Polynucleotide, lipid nanoparticle compositions and methods for delivering an exogenous nucleic acid to a cell and for in vivo delivery of an exogenous nucleic acid to a cell in an individual

Description

1 / 100 “POLYNUCLEOTIDE, LIPID NANOPARTICLE COMPOSITIONS AND METHODS FOR DELIVERING AN EXOGENOUS NUCLEIC ACID TO A CELL AND FOR IN VIVO DELIVERY OF AN EXOGENOUS NUCLEIC ACID TO A CELL IN AN INDIVIDUAL” Cross-reference to related orders

[001] This application claims the benefit of U.S. Provisional Patent Applications No. 63 / 494,303, filed April 5, 2023, and No. 63 / 610,715, filed December 15, 2023, each of which is incorporated herein by reference in its entirety. Reference to the Electronically Submitted Sequence Listing

[002] This application contains a Sequence Listing that was submitted in XML format via the Patent Center and is incorporated herein by reference in its entirety. The said XML copy, created on April 1, 2024, is named “POTH-082_001WO_SeqList” and is 94,499 bytes in size. Field

[003] This disclosure refers generally to polynucleotides for transposase expression, in particular, piggyBac transposase expression polynucleotide mRNAs comprising a piggyBac transposase coding sequence comprising hyperactive mutations and modified 5'- and 3'-UTR sequences to enhance piggyBac transposase expression, integration / excision activity and / or reduce the in vivo immunogenicity of the encoded transposase. Background

[004] Transposases can be used to introduce non-endogenous DNA sequences into genomic DNA and are, in many ways, advantageous compared to other gene editing methods. However, there is still an unmet need for transposases that possess enhanced integration / excision activity and reduced in vivo immunogenicity. Petition 870250091226, dated 06 / 10 / 2025, pp. 102 / 224 2 / 100 for in vivo delivery of nucleic acids. It has been previously reported that incorporation of human cytochrome b-245 alpha polypeptide (CYBA) UTRs into an mRNA sequence significantly increased protein levels for certain mRNAs without altering mRNA stability (see, for example, Ferizi, M., Aneja, M., Balmayor, E. et al. Sci Rep 6, 39149 (2016)). Importantly, many miRNAs have been shown to be expressed in hematopoietic lineages and to act as essential regulators of transcriptional programs for normal hematopoiesis, including self-renewal, differentiation, and HSC function (e.g., see Lu et al. (2013) Cell Res 23, 1356-1368 (2013)). Brief Description

[005] In one aspect, a polynucleotide is provided herein, comprising in the 5' to 3' direction: (i) a 5'-UTR of hemoglobin beta (HBB), (ii) a sequence encoding a nuclear localization signal (NLS), (iii) a nucleic acid sequence encoding a piggyBac transposase, (iv) a 3'-UTR comprising one or more nucleic acid sequences comprising a 3'-UTR element of human cytochrome b-245 (CYBA) alpha polypeptide and one or more miR-142-3p binding sites, and (v) a polyA tail. In some embodiments, the piggyBac transposase comprises the five selected hyperactive mutations I30V; G165S; M226F, M282V and N538K (i.e., the amino acid sequence shown in SEQ ID NO: 14). In some embodiments, the nucleic acid sequence encoding the piggyBac transposase comprises the nucleic acid sequence shown in SEQ ID NO: 2.

[006] In some embodiments, NLS is an NLS SV40 comprising the amino acid sequence shown in SEQ ID NO: 8.

[007] In some embodiments, one or more elements of CYBA's 3'UTR each comprise the nucleic acid sequence shown in SEQ ID NO: 3. In some embodiments, 3'-UTR comprises Petition 870250091226, dated 06 / 10 / 2025, pp. 103 / 224 3 / 100 at least two tandem nucleic acid sequences encoding a 3'-UTR element of CYBA, which are separated by a linker sequence. In some embodiments, the tandem nucleic acid sequences encoding a 3'-UTR element of CYBA each comprise the nucleic acid sequence shown in SEQ ID NO. 4.

[008] In some embodiments, one or more miR-142-3p binding sites each comprise the nucleic acid sequence shown in SEQ ID NO: 5. In some embodiments, each of the one or more miR-142-3p binding sites comprises the nucleic acid sequence ACACTAC. In some embodiments, 3'-UTR comprises four miR-142-3p binding sites. In some embodiments, the polynucleotide further comprises a linker sequence located between each of the four miR-142-3p binding sites. In some embodiments, the linker sequence comprises the nucleic acid sequence shown in SEQ ID NO. 6.

[009] In some embodiments, HBB 5'-UTR comprises the nucleic acid sequence shown in SEQ ID NO: 1.

[0010] In some embodiments, the polyA tail is an 80X polyA tail comprising the nucleic acid sequence shown in SEQ ID NO: 7.

[0011] In some embodiments, the polynucleotide comprises the nucleic acid sequence presented in SEQ ID NO: 10.

[0012] In some embodiments, the polynucleotide is a DNA molecule. In some embodiments, the polynucleotide is an RNA molecule. In some embodiments, the RNA molecule is an mRNA molecule. In some embodiments, the mRNA comprises a 5'-CAP. In some embodiments, the 5'CAP is a 5' CleanCap.

[0013] In another aspect, a composition of lipid nanoparticles (LNPs) comprising a Petition 870250091226, dated 06 / 10 / 2025, pp. 104 / 224 4 / 100 polynucleotide described herein.

[0014] In another aspect, a method for delivering an exogenous nucleic acid to a cell is provided in this document, comprising: introducing into the cell a DNA transposon comprising an exogenous nucleic acid; and an mRNA comprising in the 5' to 3' direction: (i) a 5'-UTR of HBB, a nucleic acid sequence encoding an NLS, (ii) a nucleic acid sequence encoding a piggyBac transposase comprising five hyperactive mutations, (iii) a 3'-UTR comprising two or more tandem nucleic acid sequences comprising a 3'-UTR element of CYBA and four miR-142-3p binding sites, and (iv) a polyA tail;and in which the piggyBac transposase is expressed in the cell and integrates the transposon comprising the exogenous nucleic acid into a TTAA integration site in the cell genome, wherein the expressed piggyBac transposase exhibits enhanced integration and / or excision activity compared to a piggyBac transposase comprising four or fewer hyperactive mutations.

[0015] In another aspect, a method is provided in the present document for the in vivo delivery of an exogenous nucleic acid to a cell in an individual, comprising: co-introducing into the individual a DNA transposon comprising an exogenous nucleic acid; and an mRNA comprising in the 5' to 3' direction: a 5'-UTR of HBB, an NLS coding sequence, a piggyBac transposase coding sequence comprising five hyperactive mutations, a 3'-UTR comprising a 3'-UTR element of CYBA in tandem upstream of the sequences for four miR142-3p binding sites, and a polyA tail; and wherein a cell in the individual takes up the transposon and expresses the piggyBac transposase in the cell and integrates the transposon comprising the exogenous nucleic acid into a TTAA integration site in the genome of the individual's cell, wherein the expressed piggyBac transposase exhibits reduced immunogenicity in the individual compared to an mRNA encoding Petition 870250091226, dated 06 / 10 / 2025, pp. 105 / 224 5 / 100 a piggyBac transposase without the sequences for four miR142-3p binding sites. Brief description of the Figures

[0016] FIGURE 1A shows a diagram illustrating an illustrative polynucleotide for expressing a piggyBac transposase comprising, in the 5' to 3' direction: a 5'-UTR of HBB; a coding sequence for a nuclear localization sequence (NLS); a piggyBac transposase coding sequence encoding four hyperactive mutations; a 3'UTR of HBB and a polyA tail.

[0017] FIGURE 1B shows a schematic illustrating a polynucleotide for expressing a piggyBac transposase comprising, in the 5' to 3' direction: a 5'-UTR of HBB; a coding sequence for a nuclear localization sequence (NLS); a piggyBac transposase coding sequence encoding five hyperactive mutations; a 3'UTR comprising tandem CYBA 3'-UTR sequences and sequences for four miR-142-3p binding sites, and a polyA tail.

[0018] FIGURE 2 is an illustration of the GFP episomal excision reporter construct.

[0019] FIGURE 3A shows a schematic of two polynucleotides expressing transposase comprising, in the 5' to 3' direction: a 5'-UTR of HBB; an NLS coding sequence; a piggyBac transposase coding sequence encoding four hyperactive mutations; a 3'-UTR of HBB, a polyA tail, and a polynucleotide comprising, in the 5' to 3' direction: a 5'-UTR of HBB; a coding sequence for a nuclear localization sequence (NLS); a piggyBac transposase coding sequence encoding four hyperactive mutations; a 3'-UTR of CYBA in tandem. SPBv3.1-HBB is the same as shown in FIGURE 1. SPB-HBB / 2xCYBA is an intermediate construct. Petition 870250091226, dated 06 / 10 / 2025, pp. 106 / 224 6 / 100 that does not possess the fifth hypermutation (M226F) and the miR-1423p binding sites compared to the construct shown in FIGURE 1.

[0020] Figure 3B shows the results of an excision reporter assay showing a time course of GFP expression in HepG2 transposed cells using polynucleotides expressing the transposase from Figure 3A, or a catalytically killed transposase (“CD-SPB”) as a negative control.

[0021] FIGURE 4 shows a graph of serum Factor VIII (hFVIII) levels on Day 6 (as % of normal level) in young BALB / c mice that were co-administered LNP compositions encapsulating a DNA transposon comprising an FVIII expression cassette and an LNP composition encapsulating one of the polynucleotides expressing the mRNA transposase of Figure 3A, or a catalytically killed (CD) transposase as a negative control.

[0022] FIGURE 5 shows a graph of serum Factor VIII (hFVIII) levels on Day 6 (as % of normal level) in adult C57BL / 6 mice that were co-administered LNP compositions encapsulating a DNA transposon comprising an FVIII expression cassette and an LNP composition encapsulating one of the polynucleotides expressing the mRNA transposase of Figure 3A.

[0023] FIGURE 6 shows the results of an excision reporter assay showing a 42-hour time course of GFP expression in HepG2 transposed cells using polynucleotides expressing transposase without (w / o) or containing (+miR-142-3p BS) four miR-1423p binding sites on the 3'-UTR or a catalytically killed transposase (CD-SPB) as a negative control.

[0024] FIGURE 7 shows the results of a reporter expression assay of GFP showing an 18-hour time course of Petition 870250091226, dated 06 / 10 / 2025, page 107 / 224 7 / 100 expression of GFP in K562 cells (a hematopoietic cell) nucleofected with polynucleotides expressing GFP without (w / o) or containing (+miR-1423p BS) four miR-142-3p binding sites on the 3'-UTR.

[0025] FIGURE 8 shows the results of an excision reporter assay, showing a 42-hour time course of GFP expression in K562 transposed cells using polynucleotides expressing transposase without (w / o) or containing (+miR-142-3p BS) four miR-142-3p binding sites on the 3'-UTR.

[0026] FIGURE 9 shows a graph of serum Factor VIII (hFVIII) levels on Day 6 (as % of normal level) in juvenile BALB / c mice that were co-administered LNP compositions encapsulating a DNA transposon containing an FVIII expression cassette and an LNP composition encapsulating one of the mRNA transposase-expressing polynucleotides from Figure 3A, or the mRNA transposase-expressing polynucleotides from Figure 3A, also containing sequences for four miR-142-3p binding sites in the 3'-UTR, or a catalytically killed transposase as a negative control.

[0027] FIGURE 10 shows the number of piggyBac transposase-reactive T cells (as # of IFN gamma positive cells / 10e6 splenocytes) on Day 28 in juvenile BALB / c mice to which an LNP composition was co-administered encapsulating a DNA transposon comprising an FVIII expression cassette and an LNP composition encapsulating one of the transposase-expressing mRNA polynucleotides in Figure 3A, the transposase-expressing mRNA polynucleotides in Figure 3A additionally comprising sequences for four miR-142-3p binding sites in the 3'-UTR, or a catalytically killed transposase as a negative control.

[0028] FIGURE 11 shows the number of T cells reactive to Petition 870250091226, dated 06 / 10 / 2025, pp. 108 / 224 8 / 100 piggyBac transposase (as # of IFN gamma positive cells / 10e6 splenocytes) on Day 21 in adult C57BL / 6 mice co-administered with LNP compositions encapsulating a DNA transposon comprising an FVIII expression cassette and an LNP composition encapsulating one of the transposase-expressing mRNA polynucleotides of Figure 3A, the transposase-expressing mRNA polynucleotides of Figure 3A additionally comprising sequences for four miR-142-3p binding sites in 3'UTR, or a catalytically killed transposase as a negative control. '1 dose' groups received LNP compositions on Day 1 of the study; '3 dose' groups received LNP compositions repeatedly on Days 1, 7, and 14 of the study.

[0029] FIGURE 12 shows a schematic representation of the dual reporter plasmid design used to confirm excision and integration rates using each mutant transposon. Using a GFP H-2Kk transposon reporter (Reporter 1), an increase in H-2Kk expression is observed if there is an increase in transposon excision. Using Reporter 2, an increase in GFP expression is observed if there is an increase in transposon integration. In an alternative Reporter 2 design, an increase in Firefly luciferase expression is observed if there is an increase in transposon excision, and an increase in NanoLuc is observed if there is an increase in transposon integration.

[0030] FIGURE 13 shows a schematic representation of a GFP H-2Kk transposon reporter (Reporter 1). The structural features of the transposon are shown in both a circular and a linear map. An increase in H-2Kk expression is observed if there is an increase in transposon excision, and an increase in GFP is observed if there is an increase in transposon integration.

[0031] FIGURE 14 is a schematic representation of a Petition 870250091226, dated 06 / 10 / 2025, pp. 109 / 224 9 / 100 reporter transposon Nano Luc of the Firefly luciferase. The structural features of the transposon are shown in both a circular and a linear map. Firefly luciferase expression is observed if there is an increase in transposon excision, and an increase in NanoLuc is observed if there is an increase in transposon integration.

[0032] FIGURE 15A shows the results of a luciferase dual integration / excision reporter assay in K562 cells, using a DNA transposon comprising a luciferase dual integration / excision reporter construct comprising wild-type LE and RE ITRs, or a wild-type 35TCC LE ITR and a wild-type RE ITR, and a DNA plasmid comprising transposase-expressing polynucleotides encoding a piggyBac transposase comprising 4 (“SPB”) or 5 (“Hiper”) hyperactive mutations, or no transposase-expressing polynucleotide encoding a piggyBac transposase (no SPB) as a negative control.

[0033] FIGURE 15B shows the results of a luciferase dual integration / excision reporter assay in 293T cells, using a DNA transposon comprising a luciferase dual integration / excision reporter construct comprising wild-type LE and RE ITRs, or a wild-type 35TCC LE ITR and a wild-type RE ITR, and a DNA plasmid comprising transposase-expressing polynucleotides encoding a piggyBac transposase comprising 4 (“SPB”) or 5 (“Hiper”) hyperactive mutations, or no transposase-expressing polynucleotide encoding a piggyBac transposase (no SPB) as a negative control.

[0034] FIGURE 16 shows the results of a luciferase double integration / excision reporter assay in juvenile BALB / c mice that were co-administered LNP compositions encapsulating a DNA transposon comprising a luciferase double integration / excision reporter construct comprising LE and ER type ITRs. Petition 870250091226, dated 06 / 10 / 2025, pp. 110 / 224 10 / 100 wild-type or a wild-type 35TCC LE ITR and a wild-type ER ITR, and an LNP composite encapsulating an mRNA expressed from one of the mRNA transposase-expressing polynucleotides of Figure 1, or a catalytically killed transposase as a negative control.

[0035] Figure 17A shows a comparison of the activity of two versions of SPB, measured by transposition effectiveness, B2M knockout and T cell expansion.

[0036] Figure 17B shows a comparison of the activity of two versions of SPB, measured by transposition effectiveness, B2M knockout, and T-cell expansion.

[0037] Figure 17C shows a comparison of the activity of two versions of SPB, measured by transposition effectiveness, B2M knockout and T cell expansion.

[0038] Figure 17D shows a comparison of the activity of two versions of SPB, measured by transposition effectiveness, B2M knockout, and T-cell expansion.

[0039] Figure 17E shows a comparison of the activity of two versions of SPB, measured by transposition effectiveness, B2M knockout and T cell expansion. Detailed Description

[0040] Polynucleotides for expressing transposases are provided in this document, in particular, polynucleotide mRNAs for expressing piggyBac transposases, comprising a piggyBac transposase coding sequence with hyperactive mutations and modified 5' and 3'UTR sequences to enhance the expression, activity and reduce the in vivo immunogenicity of the encoded transposase. Polynucleotides that Express PiggyBac Transposase

[0041] In certain aspects of disclosure, the polynucleotide that Petition 870250091226, dated 06 / 10 / 2025, pp. 111 / 224 11 / 100 encodes the transposase coding sequence and is a polynucleotide comprising in the 5' to 3' direction: (i) a 5'-UTR of HBB, (ii) a nucleic acid sequence encoding an SV40 NLS, (iii) a nucleic acid sequence encoding a piggyBac transposase comprising five hyperactive mutations, (iv) a 3'-UTR comprising a CYBA 3'-UTR element tandem upstream of a nucleic acid sequence encoding four miR-142-3p binding sites, and (v) an 80X polyA tail (Figure 1).

[0042] In certain respects, the 5'-UTR is a 5'-UTR of HBB. In certain respects, the 5'-UTR sequence of HBB comprises, or essentially consists of, the nucleic acid sequence: ACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC (SEQ ID NO. 1). In some embodiment, the 5'-UTR sequence of HBB comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence shown in SEQ ID NO: 1.

[0043] In certain respects, the nucleic acid sequence encodes a piggyBac transposase comprising the following five hyperactive mutations: I30V; G165S; M226F, M282V and N538K (for example, the piggyBac transposase comprising the sequence shown in SEQ ID NO: 45).

[0044] In some embodiments, a piggyBac transposase comprising the five hyperactive mutations I30V; G165S; M226F, M282V and N538K comprises the sequence with SEQ ID NO: 45 (the underlined sequence in bold is a nuclear localization sequence and may be omitted; residue numbering for mutation purposes begins at residue 12). MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQ Petition 870250091226, dated 06 / 10 / 2025, pp. 112 / 224 12 / 100 SDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNK HCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVK WTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDR SLSMVYVSVMSRDRFDFLIRCLRFDDKSIRPTLRENDVFTPVRKIWDLFIHQCI QNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMING MPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEP YKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVY LLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWP MALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLE APTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASC KKCKKVICREHNIDMCQSCF (SEQ ID NO: 45).

