Feb 28; 50(4): 1888-1907) which could hypothetically impact a subsequent administration of R2 elements for gene insertion.

[0033] Prior to the instant invention, therefore, there was conventional wisdom that repeated dosing of a ribosomal retrotransposon element, such as R2 and R8 elements, may not effectively increase overall editing efficiency.

[0034] Surprisingly and unexpectedly, however, the instant inventors have herein demonstrated that when a subsequent dose was with a different R2 element, there was no interference between the first dosing and the second dosing, since each of them resulted in the same editing rate (Example 1 and FIG. 1). Yet more surprisingly, when the same R2 element was dosed twice, the second dosing also led to substantially the same level of editing as the first dosing (Example 2 and FIG. 2). Moreover, such “repeats” can be sequential (Example 3, FIG. 3) or concurrent (Example 4, FIG. 4), with different transgenes. These data therefore demonstrate that, unlike CRISPR-based editing technologies, retrotransposon-based editing can achieve proportionally increased editing efficiencies with repeated dosing.

[0035] In accordance with one embodiment of the present disclosure, therefore, provided is a method for integrating an exogenous sequence to the genome of a mammalian cell. In some embodiments, the method entails introducing to the cell a first amount of a first R element that encloses a first exogenous sequence, and subsequently introducing to the cell a second amount of a second R element that encloses a second exogenous sequence.

[0036] In another embodiment, provided is a method for integrating one or more exogenous sequence(s) to the genome of a mammalian cell, which entails introducing to the cell a first amount of one or more polynucleotide(s) comprising a first ribosomal non-long terminal repeat (LTR) retrotransposon (R element) which comprises a first exogenous sequence, and, introducing to the cell a second amount of one or more polynucleotide(s) comprising a second R element which comprises a second exogenous sequence. In some embodiments, the first introduction and the second introduction are concurrent or are conducted in the same transduction or transfection. In some embodiments, the first exogenous sequence is different from the second exogenous sequence.

[0037] In some embodiments, the first exogenous sequence is different from the second exogenous sequence, and thus two different sequences can be integrated to the target genome. In some embodiments, the first exogenous sequence is the same as the second exogenous sequence, and thus the overall number of successful integration occurrences for the exogenous sequence is increased.

[0038] In some embodiments, the first R element is different from the second R element. Different R elements likely have different integration sites in the target mammalian genome, and thus the two integration events introduce exogenous sequences to different sites within the target genome. As the total number of possible target sites for each R element may be limited in a target genome, such a method allows higher ceiling of integration occurrences.

[0039] In some embodiments, the first R element is the same as the second R element. In In some embodiments, the first R element and the second R element have the same target site within the genome of the mammalian cell. In some aspects of these embodiments, the first exogenous sequence is also the same as the second exogenous sequence. In other words, the two steps constitute repeats of the same desired integration which leads to a higher overall successrate. In some embodiments, both the first exogenous sequence and the second exogenous sequence are integrated (or inserted) into the genome of the same cell.

[0040] In some embodiments, following the second introduction, a third, a fourth or even more repeats can be carried out, with the same or different R elements / exogenous sequences.

[0041] In some embodiments, between each adjacent rounds of introduction, a sufficient amount of time is allocated to allow sufficient transfection / transduction and integration. In some embodiments, the time between them is 2 hours or longer, or 4 hours, 6 hours, 8 hours, 12 hours, 16 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 years, 2 years, 5 years, or 10 years.

[0042] As noted before, there are at least nine different types of R elements, including Rl, R2, R4, R5, R6, R7, R8, R9, and RT. Among them, Rl, R2, R4, R5, R6, R9, and RT elements insert into 28S rDNA, whereas R7 and R8 elements insert into 18S rDNA. Each of these is within the scope of the present disclosure.

[0043] There are sufficient number of examples for each type of R elements. R2 is described in more details herein as an example. Several lineages of R2 have been maintained for a long time in animals. Four clades (supergroups) of R2 exist. R2A, R2B, R2C, and R2D show independent lineages in the phylogenetic tree based on their reverse transcriptase sequences. They have a distinct number and type of zinc-fingers proximal to the reverse transcriptase domain. R2A has three zinc-fingers, two CCHH type and one CCHC type. R2B has two zinc-fingers, one CCHH type and one CCHC type

[0018] , R2C also has two zinc-fingers although both of them are of the CCHH type. R2D has only one zinc-finger, which is of the CCHH type. These zinc-fingers are responsible for target recognition, and interestingly the contribution of each zinc-finger to target recognition is different between clades.

