AAV piggyBac transposon polynucleotide compositions and methods of use thereof

Abstract

The present disclosure relates to compositions and methods for treating phenylketonuria (PKU). In particular, the disclosure relates to LNP compositions comprising an AAV-piggyBac transposon polynucleotide vector and a nucleic acid encoding a transposase, and methods of using the compositions to treat PKU.

Description

[Technical Field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 486,168, filed February 21, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (POTH-078_001WO_SeqListing_ST26.xml; size 70,664 bytes; created February 12, 2024) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates generally to novel AAV piggyBac transposon polynucleotides comprising the human phenylalanine hydroxylase (PAH) gene, AAV piggyBac vectors comprising the polynucleotides, compositions comprising the AAV piggyBac vector and lipid nanoparticles ("LNPs") comprising mRNA encoding the transposase, methods for preparing these polynucleotides and LNPs, and the use of these AAV piggyBac vectors and LNPs for gene therapy applications, particularly for the treatment of phenylketonuria (PKU). [Background technology]

[0004] background There has been a long-felt, yet unmet, need in the art for compositions and methods for delivering nucleic acids to cells and for genetically modifying cells in vivo, ex vivo, and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides an improved AAV piggyBac transposon polynucleotide composition containing the human phenylalanine hydroxylase (PAH) gene, and a method for delivering the polynucleotide composition in an AAV piggyBac vector in combination with a lipid nanoparticle (LNP) composition containing mRNA encoding a transposase, such as Super PiggyBac transposase (SPB), to cells, including hepatocytes, in vivo with high efficiency and low toxicity. Thus, the compositions and methods of the present disclosure have potential applications as gene therapy therapeutics for treating phenylketonuria (PKU).

[0005] The present disclosure provides AAV piggyBac transposon polynucleotides comprising a transgene encoding a codon-optimized and modified human phenylalanine hydroxylase (PAH) gene, AAV piggyBac vector compositions comprising the polynucleotides, and methods of using the AAV piggyBac vector compositions in combination with LNP compositions comprising at least one mRNA encoding a transposase, e.g., SPB, for the treatment of phenylketonuria (PKU). The compositions and methods are described in further detail herein. Summary of the Invention

[0006] Summary of the Invention The present disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first piggyBac ITR sequence; c) a first piggyBac 3'UTR; d) a first insulator sequence; e) at least one promoter sequence; f) at least one intron sequence; g) at least one transgene sequence encoding human phenylalanine hydroxylase (PAH); h) a polyA sequence; i) a second insulator sequence; j) a first piggyBac 5'UTR sequence; k) a second piggyBac ITR sequence; l) at least one DNA spacer sequence; and m) a second AAV ITR sequence.

[0007] The present disclosure provides a transgene sequence encoding a human PAH gene comprising, from 5' to 3' direction, a) a first AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 1; b) a first piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 2; c) a first piggyBac 3'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 3; d) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 4; e) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 7; f) a synthetic intron sequence comprising the nucleic acid sequence of SEQ ID NO: 17; g) a transgene sequence encoding a codon-optimized and modified human PAH gene comprising the nucleic acid sequence of SEQ ID NO: 9; h) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 11; i) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 12; j) a piggyBac 5'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 13; k) a second piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 14; l) a DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 15; and m) a second AAV An adeno-associated virus (AAV) piggyBac transposon polynucleotide containing an ITR sequence is provided.

[0008] In some embodiments, the AAV piggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:18.

[0009] The present disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first piggyBac ITR sequence; c) a first piggyBac 3'UTR; d) a first insulator sequence; e) at least one enhancer sequence; f) at least one promoter sequence; g) at least one transgene sequence encoding human phenylalanine hydroxylase (PAH); h) a first 3'UTR; i) a polyA sequence; j) a second insulator sequence; k) a first piggyBac 5'UTR; l) a second piggyBac ITR sequence comprising a 35TCC mutation; m) at least one DNA spacer sequence; and n) a second AAV ITR sequence.

[0010] The present disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in the 5' to 3' direction: a) a first AAV ITR sequence; b) a first piggyBac ITR sequence; c) a first piggyBac 3'UTR; d) a first insulator sequence; e) a 3xhSERPINA1 enhancer; f) a TTR enhancer; g) a TTRm promoter sequence; h) an MVM intron; i) a transgene sequence comprising a nucleic acid sequence encoding a codon-optimized and modified human PAH gene; j) an AES-mtRNR 3'UTR; k) a polyA sequence; l) a second insulator sequence; m) a first piggyBac 5'UTR; n) a second piggyBac ITR sequence comprising a 35TCC mutation; o) a DNA spacer sequence; and p) a second AAV ITR sequence.

[0011] The present disclosure provides a transgene sequence encoding a human PAH gene comprising, from 5' to 3' direction, a) a first AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO:1; b) a first piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO:2; c) a first piggyBac 3'UTR sequence comprising the nucleic acid sequence of SEQ ID NO:3; d) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO:4; e) a first enhancer sequence comprising the nucleic acid sequence of SEQ ID NO:5; f) a second enhancer sequence comprising the nucleic acid sequence of SEQ ID NO:6; g) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO:7; h) an intron sequence comprising the nucleic acid sequence of SEQ ID NO:8; i) a transgene sequence encoding a codon-optimized and modified human PAH gene comprising the nucleic acid sequence of SEQ ID NO:9; j) an AES-mtRNR 3'UTR comprising the nucleic acid of SEQ ID NO:10; k) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO:11; l) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO:12; m) a piggyBac 3'UTR comprising the nucleic acid sequence of SEQ ID NO:13. Provided is an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising: a 5' UTR sequence; n) a second piggyBac ITR sequence comprising a 35TCC mutation, the second piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 19; o) a DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 15; and p) a second AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 16.

[0012] In some embodiments, the AAV piggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO:21.

[0013] The present disclosure provides a vector comprising any one of the AAV piggyBac transposon polynucleotides of the present disclosure.In some embodiments, the vector is an AAV viral vector.In some embodiments, the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 viral vector.In some embodiments, the AAV viral vector is an AAV8 or AAV9 viral vector.

[0014] The present disclosure provides a pharmaceutical composition comprising any one of the vectors of the present disclosure.

[0015] The present disclosure provides compositions comprising: 1) any one of the vectors of the present disclosure; and 2) at least one LNP composition comprising at least one mRNA molecule encoding a transposase. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

[0016] In some embodiments, at least one LNP composition comprises about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis.

[0017] The present disclosure provides a pharmaceutical composition comprising any one of the compositions of the present disclosure.

[0018] The present disclosure provides a method of treating phenylketonuria (PKU) in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a composition comprising: a) a polynucleotide, vector, or pharmaceutical composition of any one of the preceding claims; and b) at least one LNP composition comprising at least one mRNA molecule encoding a transposase.

[0019] In some embodiments, at least one LNP composition comprises about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase. In some embodiments, the transposase is a Super piggyBac™ (SPB) transposase. [Brief explanation of the drawings]

[0020] The above and further features will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.

[0021] [Figure 1] FIG. 1 is a schematic diagram of an exemplary AAV piggyBac transposon polynucleotide comprising a codon-optimized and modified human phenylalanine hydroxylase (PAH) gene of the present disclosure.

[0022] [Figure 2] FIG. 2 is a schematic diagram of a second exemplary AAV piggyBac transposon polynucleotide comprising a codon-optimized and modified human PAH gene of the present disclosure.

[0023] [Figure 3A]Figure 3A is a digital image of a Western blot of HA-PAH transgene expression levels in wild-type mice treated intravenously with LNP compositions containing vehicle, low-dose, and high-dose AAV piggyBac transposon polynucleotide and mRNA encoding SPB or catalytically inactive SPB, or mRNA encoding a combination of SPBs via hydrodynamic delivery (HDD).

[0024] [Figure 3B] FIG. 3B is a graph showing the amount of HA-tagged PAH expression / mg protein for samples from vehicle-, treated, and HDD-treated mice from FIG. 3A.

[0025] [Figure 4] FIG. 4 is a graph showing the relative percentage of hepatocytes expressing human PAH in treated mice administered a low or high dose of an AAV piggyBac transposon vector and an LNP composition containing mRNA encoding SPB or mRNA encoding catalytically inactive SPB, or mRNA encoding SPB via hydrodynamic delivery (HDD).

[0026] [Figure 5A] Figure 5A is a graph showing episomal and cumulative viral copy number (VCN) in hepatocytes isolated from mice treated with vehicle, a low dose of AAV piggyBac transposon vector alone, an LNP composition comprising a low dose of AAV piggyBac transposon vector and mRNA encoding SPB, a high dose of AAV piggyBac transposon vector alone, an LNP composition comprising a high dose of AAV piggyBac transposon vector and mRNA encoding catalytically inactive SPB and a high dose of AAV piggyBac transposon vector, and an LNP composition comprising mRNA encoding SPB.

[0027] [Figure 5B]FIG. 5B is a magnified graph showing only the integrated VCN of the sample shown in FIG. 5A.

[0028] [Figure 6A] Figure 6A shows a graph of serum Phe levels (μM) at days 0, 7, 14, and 28 post-administration in male BALB PAHenu mice treated with vehicle, an LNP composition comprising an AAV piggyBac transposon vector (construct #1) and mRNA encoding SPB, an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding catalytically inactive SPB, or control wild-type mice.

[0029] [Figure 6B] Figure 6B shows a graph of serum Phe levels (μM) at days 0, 7, 14, and 28 post-administration in female BALB PAHenu mice treated with vehicle, an LNP composition comprising an AAV piggyBac transposon vector (construct #1) and mRNA encoding SPB, an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding catalytically inactive SPB, or control wild-type mice.

[0030] [Figure 7A] Figure 7A shows a graph of serum Phe levels (μM) at days 0, 7, 14, and 28 post-administration in male BALB PAHenu mice treated with vehicle, an LNP composition comprising an AAV piggyBac transposon vector (construct number 2) and mRNA encoding SPB, an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding catalytically inactive SPB, or control wild-type mice.

[0031] [Figure 7B]Figure 7B shows a graph of serum Phe levels (μM) at days 0, 7, 14, and 28 post-administration in female BALB PAHenu mice treated with vehicle, an LNP composition comprising an AAV piggyBac transposon vector (construct number 2) and mRNA encoding SPB, an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding catalytically inactive SPB, or control wild-type mice.

[0032] [Figure 8A] Figure 8A is a graph showing the amount of HA-tagged PAH expression / mg protein in samples from wild-type young mice treated intravenously with vehicle, an LNP composition comprising an AAV piggyBac transposon polynucleotide and an mRNA encoding catalytically inactive SPB, or an LNP composition comprising an AAV piggyBac transposon polynucleotide and an mRNA encoding SPB.

[0033] [Figure 8B] Figure 8B is a graph showing episomal and integrated viral copy numbers (VCN) in hepatocytes isolated from mice treated with vehicle, an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding a catalytically inactive SPB, and an LNP composition comprising an AAV piggyBac transposon vector and mRNA encoding SPB. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description Compositions of the Disclosure—AAV piggyBac Transposon Polynucleotides

[0035] The present disclosure provides an AAV piggyBac transposon polynucleotide comprising a human PAH gene. In some embodiments, the nucleotide sequence of the PAH gene is codon-optimized to improve expression of the encoded human PAH enzyme. In some embodiments, the nucleotide sequence of the human PAH gene is modified to remove any internal TTAA sites; any undesired restriction enzyme recognition sites; and any potential cryptic splice sites for cloning the modified sequence into an AAV piggyBac transposon vector. In some embodiments, the nucleotide sequence of the human PAH gene is codon-optimized and further modified as described above. Two exemplary AAV piggyBac transposon polynucleotides comprising codon-optimized and modified PAH genes are shown schematically in Figures 1 and 2, respectively.

[0036] In certain embodiments of the present disclosure, the AAV piggyBac transposon polynucleotide comprises, from 5' to 3', a) a first AAV ITR sequence; b) a first piggyBac ITR sequence; c) a piggyBac 3'UTR sequence; d) a first insulator sequence; e) a TTRm promoter sequence; f) a synthetic intron sequence; g) a transgene sequence comprising a nucleic acid sequence encoding a human PAH gene; h) a polyA sequence; i) a second insulator sequence; j) a piggyBac 5'UTR sequence; k) a second piggyBac ITR; l) a DNA spacer sequence; and m) a second AAV ITR sequence.

[0037] In one embodiment, the first AAV ITR sequence comprises the nucleic acid of SEQ ID NO: 1. In one embodiment, the first piggyBac ITR sequence comprises the nucleic acid of SEQ ID NO: 2. In one embodiment, the first piggyBac 3'UTR sequence comprises the nucleic acid of SEQ ID NO: 3. In one embodiment, the first insulator sequence comprises the nucleic acid of SEQ ID NO: 4. In one embodiment, the TTRm promoter sequence comprises the nucleic acid of SEQ ID NO: 7. In one embodiment, the synthetic intron sequence comprises the nucleic acid of SEQ ID NO: 17. In one embodiment, the transgene PAH nucleotide sequence further comprises a hemagglutinin (HA) tag. In one embodiment, the transgene PAH nucleotide sequence is codon-optimized. In one embodiment, the transgene PAH nucleotide sequence is modified. In one embodiment, the transgene PAH nucleotide sequence comprises a hemagglutinin tag, is codon-optimized, and is further modified as disclosed herein. In one embodiment, the nucleic acid sequence encoding the human PAH gene sequence comprises the nucleic acid of SEQ ID NO: 9. In one embodiment, the polyA sequence comprises the nucleic acid of SEQ ID NO: 11. In one embodiment, the second insulator sequence comprises the nucleic acid of SEQ ID NO: 12. In one embodiment, the piggyBac 5'UTR sequence comprises the nucleic acid of SEQ ID NO: 13. In one embodiment, the second piggyBac ITR comprises the nucleic acid of SEQ ID NO: 14. In one embodiment, the DNA spacer sequence comprises the nucleic acid of SEQ ID NO: 15. In one embodiment, the second AAV ITR sequence comprises the nucleic acid of SEQ ID NO: 16.

[0038] In certain embodiments of the present disclosure, the first AAV piggyBac transposon polynucleotide comprises, from 5' to 3', a) a first AAV ITR sequence comprising the nucleic acid of SEQ ID NO: 1; b) a first piggyBac ITR sequence comprising the nucleic acid of SEQ ID NO: 2; c) a piggyBac 3'UTR comprising the nucleic acid of SEQ ID NO: 3; d) a first insulator sequence comprising the nucleic acid of SEQ ID NO: 4; e) a TTRm promoter sequence comprising the nucleic acid of SEQ ID NO: 7; f) a synthetic intron sequence comprising the nucleic acid of SEQ ID NO: 17; g) a transgene sequence comprising a nucleic acid sequence encoding a hemagglutinin (HA)-tagged, codon-optimized and modified human PAH gene comprising the nucleic acid of SEQ ID NO: 9; h) a polyA sequence comprising the nucleic acid of SEQ ID NO: 11; i) a second insulator sequence comprising the nucleic acid of SEQ ID NO: 12; j) a piggyBac 5'UTR sequence comprising the nucleic acid of SEQ ID NO: 13; k) a second piggyBac 5'UTR sequence comprising the nucleic acid of SEQ ID NO: 14. ITR; l) a DNA spacer sequence comprising the nucleic acid of SEQ ID NO:15; and m) a second AAV ITR sequence comprising the nucleic acid of SEQ ID NO:16.

