Improved synthesis-mediated transgene insertion

Abstract

Provided herein are compositions and methods for improved synthesis-mediated transgene insertion. The methods can include modulation of DNA repair pathways to inhibit stable gene insertion without base-pairing between the genome-side and insertion-side DNA sequences, as useful with other methods based on host cell DNA repair pathway manipulation to improve functional full-length intact transgene synthesis.

Description

IMPROVED SYNTHESIS-MEDIATED TRANSGENE INSERTIONCROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application 63 / 648,622, filed May 16, 2024, which is incorporated by reference herein in its entirety for all purposes.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant number HL 156819 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Target primed reverse transcription (TPRT) inserts a nucleic acid sequence into a subject genome using non-long terminal repeat (non-LTR) retrotransposons. The insertion of an exogenous nucleic acid sequence into a subject via TPRT faces challenges. Additional methods and systems are needed to improve the fidelity of TPRT methods and systems.SUMMARY

[0004] Certain aspects and embodiments of the present disclosure include:

[0005] 1. A method of improved synthesis-mediated transgene sequence insertion, comprising modulating DNA synthesis to inhibit stable gene insertion without base-pairing between the genome-side and insertion-side DNA sequences.

[0006] 2. A method herein, wherein the sequence insertion comprises a step of joining the first strand cDNA to upstream target site using annealing lengths of preferably 10-40 bp (or 20-30 bp, or more than 40 bp), by a mechanism different from microhomology-mediated joining (mediated by formation of 1-8 bp) and RAD52-dependent single strand annealing or RAD51- dependent strand invasion that can initiate homologous recombination (favored by 50+ nucleotides of complementary base-pairing potential).

[0007] 3. A method herein, wherein the method is independent of homologous recombination.

[0008] 4. A method herein, wherein the method inhibits stable sequence insertion without formation of approximately 10-40 (or 20-30) or more base-pairs between DNA strands, whether template for the new sequence synthesis is introduced to cells directly as DNA or introduced as RNA to template DNA synthesis, to make stable sequence insertion dependent on formation of at least 10-40 (or 20-30) base-pairs between the genome-side and insertion- side DNA sequences.

[0009] 5. A method herein, wherein the synthesis-mediated gene insertion comprises second- strand cDNA synthesis, and second-strand synthesis requires a cDNA with upstream target-site base-pairing potential, wherein the insertion may be at any intended native or non-native target site.

[0010] 6. A method herein, wherein the insertion is initiated by a non-LTR retrotransposon protein at its native target site (e.g. rDNA) or elsewhere, or at any non-LTR retrotransposon insertion site with or without retargeting.[Oil] 7. A method to improve synthesis-mediated gene insertion, comprising modulating second-strand synthesis (transgene 5’ junction formation and cDNA-complementary strand synthesis) to reduce or eliminate stable insertion of a 5 ’-truncated transgene.

[0012] 8. A method to inhibit unintended template RNA use for stable transgene insertion, achieved by second-strand synthesis requirement for base-pairing of cDNA and upstream insertion-site sequence.

[0013] 9. A method to reduce or prevent off-target insertions by modulating 5’ junction formation and second-strand synthesis, achieved by a requirement for base-pairing of cDNA and upstream insertion-site sequence.

[0014] 10. A method to reduce or eliminate cDNA snap-back synthesis prior to 5’ transgene junction formation by modulating host cell factors, including but not limited to POLQ and 5’ junction formation and second-strand synthesis factors.

[0015] 11. A method herein, wherein the modulating is achieved by a combination one or more or all of the following transgene 5 ’ junction formation and / or second-strand synthesis mechanisms:

[0016] inhibition of DNA synthesis by Polymerase theta (encoded by human POLQ),

[0017] inhibition of recruitment of Polymerase alpha Primase complex, such as mediated by inactivation of Shieldin or other factor(s) composing, associated with, or activating the Shieldin complex (e.g. SHLD 1, SHLD 2, SHLD 3, REV7 / MAD2L27),

[0018] inhibition of recruitment of Polymerase alpha Primase complex, such as mediated by inactivation of CTC1 or other factor(s) composing, associated with, or activating the CST complex (e.g. CTC1, STN1, TEN1),

[0019] inhibition of DNA binding by SHLD2 and / or CTC1 by any approach, including but not exclusively by dominant-negative Shieldin or CST subunit expression or small interfering RNA expression or small molecule drugs,

[0020] inhibition of non-homologous end-joining mechanisms described above and in general, to disfavor annealing-independent transgene 5’ junction formation by targeting cellular factors including but not limited to TOPBP 1, or

[0021] inhibition of non-homologous end-joining mechanisms involving polymerases mu (POLM), lambda (POLL) and / or theta (POLQ).

[0022] 12. A method herein, where end-resection factors including but not limited toRBBP8 / QIP promote the efficiency of stable transgene insertion dependent on base-pairing of cDNA and target site for Anneal junction formation, such that increase in expression or function of these factors will improve the efficiency of Anneal junction formation.

[0023] 13. A method herein, wherein manipulation of second-strand synthesis and associated transgene 5’ junction formation limit unwanted repair of partial second-strand synthesis products to produce transgene internal gaps.

[0024] 14. A method herein, for enabling full-length transgene insertion by providing factors that promote the Anneal pathway of transgene 5’ junction formation.

[0025] The invention can encompass all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0026] In one aspect, the present disclosure provides a system comprising (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide; (c) a polymerase theta (POLQ) inhibitor; and (d) a Shieldin complex inhibitor or a CTC1-STN1-TEN1 (CST) complex inhibitor.

[0027] In some embodiments, the POLQ inhibitor comprises a small molecule inhibitor. In some embodiments, the POLQ inhibitor comprises ART558. In some embodiments, the POLQ inhibitor comprises an siRNA targeting an mRNA encoding a POLQ gene. In some embodiments, the system comprises a Shieldin inhibitor and the Shieldin complex inhibitor comprises a small molecule inhibitor. In some embodiments, the system comprises a Shieldin inhibitor and the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a REV7 gene. In some embodiments, the system comprises a Shieldin inhibitor and the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a SHLD1 gene, a SHLD2 gene, or a SHLD3 gene. In some embodiments, the system comprises a CST complex inhibitor and the CST complex inhibitor comprises a small molecule inhibitor. In some embodiments, the system comprises a CST complex inhibitor and the CST complex inhibitor comprises an siRNA targeting a CTC1 gene. In some embodiments, the system comprises a CST complex inhibitor and the CST complex inhibitor comprises an siRNA targeting a STN 1 gene. In some embodiments, the system comprises a CST complex inhibitor and the CST complex inhibitor comprises an siRNA targeting a TEN1 gene.

[0028] In some embodiments, the first nucleic acid comprises a pseudouridine or a Nl- methylpseudouridine. In some embodiments, the 5 ’ module of the first nucleic acid comprises an rDNA sequence. In some embodiments, the rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some embodiments, the rDNA sequence has a length of from 10-40 nucleotides. In some embodiments, the 3’ module of the first nucleic acid further comprises an additional rDNA sequence. In some embodiments, the additional rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some embodiments, the additional rDNA sequence has a length of from 4-40 nucleotides. In some embodiments, the 5’ module further comprises a ribozyme sequence. In some embodiments, the 3’ module further comprises a polyA sequence.

[0029] In some embodiments, the system further comprises an RNaseH activity enhancer. In some embodiments, the RNaseH activity enhancer comprises mRNA encoding RNaseH.

[0030] In some embodiments, the present disclosure provides a cell comprising the system. In some embodiments, the cell further comprises genomic DNA comprising a 28S rDNA target site. In some embodiments, the template sequence encodes a transgene. In some embodiments, the cell comprises non-native RNaseH or mRNA encoding non-native RNaseH.