[0045] In some embodiment, the piggyBac transposase comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence presented in SEQ ID NO: 45

[0046] In certain respects, the nucleic acid sequence encoding the piggyBac transposase comprising mutations I30V; G165S; M226F, M282V and N538K comprises or consists of the sequence:

[0047] ATGGCTCCCAAGAAGAAGCGGAAAGTTGGCGGCGG AGGCAGCAGCCTGGATGATGAGCATATTCTGAGCGCCCTGCTGCAGAGC GACGATGAACTCGTGGGCGAAGATAGCGACAGCGAGGTGTCCGATCACG TGTCCGAGGATGACGTGCAGTCCGATACCGAGGAAGCCTTCATCGACGAG GTGCACGAAGTGCAGCCTACAAGCAGCGGCAGCGAGATCCTGGACGAGC AGAATGTGATCGAGCAGCCAGGATCTAGCCTGGCCAGCAACAGAATCCTG ACACTGCCCCAGAGAACCATCCGGGGCAAGAACAAGCACTGCTGGTCCAC CAGCAAGAGCACCAGACGGTCTAGAGTGTCTGCCCTGAACATCGTGCGAA Petition 870250091226, dated 06 / 10 / 2025, pp. 113 / 224 13 / 100 GCCAGAGGGGCCCTACCAGAATGTGCCGGAACATCTACGACCCTCTGCTG TGCTTCAAGCTGTTCTTCACCGACGAGATCATCTCCGAGATCGTGAAGTGG ACCAACGCCGAGATCAGCCTGAAGCGGAGAGAATCCATGACCAGCGCCA CCTTCAGAGACACCAACGAGGACGAGATCTACGCCTTCTTCGGCATCCTG GTCATGACAGCCGTGCGGAAGGACAACCACATGAGCACCGACGACCTGTT CGACCGCAGCCTGTCTATGGTGTACGTGTCCGTGATGAGCCGGGACAGAT TCGACTTCCTGATCCGGTGCCTGCGGTTCGACGACAAGTCCATCAGACCC ACACTGCGCGAGAACGACGTGTTCACACCTGTGCGGAAGATCTGGGACCT GTTCATCCACCAGTGCATCCAGAACTACACCCCTGGCGCTCACCTGACCA TCGACGAACAGCTGCTGGGCTTCAGAGGCAGATGCCCCTTCAGAGTGTAC ATCCCCAACAAGCCCTCTAAGTACGGCATCAAGATCCTGATGATGTGCGA CAGCGGCACCAAGTACATGATCAACGGCATGCCCTACCTCGGCAGAGGCA CCCAAACAAATGGCGTGCCACTGGGCGAGTACTACGTGAAAGAACTGAGC AAGCCTGTGCACGGCAGCTGCAGAAACATCACCTGTGACAACTGGTTTAC CAGCATTCCCCTGGCCAAGAACCTGCTGCAAGAACCCTACAAGCTGACAA TCGTGGGCACCGTGCGGAGCAACAAGAGGGAAATTCCCGAGGTGCTGAA GAACTCTCGGAGCAGACCTGTGGGCACCAGCATGTTCTGCTTCGACGGAC CTCTGACACTGGTGTCCTACAAGCCCAAGCCTGCCAAGATGGTGTACCTG CTGAGCAGCTGTGACGAGGACGCCAGCATCAATGAGAGCACCGGCAAGCCCCAGATGGTCATGTACTACAACCAGACCAAAGGCGGCGTGGACACCCTG GATCAGATGTGCAGCGTGATGACCTGCAGCAGAAAGACCAACAGATGGCC CATGGCTCTGCTGTACGGCATGATCAATATCGCCTGCATCAACAGCTTCAT CATCTACAGCCACAACGTGTCCAGCAAGGGCGAGAAGGTGCAGAGCCGG AAGAAATTCATGCGGAACCTGTACATGAGCCTGACCAGCAGCTTCATGAG AAAGCGGCTGGAAGCCCCTACACTGAAGAGATACCTGCGGGACAACATCA GCAACATCCTGCCTAAAGAGGTGCCCGGCACCAGCGACGATAGCACAGA GGAACCCGTGATGAAGAAGAGGACCTACTGCACCTACTGTCCCAGCAAGA TCCGGCGGAAGGCCAACGCCAGCTGCAAAAAGTGCAAGAAAGTGATCTGC Petition 870250091226, dated 06 / 10 / 2025, pp. 114 / 224 14 / 100 CGCGAGCACAACATCGATATGTGCCAGAGCTGCTTCtga (SEQ ID NO. 2). In some embodiment, the nucleic acid sequence encoding the piggyBac transposase comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least less than 99% identical to the nucleic acid sequence shown in SEQ ID NO: 2.

[0048] In certain respects, the nucleic acid sequence encodes a piggyBac transposase comprising the following four hyperactive mutations: I30V, G165S, M282V, and N538K.

[0049] In some embodiments, a piggyBac transposase comprising the four hyperactive mutations I30V, G165S, M282V, and N538K comprises the sequence with SEQ ID NO: 46 (the underlined sequence in bold is a nuclear localization sequence and may be omitted; residue numbering for mutation purposes begins at residue 12). MAPKKKRKVGGGGSSLDDEHILSALLQSDDELVGEDSDSEVSDHVSEDDVQ SDTEEAFIDEVHEVQPTSSGSEILDEQNVIEQPGSSLASNRILTLPQRTIRGKNK HCWSTSKSTRRSRVSALNIVRSQRGPTRMCRNIYDPLLCFKLFFTDEIISEIVK WTNAEISLKRRESMTSATFRDTNEDEIYAFFGILVMTAVRKDNHMSTDDLFDR SLSMVYVSVMSRDRFDFLIRCLRMDDKSIRPTLRENDVFTPVRKIWDLFIHQCI QNYTPGAHLTIDEQLLGFRGRCPFRVYIPNKPSKYGIKILMMCDSGTKYMING MPYLGRGTQTNGVPLGEYYVKELSKPVHGSCRNITCDNWFTSIPLAKNLLQEP YKLTIVGTVRSNKREIPEVLKNSRSRPVGTSMFCFDGPLTLVSYKPKPAKMVY LLSSCDEDASINESTGKPQMVMYYNQTKGGVDTLDQMCSVMTCSRKTNRWP MALLYGMINIACINSFIIYSHNVSSKGEKVQSRKKFMRNLYMSLTSSFMRKRLE APTLKRYLRDNISNILPKEVPGTSDDSTEEPVMKKRTYCTYCPSKIRRKANASC KKCKKVICREHNIDMCQSCF (SEQ ID NO: 46).

[0050] In some embodiment, the transposase piggyBac Petition 870250091226, dated 06 / 10 / 2025, pp. 115 / 224 15 / 100 comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence shown in SEQ ID NO: 46.

[0051] In certain respects, the NLS sequence comprises, or consists essentially of, a nucleic acid sequence encoding the amino acid sequence PKKKRKV (SEQ ID NO: 8). In certain respects, the nucleic acid encoding NLS comprises, or consists of, the nucleic acid sequence: CCCAAGAAGAAGCGGAAAGTT (SEQ ID NO: 9).

[0052] In certain respects, the 80X pulley tail comprises, or consists of, the following sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 7).

[0053] In certain respects, the polynucleotide for expressing a piggyBac transposase comprises the nucleic acid sequence shown in SEQ ID NO: 10. In some embodiment, the polynucleotide for expressing a piggyBac transposase comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence shown in SEQ ID NO: 10. Element 3'-UTR of CYBA

[0054] In certain respects, polynucleotides expressing transposase comprise one or more nucleic acid sequence(s) encoding a 3'-UTR element of CYBA.

[0055] In certain respects, the nucleic acid that codes for a Petition 870250091226, dated 06 / 10 / 2025, pp. 116 / 224 The 16 / 100 element of 3'-UTR of CYBA comprises, or consists of, the nucleic acid sequence: CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTTGCAATAAATG CAGCGAAGCCGGGA (SEQ ID NO. 3). In some embodiment, the nucleic acid encoding a 3'-UTR element of CYBA comprises a nucleic acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence shown in SEQ ID NO: 3.

[0056] In certain aspects, the polynucleotide comprises tandem nucleic acid sequences, each encoding a 3'-UTR element of CYBA. In some embodiments, two tandem nucleic acid sequences encoding 3'-UTR elements of CYBA are separated by a linker sequence. In certain aspects, the tandem nucleic acid sequences encoding 3'-UTR elements of CYBA separated by a linker sequence comprise, or consist of, the sequence: CCTCGCCCCGGACCTGCCCTCCCGCCAGGTGCACCCACCTGCAATAAATG CAGCGAAGCCGGGAGAATTCCCTCGCCCCGGACCTGCCCTCCCGCCAGG TGCACCCACCTGCAATAAATGCAGCGAAGCCGGGA (SEQ ID NO. 4). miR-142-3P binding sites

[0057] The transposase-encoding polynucleotides described in this document comprise one or more miR-142-3p binding sites in the 3'-UTR of the encoded mRNAs. MicroRNAs (miRNAs) are a group of non-coding RNAs (-22 nt) that can silence gene expression by binding to the 3' untranslated region (UTR) or the coding region of target mRNAs to promote mRNA destabilization or inhibit protein translation. Petition 870250091226, dated 06 / 10 / 2025, pp. 117 / 224 17 / 100

[0058] In some embodiments, the miR-142-3p binding site comprises the ACACTAC sequence. In certain respects, the miR142-3p sequence comprises, or consists of, the nucleic acid sequence: TCCATAAAGTAGGAAACACTACA (SEQ ID NO. 5).

[0059] In certain respects, the 3'-UTR comprises four miR-142-3p binding sites. In certain respects, the four miR142-3p target sequences are separated by three linker sequences. In certain respects, the four miR-142-3p target sequences separated by three linker sequences comprise, or consist of, the sequence: TCCATAAAGTAGGAAACACTACACGATTCCATAAAGTAGGAAACACTACAA CCGGTTCCATAAAGTAGGAAACACTACATCACTCCATAAAGTAGGAAACAC TACA (SEQ ID NO. 6). piggyBac ITR sequences

[0060] PiggyBac transposases recognize transposon-specific inverted terminal repeat sequences (ITRs) at the transposon ends and insert the content between the ITRs into the 5'-TTAA-3' sequence within a chromosomal site (a TTAA target sequence) or into the 5'-TTAT-3' sequence within a chromosomal site (a TTAT target sequence). The target PB sequence or a piggyBac-like transposon (PBL) may comprise or consist of 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3'5'-TTCT-3' and 5'-TTTT-3'.The PB transposon system has no payload limit for the genes of interest (i.e., no limit as to the size of the gene of interest) that can be included. Petition 870250091226, dated 06 / 10 / 2025, pp. 118 / 224 18 / 100 included among the ITRs.

[0061] In some respects, the DNA transposon comprises a piggyBac ITR sequence. In some respects, the DNA transposon comprises a first piggyBac ITR sequence, wherein the first piggyBac sequence is a left-end (LE) piggyBac ITR sequence. In some respects, the LE piggyBac ITR sequence is a minimal LE piggyBac ITR sequence. In some respects, the minimal LE piggyBac ITR is a 35 bp LE piggyBac ITR sequence comprising SEQ ID NO: 11.

[0062] In some embodiments, the minimum ITR is a minimum ITR of LE comprising the sequence CCCTAGAAAGATAGTCTGCGTAAAATTGACGCATG (SEQ ID NO: 14).

[0063] In some respects, the piggyBac LE ITR sequence is a superminimal piggyBac LE ITR sequence. In some respects, the DNA transposon comprises a second piggyBac ITR sequence, wherein the second piggyBac sequence is a piggyBac right-end (RE) ITR sequence. In some respects, the piggyBac RE ITR sequence is a minimal piggyBac RE ITR sequence. In some respects, the minimal piggyBac RE ITR is a 63 bp piggyBac RE ITR sequence comprising SEQ ID NO: 12. In some embodiments, the minimal ITR is a minimal RE ITR comprising the sequence CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGCGTAAA ATTGACGCATG (SEQ ID NO: 15). In some embodiments, the LE piggyBac ITR sequence is a superminimal LE piggyBac ITR sequence comprising the sequence: CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCA (SEQ ID NO: 16); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGAT (SEQ ID Petition 870250091226, de 06 / 10 / 2025, pág. 119 / 224 19 / 100 NO: 17); CCCTAGAAAGATAATCATATTGTGACGTACGTTA (SEQ ID NO: 18); CCCTAGAAAGATAATCATATTGTGACGTA (SEQ ID NO:19); CCCTAGAAAGATAATCATATTGTG (SEQ ID NO:20); CCCTAGAAAGATAATCATA (SEQ ID NO: 21); CCCTAGAAAGATAATCA (SEQ ID NO: 22); CCCTAGAAAGATAATC (SEQ ID NO:23); CCCTAGAAAGATAATCAT (SEQ ID NO: 24); CCCTAGAAAGATAATCATAT (SEQ ID NO: 25); CCCTAGAAAGATAATCATATT (SEQ ID NO: 26); CCCTAGAAAGATAATCATATTG (SEQ ID NO:27); CCCTAGAAAGATAATCATATTGT (SEQ ID NO:28); CCCTAGAAAGATAATCATATTGTGA (SEQ ID NO:29); CCCTAGAAAGATAATCATATTGTGAC (SEQ ID NO:30); CCCTAGAAAGATAATCATATTGTGACG (SEQ ID NO:31); CCCTAGAAAGATAATCATATTGTGACGT (SEQ ID NO:32); CCCTAGAAAGATAATCATATTGTGACGTAC (SEQ ID NO:33); CCCTAGAAAGATAATCATATTGTGACGTACG (SEQ ID NO:34); CCCTAGAAAGATAATCATATTGTGACGTACGT (SEQ ID NO:35); CCCTAGAAAGATAATCATATTGTGACGTACGTT (SEQ ID NO:36); CCCTAGAAAGATAATCATATTGTGACGTACGTTAA (SEQ ID NO:37); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAA (SEQ ID NO: 38); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAG (SEQ ID NO: 39); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGA (SEQ ID NO: 40); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATA (SEQ ID NO: 41); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAA (SEQ ID NO: 42); CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAAT (SEQ ID NO: 43); or CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATC (SEQ ID NO: 44).

[0064] In some respects, the DNA transposon comprises a second piggyBac ITR sequence, in which the second sequence of Petition 870250091226, dated 06 / 10 / 2025, pp. 120 / 224 20 / 100 piggyBac is a left-end (LE) ITR sequence of piggyBac comprising a 35TCC mutation. The piggyBac ITR sequence comprising the 35TCC mutation was described in International Publication Application PCT No. WO / 2023 / 060088, which is incorporated herein by reference in its entirety for examples of piggyBac ITR sequences that may be used in the compositions and methods described herein. The 35TCC mutation comprises a G to T substitution at position 31 of the ITR and an A to C substitution at position 33 to create the 35TCC ITR variant. In certain respects, the 35TCC ITR variant is a complete LE ITR sequence comprising SEQ ID NO: 13.

[0065] In some respects, a piggyBac ITR sequence may comprise any piggyBac ITR sequence known in the art. For example, a piggyBac ITR sequence, such as a first piggyBac ITR sequence and / or a second piggyBac ITR sequence in a transposon, may comprise, consist essentially of, or consist of a Sleeping Beauty transposon ITR, a Helraiser transposon ITR, a Tol2 transposon ITR, a TcBuster transposon ITR, or any combination thereof. Lipid Nanoparticles

[0066] This disclosure provides a transposase-expressing polynucleotide, as described herein, encapsulated in a lipid nanoparticle (LNP) for in vivo delivery of the transposase-expressing polynucleotide. In some embodiments, the LNP composition comprises at least one cationic lipid and at least one transposase-encoding polynucleotide molecule. In some aspects, a lipid nanoparticle may further comprise at least one structural lipid. In some aspects, a lipid nanoparticle may comprise Petition 870250091226, dated 06 / 10 / 2025, pages 121 / 224 21 / 100 additionally comprising at least one phospholipid. In some respects, a lipid nanoparticle may additionally comprise at least one PEGylated lipid.

[0067] Consequently, the present disclosure provides LNP compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid and at least one PEGylated lipid. Cationic Ionizable Bioreducible Lipids

[0068] In some respects, a cationic lipid can be a bioreducible ionizable cationic lipid. Thus, the present disclosure provides compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid.

[0069] In some aspects of the compositions and methods of the present disclosure, a bioreducible ionizable cationic lipid for use in the LNP compositions may be C12-200 (1,1'-[[2-[4-[2-[[2-[bis(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-1-piperazinyl]ethyl]imino]bis2-dodecanol). In some embodiments, an illustrative LNP composition comprising C12-200 for encapsulating a DNA molecule is: 35% C12-200; 41.84% DOPE; 20% CHOL; and 3.16% DM-PEG. In some embodiments, an illustrative LNP composition comprising C12-200 for encapsulating an RNA molecule is: 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG.

[0070] In some aspects of the compositions and methods of the present disclosure, the bioionizable cationic lipid is a terpene lipidoid with the structure: Petition 870250091226, dated 06 / 10 / 2025, pages 122 / 224 22 / 100 Compound X

[0071] Methods for the preparation of Compound X and the preparation of LNP compositions comprising Compound X have been previously described in jointly owned International PCT Application No. PCT / US2023 / 61005. In some embodiments, illustrative LNP compositions comprising Compound X for encapsulating a nucleic molecule are: 41.84% Compound X; 45.85% DOPE; 10% CHOL; and 2.7% DM-PEG or 33.5% Compound X; 32% DOPE; 33.5% CHOL; and 1% DM-PEG.

[0072] In some aspects of the compositions and methods of the present disclosure, a bioreducible ionizable cationic lipid for use in LNP compositions may be ssPalmO-Ph-P4C2. As would be appreciated by those skilled in the art, ssPalmO-Ph-P4C2 has the following structure:

[0073] As would be appreciated by those skilled in the art, ssPalmOPh-P4C2 may also be referred to as Coatsome® SS-OP, ssPalmO-PheP4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe and ssPalmO-Ph. Thus, ssPalmOPh-P4C2, Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe and ssPalmO-Ph are used interchangeably in this document to refer to the bioreducible ionizable cationic lipid with the chemical structure shown in Formula I. Petition 870250091226, dated 06 / 10 / 2025, pages 123 / 224 23 / 100

[0074] Without wanting to limit ourselves to theory, three specific segments of ssPalmO-Ph-P4C2 facilitate its biodegradation. First, the tertiary amine of each piperdine ring is a cationic charge unit sensitive to acidic pH. After endocytosis, the tertiary amine portions become positively charged in response to the acidic intracellular endosomal compartment. These are now able to interact with and destabilize the membrane, leading to endosomal escape. Once in the cytosol, the disulfide bond is susceptible to reduction by glutathione, generating two free sulfhydryl groups. The resulting high concentration of free thiols further leads to a nucleophilic reaction, and the particle undergoes self-degradation / collapse via thioesterification, releasing the payload into the cytosol.This is defined as HyPER or Hydrolysis accelerated by the intra-particle enrichment of the reagent and potentially eliminates the potentially toxic side effects of cationic lipids in general.

[0075] As used in this document, the term bioreducible ionizable cationic lipid is used in its broadest sense to refer to a cationic lipid comprising: at least one tertiary amine, at least one disulfide group, at least one group comprising a linkage susceptible to thioesterification cleavage, and additionally comprising at least two saturated or unsaturated hydrocarbon chains. Illustrative bioreducible ionizable cationic lipids include, among others, those described in Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the contents of which are incorporated herein by reference in their entirety.

[0076] In some respects, a bioreducible ionizable cationic lipid may comprise at least two tertiary amines. In some respects, at least one tertiary amine may be a substituted piperidinyl group. In some respects, each tertiary amine may be a group Petition 870250091226, dated 06 / 10 / 2025, pages 124 / 224 24 / 100 substituted piperidinyl. In some respects, a bioreducible ionizable cationic lipid may comprise at least one disulfide bond. In some respects, the sulfur atoms of the disulfide bond are linked to the nitrogen of the piperidinyl ring via an alkylene group, thus forming two tertiary amine groups flanking the disulfide bond. In some respects, at least one of the alkylene groups is an ethylene group. In some respects, each of the alkylene groups is an ethylene group.

[0077] In some respects, at least one group comprising a linkage susceptible to thioesterification cleavage may be a phenyl ester group. In some respects, a bioreducible ionizable cationic lipid may comprise at least two phenyl ester groups. In some respects, at least one of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some respects, each of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some respects, an unsaturated hydrocarbon chain may be an octadecene. In some respects, an octadecene may be (Z)octadec-9-ene. In some respects, an (Z)-octadec-9-ene group may be attached to a phenyl ester group of the bioreducible ionizable cationic lipid.

[0078] Illustrative bioreducible ionizable lipids and methods for preparing such lipids useful in the methods of the present invention include those disclosed in International Patent Application No. PCT / JP2016 / 052690, published as WO / 2016 / 121942, the contents of which are incorporated herein by reference in their entirety for examples of bioreducible ionizable lipids that can be used in the compositions and methods described herein. Consequently, the present disclosure provides compositions comprising at least one lipid nanoparticle, wherein at least one Petition 870250091226, dated 06 / 10 / 2025, pages 125 / 224 25 / 100 lipid nanoparticle comprises any of the bioreducible ionizable cationic lipids presented in document WO / 2016 / 121942.