[0039] In certain embodiments, the AAV piggyBac transposon polynucleotide comprises, from 5' to 3', a) a first AAV ITR sequence (SEQ ID NO:1); b) a first piggyBac ITR sequence (SEQ ID NO:2); c) a piggyBac 3'UTR sequence (SEQ ID NO:3); d) a first insulator sequence (SEQ ID NO:4); e) a TTRm promoter region comprising a 3xhSERPINA1 enhancer (SEQ ID NO:5), a TTR enhancer (SEQ ID NO:6), a TTRm promoter sequence (SEQ ID NO:7), and an MVM intron (SEQ ID NO:8); f) a transgene sequence comprising a nucleic acid sequence encoding a hemagglutinin (HA)-tagged, codon-optimized, and modified human PAH gene (SEQ ID NO:9) followed by an AES-mtRNR 3'UTR (SEQ ID NO:10); g) a polyA sequence (SEQ ID NO:11); h) a second insulator sequence (SEQ ID NO:12), a piggyBac 5'UTR sequence (SEQ ID NO:13), a second piggyBac 3'UTR sequence comprising a 35TCC mutation. ITR sequence (SEQ ID NO: 19); j) DNA spacer sequence (SEQ ID NO: 20); and k) second AAV ITR sequence (SEQ ID NO: 16).

[0040] Compositions of the Disclosure—Lipid Nanoparticles

[0041] The present disclosure provides a composition comprising at least one lipid nanoparticle, comprising at least one cationic lipid and at least one nucleic acid molecule.In some embodiments, the lipid nanoparticle can further comprise at least one structural lipid.In some embodiments, the lipid nanoparticle can further comprise at least one phospholipid.In some embodiments, the lipid nanoparticle can further comprise at least one PEGylated lipid.

[0042] Accordingly, the present disclosure provides a composition 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.

[0043] Bioreducible ionizable cationic lipids

[0044] In some aspects, the cationic lipid can be a bioreducible, ionizable cationic lipid.

[0045] Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid.

[0046] As used herein, the term "bioreducible ionizable cationic lipid" is used in its broadest sense and refers to a cationic lipid that contains at least one tertiary amine, at least one disulfide group, at least one group containing a bond susceptible to cleavage by thioesterification, and further contains at least two saturated or unsaturated hydrocarbon chains. Exemplary bioreducible ionizable cationic lipids include, but are not limited to, those described in Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the entire contents of which are incorporated herein by reference.

[0047] Further exemplary bioreducible ionizable cationic lipids useful in the methods of the present disclosure and methods for preparing such lipids include those disclosed in International Patent Application No. PCT / JP2016 / 052690, published as WO 2016 / 121942, and International Patent Application No. PCT / JP2019 / 012302, published as WO 2019 / 188867, the contents of each of which are incorporated herein by reference in their entirety.

[0048] Accordingly, the present disclosure provides compositions comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises any one of the bioreducible ionizable cationic lipids described in WO 2016 / 121942 and WO 2019 / 188867.

[0049] Accordingly, the present disclosure provides a composition 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.

[0050] In some embodiments, the bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2, having the following structure: (Formula I) JPEG2026507612000002.jpg27170

[0051] See Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the entire contents of which are incorporated by reference.

[0052] Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle comprising at least one bioreducible ionizable cationic lipid, wherein the at least one bioreducible ionizable cationic lipid comprises ssPalmO-Ph-P4C2.

[0053] As will be understood by those skilled in the art, ssPalmO-Ph-P4C2 may also be referred to as Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph. Accordingly, ssPalmO-Ph-P4C2, Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph are used interchangeably herein to refer to the bioreducible, ionizable cationic lipid having the chemical structure shown in Formula I.

[0054] As described herein, the LNP compositions of the present disclosure, which comprise at least one bioreducible ionizable cationic lipid, advantageously exhibit significantly reduced toxicity in animals compared to LNP compositions comprising non-bioreducible ionizable cationic lipids.In particular, administration of the LNP compositions of the present disclosure surprisingly does not result in any weight loss.In some embodiments, certain LNP compositions of the present disclosure are so non-toxic that animals administered the LNP actually gain weight, even at doses of LNP that exceed the lethal dose of LNP compositions comprising non-bioreducible ionizable cationic lipids.

[0055] LNP component

[0056] In some embodiments, LNPs of the disclosure can 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 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 on a molar basis.

[0057] In some embodiments, the LNPs of the disclosure have a molecular weight of 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 The lipid composition can comprise 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.

[0058] In some embodiments, the LNPs of the disclosure can 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 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 structured lipid on a molar basis.

[0059] In some embodiments, LNPs of the disclosure can 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% on a molar basis of at least one structured lipid.

[0060] In some embodiments, the LNPs of the disclosure can 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 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 phospholipid on a molar basis.

[0061] In some embodiments, LNPs of the disclosure can 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 phospholipid on a molar basis.

[0062] In some embodiments, the LNPs of the present disclosure can comprise, on a molar basis, 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 2.5%, or about 5% of at least one PEGylated lipid.

[0063] In some embodiments, LNPs of the present disclosure can 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 on a molar basis.

[0064] structured lipids

[0065] In some embodiments, the structured lipids can be steroids. In some embodiments, the structured lipids can be sterols. In some embodiments, the structured lipids can comprise cholesterol. In some embodiments, the structured lipids can comprise ergosterol. In some embodiments, the structured lipids can be phytosterols.

[0066] phospholipids

[0067] As used herein, the term "phospholipid" is used in its broadest sense to refer to any amphipathic molecule containing a polar (hydrophilic) head group containing phosphate and two hydrophobic fatty acid chains.

[0068] In some embodiments of the lipid nanoparticles of the present disclosure, the phospholipid can include dioleoylphosphatidylethanolamine (DOPE).

[0069] In some embodiments of the lipid nanoparticles of the present disclosure, the phospholipid can include DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0070] In some embodiments of the lipid nanoparticles of the present disclosure, the phospholipid can include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine).

[0071] In some embodiments, the phospholipid is selected from the group consisting of 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[phospho-rac-(1-glycerol)(sodium salt)), DLOPC (1,2-dilinoleoyl-sn -glycero-3-phosphocholine), DLPA-NA (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)) (sodium salt)), DLPS-NA (1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt)), DMPA-NA (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt)), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DMPG-NH4 (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), 1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (ammonium salt)), DMPG-NH4 / NA (1,2-Dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (sodium / ammonium salt)), DMPS-NA (1,2-Dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DOPA-NA (1,2-Dioleoyl-sn-glycero-3-phosphate (sodium salt)), DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-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)), 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[phospho-rac-(1-glycerol)(sodium salt)), DSPG-NH4 (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DSPS-NA(1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt)), EPC (egg PC), HEPC (hydrogenated egg PC), HSPC (hydrogenated soybean 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-derived 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-glycero-1)] (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.

[0072] PEGylated lipids

[0073] As used herein, the term "PEGylated lipid" refers to any lipid modified (e.g., covalently linked) to at least one polyethylene glycol molecule. In some embodiments, the PEGylated lipid can comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (hereinafter referred to as DMG-PEG2000).

[0074] nucleic acid

[0075] In some embodiments, the lipid nanoparticles can contain at least one nucleic acid molecule. In some embodiments, the lipid nanoparticles can contain multiple nucleic acid molecules. In some embodiments, at least one nucleic acid molecule or multiple nucleic acid molecules can be formulated in the lipid nanoparticles.

[0076] In some embodiments, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some embodiments, the non-naturally occurring nucleic acid molecule can contain at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some embodiments, the nucleic acid molecule can be a modified nucleic acid molecule. In some embodiments, the modified nucleic acid molecule can contain at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0077] In some embodiments, the lipid nanoparticles can comprise lipids and nucleic acids in a specific ratio (weight / weight).

[0078] In some embodiments, lipid nanoparticles comprising at least one nucleic acid molecule have a lipid to lipid 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 The lipids and nucleic acids can be contained in a lipid:nucleic acid (weight / weight) ratio of 85:1, or from about 80:1 to about 90:1, or from about 85:1 to about 95:1, or from about 90:1 to about 100:1, or from about 95:1 to about 105:1, or from about 100:1 to about 110:1, or from about 105:1 to about 115:1, or from about 110:1 to about 120:1, or from about 115:1 to about 125:1, or from about 120:1 to about 130:1, or from about 125:1 to about 135:1, or from about 130:1 to about 140:1, or from about 135:1 to about 145:1, or from about 140:1 to about 150:1.

[0079] In some embodiments, the lipid nanoparticles have a lipid to lipid 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, Alternatively, the lipids and nucleic acid may be present in a lipid:nucleic acid (weight / weight) ratio of 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 about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, or about 200:1.

[0080] In some embodiments, the lipid nanoparticles can comprise lipids and nucleic acid at a lipid:nucleic acid (wt / wt) ratio of about 10:1, or about 17.5:1, or about 25:1.

[0081] In some embodiments, nucleic acid molecule can be RNA molecule.Therefore, in some embodiments, lipid nanoparticle can comprise at least one RNA molecule.In some embodiments, RNA molecule can be mRNA molecule.In some embodiments, mRNA molecule can comprise 5'-CAP.

[0082] In some embodiments, mRNA molecules can be capped using any method and / or capping moiety known in the art. mRNA molecules can be capped with an m7G(5')ppp(5')G moiety. The m7G(5')ppp(5')G moiety is also referred to herein as "CapO." mRNA molecules can be capped with a CleanCap® moiety. The CleanCap® moiety can comprise an m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. The CleanCap® moiety can comprise an m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. mRNA molecules can be capped with an anti-reverse cap analog (ARCA®) site. The ARCA® moiety can comprise an m7(3'-0-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a CleanCap® 3'OMe moiety (CleanCap® + ARCA®).

[0083] In some embodiments, the mRNA molecule can include at least one modified nucleic acid.

[0084] Modified nucleic acids include 5-methoxyuridine (5moU), N1-methylpseudouridine (me 1 These may include, but are not limited to, ψ), pseudouridine (Y), and 5-methylcytidine (5-MeC).

[0085] In some embodiments, the nucleic acid molecule can be a DNA molecule. Thus, in some embodiments, the lipid nanoparticle can comprise at least one DNA molecule. In some embodiments, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid. In some embodiments, the lipid nanoparticle can comprise a DNA plasmid. In some embodiments, the DNA molecule can be a linearized DNA molecule, such as, but not limited to, a linearized DNA plasmid. In some embodiments, the DNA molecule can be a DoggyBone DNA molecule. In some embodiments, the DNA molecule can be a DNA nanoplasmid.

[0086] The DNA plasmid may be at least about 0.25 kb in length, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb. kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or less or at least about 7.75 kb, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least It can be about 11.5 kb, or at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb or at least about 15.0 kb.

[0087] LNP composition

[0088] In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one structural lipid. In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one PEGylated lipid. In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0089] In some embodiments, at least one structured lipid can be a mixture of two structured lipids. In some embodiments, at least one PEGylated lipid can be a mixture of two PEGylated lipids.

[0090] In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one PEGylated lipid, or any combination thereof. In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0091] In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, and at least one PEGylated lipid. In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0092] In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one phospholipid, at least one PEGylated lipid, or any combination thereof. In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0093] In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid. In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0094] In some embodiments, the nucleic acid molecule is an RNA molecule.Therefore, in some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule can comprise about 54% ssPalmO-Ph-P4C2 by molar basis, about 35% cholesterol by molar basis, about 10% DOPC by molar basis, and about 1% DMG-PEG2000 by molar basis, and the lipid nanoparticles further comprise at least one mRNA molecule.In some embodiments, the mRNA molecule further comprises 5'-CAP.In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle can be about 100:1 (w / w).

[0095] In some embodiments, lipid nanoparticles comprising at least one nucleic acid molecule can comprise about 44% to 64% ssPalmO-Ph-P4C2 on a molar basis, about 25% to 45% cholesterol on a molar basis, about 0.1% to 20% DOPC on a molar basis, and about 0.1% to 11% DMG-PEG2000 on a molar basis, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, lipid nanoparticles containing at least one nucleic acid molecule can comprise about 49%-59% ssPalmO-Ph-P4C2 on a molar basis, about 30%-40% cholesterol on a molar basis, about 5%-15% DOPC on a molar basis, and about 0.5%-6% DMG-PEG2000 on a molar basis, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the mRNA molecule further comprises a 5'-CAP. In some embodiments, the lipid to nucleic acid ratio in the at least one nanoparticle can be about 75:1 to about 100:1 (w / w).

[0096] In some embodiments, the nucleic acid molecule is a DNA molecule. Accordingly, the present disclosure provides lipid nanoparticles comprising at least one nucleic acid molecule, which may comprise about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis, and further comprising at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed-end DNA (see WO 2020 / 154645). In some embodiments, the lipid to nucleic acid ratio in the nanoparticle may be about 100:1 (w / w).

[0097] In some embodiments, lipid nanoparticles comprising at least one nucleic acid molecule can comprise about 44% to 64% ssPalmO-Ph-P4C2 on a molar basis, about 25% to 45% cholesterol on a molar basis, about 0.1% to 20% DOPC on a molar basis, and about 0.1% to 11% DMG-PEG2000 on a molar basis, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one DNA molecule. In some embodiments, lipid nanoparticles containing at least one nucleic acid molecule can comprise approximately 49%-59% ssPalmO-Ph-P4C2 on a molar basis, approximately 30%-40% cholesterol on a molar basis, approximately 5%-15% DOPC on a molar basis, and approximately 0.5%-6% DMG-PEG2000 on a molar basis, wherein the at least one lipid nanoparticle contains at least one nucleic acid molecule, and the at least one nucleic acid molecule contains at least one DNA molecule. In some embodiments, the at least one DNA molecule can be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule can be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule can be a covalently closed-end DNA. In some embodiments, the lipid to nucleic acid ratio in the at least one nanoparticle can be approximately 75:1 to approximately 100:1 (w / w).

[0098] Exemplary LNP compositions useful in the methods of the present disclosure and methods for preparing such LNP compositions include those disclosed in International Patent Application No. PCT / US2022 / 017570, published as WO 2022 / 182792, the contents of which are incorporated herein by reference in their entirety.

[0099] Accordingly, the present disclosure provides LNP compositions comprising any one of the LNP compositions described in WO 2022 / 182792.

[0100] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform, hi some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0101] In some embodiments, a microfluidic mixing platform can produce lipid nanoparticles of the present disclosure by combining a miscible solvent phase containing the lipid components of the nanoparticle with an aqueous phase containing the cargo of the lipid nanoparticle (e.g., nucleic acid, DNA, mRNA, etc.) using a microfluidic device. In some embodiments, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow the two phases to mix immediately. As the two phases move under laminar flow through the microfluidic channel, microscopic features within the channel allow for controlled, uniform mixing to produce lipid nanoparticles of the present disclosure.

[0102] In some embodiments, microfluidic mixing platforms include, but are not limited to, NanoAssemblr® Spark (Precision NanoSystems), NanoAssemblr® Ignite™ (Precision NanoSystems), NanoAssemblr® Benchtop (Precision NanoSystems), NanoAssemblr® Blaze (Precision NanoSystems), or NanoAssemblr® GMP System (Precision NanoSystems).

[0103] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0104] In some embodiments, lipid nanoparticles of the present disclosure can be produced using a T-mixer that mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0105] In some embodiments, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform that mixes a miscible solvent phase with an aqueous phase in a ratio (solvent:water, v / v) of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10.

[0106] In some embodiments, lipid nanoparticles of the present disclosure can be produced using a T-mixer, which mixes a miscible solvent phase with an aqueous phase in a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solvent:aqueous, v:v.

[0107] Compositions of the Disclosure—Adeno-Associated Virus (AAV) piggyBac Transposon Polynucleotides

[0108] The present disclosure provides compositions comprising an adeno-associated virus (AAV) piggyBac transposon polynucleotide.

[0109] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one AAV inverted terminal repeat (ITR) sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one piggyBac ITR sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one insulator sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one enhancer sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one promoter sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one transgene sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one polyA sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one DNA spacer sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one piggyBac 3'UTR sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one piggyBac 5' UTR sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one 3' UTR sequence. In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one intron sequence.

[0110] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0111] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0112] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by a second AAV ITR sequence.

[0113] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence, with any combination of at least one promoter sequence, at least one transgene sequence, and at least one polyA sequence between the first and second insulator sequences.

[0114] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from 5' to 3', a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence, wherein between the first insulator sequence and the second insulator sequence is any combination of at least one promoter sequence, at least one transgene sequence, and at least one polyA sequence.