[0044] R2 is inserted at a specific site within the 28S rRNA genes. It is dependent on the target specific cleavage by the endonuclease encoded by R2. The bottom strand (antisense strand) cleavage is strictly determined, while the top strand (sense strand) shows some variations of cleavage sites, which are determined by the target site alterations upon insertion.

[0045] A good number of example R2 elements are known in the art, some of which are listed in Table A below.Table A. Example R2 Elements* Kojima et al., PLOS One, DOI:10.1371 / journal. pone.0163496 September 23, 2016.

[0046] It is appreciated that introduction of an R element with an exogenic sequence into a target cell can be done in cis or trans configurations, without limitation. In an example cis configuration, the exogenous sequence can be packaged downstream of the native open reading frame (ORF) of the R element, both of which together flanked by the R element’s 5’ and 3’ untranslated regions (UTRs). In such a configuration, both the ORF and the exogenous sequences can be integrated into the target genome. In some embodiments, the ORF and the exogenous sequence may have the same orientation. In some embodiments, the ORF and the exogenous sequence may have opposite orientations.

[0047] In an example trans configuration, the exogenous sequence is enclosed by the 5’ and 3’ UTRs and the ORF can be encoded by a separate construct. Expression of the ORF enablesintegration of the exogenous sequence to the target genome, but the ORF itself may not be integrated.Engineered R Elements

[0048] Engineered R elements have also been made. Each R element includes a DNA-binding domain, which typically includes one or more zinc finger domains. When the zinc finger domains are substituted with other DNA-binding domains (hence a “Heterologous Targeting Domain”), the resulting engineered R element can maintain the site-specific integration capability, whereas the target site may change depending on the specificity of the heterologous targeting domain.

[0049] A “DNA-binding domain” refers to a protein domain capable of binding a nucleic acid sequence of interest, preferably a double strand nucleic acid molecule. The DNA binding domain recognizes and binds nucleic acid at specific polynucleotide sequences, further referred to as “nucleic acid target sequence.” The DNA-binding domain comprises any of the DNA-binding domain known in the art, such as transcription activator-like effector (TALE), homeodomain protein, a zinc finger, a helix-turn-helix, a leucine zipper, and a DNA-binding domain of a homing endonuclease, a transposon or retrotransposon. In certain embodiments, the DNA- binding domain recognizes a target DNA sequence in the genome.

[0050] A “homeodomain protein” consists of three linked alpha helices (helices 1, 2 and 3). Helices 2 and 3 are arranged in a conspicuous helix turn helix motif. A 60 amino acid long region (homeodomain) within helix 3 binds specifically to DNA segments that contain the sequence 5’ATTA3’. In animals, there are 16 major classes of homeodomain protein, ANTP, PRD, PRD-LIKE, POU, HNF, CUT (with four subclasses: ONECUT, CUX, SATB, and CMP), LIM, ZF, CERS, PROS, SIX / SO, plus the TALE superclass with the classes IRO, MKX, TGIF, PBC, and MEIS. Further homeodomain proteins include but not limited to HEX motif, EH1 motif, Octapeptide / Hep / EHl / TN / GEH motif, WRPW motif, TUP1, OAR motif, CUT domain, such as ONECUT, CUX, SATB, CMP domains, COMPASS domain, HNF1A (LFB1) transcription factor, POU, OCT-1, OCT-2, Pit 1, LIM domain, PROSPERO (PROS), SIX / SO and CERS (LASS).

[0051] The “zinc finger” (ZF) includes at least nine types, C2H2, C2HC, C3H, C4, C6, C3HC4, C2HC5, C4HC3, and C8, in which C and H represent cysteine and histidine, respectively. C2H2 zinc finger is a loop of 12 amino acids with two cysteines and two histidines at the base of the loop (Cys2His2 zinc finger motif consisting of an a helix and an antiparallel P sheet) that tetrahedrally coordinate a zinc ion. C2H2 zinc finger is represented by TFIIIA and further comprises Adnp, Tshz, Zeb, Zfhx and Zhx. C2HC-typezinc finger domains, also referred to as retroviral -type (RT) zinc finger sequences, are required for viral genome packaging and RNA or single-stranded DNA binding in eukaryotes. The C3H family proteins are divided into 18 groups based on the different amino acid spacing numbers between C and H in zinc finger motif. C4 finger has one alpha helix and contains a zinc atom bound to four cysteine amino acids. The C4 zinc finger contains a 70 amino acid long region near the zinc atom that binds specifically to DNA segments. The C4 family includes seven types: GATA, FYVE, TimlO / DDP, LSD1, A20, TFIIB, and Zn-finger in Ran-binding protein. C3HC4-type finger, also termed RING finger, can be categorized into seven types with different conserved motifs, such as RING- 112, RING-HC, RING-v, RING-D, RING-S / T, RING-G and RING-C2. The C2HC5 motif, also referred to as LIM. More information on the zinc finger can be found in the prior art publication, such as Li et al., International Journal of Molecular Sciences, (2020) 21(4): 1361. Further examples of the zinc finger proteins include Spl, Glucocorticoid receptor, estrogen receptor, progesterone receptor, thyroid hormone receptor (erbA), retinoid acid receptor, and the vitamin D3 receptor.