[0079] Thus, the present disclosure provides compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid and at least one PEGylated lipid.

[0080] In some respects, the bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2, having the structure shown in Formula I (see Akita et al., (2020) Biol. Phar. Bull. 43:1617 - 1625, the contents of which are incorporated by reference in full).

[0081] As described in this document, the LNP compositions of this disclosure comprising at least one bioreducible ionizable cationic lipid advantageously exhibit significantly reduced toxicity in animals compared with LNP compositions comprising non-bioreducible ionizable cationic lipids. In particular, administration of the LNP compositions of this disclosure surprisingly does not result in any loss of body weight. In fact, the LNP compositions of this disclosure are so non-toxic that animals to which the LNPs are administered gain body weight, even when administered amounts of LNPs that exceed the lethal dose of the LNP compositions comprising non-bioreducible ionizable cationic lipids. LNP Components

[0082] In some respects, an LNP of this disclosure may comprise about 2.5%, or about 5%, or about 7.5%, or about 10%, or about 12.5%, or about 15%, or about 17.5%, or about Petition 870250091226, dated 06 / 10 / 2025, pp. 126 / 224 26 / 100 20%, or about 22.5%, or about 25%, or about 27.5%, or about 30%, or about 32.5%, or about 35%, or about 37.5%, or about 40%, or about 42.5%, or about 45%, or about 47.5%, or about 50%, or about 52.5%, or about 55%, or about 57.5%, or about 60%, or about 62.5%, or about 65%, or about 67.5%, or about 70% of at least one bioreducible ionizable cationic lipid in moles.

[0083] In some respects, a non-profit portion of this disclosure may comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one bioreducible ionizable cationic lipid in moles.

[0084] In some respects, an LNP of this disclosure may comprise approximately 2.5%, or approximately 5%, or approximately 7.5%, or approximately 10%, or approximately 12.5%, or approximately 15%, or approximately 17.5%, or approximately 20%, or approximately 22.5%, or approximately 25%, or approximately 27.5%, or approximately 30%, or approximately 32.5%, or approximately 35%, or approximately 37.5%, or approximately 40%, or approximately 42.5%, or approximately 45%, or approximately 47.5%, or approximately 50%, or approximately 52.5%, or approximately 55%, or approximately 57.5%, or approximately 60%, or approximately 62.5%, or approximately 65%, or about 67.5%, or about 70% of at least Petition 870250091226, dated 06 / 10 / 2025, pages 127 / 224 27 / 100 a structural lipid in moles.

[0085] In some respects, a non-profit portion of this disclosure may comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one structural lipid in moles.

[0086] In some respects, an LNP of this disclosure may comprise approximately 2.5%, or approximately 5%, or approximately 7.5%, or approximately 10%, or approximately 12.5%, or approximately 15%, or approximately 17.5%, or approximately 20%, or approximately 22.5%, or approximately 25%, or approximately 27.5%, or approximately 30%, or approximately 32.5%, or approximately 35%, or approximately 37.5%, or approximately 40%, or approximately 42.5%, or approximately 45%, or approximately 47.5%, or approximately 50%, or approximately 52.5%, or approximately 55%, or approximately 57.5%, or approximately 60%, or approximately 62.5%, or approximately 65%, or about 67.5%, or about 70% of at least one phospholipid by mole.

[0087] In some respects, a non-profit (NPP) of this disclosure may comprise at least about 2.5%, or at least about 5%, or at least about 7.5%, or at least about 10%, or at least about 12.5%, or at least about 15%, or at least about 17.5%, or at least about 20%, or at least about 22.5%, or at least about Petition 870250091226, dated 06 / 10 / 2025, pp. 128 / 224 28 / 100 25%, or at least about 27.5%, or at least about 30%, or at least about 32.5%, or at least about 35%, or at least about 37.5%, or at least about 40%, or at least about 42.5%, or at least about 45%, or at least about 47.5%, or at least about 50%, or at least about 52.5%, or at least about 55%, or at least about 57.5%, or at least about 60%, or at least about 62.5%, or at least about 65%, or at least about 67.5%, or at least about 70% of at least one phospholipid in moles.

[0088] In some respects, an LNP of the present disclosure may comprise about 0.25%, or about 0.5%, or about 0.75%, or about 1.0%, or about 1.25%, or about 1.5%, or about 1.75%, or about 2.0%, or at least about 2.5%, or about 5% of at least one PEGylated lipid by moles.

[0089] In some respects, an LNP of the present disclosure may comprise at least about 0.25%, or at least about 0.5%, or at least about 0.75%, or at least about 1.0%, or at least about 1.25%, or at least about 1.5%, or at least about 1.75%, or at least about 2.0%, or at least about 2.5%, or at least about 5% of at least one PEGylated lipid by moles. Structural Lipids

[0090] In some respects, a structural lipid may be a steroid. In some respects, a structural lipid may be a sterol. In some respects, a structural lipid may comprise cholesterol. In some respects, a structural lipid may comprise ergosterol. In some respects, a structural lipid may be a phytosterol. Phospholipid

[0091] As used in this document, the term phospholipid is used in its broadest sense to refer to any Petition 870250091226, dated 06 / 10 / 2025, pp. 129 / 224 29 / 100 amphiphilic molecule comprising a polar (hydrophilic) head group that includes phosphate and two hydrophobic fatty acid chains. In some respects, a phospholipid may comprise dileoylphosphatidylethanolamine (DOPE). In some respects, a phospholipid may comprise DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl-sn-glycero-3-phosphate (Sodium Salt)), DEPC (1,2-dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-dierucoyl-sn-glycero-3-phosphorac-(1-glycerol) (Sodium Salt)), DLOPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine), DLPANA (1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt)), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DLPE (1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt)), DLPG-NH4 (1,2-Dilauroyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Ammonium Salt)), DLPS-NA (1,2-Dilauroyl-sn-glycero-3-phosphoserine (Sodium Salt)),DMPA-NA (1,2-Dimiristoyl-sn-glycero-3-phosphate (Sodium Salt)), DMPC (1,2-Dimiristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-Dimiristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-Dimiristoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Sodium Salt)), DMPG-NH4 (1,2-Dimiristoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Ammonium Salt)), DMPGNH4 / NA (1,2-Dimiristoyl-sn-glycero-3[Phospho-rac-(1-glycerol) (Salt of, Sodium / Ammonium), DMPS-NA (1,2-Dimiristoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DOPA-NA (1,2-Dileoyl-sn-glycero-3-phosphate (Sodium Salt)), DOPC (1,2-Dileoyl-sn-glycero-3-phosphocholine), DOPE (1,2-Dileoyl-sn-glycero-3-phosphoethanolamine), DOPG-NA (1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol) (Sodium Salt)), DOPS-NA (1,2-dioleoyl-sn-glycero-3-phosphoserine (Sodium Salt)), DPPA-NA (1,2-dipalmitoyl-sn-glycero-3-phosphate (Sodium Salt)), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DPPG-NH4 (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol) (ammonium salt)), DPPS-NA (1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt)), Petition 870250091226, dated 06 / 10 / 2025, pages 130 / 224 30 / 100 DSPA-NA (1,2-distearoyl-sn-glycero-3-phosphate (Sodium Salt)), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1,2-distearoyl-sn-glycero-3[phosphorac-(1-glycerol) (Sodium Salt)), DSPG-NH4 (1,2-distearoyl-sn-glycero-3[phosphorac-(1-glycerol) (Ammonium Salt)), DSPS-NA (1,2-distearoyl-sn-glycero-3-phosphoserine (Sodium Salt)), EPC (Egg PC), HEPC (Hydrogenated Egg PC), HSPC (Hydrogenated Soy PC), LYSOPC MYRISTIC (1-myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (1-palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (1-stearoyl-sn-glycero-3-phosphocholine), Milk Sphingomyelin (MPPC;1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), POPG-NA (1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)] (Sodium Salt)), PSPC (1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), SOPC (1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SPPC (1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combination thereof. PEGylated Lipid

[0092] As used in this document, the term PEGylated lipid is used to refer to any lipid that is modified (e.g., covalently linked to) at least one polyethylene glycol molecule. In some respects, a PEGylated lipid may comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, hereinafter referred to as DMG-PEG2000. Nucleic Acids

[0093] In some respects, a lipid nanoparticle may comprise at least one polynucleotide molecule that expresses transposase. In some respects, a lipid nanoparticle may comprise Petition 870250091226, dated 06 / 10 / 2025, pp. 131 / 224 31 / 100 a plurality of polynucleotide molecules expressing transposase. In some respects, at least one polynucleotide molecule expressing transposase or a plurality of polynucleotide molecules expressing transposase can be formulated into a lipid nanoparticle.

[0094] In some respects, a nucleic acid molecule may be a synthetic polynucleotide molecule expressing transposase. In some respects, a polynucleotide molecule expressing transposase may be a non-naturally occurring nucleic acid molecule. In some respects, a non-naturally occurring nucleic acid molecule may comprise at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide may be any non-naturally occurring nucleotide known in the art. In some respects, a nucleic acid molecule may be a modified polynucleotide molecule expressing transposase. In some respects, a modified nucleic acid molecule may comprise at least one modified nucleotide. The at least one modified nucleotide may be any modified nucleic acid known in the art.

[0095] In some respects, a lipid nanoparticle may comprise lipid and nucleic acid in a specific ratio (weight / weight).

[0096] In some respects, a lipid nanoparticle comprising at least one nucleic acid may comprise lipid and nucleic acid in a ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or about 75:1 to about 85:1, or about Petition 870250091226, dated 06 / 10 / 2025, pp. 132 / 224 32 / 100 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or approximately 115:1 up to approximately 130:1, or approximately 125:1 up to approximately 140:1, or approximately 135:1 up to approximately 150:1, lipid:nucleic acid, weight / weight.

[0097] 125:1, or approximately 120:1 up to approximately 135:1, or approximately 130:1 until approximately 145:1, or about 140:1 up to about lipid In some respects, a nanoparticle comprises lipid and nucleic acid in a ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1, or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or approximately 125:1, or approximately 130:1, or approximately 135:1, or approximately 140:1, or approximately 145:1, or approximately 150:1, or approximately 200:1, lipid: nucleic acid, weight / weight.

[0098] In some respects, a lipid nanoparticle may comprise lipid and nucleic acid in a ratio of about 10:1, or about 17.5:1, or about 25:1, lipid:nucleic acid, weight / weight.

[0099] In some respects, a nucleic acid molecule can be an RNA molecule. Thus, in some respects, a lipid nanoparticle can comprise at least one polynucleotide expressing transposase, wherein the polynucleotide molecule is an RNA molecule. In some respects, an RNA molecule can be an mRNA molecule. In some respects, an mRNA molecule can comprise a 5'-CAP.

[00100] In some respects, an mRNA molecule may have capping added using any known capping method and / or moiety in Petition 870250091226, dated 06 / 10 / 2025, pp. 133 / 224 33 / 100 technique. An mRNA molecule may have an added cap with the m7G(5')ppp(5')G moiety. An m7G(5')ppp(5')G moiety is also referred to in this document as a Cap0. An mRNA molecule may have an added cap with a CleanCap® moiety. A CleanCap® moiety may comprise an m7G(5')ppp(5')(2'OMeA) moiety (CleanCap® AG). A CleanCap® moiety may comprise an m7G(5')ppp(5')(2'OMeG) moiety (CleanCap® GG). An mRNA molecule may have an added cap with an antireverse cap analog moiety (ARCA®). An ARCA® moiety may comprise an m7(3'-O-methyl)G(5')ppp(5')G moiety. An mRNA molecule may have an added cap with a CleanCap® 3'OMe portion (CleanCap®+ARCA®).

[00101] In some respects, the mRNA molecule comprising a polynucleotide expressing transposase, as described herein, is prepared according to the method in Example 10.

[00102] In some respects, an mRNA molecule may comprise at least one modified nucleic acid.

[00103] Modified nucleic acids may include, but are not limited to, 5-methoxyuridine (5moU), N-methylpseudouridine (meE), pseudouridine (Ψ), 5-methylcytidine (5-MeC). DNA Editing Compositions

[00104] This disclosure also provides gene-editing compositions and cells comprising the gene-editing compositions. The gene-editing composition may comprise a nucleic acid sequence encoding a DNA-binding domain and a nucleic acid sequence encoding a nuclease protein or a nuclease domain thereof. The sequence encoding a nuclease protein or the sequence encoding a nuclease domain thereof may comprise a DNA sequence, an RNA sequence, or a combination thereof. The nuclease or nuclease domain thereof may comprise one or more proteins. Petition 870250091226, dated 06 / 10 / 2025, pages 134 / 224 34 / 100 CRISPR / Cas, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and an endonuclease.

[00105] The nuclease or nuclease domain thereof may comprise a nuclease-inactivated Cas protein (dCas) and an endonuclease. The endonuclease may comprise a Clo051 nuclease or a nuclease domain thereof. The gene-editing composition may comprise a fusion protein. In some embodiments, the fusion protein comprises a nuclease-inactivated Cas9 protein (dCas9) and a Clo051 nuclease or a Clo051 nuclease domain. The gene-editing composition may further comprise a guide sequence. The guide sequence comprises an RNA sequence.

[00106] Disclosure provides compositions comprising a Cas9 operatively linked to an effector. Disclosure provides a fusion protein comprising, essentially consisting of, or consisting of a DNA localization component and an effector molecule, wherein the effector comprises a Cas9. A Disclosure Cas9 construct may comprise an effector comprising a type IIS endonuclease.

[00107] In some embodiments, a gene-editing composition comprises an inactivated Cas9 (dSaCas9) operatively linked to an effector. Disclosure provides a fusion protein comprising a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dSaCas9). An inactivated Cas9 (dSaCas9) construct from disclosure may comprise an effector comprising a type IIS endonuclease. A dSaCas9 comprises the amino acid sequence SEQ ID NO: 48, which includes a D10A mutation and an N580A mutation to inactivate the catalytic site.

[00108] The disclosure provides compositions comprising an inactivated Cas9 (dCas9) operatively linked to an effector. The disclosure Petition 870250091226, dated 06 / 10 / 2025, pages 135 / 224 35 / 100 additionally provides a fusion protein comprising a DNA localization component and an effector molecule, wherein the effector comprises an inactivated Cas9 (dCas9). An inactivated Cas9 (dCas9) disclosure construct may comprise an effector comprising a type IIS endonuclease.

[00109] dCas9 can be isolated from or derived from Streptococcus pyogenes. dCas9 may comprise a dCas9 with substitutions at amino acid positions 10 and 840, which inactivate the catalytic site. In some respects, these substitutions are D10A and H840A. dCas9 may comprise the amino acid sequence SEQ ID NO: 49 or SEQ ID NO: 50.

[00110] In some embodiments, the C-terminal of dCas9 or its inactivated nuclease domain is linked to the N-terminal of the Clo051 polypeptide or its nuclease domain via a selected GGGGS peptide linker sequence (SEQ ID NO: 60).

[00111] In some embodiments, the Clo051 nuclease domain comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments, the Clo051 nuclease domain comprises at least one amino acid substitution with respect to SEQ ID NO: 51. In some aspects, the amino acid substitution occurs in the alpha-helix-loop domain of the Clo051 nuclease. In some aspects, the amino acid substitution is at position 35, 37, 60, 92, 98, 100, or 146 of SEQ ID NO: 51. In some aspects, the amino acid substitution is at position 37 of SEQ ID NO: 51. In some aspects, the amino acid substitution is at positions 37 and 92 of SEQ ID NO: 51.

[00112] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise the amino acid sequence of SEQ ID NO: 52. The illustrative dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 53. The nucleic acid encoding the dCas9-Clo051 fusion protein may be Petition 870250091226, dated 06 / 10 / 2025, pp. 136 / 224 36 / 100 DNA or RNA.

[00113] An illustrative dCas9-Clo051 (Cas-CLOVER) fusion protein may comprise the amino acid sequence of SEQ ID NO: 54. The illustrative dCas9-Clo051 fusion protein may be encoded by a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 55. The nucleic acid encoding the dCas9-Clo051 fusion protein may be DNA or RNA.

[00114] An illustrative dCas9-Clo051 fusion protein (Cas-CLOVER) of the present disclosure may further comprise at least one nuclear localization sequence (NLS). In some embodiments, the dCas9-Clo051 fusion protein of the disclosure comprises two nuclear localization sequences. In some embodiments, the NLS is at the N-terminal end of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051). In some embodiments, the NLS is at the C-terminal end of the dCas9-Clo051 fusion protein (dCas9-Clo051-NLS). In some embodiments, the NLS is at both the N-terminal and C-terminal ends of the dCas9-Clo051 fusion protein (NLS-dCas9-Clo051-NLS or wild-type Cas-CLOVER or dspCas9 Ca-CLOVER).

[00115] The NLS-dCas9-Clo051-NLS fusion protein (“wild-type CasCLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 CasCLOVER”) may comprise the amino acid sequence of SEQ ID NO: 56, wherein the NLS amino acid sequence is bold and underlined and the linker is bold and italicized): MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDSKQNRLFEMKVL ELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDNFGIIVDTKAYSEGYSLPISQAD EMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRR LSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY GGGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG Petition 870250091226, dated 06 / 10 / 2025, pp. 137 / 224 37 / 100 ETAEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSG EQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRRPLIETNGETGEIVWDKGRDFA TVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDGSPKKKRKVSS (SEQ ID NO: 56).

[00116] In some embodiments, the NLSdCas9-Clo051-NLS fusion protein (“wild-type Cas-CLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas-CLOVER”) comprises a sequence of Petition 870250091226, dated 06 / 10 / 2025, pages 138 / 224 38 / 100 amino acids that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 56.

[00117] In some embodiments, the NLSdCas9-Clo051-NLS fusion protein (“wild-type Cas-CLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas-CLOVER”) comprises the amino acid sequence shown in SEQ ID NO: 56 with one, two, three, four, or five conservative amino acid substitutions.