[0031] In some embodiments, the present disclosure provides a method of editing a cell, the method comprising administering to the cell the system, wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) and (d) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

[0032] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide; (c) a polymerase theta (POLQ) inhibitor; and (d) an RNaseH activity enhancer; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) and (d) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some embodiments, the POLQ inhibitor comprises a small molecule inhibitor. In some embodiments, the POLQ inhibitor comprises ART558. In some embodiments, the POLQ inhibitor comprises an siRNA targeting an mRNA encoding a POLQ gene. In some embodiments, administering (d) the RNaseH activity enhancerinduces a higher expression of RNaseH in the cell, compared to that upon administering to a corresponding cell with (a)-(c) alone.

[0033] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a DNA topoisomerase II binding protein 1 (TOPBP1) inhibitor; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

[0034] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a C -terminal-binding protein interacting protein (CtIP) activity enhancer; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) enhances full-length second-strand synthesis for insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0035] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) an Upstream Binding Transcription Factor (UBTF) or a third nucleic acid encoding the UBTF; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0036] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the templatesequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a modulator of RNA Polymerase I activity or a third nucleic acid encoding the modulator of RNA Polymerase I activity; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0037] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a modulator of a nucleolar architecture or a third nucleic acid encoding the modulator of the nucleolar architecture; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone. In some embodiments, the modulator comprises a Lamin A inhibitor.

[0038] In some aspects, the first nucleic acid comprises a pseudouridine or a Nl- methylpseudouridine. In some aspects, the 5’ module of the first nucleic acid comprises an rDNA sequence. In some aspects, the rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some aspects, the rDNA sequence has a length of from 10-40 nucleotides. In some aspects, the 3’ module of the first nucleic acid further comprises an additional rDNA sequence. In some aspects, the additional rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some aspects, the additional rDNA sequence has a length of from 4-40 nucleotides. In some aspects, the 5’ module further comprises a ribozyme sequence. In some aspects, the 3’ module further comprises a polyA sequence. In some aspects, the template sequence encodes a transgene.INCORPORATION BY REFERENCE

[0039] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Fig. 1. Target-primed reverse transcription for new gene insertion to the genome. Thin and dashed lines are RNA, thick lines are DNA. EN, endonuclease. RT, reverse transcriptase.

[0041] Fig. 2. Representative “precise RNA mediated insertion of transgenes” (PRINT) mRNA transcription vector and product RNA. Digested DNA vector is transcribed by T7 RNA Polymerase (IVT). Representative but not fully inclusive features of a PRINT mRNA may be a 5 ’ AG dinucleotide cap, 5 ’ and 3 ’ UTR sequences suited to the desired level of translation and mRNA half-life, a vector-encoded poly-adenosine tail (e.g. AjoLinkerioA o or A30), and substitution of uridine with 1 -methyl -pseudouridine. The ORF encodes a protein with features such as zinc fingers (ZF) and zinc knuckle (ZK), Myb DNA binding domain (Myb), RNA binding surface (RBD). The RT and EN domain active sites may be mutated to cripple the RT (RTD), cripple the EN (END), or tune down EN (ENT).

[0042] Fig. 3. Representative PRINT template RNA transcription vector and product RNA. Digested DNA vector is transcribed by T7 RNA Polymerase (IVT). Representative but not fully inclusive features of a PRINT template RNA are a 5’ leader sequence, upstream rDNA target site sequence (e.g. R28, 28 nucleotides), a ribozyme (Rz) that can be 5’ of the rDNA sequence or encompass the rDNA sequence, a transgene payload such as a protein expression cassette with a promoter and polyadenylation signal sequence (PA), and a 3’ module with protein binding surface (e.g. GeFo 3’UTR), downstream rDNA (e.g. R4, 4 nucleotides), and a stabilizing sequence (e.g. A22, ~22 adenosines). Template RNAs can have substitution of uridine (e.g. with pseudouridine or 1-methyl-pseudouridine).

[0043] Fig. 4. Categories of transgene 5’ junction formation. DNA is solid lines (target site DNA is linear and cDNA is an arc), template RNA is dashed line, and a subset of base pairings is illustrated with thin vertical lines. If the cDNA 3’ end or sequence internal to the 3’ end has sufficient length of complementarity to the upstream target site top / second strand close to the position of first / bottom- strand nicking, the cDNA 3’ end can make a 5’ junction by the “Anneal” mechanism. Other 5 ’ junctions form by loin mechanisms with or without microhomology pairing.

[0044] Fig. 5. Types of transgene 5’ junction detected by whole genome sequencing after PRINT.

[0045] Fig. 6. High-throughput screen for host-cell factors important for PRINT transgene insertion. The GFP template and mCherry template RNAs differ in the presence or absence of template 5’ rRNA sequence that gives the first-strand cDNA complementarity or lack ofcomplementarity to the upstream target site, and thus ability to use the Anneal and Join versus only Join pathways for 5’ junction formation and second-strand synthesis. D, day

[0046] Figs. 7A-C. High-throughput screen results from siRNA gene expression knock-down to detect host cell factor influences on PRINT insertion efficiency, using co-transfected template RNAs either without 5’ rRNA sequence (results plotted on x-axis, GFP) or with 5’ template rRNA sequence to generate cDNA base-pairing potential with upstream target-site (results plotted on y-axis, mCherry). If an siRNA differentially alters the ability of the two template RNAs to support PRINT, the data point for that siRNA will be off the diagonal line. (A) A major differential influence that reduces Join more than Anneal 5’ junction formation is the depletion of DNA polymerase theta (POLQ gene) observed using both siRNA knock-down (screen results shown, and separate siRNAs validate screen results) and a small-molecule drug that inhibits polymerase activity (e.g. ART558). (B) A repeat screen performed in the presence of ART558 to inhibit Polymerase theta revealed additional host cell factor contributions to PRINT. A major differential influence that reduces Join more than Anneal transgene 5’ junction formation is the depletion of factors involved in Shieldin / CST directed top-strand fill-in. (C) The repeat screen performed in the presence of ART558 to inhibit Polymerase theta also revealed factors that reduce Anneal more than Join transgene 5’ junction formation, such as CtIP (RBBP8 gene).

[0047] Figs. 8A-B. Secondary assays of depletions or inhibition of host cell factors required for Join pathways of transgene 5’ junction formation, assayed using co-transfected template RNAs either with 5’ rRNA sequence (A) or without 5’ rRNA sequence (B). Factors that decrease Join pathways of transgene 5’ junction formation were tested alone or in combination. siNEG is siRNA negative control, REV7 encodes a subunit of Shieldin, CTC1 encodes a subunit of CST, and Polymerase theta chemical inhibitor was added or not as indicated. siRNAs that reduce abundance of REV7 and CTC 1 have minimal impact on transgene insertion by the Anneal pathway but in combination with POLQ inhibition cripple Join 5’ junction formation.

[0048] Figs. 9A-D. Whole genome sequencing (WGS) analysis of transgene 5’ junction formation. FL (full-length) is transgene insertion with complete transgene sequence, whereas Trunc indicates 5’ truncation. Each panel is from PRINT with a template RNA containing 5’ rRNA sequence that can support Anneal junction formation (R28 or TCA 5’ module).ART558 is a chemical inhibitor of POLQ polymerase activity; this was added or for comparison its solvent alone (DMSO). (A,B) Join junction formation necessary for 5’-truncated transgene insertions was inhibited by host factor manipulation, suppressing 5’ truncated transgene insertions. (C) Anneal junction formation factors such as CtIP are limiting for stable full-length transgeneinsertions. (D) TOPBP1 siRNA versus negative control siNEG shows that TOPBP1 depletion increases percentage of transgene 5 ’ junctions that form using the Anneal pathway.

