Compositions and methods for the treatment of metabolic liver disorders

Abstract

To provide compositions and methods for the treatment of metabolic liver disorders. [Solution] This disclosure relates to compositions and methods for the treatment of metabolic liver disorders. The compositions and methods may include adeno-associated virus (AAV)piggyBac polynucleotides containing a trans gene. The trans gene may include ornithine transcarbamylase (OTC) or methylmalonyl-CoA mutase (MUT1).

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and benefit to U.S. Provisional Application No. 62 / 985,047, filed on 4 March 2020, and U.S. Provisional Application No. 63 / 121,488, filed on 4 December 2020. The contents of each of the aforementioned patent applications are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted via EFS-Web in ASCII format, the entire listing of which is incorporated herein by reference. This ASCII copy, created on March 3, 2021, is named "POTH-058_001WO_SeqList.txt" and is approximately 329KB in size. [Background technology]

[0003] Hereditary metabolic disorders, also known as congenital metabolic disorders, are medical conditions caused by genetic defects that are most commonly inherited from parents. Normal metabolism requires a series of complex chemical reactions that cells and organisms use to convert food and other nutrients into essential compounds and energy. These chemical reactions are also used to break down and remove unwanted substances, including toxic ones. Genetic defects that cause hereditary metabolic disorders often result in a deficiency in the activity of specific enzymes within one or more metabolic pathways. This deficiency can not only lead to the accumulation of potentially toxic substances but can also limit the subject's ability to synthesize essential compounds. Hundreds of hereditary metabolic disorders have been characterized, including those that primarily affect the liver. Hereditary metabolic disorders affecting the liver include urea cycle disorders (UCD) and methylmalonic acidemia (MMA).

[0004] Urea cycle disorders (UCDs) are caused by genetic mutations that result in defects in the metabolism of nitrogen, which is produced by the breakdown of proteins and other nitrogen-containing molecules. UCDs are commonly caused by a severe or complete deficiency of the activity of one of the first four enzymes of the urea cycle—carbamoyl phosphate synthase I (CPSI), ornithine transcarbamylase (OTC), argininosuccinate synthase deficiency (ASS), and argininosuccinate lyase deficiency (ASL)—or the cofactor product N-acetylglutamate synthase (NAGS), leading to the accumulation of ammonia and other precursor metabolites. UCDs are usually diagnosed in newborns, but late-onset UCDs have been reported. UCDs can lead to brain damage, cognitive impairment, and even death. In fact, it is hypothesized that up to 20% of sudden infant death syndrome (SIDS) cases may be attributable to hereditary metabolic disorders such as UCDs.

[0005] Current treatment for UCD focuses on the acute control of hyperammonemia, a common symptom of UCD. Hyperammonemia is highly neurotoxic and requires intensive therapeutic intervention, including venous-venous hemofiltration. Currently, long-term treatment for UCD relies on alternative pathway therapy, strict dietary protein restriction, supplementation of urea cycle intermediates, and strict avoidance of catabolism. Patients with UCD often require liver transplantation. However, preventing recurrence of hyperammonemia before liver transplantation can be difficult. Therefore, improved compositions and methods for the treatment of UCD are needed in the art.

[0006] Other metabolic disorders that affect the liver include methylmalonic acidemia (MMA), an autosomal recessive disorder (also known as methylmalonic aciduria). MMA disrupts normal amino acid metabolism. The genotype of methylmalonic acidemia causes defects in the metabolic pathway that regulates the conversion of methylmalonyl-CoA to succinyl-CoA by the enzyme methylmalonyl-CoA mutase. As a result of this condition, certain fats and proteins cannot be properly digested, leading to the accumulation of toxic levels of methylmalonic acid in the blood. Isolated methylmalonic acidemia is caused by a change in one of five genes: MMUT, MMAA, MMAB, MMADHC, or MCEE. Methylmalonic acidemia with homocystinuria is caused by mutations in the MMADHC, LMBRD1, and ABCD4 genes.

[0007] There is no specific treatment for methylmalonic acidemia. Treatment is currently limited to symptom management and includes the active treatment of decompensated events, a special protein-restricted diet, vitamin B12 supplementation for vitamin B12-responsive subtypes, drug treatments such as carnitine, and the avoidance of stress factors (such as fasting and illness) that can cause decompensated events. Liver or kidney transplantation (or both) has been shown to help some patients. These transplants provide the body with new cells that help break down methylmalonic acid normally.

Summary of the Invention

Problems to be Solved by the Invention

[0008] Previous attempts to develop gene therapies for the treatment of genetic metabolic disorders, including hereditary metabolic disorders of the liver, have faced the problem of being unable to achieve long-term expression of the delivered transgene in the target tissue. This problem is particularly prominent in rapidly dividing tissues such as the liver of young individuals. Existing gene therapy vectors such as AAV vectors have the problem that the introduced transgene is expressed only for a short period because integration into the host genome is impossible. In response to this long-felt need in the art, the compositions and methods of the present disclosure provide a solution by providing a transposon / transposase-based AAV vector that results in long-term expression of the delivered transgene in the target tissue.

Means for Solving the Problems

[0009] Summary 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 comprising the nucleic acid sequence of SEQ ID NO: 3; b) a first piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125; c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 7; d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 126; e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 22; f) a polyA sequence comprising the nucleic acid sequence of SEQ ID NO: 97; g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 8; h) a second piggyBac ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 96; i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 129; and j) a second AAV ITR sequence comprising the nucleic acid sequence of SEQ ID NO: 4.

[0010] The present disclosure provides an AAV piggyBac transposon polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 138.

[0011] This disclosure provides an AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 3, b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125, c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 7, d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 132, e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 22, f) a poly-A sequence comprising the nucleic acid sequence of SEQ ID NO: 97, g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 8, h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 96, i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 130, and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 4.

[0012] This disclosure provides an AAVpiggyBac transposon polynucleotide containing the nucleic acid sequence of Sequence ID No. 139.

[0013] This disclosure provides an adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 3; b) a first piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 125; c) a first insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 7; d) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 13; e) at least one transgene sequence comprising the nucleic acid sequence of SEQ ID NO: 22; f) a poly-A sequence comprising the nucleic acid sequence of SEQ ID NO: 97; g) a second insulator sequence comprising the nucleic acid sequence of SEQ ID NO: 8; h) a second piggyBacITR sequence comprising the nucleic acid sequence of SEQ ID NO: 96; i) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 131; and j) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 4.

[0014] This disclosure provides an AAVpiggyBac transposon polynucleotide comprising the nucleic acid sequence of Sequence ID No. 140.

[0015] This disclosure provides an AAV transposase polynucleotide comprising the following in the 5' to 3' direction: a) a first AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 127; b) at least one promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 126; c) at least one transposase sequence comprising the nucleic acid sequence of SEQ ID NO: 48; d) a poly-A sequence comprising the nucleic acid sequence of SEQ ID NO: 136; e) at least one DNA spacer sequence comprising the nucleic acid sequence of SEQ ID NO: 137; and f) a second AAVITR sequence comprising the nucleic acid sequence of SEQ ID NO: 4.

[0016] This disclosure provides an AAV transposase polynucleotide comprising the nucleic acid sequence of Sequence ID No. 144.

[0017] This disclosure provides a vector comprising at least one of the AAVpiggyBac transposon polynucleotides of this disclosure. In some embodiments, the vector may be a viral vector. In some embodiments, the viral vector may be an AAV viral vector. In some embodiments, the AAV viral vector may be an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 viral vector. In some embodiments, the AAV viral vector may be an AAV-KP-1 or AAV-NP59 viral vector. In some embodiments, the AAV viral vector may be an AAV-KP-1 viral vector.

[0018] This disclosure provides a composition comprising at least one vector of this disclosure.

[0019] This disclosure provides a method for treating at least one metabolic liver disorder (MLD) in a subject requiring such treatment, comprising administering to the subject at least one therapeutically effective dose of the polynucleotides, vectors, or compositions of the presented disclosure.

[0020] The present disclosure provides a method for treating at least one MLD in a subject in need thereof, comprising administering to the subject: a) the AAVpiggyBac transposon polynucleotide of the present disclosure, or a vector or composition comprising the AAVpiggyBac transposon polynucleotide of the present disclosure; and b) the AAV transposase polynucleotide of the present disclosure, or a vector or composition comprising the AAV transposase polynucleotide of the present disclosure.

[0021] This disclosure provides the use of the polynucleotides, vectors, or compositions of this disclosure for the treatment of at least one MLD in a subject requiring such treatment, wherein the polynucleotides, vectors, or compositions are for administering at least one therapeutically effective dose to the subject.