[0052] A “leucine zipper” consists of an alpha helix that contains a region in which every seventh amino acid is leucine, which has the effect of lining up all the leucine residues on one side of the alpha helix. The leucine residues allow for the dimerization of the two lecine zipper proteins and formation of Y shaped dimer. Dimerization may occur between two of the same proteins (homodimers, e.g., Jun-Jun) or two different proteins (heterodimers, e.g., Fos-Jun). A leucine zipper contains a 20 amino acid long region that binds specifically to DNA segments. Examples of the Leucine Zipper proteins include but not limited to CCAAT / enhancer binding protein (C / EBP), Cyclic AMP response element binding protein (CREB), Finkel osteogeneic sarcoma virus (Fos) protein, Jun, GCN4, and HSF.

[0053] A “helix loop helix” (HLH) or “helix-turn-helix” consists of a short alpha helix connected by a loop to a loner alpha helix. The loop allows for dimerization of two HLH proteins and formation of Y shaped dimer. The dimerization may occur between two of the same proteins (homo dimers) or two different proteins (heterodimers). Example of HLH domains include but not limited to MyoD, Myc, Pho4, SREBP-la, and Max-Mad.

[0054] “Transcription Activator like Effectors” (TALE) are proteins that are encoded by phytopathogenic bacteria of the genus Xanthomonas and Ralstonia to influence the gene expression of host plant cells during bacterial infection. These proteins are helix-loop-helix- containing transcriptional factors that comprise a DNA binding region and an N-terminal domain that appears to interact with the bacterial transport machinery for introducing the protein into the plant cell. The C-terminal domain of the TALE protein seems to interact with the plant host's transcriptional machinery to induce expression of sets of plant genes that are beneficial to the invading bacteria. The DNA binding portion of the proteins is found in the middle section of the protein and is made of an array of repeat units, each approximately 33-35 amino acids in length, which have been shown to be responsible for interacting with the target DNA. In a preferred embodiment, said DNA-binding domain is derived from a TALE engineered to bind a specific nucleic acid target sequence. Unlimited examples of the TALE include KNOX and BEL, such as PBC, MEIS, PREP, IRO, MKX and TGIF.

[0055] A “homing endonuclease (HEs)” or meganuleases (MNs), are sequence-specific endonucleases with large cleavage sites (14-25 bp) that can create double-stranded breaks at specific locations. The homing endonucleases are encoded by mobile genetic elements that induce recombination in a process called homing. The homing endonucleases genes (HEGs) that have invaded many genomic niches including group I and group II introns. Some HEGs can move from an ORF-containing intron to an “ORF -less” intron. There are four major families of homing endonucleases (HEs), naming is based on conserved amino acid motifs: the H-N-H, HIS- CYS, LAGLID ADG, and GIY-YIG families of HEs. The LAGLIDADG family of HEs is the most frequently encountered among group I introns. However GIY-YIG endonucleases have been identified within numerous group I introns, and LAGLIDADG HEs and the H-N-H domain are present within the ORFs of some group II introns. Additional HE-like proteins have been described, the PD-(D / E)XK HEs are found in bacterial tRNA group I introns, the very-shortpatch repair (Vsr) endonucleases (a predicted family of phage HEs based on metagenomic), and the Holliday junction resolvase-like HEs found in some phage introns. In certain embodiments of the present disclosure, the homing endonuclease comprises Pl-Scel, Pl-Pful, I-Crel, I-Ceul, I- Dmol, F-TevI, F-TevII, I-TevI, I-TevII, I-Ppol, I-Dirl, I-Njal, I-NanI, I-Nitl, I-SceV, I-SceVI and I-Llal, I-Hmul, I-HmuII, I-TevIII and I-Cmoel. In a particular embodiment, the homing endonuclease is I-Ppol (Intron-Encoded Endonuclease).

[0056] Retrotransposon is a class of eucaryotic genes capable of replicating to new locations within their own genome through an RNA intermediate. Retrotransposons are widespread in metazoan genomes. The proportion of the genome that contains retrotransposons often exceeds that containing DNA transposons, particularly in higher vertebrates such as humans. Most retrotransposons integrate into random sites of the host genome, but some have a sequence preference. In particular, a few non-LTR subclades integrate into the genome in a highly sequence-specific manner. By leaving their 5’ and / or 3’ ends of genomic copies (usually with a poly (A) stretch and target-site duplication (TSD)), the site-specific non-LTR retrotransposons can be searched throughout the DNA database.