[0049] Fig. 10. WGS analysis showing that CtIP depletion by siRNA influences Join junction formation mediated by Polymerase theta, which is characterized by microhomology signature at the transgene 5’ junction. Additional experiments confirm that CtIP is limiting for full-length transgene insertion by compromising both Anneal junction formation and Polymerase theta Join junction formation, which favors full-length insertions more than the Shieldin / CST pathway of Join junction formation.

[0050] Figs. 11 A-B. Volcano plots of high-throughput screening siRNA gene expression knock-downs demonstrating that depletion of various host cell factors compromised PRINT efficiency. The x-axes values are normalized to the negative control siRNA, which was assigned a value of 1.0. The y-axes are significance of the deviation from siRNA negative control, with increasing vertical scale indicating increasing significance. Data for each of the two template RNAs co-transfected in the PRINT siRNA screens are plotted separately: (A) shows template RNA with 5’ rRNA (mCherry transgene) and (B) shows template RNA without 5’ rRNA and thus without cDNA 3’ base-pairing potential to upstream rDNA (GFP transgene). Note the severe negative impact of siRNA depletion of UBTF using both templates, compared to the more template-selective impact of POLQ or TOPBP 1 depletion that reduced use of template RNA lacking 5’ rRNA.

[0051] Fig. 12. Number of gaps (y-axis) per number of transgenes sequenced (x-axis) was lower using template RNAs synthesized with pseudouridine or N1 -methylpseudouridine in replacement of physiological uridine. Dashed lines are fits to the collection of datapoints for a particular uridine. Data points are independent experiment WGS data.

[0052] Fig. 13. Gap lengths generally decreased in parallel with decrease in number of gaps. Gap length analysis used the same WGS datasets as used for data points in Figure 12.

[0053] Fig. 14. Number of gaps increases with RNaseH depletion (si) and decreases with RNaseH overexpression (OE). Linear line fits to WGS analysis were done and the difference in slope between lines for different PRINT conditions is given as the residual from linear fit. Increase indicates more gaps, while decrease indicates fewer gaps.

[0054] Fig. 15. Inhibition of snap-back cDNA synthesis by inhibition of POLQ. Under 3 different PRINT conditions, snap-back transgene 5’ junctions were quantified relative to total 5’ transgene junctions. The 3 sets of bars are the three different conditions; within each set, PRINT was performed in parallel for cells with addition of POLQ inhibitor (ART558) or addition of the solvent for the small molecule inhibitor (DMSO).DETAILED DESCRIPTIONOverview

[0055] Described herein are systems and methods for improved synthesis-mediated transgene sequence insertion. Traditional approaches for transgene addition to the human genome use virus vectors that infect cells and result in insertion of the entire virus vector contents at a semirandom site. Other methods use delivered donor DNA with a delivered recombinase-like enzyme to mediate integration, or use delivered donor DNA to serve as a homologous recombination template at a double-stranded DNA break made by a delivered enzyme. All of these virus-based and DNA-donor- based methods are severely limited by adaptive and innate immune responses, delivery challenges, and unwanted mutagenesis or other genome damage.

[0056] We developed an RNA-only method for precise RNA-mediated insertion of transgenes (PRINT), which exploits a site-specific non-LTR retrotransposon protein for reverse transcription of new content into the human genome at a specific multicopy safe-harbor locus (Zhang, Van Treeck et al. Nature Biotechnology 2024), which is incorporated by reference herein in its entirety. The non-LTR retrotransposon protein nicks the genomic target-site first strand (the so-called bottom strand) and performs target-primed reverse transcription (TPRT) using the nick 3 ’OH as a primer for cDNA synthesis on the protein-bound RNA template (Figure 1). Two types of RNAs are combined for delivery to cells to accomplish PRINT: an mRNA encoding a protein from a non-LTR R2 retrotransposon with nicking endonuclease (EN) and reverse transcriptase (RT) activities, and a template RNA that encodes the transgene payload with flanking 5’ and 3’ modules that provide biostability, R2p binding, and stimulation of TPRT (Figures 2 and 3).

[0057] R2 retrotransposon protein (R2p) currently useful for PRINT is avian - for example from white-throated sparrow (ZoAl) or zebrafinch (TaGu). These R2p bind to the 3’ end of an avian R2 3’UTR (Palm et al 2024 RNA. 30(9): 1227-1245. doi: 10.1261 / rna.080031.124, Thawani et al bioRxiv 2024 doi: https: / / doi.Org / 10.l 101 / 2024. 11.11 .6231 12). which is incorporated by reference herein in its entirety. The RNA motif for R2p binding is in the 3 ’ module of a PRINT template RNA. A complete template RNA 3’ module contains an R2p binding sequence, such as from the medium ground finch (GeFo) R2, with an added “3’ tail” of typically 4 nucleotides complementary to the target-site primer (R4) and a poly-adenosine tract (A22); altogether this 3’ module is indicated as GeFo3_R4A22 (Zhang, Van Treeck et al. Nature Biotechnology 2024; Palm et al. Methods Enzymol. 2024;705:1-24. doi:10.1016 / bs.mie.2024.07.007), which is incorporated by reference herein in its entirety. The 3’ end of GeFo3 has the required 3 ’ UTR sequence, often termed GeFo98 or GeFo68 for the 98 or68 nucleotides of 3’UTR sequence, respectively. GeFo68 folds into a 5’ pseudoknot and 3’ hairpin flanked by single-stranded regions (Thawani et al bioRxiv 2024 doi: https:. / / doi.org / 10. 1101 / 2024. 11.11 .62311 ), which is incorporated by reference herein in its entirety. Template RNAs also can have a 5’ module that can confer biostability to the template RNA and can include upstream target-site sequence (e.g. 28 nucleotides in R28, Figure 3) that allows the cDNA 3’ end to base-pair with the upstream target site (Zhang, Van Treeck et al. Nature Biotechnology 2024; Palm et al 2024 RNA. 30(9): 1227- 1245. doi: 10. 1261 / rna.O8OO31.124), which is incorporated by reference herein in its entirety.

[0058] The R2p and template RNA can result in precise target-site nicking and TPRT, as evident from biochemical assays using purified recombinant protein, RNA, and annealed targetsite DNA oligonucleotides. R2p can also nick the second strand (the so-called top strand, Figure 1) near the position of the first-strand nick (Lee, Horton, Van Treeck, McIntyre, Collins Cell Reports 2024). However, R2p appears not to support second-strand synthesis using the nicked second strand as a primer. Therefore, DNA repair machineries encoded by the host genome could play a major role in second-strand synthesis. Of note: there is no prior art that implicates a second-strand synthesis mechanism for any non-LTR retrotransposon, including R2 retrotransposons. As described herein, control of second-strand synthesis can improve productive PRINT transgene insertion if the cellular factors involved can be identified and manipulated.

[0059] The transgene 5’ junction with upstream rDNA is created predominantly by an “Anneal” pathway or a “Join” pathway of DNA repair, and the latter sometimes has a signature of microhomology at the Join junction (Figures 4 and 5). Template RNAs with a 5’ module including a particular region of upstream target site sequence that we have defined, such as R28 (28 nucleotides immediately upstream of the first-strand nick), preferentially use the Anneal pathway. In contrast, template RNAs with a 5 ’ module lacking upstream target site sequence and therefore no cDNA 3’ end potential to make a transgene 5’ junction by the Anneal pathway must use a Join pathway for stable transgene insertion. Also 3 ’-truncated cDNAs (e.g. cDNAs copied from 5’ truncated templates) that lack upstream target site homology must use a Join pathway for stable transgene insertion.

[0060] The ability to introduce PRINT dependence on the Anneal pathway would preclude non-productive 5 ’-truncated insertions and also off-target insertions, since the off-target site would not have R28 homology. The TPRT first-strand cDNA sequence that can anneal to upstream target-site sequence can be anywhere 5’ of the intended transgene payload, either at the cDNA 3’ end or internally, with the latter bene fitting from a slightly longer region of target site base-pairing.