[00118] The nucleic acid that codes for the NLSdCas9-Clo051-NLS fusion protein (“wild-type Cas-CLOVER”, or “Cas-CLOVER v2”, or “CCv2”, or “dspCas9 Cas-CLOVER”) can be DNA or RNA. In some embodiments, a dCas9-Clo051 fusion protein comprising two NLS regions is encoded by an mRNA sequence comprising the sequence shown in SEQ ID NO: 57 or a DNA sequence comprising the sequence shown in SEQ ID NO: 61: atggctcccaagaagaagcggaaggtcGAGGGCATCAAGAGCAACATCAGCCTGCTG AAGGACGAGCTGAGAGGCCCAGATCAGCCACATCTCCCACGAGTACCTGAG CCTGATCGACCTGGCCTTCGACcccAAGCAGAACCGGCTGTTCGAGATGAA GGTGCTGGAACTGCTGGTCAACGAGTACGGCTTCAAGGGCAGACACCTC GGCGGCAGCAGAAAGCCTGATGGCATCGTGTACAGCACCACACTCGAGG ACAACTTCGGCATCATCGTGGACACCAAGGCCTACAGCGAGGGCTACAGC CTGCCTATCTCTCAGGCCGACGAGATGGAAAGATACGTGCGCGAGAACAG CAACCGCGACGAGGAAGTGAACCCCAACAAGTGGTGGGAGAACTTCAGC Petition 870250091226, dated 06 / 10 / 2025, pp. 139 / 224 39 / 100 GAGGAAGTCAAAAAGTACTACTTCGTGTTCATCAGCGGCAGCTTTAAGGG CAAGTTCGAGGAACAGCTGCGGCGGCTGTCTATGACCACAGGCGTTAACG GCAGCGCCGTGAACGTGGTCAATCTGCTGCTGGGCGCCGAGAAGATTAG AAGCGGCGAGATGACCATCGAGGAACTGGAACGGGCCATGTTCAACAACA GCGAGTTCATCCTGAAGTACggcggaggcggcagcgacaagaagtactctatcggactggcc atcggcaccaactctgttggatgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaagtg ctgggcaacaccgaccggcacagcatcaagaagaatctgatcggcgccctgctgttcgactctggcgaaac agccgaagccaccagactgaagagaaccgccagacggcggtacaccagaagaaagaaccggatctgct acctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtcct tcctggtggaagaggacaagaagcacgagcggcaccccatcttcggaaatatcgtggacgaggtggcctac cacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctg cgactgatctatctggccctggctcacatgatcaagttccggggccacttcctgatcgagggcgacctgaatcct gacaactccgacgtggacaagctgttcatccagctggtgcagacctacaatcagctgttcgaagagaatccc atcaacgcctctggcgtggacgccaaagccatcctgtctgccagactgagcaagagcagacggctggaaa acctgatcgctcagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgtctctgggcctgacacctaacttcaagtccaacttcgatctggccgaggatgccaaactgcagctgtccaaggacacctacgac gacgacctggataacctgctggcccagatcggcgatcagtacgccgacttgtttctggccgccaagaacctgt ctgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcacaaaggcccctctgagcgcctcta tgatcaagagatacgacgagcaccaccaggatctgaccctgctgaaagctctcgtcaggcagcagctgcca gagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgatggcggagccag ccaagaggaattctacaagttcatcaagcccatcctcgagaagatggacggcacagaggaactgctcgtga agctgaacagagaggacctgctgcggaagcagcggaccttcgacaatggctctatccctcaccagatccac ctgggagagctgcacgccattctgcggagacaagaggacttttacccattcctgaaggacaaccgggaaaa gattgagaagatcctgaccttcaggatcccctactacgtgggaccactggccagaggcaatagcagattcgc ctggatgaccagaaagagcgaggaaaccatcacaccctggaacttcgaagaggtggtggacaagggcgc cagcgctcagtccttcatcgagcggatgaccaatttcgacaagaatctgcccaacgagaaagtgctgcccaa gcactccctgctgtacgagtacttcaccgtgtacaacgagctgaccaaagtgaaatacgtgaccgagggaat gagaaagcccgcctttctgtccggcgagcagaaaaaggccatcgtggatctgctgttcaagaccaaccgga Petition 870250091226, dated 06 / 10 / 2025, pp. 140 / 224 40 / 100 aagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatcagc ggcgtggaagatcggttcaatgccagcctgggcacataccacgatctgctgaaaattatcaaggacaagga cttcctggacaacgaggaaaacgaggacatccttgaggacatcgtgctgaccctgacactgttcgaggacag agagatgatcgaggaaaggctgaaaacatacgcccacctgttcgacgacaaagtcatgaagcaactgaag cggcggcgctacacaggctggggcagactgtctagaaagctgatcaacggcatccgggacaagcagtccg gcaagaccatcctggactttctgaagtccgacggcttcgccaacagaaacttcatgcagctgattcacgacga cagcctcaccttcaaagaggacattcagaaggcccaggtttccggccagggcgattctctgcacgagcacatt gccaatctggccggctctcccgccattaagaagggcattctgcagacagtgaaagtggtggatgagctggtca aagtgatggggagacacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacacag aagggccagaagaactcccgcgagagaatgaagcggatcgaagagggaatcaaagagctggggagcc agatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctcca gaacggccgggatatgtacgtggaccaagagctggacatcaaccgcctgagcgactacgatgtggacgcta tcgtgccccagtcttttctgaaagatgactccatcgacaacaaggtgctgaccagaagcgataagaaccggggcaagagcgacaacgtgccctctgaagaggtcgtgaagaagatgaagaactactggcgacagctgctgaa cgccaagctgattacccagcggaagttcgataacctgaccaaggccgagagaggcggcctgtctgaactgg ataaggccggcttcatcaagagacagctggtggaaacccggcagatcaccaaacacgtggcacagattctg gactcccggatgaacaccaaatacgatgagaacgacaaactgatccgggaagtgaaagtcatcaccctga agtccaagctggtgtccgatttccggaaggatttccagttctacaaagtgcgggaaatcaacaactaccatcac gcccacgacgcctacctgaatgccgttgttggaacagccctgatcaagaagtatcccaagctggaaagcga gttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaagagattggaa aggctaccgccaaatacttcttctactccaacatcatgaactttttcaagacagagatcaccctcgccaacggc gagatcagaaagcggcctctgatcgagacaaacggcgaaaccggcgagattgtgtgggataagggcaga gactttgccacagtgcggaaggtgctcagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagac aggcggcttcagcaaagagtccattctgcctaagcggaactccgacaagctgatcgcccggaagaaggact gggaccccaagaaatacggcggcttcgatagccctaccgtggcctattctgtgctggtggtggccaaagtgga aaagggaaagtccaagaagctcaagagcgtcaaagaactcctgggcatcaccatcatggaacggtccagcttcgagaagaaccctatcgactttctggaagccaagggctacaaagaagtcaagaaggacctgatcaa gctccccaagtacagcctcgagctggaaaatggccggaagcggatgctggcttctgctggcgaactgca Petition 870250091226, of 06 / 10 / 2025, p. 141 / 224 41 / 100 gaagggaaacgaactgccctgctagcaaattgtgaacttcctgtacctggccagccactatgagaagct gaagggcagccccgagacaatgagcagaagcagctttcgtcgagcagcacaagcactacctgacga gatcatcgagcagatctccgagttctccaagagagtgatcctggccgacgccaacctggacaaggttctgtcc gcctacaacaaccgggataagcccatcagagagcaggcgagaatatcatccacctgtttaccctgac caacctgggagcccctgccgccttcaagtacttcgacccaccacc aagaagtgctggacgccacactgatccaccagagcatcaccggcctcgagacacggatcgatctgtctc agcttggaggcgacggcagccctaagaagagagaaaggtttccagctaataa (SEQ ID NO: 61).

[00119] In some respects, NLS-dCas9-Clo051-NLS (“wild-type CasCLOVER”) comprises at least one amino acid substitution with respect to SEQ ID NO: 56. In some respects, the amino acid substitution is located in the Clo051 domain of NLS-dCas9-Clo051-NLS.

[00120] In some respects, NLS-dCas9-Clo051-NLS of SEQ ID NO: 56 may comprise at least one substitution at amino acid positions 42, 44, 67, 105, 107, or 153. In some respects, the amino acid substitutions are F42E, F42D, S44E, S44P, R67E, I105Q, Q107A, Q107E, Q107H, Q107D, and / or K153D. In some respects, the amino acid substitution is S44P.

[00121] An illustrative S44P NLS-dCas9Clo051-NLS mutant fusion protein (“S44P Cas-CLOVER”, or “S44P CC”, or “S44P”, or “Cas-CLOVERv3”, or “CCv3”) may comprise the amino acid sequence of SEQ ID NO: 58, wherein the NLS amino acid sequence is bold and underlined and the linker is bold and italicized: MAPKKKRKVEGIKSNISLLKDELRGQISHISHEYLSLIDLAFDPKQNRLFEMKVL ELLVNEYGFKGRHLGGSRKPDGIVYSTTLEDFGIIVDTKAYSEGYSLPISQAD EMERYVRENSNRDEEVNPNKWWENFSEEVKKYYFVFISGSFKGKFEEQLRR LSMTTGVNGSAVNVVNLLLGAEKIRSGEMTIEELERAMFNNSEFILKY GGGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSG ETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE Petition 870250091226, de 06 / 10 / 2025, pág. 142 / 224 42 / 100 DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKLVDSTDKADLRIYLALAMHIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSK SRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYD DDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEH HQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEK MDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDN REKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQ SFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKKYVTEGMRKPAFLSG EQKKAIVDLLFKTTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLK RRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFK EDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENI VIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRG KSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIK RQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQF YKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSE QEIGKATAKYFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGF DSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSI TGLYETRIDLSQLGGDGSPKKKRKVSS (SEQ ID NO: 58)

[00122] In some embodiments, the mutant S44P NLSdCas9-Clo051-NLS fusion protein (“S44P Cas-CLOVER”, or “S44P CC”, or “S44P”, or “Cas-CLOVERv3”, or “CCv3”) comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, Petition 870250091226, dated 06 / 10 / 2025, pp. 143 / 224 43 / 100 at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 56. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence shown in SEQ ID NO: 58.

[00123] In some embodiments, the mutant S44P NLSdCas9-Clo051-NLS fusion protein (“S44P Cas-CLOVER”, or “S44P CC”, or “S44P”, or “Cas-CLOVERv3”, or “CCv3”) comprises the amino acid sequence shown in SEQ ID NO: 58 with one, two, three, four, or five conservative amino acid substitutions.

[00124] The Cas-CLOVER v3 fusion protein can be encoded by a polynucleotide comprising the nucleic acid sequence SEQ ID NO: 59. The nucleic acid encoding the dCas9-Clo051 fusion protein can be DNA or RNA: acauuugcuucugacacaacuguguucacuagcaaccucaaacagacaccaagcuugccaccaug gcucccaagaagaagcggaaggucGAGGGCAUCAAGAGCAACAUCAGCCUGCUGA AGGACGAGCUGAGAGGCCAGAUCAGCCACAUCUCCCACGAGUACCUGAG CCUGAUCGACCUGGCCUUCGACcccAAGCAGAACCGGCUGUUCGAGAUG AAGGUGCUGGAACUGCUGGUCAACGAGUACGGCUUCAAGGGCAGACAC CUCGGCGGCAGCAGAAAAGCCUGAUGCCAUCGUACAGCACCACACUC GAGGACAACUUCGGCAUCAUCGUGGACACCAAGGCCUACAGCGAGGGC UACAGCCUGCCUAUCUCUCAGGCCGACGAGAUGGAAAGAUACGUGCGC GAGAACAGCAACCGCGACGAGGAAGUGAACCCCAAGUGGUGGGAGA ACUUCAGCGAGGAAGUCAAAAAGUACUACUUCGUGUUCAUCAGCGGCAG CUUUAAGGGCAAGUUCGAGGAACAGCUGCGGCGGCUGUCUAUGACCAC AGGCGUUAACGGCAGCGCCGUGAACGUGGUCAAUCUGCUGCUGGGCGC Petition 870250091226, dated 06 / 10 / 2025, pages 144 / 224 44 / 100 CGAGAAGAUUAGAAGCGGCGAGAUGACCAUCAGGAACUGGGACGGGC CAUGUUCAACAACAGCGAGUUCAUCCUGAGUAACggcggaggcggcagcgacaa gaaguacucuaucggacuggccaucggcaccaacucuguuggaugggccgugaucaccgacgagu acaaggugcccagcaagaaauucaaagugcuggcaacaccgaccggcacagcaucaagaaau cugaucggcccugcuguucgacucuggcgaaacagccgaagccaccagacugaagaaccgcc agacggcguacaccagaagaaagaaccgcuaccugcaagaauccuucagcaacgagau ggccaagguggacgacagcuucucagagagugaguccucuggugaagagaccacccaucuacuucggagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagagaga ucuggcccuggcucacaugaucaaguuccgggggccaacuuccugaucgagggcgaccugaauccug aaacucccgacguggacaagcuguucaucacgcuggugcagaccuacaaucagcucuggagaag aaucccaucaacgccucuggcguggagcgccaaagccauccugucuggacagaacgcaagacagacggcuggagaaaccugaucgcucagcugcccgggccagaagaagggccuguucggcaaccug uugcccugucucugggccugacaccuaaacuacaaguccaacucgaucugggccgaggaugccaaa cugcagcuguccaaggacaccuacgacgacgaccugguaaaccugcuggcccagaucggcgaucaguacgccgacuuguuucuggccgccaagaaccugucugacgccauccugcugagcgacauccuga gagugaacaccgagaucacaaaggccccucugagcgccucuaugaucaagagauacgacgagcac caccaggaucugacccugcugaaagcucucgucaggcagcagcugccagagaaguacaaagagau uuucuucgaccagagcaagaacggcuacgccggcuacauugauggcggagccagccaagaggaau ucuacaaguucaucaagcccauccucgagaagauggacggcacagaggaacugcucgugaagcug aacagagaggaccugcugcggaagcagcggaccuucgacaauggcucuaucccucaccagaucca ccugggagagcugcacgccauucugcggagacaagaggacuuuuacccauuccugaaggacaacc gggaaaagauugagaagauccugaccuucaggauccccuacuacgugggaccacuggccagaggc aauagcagauucgccuggaugaccagaaagagcgaggaaaccaucacacccuggaacuucgaaga ggugguggacaagggcgccagcgcucaguccuucaucgagcggaugaccaauuucgacaagaauc ugcccaacgaaagugcugcccaagcacucccugcuguacgaguacuucaccguguacaacgag cugaccaaagugaaauacgugaccgagggaaugagaaagcccgccuuucuguccggcgagcagaa aaaggccaucguggaucugcuguucaagaccaaccggaaagugaccgugaagcagcugaaagagg Petition 870250091226, dated 06 / 10 / 2025, pages 145 / 224 45 / 100 acuacuucaagaaaaucgagugcuucgacuccguggaaaucagcggcguggaagaucgguucaau gccagccugggcacauaccacgaucugcugaaaauuaucaaggacaaggacuuccuggacaacga ggaaaacgaggacauccuugaggacaucgugcugacccugacacuguucgaggacagagagauga ucgaggaaaggcugaaaacauacgcccaccuguucgacgacaaagucaugaagcaacugaagcgg cggcgcuacacaggcuggggcagacugucuagaaagcugaucaacggcauccgggacaagcaguc cggcaagaccauccuggacuuucugaaguccgacggcuucgccaacagaaacuucaugcagcuga uucacgacgacagccucaccuucaaagaggacauucagaaggcccagguuuccggccagggcgau ucucugcacgagcacauugccaaucuggccggcucucccgccauuaagaagggcauucugcagac agugaaagugguggaugagcuggucaaagugauggggagacacaagcccgagaacaucgugauc gaaauggccagagagaaccagaccacacagaagggccagaagaacucccgcgagagaaugaagcg gaucgaagagggaaucaaagagcuggggagccagauccugaaagaacaccccguggaaaacaccc agcugcagaacgagaagcuguaccuguacuaccuccagaacggccgggauauguacguggaccaa gagcuggacaucaaccgccugagcgacuacgauguggacgcuaucgugccccagucuuuucugaa agaugacuccaucgacaacaaggugcugaccagaagcgauaagaaccggggcaagagcgacaacg ugcccucugaagaggucgugaagaagaugaagaacuacuggcgacagcugcugaacgccaagcugauuacccagcggaaguucgauaaccugaccaaggccgagagaggcggccugucugaacuggauaa ggccggcuucaucaagagacagcugguggaaacccggcagaucaccaaacacguggcacagauuc uggacucccggaugaacaccaaauacgaugagaacgacaaacugauccgggaagugaaagucauc accugaaguccaagcugguguccgauuuccggaaggauuuccaguucuacaaagugcgggaaau caacaacuaccaucacgcccacgacgccuaccugaaugccguuguuggaacagcccugaucaagaa guaucccaagcuggaaagcgaguucguguacggcgacuacaagguguacgacgugcggaagauga ucgccaagagcgagcaagagauuggaaaggcuaccgccaaauacuucuucuacuccaacaucaug aacuuuuucaagacagagaucacccucgccaacggcgagaucagaaagcggccucugaucgagac aaacggcgaaaccggcgagauugugugggauaagggcagagacuuugccacagugcggaaggug cucagcaugccccaagugaauaucgugaaaaagaccgaggugcagacaggcggcuucagcaaaga guccauucugccuaagcggaacuccgacaagcugaucgcccggaagaaggacugggaccccaaga aauacggcggcuucgauagcccuaccguggccuauucugugcuggugguggccaaaguggaaaag ggaaaguccaagaagcucaagagcgucaaagaacuccugggcaucaccaucauggaacgguccag Petition 870250091226, dated 06 / 10 / 2025, pages 146 / 224 46 / 100 cucgagagaaccuaucgacuucuggagccaagggcuagaagucaaaaccugaaccuga ucaaccuccccaaguecagcugcugcugcuggaaaoggccgagcggcuggcuucugcuggcuacucuacuacuacuauguaaux ggccagccacuaugagaagcugaagggcagccccgaggacaugagagcagcagcuuuucgucg agcagcaagcacuaccuggacgagagauc cuggccgacgccaaccuggaaccucuguccgccuacaagcccaccggcagauag gagcaggccgagaauaucauccaccuguuuacccugaccaccaccugggagccccugccgcuucag uacuucgaccaccaccaccaucgaccgaagcgcuaccagcaccacggcuggacgccacacug cagcccuaagagagagaaagguuccagcuaauaaggcggcgcccucgccccgcggacugccu cccgggggcacccugcauaaaugcgcgcgcgggauucccucgccgcgcgcgcgcgcgcgccg IDaAaaaaaaaaaaaaaaaaaaaaaaaaaaaaa’’aaaaaaaaaaaaaaaaaaaaaaaaaaa’aaaaoaaaaaaoooaaaaaaaaaaaaaasiaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaar’5aaaa)

[00125] As used herein, the term guide sequence or spacer in the context of a Cas-Clover system or a CRISPR-Cas9 system comprises any polynucleotide molecule that has sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct the sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. The guide sequence may comprise RNA and DNA polynucleotides. The guide sequence may form a duplex with a target sequence. The duplex may be a DNA duplex, an RNA duplex, or an RNA / DNA duplex. The terms guide molecule, guide RNA, gRNA, single guide RNA, and sgRNA are used interchangeably herein to refer to RNA-based molecules that are capable of Petition 870250091226, dated 06 / 10 / 2025, pp. 147 / 224 47 / 100 form a complex with a Cas-Clover or CRISPR-Cas protein and comprise a guide sequence with sufficient complementarity to a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of the complex to the target nucleic acid sequence. The guide molecule or guide RNA may comprise RNA-based molecules with one or more chemical modifications (e.g., by chemical linking of two ribonucleotides or by substitution of one or more ribonucleotides for one or more deoxyribonucleotides), as described in this document. The guide sequence may also partially comprise RNA- and DNA-based nucleotides, in which the molecule is chimeric for RNA- and DNA nucleobases (e.g., containing ribose or deoxyribose sugars).

[00126] The Cas-Clover system or the CRISPR / Cas9-based system may include two or more gRNAs, where the gRNAs target different DNA sequences. The target DNA sequences may overlap. The target sequence or protospacer is followed by a PAM sequence at the 3' end of the protospacer. Different CRISPR Type II systems have different PAM requirements. For example, the S. pyogenes Type II system uses an NGG sequence, where N can be any nucleotide.

[00127] The guide RNA or guide RNA of a Cas-Clover protein or a CRISPR-Cas protein may comprise a tracr-mate sequence (encompassing a direct repeat in the context of an endogenous CRISPR system) and a guide sequence (also called a spacer in the context of an endogenous CRISPR system). In some embodiments, the Cas-Clover or CRISPR-Cas system or complex, as described herein, does not comprise and / or does not depend on the presence of a tracr sequence. In certain embodiments, the guide molecule may comprise, consist essentially of, or consist of a fused direct repeat sequence. Petition 870250091226, dated 06 / 10 / 2025, pages 148 / 224 48 / 100 or connected to a guide sequence or spacer sequence.

[00128] In certain embodiments, the guide sequence or spacer length of the guide molecules is 15 to 50 nucleotides long. In certain embodiments, the guide RNA spacer length is at least 15 nucleotides long. In certain embodiments, the spacer length is 15 to 17 nucleotides long, 17 to 20 nucleotides long, 20 to 24 nucleotides long, 23 to 25 nucleotides long, 24 to 27 nucleotides long, 27 to 30 nucleotides long, 30 to 35 nucleotides long, or greater than 35 nucleotides long.