[0022] This disclosure provides a combination of a) a vector or composition comprising the AAVpiggyBac transposon polynucleotide of this disclosure, or the AAVpiggyBac transposon polynucleotide of this disclosure, and b) an AAV transposase polynucleotide of this disclosure, or a vector or composition comprising the AAV transposase polynucleotide of this disclosure, for use in the treatment of at least one MLD in a subject requiring such treatment.

[0023] In some embodiments, at least one MLD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof. In some embodiments, MLD is OTC deficiency.

[0024] Any of the above embodiments can be combined with other embodiments.

[0025] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure pertains. In this specification, singular forms include plural forms unless the context explicitly indicates otherwise. For example, the terms “a,” “an,” and “the” are understood to be singular or plural, and the term “or” is understood to be inclusive. For example, “element” means one or more elements. Throughout this specification, the word “comprising,” or variations such as “comprises” or “comprising,” is understood to mean including the elements, integers, or processes, or groups of elements, integers, or processes described herein, but not excluding any other elements, integers, or processes, or groups of elements, integers, or processes. "Approximately" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clearly indicated in the context, all figures provided herein are qualified by the term "approximately."

[0026] Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this disclosure, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. References cited herein are not considered to be prior art of the claimed invention. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are for illustrative purposes only and are not intended to limit the invention. Other features and advantages of this disclosure will be apparent from the following detailed description and claims.

[0027] The above and further features will be more clearly understood from the following detailed description in conjunction with the attached drawings. [Brief explanation of the drawing]

[0028] [Figure 1] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0029] [Figure 2] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0030] [Figure 3A] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0031] [Figure 3B] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0032] [Figure 4A] This is a schematic diagram of an exemplary AAV transposase polynucleotide in this disclosure.

[0033] [Figure 4B]This is a schematic diagram of an exemplary AAV transposase polynucleotide in this disclosure.

[0034] [Figure 5] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0035] [Figure 6] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0036] [Figure 7] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0037] [Figure 8] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0038] [Figure 9] This is a schematic diagram of an exemplary AAVpiggyBac transposon polynucleotide in the present disclosure.

[0039] [Figure 10] This graph shows the BLI measured in mice treated with various viral vectors of this disclosure.

[0040] [Figure 11] This graph shows the BLI measured in mice treated with various viral vectors of this disclosure at various concentrations.

[0041] [Figure 12] This graph shows the BLI measured in Otcspf-ash mice treated with various viral vectors of this disclosure.

[0042] [Figure 13]This is a series of graphs showing the amount of non-integrated vector copies per diploid genome for various viral vectors of the present disclosure administered to Otcspf-ash mice.

[0043] [Figure 14] This is a series of graphs showing the amount of non-integrated vector copies per diploid genome and integrated vector copies per diploid genome for various viral vectors of the present disclosure administered to Otcspf-ash mice.

[0044] [Figure 15] This is a series of graphs showing the levels of human OTC mRNA and SPB mRNA relative to the level of mouse OTC mRNA in Otcspf-ash mice treated with the viral vector of this disclosure.

[0045] [Figure 16] This graph shows the correlation between human OTC mRNA or SPB mRNA and the total vector copy number per diploid genome in Otcspf-ash mice treated with the viral vector of this disclosure.

[0046] [Figure 17] This graph shows the survival rate in a mouse model of inducible hyperammonemia treated with the viral vector of this disclosure.

[0047] [Figure 18] This graph shows the ammonia concentration in plasma obtained from an inducible hyperammonemia mouse model treated with the viral vector of this disclosure.

[0048] [Figure 19] This graph shows the liver BLI measured in mice treated with various viral vectors of this disclosure.

[0049] [Figure 20]This graph shows the amount of human OTC mRNA relative to the level of mouse OTC mRNA in mice treated with the viral vector of this disclosure.

[0050] [Figure 21] This graph shows the amount of SBPmRNA relative to the level of mouse OTCmRNA in mice treated with the viral vector of this disclosure.

[0051] [Figure 22] This graph shows the amount of human OTC protein relative to the level of mouse OTC protein in mice treated with the viral vector of this disclosure.

[0052] [Figure 23] This graph shows the BLI measured in mice treated with various viral vectors of this disclosure.

[0053] [Figure 24] This graph shows the amount of human OTC mRNA relative to the level of mouse OTC mRNA in mice treated with the viral vector of this disclosure.

[0054] [Figure 25] This graph shows the amount of SBPmRNA relative to the level of mouse OTCmRNA in mice treated with the viral vector of this disclosure.

[0055] [Figure 26] This graph shows the amount of human OTC protein relative to the level of mouse OTC protein in mice treated with the viral vector of this disclosure.

[0056] [Figure 27] This document shows immunohistochemical analysis of hepatocytes isolated from mice treated with the viral vector of this disclosure. [Modes for carrying out the invention]

[0057] Detailed explanation This disclosure provides, but is not limited to, compositions and methods for the treatment of metabolic liver disorders, including urea cycle disorders (UCD). These compositions and methods are described in more detail herein.

[0058] Disclosure composition

[0059] Adeno-associated virus (AAV) piggyBac transposon polynucleotide

[0060] This disclosure provides a composition comprising adeno-associated virus (AAV) piggyBac transposon polynucleotide.

[0061] In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one AAV reverse-end repeat (ITR) sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one piggyBacITR sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one insulator sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one promoter sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one transgene sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one poly(A) sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one self-cleaving peptide sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one DNA spacer sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one Int6F sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may contain at least one Int6P1 sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may contain at least one Int6R sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may contain at least one JctR sequence. In some embodiments, the AAVpiggyBac transposon polynucleotide may contain at least one MCS sequence.

[0062] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0063] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence in the 5' to 3' direction.

[0064] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, and then a second AAVITR sequence.

[0065] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence, wherein any combination of at least one promoter sequence, at least one transgene sequence, at least one self-cleaving peptide sequence, and at least one polyA sequence exists between the first and second insulator sequences.

[0066] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a second insulator sequence, and a second piggyBacITR sequence, where any combination of at least one promoter sequence, at least one transgene sequence, at least one self-cleaving peptide sequence, and at least one poly(A) sequence exists between the first and second insulator sequences.

[0067] In some embodiments, the AAVpiggyBac transposon polynucleotide may comprise a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, and then a second AAVITR sequence, wherein between the first and second insulator sequences, there is any combination of at least one promoter sequence, at least one transgene sequence, at least one self-cleaving peptide sequence, and at least one polyA sequence.

[0068] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0069] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0070] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR 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 piggyBacITR sequence, and then a second AAVITR sequence.

[0071] In the non-limiting example of the AAVpiggyBac transposon polynucleotide described above, at least one promoter sequence may include a hybrid liver promoter (HLP), and at least one transgene sequence may include a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 2.

[0072] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, at least one DNA spacer sequence, and a second AAVITR sequence.

[0073] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, at least one DNA spacer sequence, and a second AAVITR sequence.

[0074] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR 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 piggyBacITR sequence, followed by at least one DNA spacer sequence, and then a second AAVITR sequence.

[0075] In the non-limiting example of the AAVpiggyBac transposon polynucleotide described above, at least one promoter sequence may include a hybrid liver promoter (HLP), and at least one transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 3A.

[0076] In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one DNA spacer sequence between a second piggyBacITR sequence and a second AAVITR sequence.

[0077] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

[0078] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, at least one transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

[0079] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR 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 piggyBacITR sequence, followed by a second AAVITR sequence, and followed by at least one DNA spacer sequence.

[0080] In the non-limiting example of the AAVpiggyBac transposon polynucleotide described above, at least one promoter sequence may include a hybrid liver promoter (HLP), and at least one transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 3B.

[0081] In some embodiments, the AAVpiggyBac transposon polynucleotide may include at least one DNA spacer after the second AAVITR sequence. A non-limiting example of an AAVpiggyBac transposon polynucleotide having at least one DNA spacer after the second AAVITR sequence is shown in Figure 3B.

[0082] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, at least one self-cleaved peptide sequence, at least a second transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0083] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, at least one self-cleaving peptide sequence, at least a second transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0084] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by at least one promoter sequence, followed by a first transgene sequence, followed by at least one self-cleaving peptide sequence, followed by at least a second transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, and then a second AAVITR sequence.

[0085] In the aforementioned non-limiting example of the AAVpiggyBac transposon polynucleotide, at least one promoter sequence may include a hybrid liver promoter (HLP), the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, at least one self-cleaving peptide sequence may include one encoding a T2A peptide, and at least a second transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 5.