[0061] The lack of insight about how second-strand synthesis occurs (i.e. how the TPRT cDNA-complementary strand is synthesized) potentially limits the general applicability of PRINT and limits the fraction of insertions that are full-length transgenes. This limitation can be a hurdle for transitioning PRINT, and of note also any other RT-mediated genome insertion method, into clinical therapy, especially since the potential for transgene 5’ truncation increases as transgene payload length increases. As described herein, we elucidated transgene 5’ junction formation and second-strand synthesis mechanisms. As described herein, we used this knowledge of mechanisms that generate the cDNA-complementary second DNA strand that is necessary for stable transgene insertion to force transgene 5’ junction reliance on the Anneal pathway, which suppresses transgene 5 ’-truncation and off-target insertion, and also to reduce alterations to the intended transgene content such as by cDNA snap-back synthesis prior to transgene 5’ junction formation (Figure 5) or gap formation (internal transgene sequence deletion) during second-strand synthesis due to action of host cell DNA repair pathways (Zhang, Van Treeck et al. Nature Biotechnology 2024), which is incorporated by reference herein in its entirety.

[0062] Relevant Literature includes: Zhang, Van Treeck et al. Nature Biotechnology 2024, doi.org / 10. 1038 / s41587-024-02137-y; Lee et al. Cell Reports 2024 doi: 10.1016 / j.celrep.2024.114239; Palm et al. RNA 2024 doi: 10.1261 / ma.080031.124; Palm et al. Methods Enzymol. 2024;705:1-24. doi: 10.1016 / bs.mie.2024.07.007), which is incorporated by reference herein in its entirety.

[0063] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes. Systems and Compositions

[0064] In some aspects, described herein are systems for improved synthesis-mediated transgene sequence insertion. In some aspects, the present disclosure provides a system for nucleic acid insertion comprising (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide. The system can further comprise one or more compositions capable of suppressing a Join junction formation or a snap-back synthesis prior to transgene junctionformation, for example, as indicated in FIG. 4. The system can further comprise one or more compositions capable of enhancing an Anneal pathway for nucleic acid insertion, for example, as shown in FIG. 5. Examples of compositions that can help suppress a Join junction formation or a snap-back synthesis and / or enhancing an Anneal pathway for nucleic acid insertion include a polymerase theta (POLQ) inhibitor, Shieldin complex inhibitor, a CTC1-STN1-TEN1 (CST) complex inhibitor, a RNaseH activity enhancer, a DNA topoisomerase II binding protein 1 (TOPBP1) inhibitor, a C-terminal-binding protein interacting protein (CtIP) activity enhancer, or a combination thereof. The system can further comprise one or more compositions capable of improving efficiency of nucleic acid insertion of the template sequence into a target nucleic acid region in the genome of a cell. Examples of compositions that can help improve efficiency of nucleic acid insertion include an Upstream Binding Transcription Factor (UBTF) or a nucleic acid encoding an UBTF, or a modulator of a nucleolar architecture or a nucleic acid encoding a modulator of the nucleolar architecture.

[0065] In some aspects, the system further comprises a polymerase theta (POLQ) inhibitor. The POLQ inhibitor can comprise a small molecule inhibitor, for example, ART558. Alternatively, the POLQ inhibitor can comprise an siRNA targeting an mRNA encoding a POLQ gene. The system can further comprise a Shieldin complex inhibitor or a CTC1-STN1- TEN 1 (CST) complex inhibitor. The Shieldin complex inhibitor can comprise a small molecule inhibitor. In some cases, the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a REV7 gene. In some cases, the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a SHLD1 gene, an siRNA targeting an mRNA encoding SHLD2 gene, an siRNA targeting an mRNA encoding a SHLD3 gene, or a combination thereof. The system can comprise a mutant version of POLQ or a nucleic acid (e.g., mRNA) encoding a mutant version of POLQ.

[0066] The CST complex inhibitor can comprise a small molecule inhibitor. In some cases, the CST complex inhibitor comprises an siRNA targeting a CTC1 gene. In other cases, the CST complex inhibitor comprises an siRNA targeting a STN1 gene. In further cases, the CST complex inhibitor comprises an siRNA targeting a TEN 1 gene. The system can comprise a mutant version of a CST complex component or a nucleic acid (e.g., mRNA) encoding a mutant version of a CST complex component.

[0067] The system can further comprise a DNA topoisomerase II binding protein 1 (TOPBP 1) inhibitor. The TOPBP1 inhibitor can be a small molecule or an siRNA targeting a TOPBP1 gene. In some embodiments, the system comprises a POLQ inhibitor and a TOPBP1 inhibitor. In some embodiments, the system comprises a CST complex inhibitor and a TOPBP1 inhibitor. In some embodiments, the system comprises a polymerase theta (POLQ) inhibitor, a Shieldincomplex inhibitor or a CST complex inhibitor, and a TOPBP1 inhibitor. The system can comprise a mutant version of TOPBPlor a nucleic acid (e.g., mRNA) encoding a mutant version of TOPBPl.

[0068] The system can further comprise an RNaseH activity enhancer. The RNaseH activity enhancer can be a small molecule, a protein or peptide, or a nucleic acid (e.g., mRNA) encoding RNaseH. In some cases, the RNaseH activity enhancer is a component capable of increasing RNaseH expression in a cell. The RNaseH activity enhancer can comprise an inducer or transcription activator that is capable of enhancing expression of RNaseH. In some cases, the RNaseH activity enhancer comprises a combination of a vector encoding RNaseH operatively controlled by a promoter and an inducer or transcription activator that is capable of enhancing expression of RNaseH in a cell. The RNaseH can be a non-native RNaseH or nucleic acid encoding non-native RNaseH that is introduced to the cell. In some embodiments, the system comprises a POLQ inhibitor and an RNaseH activity enhancer. In some embodiments, the system further comprises at least one, at least two, or at least three selected from a POLQ inhibitor, a Shieldin complex inhibitor, or a CST complex inhibitor, and a TOPBP1 inhibitor.

[0069] The system can further comprise a C-terminal-binding protein interacting protein (CtIP) activity enhancer. The CtIP activity enhancer can be a small molecule, a protein or peptide, or a nucleic acid (e.g., mRNA) encoding CtIP. In some cases, the CtIP activity enhancer is a component capable of increasing CtIP expression in a cell. The CtIP activity enhancer can comprise an inducer or transcription activator that is capable of enhancing expression of CtIP. In some cases, the CtIP activity enhancer comprises a combination of a vector encoding CtIP operatively controlled by a promoter and an inducer or transcription activator that is capable of enhancing expression of CtIP in a cell. The CtIP can be a non-native CtIP or nucleic acid encoding non-native CtIP that is introduced to the cell. In some embodiments, the system comprises an CtIP activity enhancer and an RNaseH activity enhancer. In some embodiments, the system further comprises at least one, at least two, at least three selected from a POLQ inhibitor, a Shieldin complex inhibitor, or a CST complex inhibitor, and a TOPBP 1 inhibitor.

[0070] In some embodiments, the system further comprises an Upstream Binding Transcription Factor (UBTF) or a nucleic acid encoding the UBTF. In some cases, the system comprises a UBTF activity enhancer. The UBTF activity enhancer can be a small molecule, a protein or peptide, or a nucleic acid (e.g., mRNA) encoding UBTF. In some cases, the UBTF activity enhancer is a component capable of increasing UBTF expression in a cell. The UBTF activity enhancer can comprise an inducer or transcription activator that is capable of enhancing expression of UBTF. In some cases, the UBTF activity enhancer comprises a combination of a vector encoding UBTF operatively controlled by a promoter and an inducer or transcriptionactivator that is capable of enhancing expression of UBTF in a cell. The UBTF can be a nonnative UBTF or nucleic acid encoding non-native UBTF that is introduced to the cell.