[00129] In some realizations, the guide sequence has 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 nucleotides in length.

[00130] As described above, the Cas-Clover system and the CRISPR / Cas9 system utilize one or more target gRNAs that provide the target for the Cas-Clover system and the CRISPR / Cas9-based system. The gRNA can be a fusion of two non-coding RNAs: a crRNA and a tracrRNA. The sgRNA can target any desired DNA sequence by changing the sequence encoding a 20 bp protospacer, which confers target specificity through complementary base pairing with the desired target DNA. The gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system. This duplex, which may include, for example, a crRNA of Petition 870250091226, dated 06 / 10 / 2025, pp. 149 / 224 Composed of 49 / 100 nucleotides and a 75-nucleotide tracrRNA, it acts as a guide for Cas9 to cleave the target nucleic acid. Expression Vectors and Host Cells

[00131] Disclosure also refers to vectors that include disclosure polynucleotides, host cells that are genetically engineered with recombinant vectors, and the production of at least one protein structure by recombinant techniques, as is well known in the art. See, for example, Sambrook, et al., supra; Ausubel, et al., supra, each incorporated herein by reference.

[00132] Polynucleotides can optionally be attached to a vector containing a selectable marker for propagation into a host. Generally, a plasmid vector is introduced into a precipitate, such as a calcium phosphate precipitate, or into a complex with a loaded lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

[00133] The DNA insert must be operatively ligated to an appropriate promoter. In some embodiments, the promoter is an EF1α promoter. The expression constructs will additionally contain transcription initiation and termination sites and, in the transcript region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiation codon at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for expression in mammalian or eukaryotic cells.

[00134] Expression vectors may include at least one selectable marker. Such markers include, for example, but are not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), Petition 870250091226, dated 06 / 10 / 2025, pages 150 / 224 50 / 100 hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring methotrexate resistance), mycophenolic acid or glutamine synthetase (GS, U.S. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, as well as ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B or tetracycline resistance genes for culture in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein in full by reference). Appropriate culture media and conditions for the host cells described above are known in the art. Suitable vectors will be readily apparent to those skilled in the art.The introduction of a vector construct into a host cell can be effected by transfection with calcium phosphate, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are described in the art, as in Sambrook, supra, Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15 and 16.

[00135] Expression vectors may include at least one selectable cell surface marker for isolating cells modified by disclosure compositions and methods. Selectable disclosure cell surface markers comprise surface proteins, glycoproteins, or protein clusters that distinguish one cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by a disclosure composition or method from those not modified by a disclosure composition or method. Such cell surface markers include, for example, but are not limited to, designation cluster proteins or sorting determinants. Petition 870250091226, dated 06 / 10 / 2025, pp. 151 / 224 51 / 100 (often abbreviated as CD), as a truncated or full-length form of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers additionally include the suicide gene marker RQR8 (Philip B et al. Blood. August 21, 2014; 124(8):1277-87).

[00136] Expression vectors may include at least one drug resistance marker selectable for isolation from cells modified by the disclosure compositions and methods. Disclosure-selectable drug resistance markers may comprise Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2 wild-type or mutant, or any combination thereof.

[00137] Those skilled in the art are aware of the numerous expression systems available for the expression of a nucleic acid encoding a protein of the present disclosure. Alternatively, the nucleic acids of the present disclosure can be expressed in a host cell by activation (by manipulation) in a host cell containing endogenous DNA encoding a protein structure of the present disclosure. Such methods are well known in the art, for example, as described in U.S. Patents Nos. 5,580,734, 5,641,670, 5,733,746 and 5,733,761, which are incorporated herein by reference.

[00138] Cell cultures useful for the production of protein structures, specific portions or variants thereof, are illustrative of bacterial, yeast and mammalian cells, as known in the art. Mammalian cell systems are frequently presented in the form of cell monolayers, although mammalian cell suspensions or bioreactors may also be used. Several suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including COS cell lines. Petition 870250091226, dated 06 / 10 / 2025, pages 152 / 224 52 / 100 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610), and BSC-1 (e.g., ATCC CRL-26), Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8653 cells, SP2 / 0-Ag14 cells, 293 cells, HeLa cells, and similar cells, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2 / O-Ag14 cells (ATCC Accession Number CRL-1851). In a preferred aspect, the recombinant cell is a P3X63Ab8.653 or SP2 / O-Ag14 cell.

[00139] Expression vectors for these cells may include one or more of the following expression control sequences, such as, but not limited to, an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (U.S. Patents Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alpha promoter (U.S. Patent No. 5,266,491), at least one human promoter; an enhancer and / or information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., a large SV40 polyAT Ag addition site), and transcriptional termination sequences. See, for example, Ausubel et al., supra; Sambrook et al., supra.Other cells useful for the production of nucleic acids or proteins of the present disclosure are known and / or available, for example, in the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or in other known or commercial sources.

[00140] When eukaryotic host cells are employed, polyadenylation or transcription terminator sequences are Petition 870250091226, dated 06 / 10 / 2025, pages 153 / 224 53 / 100 are typically incorporated into the vector. An example of a terminator sequence is the polyadenylation sequence of the bovine growth hormone gene. In some embodiments, the polyA sequence is a polyA sequence of SV40.

[00141] Sequences for precise transcript splicing may also be included. An example of a splicing sequence is the SV40 VP1 intron (Sprague et al., J. Virol. 45:773-781 (1983)). In addition, genetic sequences to control replication in the host cell may be incorporated into the vector, as known in the art.

[00142] The plasmid constructs described herein can be used to deliver nucleic acids encoding the transposase domains or fusion proteins described herein to a cell.

[00143] The transposase domains and fusion proteins described herein can also be delivered to a cell using mRNA constructs. Thus, in one embodiment, an mRNA sequence encoding a transposase domain or a fusion protein described herein is provided herein. Such mRNA sequences can be delivered to a cell using a nanoparticle, for example, a lipid nanoparticle. Examples of lipid nanoparticles are described, for example, in International Patent Applications No. WO 2022 / 087148, No. WO 2022 / 182792 and No. WO 2023 / 141576, each of which is incorporated herein by reference in its entirety for examples of lipid nanoparticles that can be used to deliver mRNA constructs encoding the fusion proteins or transposase domains described herein. CELLS AND MODIFIED CELLS

[00144] The transposase-expressing polynucleotides described in this document can be used in conjunction with a Petition 870250091226, dated 06 / 10 / 2025, pp. 154 / 224 54 / 100 transposon for modifying cells. The transposon may be a piggyBac™ (PB) transposon. In some embodiments, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB) transposase. Non-limiting examples of PB transposons are described in detail in U.S. Patent No. 6,218,182; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643 and PCT Publication No. WO 2010 / 099296, the contents of each of which are incorporated herein by reference in their entirety for examples of transposases that may be delivered to cells using the polynucleotides described herein. Transposons can comprise a nucleic acid that codes for a therapeutic protein or a therapeutic agent.Examples of therapeutic proteins include those disclosed in PCT Publications No. WO 2019 / 173636 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety.

[00145] Thus, modified cells comprising one or more transposons and a transposase or fusion proteins encoded by a polynucleotide expressing transposase or fusion proteins described herein are provided in this document. The cells and modified cells of the disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells.

[00146] A cell modified using a polynucleotide expressing transposase described in this document may be a germline cell or a somatic cell. Disclosure cells and modified cells may be immune cells, for example, lymphoid progenitor cells, natural killer (NK) cells, T lymphocytes (T cells), memory T stem cells (Tscm cells), central memory T cells (Tcm cells), stem cell-like T cells, B lymphocytes (B cells), antigen-presenting cells Petition 870250091226, dated 06 / 10 / 2025, pages 155 / 224 55 / 100 antigens (APCs), cytokine-induced killer cells (CIKs), myeloid progenitor cells, neutrophils, basophils, eosinophils, monocytes, macrophages, platelets, erythrocytes, red blood cells (RBCs), megakaryocytes, or osteoclasts. The modified cell can be differentiated, undifferentiated, or immortalized. The undifferentiated modified cell can be a stem cell. The undifferentiated modified cell can be an induced pluripotent stem cell. The modified cell can be a T cell, a hematopoietic stem cell, a natural killer cell, a macrophage, a dendritic cell, a monocyte, a megakaryocyte, or an osteoclast. The modified cell can be modified while quiescent, activated, resting, in interphase, prophase, metaphase, anaphase, or telophase. The modified cell can be fresh, cryopreserved, in bulk, separated into subpopulations, from whole blood, from leukapheresis, or from an immortalized cell line.A detailed description for isolating cells from a leukapheresis product or blood is provided in PCT Publications No. WO 2019 / 173636 and PCT / US2019 / 049816.

[00147] The disclosure methods may modify and / or produce a population of modified T cells, in which at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% or any percentage among the plurality of modified T cells in the population express one or more cell surface marker(s) of a memory T stem cell (Tscm) or a Tscm-like cell; and wherein one or more cell surface marker(s) comprise(s) CD45RA and CD62L. The cell surface markers may comprise one or more Petition 870250091226, dated 06 / 10 / 2025, pages 156 / 224 56 / 100 of CD62L, CD45RA, CD28, CCR7, CD127, CD45RO, CD95, CD95 and IL-2Rp. Cell surface markers may comprise one or more of CD45RA, CD95, IL-2Rp, CCR7 and CD62L.

[00148] This disclosure provides methods for expressing a CAR on the surface of a cell. The method comprises (a) obtaining a cell population; (b) contacting the cell population with a composition comprising a CAR or a sequence encoding the CAR, under conditions sufficient to transfer the CAR across the cell membrane of at least one cell in the cell population, thereby generating a modified cell population; (c) culturing the modified cell population under conditions suitable for integration of the sequence encoding the CAR; and (d) expanding and / or selecting at least one cell from the modified cell population that expresses the CAR on the cell surface. A more detailed description of the methods for expressing a CAR on the surface of a cell is disclosed in PCT Publications No. WO 2019 / 049816 and No. WO 2020 / 051374, each of which is incorporated herein by reference in its entirety.

[00149] This disclosure provides a cell or a population of cells wherein the cell comprises a composition comprising (a) an inducible transgenic construct comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, such as a CAR, wherein, after integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous receptor is expressed, and wherein the exogenous receptor, upon binding to a ligand or antigen, transduces an intracellular signal that directly or indirectly targets the inducible promoter regulating the expression of the inducible transgene (a) to modify gene expression. In some embodiments, the CAR targets Petition 870250091226, dated 06 / 10 / 2025, pages 157 / 224 57 / 100 MUC1C.

[00150] The disclosure further provides a composition comprising the modified, expanded and selected cell population from the methods described herein.

[00151] Modified disclosure cells (e.g., CAR T cells) can be further modified to enhance their therapeutic potential. Alternatively or additionally, the modified cells can be further modified to make them less sensitive to immunological and / or metabolic checkpoints, for example, by blocking and / or diluting specific checkpoint signals delivered to the cells (e.g., checkpoint inhibition) naturally within the immunosuppressive tumor microenvironment.

[00152] Disclosure-modified cells (e.g., CAR T cells) may be further modified to silence or reduce the expression of (i) one or more gene(s) encoding(s) inhibitory checkpoint signal receptor(s); (ii) one or more gene(s) encoding(s) intracellular proteins involved in checkpoint signaling; (iii) one or more gene(s) encoding(s) a transcription factor that hinders the effectiveness of a therapy; (iv) one or more gene(s) encoding(s) a cell death or apoptosis receptor; (v) one or more gene(s) encoding(s) a metabolic detection protein; (vi) one or more gene(s) encoding(s) proteins that confer sensitivity to a cancer therapy, including a monoclonal antibody; and / or (vii) one or more gene(s) encoding(s) a growth advantage factor.Non-limiting examples of genes that can be modified to silence or reduce expression or to repress a function include, but are not limited to, illustrative inhibitory checkpoint signals, intracellular proteins, transcription factors, cell death or apoptosis receptors, metabolic sensing proteins, proteins that... Petition 870250091226, dated 06 / 10 / 2025, pages 158 / 224 58 / 100 confer sensitivity to cancer therapy and growth advantage factors that are disclosed in PCT Publication No. WO 2019 / 173636, which is incorporated herein by reference in its entirety.

[00153] Modified disclosure cells (e.g., CAR T cells) can be further modified to express a modified / chimeric checkpoint receptor. The modified / chimeric checkpoint receptor may comprise a null receptor, a decoy receptor, or a negative dominant receptor. Illustrative examples of null, decoy, or negative dominant intracellular receptors / proteins include, but are not limited to, downstream signaling components of an inhibitory checkpoint signal, a transcription factor, a cytokine or cytokine receptor, a chemokine or chemokine receptor, a cell death or apoptosis receptor / ligand, a metabolic sensor molecule, a protein that confers sensitivity to a cancer therapy, and an oncogene or tumor suppressor gene.Non-limiting examples of cytokines, cytokine receptors, chemokines, and chemokine receptors are disclosed in PCT Publication WO 2019 / 173636, which is incorporated herein by reference in its entirety for examples of receptors and proteins that may be expressed in the cells disclosed herein.

[00154] In some aspects of the present disclosure, the cells of the present disclosure can be modified to decrease the expression of B2M. In some aspects of the present disclosure, the cells of the present disclosure can be modified to decrease the expression of CD3. The expression of B2M and / or CD3 can be reduced using the gene-editing compositions disclosed in this document to target these genes. For example, cells can be modified using one of the CasClover enzymes disclosed in this document in conjunction with gRNAs that target B2M and / or CD3. Petition 870250091226, dated 06 / 10 / 2025, pp. 159 / 224 59 / 100

[00155] Genome modification may involve the introduction of a nucleic acid sequence, a transgene, and / or a genome editing construct into a cell ex vivo, in vivo, in vitro, or in situ to stably integrate a nucleic acid sequence, transiently integrate a nucleic acid sequence, produce site-specific integration of a nucleic acid sequence, or produce biased integration of a nucleic acid sequence. The nucleic acid sequence may be a transgene.

[00156] Stable chromosome integration can be random integration, site-specific integration, or skewed integration. Formulations, Dosages and Methods of Administration

[00157] This disclosure provides formulations, dosages, and methods for administering the compositions and cells described herein. In one aspect, a pharmaceutical composition comprising a polynucleotide for expressing a transposase described herein and a pharmaceutically acceptable carrier is provided herein. In another aspect, a pharmaceutical composition comprising a modified cell described herein and a pharmaceutically acceptable carrier is provided herein.

[00158] The disclosed pharmaceutical compositions and compounds may comprise at least one of any suitable auxiliaries, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Pharmaceutically acceptable auxiliaries are preferred. Non-limiting examples and methods of preparation of such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington's Pharmaceutical Petition 870250091226, dated 06 / 10 / 2025, pp. 160 / 224 60 / 100 Sciences, 18th edition, Mack Publishing Co. (Easton, Pa.) 1990 and in the “Physician's Desk Reference”, 52nd edition, Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable vehicles may be routinely selected that are suitable for the mode of administration, solubility and / or stability of the protein structure, fragment composition or variants, as well known in the art or as described herein.

[00159] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra- and oligosaccharides; derived sugars, such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which may be present individually or in combination, comprising, alone or in combination, from 1% to 99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein and the like. Representative amino acid / protein components, which can also function as buffers, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and similar compounds.A preferred amino acid is glycine.

[00160] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myo-inositol and the like. Preferably, the carbohydrate excipients are mannitol, trehalose and / or raffinose. Petition 870250091226, dated 06 / 10 / 2025, pp. 161 / 224 61 / 100

[00161] The compositions may also include a buffer or pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include salts of organic acids, such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferred buffers are salts of organic acids, such as citrate.

[00162] In addition, the disclosed compositions may include polymeric excipients / additives, such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as TWEEN 20 and TWEEN 80), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[00163] Many known and developed methods can be used to administer therapeutically effective amounts of the compositions or pharmaceutical compositions disclosed in this document. Non-limiting examples of administration methods include bolus, buccal, infusion, intra-articular, intrabronchial, intra-abdominal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal, or vaginal. In preferred embodiments, a composition comprising a Petition 870250091226, dated 06 / 10 / 2025, pages 162 / 224 The 62 / 100 modified cell described in this document is administered intravenously, for example, by intravenous infusion.

[00164] A composition of the present disclosure may be prepared for parenteral use (subcutaneous, intramuscular or intravenous) or any other administration, particularly in the form of liquid solutions or suspensions. For parenteral administration, a composition disclosed herein may be formulated as a solution, suspension, emulsion, particle, powder or lyophilized powder in combination, or supplied separately, with a pharmaceutically acceptable parenteral vehicle. Formulations for parenteral administration may contain as common excipients water or sterile saline solution, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes and the like. Aqueous or oily suspensions for injection may be prepared using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.Agents for injection or infusion may be a non-toxic, non-orally administerable diluent, such as an aqueous solution, a sterile injectable solution, or a suspension in a solvent. Water, Ringer's solution, isotonic saline solution, etc., are permitted as vehicles or solvents; sterile volatile oil may be used as a common solvent or suspension solvent. For these purposes, any type of volatile oil and fatty acid may be used, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or triglycerides. Parenteral administration is known in the art and includes, among others, conventional means of injection, a needleless injection device under gas pressure, as described in U.S. Patent No. 5,851,198, and a laser-piercing device, as described in U.S. Patent No. 5,839,446.

[00165] It may be desirable to deliver the polynucleotides Petition 870250091226, dated 06 / 10 / 2025, pp. 163 / 224 63 / 100 doses are released to the individual over extended periods, for example, from one week to one year following a single administration. Various dosage forms, including slow-release, depot, or implantable forms, can be used.For example, a dosage form may contain a pharmaceutically acceptable non-toxic salt of compounds that have a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid, such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acids, polygalacturonic acid and the like; (b) a salt with a polyvalent metallic cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from, for example, N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), for example, a zinc tannate salt.Furthermore, the disclosed compounds, or preferably a relatively insoluble salt, such as those described above, may be formulated into a gel, for example, an aluminum monostearate gel with, for example, sesame oil, suitable for injection. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of slow-release depot formulation for injection would contain the compound or salt dispersed for encapsulation in a slow-degrading, non-toxic, and non-antigenic polymer, such as a polylactic acid / polyglycolic acid polymer, for example, as described in U.S. Patent No. 3,773,919. The compounds, or preferably relatively insoluble salts, such as those described above, may also be formulated into silastic pellets with a cholesterol matrix, particularly for use in animals.Additional slow-release, depot, or implant formulations, for example, gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed.,). Petition 870250091226, dated 06 / 10 / 2025, pages 164 / 224 64 / 100 Marcel Dekker, Inc., NY, 1978).

[00166] The disclosure provides the use of a disclosed composition or pharmaceutical composition for the treatment of a disease or disorder in a cell, tissue, organ, animal or individual, as known in the art or as described herein, using the disclosed compositions and pharmaceutical compositions, for example, by administering or bringing into contact the cell, tissue, organ, animal or individual with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the individual is a mammal. Preferably, the individual is human. The terms individual and patient are used interchangeably herein.

[00167] In some aspects, the treatment of a disease or disorder comprises adoptive cell therapy. For example, in one aspect, disclosure provides modified cells that express a chimeric antigen receptor (CAR). The modified cells may be allogeneic or autologous to the patient. In some preferred embodiments, the modified cell is an allogeneic cell. In some embodiments, the modified cell is an autologous T cell or a modified autologous CAR T cell. In some preferred embodiments, the modified cell is an allogeneic T lymphocyte or a modified allogeneic CAR T lymphocyte.