[0115] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by a second AAV ITR sequence, with any combination of at least one promoter sequence, at least one transgene sequence, and at least one polyA sequence between the first and second insulator sequences.

[0116] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0117] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, in the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0118] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by a second AAV ITR sequence.

[0119] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one DNA spacer sequence between the second piggyBac ITR sequence and the second AAV ITR sequence.

[0120] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0121] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0122] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by at least one DNA spacer sequence, followed by the second AAV ITR sequence.

[0123] In a non-limiting example of a preceding AAV piggyBac transposon polynucleotide, the at least one transgene sequence can comprise a nucleic acid sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide. In certain embodiments, the nucleotide sequence encoding the hPAH is codon-optimized. In certain embodiments, the codon-optimized PAH gene is further modified to remove i) any internal TTAA sites; ii) any undesirable restriction enzyme recognition sites; and iii) potential cryptic splice sites for cloning the modified sequence into an AAV piggyBac transposon vector. This non-limiting example of an AAV piggyBac transposon polynucleotide is shown in Figure 1.

[0124] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one enhancer sequence, at least one promoter sequence, a first transgene sequence, at least one 3' UTR, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0125] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one enhancer sequence, at least one promoter sequence, a first transgene sequence, at least one 3' UTR, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, and a second AAV ITR sequence.

[0126] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by at least one enhancer sequence, followed by at least one promoter sequence, followed by a first transgene sequence, followed by at least one 3' UTR, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by a second AAV ITR sequence.

[0127] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise at least one DNA spacer sequence between the second piggyBac ITR sequence and the second AAV ITR sequence.

[0128] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one enhancer sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0129] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, at least one enhancer sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0130] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by at least one enhancer sequence, followed by at least one promoter sequence, followed by at least one transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by at least one DNA spacer sequence, followed by the second AAV ITR sequence.

[0131] In some embodiments, an AAV piggyBac transposon polynucleotide can comprise two or more enhancer sequences. In some embodiments in which an AAV piggyBac transposon polynucleotide comprises two or more enhancer sequences, the enhancer sequences can be the same or the enhancer sequences can be different.

[0132] The AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a first enhancer sequence, a second enhancer sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0133] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, from the 5' to 3' direction, a first AAV ITR sequence, a first piggyBac ITR sequence, a first insulator sequence, a first enhancer sequence, a second enhancer sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBac ITR sequence, at least one DNA spacer sequence, and a second AAV ITR sequence.

[0134] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise a first AAV ITR sequence, followed by a first piggyBac ITR sequence, followed by a first insulator sequence, followed by a first enhancer sequence, followed by a second enhancer sequence, followed by at least one promoter sequence, followed by at least one transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBac ITR sequence, followed by at least one DNA spacer sequence, followed by the second AAV ITR sequence.

[0135] In a non-limiting example of a preceding AAV piggyBac transposon polynucleotide, the at least one transgene sequence can comprise a nucleic acid sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide. In certain embodiments, the nucleotide sequence encoding the hPAH is codon-optimized. In certain embodiments, the codon-optimized PAH gene is further modified to remove i) any internal TTAA sites; ii) any undesirable restriction enzyme recognition sites; and iii) potential cryptic splice sites for cloning the modified sequence into an AAV piggyBac transposon vector. This non-limiting example of an AAV piggyBac transposon polynucleotide is shown in Figure 2.

[0136] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:18.

[0137] In some embodiments, the AAV piggyBac transposon polynucleotide can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:21.

[0138] AAV ITR sequences

[0139] In some embodiments, the AAV ITR sequences can comprise any AAV ITR sequence known in the art. In some embodiments, the AAV ITR sequences can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to any one of the sequences set forth in SEQ ID NOs: 1 and 16.

[0140] In some embodiments, the first AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:1, and the second AAV ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:16.

[0141] piggyBac ITR sequences

[0142] In some embodiments, the piggyBac ITR sequence can comprise any piggyBac ITR sequence known in the art. In some embodiments, the piggyBac ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of the sequences set forth in SEQ ID NOs: 2 and 14.

[0143] In some embodiments, the first piggyBac ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:2, and the second piggyBac ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:14.

[0144] In some embodiments of the methods of the present disclosure, the piggyBac ITR sequences, such as the first piggyBac ITR sequence and / or the second piggyBac ITR sequence in an AAV piggyBac transposon, can comprise, consist essentially of, or consist of Sleeping Beauty transposon ITRs, Helraiser transposon ITRs, Tol2 transposon ITRs, TcBuster transposon ITRs, or any combination thereof.

[0145] In some embodiments, the piggyBac ITR sequences of the present disclosure are selected from the following sequences: 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'-T It may be flanked on one or both ends by at least one of AAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-C AAA-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'. In some embodiments, the piggyBac ITR sequences can be flanked by 5'-TTAA-3'.Thus, any AAV piggyBac transposon polynucleotide of the present disclosure can further comprise any one of the following: 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'-T AAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-C AAA-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.

[0146] Mutant piggyBac ITR sequences having at least one nucleic acid substitution compared to the wild-type ITR of the piggyBac transposon can enhance the efficacy and efficiency of transposition. An exemplary mutant piggyBac ITR sequence includes the 35TCC mutation. Further exemplary mutant piggyBac ITR sequences include those disclosed in International Patent Application No. PCT / US2022 / 77544, the contents of which are incorporated herein by reference in their entirety.

[0147] In some embodiments, a mutant piggyBac ITR sequence containing a 35TCC mutation can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:19.

[0148] In some embodiments, the first piggyBac ITR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:2, and the second piggyBac ITR sequence can comprise a 35TCC mutation and can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:19.

[0149] piggyBac 3'UTR sequence

[0150] In some embodiments, the piggyBac 3'UTR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:3.

[0151] piggyBac 5'UTR sequence

[0152] In some embodiments, the piggyBac 5'UTR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:13.

[0153] Insulator array

[0154] In some embodiments, the insulator sequence can comprise any insulator sequence known in the art. In some embodiments, the insulator sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to any of the sequences set forth in SEQ ID NOs: 4 and 12.

[0155] In some embodiments, the first insulator sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:4, and the second insulator sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any of SEQ ID NO:12.

[0156] Promoter sequence

[0157] In some aspects, the promoter sequence can include any promoter sequence known in the art, hi some aspects, the promoter sequence can include any liver-specific promoter sequence known in the art.

[0158] In some embodiments, the promoter sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:7.

[0159] In some embodiments, the promoter sequence can comprise a TTRm promoter sequence, which can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:7.

[0160] Transgene sequence

[0161] In some embodiments, the transgene sequence can comprise a nucleic acid sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide. In some embodiments, the nucleic acid sequence encoding the hPAH polypeptide can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:9.

[0162] In some embodiments, the transgene sequence can include a tag, such as a hemagglutinin (HA) tag. Such tags are well known in the art and are useful for protein purification.

[0163] In some aspects, the transgene sequence can be codon optimized according to methods known in the art.

[0164] In some embodiments, a nucleic acid sequence encoding a polypeptide (e.g., hPAH) can be a codon-optimized nucleic acid sequence that encodes the polypeptide. A codon-optimized nucleic acid sequence encoding a polypeptide can comprise, consist essentially of, or consist of a nucleic acid sequence that is 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or less (or any percentage therebetween) identical to a wild-type human nucleic acid sequence encoding the polypeptide.

[0165] In some embodiments, SEQ ID NO: 9 is a unique codon-optimized nucleic acid sequence that can be included in the polynucleotides, vectors and compositions of the disclosure.

[0166] In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide, such as that set forth in SEQ ID NO:9, may contain no donor splice sites. In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide may contain about one or fewer, or about two or fewer, or about three or fewer, or about four or fewer, or about five or fewer, or about six or fewer, or about seven or fewer, or about eight or fewer, or about nine or fewer, or about ten or fewer donor splice sites. In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide contains at least one, or at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight, or at least nine, or at least ten fewer donor splice sites compared to a wild-type human nucleic acid sequence encoding the polypeptide. Without wishing to be bound by theory, removal of donor splice sites in a codon-optimized nucleic acid sequence may unexpectedly and unpredictably increase expression of the polypeptide in vivo because cryptic splicing is prevented. Furthermore, cryptic splicing can differ between different subjects, meaning that the expression levels of polypeptides containing donor splice sites can vary unpredictably between different subjects.

[0167] In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide, such as that set forth in SEQ ID NO:9, can have a GC content that differs from the GC content of a wild-type human nucleic acid sequence encoding the polypeptide. In some embodiments, the GC content of a codon-optimized nucleic acid sequence encoding a polypeptide is more evenly distributed throughout the nucleic acid sequence compared to a wild-type human nucleic acid sequence encoding the polypeptide. Without wishing to be bound by theory, by distributing the GC content more evenly throughout the nucleic acid sequence, the codon-optimized nucleic acid sequence exhibits a more uniform melting temperature ("Tm") across the length of the transcript. Uniformity of melting temperature unexpectedly results in increased expression of the codon-optimized nucleic acid in a human subject, as transcription and / or translation of the nucleic acid sequence occurs with fewer stalls of polymerases and / or ribosomes.

[0168] In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide, such as that set forth in SEQ ID NO:9, exhibits at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 50%, at least a 75%, at least a 100%, at least a 200%, at least a 300%, at least a 500%, or at least a 1000% increase in expression in a human subject compared to a wild-type or non-codon-optimized nucleic acid sequence encoding the polypeptide.

[0169] In some embodiments, at least one transgene sequence may be operably linked to at least one promoter sequence present in the same polynucleotide.

[0170] Poly(A) sequence

[0171] In some embodiments, the polyA sequence can comprise any polyA sequence known in the art. Non-limiting examples of polyA sequences include, but are not limited to, the SV40 polyA sequence. In some embodiments, the insulator sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the sequence set forth in SEQ ID NO:11.

[0172] DNA spacer sequence

[0173] In some embodiments, the DNA spacer sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of the sequences set forth in SEQ ID NOs: 15 and 20.

[0174] The DNA spacer sequence can be positioned anywhere within the AAV piggyBac transposon polynucleotide or the AAV piggyBac transposase polynucleotide. In some embodiments, the DNA spacer sequence is positioned between the second piggyBac ITR and the 3' AAV ITR.

[0175] Enhancer sequence

[0176] In some aspects, the enhancer sequence can include any enhancer sequence known in the art, hi some aspects, the enhancer sequence can include any liver-specific enhancer sequence known in the art.

[0177] In some embodiments, the enhancer sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to any one of the sequences set forth in SEQ ID NOs: 5 and 6.

[0178] In some embodiments, the enhancer sequence can comprise a 3xhSERPINA1 enhancer sequence. The 3xhSERPINA enhancer sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:5.

[0179] In some embodiments, the enhancer sequence can comprise a TTR enhancer sequence, which can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:6.

[0180] 3'UTR sequence

[0181] In some embodiments, the 3' UTR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:10.

[0182] In some embodiments of the preceding transposon, the first 3' UTR sequence can be an AES-mtRNR 3' UTR sequence. The AES-mtRNR 3' UTR sequence can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:10.

[0183] Intron sequences

[0184] In some embodiments, the intron sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to any one of the sequences set forth in SEQ ID NOs: 8 and 17.

[0185] In some embodiments, the intron sequence can comprise an MVM intron sequence, which can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to SEQ ID NO:8.

[0186] In some embodiments, the intron sequence can comprise a synthetic intron sequence, which can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to SEQ ID NO: 17.

[0187] Transposase sequence

[0188] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding any transposase polypeptide known in the art. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a piggyBac® (PB) transposase polypeptide. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a piggyBac-like® (PBL) transposase polypeptide. In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Super piggyBa® (SPB) transposase polypeptide.

[0189] Non-limiting examples of PB transposons and PB, PBL and SPB transposases are described in detail in U.S. Patent Nos. 6,218,182; 6,962,810; 8,399,643 and PCT Publication No. WO 2010 / 099296.

[0190] PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of transposons and insert their contents between the ITRs of the sequence 5'-TTAA-3' within the chromosomal site (TTAA target sequence). The target sequences of PB and PBL transposons are 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', and 5'-TTAC-3'. The PB or PBL transposon system can include or consist of the following ITRs: AAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-AAAT-3', 5'-AATC-3', 5'-ACAA-3', 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 or PBL transposon system does not limit the payload of the gene of interest that can be included between the ITRs.

[0191] Exemplary amino acid sequences of one or more PB, PBL, and SPB transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; and U.S. Patent No. 8,399,643. In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:22.

[0192] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 22. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 22, wherein the amino acid substitution at position 30 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 can be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:23.

[0193] In certain embodiments, where the transposase comprises the above-described mutations at positions 30, 165, 282 and / or 538, the PB, PBL and SPB transposases comprise the mutations at positions 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 260, 262, 264, 266, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 3 , 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570 and 591, and are described in more detail in PCT Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816.

[0194] In preferred embodiments, the PB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:24.

[0195] The PB or PBL transposase can comprise or consist of an amino acid sequence having an amino acid substitution at two or more, three or more, or each of positions 29, 164, 281, and / or 537 of the sequence of SEQ ID NO: 24. The transposase can be an SPB transposase comprising or consisting of the amino acid sequence of SEQ ID NO: 24, wherein the amino acid substitution at position 29 can be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 164 can be a substitution of serine (S) for glycine (G), the amino acid substitution at position 281 can be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 537 can be a substitution of lysine (K) for asparagine (N). In preferred embodiments, the SPB transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:25.

[0196] In certain embodiments, where the transposase comprises the above-described mutations at positions 29, 164, 281 and / or 537, the PB, PBL and SPB transposases comprise the mutations at positions 2, 45, 81, 102, 118, 124, 176, 179, 184, 186, 199, 206, 208, 225, 234, 239, 240, 242, 257 of the sequence of SEQ ID NO:24 or SEQ ID NO:25. , 295, 297, 310, 314, 318, 326, 327, 339, 420, 435, 455, 469, 485, 502, 551, 569 and 590, and are described in more detail in PCT Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816.

[0197] The PB, PBL, or SPB transposase can be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in further detail in PCT Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insect Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. B ADI 1135).

[0198] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185; U.S. Patent No. 6,962,810; U.S. Patent No. 8,399,643; and International Publication No. WO 2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0199] In some embodiments, PB, PBL or SPB transposase can be fused to a nuclear localization signal. Examples of PB, PBL or SPB transposase fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643 and International Publication No. WO 2019 / 173636. The nuclear localization signal can comprise, consist essentially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage therebetween) identical amino acid sequence of SEQ ID NO: 26. The nuclear localization signal can be encoded by a nucleic acid sequence comprising, consisting essentially of, or consisting of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO:27.

[0200] In some embodiments, the nuclear localization signal can be fused to the PB, PBL, or SPB transposase using a G4S linker located between the NLS and the PB, PBL, or SPB. The G4S linker can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 28. The G4S linker can be encoded by a nucleic acid sequence that comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 29.

[0201] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding an SBP transposase polypeptide fused to an NLS, wherein the SBP transposase polypeptide fused to an NLS comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 30. In some embodiments, the nucleic acid sequence encoding the SBP transposase polypeptide fused to an NLS can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of the sequences set forth in SEQ ID NO: 31.

[0202] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding an SBP transposase polypeptide fused to an NLS, wherein the SBP transposase polypeptide fused to an NLS comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 32. In some embodiments, the nucleic acid sequence encoding the SBP transposase polypeptide fused to an NLS can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of the sequences set forth in SEQ ID NO: 33.

[0203] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Sleeping Beauty transposase polypeptide (e.g., as disclosed in U.S. Pat. No. 9,228,180). In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Hyperactive Sleeping Beauty (SB100X) transposase polypeptide. In some embodiments, the Sleeping Beauty transposase comprises or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 34 or 35. In preferred embodiments, the hyperactive Sleeping Beauty (SB100X) transposase comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to SEQ ID NO: 36 or 37.