[0086] In another non-limiting example of the aforementioned AAVpiggyBac transposon polynucleotide, at least one promoter sequence may include a thyroxine-binding globulin (TBG) promoter, the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, at least one self-cleaving peptide sequence may include one encoding a T2A peptide, and at least a second transgene sequence may include a luciferase sequence (e.g., NanoLuc). This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 8.

[0087] In the non-limiting example of the AAVpiggyBac transposon polynucleotide described above, at least one promoter sequence may include a hybrid liver promoter (HLP), the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, at least one self-cleaving peptide sequence may include one encoding a T2A peptide, and at least a second transgene sequence may include a nucleic acid sequence encoding an inducible caspase-9 (iCAS9) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 7.

[0088] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a first promoter sequence, a first transgene sequence, at least a second promoter sequence, at least a second transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0089] The AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, a first promoter sequence, a first transgene sequence, at least a second promoter sequence, at least a second transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0090] The AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by a first promoter sequence, followed by a first transgene sequence, followed by at least a second promoter sequence, followed by at least a second transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, and then a second AAVITR sequence.

[0091] In the non-limiting example of the AAVpiggyBac transposon polynucleotide described above, the first promoter sequence may include a hybrid liver promoter (HLP), the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, at least the second promoter sequence may include an HLP, and at least the second transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 6.

[0092] In some embodiments, the AAVpiggyBac transposon polynucleotide may contain two or more transgene sequences. In some embodiments, where the AAVpiggyBac transposon polynucleotide contains two or more transgene sequences, the individual transgene sequences may be separated by self-cleaving peptide sequences. In some embodiments, where the AAVpiggyBac transposon polynucleotide contains two or more self-cleaving peptide sequences, the self-cleaving peptide sequences may be the same or different.

[0093] In some embodiments, the AAVpiggyBac transposon polynucleotide may include two or more transgene sequences. In some embodiments in which the AAVpiggyBac transposon polynucleotide includes two or more transgene sequences, the AAVpiggyBac transposon may include multiple copies of a nucleic acid sequence encoding the same polypeptide. In an unrestricted example, the AAVpiggyBac transposon polynucleotide may include a first transgene sequence and a second transgene sequence, wherein the first and second transgene sequences each include a nucleic acid encoding an ornithine transcarbamylase (OTC) polypeptide.

[0094] In some embodiments, the AAVpiggyBac transposon polynucleotide may contain two or more promoter sequences. In some embodiments, the AAVpiggyBac transposon polynucleotide may contain two or more promoter sequences, and the promoter sequences may be the same or different.

[0095] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a polyA sequence, a second insulator sequence, and a second AAVITR sequence.

[0096] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a polyA sequence, a second insulator sequence, and a second AAVITR sequence.

[0097] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by at least one promoter sequence, followed by a first transgene sequence, followed by a first self-cleaving peptide sequence, followed by a second transgene sequence, followed by at least a second self-cleaving peptide sequence, followed by at least a third transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, and then a second AAVITR sequence.

[0098] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0099] In some embodiments, the AAVpiggyBac transposon polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a first transgene sequence, a first self-cleaving peptide sequence, a second transgene sequence, at least a second self-cleaving peptide sequence, at least a third transgene sequence, a polyA sequence, a second insulator sequence, a second piggyBacITR sequence, and a second AAVITR sequence.

[0100] In some embodiments, the AAVpiggyBac transposon polynucleotide may include a first AAVITR sequence, followed by a first piggyBacITR sequence, followed by a first insulator sequence, followed by at least one promoter sequence, followed by a first transgene sequence, followed by a first self-cleaving peptide sequence, followed by a second transgene sequence, followed by at least a second self-cleaving peptide sequence, followed by at least a third transgene sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second piggyBacITR sequence, and then a second AAVITR sequence.

[0101] In the aforementioned non-limiting example of the AAVpiggyBac transposon polynucleotide, at least one promoter sequence may include a hybrid liver promoter (HLP), the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, the second transgene sequence may include a fluorescent protein sequence (e.g., GFP or eGFP), and at least a third transgene sequence may include a luciferase sequence (e.g., NanoLuc). In this non-limiting example, both the first self-cleaving peptide sequence and at least the second self-cleaving peptide sequence may include nucleic acid sequences encoding a T2A peptide or a GSG-T2A peptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 1.

[0102] In another non-limiting example of the AAVpiggyBac transposon polynucleotide described above, at least one promoter sequence may include an LP1 promoter, the first transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide, the second transgene sequence may include a fluorescent protein sequence (e.g., GFP or eGFP), and at least a third transgene sequence may include a luciferase sequence (e.g., NanoLuc). In this non-limiting example, both the first self-cleaving peptide sequence and at least the second self-cleaving peptide sequence may include nucleic acid sequences encoding a T2A peptide or a GSG-T2A peptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 8.

[0103] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise, 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 shown in Sequence ID No. 104.

[0104] AAVITR sequence

[0105] In some embodiments, the AAVITR sequence may include any AAVITR sequence known in the art. In some embodiments, the AAVITR sequence may include, essentially consist 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 of the sequences shown in SEQ ID NOs. 1-4, 93-94, 105-106, and 127.

[0106] In some embodiments, the first AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 1, and the second AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 2.

[0107] In some embodiments, the first AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 3, and the second AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 4.

[0108] In some embodiments, the first AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 93, and the second AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 94.

[0109] In some embodiments, the first AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 105, and the second AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 106.

[0110] In some embodiments, the first AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 127, and the second AAVITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 4.

[0111] piggyBacITR array

[0112] In some embodiments, a piggyBacITR sequence may include any piggyBacITR sequence known in the art. In some embodiments, a piggyBacITR sequence may include, essentially consist 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 of the sequences shown in SEQ ID NOs. 5-6, 86-90, 95-96, and 125.

[0113] In some embodiments, the first piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 5, and the second piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 6.

[0114] In some embodiments, the first piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 6, and the second piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 5.

[0115] In some embodiments, the first piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 95, and the second piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 96.

[0116] In some embodiments, the first piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 125, and the second piggyBacITR sequence comprises, essentially comprises, or may comprise, 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 sequence number 96.

[0117] In some embodiments of the methods of the present disclosure, the piggyBacITR sequences within the AAVpiggyBac transposon, for example, a first piggyBacITR sequence and / or a second piggyBacITR sequence, include, essentially consist of, or comprise the Sleeping Beauty transposon ITR, the Hellraiser transposon ITR, the Tol2 transposon ITR, the TcBuster transposon ITR, or any combination thereof.

[0118] In some embodiments, the piggyBacITR sequence of the present disclosure may be flanked at either or both ends by at least one of 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'- TAAA-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'. In some embodiments, the piggyBacITR sequence may be flanked to 5'-TTAA-3'.Accordingly, any AAV transposase polynucleotide, AAVpiggyBac transposon polynucleotide, and / or any liver nanoplasmid of the present disclosure may 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', flanking to a piggyBac ITR sequence. 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'-TGAA-3', 5'-AAAT-3', 5'-AA TC-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'-TA AG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', 5'-TTTT-3'.

[0119] Insulator Array

[0120] In some embodiments, the insulator sequence may include any insulator sequence known in the art. In some embodiments, the insulator sequence may include, essentially consist 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 of the sequences shown in SEQ ID NOs. 7-8, 77-80, and 91-92.

[0121] In some embodiments, the first insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 7, and the second insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 8.

[0122] In some embodiments, the first insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 77, and the second insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 78.

[0123] In some embodiments, the first insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 79, and the second insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 80.

[0124] In some embodiments, the first insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 91, and the second insulator sequence comprises, essentially comprises, or may comprise, 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 sequence number 92.

[0125] Promoter array

[0126] In some embodiments, the promoter sequence may include any promoter sequence known in the art. In some embodiments, the promoter sequence may include any liver-specific promoter sequence known in the art.

[0127] In some embodiments, the promoter sequence comprises, essentially comprises, or may comprise, 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 of the sequences shown in SEQ ID NOs. 9-16, 69, 107, 126, 132, 145, and 146.

[0128] In some embodiments, the promoter sequence may include a hybrid liver promoter (HLP). The HLP may contain, essentially consist 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 sequence numbers 9, 107, or 126.

[0129] In some embodiments, the promoter sequence may include an LP1 promoter. The LP1 promoter may include, essentially consist 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 sequence number 10 or 132.

[0130] In some embodiments, the promoter sequence may include leukocyte-specific expression of the pp52(LSP1) long promoter. The LSP1 long promoter may contain, essentially consist 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 SEQ ID NO: 11.