[0057] There are five clades of restriction enzyme-like endonuclease (RLE)-encoding elements based on their RT sequence similarity. Most of three clades (NeSL, R2, and R4) are target specific and two clades (HERO and CRE) have some site-specific elements. R2 (R2 clade) was found at a specific sequence in the 28S rDNA of many invertebrate and vertebrate species. R4 in Ascaris and Dong (R4 clade) were found at another site of the 28S rDNA and microsatellite TAA repeats, respectively.

[0058] In contrast to RLE-encoding elements, most of the apurinic / apyrimidinic endonuclease (APE)-encoding non-LTR elements do not insert themselves in a sequence-specific manner, but do have weak target-site specificity, e.g., human LI for TAAA repeats. However, two clades of APE-encoding non-LTR elements, Txl and Rl, are known to be sequence-specific.

[0059] Among the site-specific non-LTR retrotransposons, Rl clade (R1 / R6 / R7 / RT), R2 clade (R2 / R8 / R9), R4 clade (R4) and NeSl clade (R5) target the rRNA genes, whereas R7 and R8 target the 18S rDNA, and Rl, R2, R4, R5, R6, R9, and RT target the 28S rDNA. All R-elementtarget sites within 28S and 18S rDNA are highly conserved among organisms (see Fujiwara et al., Microbiology Spectrum, (2015) Vol. 3, Issue 2).

[0060] In a preferred embodiment, the retrotransposon is rDNA specific and comprises Rl, R2, R4, R5, R6, R7, R8, R9 and RT. In certain embodiments, the heterologous DNA-binding domain provided herein comprises the DNA-binding domain of any one of the Rl, R2, R4, R5, R6, R7, R8, R9 and RT.

[0061] In certain embodiments, the one or more N-terminal ZnF domains is replaced with a CRISPR Cas protein. In certain embodiments, the CRISPR Cas protein comprises Cas9, Casl2a / Cpfl and Casl3. The clustered regularly interspaced short palindromic repeat (CRISPR) / Cas mediated genome editing technology has seen wild use since its invention due to its simplicity and efficiency, leading to a new era of genetic engineering and gene therapy. The targeting and cleavage are achieved by a single CRISPR RNA (“crRNA”)-bound Cas protein in Class 2 CRISPR-Cas systems. Class 2 systems can be subdivided into Type II Cas9 and Type V Cas 12 that target DNA, as well as Type VI Cas 13 that target RNA. Cas9 and Casl2a / Cpfl have been well studied in the past and broadly harnessed for gene editing in various cell types and organisms in prokaryotes and eukaryotes. Both Cas9 and Casl2 systems utilize guide RNA to recognize the target site and protospacer adjacent motifs (PAMs) to determine the cleavage site and generate double-stranded breaks. However, the binding and cleavage of DNA by Cas9 and Casl2 are quite different. Cas9 recognizes a 3’-G-rich PAM and produces blunt ends cleaved by the RuvC and HNH domains, whereas Cas 12 recognizes a 5’ T-rich PAM and produces staggered ends cleaved solely by the RuvC domain. Pre-assembled Cas 13 and crRNA recognizes target RNAs. Upon RNA-binding, Cas 13 will undergo a conformational change and induce the catalytic activity of its nuclease domains, resulting in the cleavage of target transcripts. In certain embodiments, the gRNA or the crRNA can be designed to target a DNA sequence in the genome. In certain embodiments, the Cas protein comprises an inactive nuclease domain. In certain embodiments, the Cas protein does not comprise an active nuclease domain.

[0062] In certain embodiments, the guide RNA (“gRNA”) comprises two short, non-coding RNA species referred to as crRNA and trans-acting RNA (“tracrRNA”). In an exemplary system, the gRNA forms a complex with a Cas protein of the present disclosure. The gRNA:Cas proteincomplex binds a target polynucleotide sequence in the genome. The gRNA can be supplied separately that works with the Cas protein in guiding the Cas protein (or the fusion) to a target location on a genomic sequence.

[0063] In some embodiments, the endonuclease (EN) of the native R element can also be substituted with a heterologous endonuclease.

[0064] An endonuclease is an enzyme that cleaves internal phosphodiester bonds of polynucleotides. These enzymes are either specific or non-specific to the sequences being cleaved. The endonucleases that are specific to a particular sequence are termed restriction endonucleases, which cleave large DNA molecules at specific sequences of four to six nucleotides.