[00168] In some embodiments, the disease or disorder treated according to the methods described herein is cancer. Non-limiting examples of cancer include leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), acute myeloid leukemia, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodysplastic syndrome (MDS), lymphoma, Hodgkin's disease, malignant lymphoma, non-Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, sarcoma of Petition 870250091226, dated 06 / 10 / 2025, pages 165 / 224 65 / 100 Kaposi's lymphoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome / hypercalcemia of malignancy, solid tumors, bladder cancer, breast cancer, colorectal cancer, endometrial cancer, head cancer, neck cancer, hereditary nonpolyposis carcinoma, Hodgkin's lymphoma, liver cancer, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, testicular cancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma, metastatic disease, cancer-related bone resorption, cancer-related bone pain, and similar conditions.

[00169] In another non-limiting example, the present disclosure provides methods for treating a metabolic disorder of the liver in an individual, comprising administering to the individual: a) at least a therapeutically effective amount of at least one composition comprising a transposon of the present disclosure comprising a sequence encoding a therapeutic polypeptide; and b) at least a therapeutically effective amount of a composition comprising a nucleic acid sequence encoding at least one transposase. In some respects, the metabolic disorder of the liver may be Ornithine Transcarbamylase (OTC) Deficiency and the at least one therapeutic protein may comprise the ornithine transcarbamylase (OTC) polypeptide. In some respects, the metabolic disorder of the liver may be methylmalonic acidemia (MMA) and the at least one therapeutic protein may comprise a methylmalonyl-CoA mutase (MUT1) polypeptide.

[00170] In a non-limiting example, the present disclosure provides methods for treating a hemophilic disease in an individual, the methods comprising administering to the individual: at least a therapeutically effective amount of at least one composition comprising a transposon of the present disclosure comprising a Petition 870250091226, dated 06 / 10 / 2025, pages 166 / 224 66 / 100 sequence encoding a therapeutic polypeptide; and b) at least a therapeutically effective amount of a composition comprising a nucleic acid sequence encoding at least one transposase. In some aspects, hemophilia disease may be hemophilia A and at least one therapeutic protein may comprise Factor VIII. In some aspects, hemophilia disease may be hemophilia B and at least one therapeutic protein may comprise Factor IX.

[00171] In a non-limiting example, the present disclosure provides methods for treating phenylketonuria (PKU) in an individual, the methods comprising administering to the individual: a) at least a therapeutically effective amount of at least one composition comprising a transposon of the present disclosure comprising a sequence encoding the phenylalanine hydroxylase gene; and b) at least a therapeutically effective amount of a composition comprising a nucleic acid sequence encoding at least one transposase.

[00172] In a non-limiting example, the present disclosure provides methods for treating a disease or disorder in an individual by administering to the individual in need thereof a therapeutically effective amount of an LNP composition comprising a DNA transposon encoding a therapeutic protein and a polynucleotide mRNA expressing transposase encoding a piggyBac transposase, as described herein. In some embodiments, the disease or disorder is cancer, a liver disease or disorder, a urea cycle disorder, a metabolic liver disorder, or a hemophilia disease.

[00173] In a non-limiting example, the present disclosure provides methods for treating a disease or disorder in an individual by administering to the individual in need thereof a therapeutically effective amount of a first LNP composition comprising Petition 870250091226, dated 06 / 10 / 2025, pp. 167 / 224 67 / 100 a DNA transposon encoding a therapeutic protein and a second LNP composition comprising a polynucleotide mRNA expressing a transposase encoding a piggyBac transposase. In some embodiments, the disease or disorder is cancer, a liver disease or disorder, a urea cycle disorder, a metabolic liver disorder, or a hemophilia disease.

[00174] In a non-limiting example, the present disclosure provides methods for treating a disease or disorder in an individual by administering to the individual in need thereof a therapeutically effective amount of a first LNP composition comprising a DNA transposon encoding a therapeutic protein comprising a superminimal ITR and a second LNP composition comprising an mRNA encoding a piggyBac transposase. In some embodiments, the disease or disorder is cancer, a liver disease or disorder, a urea cycle disorder, a metabolic liver disorder, or a hemophilia disease. In some embodiments, the disease or disorder is an autoimmune disease.In one embodiment, autoimmune disease includes autoimmune neutropenia, Guillain-Barré syndrome, epilepsy, autoimmune encephalitis, Isaacs syndrome, nevus syndrome, pemphigus vulgaris, pemphigus decidua, bullous pemphigoid, acquired epidermolysis bullosa, gestational pemphigoid, mucous membrane pemphigoid, antiphospholipid antibody syndrome, autoimmune anemia, myasthenia gravis, autoimmune Graves' disease, thyroid eye disease (TED), Goodpasture syndrome, multiple sclerosis, rheumatoid arthritis, lupus, idiopathic thrombocytopenic purpura (ITP), warm autoimmune hemolytic anemia (WAIHA), chronic inflammatory demyelinating polyneuropathy (CIDP), lupus nephritis, or membranous nephropathy.

[00175] The dosage of a pharmaceutical composition to be administered to an individual may vary depending on known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration. Petition 870250091226, dated 06 / 10 / 2025, pp. 168 / 224 68 / 100 of administration; age, health and weight of the recipient; nature and extent of symptoms, type of concomitant treatment, frequency of treatment and desired effect.

[00176] In aspects where the compositions to be administered to an individual who needs them are modified cells as disclosed in this document, between approximately 1x103 and approximately 1x104 cells; between approximately 1x104 and approximately 1x105 cells; between approximately 1x105 and approximately 1x106 cells; between approximately 1x106 and approximately 1x107 cells; between approximately 1x107 and approximately 1x108 cells; between approximately 1x108 and approximately 1x109 cells; between about 1x10⁹ and about 1x10¹⁰ cells, between about 1x10¹⁰ and about 1x10¹¹ cells, between about 1x10¹¹ and about 1x10¹² cells, between about 1x10¹² and about 1x10¹³ cells, between about 1x10¹³ and about 1x10¹⁴ cells, between about 1x10¹⁴ and about 1x10¹⁵ cells, between about 1x10¹⁵ and about 1x10¹⁶ cells, between about 1x10¹⁶ and about 1x10¹⁷ cells, between about 1x10¹⁷ and about 1x10¹⁸ cells, between about 1x10¹⁸ and about 1x10¹⁹ cells; Between approximately 1x10¹⁹ and approximately 1x10²⁰ cells can be administered.In some embodiments, the cells are administered at a dose of approximately 5x10⁶ to approximately 25x10⁶ cells.

[00177] In other embodiments, the cell dosage may depend on the person's body weight, for example, between about 1x103 and about 1x104 cells; between about 1x104 and about 1x105 cells; between about 1x105 and about 1x106 cells; between about 1x106 and about 1x107 cells; between about 1x107 and about 1x108 cells; between about 1x108 and about 1x109 cells; between approximately 1x10⁹ and approximately 1x10¹⁰ cells, between approximately 1x10¹⁰ and approximately 1x10¹¹ cells, between approximately 1x10¹¹ and approximately 1x10¹² cells, between approximately 1x10¹² and approximately 1x10¹³ cells, between approximately 1x10¹³ and approximately 1x10¹⁴ cells, between approximately 1x10¹⁴ and approximately 1x10¹⁵ cells, between approximately 1x10¹⁵ and approximately 1x10¹⁶ cells, between approximately 1x10¹⁶ and approximately 1x10¹⁷ Petition 870250091226, dated 06 / 10 / 2025, pp. 169 / 224 69 / 100 cells, between approximately 1x1017 and approximately 1x1018 cells, between approximately 1x1018 and approximately 1x1019 cells; or between approximately 1x1019 and approximately 1x1020 cells can be administered per kg of the individual's body weight.

[00178] A more detailed description of the excipients, formulations, dosages and pharmaceutically acceptable methods of administration of the disclosed compositions and pharmaceutical compositions is published in PCT Publication No. WO 2019 / 049816. Kits

[00179] In another aspect, a kit comprising one or more compositions comprising a polynucleotide expressing transposase described in this document and a DNA transposon is provided in the present document. In some embodiments, the one or more compositions comprising a polynucleotide expressing transposase mRNA described in this document and a DNA transposon is an LNP composition. In some embodiments, the one or more compositions comprise a first LNP composition comprising a polynucleotide expressing transposase mRNA described in this document and a second LNP composition comprising a DNA transposon. Definitions

[00180] As used throughout the disclosure, the singular forms “a / an”, “and” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a method includes a plurality of such methods and reference to a dose includes reference to one or more doses and equivalents known to those skilled in the art, and so forth.

[00181] The term about or approximately means within an acceptable range of error for the particular value, as determined by a person skilled in the art, which will depend in part on how the Petition 870250091226, dated 06 / 10 / 2025, pp. 170 / 224 70 / 100 value is measured or determined, for example, the limitations of the measurement system. For example, “about” may mean within 1 or more standard deviations. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with regard to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times, and more preferably within 2 times, of a value. When particular values ​​are described in the application and claims, unless otherwise indicated, the term about should be assumed to mean within an acceptable error range for the particular value.

[00182] Disclosure provides compositions of isolated or substantially purified polynucleotides or proteins. An isolated or purified polynucleotide or protein, or its biologically active moiety, is substantially or essentially free of components that normally accompany or interact with the polynucleotide or protein as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Ideally, an isolated polynucleotide is free of sequences (optimally protein-coding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.For example, in several respects, the isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flanks the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A substantially cellular material-free protein includes protein preparations with... Petition 870250091226, dated 06 / 10 / 2025, pp. 171 / 224 71 / 100 less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of contaminating protein. When the dissemination protein or biologically active portion thereof is produced recombinantly, the ideal culture medium contains less than about 30%, 20%, 10%, 5% or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[00183] The disclosure provides fragments and variants of the disclosed DNA sequences and proteins encoded by those DNA sequences. As used throughout the disclosure, the term fragment refers to a portion of the DNA sequence or a portion of the amino acid sequence and therefore to the protein it encodes. Fragments of a DNA sequence comprising coding sequences may encode protein fragments that retain the biological activity of the native protein and therefore the DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, fragments of a DNA sequence that are useful as hybridization probes generally do not encode proteins that retain biological activity or that do not retain promoter activity.Thus, fragments of a DNA sequence can range from at least about 20 nucleotides, to about 50 nucleotides, to about 100 nucleotides, and up to the full-length polynucleotide of the fragment.

[00184] The nucleic acids or proteins of the disclosure can be constructed by a modular approach, including the pre-assembly of monomer units and / or repeat units into target vectors that can be subsequently assembled into a final target vector. The polypeptides of the present disclosure may comprise repeat monomers of the present disclosure and can be constructed by a modular approach, pre-assembling repeat units into target vectors that can be subsequently assembled into a final target vector. The present disclosure provides the polypeptide Petition 870250091226, dated 06 / 10 / 2025, pp. 172 / 224 72 / 100 produced by this method, as well as nucleic acid sequences encoding these polypeptides. This disclosure provides host organisms and cells comprising nucleic acid sequences encoding the polypeptides produced by this modular approach.

[00185] The term comprising is intended to mean that the compositions and methods include the elements mentioned, but do not exclude others. Consisting essentially of, when used to define compositions and methods, means excluding other elements of any essential significance to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants or inert carriers. Consisting of means excluding more than trace elements from other substantial ingredients and steps of the method. The aspects defined by each of these transitional terms are within the scope of this disclosure.

[00186] As used in this document, expression refers to the process by which polynucleotides are transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, the expression may include mRNA splicing in a eukaryotic cell.

[00187] Gene expression refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by the translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, Petition 870250091226, dated 06 / 10 / 2025, pp. 173 / 224 73 / 100 for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation and glycosylation.

[00188] Modulation or regulation of gene expression refers to a change in the activity of a gene. Modulation of expression may include, but is not limited to, gene activation and repression.

[00189] The term “operatively linked” or its equivalents (e.g., “operatively bound”) means that two or more molecules are positioned relative to each other in such a way that they are capable of interacting to affect a function attributable to one or both molecules or to a combination thereof. In the context of nucleic acids, a promoter may be operatively linked to a nucleotide sequence encoding a transposable domain or fusion protein described in this document, placing the expression of the nucleotide sequence under the control of the promoter.

[00190] Non-covalently linked components and methods for their production and use are disclosed. The various components can assume a variety of different forms, as described in this document. For example, non-covalently linked (i.e., operatively linked) proteins can be used to allow temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows functional association only or mainly in circumstances where such association is necessary for the desired activity. The binding can last long enough to allow the desired effect.

[00191] A method is disclosed for targeting proteins to a specific locus in an organism's genome. The method may comprise the steps of providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the molecule Petition 870250091226, dated 06 / 10 / 2025, pp. 174 / 224 74 / 100 effector molecules are capable of operative bonding via a non-covalent bond.

[00192] A target site or target sequence is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided there are sufficient conditions for binding.

[00193] The terms nucleic acid, oligonucleotide, or polynucleotide refer to at least two covalently linked nucleotides. The representation of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of a single strand shown. A nucleic acid disclosure also encompasses substantially identical nucleic acids and complements thereof that retain the same structure or encode the same protein.

[00194] Disclosure nucleic acids can be single-stranded or double-stranded. Disclosure nucleic acids can contain double-stranded sequences, even when most of the molecule is single-stranded. Disclosure nucleic acids can contain single-stranded sequences, even when most of the molecule is double-stranded. Disclosure nucleic acids can include genomic DNA, cDNA, RNA, or a hybrid thereof. Disclosure nucleic acids can contain combinations of deoxyribonucleotides and ribonucleotides. Disclosure nucleic acids can contain combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Disclosure nucleic acids can be synthesized to comprise non-natural amino acid modifications. Disclosure nucleic acids can be obtained by chemical synthesis methods or by recombinant methods.

[00195] The nucleic acids in the disclosure, whether their complete sequence or any portion thereof, may be of non-natural origin. The nucleic acids in the disclosure may contain one or more mutations, Petition 870250091226, dated 06 / 10 / 2025, pages 175 / 224 75 / 100 substitutions, deletions, or insertions that do not occur naturally, making the entire nucleic acid sequence unnatural. The nucleic acids in this disclosure may contain one or more duplicated, inverted, or repeated sequences, the resulting sequence of which does not occur naturally, making the entire nucleic acid sequence unnatural. The nucleic acids in this disclosure may contain modified, artificial, or synthetic nucleotides that do not occur naturally, making the entire nucleic acid sequence unnatural.

[00196] Given the redundancy in the genetic code, a plurality of nucleotide sequences can code for any particular protein. All such nucleotide sequences are covered in this document.

[00197] As used throughout this disclosure, the term promoter refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing the expression of a nucleic acid in a cell. A promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or alter the spatial and / or temporal expression thereof. A promoter may also comprise distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. A promoter may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals.A promoter can regulate the expression of a gene component constitutively or differentially with respect to the cell, tissue, or organ in which the expression occurs, or with respect to the developmental stage at which the expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Representative examples of promoters include the bacteriophage T7 promoter, the bacteriophage T3 promoter, the SP6 promoter, and the operator. Petition 870250091226, dated 06 / 10 / 2025, pages 176 / 224 76 / 100 promoter lac, the tac promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV IE promoter, the EF-1 Alfa promoter, the CAG promoter, the SV40 early promoter or the SV40 late promoter and the CMV IE promoter.

[00198] As used throughout the disclosure, the term vector refers to a nucleic acid sequence containing an origin of replication. A vector can be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector can be a DNA or RNA vector. A vector can be a self-replicating extrachromosomal vector and, preferably, a DNA plasmid. A vector can comprise a combination of an amino acid with a DNA sequence, an RNA sequence, or a DNA and RNA sequence.

[00199] A conservative amino acid substitution, that is, the substitution of an amino acid by a different amino acid with similar properties (e.g., hydrophilicity, degree and distribution of charged regions), is recognized in the art as typically involving a minor alteration. These small alterations can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on consideration of its hydrophobicity and charge. Amino acids with similar hydropathic indices can be substituted and still retain protein function. In one aspect, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in the retention of the biological function of proteins.Considering the hydrophilicity of amino acids within a peptide allows for the calculation of the highest local average hydrophilicity of that peptide, a useful measure reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated. Petition 870250091226, dated 06 / 10 / 2025, pp. 177 / 224 77 / 100 in full in this document by reference.

[00200] The substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of each amino acid. In line with this observation, it is understood that amino acid substitutions compatible with biological function depend on the relative similarity of the amino acids, and particularly of the side chains of these amino acids, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[00201] As used in this document, “conservative” amino acid substitutions can be defined as set forth in Table 1, Table 2, and Table 3 below. In some respects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions that have been introduced by modification of polynucleotides encoding dissemination polypeptides. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of the protein. A conservative substitution is the replacement of one amino acid with another amino acid that has similar properties. Illustrative conservative substitutions are presented in Table 1. Table 1: Conservative Substitutions I Side chain characteristics: Aliphatic amino acid: Nonpolar; GAPILVF: Polar - uncharged; CSTMNQ: Polar - charged; DEKR: Aromatic; HFWY: Other; NQDE Petition 870250091226, dated 06 / 10 / 2025, pp. 178 / 224 78 / 100

[00202] Alternatively, the conservative amino acids can be grouped as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77) as shown in Table 2. Table 2: Conservative Substitutions II Side Chain Characteristics Amino Acid Nonpolar (hydrophobic) Aliphatic: ALIVP Aromatic: FWY Sulfur-containing: M Borderline: GY Polar uncharged Hydroxyl: STY Amides: NQ Sulfhydryl: C Borderline: GY Positively Charged (Basic): KRH Negatively Charged (Acidic): DE

[00203] Alternatively, illustrative conservative substitutions are shown in Table 3. Table 3: Conservative Substitutions III Original Residue Illustrative Substitution Ala (A) Val Leu Ile Met Arg (R) Lys His Asn (N) Gln Asp (D) Glu Cys (C) SerThr Gln (Q) Asn Glu (E) Asp Gly (G) Ala Val Leu Pro His (H) Arg Ile (I) Leu Met Valu Ala (H) Phe Lys (K) Arg His Met (M) Leu Ile Val Ala Phe (F) Trp Tyr Ile Pro (P) Gly Ala Val Leu Ile Ser(S) Thr Thr (T) Ser Trp (W) Tyr Phe Ile Tyr (Y) Trp Phe Thr Ser Val (V) Ile Leu Met Ala Petition 870250091226, of 06 / 10 / 2025, p. 179 / 224 79 / 100

[00204] Disclosure polypeptides and proteins, whether their entire sequence or any portion thereof, may be of unnatural origin. Disclosure polypeptides and proteins may contain one or more mutations, substitutions, deletions, or insertions that do not occur naturally, making the entire amino acid sequence unnatural. Disclosure polypeptides and proteins may contain one or more duplicated, inverted, or repeated sequences, the resulting sequence of which does not occur naturally, making the entire amino acid sequence unnatural. Disclosure polypeptides and proteins may contain modified, artificial, or synthetic amino acids that do not occur naturally, making the entire amino acid sequence unnatural.

[00205] As used throughout the disclosure, the identity between two sequences can be determined using the stand-alone executable program BLAST for comparison of two sequences (bl2seq), which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site, using the standard parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms identical or identity, when used in the context of two or more nucleic acid or polypeptide sequences, refer to a specified percentage of residues that are the same in a specific region of each of the sequences. In some embodiments, sequence identity is determined along the entire length of a sequence.The percentage can be calculated by optimally aligning the two sequences, comparing them in the specified region, determining the number of positions where the identical residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of identity. Petition 870250091226, dated 06 / 10 / 2025, pages 180 / 224 80 / 100 sequence. In cases where the two sequences have different lengths or the alignment produces one or more staggered ends and the specified comparison region includes only a single sequence, the residues of the single sequence are included in the denominator but not in the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identification can be performed manually or using a computational sequencing algorithm such as BLAST or BLAST 2.0.

[00206] In certain embodiments, if a sequence has a certain sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) with a given SEQ ID NO, the sequence and the sequence of the SEQ ID NO have the same length. In certain embodiments, if a sequence has a certain sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) with a given SEQ ID NO, the sequence and the sequence of the SEQ ID NO differ only due to conservative amino acid substitutions.