[0131] In some embodiments, the promoter sequence may include a thyroxine-binding globulin (TBG) promoter. The TBG promoter may contain, essentially consist 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 SEQ ID NO: 12.

[0132] In some embodiments, the promoter sequence may include a wTBG promoter. The wTBG promoter may include, essentially consist of, or consist of a nucleic acid sequence that is identical to SEQ ID NO: 13 by at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between).

[0133] In some embodiments, the promoter sequence may include a liver combinatorial bundle (HCB) promoter. The HCB promoter may contain, essentially consist 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 SEQ ID NO: 14.

[0134] In some embodiments, the promoter sequence may include a 2xApoE-hAAT promoter. The 2xApoE-hAAT promoter may contain, essentially consist 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 SEQ ID NO: 15.

[0135] In some embodiments, the promoter sequence may include leukocyte-specific expression of the pp52(LSP1)+ chimeric intron promoter. The LSP1+ chimeric intron promoter may contain, essentially consist 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 SEQ ID NO: 16.

[0136] In some embodiments, the promoter sequence may include a cytomegalovirus (CMV) promoter. The CMV promoter may contain, essentially consist 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 SEQ ID NO: 69.

[0137] In some embodiments, the promoter sequence may include a TTR promoter. The TTR promoter may contain, essentially consist 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 SEQ ID NO: 145.

[0138] In some embodiments, the promoter sequence may include a TTRm promoter. The TTRm promoter may contain, essentially consist 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 SEQ ID NO: 146.

[0139] trans gene sequence

[0140] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. In some embodiments, the transgene sequence may include a nucleic acid sequence encoding a MUT1 polypeptide, where the MUT1 polypeptide includes, essentially consists of, or comprises 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 SEQ ID NOs. 17, 18, 121, or 122. In some embodiments, the acid sequence encoding the MUT1 polypeptide comprises, essentially comprises, or may comprise 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 of the sequences shown in SEQ ID NOs. 19, 20, or 111-120.

[0141] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, the transgene sequence may include a nucleic acid sequence encoding an OTC polypeptide, where the OTC polypeptide comprises, essentially comprises, or comprises 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 SEQ ID NOs. 21, 81, 123, or 124. In some embodiments, the acid sequence encoding the OTC polypeptide may comprise, essentially comprises, or comprises 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 of the sequences shown in SEQ ID NOs. 22, 23, 82, and 83.

[0142] In some embodiments, the transgene sequence may include a nucleic acid sequence encoding the iCAS9 polypeptide. In some embodiments, the transgene sequence may include a nucleic acid sequence encoding the iCAS9 polypeptide, where the iCAS9 polypeptide comprises, essentially comprises, or consists 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 SEQ ID NO: 24 or 84. In some embodiments, the acid sequence encoding the iCAS9 polypeptide may comprise, essentially comprises, or consists 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 either the sequence shown in SEQ ID NO: 25 or 85.

[0143] In some embodiments, the transgene sequence can be codon-optimized according to methods known in the art.

[0144] In some embodiments, a nucleic acid sequence encoding a polypeptide (e.g., OTC, MUT1, etc.) can be a codon-optimized nucleic acid sequence encoding a polypeptide. A codon-optimized nucleic acid sequence encoding a polypeptide may include, essentially consist of, or consist of a nucleic acid sequence that is identical to, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (or any percentage in between) or less of a wild-type human nucleic acid sequence encoding a polypeptide.

[0145] Sequence IDs 19, 20, 22, 23, 82, and 83 are unique codon-optimized nucleic acid sequences that can be included in the polynucleotides, vectors, and compositions of this disclosure.

[0146] In some embodiments, codon-optimized nucleic acid sequences encoding polypeptides, such as those shown in SEQ ID NOs. 19, 20, 22, 23, 82, and 83, may not contain donor splice sites. In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide may contain approximately 1, or approximately 2, or approximately 3, or approximately 4, or approximately 5, or approximately 6, or approximately 7, or approximately 8, or approximately 9, or approximately 10 or fewer donor splice sites. In some embodiments, a codon-optimized nucleic acid sequence encoding a polypeptide may contain at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 fewer donor splice sites compared to a wild-type human nucleic acid sequence encoding a polypeptide. While we do not wish to be bound by theory, the removal of donor splice sites in codon-optimized nucleic acid sequences may unexpectedly and unforeseenly increase polypeptide expression in vivo because hidden splicing is prevented. Furthermore, hidden splicing can vary between different subjects, meaning that the expression levels of polypeptides containing donor splice sites can vary unpredictably between different subjects.

[0147] In some embodiments, codon-optimized nucleic acid sequences encoding polypeptides, such as those shown in SEQ ID NOs: 19, 20, 22, 23, 82, and 83, may have a different GC content than wild-type human nucleic acid sequences encoding polypeptides. In some embodiments, the GC content of codon-optimized nucleic acid sequences encoding polypeptides is more uniformly distributed across the nucleic acid sequence compared to wild-type human nucleic acid sequences encoding polypeptides. While we do not wish to be constrained by theory, by more uniformly distributing the GC content across the nucleic acid sequence, codon-optimized nucleic acid sequences exhibit a more uniform melting temperature ("Tm") over the length of the transcript. This uniformity of melting temperature leads to transcription and / or translation of the nucleic acid sequence with less polymerase and / or ribosome stalling, resulting in an unexpected increase in the expression of codon-optimized nucleic acids in human subjects.

[0148] In some embodiments, codon-optimized nucleic acid sequences encoding polypeptides, such as those shown in SEQ ID NOs: 19, 20, 22, 23, 82, and 83, exhibit at least 5%, at least 100%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased expression in human subjects compared to wild-type or non-codon-optimized nucleic acid sequences encoding polypeptides.

[0149] In some embodiments, at least one transgene sequence can be operably ligated to at least one promoter sequence located on the same polynucleotide.

[0150] Poly-A array

[0151] In some embodiments, the polyA sequence may include 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 polyA sequence may include, essentially consist 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 of the sequences shown in SEQ ID NOs. 26-27, 97, 108, 128, and 136.

[0152] Self-cleaving peptide sequence

[0153] In some embodiments, the self-cleaving peptide sequence may include any self-cleaving peptide sequence known in the art. In some embodiments, the self-cleaving peptide sequence may include 2A self-cleaving peptide sequences known in the art. Non-limiting examples of self-cleaving peptides include T2A peptides, GSG-T2A peptides, E2A peptides, GSG-E2A peptides, F2A peptides, GSG-F2A peptides, P2A peptides, or GSG-P2A peptides.

[0154] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the T2A peptide. In some embodiments, the self-cleaving peptide sequence includes, essentially comprises, or consists 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 SEQ ID NO: 28.

[0155] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the GSG-T2A peptide. In some embodiments, the self-cleaving peptide sequence may include, essentially consist of, a nucleic acid sequence encoding the GSG-T2A peptide, where the GSG-T2A peptide includes, or essentially consists 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 SEQ ID NOs. 20. In some embodiments, the nucleic acid sequence encoding the GSG-T2A peptide may include, or essentially 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 SEQ ID NOs. 30-32 and 135.

[0156] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the E2A peptide. In some embodiments, the self-cleaving peptide sequence may include, essentially consist of, or comprise 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 SEQ ID NO: 33.

[0157] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the GSG-E2A peptide. In some embodiments, the self-cleaving peptide sequence may include, essentially consist of, or comprise 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 SEQ ID NO: 34.

[0158] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the F2A peptide. In some embodiments, the self-cleaving peptide sequence includes a nucleic acid sequence encoding the F2A peptide, wherein the F2A peptide includes, essentially consists of, or comprises 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 SEQ ID NO: 35.

[0159] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the GSG-F2A peptide. In some embodiments, the self-cleaving peptide sequence may include, essentially consist of, or comprise 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 SEQ ID NO: 36.

[0160] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding a P2A peptide. In some embodiments, the self-cleaving peptide sequence includes a nucleic acid sequence encoding a P2A peptide, wherein the P2A peptide includes, essentially consists of, or comprises 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 SEQ ID NO: 37.

[0161] In some embodiments, the self-cleaving peptide sequence may include a nucleic acid sequence encoding the GSG-P2A peptide. In some embodiments, the self-cleaving peptide sequence may include, essentially consist of, or comprise 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 SEQ ID NO: 38.

[0162] DNA spacer sequence

[0163] In some embodiments, the DNA spacer sequence comprises, essentially comprises, or may comprise 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 of the nucleic acid sequences shown in SEQ ID NOs. 103, 109, 129-131, and 137.