[0065] In some embodiments the endonuclease is a eukaryotic endonuclease. Non-limiting examples include Neurospora endonuclease, SI nuclease, Pl -nuclease, Mung bean nuclease I, Ustilago nuclease, DNase I, AP endonuclease and Endo R. Other examples include Fokl nuclease, type-II restriction 1 -like endonucleases (RLE-type nuclease), and RLE-type endonucleases (REL).

[0066] Many known transposons and retrotransposons also include related endonucleases. In some embodiments, the heterologous endonuclease is one from Rl, R2, R4, R5, R6, R7, R8, R9 or RT.Exogenous Sequences and Other Components

[0067] An R element can package one or more exogenous sequences for integration to a target genome, particularly in a target mammalian cell, such as a human cell. In a preferred embodiment, the exogenous sequence encodes a protein, such as a therapeutic protein.

[0068] A wild-type R element, such as an R2 element, includes an ORF that has the required EN and RT activities. It has been shown, however, that for the R2 element to work, the ORF can be expressed from a separate polynucleotide, which enables the “empty” R2 retrotransposon to include a large exogenous sequence for delivery to the target cell. Alternatively, the exogenous sequence can be included on the same polynucleotide as the ORF for RT and EN. In someembodiments, when the exogenous sequence and the R2 element’s native ORF are on the same polynucleotides, they are in different orientations (i.e., their translation starts from opposite directions).

[0069] In some embodiments, the sequence to be integrated, which includes the exogenous sequence, is flanked by sequences such as a 5’ untranslated region (UTR) and a 3’ UTR. In some embodiments, the 5 ’UTR and the 3 ’UTR are native to the corresponding R2.

[0070] In some embodiments, the exogenous sequence to be integrated is adjacent to a downstream flanking sequence that is 3’ to the exogenous sequence. Addition of a downstream flanking sequence can improve the retrotransposition efficiency. In certain embodiments, the protein-coding sequence may comprise, at the 3’ end, one or more consecutive nucleotides (rDNA sequences) of the “downstream sequence” starting from the EN cleavage site on the target 28 S rDNA gene. The protein-coding sequence may include, at the 3’ end of the gene encoding sequence, different lengths of the downstream sequence depending on the type of polynucleotide or vector used for delivery.

[0071] In some embodiments, the exogenous sequence to be integrated is adjacent to an upstream flanking sequence that is 5’ to the exogenous sequence. Addition of an upstream flanking sequence may also improve the retrotransposition efficiency. In certain embodiments, the exogenous sequence may include, at the 5’ end, one or more consecutive nucleotides (rDNA sequences) of the “upstream sequence” starting from the EN cleavage site on the target 28 S rDNA gene. The protein-coding sequence may include, at the 5’ end of the gene encoding sequence, different lengths of the downstream sequence depending on the type of polynucleotide or vector used for delivery.

[0072] In some embodiments, the exogenous sequence encodes a therapeutic protein. In some embodiments, the protein is a therapeutic protein that can be helpful in treating a disease or condition in a patient. Examples include:- Insulin - used to treat diabetes- Phenylalanine hydroxylase - used to treat PKU (Phenylketonuria) disease- Erythropoietin - used to treat anemia- Filgrastim (G-CSF) - used to treat neutropenia- Interferons - used to treat cancer, multiple sclerosis, and viral infections- Tumor necrosis factor (TNF) inhibitors - used to treat autoimmune diseases, such as rheumatoid arthritis and Crohn's disease- Monoclonal antibodies - used to treat cancer, autoimmune diseases, and some viral infections- Follitropin alfa - used to treat infertility- Coagulation factor - used to treat bleeding disorders, such as Hemophilia- Enzyme replacement therapy enzymes - used to treat lysosomal storage disorders, such as Gaucher's disease- Growth hormone - used to treat growth hormone deficiency.Gene Delivery and Treatments

[0073] The method disclosed here are suitable for delivering exogenous sequences, at high efficiency, to a target cell, such as mammalian cells. As the delivery results in target sequence (28S rDNA)-specific integration, it can be highly safe as well. This new technology, therefore, has promising therapeutic uses as well.

[0074] In some embodiments, the one or more polynucleotides of the R elements are provided as DNA which can be translated into the desired RNA for the R elements. In some embodiments, the DNA is provided on a vector, such as plasmid or viral vector. In some embodiments, the one or more polynucleotides are provided as RNA directly.

[0075] RNAs may be synthesized according to any of a variety of known methods. For example, the RNAs may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (c. ., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions will vary according to the specific application.