[00207] As used throughout the disclosure, the term endogenous refers to the nucleic acid or protein sequence naturally associated with a target gene or a host cell into which it is introduced.

[00208] As used throughout the disclosure, the term exogenous refers to a nucleic acid or protein sequence not naturally associated with a target gene or a host cell into which it is introduced, including unnaturally occurring multiple copies of a naturally occurring nucleic acid, for example, a DNA sequence, or a naturally occurring nucleic acid sequence located at an unnaturally occurring genomic locus.

[00209] The disclosure provides methods for introducing a polynucleotide construct comprising a DNA sequence into a Petition 870250091226, dated 06 / 10 / 2025, pp. 181 / 224 81 / 100 host cell. By introduction is meant presenting the polynucleotide construct to the cell in such a way that the construct has access to the interior of the host cell. Disclosure methods do not depend on a particular method for introducing a polynucleotide construct into a host cell, but only that the polynucleotide construct has access to the interior of a host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[00210] All documents cited in this document, including any patent or application referenced or related, are incorporated herein by reference in their entirety for all purposes, unless expressly excluded or otherwise limited. Citation of any document is not an admission that it constitutes prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Furthermore, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition ascribed to that term in this document shall prevail. Examples

[00211] The examples in this section are provided for illustrative purposes only and are not intended to limit the invention. Example 1: Effect of 3'UTR Composition on PiggyBac Transposase Expression and Excision Activity In Vitro in Hepatocytes

[00212] The effect of 3'-UTR composition on piggyBac transposase expression and excision activity was examined by constructing and Petition 870250091226, dated 06 / 10 / 2025, pages 182 / 224 82 / 100 Comparison of two polynucleotides encoding piggyBac transposase comprising different 3'-UTR sequences. The first polynucleotide encoding piggyBac transposase comprised, in the 5' to 3' direction: a 5'UTR of HBB, an NLS coding sequence, a piggyBac transposase coding sequence comprising four hyperactive mutations at amino acids I30V; G165S; M282V and N538K, a 3'-UTR of HBB and a polyA tail. The second polynucleotide encoding piggyBac transposase comprised, in the 5' to 3' direction: a 5'-UTR of HBB, an NLS coding sequence, a piggyBac transposase coding sequence comprising four hyperactive mutations at amino acids I30V; G165S; M282V and N538K, a 3'-UTR comprising a CYBA 3'-UTR element in tandem joined by a linker string and a pulley tail (FIGURE 3A).

[00213] The excision activity of the two polynucleotides encoding transposase comprising a tandem HBB 3'-UTR or CYBA 3'-UTR element was determined using an episomal excision reporter plasmid (FIGURE 2). Briefly, the episomal excision reporter plasmid comprises an EF-1a promoter controlling the expression of a GFP gene followed by a polyA sequence. The GFP coding sequence was disrupted by the insertion of a minitransposon, which prevents the expression of functional GFP. If a transposase exhibits excision activity, excision of the minitransposon inserted by the transposase restores the full-length GFP coding sequence, resulting in intracellular GFP expression. A catalytically killed piggyBac transposase, without any appreciable transposase excision activity, was used as a negative control. On Day 0, 75,000 HepG2 cells were seeded into 48-well plates.On Day 1, 125 ng of mRNA encoding the transposase (e.g., HBB 3'-UTR or 2X-CYBA 3'-UTR) and 375 ng of donor plasmid. Petition 870250091226, dated 06 / 10 / 2025, pages 183 / 224 83 / 100 of transposon-interrupted GFP cells were introduced into each well of a 48-well plate, and the cells were cotransfected with Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's instructions. GFP expression was monitored over a 48-hour time course using Incucyte. The results are shown in FIGURE 3B.

[00214] As shown in FIGURE 3B, polynucleotides encoding the piggyBac transposase, comprising the 3'-UTR of HBB or the 3'UTR of 2X CYBA, were able to express the piggyBac transposase-encoded mRNA in cells and excise the disruptive reporter minitransposon. Minitransposon excision restores a full-length GFP gene, resulting in intracellular GFP expression. Polynucleotides encoding the transposase, comprising the 3'-UTR of 2X CYBA, demonstrated approximately two-fold enhanced GFP expression in the HepG2 hepatocyte cell line compared to polynucleotides encoding the transposase, comprising the 3'-UTR of HBB. Cells transfected with polynucleotides encoding the catalytically killed piggyBac transposase negative control exhibited little or no GFP expression. Example 2: Effect of 3'UTR Composition on In Vivo Expression and EXCISION Activity of PIGGYBAC TRANSPOSASE IN YOUNG MICE

[00215] In a second experiment, polynucleotides encoding the transposase, comprising the 3'-UTR of 2X CYBA, demonstrated enhanced serum levels of Factor VIII (FVIII) in young mice, compared to polynucleotides encoding the transposase, comprising the 3'-UTR of HBB.

[00216] On Day 1, juvenile wild-type BALBc mice (N = 4 / group) received a single intravenous injection of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12-200; 41.84% DOPE; Petition 870250091226, dated 06 / 10 / 2025, pages 184 / 224 84 / 100 20% CHOL; and 3.16% DM-PEG) encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a specific liver promoter; or co-administered a single dose of 0.25 mg / kg of the Factor VIII LNP composition and a dose of 0.35 mg / kg of an LNP composition (C12-D15 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating an mRNA transposase-encoding polynucleotide comprising an HBB 3'-UTR or a 2X CYBA 3'-UTR (or an mRNA transposase-encoding polynucleotide comprising a nucleic acid encoding a catalytically killed transposase as a negative control). On Day 6, serum Factor VIII levels were determined from blood samples of treated and control animals using a Factor VIII ELISA. The results are shown in FIGURE 4.

[00217] As shown in FIGURE 4, the transposase-encoding polynucleotides comprising the 3'-UTR of 2X CYBA conferred a 42% increase in serum Factor VIII levels in young mice compared to the transposase-encoding polynucleotides comprising the 3'-UTR of HBB. Example 3: Effect of 3'-UTR Composition on In Vivo Expression and Excision Activity of PiggyBac Transposase in Adult Mice

[00218] In a related experiment, the polynucleotides encoding the 3'-UTR of 2X CYBA transposase demonstrated enhanced serum levels of Factor VIII (FVIII) in adult mice compared to the polynucleotides encoding the 3'-UTR of HBB transposase.

[00219] On Day 1, adult wild-type C57BL / 6 mice (N = 4 / group) received a single intravenous injection of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12-200; 41.84% of Petition 870250091226, dated 06 / 10 / 2025, pages 185 / 224 85 / 100 DOPE; 20% CHOL; and 3.16% DM-PEG) encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a liver-specific promoter; or co-administered a single dose of 0.25 mg / kg of the Factor VIII LNP composition and a dose of 0.5 mg / kg of an LNP composition (C12-D15 - 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating an mRNA transposase-encoding polynucleotide comprising a 3'UTR of HBB or a 3'-UTR of 2X CYBA. On Day 6, serum Factor VIII levels were determined from blood samples collected from treated and control animals using a Factor VIII ELISA. The results are shown in FIGURE 5.

[00220] As shown in FIGURE 5, the polynucleotide encoding the 3'-UTR-comprising transposase of 2X CYBA conferred a 38% increase in serum Factor VIII levels in adult mice compared to the polynucleotide encoding the 3'-UTR-comprising transposase of HBB. Example 4: Effect of Adding miR-142-3p Binding Sites to the 3'-UTR of PIGGYBAC TRANSPOSASE POLYNUCLEOTIDES on IN VITRO EXPRESSION and Excision Activity in Hepatocytes

[00221] The effect of adding miR-142-3p binding sites to the 3'UTR in piggyBac transposase mRNA on in vitro expression and excision activity in hepatocytes was examined using HepG2 cells.

[00222] In summary, on Day 0, 75,000 HepG2 cells were seeded into 48-well plates. On Day 1, 125 ng of mRNA encoding the transposase containing or not four miR-142-3p binding sites in the 3'-UTR and 375 ng of the transposon-disrupted GFP donor plasmid described in Example 1 were introduced into each well of the 48-well plate, and the cells were co-transfected with Lipofectamine 3000 (Thermo Fisher) reagent. Petition 870250091226, dated 06 / 10 / 2025, pages 186 / 224 86 / 100 according to the manufacturer's instructions. GFP expression was monitored for a period of 40 hours using Incucyte. The results are shown in FIGURE 6.

[00223] As shown in FIGURE 6, the addition of four miR-142-3p binding sites separated by 3'-UTR linker sequences to the piggyBac transposase had no observable effect on the excision activity of the encoded transposase over a 40-hour period, compared to 3'-UTR without the four miR-142-3p binding sites. Example 5: Addition of miR-142-3p binding sites to the 3'-UTR of an mRNA encoding GFP decreases GFP expression in hematopoietic cells.

[00224] The effect of adding miR-142-3p binding sites to the 3'UTR of an mRNA encoding GFP was examined by constructing and comparing two DNA plasmids comprising a GFP gene encoding mRNAs comprising or not comprising four miR-142-3p binding sites within the 3'-UTR. The first mRNA encoding GFP comprises the miR-142-3p binding sites and the second mRNA encoding GFP does not possess the miR-142-3p binding sites.

[00225] On Day 0, approximately 250,000 K562 cells were nucleofected using 20 µl of SF buffer and FF-120 program. Each reaction contained 830 ng of mRNAs encoding GFP, either including or not including four miR-142-3p binding sites in the 3'-UTR. GFP expression in K562 cells transfected with each mRNA was monitored for 18 hours using Incucyte. The results are shown in FIGURE 7.

[00226] As shown in FIGURE 7, GFP expression in K562 cells nucleofected with mRNA encoding GFP without the four miR-142-3p binding sites is detected approximately four hours after nucleofection and reaches a peak expression level approximately 16 hours after transfection. In contrast, GFP expression in transfected K562 cells Petition 870250091226, dated 06 / 10 / 2025, pp. 187 / 224 The 87 / 100 GFP-encoding mRNA, comprising the four miR-142-3p binding sites, remains essentially undetectable during the 18-hour monitoring period after nucleofection, resulting in a 99% inhibition of GFP expression in hematopoietic cells. Example 6: Addition of miR-142-3p Binding Sites to the 3'-UTR in PIGGYBAC TRANSPOSASE POLYNUCLEOTIDES DECREASES THE EXPRESSION AND EXCESSIVE ACTIVITY OF PIGGYBAC TRANSPOSASE IN HEMATOPOIETIC CELLS

[00227] The effect of adding miR-142-3p binding sites to the 3'UTR in piggyBac transposase mRNA on in vitro expression and excision activity in hematopoietic cells was analyzed.

[00228] Two mRNAs encoding a piggyBac transposase comprising or not four miR-142-3p binding sites with the 3'UTR were constructed, and the transposon excision activity of the encoded transposases was determined in K562 hematopoietic cells using the episomal excision reporter plasmid system described in FIGURE 2.

[00229] On Day 0, approximately 250,000 K562 cells were nucleofected using 20 µl of SF buffer and FF-120 program. Each reaction contained 208 ng of mRNAs encoding the piggyBac transposase, with or without four miR-142-3p binding sites within the 3'-UTR, and 400 ng of the transposon-disrupted GFP donor plasmid described in Example 1. GFP expression in nucleofected K562 cells was monitored for 42 hours using Incucyte. The results are shown in FIGURE 8.

[00230] As shown in FIGURE 8, transposase-mediated GFP reporter expression in K562 cells transfected with mRNA encoding piggyBac transposase without the four miR-142-3p binding sites is detected approximately twelve hours after transfection and increases linearly. Petition 870250091226, dated 06 / 10 / 2025, pp. 188 / 224 88 / 100 up to a peak expression level at the end of the 42-hour time course post-transfection. In contrast, transposase-mediated GFP reporter expression in K562 cells nucleofetated with mRNA encoding piggyBac transposase, comprising the four miR-142-3p binding sites, remains essentially undetectable until approximately 18 hours post-transfection monitoring and exhibited a 79% inhibition of piggyBac transposase excision activity in hematopoietic cells at the end of the 42-hour monitoring period. Example 7: Effect of 3'-UTR Composition on In Vivo Expression of PiggyBac Transposase, Excision Activity, and T-Cell Reactivity in Young Mice

[00231] The effect of adding miR-142-3p binding sites to the 3'UTR of an mRNA encoding piggyBac transposase was examined in vivo by constructing and comparing two mRNAs encoding a piggyBac transposase with an HBB 3'-UTR or a 2X CYBA 3'-UTR (illustrated in Figure 3A) comprising or not four miR-142-3p binding sites in the 3'-UTR.

[00232] On Day 1, juvenile wild-type BALBc mice (N = 4 / group) received by intravenous injection a single dose of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12-200; 41.84% DOPE; 20% CHOL; and 3.16% DM-PEG) encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a liver-specific promoter; or co-administered a single dose of 0.25 mg / kg of the Factor VIII LNP composition and a dose of 0.35 mg / kg of an LNP composition (C12-D15 - 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating a polynucleotide encoding the piggyBac mRNA transposase comprising an HBB 3'-UTR with or without four miR-142-3p binding sites or a polynucleotide encoding the piggyBac mRNA transposase comprising Petition 870250091226, dated 06 / 10 / 2025, pp. 189 / 224 89 / 100 a 3'-UTR of 2X CYBA comprising or not four miR142-3p binding sites (or an mRNA transposase-encoding polynucleotide comprising a nucleic acid encoding a catalytically killed transposase as a negative control). On Day 6, serum Factor VIII levels were determined from blood samples of untreated, treated, and control animals using a Factor VIII ELISA. The results are shown in FIGURE 9. As shown in FIGURE 9, the addition of the four miR-142-3p binding sites to mRNA transposase-encoding polynucleotides comprising an HBB 3'-UTR or a 2X CYBA 3'-UTR did not exhibit an effect on serum Factor VIII levels in young mice compared to mRNA transposase-encoding polynucleotides lacking the four miR-142-3p binding sites.

[00233] In a second experiment, the effect of adding miR-142-3p binding sites to the 3'-UTR of an mRNA encoding piggyBac transposase on the number of piggyBac transposase-reactive T cells was examined in vivo by constructing and comparing mRNAs encoding a piggyBac transposase with an HBB 3'-UTR or a 2X CYBA 3'-UTR (illustrated in Figure 3A) comprising or not four miR142-3p binding sites in the 3'-UTR.

[00234] On Day 1, juvenile wild-type BALBc mice (N = 4 / group) received by intravenous injection a single dose of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12-200; 41.84% DOPE; 20% CHOL; and 3.16% DM-PEG) encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a liver-specific promoter; or co-administered a single dose of 0.25 mg / kg of the Factor VIII LNP composition and a dose of 0.35 mg / kg of an LNP composition (C12-D15 - 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating a polynucleotide Petition 870250091226, dated 06 / 10 / 2025, pages 190 / 224 90 / 100 encoding the mRNA piggyBac transposase comprising an HBB 3'-UTR with or without four miR-142-3p binding sites or a polynucleotide encoding the mRNA piggyBac transposase comprising a 2X CYBA 3'-UTR with or without four miR142-3p binding sites (or a polynucleotide encoding the mRNA transposase comprising a nucleic acid encoding a catalytically killed transposase as a negative control).

[00235] On Day 28, treated and control mice were euthanized, their spleens were extracted, splenocytes were isolated, and the number of piggyBac transposase-reactive T cells (IFN gamma / 10e6 splenocytes positive T cells) was calculated using an ELISpot assay in treated and control animals. The results are shown in FIGURE 10.

[00236] As shown in FIGURE 10, the addition of the four miR-142-3p binding sites to the mRNA transposase-encoding polynucleotides, comprising a 3'UTR of HBB or a 3'-UTR of 2X CYBA, resulted in a decrease in the number of IFN-gamma-positive T cells in young mice, compared with the corresponding mRNA transposase-encoding polynucleotides without the four miR-142-3p binding sites. Example 8: Effect of 3'UTR Composition on T Cell Reactivity after Repeated Administration of PiggyBac Transposase Polynucleotides in Adult Mice

[00237] The effect of adding miR-142-3p binding sites to the 3'UTR of an mRNA encoding piggyBac transposase on the number of piggyBac transposase-reactive T cells after single or repeated administration was examined in vivo by constructing and comparing mRNAs encoding a piggyBac transposase with a 2X CYBA 3'-UTR (illustrated in Figure 3A) comprising or not four miR-142-3p binding sites in the 3'-UTR. Petition 870250091226, dated 06 / 10 / 2025, pp. 191 / 224 91 / 100

[00238] On Day 1, adult wild-type C57BL / 6 mice (N= 5 / group) were co-administered by intravenous injection a single dose of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12200; 41.84% DOPE; 20% CHOL; and 3.16% DM-PEG) encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a liver-specific promoter and a dose of 0.5 mg / kg of an LNP composition (C12-D15 - 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating: a polynucleotide encoding the piggyBac mRNA transposase comprising a 3'-UTR of HBB; a polynucleotide encoding the mRNA piggyBac transposase comprising a 2X CYBA 3'-UTR in tandem or a polynucleotide encoding the mRNA piggyBac transposase comprising a 2X CYBA 3'-UTR and comprising four miR-142-3p binding sites.

[00239] In addition, on Day 1, a group of mice received a dose of 0.25 mg / kg of an LNP composition encapsulating a DNA transposon comprising a nucleic acid encoding a Factor VIII gene modified under the control of a liver-specific promoter and a dose of 0.5 mg / kg of an LNP composition encapsulating: an mRNA polynucleotide encoding piggyBac transposase comprising an HBB 3'-UTR; an mRNA polynucleotide encoding piggyBac transposase comprising a tandem 2X CYBA 3'-UTR; or an mRNA polynucleotide encoding piggyBac transposase comprising a 2X CYBA 3'-UTR and comprising four miR-1423p binding sites.On Day 7, the treated mice received a further dose by co-administration of a 0.15 mg / kg dose of an LNP composition encapsulating a DNA transposon and a 0.5 mg / kg dose of the LNP composition encapsulating the same polynucleotide encoding the mRNA piggyBac transposase, and on Day 14, the treated mice received a... Petition 870250091226, dated 06 / 10 / 2025, pages 192 / 224 92 / 100 third dose of 0.5 mg / kg of the LNP composition encapsulating the same polynucleotide that encodes the piggyBac transposase of the mRNA.

[00240] On Day 21, treated and control mice were euthanized, their spleens were removed, splenocytes were isolated, and the number of piggyBac transposase-reactive T cells (IFN gamma / 10e6 splenocytes positive T cells) was calculated using an ELISpot assay in treated and control animals. The results are shown in FIGURE 11.

[00241] As shown in FIGURE 11, the addition of the four miR-142-3p binding sites to the mRNA transposase-encoding polynucleotides resulted in a substantial reduction in the number of IFN-gamma-positive T cells after repeated administration in adult mice, compared with repeated administration of the mRNA transposase-encoding polynucleotides without the four miR-142-3p binding sites. Example 9: Effect of the Number of Hyperactive Mutations and ITR Composition on the Integration and Excision Activity of the Polynucleotide Transposase PiggyBac

[00242] The integration and excision activities of piggyBac transposases encoding a piggyBac transposase comprising four hyperactive mutations (I30V; G165S; M282V and N538K) or five hyperactive mutations (I30V; G165S; M226F, M282V and N538K) were determined using transposons comprising left-end (LE) and right-end (RE) wild-type piggyBac ITRs or a RE wild-type piggyBac ITR and a LE piggyBac ITR comprising a 35TCC mutation. The reporter systems used to test integration or excision are shown in FIGURES 12 to 14. FIGURE 12 shows a schematic of the assays and FIGURES 13 and 14 show the vector map of the plasmids used.