[0164] The DNA spacer sequence can be located at any position within the AAVpiggyBac transposon polynucleotide or the AAVpiggyBac transposase polynucleotide.

[0165] Int6F array

[0166] In some embodiments, the Int6F sequence comprises, essentially comprises, or may comprise 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 sequence number 98. In some embodiments, the Int6F sequence may be located between the polyA sequence and a second insulator sequence.

[0167] Int6P1 Array

[0168] In some embodiments, the Int6P1 sequence comprises, essentially comprises, or may comprise 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 sequence number 99. In some embodiments, the IntP1 sequence may be located between the polyA sequence and a second insulator sequence.

[0169] Int6R array

[0170] In some embodiments, the Int6R sequence comprises, essentially comprises, or may comprise 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 sequence number 100. In some embodiments, the Int6R sequence may be located between the polyA sequence and a second insulator sequence.

[0171] JctR sequence

[0172] In some embodiments, the JctR sequence comprises, essentially comprises, or may comprise 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 sequence number 101. In some embodiments, the JctR sequence may be located between a second piggyBacITR sequence and a second AAVITR sequence.

[0173] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise, 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 SEQ ID NO: 138.

[0174] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise 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 SEQ ID NO: 139.

[0175] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise, 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 SEQ ID NO: 140.

[0176] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise 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 SEQ ID NO: 141.

[0177] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise, 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 SEQ ID NO: 142.

[0178] In some embodiments, the AAVpiggyBac transposon polynucleotide comprises, essentially comprises, or may comprise, 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 SEQ ID NO: 143.

[0179] MCS sequence

[0180] In some embodiments, the MCS sequence comprises, essentially comprises, or may comprise 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 sequence number 102. In some embodiments, the MCS sequence may be located between a second piggyBacITR sequence and a second AAVITR sequence.

[0181] AAV Transposase Polynucleotide

[0182] This disclosure provides compositions comprising AAV transposase polynucleotides.

[0183] In some embodiments, an AAV transposase polynucleotide may include at least one AAV reverse-end repeat (ITR) sequence. In some embodiments, an AAV transposase polynucleotide may include at least one promoter sequence. In some embodiments, an AAV transposase polynucleotide may include at least one transposase sequence. In some embodiments, an AAV transposon polynucleotide may include at least one poly(A) sequence. In some embodiments, an AAV transposon polynucleotide may include at least one DNA spacer sequence.

[0184] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, and a second AAVITR sequence.

[0185] In some embodiments, the AAV transposase polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, and a second AAVITR sequence.

[0186] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, followed by at least one promoter sequence, followed by at least one transposase sequence, followed by a polyA sequence, and then a second AAVITR sequence.

[0187] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, at least one DNA spacer sequence, and a second AAVITR sequence.

[0188] In some embodiments, the AAV transposase polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, at least one DNA spacer sequence, and a second AAVITR sequence.

[0189] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, followed by at least one promoter sequence, followed by at least one transposase sequence, followed by a polyA sequence, followed by at least one DNA spacer sequence, and then a second AAVITR sequence.

[0190] In the non-limiting examples of the AAV transposase polynucleotides described above, at least one promoter sequence may include a hybrid liver promoter (HLP), and at least one transposase sequence may include a nucleic acid sequence encoding the SuperpiggyBac® (SPB) transposase polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 4A.

[0191] In some embodiments, the AAV transposase polynucleotide may include at least one DNA spacer sequence between the polyA sequence and the second AAVITR sequence, as shown in the non-limiting example in Figure 4A.

[0192] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

[0193] In some embodiments, the AAV transposase polynucleotide may include, in the 5' to 3' direction, a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, a second AAVITR sequence, and at least one DNA spacer sequence.

[0194] In some embodiments, the AAV transposase polynucleotide may include a first AAVITR sequence, followed by at least one promoter sequence, followed by at least one transposase sequence, followed by a polyA sequence, followed by a second AAVITR sequence, and followed by at least one DNA spacer sequence.

[0195] In the non-limiting examples of the AAV transposase polynucleotides described above, at least one promoter sequence may include a hybrid liver promoter (HLP), and at least one transposase sequence may include a nucleic acid sequence encoding the SuperpiggyBac® (SPB) transposase polypeptide. This non-limiting example of the AAVpiggyBac transposon polynucleotide is shown in Figure 4B.

[0196] In some embodiments, the AAV transposase polynucleotide may include at least one DNA spacer sequence after the second AAVITR sequence, as shown in the non-limiting example in Figure 4B.

[0197] In some embodiments, the AAV transposase polynucleotide comprises, essentially comprises, or may comprise 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 shown in SEQ ID NO: 110.

[0198] In some embodiments, the AAV transposase polynucleotide comprises, essentially comprises, or may comprise, 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 shown in SEQ ID NO: 144.

[0199] transposase sequence

[0200] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding any transposase polypeptide known in the art. In some embodiments, the transposase sequence may include a nucleic acid sequence encoding the piggyBac(PB) transposase polypeptide. In some embodiments, the transposase sequence may include a nucleic acid sequence encoding the piggyBac-like(PBL) transposase polypeptide. In some embodiments, the transposase sequence may include a nucleic acid sequence encoding the SuperpiggyBac(SPB) transposase polypeptide.

[0201] PB transposons and non-limiting examples of PB, PBL, and SPB transposases are described in detail in U.S. Patents 6,218,182, 6,962,810, 8,399,643, and PCT Publication No. WO2010 / 099296.

[0202] PB, PBL, and SPB transposases recognize transposon-specific reverse terminal repeats (ITRs) at the ends of transposons and insert their contents between the ITRs at the 5'-TTAA-3' position within a chromosomal region (TTAA target sequence). The target sequences of PB or 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', 5'-TAAA-3', 5'-TCTC-3', 5'-TGAA-3', 5'-A AAT-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' may be included or comprised of AAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-ATAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. There are no payload limitations on the target gene that can be included between ITRs in PB or PBL transposon systems.

[0203] 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 a preferred embodiment, a PB transposase comprises or consists of an amino 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 SEQ ID NO: 39.

[0204] PB or PBL transposases may contain or consist of an amino acid sequence having two or more, three or more, or each of the positions at positions 30, 165, 282, and / or 538 of the sequence of SEQ ID NO: 39. The transposase may also be an SPB transposase containing or consisting of the amino acid sequence of the sequence of SEQ ID NO: 39, where the amino acid substitution at position 30 may be a substitution of isoleucine (I) with valine (V), the amino acid substitution at position 165 may be a substitution of glycine (G) with serine (S), the amino acid substitution at position 282 may be a substitution of methionine (M) with valine (V), and the amino acid substitution at position 538 may be a substitution of asparagine (N) with lysine (K). In a preferred embodiment, the SPB transposase comprises or consists of an amino 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 SEQ ID NO: 40.

[0205] In certain embodiments in which the transposase contains the above mutations at positions 30, 165, 282, and / or 538, the PB, PBL, and SPB transposases may further contain amino acid substitutions at one or more positions of 3, 46, 82, 103, 119, 125, 177, 180, 185, 187, 200, 207, 209, 226, 235, 240, 241, 243, 258, 296, 298, 311, 315, 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591 of the sequence of SEQ ID NO. 39 or SEQ ID NO. 40, which are described in detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816.

[0206] In a preferred embodiment, the PB transposase comprises or consists of an amino 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 SEQ ID NO: 41.

[0207] PB or PBL transposases may contain or consist of an amino acid sequence having two or more, three or more, or each of the following amino acid substitutions at positions 29, 164, 281, and / or 537 of sequence 41. The transposase may also be an SPB transposase containing or consisting of the amino acid sequence of sequence 41, where the amino acid substitution at position 29 may be a substitution of isoleucine (I) by valine (V), the amino acid substitution at position 164 may be a substitution of glycine (G) by serine (S), the amino acid substitution at position 281 may be a substitution of methionine (M) by valine (V), and the amino acid substitution at position 537 may be a substitution of asparagine (N) by lysine (K). In a preferred embodiment, the SPB transposase comprises or consists of an amino 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 SEQ ID NO: 42.

[0208] In certain embodiments in which the transposase contains the above mutations at positions 29, 164, 281, and / or 537, the PB, PBL, and SPB transposases may further contain amino acid substitutions at one or more positions of 2, 45, 81, 102, 118, 124, 176, 179, 184, 186, 199, 206, 208, 225, 234, 239, 240, 242, 257, 295, 297, 310, 314, 318, 326, 327, 339, 420, 435, 455, 469, 485, 502, 551, 569, and 590 of the sequence of SEQ ID NO. 41 or SEQ ID NO. 42, which are described in more detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816.