[0071] In other embodiments, the system further comprises a UBTF inhibitor (e.g., a small molecule or an siRNA targeting a UBTF gene).

[0072] The system can further comprise a modulator of RNA Polymerase I activity or a nucleic acid encoding the modulator of RNA Polymerase I activity. For example, the modulator can comprise a small molecule inhibitor or an siRNA inhibitor. The system can comprise the mutant version of RNA Polymerase I or a nucleic acid (e.g., mRNA) encoding the mutant version of RNA Polymerase I.

[0073] The system can further comprise a modulator of a nucleolar architecture or a third nucleic acid encoding the modulator of the nucleolar architecture. For example, the modulator can comprise a Lamin A inhibitor. The Lamin A inhibitor can be a small molecule or an siRNA targeting a LMNA gene. The system can comprise the mutant version of Lamin A or a nucleic acid (e.g., mRNA) encoding the mutant version of Lamin A.

[0074] In some aspects, the first nucleic acid comprises a pseudouridine or a N1 - methylpseudouridine. The 5 ’ module of the first nucleic acid can comprises an rDNA sequence. The rDNA sequence can comprise, for example, a portion of a 28S rDNA sequence or a reverse complement thereof. In some embodiments, the rDNA sequence has a length of from 10-40 nucleotides. In some embodiments, the 3’ module of the first nucleic acid further comprises an additional rDNA sequence. The additional rDNA sequence can comprise, for example, a portion of a 28S rDNA sequence or a reverse complement thereof. In some embodiments, the additional rDNA sequence has a length of from 4-40 nucleotides. In some embodiments, the 5’ module further comprises a ribozyme sequence. In some embodiments, the 3’ module further comprises a polyA sequence.

[0075] In some aspects, the R2 retroelement polypeptide, the 3’ UTR, or the 5’ UTR can be derived from a white- throated sparrow Zonotrichia albicollis (ZoAl), a zebrafinch Taeniopygia guttata (TaGu), or the medium ground finch Geospiza fords (GeFo).

[0076] In some embodiments, the present disclosure provides a cell comprising a system described herein. The cell can further comprise genomic DNA comprising a 28S rDNA target site. In some embodiments, the template sequence encodes a transgene. In some embodiments, the cell comprises non-native RNaseH or mRNA encoding non-native RNaseH. In some embodiments, the cell comprises non-native CtlP or mRNA encoding non-native CtlP. In some embodiments, the cell comprises non-native UBTF or mRNA encoding non-native UBTF.Methods

[0077] In some aspects, the present disclosure provides a method for editing a cell by administering to the cell (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence; and (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, wherein the administering results in insertion of a template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell.

[0078] In some aspects, the method comprises administering to the cell a system or composition described elsewhere herein, for example, to improve synthesis-mediated transgene sequence insertion. This can involve modulating DNA synthesis, for example, by suppressing a Join junction formation or a snap-back synthesis prior to transgene junction formation. In some cases, the method inhibits stable gene insertion without base-pairing between the genome-side and insertion- side DNA sequence. In some cases, the method inhibits stable gene insertion with only microhomology or only 1-8 base-pair (bp) homology between the genome-side and insertion-side DNA sequence. Suppressing a Join junction formation can suppress a homologyindependent repair mechanism or a microhomology (e.g., 1-8 bp homology) repair mechanism, for example, as indicated in FIG. 4. In some cases, the method comprises suppressing snap-back synthesis, for example, as indicated in FIG 5. In some cases, the method modulates DNA synthesis by enhancing an Anneal pathway for gene insertion, for example, as shown in FIG. 5. The Anneal pathway can comprise homology lengths of greater than 8 bp, at least 10 bp, at least 20 bp, at least 30 bp, or at least 40 bp. In some cases, the Anneal pathway comprises homology lengths of 10-40 bp, 20-40 bp, or 20-30 bp. In some aspects, the present disclosure provides methods for suppressing base-paring-independent pathways for transgene 5’ junction formation that are mediated by the DNA repair pathways demonstrated herein or others in other cell types or organisms. As just one example method, transgene insertions other than by the Anneal pathway for 5’ junction formation and second-strand synthesis (such as the Join junction pathway), could be suppressed by co-delivery of PRINT RNAs with dominant- negative mRNAs and / or pharmaceuticals (e.g. small molecule drugs and / or oligonucleotides that target DNA repair pathways) that inhibit one or more of the pathways that permit formation of other types of transgene 5’ junction (Figure 5). In some aspects, the present disclosure also provides methods of using PRINT in cells and tissues that are less dependent on DNA repair pathways that mediate “Join” junctions.

[0079] In some aspects, suppressing a Join junction formation or snap-back synthesis or enhancing an Anneal pathway prevents or helps suppress deletion of a portion of the templatesequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some cases, suppressing a Join junction formation or snap-back synthesis or enhancing an Anneal pathway prevents or helps suppress a 5’ truncation of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In other cases, suppressing a Join junction formation or snap-back synthesis or enhancing an Anneal pathway prevents or helps suppress a gap formation in the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some cases, the method modulates a second-strand synthesis activity. This can involve improving the fidelity of second-strand synthesis to achieve a stable full-length template sequence synthesis outcome. In some cases, the method improves the efficiency of second-strand synthesis.

[0080] The methods described herein can suppress deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region or gap formation in a transgene that is inserted. This can comprise influencing or modulating the activity or expression of host factors relevant to second-strand synthesis. As one example method, intact transgene insertions could be increased by design of template RNA sequence, nucleotide content, chemistry, or other variables. As a second example method, PRINT RNAs (e.g., the first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence and / or the second nucleic acid encoding the R2 retroelement polypeptide) could be co-delivered with mRNAs that encode human or non-native RNaseH, RNaseH-like, or RNA-DNA unwindase activities, or that encode single-stranded DNA protective factors such as Replication Protein A (RPA) for which siRNA-mediated depletion reduces functional transgene insertion efficiency. As a third example, R2p amino acid sequence could be directly fused or non-covalently linked to factors that improve use of full-length cDNAs generated by TPRT. As a fourth example, formation of the 5’ transgene junction could be designed to trigger recruitment of a second-strand synthesis machinery with improved strand-displacement activity or other feature that promotes the desired stable transgene synthesis outcome. In some aspects, the present disclosure also provides methods of using PRINT in cells and tissues that support full-length second-strand synthesis.

[0081] In some aspects, the methods described herein improves PRINT efficiency. In some aspects, the present disclosure also provides methods for decreasing or increasing rDNA targetsite amenability to PRINT, for example, by changing RNA Polymerase I transcription and / or rDNA methylation and histone content. As one example method, transgene insertions can be increased in efficiency by co-delivery of PRINT RNAs with mRNA encoding an Upstream Binding Transcription Factor (UBTF) or other modulators of rDNA chromatin state. As a second example method, transgene insertions could be tuned in efficiency by modulation ofRNA Polymerase I activity, using delivered mRNA, siRNA, or other reagents such as small molecule drug RNAP I inhibitors. As a third example method, transgene insertions could be tuned in efficiency by co-delivery of PRINT RNAs with siRNA against Lamin A, or treatments such as delivery of an mRNA encoding the mutant version of Lamin A named progerin, to affect nucleolar architecture. In some aspects, the present disclosure also provides methods of using PRINT in cells and tissues that are natively, or are treated to become, relatively higher in amenable PRINT target sites.

[0082] In some aspects, the method comprises administering (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide; and (c) a polymerase theta (POLQ) inhibitor. The administering of the polymerase theta (POLQ) inhibitor can suppress a Join junction formation, which can prevent deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some aspects, the POLQ inhibitor comprises a small molecule inhibitor. In some embodiments, the POLQ inhibitor comprises ART558. In some embodiments, the POLQ inhibitor comprises an siRNA targeting an mRNA encoding a POLQ gene.