[0204] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Helitron transposase polypeptide (e.g., as disclosed in WO 2019 / 173636). In some embodiments, the Helitron transposase polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 38 or 39.

[0205] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a Tol2 transposase polypeptide (e.g., as disclosed in WO 2019 / 173636). In some embodiments, the Tol2 transposase polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 40 or 41.

[0206] In some embodiments, the transposase sequence can comprise a nucleic acid sequence encoding a TcBuster transposase polypeptide (e.g., as disclosed in WO 2019 / 173636) or a mutant TcBuster transposase polypeptide (described in more detail in PCT Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816). In some embodiments, the TcBuster transposase polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 42 or 43. A polynucleotide encoding a TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0207] Vectors of the present disclosure

[0208] The present disclosure provides a composition comprising a vector, wherein the vector comprises at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide. A vector comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide is referred to herein as an "AAV piggyBac transposon vector."

[0209] The present disclosure provides compositions comprising a vector, wherein the vector comprises at least one AAV transposase polynucleotide. A vector comprising at least one AAV transposase polynucleotide is referred to herein as an "AAV transposase vector."

[0210] The vector of the present disclosure can be a viral vector or a recombinant vector.The viral vector can comprise a sequence isolated or derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or any combination thereof.The viral vector can comprise a sequence isolated or derived from an adeno-associated virus (AAV).The viral vector can comprise a recombinant AAV (rAAV).

[0211] Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids comprising the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, AAV8, AAV9, rAAV-LK03, AAV-KP-1 (also known as AAV-KP1, described in detail in Kerun el al. JCI Insight, 2019;4(22):el31610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018;26(1):289-303).

[0212] The present disclosure provides a composition comprising a plurality of AAV-KP-1 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide.The present disclosure provides a composition comprising a plurality of AAV-NP59 particles comprising at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide.

[0213] The present disclosure provides compositions comprising multiple AAV-NP59s (described in detail in Paulk et al. Molecular Therapy, 2018;26(1):289-303).

[0214] The present disclosure provides a composition comprising a plurality of AAV8 particles containing at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide, and an LNP composition comprising an mRNA encoding a transposase. In certain embodiments, the mRNA molecule further comprises a 5'-CAP. In certain embodiments, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase. In certain embodiments, the transposase is a Super piggyBac™ (SPB) transposase. In certain embodiments, at least one LNP composition comprises about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis.

[0215] The present disclosure provides a composition comprising a plurality of AAV9 particles containing at least one adeno-associated virus (AAV) piggyBac transposon polynucleotide, and an LNP composition comprising an mRNA encoding a transposase. In certain embodiments, the mRNA molecule further comprises a 5'-CAP. In certain embodiments, the transposase is piggyBac™ (PB) transposase, piggyBac-like (PBL) transposase, Super piggyBac™ (SPB) transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or a mutant TcBuster transposase. In certain embodiments, the transposase is Super piggyBac™ (SPB) transposase. In certain embodiments, at least one LNP composition comprises about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis.

[0216] The viral vectors and viral particles of the present disclosure can be produced using standard methods known in the art.

[0217] The cell delivery compositions (e.g., polynucleotides, vectors) disclosed herein can include nucleic acids encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication No. WO 2019 / 173636 and International Patent Application No. PCT / US2019 / 049816. Therapeutic proteins can also include, but are not limited to, any one of the polypeptides described herein as part of a transgene sequence (e.g., hPAH).

[0218] Formulations, dosages and modes of administration

[0219] The present disclosure provides formulations, dosages and methods of administration of the compositions described herein.

[0220] The disclosed compositions and pharmaceutical compositions can further comprise at least one of any suitable auxiliary agent, such as, but not limited to, a diluent, a binder, a stabilizer, a buffer, a salt, a lipophilic solvent, a preservative, an adjuvant, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples of such sterile solutions and methods for their preparation are well known in the art, such as, but not limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 and "Physician's Desk Reference," 52nd ed., Medical Economics (Montvale, NJ) 1998.

[0221] Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and / or stability of the protein scaffold, fragment or variant compositions known in the art or described herein.

[0222] For example, the disclosed LNP compositions of the present disclosure can further comprise a diluent. In some embodiments, the diluent can be phosphate buffered saline (PBS). In some compositions, the diluent can be sodium acetate.

[0223] 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; derivatized sugars, such as alditols, aldonic acids, and esterified sugars; and polysaccharides or sugar polymers), which can be present alone or in combination and may comprise 1 to 99.99% by weight or volume, alone or in combination. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (EISA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. One preferred amino acid is glycine.

[0224] Non-limiting examples of carbohydrate excipients suitable for use include monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myo-inositol, etc. Preferably, the carbohydrate excipient is mannitol, trehalose, and / or raffinose.

[0225] The composition can also contain a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, and phthalic acid, as well as Tris, tromethamine hydrochloride, and phosphate buffers. Preferred buffers are organic acid salts such as citrate.

[0226] Additionally, the disclosed compositions can include polymeric excipients / additives such as polyvinylpyrrolidone, Ficoll (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycol, 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).

[0227] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein.Non-limiting examples of administration modes include bolus, buccal, injection, intra-articular, intrabronchial, intraperitoneal, intravesical, intrachondral, intracavity, intracavity, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intralesional, intramuscular, intramyocardial, intranasal, intraocular, intramedullary, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intravesical, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intratumoral, intravenous, intravesical, oral, parenteral, rectal, sublingual, subcutaneous, transdermal or intravaginal means.

[0228] The compositions of the present disclosure may be used for parenteral (subcutaneous, intramuscular, or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories; for buccal or sublingual administration, for example, but not limited to, in the form of tablets or capsules; or intranasally, such as, but not limited to, in the form of powders, nasal sprays, or aerosols or certain pharmaceutical agents; or with chemical enhancers such as dimethyl sulfoxide to modify skin structure or increase drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, D.S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994), or oxidizing agents that allow for application of protein and peptide-containing formulations to the skin (WO 98 / 53847), or the application of electric fields to create transient transport pathways, such as electroporation, or to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0229] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, granules, powder, or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle, or may be provided separately. Parenteral formulations may contain common excipients such as sterile water or saline, polyalkylene glycols such as polyethylene glycol, vegetable oils, hydrogenated naphthalenes, etc. Aqueous or oily suspensions for injection can be prepared according to known methods using appropriate emulsifiers or wetting agents and suspending agents. Injectable preparations may be non-toxic, non-oral diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, etc., and sterile fixed oils can be used as common solvents or suspension media. For these purposes, any type of fixed oil or fatty acid, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic mono-, di-, or triglycerides, can be used. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas-pressurized needleless injection devices such as those described in U.S. Pat. No. 5,851,198, and laser perforation devices such as those described in U.S. Pat. No. 5,839,446.

[0230] Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and nonionic surfactants, such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall, and enzyme inhibitors (e.g., pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. Formulations for delivering hydrophilic agents, including proteins and protein backbones, and combinations of at least two surfactants intended for oral, buccal, mucosal, nasal, pulmonary, transvaginal, or rectal administration are described in U.S. Patent No. 6,309,663. The active ingredient compound in a solid dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, rafmose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides. These dosage forms can also contain other types of additives, such as inert diluents, lubricants, preservatives, antioxidants, such as magnesium stearate, parabens, preservatives, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, perfumes, etc.

[0231] Tablets and pills can be further processed into enteric-coated preparations. Liquid preparations for oral administration include pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, solutions, etc. These preparations can contain inert diluents commonly used in the field, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carrier compounds described in U.S. Pat. Nos. 5,879,681 and 5,871,753, which are used for oral delivery of biologically active agents, are known in the art.

[0232] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower respiratory tract of the lungs or paranasal sinuses. The compositions or pharmaceutical compositions can be delivered by any of a variety of inhalation or nasal devices known in the art for administering therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations in a patient's paranasal sinuses or alveoli, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers, etc.), dry powder generators, atomizers, and the like. All such devices can employ formulations suitable for aerosolizing the compositions or pharmaceutical compositions described herein. Such aerosols can be composed of solutions (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be produced by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In metered-dose inhalers (MDIs), a propellant, the compositions or pharmaceutical compositions described herein, and excipients or other additives are contained in a canister as a mixture with a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol containing particles preferably in the size range of less than about 10 pm, preferably about 1 pm to about 5 pm, and most preferably about 2 pm to about 3 pm. A more detailed description of pulmonary administration, formulations, and related devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0233] For absorption through mucosal surfaces, the composition comprises an emulsion containing a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Patent No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, etc. Formulations for nasal administration may be solid and may contain excipients such as lactose, and may be aqueous or oily solutions for nasal drops. For buccal administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc. (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO 2019 / 049816.

[0234] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device such as a liposome or polymer nanoparticle, microparticle, microcapsule, or microsphere (collectively referred to as microparticles unless otherwise specified). Several suitable devices are known, including microparticles made of polyhydroxy acids such as polylactic acid, polyglycolic acid and its copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, as well as synthetic polymers such as collagen, polyamino acids, natural polymers such as albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication No. WO 2019 / 049816.

[0235] It may be desirable to administer the disclosed compounds to a subject for an extended period of time, for example, from one week to one year after a single administration. Various sustained-release, depot, and implant formulations are available. For example, the dosage form may include a pharmaceutically acceptable non-toxic salt of a compound that is poorly soluble in body fluids, such as (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 acid, polygalacturonic acid, etc.; (b) a salt with a polyvalent metal cation, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or a salt with an organic cation, such as formed from N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate. Furthermore, the disclosed compounds, or preferably relatively insoluble salts such as those just described, can be formulated into gels suitable for injection, such as aluminum monostearate gels containing sesame oil. Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like. Another type of sustained-release depot formulation for injection contains the compound or salt dispersed for encapsulation in a slowly degrading, non-toxic, non-antigenic polymer such as polylactic acid / polyglycolic acid polymer, as described, for example, in U.S. Pat. No. 3,773,919. Such compounds, or preferably relatively insoluble salts, can also be formulated into cholesterol-matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gas or liquid liposomes, are known in the literature (U.S. Pat. No. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems," J.R. Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0236] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, California (2000); Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ. Preferred doses can optionally include about 0.1-99 and / or 100-500 mg / kg / administration, or any range, value, or fraction thereof, or can achieve a serum concentration of about 0.1-5000 pg / ml per single or multiple administration, or per any range, value, or fraction thereof. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are from about 1 mg / kg to about 3, about 6, or about 12 mg / kg of subject body weight.

[0237] Alternatively, dosages can vary depending on known factors such as the pharmacodynamic properties of the particular drug, its mode and route of administration, the recipient's age, health, and weight, the nature and severity of symptoms, the type of concurrent treatment, the frequency of treatment, and the desired effect. Typically, the dosage of the active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. To achieve the desired results, dosages of 0.1 to 50, preferably 0.1 to 10 milligrams per kilogram per administration, or sustained-release forms, are usually effective.

[0238] As a non-limiting example, human or animal treatment may be provided as a single or periodic dose of about 0.1-100 mg / kg per day, or any range, value, or fraction thereof, of a composition or pharmaceutical composition disclosed herein, at least once on days 1-40, or alternatively or additionally, at least once on weeks 1-52, or alternatively or additionally, at least once on years 1-20, or any combination thereof, using a single dose, an infusion, or multiple doses.

[0239] Dosage forms suitable for internal administration generally contain from about 0.001 milligrams to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is typically present in an amount of from about 0.5 to 99.999% by weight based on the total weight of the composition.

[0240] An effective amount, as performed and determined using known methods described herein or known in the relevant art, can include an amount of about 0.001 to about 500 mg / kg per single (e.g., bolus), multiple, or continuous administration, or an amount to achieve a serum concentration of 0.01 to 5000 pg / ml per single, multiple, or continuous administration, or any effective range or value therein.

[0241] In embodiments where the composition administered to a subject in need thereof is a modified cell disclosed herein, the cells are about 1 x 10 3 ~1×10 15 1 x 10 cells 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells; approximately 1 x 10 5 ~1×10 10 cells; approximately 1 x 10 6 ~1×10 9 cells; approximately 1 x 10 6 ~1×10 8 cells; approximately 1 x 10 6 ~1×10 7 cells; or approximately 1 x 10 6 ~25×10 6In one embodiment, the cells may be about 5 x 10 6 ~25×10 6 It is administered in individual cells.

[0242] A more detailed description of the pharmaceutically acceptable excipients, formulations, dosages and methods of administration of the disclosed compositions and pharmaceutical compositions is disclosed in PCT Publication No. WO 2019 / 049816.

[0243] Any of the uses and methods of the present disclosure can include administering an effective amount of any of the compositions or pharmaceutical compositions disclosed herein to a cell, tissue, organ, animal, or subject in need of such modulation, treatment, or therapy. Such methods can optionally further include co-administration or combination therapy to treat such a disease or disorder, where the administration of any of the compositions or pharmaceutical compositions disclosed herein further includes administering at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical) before and / or after the simultaneous administration.

[0244] In some embodiments, after administration, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG). In certain embodiments, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is intravenous injection or infusion. In certain embodiments, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0245] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is one of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 doses or any number therebetween that are produced simultaneously. In some embodiments, when the composition is autologous or allogeneic cells, the dose is sufficient to allow the cells to engraft and / or persist for a sufficient time to treat a disease or disorder.

[0246] Methods of the present disclosure

[0247] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting the cell, tissue, organ, animal, or subject with a therapeutically effective amount of the composition or pharmaceutical composition. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0248] The present disclosure provides methods for treating phenylketonuria (PKU) in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a composition comprising: (a) a polynucleotide, vector, or pharmaceutical composition described in any one of the preceding claims; and (b) at least one LNP composition comprising at least one mRNA molecule encoding a transposase. In certain embodiments, the mRNA molecule further comprises a 5'-CAP. In certain embodiments, the transposase is piggyBac™ (PB) transposase, piggyBac-like (PBL) transposase, Super piggyBac™ (SPB) transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or a mutant TcBuster transposase. In certain embodiments, the transposase is Super piggyBac™ (SPB) transposase. In certain embodiments, at least one LNP composition comprises about 54% ssPalmO-Ph-P4C2 on a molar basis, about 35% cholesterol on a molar basis, about 10% DOPC on a molar basis, and about 1% DMG-PEG2000 on a molar basis.

[0249] The present disclosure provides at least one composition of the present disclosure for use in treating PKU in a subject, wherein the at least one composition is for administration to the subject in at least one therapeutically effective amount.

[0250] The present disclosure provides use of at least one composition of the present disclosure for the manufacture of a medicament for treating PKU in a subject, wherein the at least one composition is for administration to the subject in at least one therapeutically effective amount.

[0251] The present disclosure provides methods for treating at least one disease in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0252] In some embodiments, the nucleic acid molecules formulated in the compositions of the present disclosure can comprise at least one transgene sequence, hi some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein.

[0253] In some embodiments, the nucleic acid molecule formulated in the composition of the present disclosure, comprising an AAV piggyBac transposon polynucleotide, can comprise at least one transgene sequence. In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transgene sequence can comprise a nucleotide sequence encoding at least one transposon.

[0254] In some embodiments, the at least one sequence encoding at least one therapeutic protein can be a sequence encoding a human phenylalanine hydroxylase (hPAH) polypeptide, wherein the hPAH polypeptide comprises the nucleic acid sequence of SEQ ID NO: 9. In certain embodiments, the nucleotide sequence encoding hPAH is codon-optimized.

[0255] In some embodiments, the hPAH polypeptide comprises, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical (or any percentage in between) to SEQ ID NO:9.

[0256] In some embodiments of the preceding methods, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be a composition comprising at least one LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. Accordingly, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of a composition comprising a nucleic acid comprising a transposon, wherein the transposon encodes at least one therapeutic protein, and b) at least one therapeutically effective amount of the LNP of the present disclosure, wherein the LNP comprises at least one nucleotide sequence encoding at least one transposase.