[0209] PB, PBL, or SPB transposases can be isolated or derived from insects, vertebrates, crustaceans, or urochordates, as described in detail in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, PB, PBL, or SPB transposases are isolated or derived from the insect nettle moth (Trichoplusia ni) (GenBank registration number AAA87375) or the silkworm (Bombyx mori) (GenBank registration number BAD11135).

[0210] Hyperactive PB or PBL transposases are transposases that are more active than the naturally occurring variants from which they are derived. In a preferred embodiment, hyperactive PB or PBL transposases are isolated or derived from silkworms (Bombyx mori) or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patents 6,218,185, 6,962,810, 8,399,643, and WO2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent 10,041,077.

[0211] In some embodiments, PB, PBL, or SPB transposases are integration-deficient. Integration-deficient PB, PBL, or SPB transposases are transposases that can excise the corresponding transposon but incorporate the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB, PBL, or SPB transposases are disclosed in U.S. Patents 6,218,185, 6,962,810, 8,399,643, and WO2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent 10,041,077.

[0212] In some embodiments, PB, PBL, or SPB transposases can be fused to a nuclear localization signal. Examples of PB, PBL, or SPB transposases fused to a nuclear localization signal are disclosed in U.S. Patents 6,218,185, 6,962,810, 8,399,643, and WO2019 / 173636. The nuclear localization signal may consist of, essentially consist of, or comprise an amino 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 SEQ ID NO: 43. The nuclear localization signal may be encoded by a nucleic acid sequence that includes, essentially consists of, or comprises 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 sequence number 44.

[0213] In some embodiments, the nuclear localization signal can be fused to the PB, PBL, or SPB transposase using a G4S linker positioned between the NLS and the PB, PBL, or SPB. The G4S linker may contain, essentially consist of, or comprise an amino 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 SEQ ID NO: 45. The G4S linker may be encoded by a nucleic acid sequence that contains, essentially consists of, or comprises 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 SEQ ID NO: 46.

[0214] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding an SBP transposase polypeptide fused to the NLS, where the SBP transposase polypeptide fused to the NLS comprises, essentially comprises, or consists of, an amino 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 SEQ ID NO: 47. In some embodiments, the nucleic acid sequence encoding an SBP transposase polypeptide fused to the NLS may comprise, essentially comprises, or consists 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 of the sequences shown in SEQ ID NO: 48.

[0215] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding an SBP transposase polypeptide fused to the NLS, where the SBP transposase polypeptide fused to the NLS comprises, essentially comprises, or consists of, an amino 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 SEQ ID NO: 49. In some embodiments, the nucleic acid sequence encoding an SBP transposase polypeptide fused to the NLS may comprise, essentially comprises, or consists 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 of the sequences shown in SEQ ID NO: 50.

[0216] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding the Sleeping Beauty transposase polypeptide (for example, disclosed in U.S. Patent No. 9,228,180). In some embodiments, the transposase sequence may include a nucleic acid sequence encoding the hyperactivated Sleeping Beauty (SB100X) transposase polypeptide. In some embodiments, the Sleeping Beauty transposase comprises or consists of an amino 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 SEQ ID NOs. 51 and 52. In a preferred embodiment, the hyperactive Sleeping Beauty (SB100X) transposase comprises, essentially comprises, or consists of an amino 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 SEQ ID NOs. 53 and 54.

[0217] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding a helitron transposase polypeptide (e.g., disclosed in WO2019 / 173636). In some embodiments, the helitron transposase polypeptide comprises, essentially comprises, or consists of an amino 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 SEQ ID NO: 55 or 56.

[0218] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding a Tol2 transposase polypeptide (e.g., disclosed in WO2019 / 173636). In some embodiments, the Tol2 transposase polypeptide comprises, essentially comprises, or consists of an amino 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 SEQ ID NO: 57 or 58.

[0219] In some embodiments, the transposase sequence may include a nucleic acid sequence encoding a TcBuster transposase polypeptide (e.g., disclosed in WO2019 / 173636) or a mutant TcBuster transposase polypeptide (described in detail in PCT Publication WO2019 / 173636 and PCT / US2019 / 049816). In some embodiments, the TcBuster transposase polypeptide may consist of, or comprises, an amino 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 SEQ ID NO: 59 or 60. The polynucleotide encoding the TcBuster transposase may consist of or comprise a naturally occurring nucleic acid sequence or a nucleic acid sequence that is not naturally occurring.

[0220] Nanoplasmids for testing liver-specific promoters

[0221] This disclosure provides a composition comprising a nanoplasmid for testing a liver-specific promoter, referred to herein as a “liver nanoplasmid.”

[0222] In some embodiments, the liver nanoplasmid may contain at least one piggyBacITR sequence. In some embodiments, the liver nanoplasmid may contain at least one insulator sequence. In some embodiments, the liver nanoplasmid may contain at least one promoter sequence. In some embodiments, the liver nanoplasmid may contain at least one fluorescent protein sequence. In some embodiments, the liver nanoplasmid may contain at least one self-cleaving peptide sequence. In some embodiments, the liver nanoplasmid may contain at least one luciferase sequence. In some embodiments, the liver nanoplasmid may contain at least one poly(A) sequence.

[0223] The liver nanoplasmid can include a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a fluorescent protein sequence, at least one self-cleaving peptide sequence, a luciferase sequence, a polyA sequence, a second insulator sequence, and a second piggyBacITR sequence. In some embodiments, the liver nanoplasmid can include, in the 5' to 3' direction, a first piggyBacITR sequence, a first insulator sequence, at least one promoter sequence, a fluorescent protein sequence, at least one self-cleaving peptide sequence, a luciferase sequence, a polyA sequence, a second insulator sequence, and a second piggyBacITR sequence.

[0224] In some aspects of this disclosure, the transgene sequence may include a fluorescent protein sequence.

[0225] In some embodiments, the fluorescent protein sequence may include a nucleic acid sequence encoding an eGFP polypeptide. In some embodiments, the fluorescent protein sequence may include a nucleic acid sequence encoding an eGFP polypeptide, where the eGFP polypeptide comprises, essentially comprises, or consists of an amino 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 SEQ ID NO: 61 or 62. In some embodiments, the nucleic acid sequence encoding an eGFP polypeptide may comprise, essentially comprises, or consists of a nucleic acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to any of the sequences shown in SEQ ID NO: 63, 64, or 133.

[0226] In some aspects of this disclosure, the transgene sequence may include a luciferase sequence.

[0227] In some embodiments, the luciferase sequence may include a nucleic acid sequence encoding the fLuc2 polypeptide. In some embodiments, the luciferase sequence may include a nucleic acid sequence encoding the fLuc2 polypeptide, where the fLuc2 polypeptide comprises, essentially comprises, or consists of an amino 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 SEQ ID NO: 65 or 66. In some embodiments, the nucleic acid sequence encoding the eGFP polypeptide may comprise, essentially comprises, or consists 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 either the sequence shown in SEQ ID NO: 67 or 68.

[0228] In some embodiments, the luciferase sequence may include a nucleic acid sequence encoding a nanoluciferase (nLuc) polypeptide. In some embodiments, the nucleic acid sequence encoding the nLuc polypeptide may include, essentially consist 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 of the sequences shown in Sequence ID No. 134.

[0229] Vector of this disclosure

[0230] This 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 the "AAVpiggyBac transposon vector".

[0231] This disclosure provides a composition 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".

[0232] The vectors of this disclosure may be viral vectors or recombinant vectors. Viral vectors may include sequences isolated or derived from retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, or any combination thereof. Viral vectors may include sequences isolated or derived from adeno-associated viruses (AAVs). Viral vectors may include recombinant AAVs (rAAVs).

[0233] 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 also include, but are not limited to, self-complementary AAVs (scAAVs), as well as AAV hybrids containing 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, rAAV-LK03, AAV-KP-1 (also known as AAV-KP1; described in detail in Kerun et al. JCI Insight, 2019; 4(22):e131610), and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).

[0234] This disclosure provides a composition comprising a plurality of AAV-KP-1 particles, each containing at least one adeno-associated virus (AAV)piggyBac transposon polynucleotide. This disclosure provides a composition comprising a plurality of AAV-KP-1 particles, each containing at least one AAV transposase polynucleotide. This disclosure provides a composition comprising a plurality of AAV-KP-1 particles, each containing at least one adeno-associated virus (AAV)piggyBac transposon polynucleotide, and a plurality of AAV-KP-1 particles, each containing at least one AAV transposase polynucleotide.