[0083] The method comprise administering (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and a combination of (c) a POLQ inhibitor and (d) a Shieldin complex inhibitor or a CTC1-STN1-TEN1 (CST) complex inhibitor, wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell. In some cases the combination of (c) the POLQ inhibitor and (d) the Shieldin complex inhibitor or the CTC1-STN1-TEN1 (CST) complex inhibitor suppresses a Join junction formation, which can prevent deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. The Shieldin complex inhibitor can comprise a small molecule inhibitor. In some cases, the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a REV7 gene. In some cases, the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a SHLD1 gene, an siRNA targeting an mRNA encoding SHLD2 gene, an siRNA targeting an mRNA encoding a SHLD3 gene, or a combination thereof. The system can comprise a mutant version of POLQ or a nucleic acid (e.g., mRNA) encoding a mutant version of POLQ. The CST complex inhibitor can comprise a small molecule inhibitor. In some cases, the CST complex inhibitor comprises an siRNA targeting a CTC1 gene. In other cases, the CSTcomplex inhibitor comprises an siRNA targeting a STN 1 gene. In further cases, the CST complex inhibitor comprises an siRNA targeting a TEN 1 gene. The system can comprise a mutant version of a CST complex component or a nucleic acid (e.g., mRNA) encoding a mutant version of a CST complex component.

[0084] In some embodiments, the method further comprises administering a DNA topoisomerase II binding protein 1 (TOPBP1) inhibitor. Administering the TOPBP1 inhibitor can help suppress a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some embodiments, the method further comprises administering an RNaseH activity enhancer. Administering the RNaseH activity enhancer can help reduce gap formation or deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

[0085] In some embodiments, the method further comprises administering a C-terminal-binding protein interacting protein (CtIP) activity enhancer. Administering the CtIP activity enhancer can promote the Anneal pathway and promote full-length template sequence insertion.

[0086] In some embodiments, the method further comprises administering an Upstream Binding Transcription Factor (UBTF) or a nucleic acid encoding the UBTF. In other embodiments, the method further comprises administering a UBTF inhibitor. In some embodiments, the method further comprises administering a modulator of RNA Polymerase I activity or a nucleic acid encoding the modulator of RNA Polymerase I activity. In some embodiments, the method further comprises administering a modulator of a nucleolar architecture or a third nucleic acid encoding the modulator of the nucleolar architecture.

[0087] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide; (c) a polymerase theta (POLQ) inhibitor; and (d) an RNaseH activity enhancer; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell. The administering of the (c) polymerase theta (POLQ) inhibitor and (d) the RNaseH activity enhancer can suppress a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region. In some embodiments, administering the RNaseH activity enhancer induces a higher expression of RNaseH in the cell, compared to that upon administering to a corresponding cell with (a)-(c) alone.

[0088] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a TOPBP 1 inhibitor; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

[0089] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a C -terminal-binding protein interacting protein (CtIP) activity enhancer; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) enhances full-length second-strand synthesis for insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0090] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) an Upstream Binding Transcription Factor (UBTF) or a third nucleic acid encoding the UBTF; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0091] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a modulator of RNA Polymerase I activity or a third nucleic acid encoding the modulator of RNA Polymerase I activity; wherein the administering results ininsertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

[0092] In another aspect, the present disclosure provides a method of editing a cell, the method comprising administering to the cell: (a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3 ’ module downstream of the template sequence; (b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and (c) a modulator of a nucleolar architecture or a third nucleic acid encoding the modulator of the nucleolar architecture; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone. In some embodiments, the modulator comprises a Lamin A inhibitor.

[0093] In some aspects, the first nucleic acid comprises a pseudouridine or a Nl- methylpseudouridine. In some aspects, the 5’ module of the first nucleic acid comprises an rDNA sequence. In some aspects, the rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some aspects, the rDNA sequence has a length of from 10-40 nucleotides. In some aspects, the 3’ module of the first nucleic acid further comprises an additional rDNA sequence. In some aspects, the additional rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof. In some aspects, the additional rDNA sequence has a length of from 4-40 nucleotides. In some aspects, the 5’ module further comprises a ribozyme sequence. In some aspects, the 3’ module further comprises a polyA sequence. In some aspects, the template sequence encodes a transgene.EXAMPLESExample 1. Screening host-cell factors that influence mechanisms of PRINT transgene insertion

[0094] In brief, the host-cell factors that influence the mechanisms of PRINT transgene insertion 5 ’ junction formation and second-strand synthesis were queried using high-throughput screening of a transfected human retinal pigmented epithelium primary cell line (RPE-1). The initial readout was high-content imaging of PRINTed GFP and mCherry transgene expression across cells previously transfected with a library of siRNAs for individual host-factor knockdowns (Figure 6). The absolute efficiency of transgene insertions was scored, and of particular relevance the ratio of transgene insertions using template RNAs with or without 5 ’rRNA sequence (with or without ability to use the Anneal pathway for 5 ’ junction formation and second-strand synthesis) was also scored. This was achieved by co-transfection of two template RNAs: one template RNA encoded GFP reporter expression and had no upstream target-site homology, while the other template RNA encoded mCherry reporter expression and had upstream target-site homology (R28). Secondary screens included testing of distinct siRNA sequences for host-factor knock-down, additional siRNA-mediated host- factor knock-downs individually or in combinations, chemical inhibitor assays, assays in cell lines with endogenous gene disruptions, PRINT assays into target sites transplanted from rDNA, whole genome sequencing (WGS), and other assays.

[0095] Initial screening compared PRINT functional transgene insertion using template RNAs that supported Anneal and loin or only loin transgene 5’ junction formation. The initial screen implicated Polymerase theta (POLQ gene) as one mechanism for formation of the Join category of transgene 5’ junction formation (Figure 7A). Transgene insertion from co-delivered template RNAs that had upstream target site base -pairing (R28 5’ end, mCherry transgene) versus obligate use of the Join mechanism (RO 5’ end, GFP transgene) showed that Polymerase theta had a much more important influence on Join junction formation (Figure 7A). Small-molecule inhibitors of POLQ are in development as anti-cancer therapeutics. POLQ siRNA influence matched the influence of its small molecule inhibitor and also use of gene-disrupted POLQ cell lines. All of these ways to reduce Polymerase theta function similarly decreased PRINT transgene insertions using template RNAs that lacked cDNA 3 ’ base -pairing potential with upstream target-site DNA but much less severely decreased transgene insertions using template RNAs that had cDNA base-pairing potential with upstream target-site DNA.

[0096] The siRNA screen was repeated in the cell background of inhibited POLQ function. In this background, inhibition of Shieldin components, as well as their pathway-associated factors for DNA repair (including 53BP1 and its downstream factors), scored as a requirement for PRINT insertion of transgenes that lack cDNA 3’ base-pairing potential with upstream targetsite DNA (Figure 7B). Depletion of other host cell factors decreased transgene insertion using template RNAs that included 5’ template rRNA and thus cDNA 3’ base-pairing potential with upstream target-site DNA (Figure 7C), including CtIP (RBBP8 gene), which is implicated as a limiting factor for Anneal transgene 5 ’ junction formation.