[0257] In some embodiments of the preceding methods, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposon can be a composition comprising an adeno-associated virus (AAV) viral vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. Accordingly, the present disclosure provides a method for treating at least one disease in a subject, comprising administering to the subject: a) at least one therapeutically effective amount of AAV viral vector particles comprising at least one nucleic acid comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. In certain methods, the nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase is mRNA.

[0258] In some embodiments of the preceding methods, the composition comprising the AAV viral vector particles can comprise at least one AAV piggyBac transposon polynucleotide. Accordingly, the present disclosure provides methods of treating at least one disease in a subject, comprising administering to the subject: a) a therapeutically effective amount of at least one composition of AAV viral vector particles comprising at least one AAV piggyBac transposon polynucleotide and at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, and b) a therapeutically effective amount of at least one composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0259] The present disclosure further provides a method for treating at least one disease in a subject, the method comprising administering to the subject: (a) at least one therapeutically effective amount of AAV viral vector particles comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and (b) at least one therapeutically effective amount of LNPs of the present disclosure, wherein the LNPs comprise at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. In certain methods, the nucleic acid molecule comprising the nucleotide sequence encoding at least one transposase is mRNA.

[0260] In some embodiments of the preceding method, the composition comprising the AAV viral vector particle can comprise at least one AAV piggyBac transposon polynucleotide. Thus, the present disclosure provides a method for treating at least one disease in a subject, comprising: (a) administering to the subject at least one therapeutically effective amount of a composition of AAV viral vector particles comprising at least one AAV piggyBac transposon polynucleotide and at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and (b) administering to the subject at least one therapeutically effective amount of the LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. In certain methods, the nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase is mRNA.

[0261] In a non-limiting example, an AAV viral vector particle comprises at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, wherein the therapeutic protein is hPAH, and which can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% (or any percentage in between) identical to any one of SEQ ID NOs: 18 and 21.

[0262] In some embodiments of the preceding methods, a composition comprising a nucleic acid molecule comprising a transposon that includes a nucleotide sequence encoding at least one therapeutic protein and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered simultaneously. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon that includes a nucleotide sequence encoding at least one therapeutic protein and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered sequentially. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon that includes a nucleotide sequence encoding at least one therapeutic protein and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered in close temporal proximity.

[0263] As used herein, the term "proximal in time" refers to the administration of one therapeutic composition (e.g., a composition comprising a transposon) occurring within a period of time before or after the administration of another therapeutic composition (e.g., a composition comprising a transposase), such that the therapeutic effect of one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, "proximal in time" refers to the administration of one therapeutic agent occurring within a period of time before or after the administration of another therapeutic agent, such that there is a synergistic effect between one therapeutic agent and the other therapeutic agent. "Proximal in time" can vary according to various factors, including, but not limited to, the age, sex, weight, genetic background, medical condition, disease history, and treatment history of the subject to whom the therapeutic agent is administered; the disease or condition being treated or ameliorated; the therapeutic result to be achieved; the dosage, frequency, and duration of administration of the therapeutic agent; the pharmacokinetics and pharmacodynamics of the therapeutic agent; and the route(s) by which the therapeutic agent is administered. In some embodiments, "proximate in time" means within 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 8 weeks. In some embodiments, multiple administrations of one therapeutic agent can be administered in close temporal proximity to a single administration of another therapeutic agent. In some embodiments, the temporal proximity can vary during a treatment cycle or dosing regimen.

[0264] In a non-limiting example, the present disclosure provides a method for treating PKU in a subject, comprising administering to the subject: (a) at least one therapeutically effective amount of AAV viral vector particles comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and (b) at least one therapeutically effective amount of LNPs of the present disclosure, wherein the LNPs comprise at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the at least one therapeutic protein comprises a human phenylalanine hydroxylase (hPAH) polypeptide.

[0265] In some embodiments of the therapeutic methods of the present disclosure, administration of at least one composition and / or nanoparticle of the present disclosure to a subject can result in expression of an exogenous protein (e.g., a therapeutic protein, a transposase, etc.) in at least one organ and / or tissue of the subject.

[0266] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells in the tissue and / or organ.

[0267] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of a particular subset or subsets of cells in a tissue and / or organ.

[0268] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in tissues and / or organs for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0269] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in a specific subset or subsets of cells in a tissue and / or organ for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0270] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in tissues and / or organs for about 1 day or less, or about 2 days or less, or about 3 days or less, or about 4 days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less.

[0271] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in a specific subset or subsets of cells within a tissue and / or organ for about 1 day or less, or about 2 days or less, or about 3 days or less, or about 4 days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less.

[0272] The present disclosure provides a method of treating at least one disease in a subject, the method comprising: enu2 The present invention provides a method for producing a gene encoding a Pah β-glucanase (Ph β-glucanase) containing a missense mutation (F263S) that inactivates the Pah β-glucanase (Ph β-glucanase). enu2 The subject exhibits classic PKU with elevated blood phenylalanine (Phe) levels, cognitive impairment, and maternal PKU syndrome. See, for example, Charron C. et al., Molecular Therapy Vol. 11, Supplement 1, May 2005, S163-S164.

[0273] In some aspects, the present disclosure provides enu2Provided is a method for treating PKU in a subject, comprising administering to the subject: (a) at least one therapeutically effective amount of AAV viral vector particles comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and (b) at least one therapeutically effective amount of LNPs of the present disclosure, wherein the LNPs comprise at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the at least one therapeutic protein comprises a human phenylalanine hydroxylase (hPAH) polypeptide.

[0274] Cells and modified cells of the present disclosure

[0275] The cells and modified cells of the present disclosure can be mammalian cells. Preferably, the cells and modified cells are human cells. In one aspect, the cells targeted for modification using the LNP composition of the present disclosure are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells. In one embodiment, the LNP composition comprises at least one mRNA molecule encoding a transposase, and the modified cells are generated in vivo. In one embodiment, the LNP composition comprises at least one DNA molecule encoding a transposon, and the modified cells are generated in vivo. In one embodiment, the transposon comprises a nucleotide sequence encoding a therapeutic gene operably linked to a liver-specific promoter.

[0276] The cells and modified cells of the present disclosure may be somatic cells. The cells and modified cells of the present disclosure may be differentiated cells. The cells and modified cells of the present disclosure may be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells may be any type of cell. Allogeneic cells may be stem cells or may be derived from stem cells. Allogeneic cells may be differentiated somatic cells.

[0277] nucleic acid molecule

[0278] The nucleic acid molecule of the present disclosure encoding a therapeutic protein may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be the coding strand, also known as the sense strand, or the non-coding strand, also known as the antisense strand.

[0279] The isolated nucleic acid molecules of the present disclosure include nucleic acid molecules containing an open reading frame (ORF), optionally containing one or more introns, such as, but not limited to, at least one specific enzymatically active portion of a therapeutic protein; nucleic acid molecules containing a coding sequence for a therapeutic protein, and nucleic acid molecules containing a nucleotide sequence that is substantially different from the above-mentioned nucleotide sequence but still encodes a therapeutic protein described herein and / or known in the art due to the degeneracy of the genetic code. Of course, the genetic code is well known in the art. Therefore, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that encode a specific protein scaffold of the present disclosure. See, for example, Ausubel, et al., supra, and such nucleic acid variants are included in the present disclosure.

[0280] As described herein, nucleic acid molecules of the present disclosure, including nucleic acid molecules encoding a Therapeutic protein, can include, but are not limited to, those that themselves encode the amino acid sequence of an enzymatically active fragment of a Therapeutic protein; coding sequences for the entire Therapeutic protein or portions thereof; coding sequences for at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, along with additional non-coding sequences, including non-coding 5' and 3' sequences, such as transcribed, non-translated sequences that play a role in mRNA processing, including transcription, splicing, and polyadenylation signals (e.g., ribosome binding and mRNA stability); and additional coding sequences encoding additional amino acids, such as those that provide additional functionality. Thus, the sequence encoding a Therapeutic protein can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused Therapeutic protein.

[0281] Nucleic acid construction

[0282] The isolated nucleic acids of the present disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, as are known in the art.

[0283] The nucleic acid can conveniently contain sequences in addition to the polynucleotide of the present disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to facilitate isolation of the polynucleotide. Also, a translatable sequence can be inserted to facilitate isolation of the translated polynucleotide of the present disclosure. For example, a hexa-histidine marker sequence provides a convenient means for purifying the protein of the present disclosure. The nucleic acid of the present disclosure, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotide of the present disclosure.

[0284] Additional sequences can be added to such cloning and / or expression sequences to optimize function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra).

[0285] Recombinant methods for constructing nucleic acids

[0286] The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA, the construction of cDNA and genomic libraries are well known to those skilled in the art. (See, for example, Ausubel, supra; or Sambrook, supra).

[0287] Nucleic Acid Screening and Isolation Methods

[0288] Probes based on the sequences of the polynucleotides of this disclosure can be used to screen cDNA or genomic libraries. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes from the same or different organisms. Those skilled in the art will understand that varying degrees of hybridization stringency can be employed in the assay, and that either the hybridization or wash medium can be stringent. The more stringent the hybridization conditions, the higher the degree of complementarity between the probe and target must be for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent (such as formamide). For example, hybridization stringency can be conveniently varied by changing the polarity of the reaction solution, e.g., by manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies depending on the stringency of the hybridization medium and / or wash medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value therebetween, although it should be understood that minor sequence differences between the probe and primer can be compensated for by reducing the stringency of the hybridization and / or wash solutions.

[0289] Methods for amplifying RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teachings and guidance provided herein.

[0290] Known methods for amplifying DNA or RNA include polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188 (Mullis, et al.); 4,795,699 and 4,921,794 (Tabor, et al.); 5,142,033 (Innis); 5,122,464 (Wilson, et al.); 5,091,310 (Innis); 5,066,584 (Gyllensten, et al.); 4,889,818 (Gelfand, et al.); 4,994,370 (Silver, et al.)). al; 4,766,067 (Biswas); 4,656,134 (Ringold), and RNA-mediated amplification using antisense RNA to a target sequence as a template for double-stranded DNA synthesis (U.S. Pat. No. 5,130,238 (Malek, et al), trade name NASBA), the entire contents of which are incorporated herein by reference (see, for example, Ausubel, supra; or Sambrook, supra).

[0291] For example, polymerase chain reaction (PCR) technology can be used to directly amplify the sequences of the polynucleotides and related genes of the present disclosure from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods are also useful for, for example, cloning nucleic acid sequences encoding proteins to be expressed, generating nucleic acids to be used as probes to detect the presence of desired mRNA in a sample, nucleic acid sequencing, and other purposes. Examples of techniques sufficient to guide one of skill in the art through in vitro amplification methods can be found in Berger, supra; Sambrook, supra; Ausubel, supra; Mullis, et al., U.S. Pat. No. 4,683,202 (1987); and Innis, et al., PCR Protocols: A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, the Advantage-GC Genomic PCR Kit (Clontech). Additionally, to improve the yield of long PCR products, for example, T4 gene 32 protein (Boehringer Mannheim) can be used.

[0292] Synthetic methods for constructing nucleic acids

[0293] The isolated nucleic acids of the present disclosure can also be prepared by direct chemical synthesis using known methods (see, for example, Ausubel, et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization using a DNA polymerase with the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA may be limited to base sequences up to about 100 bases, but longer base sequences can be obtained by ligating shorter base sequences.

[0294] Recombinant Expression Cassette

[0295] The present disclosure further provides a recombinant expression cassette comprising a nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure, for example, a cDNA or genomic sequence encoding a protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette typically consists of a polynucleotide of the present disclosure operably linked to a transcription initiation regulatory sequence that directs transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct the expression of the nucleic acid of the present disclosure.

[0296] In some embodiments, isolated nucleic acids that function as promoters, enhancers, or other elements can be introduced into appropriate locations (upstream, downstream, or in an intron) of non-heterologous forms of the polynucleotides of the disclosure to up- or down-regulate expression of the polynucleotides of the disclosure. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion, and / or substitution.

[0297] Expression vectors and host cells

[0298] The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one therapeutic protein by recombinant techniques, as known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each incorporated herein by reference).

[0299] The polynucleotide can be optionally ligated to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and transduced into host cells.

[0300] The DNA insert must be operably linked to a suitable promoter. The expression construct further contains a transcription initiation site, a termination site, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably initiates translation at a start codon and includes 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 mammalian or eukaryotic expression.

[0301] Expression vectors preferably, but optionally, include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encoding dihydrofolate reductase and conferring resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739), blasticidin (bsd gene), resistance genes for eukaryotic cell culture, and ampicillin, zeocin (Sh bla gene) for culturing in E. coli and other bacteria or prokaryotes. Examples of suitable host cell resistance genes include those encoding cytosine, ...

[0302] The expression vector preferably includes at least one selectable cell surface marker for isolating cells modified by the disclosed compositions and methods, but this is optional. The selectable cell surface markers of the present disclosure consist of a surface protein, glycoprotein, or group of proteins that distinguish a cell or a subset of cells from another defined subset of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the disclosed compositions or methods from cells not modified by the disclosed compositions or methods. Examples of such cell surface markers include, but are not limited to, "cluster designator" or "classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or combinations thereof. Cell surface markers include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21;124(8):1277-87).

[0303] Preferably, but optionally, the expression vector includes at least one selectable drug resistance marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure can include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0304] At least one protein scaffold of the present disclosure can be expressed in modified forms, such as fusion proteins, and can include not only secretion signals but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of the protein scaffold to improve stability and persistence in host cells during purification or subsequent handling and storage. Peptide moieties can also be added to the protein scaffold of the present disclosure to facilitate purification. Such regions can be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17, and 18.

[0305] Those skilled in the art are familiar with the many expression systems that can be used to express the nucleic acid molecules that encode the proteins of the present disclosure.Alternatively, the nucleic acid of the present disclosure can be expressed in host cells by turning on (through manipulation) the endogenous DNA that contains the protein backbone of the present disclosure.Such methods are well known in the art, for example, as described in U.S. Patent No. 5,580,734, U.S. Patent No. 5,641,670, U.S. Patent No. 5,733,746 and U.S. Patent No. 5,733,761, which are incorporated herein by reference in their entirety.

[0306] Illustrative examples of cell cultures useful for producing protein scaffolds, specific portions, or variants thereof are bacteria, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of cell monolayers, although mammalian cell suspensions or bioreactors can also be used. Several suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art, including, for example, COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL1610), and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Agl4, 293 cells, HeLa cells, and the like, all readily available from the American Type Culture Collection (Manassas, Virginia) (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma cells and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Agl4 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Agl4 cells.

[0307] Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to, an origin of replication: a promoter (e.g., a late or early SV40 promoter, a CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1α promoter (U.S. Pat. No. 5,266,491), at least one human promoter; an enhancer, and / or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., the SV40 large T Ag polyA addition site), and a transcription terminator sequence. See, e.g., Ausubel et al., supra; Sambrook, supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or available, for example, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0308] When eukaryotic host cells are used, polyadenylation or transcription terminator sequences are typically incorporated into vectors. An example of a terminator sequence is the polyadenylation sequence derived from the bovine growth hormone gene. Sequences for accurate splicing of transcripts can also be included. An example of a splicing sequence is the VP1 intron derived from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). In addition, as is known in the art, gene sequences for controlling replication in host cells can be incorporated into vectors.

[0309] Amino acid code

[0310] The amino acids that make up the protein backbone of the present disclosure are often abbreviated. Amino acid designations can be indicated by designating the amino acid by its single-letter code, its three-letter code, its name, or its three-nucleotide codon, as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). Therapeutic proteins of the present disclosure can contain one or more amino acid substitutions, deletions, or additions, either spontaneous or resulting from mutations and / or human manipulation, as specified herein. Amino acids in the therapeutic proteins of the present disclosure that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity (e.g., but not limited to, at least one neutralizing activity). Sites important for maintaining the activity of a therapeutic protein can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0311] As will be understood by those skilled in the art, the present disclosure includes at least one biologically active therapeutic protein of the present disclosure. A biologically active therapeutic protein has a specific activity that is at least 20%, 30%, or 40%, preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or greater of the specific activity of the native (non-synthetic), endogenous, or related known protein scaffold. Methods for assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those skilled in the art.