[0076] The RNA may be synthesized as unmodified or modified RNA. Typically, RNAs are modified to enhance stability. Modifications of RNA can include, for example, modifications of the nucleotides of the RNA. A modified RNA can thus include, for example, backbonemodifications, sugar modifications or base modifications. In some embodiments, RNAs may be synthesized from naturally occurring nucleotides and / or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g. 1-methyl-adenine, 2-methyl -adenine, 2-methylthio-N-6- isopentenyl -adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl- cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1 -methyl -guanine, 2-methyl- guanine, 2,2-dimethyl-guanine, 7-m ethyl -guanine, inosine, 1 -methyl -inosine, pseudouracil (5- uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5 -fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-methyl-2 -thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5 -methoxy aminomethyl -2-thio-uracil, 5’- methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5- oxyacetic acid (v), 1-methyl-pseudouracil, queosine, 13-D-mannosyl-queosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7- deazaguanosine, 5 -methylcytosine and inosine. The preparation of such analogues is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530 and 5,700,642, the disclosure of which is included here in its full scope by reference.

[0077] In some embodiments, the RNAs may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5 ’-O-(l -thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.

[0078] In some embodiments, the RNAs may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2 ’-deoxy -2’ -fluoro-oligoribonucleotide (2’ -fluoro-2’ -deoxy cytidine 5 ’-triphosphate, 2’-fluoro-2’-deoxyuridine 5’- triphosphate), 2’-deoxy-2’-deamine-oligoribonucleotide (2’-amino-2’-deoxycytidine 5’- triphosphate, 2’-amino-2’-deoxyuridine 5 ’-triphosphate), 2’-O-alkyloligoribonucleotide, 2’- deoxy-2’-C-alkyloligoribonucleotide (2’-O-methylcytidine 5 ’-triphosphate, 2 ’-methyluridine 5’- triphosphate), 2’ -C -alkyloligoribonucleotide, and isomers thereof (2’-aracytidine 5 ’-triphosphate, 2’-arauridine 5 ’-triphosphate), or azidotriphosphates (2’ -azi do-2’ -deoxy cytidine 5 ’-triphosphate, 2’ -azido-2’ -deoxyuridine 5 ’-triphosphate).

[0079] In some embodiments, the RNAs may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modified nucleotide. Examples of such base-modified nucleotides include, but are not limited to, 2-amino-6-chloropurine riboside 5 ’-triphosphate, 2-aminoadenosine 5’- triphosphate, 2-thiocytidine 5 ’-triphosphate, 2-thiouridine 5 ’-triphosphate, 4-thiouridine 5’- triphosphate, 5-aminoallylcytidine 5 ’-triphosphate, 5-aminoallyluridine 5 ’-triphosphate, 5- bromocytidine 5 ’-triphosphate, 5-bromouridine 5 ’ -triphosphate, 5-iodocytidine 5 ’ -triphosphate, 5-iodouridine 5 ’-triphosphate, 5-methylcytidine 5 ’-triphosphate, 5-methyluridine 5’- triphosphate, 6-azacytidine 5 ’-triphosphate, 6-azauridine 5 ’-triphosphate, 6-chloropurine riboside 5 ’-triphosphate, 7-deazaadenosine 5 ’-triphosphate, 7-deazaguanosine 5 ’-triphosphate, 8- azaadenosine 5 ’-triphosphate, 8-azidoadenosine 5 ’-triphosphate, benzimidazole riboside 5’- triphosphate, N1 -methyl ad enosine 5’-triphosphate, N1 -methylguanosine 5 ’-triphosphate, N6- methyladenosine 5 ’-triphosphate, 06-methylguanosine 5 ’-triphosphate, pseudouridine 5’- triphosphate, puromycin 5 ’-triphosphate or xanthosine 5 ’-triphosphate.

[0080] In some embodiments, the RNAs include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’5’5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5’)ppp (5’(A,G(5’)ppp(5)A and G(5)ppp(5’)G.

[0081] In some embodiments, the delivery is in vitro. In some embodiments, the delivery is in vivo or ex vivo. In some embodiments, the retrotransposon polynucleotide is delivered by parenteral means, or locally.

[0082] The term “parenteral” as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

[0083] Delivery can be systemic or local. In addition, it may be desirable to introduce the retrotransposon polynucleotide of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.EXAMPLES

[0084] The following examples are included to demonstrate specific embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques to function well in the practice of the disclosure, and thus can be considered to constitute specific modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1: Editing Efficiency of a Second R2 Retrotransposon

[0085] This example tested the editing efficiencies following two rounds of transfections by two different R2 retrotransposons, R2-SpeciesA and R2-SpeciesB. In particular, this example examined whether the first round of editing had any detrimental impacts on the editing efficiency of the second R2.