[0097] Our unexpected siRNA screen findings led us to test additional candidate host-factor requirements for PRINT using siRNAs not part of the initial screen. Additional siRNA-mediated host-factor knock-downs, both individually and in combinations, were used with supporting chemical inhibitor assays. Also we performed PRINT assays in cell lines with target sites transplanted from rDNA to transfected plasmids or target site arrays integrated into the genomenot in rDNA, with sequencing of target-site plasmids and genomes with inserted transgenes, and other methods.Example 2. Suppressing Join pathway insertions to improve transgene insertion

[0098] This example shows that methods described herein can vanquish insertions that were not mediated by annealing of cDNA to upstream target-site sequence (Figures 8A and 8B). This was achieved for example by combinatorial inhibition of POLQ (by small-molecule drug ART538, gene knock-out, or siRNA) and inhibition of the Shieldin / CST pathway for Polymerase alpha-Primase fill-in second-strand synthesis (e.g. by siRNAs targeting REV7 and CTC1), conditions represented in panel B. Under such conditions, template RNAs with upstream rRNA and thus cDNA base-pairing to upstream target-site top strand supported PRINT, shown in FIG. 8A.

[0099] Practical applications for host factor manipulation were demonstrated by WGS analyses of transgene insertions using PRINT template RNA with 5’ rRNA sequence that support the Anneal pathway of transgene 5’ junction formation. Suppression of Join pathways reduced 5’- truncated transgene insertions (Figure 9A-B), whereas limitation of CtIP had the opposite effect (Figure 9C). Many host factors influenced the balance between Join and Anneal pathways for transgene 5’ junction formation, such as TOPBP1 (Figure 9D), which favors the Join junction formation because its absence increased Anneal junction formation. Some host factors affect more than one pathway of transgene 5’ junction formation, such as CtIP, which in addition to promoting the Anneal pathway also favors Join transgene 5’ junction formation by Polymerase theta rather than the Shieldin / CST pathway of second-strand fill-in, as evident in the decrease of microhomology signature at Join transgene 5’ junctions (Figure 10).Example 3. High-throughput screening siRNA gene expression knock-downs demonstrate that depletion of various host cell factors compromised PRINT efficiency

[0100] In this example, high-throughput screening siRNA gene expression knock-downs were performed, demonstrating that depletion of various host cell factors compromised PRINT efficiency. Other host-cell factors are implicated in PRINT efficiency independent of template 5 ’ end sequence and thus 5’ junction formation / second- strand synthesis pathways. As one example, the histone-replacement protein UBTF is required for PRINT efficiency at the rDNA target site (Figure 11). In Figures 11 A-l IB, data for each of the two template RNAs co- transfected in the PRINT siRNA screens are plotted separately: (A) shows template RNA with 5’ rRNA (mCherry transgene) and (B) shows template RNA without 5’ rRNA and thus without cDNA 3’ basepairing potential to upstream rDNA (GFP transgene). There was severe negative impact of siRNA depletion of UBTF using both templates, compared to the more template-selective impact of POLQ or TOPBP1 depletion that reduced use of template RNA lacking 5’ rRNA.The PRINT efficiency requirement for UBTF is representative of other observed requirements for “open” rDNA units, distinguished from the rDNA units silenced by DNA methylation and associated histone-containing chromatin. The number of rDNA units in each state varies with cell type, cell proliferation status, metabolic activity, demand for ribosome production, and other variables.Example 4. Host-cell factor screening showed certain factors contributed to higher fidelity of second strand synthesis for intact transgene insertion

[0101] In this example, host-cell factor screening was performed, implicating factors of importance for productive PRINT by their contribution to higher fidelity of second strand synthesis for intact transgene insertion. Second-strand synthesis sometimes creates transgenes with internal deletions (gaps). WGS done in parallel with siRNA screening revealed changes in frequency of gap formation and gap length depending on the template RNA used (example in Figures 12-13) and modulation of host factor function (example in Figure 14).

[0102] The percentage of complete transgenes lacking gaps increased using template RNAs synthesized with pseudouridine in replacement of physiological uridine (Figure 12).

[0103] The percentage of complete transgenes decreased with reduced RNase H activity, and the desired increase was attained with RNase H over-expression (Figure 14).Our combination of findings relevant to transgene gaps described above and in additional work indicates that second-strand synthesis intermediates can be susceptible to unwanted processing that results in elimination of internal regions of the first-strand cDNA. Improving release of template RNA base-paired to first-strand cDNA enables intact second-strand synthesis, as does protecting the first-strand cDNA once it is liberated from base-pairing with template RNA.Example 5. Host-cell factor screening showed certain factors reduced snap-back synthesis

[0104] In this example, host-cell factor screening also implicated factors of importance for PRINT by their contribution to reducing snap-back synthesis. A minority of PRINT transgene insertions have snap-back synthesis prior to transgene junction formation (Figure 5). WGS done in parallel with siRNA screening revealed changes in frequency of snap-back synthesis with modulation of host factor function. For example, inhibition of POLQ reduced snap-back synthesis from the cDNA 3’ end prior to rDNA 5’ junction formation (Figure 15).Example 6. Host-cell factor screening showed certain factors reduced snap-back synthesis

[0105] Host-cell factor screening also implicated factors of importance for the Anneal pathway, such as CtlP (human gene RBBP8), that boost full-length transgene insertion (Figure 10). Depending on the cell type desired to PRINT, increasing CtlP level by co-transfecting CtlP- encoding mRNA, or other method, would promote full-length transgene insertion.

Claims

CLAIMS1. A method of improved synthesis-mediated transgene sequence insertion, comprising modulating DNA synthesis to inhibit stable gene insertion without base-pairing between the genome-side and insertion-side DNA sequences.

2. A method of claim 1, wherein the sequence insertion comprises a step of joining the first strand cDNA to upstream target site using annealing lengths of preferably 10-40 bp (or 20-30 bp, or more than 40 bp), by a mechanism different from microhomology-mediated joining (mediated by formation of 1-8 bp) and RAD52-dependent single strand annealing or RAD51- dependent strand invasion that can initiate homologous recombination (favored by 50+ nucleotides of complementary base-pairing potential).

3. A method of claim 1, wherein the method is independent of homologous recombination.

4. A method of claim 1 , wherein the method inhibits stable sequence insertion without formation of approximately 10-40 (or 20-30) or more base-pairs between DNA strands, whether template for the new sequence synthesis is introduced to cells directly as DNA or introduced as RNA to template DNA synthesis, to make stable sequence insertion dependent on formation of at least 10-40 (or 20-30) base-pairs between the genome-side and insertion- side DNA sequences.

5. A method of claim 1, wherein the synthesis -mediated gene insertion comprises second-strand cDNA synthesis, and second-strand synthesis requires a cDNA with upstream target-site basepairing potential, wherein the insertion may be at any intended native or non-native target site.

6. A method of claim 1 , wherein the insertion is initiated by a non-LTR retrotransposon protein at its native target site (e.g. rDNA) or elsewhere, or at any non-LTR retrotransposon insertion site with or without retargeting.

7. A method to improve synthesis-mediated gene insertion, comprising modulating second- strand synthesis (transgene 5’ junction formation and cDNA-complementary strand synthesis) to reduce or eliminate stable insertion of a 5 ’-truncated trans gene.

8. A method to inhibit unintended template RNA use for stable transgene insertion, achieved by second-strand synthesis requirement for base-pairing of cDNA and upstream insertion- site sequence.

9. A method to reduce or prevent off-target insertions by modulating 5’ junction formation and second-strand synthesis, achieved by a requirement for base-pairing of cDNA and upstream insertion-site sequence.

10. A method to reduce or eliminate cDNA snap-back synthesis prior to 5’ transgene junction formation by modulating host cell factors, including but not limited to POLQ and 5’ junction formation and second-strand synthesis factors.