[0235] This disclosure provides a composition comprising a plurality of AAV-NP59 particles, each containing at least one adeno-associated virus (AAV)piggyBac transposon polynucleotide. This disclosure provides a composition comprising a plurality of AAV-NP59 particles, each containing at least one AAV transposase polynucleotide. This disclosure provides a composition comprising a plurality of AAV-NP59 particles, each containing at least one adeno-associated virus (AAV)piggyBac transposon polynucleotide, and a plurality of AAV-NP59 particles, each containing at least one AAV transposase polynucleotide.

[0236] The viral vectors and viral particles of this disclosure can be produced using standard methods known in the art.

[0237] In some embodiments, the AAV-KP-1 particles of the present disclosure can be produced using a KP-1 capsid vector, wherein the KP-1 capsid vector comprises at least one of the nucleic acid sequences of SEQ ID NO: 70 and SEQ ID NO: 71. In some embodiments, the AAV-KP-1 particles of the present disclosure can be produced using an AAV vector packaging plasmid, wherein the AAV vector packaging plasmid comprises at least one of the nucleic acid sequences of SEQ ID NO: 75 and SEQ ID NO: 76.

[0238] In some embodiments, the AAV-NP59 particles of the present disclosure can be generated using an NP-59 capsid vector, wherein the NP-59 capsid vector comprises at least one of the nucleic acid sequences of SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74. In some embodiments, the AAV-NP59 particles of the present disclosure can be generated using an AAV vector packaging plasmid, wherein the AAV vector packaging plasmid comprises at least one of the nucleic acid sequences of SEQ ID NO: 75 and SEQ ID NO: 76.

[0239] The cell delivery compositions disclosed herein (e.g., polynucleotides, vectors) may include therapeutic proteins or nucleic acids encoding therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT publication numbers WO2019 / 173636 and PCT / US2019 / 049816. Therapeutic proteins may also include, but are not limited to, any polypeptides described herein as part of a transgene sequence (e.g., OTC, MUT1, etc.).

[0240] Formulation, dosage, and mode of administration

[0241] This disclosure provides formulations, dosages, and methods of administration of the compositions described herein.

[0242] The disclosed compositions and pharmaceutical compositions may further include, but are not limited to, at least one of any suitable auxiliary substances, such as diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, and auxiliaries. Medicinally acceptable auxiliaries are preferred. Non-limited examples of such sterile solutions and methods for preparing them are well known in the art, for example, but are 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. A pharmaceutically acceptable carrier suitable for the administration method, solubility, and / or stability of the protein scaffold, fragment, or variant composition, as well as those well known in the art or as described herein, can be routinely selected.

[0243] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, e.g., alditol, aldonic acid, esterified sugars, etc.; and polysaccharides or sugar polymers), which can exist alone or in combination, and constitute 1 to 99.99% by weight or volume, either alone or in combination. Non-limiting examples of protein excipients include serum albumins such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acid / protein components that also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame. One preferred amino acid is glycine.

[0244] Suitable carbohydrate excipients for use are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, and sorbose; disaccharides such as lactose, sucrose, trehalose, and cellobiose; polysaccharides such as raffinose, melegitose, maltodextrin, dextran, and starch; and algitols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), and myo-inositol. Preferably, the carbohydrate excipients are mannitol, trehalose, and / or raffinose.

[0245] The composition may also contain buffers or pH adjusters, and typically the buffer is a salt prepared from an organic acid or organic base. Typical buffers include organic acid salts, such as salts of citric acid, ascorbic acid, gluconic acid, carbonate, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffer. Preferred buffers are organic acid salts such as citrate.

[0246] Furthermore, the disclosed compositions may include polymeric excipients / additives, such as polyvinylpyrrolidone, Ficol (a high-molecular-weight sugar), dextrates (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "Tween 20" or "Tween 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0247] Many known and developed methods can be used to administer a therapeutically effective amount of the compositions disclosed herein or of the pharmaceutical compositions. Non-limiting examples of administration methods include bolus, oral, infusion, intra-articular, intra-bronchial, intraperitoneal, intrasacral, intracartilaginous, intracerebellar, intraventricular, intracolonic, intracerebral, intracerebral, intracerebral, intrastomical, intraocular, intraosseous, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, rectal, intrarenal, intraretinal, intraspinal cord, intrabursal, intrathoracic, intrauterine, intratumoral, intravenous, intrabladder, oral, parenteral, rectal, sublingual, subcutaneous, percutaneous, or vaginal means.

[0248] The compositions of this disclosure may be used parenterally (subcutaneous, intramuscular, or intravenous) or for any other administration, particularly in the form of a solution or suspension; for vaginal or rectal administration in semi-solid forms such as creams and suppositories, but not limited to; for oral or sublingual administration, particularly in the form of tablets or capsules, but not limited to; or for intranasal use in the form of powders, nasal drops, aerosols, or specific drugs, but not limited to; or for use percutaneously, for example, in a gel, ointment, lotion, suspension, or percutaneous patch system, the percutaneous patch system may include a chemical enhancer such as dimethyl sulfoxide to modify the skin structure or increase the drug concentration in the percutaneous patch (Junginger, et al. In “Drug Permeation Enhancement;” Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York) 1994), or with an oxidizing agent that enables the application of a formulation containing proteins and peptides to the skin (WO98 / 53847), or with the application of an electric field such as electroporation or iontophoresis to enhance the mobility of charged drugs passing through the skin to create a transient transport pathway, or with the application of ultrasound such as sonophoresis (U.S. Patents No. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety).

[0249] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, particles, powder, or lyophilized powder together with a pharmaceutically acceptable parenteral vehicle or provided separately. Formulations for parenteral administration may include, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, hydrogenated naphthalene, etc. Aqueous or oily suspensions for injection may be prepared according to known methods using appropriate emulsifiers or humectants and suspending agents. Drugs for injection may be non-toxic and parenterally administered diluents, such as aqueous solutions, sterile injection solutions, or suspensions in solvents. Acceptable vehicles or solvents include water, Ringer's solution, isotonic saline, etc. Sterile non-volatile oils may be used as ordinary solvents or suspension solvents. For these purposes, any type of non-volatile oil and fatty acid, including natural, synthetic, or semi-synthetic fatty oils or fatty acids; natural, synthetic, or semi-synthetic monoglycerides, diglycerides, or triglycerides may be used. Parenteral administration can be carried out by conventional injection methods, including but not limited to those known in the art, such as gas-pressurized needleless injectors as described in U.S. Patent No. 5,851,198, and laser perforation devices as described in U.S. Patent No. 5,839,446.

[0250] Formulations for oral administration rely on the co-administration of adjuvants (e.g., resorcinol and nonionic surfactants, e.g., polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase intestinal wall permeability, and on the co-administration of enzyme inhibitors (e.g., pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFF), and trazilol) to inhibit enzymatic degradation. Formulations for the delivery of hydrophilic agents containing proteins and protein scaffolds, and combinations of at least two surfactants, for oral, mucosal, nasal, pulmonary, vaginal, or rectal administration are described in U.S. Patent No. 6,309,663. The active ingredient compound in solid dosage forms for oral administration may be mixed with at least one additive (including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, acacia gum, gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and glycerides). These dosage forms may also contain other types of additives, such as inert diluents, lubricants (e.g., magnesium stearate, parabens), preservatives (e.g., sorbic acid, ascorbic acid, α-tocopherol), antioxidants (e.g., cysteine), disintegrants, binders, thickeners, buffers, sweeteners, flavorings, and fragrances.

[0251] Tablets and pills can be further processed into enteric-coated preparations. Preparations for oral administration include emulsions, syrups, elixirs, suspensions, and liquids that can be used for medical purposes. These preparations may contain inert diluents commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Patent No. 4,239,754). More recently, microspheres of artificial polymers (proteinoids) of mixed amino acids have been used for drug delivery (U.S. Patent No. 4,925,673). Furthermore, carrier compounds used for oral delivery of bioactive agents are also known in the art, as described in U.S. Patents No. 5,879,681 and U.S. Patent No. 5,871,753.