[0312] In another aspect, the present disclosure relates to therapeutic proteins and fragments described herein that have been modified by the covalent attachment of an organic moiety. Such modifications can produce protein backbone fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain aspects, the hydrophilic polymer group can have a molecular weight of about 800 to about 120,000 daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group can contain about 8 to about 40 carbon atoms.

[0313] The modified therapeutic proteins and fragments of the present disclosure can include one or more organic moieties covalently attached directly or indirectly to an antibody. Each organic moiety attached to a protein backbone or fragment of the present disclosure can independently be a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" encompasses monocarboxylic and dicarboxylic acids. A "hydrophilic polymer group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, therapeutic proteins modified by the covalent attachment of polylysine are encompassed by the present disclosure. Hydrophilic polymers suitable for modifying therapeutic proteins of the present disclosure can be linear or branched, and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers modifying therapeutic proteins of the present disclosure have a molecular weight of about 800 to about 150,000 daltons as separate molecular entities. For example, PEG 5000 and PEG 20,000 (the subscripts represent the average molecular weight of the polymer in daltons) can be used. The hydrophilic polymer group can be substituted with one to about six alkyl, fatty acid, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups can be prepared using appropriate methods. For example, a polymer containing an amine group can be coupled to the carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be coupled to a hydroxyl group on the polymer.

[0314] Fatty acids and fatty acid esters suitable for modifying the therapeutic proteins of the present disclosure may be saturated or contain one or more unsaturated units. Fatty acids suitable for modifying the protein backbone of the present disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-A9-octadecanoate (C18, oleic acid), all cis-Δ5.8.1 1.14-eicosatetraenoate (C20, arachidonic acid), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a straight or branched chain lower alkyl group, which can contain from 1 to about 12, preferably from 1 to about 6, carbon atoms.

[0315] Modified therapeutic proteins and fragments can be prepared using suitable methods, for example, by reaction with one or more modifying agents. "Modifying agent," as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) containing an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), etc. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be coupled to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be directly attached to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or can be attached via a linker moiety, such as a divalent C1-C12 group in which one or more carbon atoms can be replaced with a heteroatom, such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. Modifiers containing a linker moiety can be prepared, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or reacted with maleic anhydride to cyclize the resulting product to generate an activated maleimide derivative of a fatty acid (see, for example, WO 92 / 16221 to Thompson, et al., the teachings of which are incorporated herein by reference in their entirety).

[0316] Modified therapeutic proteins of the present disclosure can be produced by reacting a protein backbone or fragment with a modifying agent. For example, an amine-reactive modifying agent, such as an NHS ester of PEG, can be used to attach organic moieties to the protein backbone in a non-site-specific manner. Modified therapeutic proteins and fragments containing organic moieties attached to specific sites of the protein scaffold of the present disclosure can be prepared using suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnok Bioeng., 56(4):456-463 (1997)) and methods such as those described in Hermanson, G.T., Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0317] definition

[0318] As used throughout this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, reference to "a dose" includes a reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0319] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system.

[0320] For example, "about" means within one or more standard deviations. Alternatively, "about" can 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 respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a value. Where particular values ​​are described in the present application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable range of error for the particular value.

[0321] However, it will be understood that the compounds disclosed herein may be presented without a specified configuration (e.g., without a specified stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, the presentation of a compound herein without a specific configuration is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0322] It should be understood that the compounds described herein include the compounds themselves, as well as their salts, and optionally their solvates.Salts can be formed, for example, between an anion on the substituted compounds disclosed herein and a positively charged group (e.g., amino).Suitable anions include chloride, bromide, iodide, sulfate, hydrogen sulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronic acid, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate), etc.

[0323] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or a biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein as found in its natural environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular material and culture medium when produced by recombinant techniques, and is substantially free of chemical precursors and other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide, optimally, protein-encoding sequences). For example, in various embodiments, an 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 sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the disclosure or a biologically active portion thereof is recombinantly produced, optimally, the culture medium contains less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0324] The present disclosure provides fragments and variants of the disclosed DNA sequences, as well as proteins encoded by these DNA sequences. As used throughout this disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence, and thus the protein encoded thereby. A fragment of a DNA sequence consisting of a coding sequence may encode a protein fragment that retains the biological activity of the native protein, as described herein, and thus retains DNA recognition or binding activity to a target DNA sequence. Alternatively, a fragment of a DNA sequence useful as a hybridization probe generally does not encode a protein that retains biological activity or promoter activity. Thus, a fragment of a DNA sequence can range from at least about 20 nucleotides, about 50 nucleotides, or about 100 nucleotides, to the full-length polynucleotide of the present disclosure.

[0325] The nucleic acids or proteins of the present disclosure can be constructed by a modular approach, involving preassembling monomeric and / or repeating units in a target vector, which can then be assembled into a final destination vector. The polypeptides of the present disclosure can be constructed by a modular approach by preassembling repeating units in a target vector, which can be composed of repeating monomers of the present disclosure, which can then be assembled into a final destination vector. The present disclosure also provides polypeptides produced by this method, and nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells containing nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0326] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0327] The term "comprising" is intended to mean that the compounds, compositions, and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements that are essential to the combination for the purpose described. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants or inert carriers. "Consisting of" is intended to mean excluding more than trace amounts of other components and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0328] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope can comprise three amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, at least 8, 9, or 10 such amino acids. Methods for determining the spatial conformation of amino acids are known in the art, and include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0329] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression includes splicing of the mRNA in a eukaryotic cell.

[0330] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or other types of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that have been modified by processes such as capping, polyadenylation, methylation, editing, and proteins that have been modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, glycosylation, and the like.

[0331] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression includes, but is not limited to, gene activation and gene repression.

[0332] The term "operatively linked" or its equivalents (e.g., "linked operatively") means that two or more molecules are positioned relative to each other so that they can interact in a manner that affects the function attributed to one or both molecules or a combination thereof.

[0333] Non-covalently linked components, as well as methods for making and using non-covalently linked components, are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for transient interactions, avoiding one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only, or primarily, under circumstances where such association is required for the desired activity. Linkage need only be for a period of time sufficient to achieve the desired effect.

[0334] A method for targeting a protein to a specific locus in the genome of an organism is disclosed. The method may include providing a DNA localization component and providing an effector molecule, wherein the DNA localization component and the effector molecule can be operably linked via a non-covalent linkage.

[0335] The term "scFv" refers to a single-chain variable fragment. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin connected by a linker peptide. The linker peptide can be about 5 to 40 amino acids, or about 10 to 30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single-chain variable fragments lack the constant Fc region found in intact antibody molecules and thus lack the consensus binding site (e.g., protein G) used to purify antibodies. The term also includes scFvs, which are intrabodies, that is, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0336] The term "single-domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single-domain antibodies are typically peptide chains of approximately 110 amino acids in length containing one variable domain (VH) of a heavy-chain antibody or common IgG. They generally have similar affinity for antigen as the whole antibody, but are more heat-resistant and stable to detergents and high concentrations of urea. Examples are those derived from camel or fish antibodies. Alternatively, single-domain antibodies can be generated from common mouse or human IgG, which has four chains.

[0337] As used herein, the terms "specifically bind" and "specific binding" refer to the ability of an antibody, antibody fragment, or nanobody to preferentially bind to a particular antigen present in a homogeneous mixture of different antigens. In some embodiments, the specific binding interaction discriminates between desired and undesired antigens in a sample. In some embodiments, by about 10-fold to 100-fold or more (e.g., greater than about 1000-fold or greater than 10,000-fold). "Specificity" refers to the ability of an immunoglobulin or immunoglobulin fragment, e.g., a nanobody, to preferentially bind to one antigenic target over different antigenic targets and does not necessarily imply high affinity.

[0338] 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 when conditions sufficient for binding are present.

[0339] The term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked. A description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid can encompass the complementary strand of a described single strand. The nucleic acids of the present disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0340] A probe of the present disclosure may consist of a single-stranded nucleic acid capable of hybridizing to a target sequence under stringent hybridization conditions. Thus, a nucleic acid of the present disclosure may refer to a probe that hybridizes under stringent hybridization conditions.

[0341] The nucleic acids of the present disclosure may be single-stranded or double-stranded. The nucleic acids of the present disclosure may be predominantly single-stranded or may contain double-stranded sequences. The nucleic acids of the present disclosure may be predominantly double-stranded or may contain single-stranded sequences. The nucleic acids of the present disclosure may comprise genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of the present disclosure may comprise a combination of deoxyribonucleotides and ribonucleotides. The nucleic acids of the present disclosure may comprise a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. The nucleic acids of the present disclosure may be synthesized to contain unnatural amino acid modifications. The nucleic acids of the present disclosure may be obtained by chemical synthesis or recombinant methods.

[0342] The nucleic acids of the present disclosure may have either their entire base sequence or a portion thereof that does not occur in nature. The nucleic acids of the present disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not occur in nature, and the entire nucleic acid sequence may not occur in nature. The nucleic acids of the present disclosure may contain one or more overlapping sequences, inverted sequences, or repeated sequences, and as a result, the sequence does not occur in nature, and the entire nucleic acid sequence may not occur in nature. The nucleic acids of the present disclosure may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that do not occur in nature, and the entire nucleic acid sequence may not occur in nature.

[0343] Given the redundancy in the genetic code, more than one nucleotide sequence may encode a particular protein, and all such nucleotide sequences are contemplated herein.

[0344] As used throughout this disclosure, the term "operably linked" refers to the expression of a gene under the control of a spatially connected promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the promoter and the gene may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. Changes in the distance between the promoter and the gene can be accommodated without impairing the function of the promoter.

[0345] As used throughout this disclosure, the term "promoter" refers to a synthetic or naturally occurring molecule capable of conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter can contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. A promoter can also contain distal enhancer or repressor elements, located as far away as thousands of base pairs from the transcription start site. Promoters are derived from viruses, bacteria, fungi, plants, insects, animals, etc. Promoters can constitutively or differentially regulate the expression of genetic components with respect to the cell, tissue or organ in which expression occurs, or the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, inducers, etc. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, EF-1α promoter, CAG promoter, SV40 early promoter or SV40 late promoter, and CMV IE promoter.

[0346] As used throughout this disclosure, the term "substantially complementary" refers to a first sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540, or more nucleotides or amino acids, or that the two sequences hybridize under stringent hybridization conditions.

[0347] As used throughout this disclosure, the term "substantially identical" refers to a first and second sequence or nucleic acids that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or more nucleotides or amino acids, where the first sequence is substantially complementary to the complement of the second sequence.

[0348] As used throughout this disclosure, the term "variant," when used to describe a nucleic acid, refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement of the referenced nucleotide sequence or a portion thereof; (iii) a nucleic acid that is substantially identical to the referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0349] As used throughout this 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 vector or an RNA vector. A vector can be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector can be composed of amino acids and DNA sequences, RNA sequences, or a combination of both DNA and RNA sequences.

[0350] As used throughout this disclosure, the term "variant," when used to describe a peptide or polypeptide, refers to a peptide or polypeptide that differs in amino acid sequence by amino acid insertions, deletions, or conservative substitutions, but retains at least one biological activity. A variant can also refer to a protein having an amino acid sequence substantially identical to a reference protein having an amino acid sequence that retains at least one biological activity.

[0351] Conservative amino acid substitutions, i.e., replacing an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are recognized in the art as typically resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of the amino acid, as understood in the art. Kyte et al., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acid takes into account its hydrophobicity and charge. Protein function can be maintained even when substituted with an amino acid having a similar hydropathic index. In some embodiments, amino acids with a hydropathic index of ±2 are substituted. Amino acid hydrophilicity can also be used to identify substitutions that maintain the biological function of a protein. Considering the hydrophilicity of amino acids in the context of a peptide allows for calculation of the peptide's greatest local average hydrophilicity, a useful index that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101 is incorporated herein by reference in its entirety.

[0352] Substitution of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, e.g., immunogenicity. Substitutions can be made with amino acids whose hydrophilicity values ​​are within ±2 of each other. Both the hydrophobicity index and hydrophilicity value of an amino acid are affected by the specific side chain of that amino acid. Consistent with this observation, it is understood that amino acid substitutions that are compatible with biological function depend on the relative similarity of amino acids, particularly their side chains, as revealed by hydrophobicity, hydrophilicity, charge, size, and other properties.

[0353] As used herein, "conservative" amino acid substitutions may be defined as shown in Tables A, B, or C below. In some embodiments, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides contain conservative substitutions introduced by modifying a polynucleotide encoding a polypeptide of the present disclosure. Amino acids can be classified according to their physical properties and contribution to the secondary and tertiary structure of proteins. A conservative substitution is the replacement of one amino acid with another amino acid with similar properties. Exemplary conservative substitutions are shown in Table 1.

[0354] [Table 1]

[0355] Alternatively, conserved amino acids can be grouped as shown in Table 2, as described in Lehninger, (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0356] [Table 2]

[0357] TIFF2026507612000005.tif38170

[0358] Alternatively, exemplary conservative substitutions are shown in Table 3.

[0359] [Table 3]

[0360] It should be understood that the polypeptides of the present disclosure are intended to include polypeptides having one or more insertions, deletions, substitutions, or any combination thereof, of amino acid residues, as well as polypeptides having modifications other than insertions, deletions, or substitutions of amino acid residues. A polypeptide or nucleic acid of the present disclosure may contain one or more conservative substitutions.

[0361] As used throughout this disclosure, the term "two or more" of the foregoing amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the described amino acid substitutions. The term "two or more" can refer to 2, 3, 4, or 5 of the described amino acid substitutions.

[0362] The polypeptides and proteins of the present disclosure may be non-naturally occurring, either in their entire nucleotide sequence or in portions thereof. The polypeptides and proteins of the present disclosure may contain one or more non-naturally occurring mutations, substitutions, deletions, or insertions, and the entire amino acid sequence may not be naturally occurring.

[0363] The polypeptides and proteins of the disclosure may contain one or more overlapping, inverted, or repeated sequences such that the sequence is not naturally occurring, and the entire amino acid sequence may not be naturally occurring. The polypeptides and proteins of the disclosure may contain modified, artificial, or synthetic amino acids that are not naturally occurring, and the entire amino acid sequence may not be naturally occurring.

[0364] As used throughout this disclosure, "sequence identity" can be determined using a standalone executable BLAST engine program (bl2seq) for blasting two sequences, which can be obtained from the National Center for Biotechnology Information (NCBI) ftp site using default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; incorporated herein by reference in its entirety). The term "identical" or "identity," when used in the context of two or more nucleic acid or polypeptide sequences, refers to a specific percentage of identical residues over a specific region of each sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences are of different lengths, or if the alignment produces sequences with one or more offset ends, and the specified comparison region contains 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 done manually or using computer alignment algorithms such as BLAST and BLAST 2.0.

[0365] As used throughout this disclosure, the term "endogenous" refers to a nucleic acid or protein sequence that is naturally associated with the target gene or host cell into which it is introduced.

[0366] As used throughout this disclosure, the term "exogenous" refers to a nucleic acid or protein sequence that is not naturally associated with the target gene or host cell into which it is introduced, and includes non-naturally occurring multiple copies of a naturally occurring nucleic acid, e.g., a DNA sequence, or a naturally occurring nucleic acid sequence located in a non-naturally occurring genomic location.

[0367] The present disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. By "introducing" it is meant presenting the polynucleotide construct in the cell in such a way that the polynucleotide construct can access the interior of the host cell. The method of the present disclosure does not depend on a specific method for introducing a polynucleotide construct into a host cell, but only on the polynucleotide construct accessing the interior of one cell of the host. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art, including, but not limited to, stable transformation, transient transformation, and virus-mediated methods. [Example]

[0368] Example 1 - Construction of an exemplary AAV piggyBac transposon polynucleotide containing a codon-optimized and modified human phenylalanine hydroxylase (PAH) gene The following is a non-limiting example demonstrating the construction of an exemplary AAV piggyBac transposon polynucleotide containing a codon-optimized and modified human PAH gene.