11. A method of any of claims 1-10, wherein the modulating is achieved by a combination one or more or all of the following transgene 5’ junction formation and / or second-strand synthesis mechanisms: inhibition of DNA synthesis by Polymerase theta (encoded by human POLQ), inhibition of recruitment of Polymerase alpha Primase complex, such as mediated by inactivation of Shieldin or other factor(s) composing, associated with, or activating the Shieldin complex (e.g. SHLD 1, SHLD 2, SHLD 3, REV7 / MAD2L27), inhibition of recruitment of Polymerase alpha Primase complex, such as mediated by inactivation of CTC1 or other factor(s) composing, associated with, or activating the CST complex (e.g. CTC1, STN1, TEN1), inhibition of DNA binding by SHLD2 and / or CTC1 by any approach, including but not exclusively by dominant-negative Shieldin or CST subunit expression or small interfering RNA expression or small molecule drugs, inhibition of non-homologous end-joining mechanisms described above and in general, to disfavor annealing-independent transgene 5’ junction formation by targeting cellular factors including but not limited to TOPBP 1, or inhibition of non-homologous end-joining mechanisms involving polymerases mu (POLM), lambda (POLL) and / or theta (POLQ).

12. A method of any of claims 1-10, wherein end-resection factors including but not limited to RBBP8 / CtIP promote the efficiency of stable transgene insertion dependent on base-pairing of cDNA and target site for Anneal junction formation, such that increase in expression or function of these factors will improve the efficiency of Anneal junction formation.

13. A method of any of claims 1-10, wherein manipulation of second-strand synthesis and associated transgene 5’ junction formation limit unwanted repair of partial second-strand synthesis products to produce transgene internal gaps.

14. A method of any of claims 1-10, for enabling full-length transgene insertion by providing factors that promote the Anneal pathway of transgene 5’ junction formation.

15. A system comprising:(a) a first nucleic acid comprising a template sequence, a 5 ’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide;(c) a polymerase theta (POLQ) inhibitor; and(d) a Shieldin complex inhibitor or a CTC1-STN1-TEN1 (CST) complex inhibitor.

16. The system of claim 15, wherein the POLQ inhibitor comprises a small molecule inhibitor.

17. The system of claim 15 or 16, wherein the POLQ inhibitor comprises ART558.

18. The system of claim 15, wherein the POLQ inhibitor comprises an siRNA targeting an mRNA encoding a POLQ gene.

19. The system of any one of claims 15-18, wherein the system comprises a Shieldin inhibitor, wherein the Shieldin complex inhibitor comprises a small molecule inhibitor.

20. The system of any one of claims 15-18, wherein the system comprises a Shieldin inhibitor, wherein the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a REV7 gene.

21. The system of any one of claims 15-18, wherein the system comprises a Shieldin inhibitor, wherein the Shieldin complex inhibitor comprises an siRNA targeting an mRNA encoding a SHLD1 gene, a SHLD2 gene, or a SHLD3 gene.

22. The system of any one of claims 15-21, wherein the system comprises the CST complex inhibitor, wherein the CST complex inhibitor comprises a small molecule inhibitor.

23. The system of any one of claims 15-21, wherein the system comprises the CST complex inhibitor, wherein the CST complex inhibitor comprises an siRNA targeting a CTC1 gene.

24. The system of any one of claims 15-21, wherein the system comprises the CST complex inhibitor, wherein the CST complex inhibitor comprises an siRNA targeting a STN1 gene.

25. The system of any one of claims 15-21, wherein the system comprises the CST complex inhibitor, wherein the CST complex inhibitor comprises an siRNA targeting a TEN1 gene.

26. The system of any one of claims 15-25, wherein the first nucleic acid comprises a pseudouridine or a N1 -methylpseudouridine.

27. The system of any one of claims 15-25, wherein the 5’ module of the first nucleic acid comprises an rDNA sequence.

28. The system of claim 27, wherein the rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof.

29. The system of claim 27 or 28, wherein the rDNA sequence has a length of from 10-40 nucleotides.

30. The system of any one of claims 27-29, wherein the 3’ module of the first nucleic acid further comprises an additional rDNA sequence.

31. The system of claim 30, wherein the additional rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof.

32. The system of claim 30 or 31, wherein the additional rDNA sequence has a length of from 4- 40 nucleotides.

33. The system of any one of claims 15-32, wherein the 5’ module further comprises a ribozyme sequence.

34. The system of any one of claims 15-33, wherein the 3’ module further comprises a polyA sequence.

35. The system of any one of claims 1-34, further comprising an RNaseH activity enhancer.

36. The system of claim 35, wherein the RNaseH activity enhancer comprises mRNA encoding RNaseH.

37. A cell comprising the system of any one of claims 15-36.

38. The cell of claim 37, further comprising a genomic DNA comprising a 28S rDNA target site.

39. The cell of claim 37 or 38, wherein the template sequence encodes a transgene.

40. The cell of any one of claims 37-39, wherein the cell comprises non-native RNaseH or mRNA encoding non-native RNaseH.

41. A method of editing a cell, the method comprising administering to the cell the system of any one of claims 15-36, wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) and (d) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

42. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide;(c) a polymerase theta (POLQ) inhibitor; and(d) an RNaseH activity enhancer; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell,wherein administering (c) and (d) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

43. The method of claim 42, wherein the POLQ inhibitor comprises a small molecule inhibitor.

44. The method of claim 42 or 43, wherein the POLQ inhibitor comprises ART558.

45. The system of claim 42, wherein the POLQ inhibitor comprises an siRNA targeting an mRNA encoding a POLQ gene.

46. The method of any one of claims 42-45, wherein administering (d) induces a higher expression of RNaseH in the cell, compared to that upon administering to a corresponding cell with (a)-(c) alone.

47. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and(c) a DNA topoisomerase II binding protein 1 (TOPBP 1) inhibitor; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) suppresses a Join junction formation, thereby preventing deletion of a portion of the template sequence or the reverse complement thereof that is inserted into the target nucleic acid region.

48. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and(c) a C -terminal-binding protein interacting protein (CtIP) activity enhancer;wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) enhances full-length second-strand synthesis for insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

49. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and(c) an Upstream Binding Transcription Factor (UBTF) or a third nucleic acid encoding the UBTF; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

50. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and(c) a modulator of RNA Polymerase I activity or a third nucleic acid encoding the modulator of RNA Polymerase I activity; wherein the administering results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein administering (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

51. A method of editing a cell, the method comprising administering to the cell:(a) a first nucleic acid comprising a template sequence, a 5’ module upstream of the template sequence, and a 3’ module downstream of the template sequence;(b) an R2 retroelement polypeptide or a second nucleic acid encoding the R2 retroelement polypeptide, and(c) a modulator of a nucleolar architecture or a third nucleic acid encoding the modulator of the nucleolar architecture; wherein the method results in insertion of the template sequence or a reverse complement thereof into a target nucleic acid region in the genome of the cell, thereby editing the cell, wherein the administering of (c) increases efficiency of the insertion of the template sequence or the reverse complement thereof, compared to that upon administering to a corresponding cell with (a) and (b) alone.

52. The method of claim 51 , wherein the modulator comprises a Lamin A inhibitor.

53. The method of any one of claims 42-52, wherein the first nucleic acid comprises a pseudouridine or a N 1 -methylpseudouridine.

54. The method of any one of claims 42-53, wherein the 5’ module of the first nucleic acid comprises an rDNA sequence.

55. The method claim 54, wherein the rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof.

56. The method of claims 54 or 55, wherein the rDNA sequence has a length of from 10-40 nucleotides.

57. The method of any one of claims 54-56, wherein the 3’ module of the first nucleic acid further comprises an additional rDNA sequence.

58. The method of claim 57, wherein the additional rDNA sequence comprises a portion of a 28S rDNA sequence or a reverse complement thereof.

59. The method of claim 57 or 58, wherein the additional rDNA sequence has a length of from 4-40 nucleotides.

60. The method of any one of claims 42-59, wherein the 5’ module further comprises a ribozyme sequence.

61. The method of claim any one of claims 42-60, wherein the 3’ module further comprises a polyA sequence.

62. The method of any one of claims 42-61 , wherein the template sequence encodes a transgene.

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