[00243] In one case, the polynucleotides that code for Petition 870250091226, dated 06 / 10 / 2025, pages 193 / 224 93 / 100 piggyBac transposases, encoding a piggyBac transposase comprising four or five hyperactive mutations, were nucleofected into K562 cells, and the cells were cotransfected with a double excision / integration luciferase reporter vector (FIGURE 14), comprising wild-type piggyBac LE and RE ITRs or a wild-type piggyBac RE ITR and a piggyBac LE ITR comprising a 35TCC mutation. The vector was designed such that an open reading frame of firefly luciferase is disrupted by an SPB transposon. Initially, firefly luciferase is not expressed, but SPB-mediated transposon excision and ongoing repair result in expression. The transposon itself expresses a destabilized nanoluc luciferase mRNA. Nanoluc expression from the episomal vector is unstable because the mRNA lacks a polyA tail and contains a 3' destabilizing element.The integration of the transposon into genomic DNA allows the mRNA to capture a polyA and perform splice of the destabilizing element using a splice donor sequence in the transposon, leading to the expression of luciferase.

[00244] K562 cells were nucleofected using 20 µl of SF buffer and FF-120 program. Each reaction contained 50 ng of the dual luciferase reporter and 500 ng of a plasmid expressing the transposase. One day after transfection, the luciferase signal was measured using Promega's dual luciferase reagents and a plate reader. The results are shown in FIGURE 15A.

[00245] As shown in Figure 15A, the addition of the hyperactive M226F mutation to the piggyBac transposase sequence resulted in an approximately 50% enhancement in integration activity for transposons comprising wild-type ITRs, while both hyperactive mutant piggyBac transposases exhibit enhanced integration activity for transposons comprising the LE 35TCC ITR. The addition Petition 870250091226, dated 06 / 10 / 2025, pages 194 / 224 94 / 100 of the hyperactive 5mmutation M226F and the LE 35TCC ITR resulted in a twofold intensification of integration activity. The addition of the hyperactive 5mmutation to the piggyBac transposase sequence intensified excision activity for transposons comprising wild-type ITRs or the LE 35TCC ITR compared to only 4 hyperactive mutations, and both hyperactive transposases exhibited intensified excision activity for transposons comprising the LE 35TCC ITR, although no further increase in excision activity was observed for the transposase comprising the M226F mutation.

[00246] In another case, polynucleotides encoding piggyBac transposase, which encode a piggyBac transposase comprising four or five hyperactive mutations, were transfected into 293T cells, and the cells were co-transfected with a double excision / integration H2Kk / GFP reporter vector (FIGURE 13), comprising wild-type piggyBac LE and RE ITRs or a wild-type piggyBac RE ITR and a piggyBac LE ITR comprising a 35TCC mutation. The vector was designed so that an open reading frame of H2Kk is interrupted by an SPB transposon. Initially, H2Kk is not expressed, but SPB-mediated transposon excision and ongoing repair result in expression. H2Kk is a cell surface protein, and its expression can be detected on the cell surface using a fluorescent anti-H2Kk antibody. The transposon itself expresses a destabilized GFP mRNA.GFP expression from the episomal vector is unstable because the mRNA lacks a polyA tail and contains a 3' destabilizing element. Integration of the transposon into genomic DNA allows the mRNA to acquire a polyA and splice the destabilizing element using a splice donor sequence in the transposon, leading to GFP expression.

[00247] On Day 0, 120,000 HEK293T cells were seeded. Petition 870250091226, dated 06 / 10 / 2025, pages 195 / 224 95 / 100 in 24-well plates. On Day 1, 10 ng of plasmid encoding the transposase (e.g., a piggyBac transposase comprising 4 or 5 hyperactive mutants) and 490 ng of the H2Kk / GFP dual reporter plasmid were introduced into specific wells of the 24-well plate, and cells were transfected using JetPrime (Polyplus) reagent according to the manufacturer's instructions. On Day 2, the transfected cells were retransferred, and the remaining cells were stained for H2Kk expression and analyzed by flow cytometry to determine the percentage of H2Kk-positive cells. Cells were retransferred again on Day 5. On Day 8, cells were analyzed by flow cytometry to determine the percentage of GFP-positive cells. The results are shown in FIGURE 15B.

[00248] As shown in FIGURE 15B, the addition of the hyperactive 5mmutation M226F showed little or no benefit to integration activity compared to the hyperactive transposase comprising only 4 hyperactive mutations; however, the addition of the hyperactive 5mmutation M226F to the piggyBac transposase sequence resulted in an approximately 50% enhancement in excision activity for transposons comprising wild-type ITRs, while both hyperactive mutant piggyBac transposases showed enhanced excision activity for transposons comprising the LE 35TCC ITR. The addition of the hyperactive 5mmutation M226F and the LE 35TCC ITR resulted in a 2.5-fold enhancement in excision activity. Example 10: Enhanced PiggyBac Transposase Polynucleotides and LE 35TCC ITR Enhance Transposase Integration and Excision Activity in Young Mice

[00249] A dual excision / integration luciferase reporter system was used to test wild-type ITRs and the effect of LE 35TCC ITR on piggyBac transposase activity, comprising 5 Petition 870250091226, dated 06 / 10 / 2025, pages 196 / 224 96 / 100 hyperactive mutations and a 3'-UTR comprising a 2X CYBA 3'-UTR tandem sequence and four miR-142-3p binding sites or 4 hyperactive mutations and a 3'-UTR of HBB transposons engineered for in vivo integration or excision. The reporter system comprises an open reading frame of firefly luciferase interrupted by a piggyBac transposon. Initially, firefly luciferase is not expressed, but transposase-mediated transposon excision and ongoing repair result in expression. The transposon itself expresses a destabilized nanoluc luciferase mRNA. NanoLuc expression from the episomal vector is unstable since the mRNA lacks a polyA tail and contains a 3' destabilizing element. The integration of the transposon into genomic DNA allows the mRNA to utilize a genomic polyA sequence and splice the destabilizing element using a splice donor sequence in the transposon, leading to luciferase expression.

[00250] On Day 1, young wild-type BALBc mice (N = 3 / group) were co-administered by intravenous injection a single dose of 0.25 mg / kg of an LNP composition (C12-D8 - 35% C12-200; 41.84% DOPE; 20% CHOL; and 3.16% DM-PEG) encapsulating the DNA double integration / excision reporter comprising a transposon containing wild-type LE and ER ITRs or a transposon containing a wild-type LE 35TCC ITR and an ER ITR and a dose of 0.5 mg / kg of an LNP composition (C12-D15 - 33.5% C12-200; 33.5% DOPE; 32% CHOL; and 1% DM-PEG) encapsulating: a polynucleotide encoding mRNA transposase piggyBac comprising 4 hyperactive mutations and a 3'-UTR of HBB or a polynucleotide encoding the transposase piggyBac mRNA, comprising 5 hyperactive mutations and a 3'-UTR comprising a 2X CYBA 3'-UTR in tandem and four miR-142-3p binding sites. As a negative control, LNP compositions were administered, Petition 870250091226, dated 06 / 10 / 2025, pp. 197 / 224 97 / 100 as described above, containing an mRNA encoding a catalytically killed version of the piggyBac transposase.

[00251] Luciferase activity was measured on Days 1, 2, 3, 7, 14, and 21 for treated and control animals, and the total flux observed for each day and construct, as shown in Figure 16. As shown in Figure 16, each polynucleotide of the piggyBac transposase was able to express transposase and, in vivo, to integrate and excise transposons comprising wild-type ITRs or LE 35TCC ITRs in young mice, although a slight enhancement of integration activity in vivo was observed for the piggyBac transposase comprising 5 hyperactive mutations and a 3'UTR comprising 2X CYBA 3'-UTR elements in tandem and four miR-142-3p binding sites. Example 11: Illustrative Method for the Preparation of PiggyBac Transposase 5'CleanCap mRNA for Use in LNP Compositions

[00252] This Example provides an illustrative method for preparing 5'CleanCap mRNA encoding an SPB transposase comprising a 5'hemagglutinin tag. The method can be used to prepare 5'CleanCap mRNA encoding additional piggyBac transposases.

[00253] The DNA plasmid pRT-HA-SPB-CC-AG encodes the Super piggyBac transposase comprising a 5'-hemagglutinin tag corresponding to amino acids 98-106 (“HA-SPB”). This plasmid was used as a template for in vitro transcription reactions to produce mRNA encoding HA-SPB additionally comprising a 5'-CAP.

[00254] Briefly, approximately 10 µg of supercoiled pRT-HASPB-CC-AG were added to a 1.5 ml Eppendorf tube containing CutSmart 1X buffer, 200 units of the restriction enzyme SpeI (New England Biolabs, Cat # R3133l) in a total volume of 100 µl. The plasmid DNA was linearized by incubation at 37°C overnight to ensure the Petition 870250091226, dated 06 / 10 / 2025, pp. 198 / 224 98 / 100 complete digestion.

[00255] The linearized plasmid was purified using a QIAquick DNA PCR purification kit (Qiagen, Cat. # 28104), according to the manufacturer's instructions, eluting the purified DNA in 40 μL of nuclease-free water. The DNA concentration of the eluate was determined using a NanoDrop microvolume spectrophotometer (ThermoFisher), according to the manufacturer's instructions.

[00256] The purified plasmid was used as a DNA template for mRNA production using the mMESSAGE mMACHINE T7 in vitro transcription kit (ThermoFisher, Cat. # AM1344), according to the manufacturer's instructions. Briefly, 100 mM stocks of the nucleotides GTP, ATP, CTP, and UTP were prepared, and 15 μl of each stock (2X final) were transferred to an Eppendorf tube containing 2X (12 μl of CleanCap® AG Reagent (m7G(5')ppp(5')(2'OMeA)pG, TriLink, Cat # N-7113) in a total volume of 100 μl.

[00257] Approximately 1.67 μg of linear DNA pRT-HA-SPBCC-AG, 20 μl of 10X T7 transcription buffer, and 20 μl of T7 RNA polymerase mixture were added to a 1.5 ml Eppendorf tube (final volume 200 μl), and the tube was incubated at 37°C for 3 hours. A 10 μl aliquot of TURBO DNase enzyme (ThermoFisher) was added, and the tube was incubated at 37°C for 15 minutes to degrade the DNA template.

[00258] A poly(A) tail was added to the 3' end of the 5'-CleanCap®-HA-SPB mRNA using a poly(A) tail kit (ThermoFisher, cat # 1350M). Briefly, the digested DNA, mRNA solution (210 μl, 100 μl of 25 mM ATP, 200 μl of 5X-E-PAP buffer, 100 μl of 25 mM MnCl2, 40 μl of E. coli Poly(A) Polymerase (E-PAP) and 350 μl of nuclease-free water (total volume 1 ml)) were combined and the reaction was allowed to proceed at 37°C for 1 h. Petition 870250091226, dated 06 / 10 / 2025, pp. 199 / 224 99 / 100

[00259] The 5'-CleanCap®-HA-SPB-poly(A) mRNA was purified using an RNeasy Midi purification kit (Qiagen, Cat # 75144) according to the manufacturer's instructions. Briefly, a 3.5 ml RLT Buffer solution was freshly prepared using 35 µl of 2-mercaptoethanol and combined with 2.5 ml of 100% ethanol, and the final mRNA product was eluted from the column using 300 µl of nuclease-free water. The average mRNA yield from this process is approximately 600–800 pg. Example 12: Enhanced PiggyBac Transposase Polynucleotides and 35TCC RE ITR Enhance Fold Expansion and Efficiency Transposition of Genetically Edited Cells During CAR-T Cell Fabrication

[00260] The effect of mRNA encoding piggyBac transposase comprising four hyperactive molecules was tested on the in vitro production of CAR-T cells. The results demonstrated an enhancement in the expansion of CAR-T cell folds and in the percentage yield of genetically edited T cells in CAR-T populations.

[00261] Briefly, mRNA encoding SPBv3.0 or SPBv4.0 was nucleofected in pan T cells isolated from four separate donors. The T cells were conucleofected with: a) a transposon comprising: a wild-type LE piggyBac ITR and a RE piggyBac ITR comprising a 35TCC mutation: a sequence encoding an anti-MUC1C CAR, an iCAS9 safety switch (described in more detail in International Patent Application Publication No. WO 2018 / 068022, incorporated herein in its entirety) and a dihydrofolate reductase selectable marker gene; b) an mRNA encoding CasCLOVER v3.0 (SEQ ID NO. 46); c) a pair of gRNAs that target beta-2 microglobulin (B2M) and a pair of gRNAs that target CD3 for knockout expression of B2M and CD3, respectively; and d) an mRNA that encodes a Petition 870250091226, dated 06 / 10 / 2025, pp. 200 / 224 100 / 100 booster molecule for expansion of transfected T cells. (as described in International Patent Application Publication No. WO 2020 / 051374, incorporated herein by reference in its entirety).

[00262] Transfected T cells from the four donors were expanded using the CD3 / CD28 / CD2 ImmunoCult T Cell Activator, selected for methotrexate resistance, and the yield and fold expansion for activated T cells from each of the four donors were determined. CD3+ transfected CAR-T cells were removed by column chromatography using anti-CD3 beads, and the T cells were subjected to flow cytometry on Days 5 and 14 to identify T cell populations: mean fluorescence intensity (MFI; transposition efficiency [TPE]) of CAR, percentage of B2M knockout; percentage of CD3 knockout, and total yield of CD3- T cells after depletion. The results are shown in FIGURES 17A to 17E.

[00263] As shown in FIGURES 17A to 17E, the mRNA encoding SPBv4.0 resulted in a 10-fold increase in T cell expansion in three of the four donors (FIGURE 17A) compared to SPBv3.0. Although SPBv4.0 did not result in an improvement in overall transposition efficiency on Day 5 compared to SPBv3.0 (FIGURE 17B), SPB4.0 resulted in a greater increase in the percentage of B2M knockout cells (FIGURE 17C) and CD3- knockout cells (FIGURE 17D) and increased the total number of CD3- CAR-T cells in three of the four donors by approximately 3 to 4 times (FIGURE 17E). This resulted in an increase in the production of desired CAR-T cells with B2M and CD3 knockout expression in the final CAR-T isolation. Petition 870250091226, dated 06 / 10 / 2025, pages 201 / 224

Claims

1 / 4 Claims 1. POLYNUCLEOTIDE, characterized by comprising in the 5' to 3' direction: (i) a 5'-UTR of hemoglobin beta (HBB), (ii) a sequence encoding a nuclear localization signal (NLS), (iii) a nucleic acid sequence encoding a piggyBac transposase, (iv) a 3'-UTR comprising one or more nucleic acid sequences comprising a 3'-UTR element of human cytochrome b-245 alpha polypeptide (CYBA) and one or more miR-142-3p binding sites, and (v) a polyA tail.

2. POLYNUCLEOTIDE, according to claim 1, characterized by the piggyBac transposase comprising the amino acid sequence shown in SEQ ID NO: 14).

3. POLYNUCLEOTIDE, according to any one of claims 1 to 2, characterized by the nucleic acid sequence encoding the piggyBac transposase comprising the nucleic acid sequence shown in SEQ ID NO:

2.

4. POLYNUCLEOTIDE, according to claim 1, characterized in that the NLS is an SV40 NLS comprising the amino acid sequence presented in SEQ ID NO:

8.

5. POLYNUCLEOTIDE, according to claim 1, characterized in that one or more 3'-UTR elements of CYBA comprise(s), each comprising the nucleic acid sequence presented in SEQ ID NO:

3.

6. POLYNUCLEOTIDE, according to claim 5, characterized in that the 3'-UTR comprises at least two tandem nucleic acid sequences encoding a CYBA 3'-UTR element, separated by a linker sequence.

7. POLYNUCLEOTIDE, according to claim 6, characterized by the tandem nucleic acid sequences encoding a 3'-UTR element of CYBA each comprising the nucleic acid sequence shown in SEQ ID NO.

4.

8. POLYNUCLEOTIDE, according to claim 1, characterized in that one or more miR-142-3p binding sites each comprise the nucleic acid sequence shown in SEQ ID NO:

5.

9. A POLYNUCLEOTIDE, according to claim 1, characterized by each having one or more miR-142-3p binding sites comprising the nucleic acid sequence ACACTAC.

10. POLYNUCLEOTIDE, according to claim 9, characterized by the 3'-UTR comprising four miR-142-3p binding sites.

11. POLYNUCLEOTIDE, according to claim 10, characterized by further comprising a linker sequence located between each of the four miR-142-3p binding sites.

12. POLYNUCLEOTIDE, according to claim 11, characterized by the linker sequence comprising the nucleic acid sequence shown in SEQ ID NO.

6.

13. POLYNUCLEOTIDE, according to claim 1, characterized in that the 5'-UTR of HBB comprises the nucleic acid sequence presented in SEQ ID NO:

1.

14. POLYNUCLEOTIDE, according to claim 1, characterized in that the polyA tail is an 80X polyA tail comprising the nucleic acid sequence presented in SEQ ID NO:

7.

15. POLYNUCLEOTIDE, according to claim 1, characterized in that the polynucleotide comprises the nucleic acid sequence presented in SEQ ID NO:

10.

16. POLYNUCLEOTIDE, according to any one of claims 1 to 15, characterized in that the polynucleotide is a DNA molecule. Petition 870250091226, dated 06 / 10 / 2025, pp. 203 / 224 3 / 4 17. POLYNUCLEOTIDE, according to any one of claims 1 to 15, characterized in that the polynucleotide is an RNA molecule.

18. POLYNUCLEOTIDE, according to claim 17, characterized in that the RNA molecule is an mRNA molecule.

19. POLYNUCLEOTIDE, according to claim 18, characterized in that the mRNA comprises a 5'-CAP.

20. POLYNUCLEOTIDE, according to claim 19, characterized in that the 5'CAP is a 5' CleanCap.

21. LIPID NANOPARTICLE (LNP) COMPOSITION, characterized by comprising the polynucleotide as defined in claim 16.

22. LIPID NANOPARTICLE (LNP) COMPOSITION, characterized by comprising the polynucleotide as defined in claim 17.

23. METHOD FOR DELIVERING AN EXOGENOUS NUCLEIC ACID TO A CELL, characterized by comprising: introducing into the cell a DNA transposon comprising an exogenous nucleic acid; and an mRNA comprising in the 5' to 3' direction: (i) a 5'-UTR of HBB, a nucleic acid sequence encoding an NLS, (ii) a nucleic acid sequence encoding a piggyBac transposase comprising five hyperactive mutations, (iii) a 3'-UTR comprising two or more tandem nucleic acid sequences comprising a 3'-UTR element of CYBA and four miR-142-3p binding sites, and (iv) a polyA tail; wherein the piggyBac transposase is expressed in the cell and integrates the transposon comprising the exogenous nucleic acid into a TTAA integration site in the cell genome, wherein the expressed piggyBac transposase exhibits Petition 870250091226, dated 06 / 10 / 2025, p.204 / 224 4 / 4 enhanced integration and / or excision activity compared to a piggyBac transposase comprising four or fewer hyperactive mutations.

24. METHOD FOR IN VIVO DELIVERY OF AN EXOGENOUS NUCLEIC ACID TO A CELL IN AN INDIVIDUAL, characterized by comprising: co-introducing into the individual a DNA transposon comprising an exogenous nucleic acid; and an mRNA comprising, in the 5' to 3' direction: a 5'-UTR of HBB, an NLS coding sequence, a piggyBac transposase coding sequence comprising five hyperactive mutations, a 3'-UTR comprising the CYBA 3'-UTR element in tandem upstream of the sequences for four miR-142-3p binding sites and a polyA tail; whereby a cell in the individual takes up the transposon and expresses the piggyBac transposase in the cell and integrates the transposon comprising the exogenous nucleic acid into a TTAA integration site in the individual's cellular genome, wherein the expressed piggyBac transposase exhibits reduced immunogenicity in the individual compared to an mRNA encoding a piggyBac transposase lacking the sequences for four miR-1423p binding sites.Petition 870250091226, dated 06 / 10 / 2025, pp. 205 / 224.

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