[0369] The nucleotide sequence of mRNA encoding the human PAH sequence (GenBank U49897.1) was codon-optimized to improve PAH expression using computer algorithms according to various manufacturer's instructions (e.g., ThermoFisher, Blue Heron Biotech, and Integrated DNA Technologies). After codon optimization, the optimized sequence was further modified to remove i) any internal TTAA sites, ii) any unwanted restriction enzyme recognition sites, and iii) potential cryptic splice sites for cloning the modified sequence into the AAV piggyBac transposon vector.

[0370] Two AAV piggyBac transposon polynucleotides containing codon-optimized and modified human PAH genes were constructed. Schematics of each construct are shown in Figures 1 and 2, respectively.

[0371] As shown in FIG. 1 , the first AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a) the first AAV ITR sequence (SEQ ID NO:1); b) the first piggyBac ITR sequence (SEQ ID NO:2); c) the piggyBac 3′ UTR sequence (SEQ ID NO:3); d) the first insulator sequence (SEQ ID NO:4); e) the TTR promoter sequence (SEQ ID NO:7); f) the synthetic intron sequence (SEQ ID NO:17); g) a transgene sequence comprising a nucleic acid sequence encoding a hemagglutinin (HA)-tagged, codon-optimized, and modified human PAH gene (SEQ ID NO:9); h) a polyA sequence (SEQ ID NO:11); i) the second insulator sequence (SEQ ID NO:12); j) the piggyBac 5′ UTR sequence (SEQ ID NO:13); k) the second piggyBac ITR (SEQ ID NO:14); l) a DNA spacer sequence (SEQ ID NO:15); and m) the second AAV ITR sequence (SEQ ID NO:16).

[0372] The first AAV piggyBac transposon polynucleotide was constructed using Gibson assembly from a single DNA fragment containing the TTRm promoter-HA-PAH gene-polyA, and a vector backbone containing ii) insulator sequences flanking the AAV ITRs, piggyBac ITRs, and UTRs. The complete nucleotide sequence of the first AAV piggyBac transposon polynucleotide is provided in SEQ ID NO:18.

[0373] As shown in FIG. 2 , the second AAV piggyBac transposon polynucleotide comprises, from 5′ to 3′, a) the first AAV ITR sequence (SEQ ID NO:1); b) the first piggyBac ITR sequence (SEQ ID NO:2); c) the piggyBac 3′UTR sequence (SEQ ID NO:3); d) the first insulator sequence (SEQ ID NO:4); e) the TTR promoter region, including the 3×hSERPINA1 enhancer (SEQ ID NO:5), the TTR enhancer (SEQ ID NO:6), the TTR promoter sequence (SEQ ID NO:7), and the MVM intron (SEQ ID NO:8); f) a transgene sequence, including a nucleic acid sequence encoding a hemagglutinin (HA)-tagged, codon-optimized, and modified human PAH gene (SEQ ID NO:9), followed by the AES-mtRNR 3′UTR (SEQ ID NO:10); g) a polyA sequence (SEQ ID NO:11); h) the second insulator sequence (SEQ ID NO:12), the piggyBac j) a 5'UTR sequence (SEQ ID NO: 13), a second piggyBac ITR sequence containing a 35TCC mutation (SEQ ID NO: 19); j) a DNA spacer sequence (SEQ ID NO: 20); and k) a second AAV ITR sequence (SEQ ID NO: 16).

[0374] A second AAV piggyBac transposon polynucleotide was constructed using Gibson assembly by combining a DNA fragment containing the TTRm promoter-HA-PAH gene-polyA sequence; a DNA fragment containing the piggyBac 5' ITR with the 35TCC mutation; and ii) a vector backbone containing an insulator sequence, piggyBac ITR, and the 3' UTR flanked by the AAV ITR. The complete nucleotide sequence of the second AAV piggyBac transposon polynucleotide is provided in SEQ ID NO:21.

[0375] Example 2 - Preparation of 5'-capped mRNA encoding Super PiggyBac transposase ("SPB") for inclusion in LNP compositions The following is a non-limiting example demonstrating the preparation of an exemplary mRNA encoding SPB that can be incorporated into an LNP composition for use in combination with the AAV piggyBac transposon polynucleotides and vectors of the present disclosure to treat PKU.

[0376] The DNA plasmid pRTb_HBB_SPBv3.1 encodes the SPB transposase containing an N-terminal SV40 nuclear localization signal (NLS) and a reconstruction containing the 5' and 3' UTRs of the human β-globin gene (HBB).

[0377] This plasmid was used as a template for an in vitro transcription reaction to produce mRNA encoding HBB-SPBv3.1, which further contained a 5'-CAP.

[0378] Briefly, approximately 300 μg of supercoiled pRTb_HBB_SPBv3.1 was added to a 15 mL conical tube containing 300 μL of CutSmart Buffer and 60 μL of restriction enzyme Bbsl-HF (New England Biolabs, catalog number R3539M) for a total volume of 3000 μL. The plasmid DNA was linearized by incubating overnight at 37°C to ensure complete digestion.

[0379] The linearized plasmid was purified using a DNA QIAquick PCR Purification Kit (Qiagen, catalog no. 28106) according to the manufacturer's instructions, and the purified DNA was eluted in 900 μL of nuclease-free water (ThermoFisher, catalog no. AM9937). The DNA concentration and purity of the eluate were determined using a NanoDrop microspectrophotometer (ThermoFisher) according to the manufacturer's instructions.

[0380] Purified plasmid was used as a DNA template to generate mRNA using a custom in vitro transcription mMESSAGE mMACHINE T7 Transcription Kit (ThermoFisher, catalog number AM1345B001) according to internal quality-controlled manufacturing batch records. Briefly, 100 mM stocks of the nucleotides GTP (ThermoFisher, catalog number R0481), ATP (ThermoFisher, catalog number R0481), UTP (ThermoFisher, catalog number R0481), and 5MeC (5-methylcytidine-5'-triphosphate) (TriLink, catalog number N-1014) and CleanCap Reagent AG (m7G(5')ppp(5')(2'OMeA)pG; Trilink, catalog number N-7113) were prepared. 1,485 μL each of ATP, UTP, and 5MeC were blended with 1,188 μL each of GTP and CleanCap Reagent AG.

[0381] 153 μg of linearized pRTb_HBB_SPBv3.1 DNA, 1,800 μL of 10X T7 RXN buffer (ThermoFisher, catalog no. AM1345B001), 1,800 μL of T7 enzyme mix (ThermoFisher, catalog no. AM1345B001), and 6,831 μL of NTP and cap blend were added to a 50 mL conical tube (final volume 18,000 μL) and incubated for 3 hours at 37°C. A 900 μL aliquot of DNase I enzyme (ThermoFisher, catalog no. AM1345B001) was added, and the tube was further incubated for 15 minutes at 37°C to degrade the DNA template.

[0382] A poly(A) tail was added enzymatically to the 3' end of the 5'-CleanCap®-HBB-SPBv3.1-5MeC mRNA. 18,000 μL of 5x EPAP Buffer (ThermoFisher, Catalog No. AM1345B001), 9,000 μL of 25 mM MnCl2 (ThermoFisher, Catalog No. AM1345B001), 9,000 μL of ATP Solution (ThermoFisher, Catalog No. AM1345B001), and 3,000 μL of E-PAP (ThermoFisher, Catalog No. AM1345B001) were added to the IVT reaction (total volume 90,000 μL) and incubated at 37°C for 1 hour. The bulk E-PAP reaction was then divided into three 125 mL PETG bottles in 30 mL aliquots.

[0383] 5'-CleanCap®-HBB-SPBv3.1-Poly(A)-5MeC mRNA was purified using the RNeasy Maxi Purification Kit (Qiagen, catalog no. 75162) according to the manufacturer's instructions. Briefly, a working stock of Buffer RLT was formulated using 178.2 mL of Buffer RLT (Qiagen, catalog no. 75162) containing 1,800 μL of 2-mercaptoethanol (Sigma, catalog no. M3148). 52.2 mL of BME+RLT solution and 37.8 mL of 100% EtOH (ThermoFisher, catalog no. BP2818) were added to each 30 mL mRNA aliquot. The purified mRNA product was eluted in 52.5 mL of nuclease-free water, and the bulk product was stored at -80°C. The DNA linearization, IVT, and mRNA purification processes were repeated until the target yield was reached.

[0384] Bulk mRNA lots were analyzed using gel electrophoresis before being combined in 500 mL PETG bottles and sampled for concentration using a NanoDrop. Lithium chloride 5X (ThermoFisher, catalog no. AM1345B001) was added to the pooled mRNA in an amount equal to one-third of the total mRNA volume, which was then divided into equal 40 mL aliquots in 50 mL conical tubes and incubated at -20°C for 45 minutes. Immediately after incubation, the conical tubes were centrifuged at 14,000 g for 30 minutes at 4°C. The mRNA pellet was washed three times with 70% EtOH (ThermoFisher, catalog no. BP8201).

[0385] The washed mRNA pellet was dried and then resuspended in nuclease-free water. The mRNA concentration was determined using a NanoDrop, and additional nuclease-free water was added as needed to further dilute the product to the target concentration. The mRNA was sterile filtered using a 0.22 μm PES SteriCup filter (Sigma, catalog number 52GPU05RE) before the final mRNA concentration and purity were measured using a NanoDrop.

[0386] Example 3 - Preparation of LNP compositions containing 5'-capped mRNA encoding Super PiggyBac transposase The following are non-limiting examples that provide exemplary methods for formulating LNP compositions comprising 5' capped mRNA encoding an SPB transposase for use in combination with the AAV piggyBac transposon vectors disclosed herein.

[0387] Individual 25 mg / ml stock solutions were prepared by solubilizing lipids in 200-proof HPLC-grade ethanol, and the stock solutions were stored at -80 °C until formulation. At the time of formulation, the lipid stock solutions were briefly equilibrated to room temperature and then placed on a hot plate maintained at a temperature range of 50–55 °C. The hot lipid stock solutions were then combined to obtain the desired final molar percentages.

[0388] A 1 mg / ml solution of 5'-CleanCap-5MeC-SPB mRNA prepared in Example 2 for incorporation into LNPs was added to 150 mM sodium acetate buffer (pH 5.2) to form a stock solution and kept on ice. The lipid phase was mixed with the aqueous mRNA phase in a microfluidic chip using a NanoAssemblr® instrument (Precision Nanosystems, Vancouver, BC, Canada) according to the manufacturer's instructions to form an LNP composition containing encapsulated SPB mRNA. The NanoAssemblr process parameters for mRNA encapsulation were a flow rate of 20 ml / min and a lipid-to-RNA ratio of 1:3 (v / v).

[0389] The resulting SPB mRNA LNP composition was then transferred to a Repligen Float-A-Lyzer dialysis device (Spectrum Chemical Mfg. Corp., California, USA) with a molecular weight cutoff (MWCO) of 8-10 kDa and processed by dialysis against 25 mM sodium acetate (dialysate:dialysis buffer volume, at least 1:200 v / v), pH 5.5, overnight at 4 °C (or alternatively, at least 4 h at room temperature) to remove 25% ethanol and achieve complete buffer exchange. If applicable, the LNP composition was further concentrated by spinning at approximately 4100 × g in an ultracentrifuge using an Amicon® Ultra-4 centrifugal filter unit, MWCO-30 kDa (Millipore Sigma, USA). Sucrose was added to the mRNA LNP to a final concentration of 5% (w / v), which was then stored at 4 °C or frozen at -80 °C until further use. The average particle size of the LNPs ranged from approximately 84 to 121 nm.

[0390] Example 4 - Compositions of the present disclosure for the treatment of PKU The following are non-limiting examples demonstrating that the AAV piggyBac transposon polynucleotide and LNP-containing compositions and methods of the present disclosure can be used in the treatment of PKU.

[0391] Adult (10-12 week old) C57BL / 6 mice (n=3 mice per group) were administered the following treatments intravenously:

[0392] Treatment No. 1: codon-optimized and modified human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particles;

[0393] Treatment No. 2: human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particles combined with LNPs encapsulating mRNA encoding a functional SPB transposase;

[0394] Treatment number 3: human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particles combined with LNPs encapsulating catalytically inactive SPB transposase.

[0395] The human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particle was an AAV viral vector particle comprising an AAV piggyBac transposon polynucleotide comprising the nucleic acid of SEQ ID NO:18.

[0396] The LNP composition containing mRNA encoding SPB transposase prepared in Example 2 contained the following components: ssPalmO-Ph-P4C2, DOPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, with a lipid:RNA ratio of 100:1 (w / w).

[0397] The catalytically inactive mRNA SPB LNP composition also contained ssPalmO-Ph-P4C2, DOPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, with a lipid:RNA ratio of 100:1 (w / w).

[0398] Mice in treatment number 1 received either 1e12GC / kg or 3e12GC / kg of the hPAH AAV piggyBac transposon vector, while mice in treatment number 2 also received 0.5mg / kg of an LNP composition containing mRNA encoding SPB, and mice in treatment number 3 also received 0.5mg / kg of an LNP composition containing mRNA encoding catalytically inactive SPB.

[0399] After 14 days of administration, liver biopsies and blood samples were collected from treated mice. To isolate genomic DNA, liver samples were mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K) and pulverized in a TissueLyser II (Qiagen) using Triple-Pure Zirconium Beads (Fisher Scientific). The homogenized tissue was then pulverized for 56 h. o The mixture was incubated at 37°C for 30 minutes and column purified using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's instructions. Final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 nm and 280 nm using a Nanodrop device. The DNA samples were used for vector copy number (VCN) quantification.

[0400] To isolate proteins, liver samples were mixed with RIPA buffer and HALT protease inhibitor (both from ThermoFisher) and ground in a TissueLyser II (Qiagen) as described above. Lysates were then centrifuged at 20,000 g for 10 min, and the supernatant was collected for ELISA quantification of HA-tagged hPAH.

[0401] Liver biopsies were fixed in formalin for 24 hours, transferred to 70% ethanol for 24 hours, embedded in paraffin, and processed for immunofluorescence detection of HA-tagged hPAH as described in Example 5.

[0402] The results of the ELISA assay are shown in Figures 3A and 3B. hPAH expression was shown to be dose-dependent (Figure 3A). Addition of LNPs containing SPB mRNA significantly increased hPAH protein expression compared to the levels observed in mice treated with AAV alone or hPAH-AAV co-delivered with a catalytically inactive SPB mutant. hPAH protein expression levels in mice co-administered with the PAH AAV piggyBac transposon polynucleotide were similar to or exceeded those observed using hydrodynamic injection (Figure 3B).

[0403] Example 5 - Immunofluorescence staining The number of transgene-positive hepatocytes in mice from the three treatment groups described in Example 4 was measured by immunofluorescence (IF) staining. Briefly, liver sections were incubated with an anti-HA primary antibody followed by a secondary antibody conjugated to a fluorescent dye. After nuclear counterstaining with DAPI, high-resolution whole-slide digital images were obtained, and HA-positive cells were measured using an AI-enhanced image analysis algorithm (Reveal Biosciences).

[0404] The results of IF image quantification are shown in Figure 4. The addition of active SPB increased the number of hPAH-expressing hepatocytes compared to the number of hPAH-expressing hepatocytes observed from mice treated with AAV alone or hPAH-AAV co-delivered with catalytically inactive SPB.

[0405] Example 6 - Determination of episomal and integrated viral copy numbers The integrated virus copy number (VCN) levels in the livers of mice from the three treatment groups described in Example 4 were measured using a combination of three different digital droplet PCR (ddPCR) assays. Briefly, the total copy number per cell was measured using an assay that binds to the transgene sequence, the episomal copy number was measured using an assay that binds to the junction between the backbone and the transgene, and the number of diploid genomes was measured using an assay that binds to the mouse HMBS genome sequence. The VCN was then calculated by subtracting the episomal copy number from the total copy number and expressed as the integrated copies of the diploid genome.