[0086] The editing efficiencies of these R2 retrotransposons, following the first round and second round of transfection were tested with a cultured human cell line 293T. Cells were grownin Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin / streptomycin (Invitrogen) in 5% CO2 at 37 °C. Cells were grown as adherent cell cultures and passaged every 72 h at a 1:5 dilution (volume of cells: final volume of medium) until confluency was reached, in order to maintain log-phase growth.

[0087] Cells were seeded at 60% confluence in 24-well cluster plates one day before transfection. For transfection, OptiMEM medium (Invitrogen) was pre-warmed to room temperature. Transfection of mRNA into cells was performed with Lipofectamine® MessengerMAX™ (Invitrogen). mRNA Lipofectamine® MessengerMAX™ Reagent (1.5 pL of each) were mixed with 25 pl OptiMEM medium and incubated at room temperature.

[0088] For each transfection, the R2 retrotransposon (WT) was provided as mRNA encapsulated in lipid nanoparticles. The mRNA included the R2 open reading frame, as well as a target sequence (z.e., a coding sequence for GFP) flanked by the R2 5’UTR and 3’UTR. A mutant inactive R2-SpeciesA retrotransposon (R2mut) and an mRNA encoding GFP (GFP) were used as controls.

[0089] The first round of transfection, along with the R2-mediated editing, was carried out for 2 days. The cells were then split and reseeded, followed by a second round of transfection with the same R2 retrotransposon, for 3 days. Genomic DNA was extracted after each round of transfection / editing, and the numbers of successful editing (i.e., integration of the GFP coding sequence into the genome) were determined by quantitative PCR (qPCR) at the target 28 S rDNA gene loci.

[0090] The results are shown in FIG. 1. Following the first round of transfection by R2- SpeciesA (WT), a significant amount of GFP integration at the expected 28S rDNA gene loci were detected (i.e., low ct value for PCR amplification of integrated sequence).

[0091] Although R2-SpeciesB targets the same recognition sequence of integration within the 28S rDNA gene, the junction sequences (28S rDNA / R2) are different from that of R2-SpeciesA. Therefore, the quantification of the second round of editing was independent of the first round (i.e., using different PCR primers). It necessarily follows transfection by the GFP mRNA control in the second round (following successful editing (WT) from the first round) exhibited no editing(ct > 40) in the second round. By contrast, WT (1stround) => WT (2ndround) exhibited successful editing in both rounds.

[0092] More interestingly, there was no visible difference between the second round of editing efficiencies of WT => WT and GFP => WT (see, in the lower panel of FIG. 1, 2ndbar from the left vs. 2ndbar from the right). This comparison, therefore, indicates that the first round of editing by R2-SpeciesA (WT) was independent of the editing by R2-SpeciesB (WT) in the second round, and consequently, that sequential administration of R2 elements can lead to greater net levels of R2 -mediated genome editing.Example 2: Editing Efficiency of Repeat Dosing

[0093] Example 1 showed that the earlier editing by R2-SpeciesA in a target cell had no impact on the subsequent editing efficiency of a different R2 (R2-SpeciesB) in the same target cell. This example tested whether the previous editing by R2-SpeciesA would impact the editing efficiency of the same R2 following a repeat dosing to the same cells.

[0094] The experimental procedure was the same as described in Example 1, except that the second transfection was with the same R2-SpeciesA retrotransposon, and the PCR detection was with the same primers to the same integration site as the first round.

[0095] The results are shown in FIG. 2. Following the first round of transfection by R2- SpeciesA (WT), a significant amount of GFP integration at the expected 28S rDNA gene loci were detected (z.e., low ct value for PCR amplification of integrated sequence). After the second round of transfection (WT => WT), the total number of integrations further increased (a lower Ct value of qPCR than that of the first round was observed, suggesting additional editing occurred). This result, therefore, indicates that the first round of transfection and editing was independent from the second round of transfection and editing, even when both R2 retrotransposons targeted the same sites within the 28S rDNA gene. In other words, this example demonstrates that the net overall editing efficiency of R2 retrotransposons can be increased effectively by repeat dosing.Example 3. Sequential Dosing of Different Constructs

[0096] This example tested sequential dosing of R2 constructs that included different transgenes.

[0097] HEK293T cells were transfected on Day 0 first by a fluorescent reporter GFP cassette, and again on Day 3 with R2 delivering either GFP, mCherry, or no additional cassette. Flow cytometry analyses were done on Day 3 and 6 to assess the integration rates of the reporter genes. Shown on the top of FIG. 3A is an image of GFP+ cells on Day 3, which is followed by additional transfections with either GFP cassette, mCherry cassette, or no additional transfection. The bottom graph summarizes the percentage of fluorescent cells in each sequential dosing category. A flow cytometry analysis in FIG. 3C demonstrates the details of the middle group. GFP and mCherry are both distributed in target cells without evidence for exclusion.