[0406] The results of this analysis are shown in Figures 5A and 5B. The number of total and integrated vectors increased with increasing AAV dose (Figure 5A). Viral vector integration was detected only in mice treated with AAV co-delivered with catalytically active SPB.

[0407] Taken together, the results shown in Examples 4, 5, and 6 suggest that the transposon containing the codon-optimized and modified PAH gene was successfully integrated into the host genome by catalytically active SPB, resulting in stable expression of PAH in transposon-transduced hepatocytes. In contrast, administration of the transposon containing the transgene alone, or coadministration of the transposon with catalytically inactive SPB transposase, showed little or no transgene integration and subsequent poorly stable expression of PAH in hepatocytes.

[0408] Example 7 - Co-administration of an AAV piggyBac transposon vector containing a codon-optimized and modified PAH gene with an LNP composition containing mRNA encoding SPB transposase enhances the expression of BTBR PAH enu Restores normal serum phenylalanine levels and hypopigmentation in mouse models The following demonstrates that co-administration of an AAV piggyBac transposon vector comprising an AAV piggyBac transposon polynucleotide containing a codon-optimized and modified hPAH gene (Construct #1; SEQ ID NO: 18) with a LNP composition comprising mRNA encoding SPB transposase significantly inhibits the proliferation and proliferation of BTBR PAH. enu 1 is a non-limiting example showing restoration of normal serum phenylalanine levels and normal hypopigmentation in a mouse model.

[0409] BTBR PAH in adult males (n = 1-3 / group) and adult females (n = 1-3 / group) enu Mice were divided into three groups: the first group received vehicle (PBS), the second group received 1e12 GC / kg of the AAV piggyBac transposon vector co-administered with 0.5 mg / kg of the ND4-2 LNP formulation of Example 3 containing a 5'-capped mRNA encoding SPB as described in Example 2, and the third group received the ND4-2 LNP formulation of Example 3 containing mRNA encoding a catalytically inactive version of SPB. Untreated adult C57BL / 6 wild-type mice (n=3 / group) were followed in parallel as a positive control to measure normal serum Phe levels.

[0410] Blood samples were collected from each group of treated and control mice on days 0, 7, 14, and 28, and serum Phe levels were quantified using a colorimetric reader (Abcam). The results are shown in Figure 6. As shown in Figure 6A, male mice treated with the ND4-2 LNP formulation containing the AAV piggyBac transposon PAH vector and mRNA encoding SPB showed a rapid and sharp decrease in serum Phe levels on day 7, approaching wild-type levels, which continued to decrease further below baseline on days 14 and 28 (Figures 6A and 6B). Female mice also showed a decrease in serum Phe levels on days 7 and 14, albeit to a lesser extent than males (Figure 6B). However, by day 28, serum Phe levels had decreased to levels observed in normal wild-type mice.

[0411] In contrast, male and female mice treated with LNP compositions containing an AAV piggyBac transposon PAH vector and catalytically inactive SPB showed little or no decrease in serum Phe levels compared with vehicle-treated mice on days 0, 7, and 14, and only a slight decrease in serum Phe levels on day 28. Vehicle-treated male and female mice showed high serum Phe levels that were maintained over 28 days.

[0412] Furthermore, the degree of recovery of hypopigmentation in treated and control mice was visually monitored for male and female mice. enu The observation of hypopigmentation recovery in mouse models is evidence of serum Phe catabolism and subsequent restoration of coat melanin production. Male and female mice treated with ND4-2 LNP formulations containing the AAV piggyBac transposon PAH vector and mRNA encoding SPB, respectively, showed a visual change in hypopigmented coat color, whereas male and female mice treated with LNP compositions containing the AAV piggyBac transposon PAH vector and catalytically inactive SPB or vehicle showed little or no visual change in coat color.

[0413] Thus, coadministration of an AAV piggyBac transposon vector containing the codon-optimized PAH gene of the present disclosure with an LNP composition containing mRNA encoding SPB in an in vivo PKU disease model reduced serum Phe levels below those of wild-type mice. In contrast, coadministration of the same AAV piggyBac transposon vector with an LNP composition containing mRNA encoding a catalytically inactive SPB transposase showed little or no reduction in serum Phe levels, similar to vehicle-treated animals. These results suggest that integration of a transposon containing a codon-optimized and modified PAH gene with SPB and subsequent stable expression of PAH in transposon-transfected hepatocytes resulted in a reduction in serum Phe levels to normal wild-type levels, whereas coadministration of a catalytically inactive SPB transposase showed little or no reduction in serum Phe levels.

[0414] In another non-limiting example, co-administration of an AAV piggyBac transposon vector comprising an AAV piggyBac transposon polynucleotide containing a codon-optimized and modified hPAH gene (Construct No. 2; SEQ ID NO: 21) with a LNP composition comprising mRNA encoding an SPB transposase also inhibits the production of BTBR PAH. enu It restores normal serum phenylalanine levels and normal hypopigmentation in a mouse model (Figures 7A and 7B).

[0415] Blood samples were collected from each group of treated and control mice on days 0, 7, 14, and 28, and serum Phe levels were quantified using a colorimetric reader (Abcam). The results are shown in Figure 7. As shown in Figure 7A, male mice treated with the ND4-2 LNP formulation containing the AAV piggyBac transposon PAH vector containing construct number 2 and mRNA encoding SPB showed a rapid and sharp decrease in serum Phe levels on day 7, approaching wild-type levels, and continued to decrease further below baseline on days 14 and 28 (Figure 7A). Female mice also showed a decrease in serum Phe levels on days 7 and 14, albeit to a lesser extent than males (Figure 7B). However, by day 28, serum Phe levels had decreased to levels observed in normal wild-type mice.

[0416] In contrast, male and female mice treated with LNP compositions containing an AAV piggyBac transposon PAH vector and catalytically inactive SPB showed little or no decrease in serum Phe levels at days 0, 7, or 14 in male or female mice, a substantial decrease at day 28 in male mice, and only a slight decrease in serum Phe levels at day 28 in female mice compared with vehicle-treated mice. Male and female mice treated with vehicle showed high serum Phe levels that were maintained over the 28-day period.

[0417] Example 8 - Compositions of the present disclosure for the treatment of PKU The following are non-limiting examples demonstrating that the AAV piggyBac transposon polynucleotide and LNP-containing compositions and methods of the present disclosure can be used in the treatment of PKU in the juvenile setting.

[0418] Young (14 day old) C57BL / 6 mice (n=3 mice per group) were administered the following treatments intravenously:

[0419] Treatment No. 1: codon-optimized and modified human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particles combined with LNP encapsulating mRNA encoding a functional SPB transposase;

[0420] Treatment No. 2: Codon-optimized and engineered human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particles combined with LNP encapsulating a catalytically inactive SPB transposase.

[0421] A third group of mice was treated with vehicle alone.

[0422] The codon-optimized and modified human phenylalanine hydroxylase (hPAH) transposon AAV viral vector particle was an AAV viral vector particle comprising an AAV piggyBac transposon polynucleotide comprising the nucleic acid of SEQ ID NO:18.

[0423] The LNP composition containing mRNA encoding SPB transposase prepared in Example 2 contained the following components: ssPalmO-Ph-P4C2, DOPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, with a lipid:RNA ratio of 100:1 (w / w).

[0424] The catalytically inactive mRNA SPB LNP composition also contained ssPalmO-Ph-P4C2, DOPC, cholesterol, and DMG-PEG2000 in a molar ratio of 54:10:35:1, with a lipid:RNA ratio of 100:1 (w / w).

[0425] For treatment #1, mice were co-administered 1e12GC / kg of hPAH AAV piggyBac transposon vector and 0.5mg / kg of LNP composition containing mRNA encoding functional SPB. For treatment #2, mice were co-administered 1e12GC / kg of hPAH AAV piggyBac transposon vector and 0.5mg / kg of LNP composition containing mRNA encoding catalytically inactive SPB.

[0426] Four weeks after administration, liver biopsies were collected from treated mice. To isolate genomic DNA, liver samples were mixed with lysis buffer (15 mg of tissue in 200 μL of lysis buffer + 10 μL of proteinase K) and pulverized in a TissueLyser II (Qiagen) using Triple-Pure Zirconium Beads (Fisher Scientific). The homogenized tissue was then pulverized for 56 h. o The mixture was incubated at 37°C for 30 minutes and column purified using the Monarch Genomic DNA Purification Kit (New England Biolabs) according to the manufacturer's instructions. Final DNA elution was performed in 50 μL of elution buffer (10 mM Tris-Cl, pH 8.5). The concentration and purity of the DNA samples were assessed by measuring absorbance at 260 nm and 280 nm using a Nanodrop device. The DNA samples were used for vector copy number (VCN) quantification.

[0427] To isolate proteins, liver samples were mixed with RIPA buffer and HALT protease inhibitor (both from ThermoFisher) and ground in a TissueLyser II (Qiagen) as described above. Lysates were then centrifuged at 20,000 g for 10 min, and the supernatant was collected for ELISA quantification of HA-tagged hPAH.

[0428] The results of the ELISA assay are shown in Figures 8A and 8B. Addition of LNPs containing functional SPB mRNA significantly increased hPAH protein expression compared to the levels observed in mice treated with hPAH-AAV co-delivered with a catalytically inactive SPB mutant (Figure 8A).

[0429] The levels of cumulative viral copy number (VCN) in the livers of mice in the two treatment groups were measured by digital droplet PCR (ddPCR) assay as described in Example 6. The results of this analysis are shown in Figure 8B. Viral vector integration was detected only in mice treated with AAV co-delivered with catalytically active SPB.

[0430] The results in this example demonstrate that SPB-mediated integration maintains PAH protein expression in the juvenile environment.

Claims

1. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in 5' to 3' direction: a) a first AAV ITR sequence; and b) a first piggyBac ITR sequence; and c) a first piggyBac 3'UTR; and d) a first insulator array; and e) at least one promoter sequence; f) at least one intron sequence; and g) at least one transgene sequence encoding human phenylalanine hydroxylase (PAH); h) a polyA sequence; and i) a second insulator sequence; and j) a first piggyBac 5'UTR sequence; and k) a second piggyBac ITR sequence; and l) at least one DNA spacer sequence; m) a second AAV ITR sequence; and An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising:

2. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in 5' to 3' direction: a) a first AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 1; b) a first piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO:2; and c) a first piggyBac 3'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 3; and d) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 4; and e) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 7; f) a synthetic intron sequence comprising the nucleic acid sequence of SEQ ID NO: 17; g) a transgene sequence encoding a codon-optimized and modified human PAH gene comprising the nucleic acid sequence of SEQ ID NO: 9; and h) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 11; i) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 12; and j) a piggyBac 5'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 13; and k) a second piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 14; and l) a DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 15; m) a second AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 16; and An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising:

3. 3. The AAV piggyBac transposon polynucleotide of claim 1 or 2, comprising the nucleic acid sequence of SEQ ID NO:

18.

4. A vector comprising the AAV piggyBac transposon polynucleotide of any one of claims 1 to 3.

5. The vector of claim 4 , wherein the vector is an AAV viral vector.

6. 6. The vector of claim 5, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector.

7. The vector of claim 6 , wherein the AAV viral vector is an AAV8 or AAV9 viral vector.

8. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in 5' to 3' direction: a) a first AAV ITR sequence; and b) a first piggyBac ITR sequence; and c) a first piggyBac 3'UTR; and d) a first insulator array; and e) at least one enhancer sequence; and f) at least one promoter sequence; g) at least one transgene sequence encoding human phenylalanine hydroxylase (PAH); h) a first 3'UTR; and i) a polyA sequence; and j) a second insulator sequence; and k) a first piggyBac 5 UTR; and l) a second piggyBac ITR sequence containing the 35TCC mutation; and m) at least one DNA spacer sequence; n) a second AAV ITR sequence; and An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising:

9. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in 5' to 3' direction: a) a first AAV ITR sequence; and b) a first piggyBac ITR sequence; and c) a first piggyBac 3'UTR; and d) a first insulator array; and e) 3xhSERPINA1 enhancer; and f) a TTR enhancer; and g) a TTRm promoter sequence; and h) an MVM intron; and i) a transgene sequence comprising a nucleic acid sequence encoding a codon-optimized and modified human PAH gene; and j) AES-mtRNR 3'UTR; and k) a polyA sequence; and l) a second insulator array; and m) a first piggyBac 5'UTR; and n) a second piggyBac ITR sequence containing a 35TCC mutation; and o) a DNA spacer sequence; and p) a second AAV ITR sequence; and An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising:

10. An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising, in 5' to 3' direction: a) a first AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 1; b) a first piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO:2; and c) a first piggyBac 3'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 3; and d) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 4; and e) a first enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 5; f) a second enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 6; and g) a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 7; h) an intron sequence comprising the nucleic acid sequence of SEQ ID NO: 8; i) a transgene sequence encoding a codon-optimized and modified human PAH gene comprising the nucleic acid sequence of SEQ ID NO: 9; and j) an AES-mtRNR 3'UTR comprising the nucleic acid of SEQ ID NO: 10; k) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 11; l) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 12; and m) a piggyBac 5'UTR sequence comprising the nucleic acid sequence of SEQ ID NO: 13; and n) a second piggyBac ITR sequence comprising a 35TCC mutation, the second piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 19; and o) a DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 15; p) a second AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 16; and An adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising:

11. 11. The AAV piggyBac transposon polynucleotide of any one of claims 8 to 10, comprising the nucleic acid sequence of SEQ ID NO:

21.

12. A vector comprising the AAV piggyBac transposon polynucleotide of any one of claims 8 to 11.

13. The vector of claim 12 , wherein the vector is an AAV viral vector.

14. 14. The vector of claim 13, wherein the AAV viral vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector.

15. The vector of claim 14, wherein the AAV viral vector is an AAV8 or AAV9 viral vector.

16. A pharmaceutical composition comprising the vector according to any one of claims 4 to 7 or 12 to 15.

17. A composition comprising: 1) a vector according to claims 4-7 or 12-15; and 2) at least one LNP composition comprising at least one mRNA molecule encoding a transposase.

18. The composition of claim 17, wherein the mRNA molecule further comprises a 5'-CAP.

19. 19. The composition of claim 17 or 18, wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

20. the at least one LNP composition comprising: About 54% ssPalmO-Ph-P4C2 on a molar basis; About 35% cholesterol on a molar basis, About 10% DOPC on a molar basis; About 1% DMG-PEG2000 on a molar basis; The composition of any one of claims 17 to 19, comprising:

21. A pharmaceutical composition comprising the composition according to claims 17 to 20.

22. 22. A method of treating phenylketonuria (PKU) in a subject in need thereof, comprising administering to the subject at least one therapeutically effective dose of a composition comprising: a) a polynucleotide, vector, or pharmaceutical composition according to any one of claims 1 to 21; and b) at least one LNP composition comprising at least one mRNA molecule encoding a transposase.

23. the at least one LNP composition comprising: About 54% ssPalmO-Ph-P4C2 on a molar basis; About 35% cholesterol on a molar basis, About 10% DOPC on a molar basis; About 1% DMG-PEG2000 on a molar basis; 23. The method of claim 22, comprising:

24. The method of claim 22 or 23, wherein the mRNA molecule further comprises a 5'-CAP.

25. The method of claims 22 to 24, wherein the transposase is piggyBac™ (PB) transposase, piggyBac-like (PBL) transposase, Super piggyBac™ (SPB) transposase, Sleeping Beauty transposase, Hyperactive Sleeping Beauty (SB100X) transposase, Helitron transposase, Tol2 transposase, TcBuster transposase, or a mutant TcBuster transposase.

26. 26. The method of claim 25, wherein the transposase is a Super piggyBac™ (SPB) transposase.

Images