[0098] A single dose control group was carried out. Untransfected cells were maintained for three days, and were transfected with either GFP cassette, mCherry cassette, or no transfection on Day 3 (FIG. 3B).

[0099] These results show that R2 can successfully repeatedly deliver same or different payloads to target cells.Example 4. Multi-Dosing in a Single Transfection

[0100] This example demonstrates successful co-delivery of two separate reporter genes in a single transfection.

[0101] HEK293T cells were concurrently transfected with two separate fluorescent reporter cassettes, GFP and mCherry, respectively (FIG. 4A). Flow cytometry analyses were done on Day 3 to assess the integration rates of the reporter genes. The bottom graph of FIG. 4A summarizes the percentages of fluorescent (single and / or dual) cells in five transfection scenarios: GFP only, 75%GFP and 25%mCherry, 50%GFP and 50%mCherry, 25%GFP and 75%mCherry, and mCherry only. Dose response was observed, corresponding to the molar ratios of two reporter cassettes. A flow cytometry analysis demonstrates the details of the middle group in A where GFP and mCherry are both distributed in target cells without evidence for exclusion.* * *

[0102] 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 invention belongs.

[0103] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0104] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0105] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0106] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0107] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0108] It is to be understood that while the disclosure has been described in conjunction with the above embodiments, that the foregoing description and examples are intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages and modifications within the scope of the disclosure will be apparent to those skilled in the art to which the disclosure pertains.

Claims

CLAIMS:

1. A method for integrating one or more exogenous sequence(s) to the genome of a mammalian cell, comprising:(a) introducing to the cell a first amount of one or more polynucleotide(s) comprising a first ribosomal non-long terminal repeat (LTR) retrotransposon (R element) which comprises a first exogenous sequence, and,(b) introducing to the cell a second amount of one or more polynucleotide(s) comprising a second R element which comprises a second exogenous sequence.

2. The method of claim 1, wherein the second introduction is subsequent to the first introduction, or the second exogenous sequence is different from the first exogenous sequence.

3. The method of claim 1 or 2, wherein each R element is selected from the group of Rl, R2, R4, R5, R6, R7, R8, R9, and RT elements.

4. The method of claim 3, wherein each R element is an R2 element or an R8 element.

5. The method of claim 3, wherein each R element is an R2 element.

6. The method of claim 5, wherein the R2 element comprises an open reading frame encoding a reverse transcriptase (RT) and an endonuclease (EN).

7. The method of claim 5 or 6, wherein the R2 element further comprises a DNA-binding domain.

8. The method of claim 7, wherein the DNA-binding domain comprises one or more zinc finger domains.

9. The method of any one of claims 5-8, wherein the R2 element further comprises a 5’ untranslated region (UTR) and a 3’UTR.

10. The method of any one of claims 5-9, wherein the R2 element further comprises an upstream sequence and a downstream sequence that are complementary, respectively, to 28S rDNA sequences upstream and downstream of a target site recognized by the R2 element.

11. The method of claim 9 or 10, wherein each exogenous sequence is flanked by the 5’UTR and 3’UTR, or by the upstream and downstream sequences.

12. The method of any preceding claim, wherein the one or more polynucleotide(s) are DNA molecule(s).

13. The method of any one of claims 1-11, wherein the one or more polynucleotide(s) are RNA molecule(s).

14. The method of claim 13, wherein the RNA molecule(s) are encapsulated in lipid nanoparticles (LNP).

15. The method of any preceding claim, wherein the second amount was introduced at least 48 hours after the first amount.

16. The method of any preceding claim, further comprising (c) after introduction of the second amount, introducing to the cell a third amount of one or more polynucleotide(s) comprising a third R element which comprises a third exogenous sequence.

17. The method of any preceding claim, wherein the introducing is by admixing the cell and the one or more polynucleotide(s) in vitro.

18. The method of any one of claims 1-16, wherein the introducing is by administering the one or more polynucleotide(s) to a mammal subject comprising the cell.

19. The method of any preceding claim, wherein the first exogenous sequence is the same as the second exogenous sequence.

20. The method of any preceding claim, wherein the first R element is the same as the second R element.

21. The method of any preceding claim, wherein the first R element is different from the second R element.

22. The method of any preceding claim, wherein the first exogenous sequence and the second exogenous sequence are integrated into the genome of the cell.

23. The method of any preceding claim, wherein the second introduction is at least 24 hours after the first introduction.

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