[0252] For pulmonary administration, the compositions or pharmaceutical compositions described herein are preferably delivered in a particle size effective to reach the lower airways or sinuses of the lungs. These compositions or pharmaceutical compositions can be delivered by any of the various inhalation or nasal devices known in the art for the administration of therapeutic agents by inhalation. These devices, which can deposit aerosolized formulations into the sinuses or alveoli of a patient, include metered-dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices can use formulations suitable for administration to distribute the compositions or pharmaceutical compositions described herein in aerosol. Such aerosols may consist of either a solution (both aqueous and non-aqueous) or solid particles. Furthermore, sprays containing the compositions or pharmaceutical compositions described herein can be produced by extruding a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered-dose inhaler (MDI), the propellant, the compositions or pharmaceutical compositions described herein, and any excipients or other additives are contained in a canister as a mixture including liquefied compressed gas. The metering valve releases the mixture as an aerosol containing particles in a size range of preferably less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. A more detailed description of pulmonary administration, formulation, and associated devices is disclosed in PCT publication number WO2019 / 049816.

[0253] For absorption through the mucosal surface, the composition comprises an emulsion comprising an aqueous continuous phase that promotes absorption through the mucosal surface by achieving mucosal adhesion of a plurality of submicron particles, a mucoadhesive polymer, a bioactive peptide, and emulsion particles (U.S. Patent No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present disclosure may include the corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain, as excipients, for example, polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid and contain, as excipient, for example, lactose, or may be an aqueous or oily solution of a nasal drop. In the case of oral administration, excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like (U.S. Patent No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. WO2019 / 049816.

[0254] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in delivery devices such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise specified). Many suitable devices are known that include microparticles made of synthetic polymers such as polyhydroxy acids, polylactic acid, polyglycolic acid, and their copolymers, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin, and other proteins, alginates, 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. WO2019 / 049816.

[0255] It may be desirable to deliver the disclosed compounds to subjects over a long period, for example, from a single dose to one week to one year. Various sustained-release, depot, or implantable dosage forms can be utilized. For example, dosage forms may include pharmaceutically acceptable nontoxic salts of compounds with low solubility in body fluids: for example, (a) acid addition salts with polybasic acids, such as phosphoric acid, sulfuric acid, citrate, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono or disulfonic acid, polygalacturonic acid, etc.; (b) salts with polyvalent metal cations, such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc., or with organic cations formed from, for example, N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), for example, zinc tannate salts. Furthermore, the disclosed compounds, or preferably relatively insoluble salts such as those described above, can be incorporated into gels suitable for injection, such as aluminum monostearate gel containing sesame oil. Particularly preferred salts include zinc salts, zinc tannate salts, and pamoate salts. Another type of sustained-release depot formulation for injection may contain compounds or salts dispersed for encapsulation in slow-degrading, non-toxic, non-antigenic polymers, such as polylactic acid / polyglycolic acid polymers, as described in, for example, U.S. Patent No. 3,773,919. The compounds, or preferably relatively insoluble salts such as those described above, can also be incorporated into cholesterol matrix silastic pellets, particularly for use in animals. Additional sustained-release, depot, or implant formulations, such as gaseous or liquid liposomes, are known in the literature (U.S. Patent No. 5,770,222, and “Sustained and Controlled Release Drug Delivery Systems”, JR Robinson ed., Marcel Dekker, Inc., NY, 1978).

[0256] Appropriate 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, Calif. (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, N.J. Preferred dosages include, optionally, about 0.1-99 and / or 100-500 mg / kg / dose, or any range, value, or portion thereof, to achieve a serum concentration of about 0.1-5000 μg / ml, or any range, value, or portion thereof, per single or multiple administrations. Preferred dosage ranges for the compositions or pharmaceutical compositions disclosed herein are about 1 mg, up to about 3 mg, about 6, or about 12 mg per kg of body weight of the subject.

[0257] Alternatively, the dosage administered may vary depending on known factors such as the pharmacodynamic properties of the particular agent, and its mode and route of administration, the age, health status, and weight of the recipient. The nature and extent of the symptoms, the type of concurrent therapy, the frequency of treatment, and the desired effect, etc. Generally, the dosage of the active ingredient can be about 0.1-100 milligrams per kilogram of body weight. To obtain the desired result, a single administration or in a sustained release form, usually 0.1-50 mg, preferably 0.1-10 mg / kg is effective.

[0258] As a non-limiting example, for the treatment of humans or animals, as a single or regular dosage of the compositions or pharmaceutical compositions disclosed herein, on at least one day of 1 to 40 days, or alternatively or additionally on at least one week of 1 to 52 weeks, or alternatively or additionally on at least one year of 1 to 20 years, or any combination thereof, single, infusion, or repeated administration may be used to provide about 0.1 to 100 mg / kg per day, or any range, value, or portion thereof.

[0259] Dosage forms suitable for internal administration generally contain from about 0.001 mg to about 500 mg of the active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient is usually present in an amount of about 0.5 to 99.999% by weight based on the total weight of the composition.

[0260] The effective amount is about 0.1 to 5000 μg / ml of serum concentration per single or multiple administrations, or any range, value, or portion thereof, achieved using known methods described herein or known in the relevant art, and may include an amount of about 0.001 to about 500 mg / kg per single administration (e.g., bolus), multiple, or continuous administration.

[0261] In embodiments where the composition administered to a subject that requires it is the modified cell disclosed herein, the cells are about 1×10 6 ~1×10 15 cells, about 1x10 4 ~1x10 12 cells, about 1x10 5 ~1x10 10 cells, about 1x10 6 ~1x10 9 cells, about 1x10 6 ~1x10 8 cells, about 1x10 6 ~1x10 7 cells, or about 1x10 6 ~25x10 6 cells can be administered. In one embodiment, the cells are about 5×10 6 ~25×10 6 cells are administered.

[0262] A more detailed description of the disclosed compositions and pharmaceutically acceptable excipients, formulations, dosages, and methods of administration of the pharmaceutical compositions is disclosed in PCT Publication No. WO2019 / 049816.

[0263] Method of using the composition disclosed herein

[0264] This disclosure provides the use of the disclosed composition or pharmaceutical composition to treat a disease or disorder in a cell, tissue, organ, animal, or subject, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject, as known in the art or as described herein. In one embodiment, the subject is a mammal. Preferably, the subject is a human. The terms “subject” and “patient” are used interchangeably herein.

[0265] This disclosure provides a method for treating at least one metabolic liver disorder (MLD) in a subject requiring the treatment, comprising administering at least one therapeutically effective dose of at least one composition of this disclosure to the subject in need.

[0266] This disclosure provides at least one composition of the present disclosure for use in the treatment of at least one metabolic liver disorder in a subject, wherein the at least one composition is to be administered to the subject in at least one therapeutically effective dose.

[0267] This disclosure provides the use of at least one composition of this disclosure for manufacturing a medicament for treating at least one metabolic liver disorder in a subject, wherein the at least one composition is to be administered to the subject in at least one therapeutically effective dose.

[0268] In some embodiments of the methods and uses described above, at least one composition of the Disclosure may include at least one AAVpiggyBac transposon vector of the Disclosure.

[0269] Accordingly, the present disclosure provides a method for treating at least one metabolic liver disorder in a subject, comprising administering at least one therapeutically effective dose of at least one AAVpiggyBac transposon vector of the present disclosure to a subject in need thereof.

[0270] This disclosure provides at least one AAVpiggyBac transposon vector for use in the treatment of at least one metabolic liver disorder in a subject, wherein the at least one AAVpiggyBac transposon vector is to be administered to a subject in at least one therapeutically effective dose.

[0271] This disclosure provides the use of at least one AAVpiggyBac transposon vector of this disclosure for manufacturing a pharmacopoeia for the treatment of at least one metabolic liver disorder in a subject, wherein the at least one AAVpiggyBac transposon vector is to be administered to a subject in at least one therapeutically effective dose.

[0272] In some embodiments of the methods and uses described above, at least one composition of the Disclosure may include at least one AAV transposase vector of the Disclosure.

[0273] Accordingly, the present disclosure provides a method for treating at least one metabolic liver disorder in a subject requiring it, comprising administering at least one therapeutically effective dose of at least one AAV transposase vector of the present disclosure to the subject requiring it.

[0274] The present disclosure provides at least one AAV transposase vector of the present disclosure for use in treating at least one metabolic liver disorder in a subject, wherein the at least one AAV transposase vector is for administration to a subject in at least one therapeutically effective amount.

[0275] The present disclosure provides the use of at least one AAV transposase vector of the present disclosure for manufacturing a medicament for treating at least one metabolic liver disorder in a subject, wherein the at least one AAV transposase vector is for administration to a subject in at least one therapeutically effective amount.

[0276] The present disclosure provides a method for treating at least one metabolic liver disorder in a subject, the method comprising administering to the subject a) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a transposon, 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, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein.

[0277] In some embodiments of the foregoing method, the composition comprising a nucleic acid molecule comprising a transposon can be any AAVpiggyBac transposon vector described herein.

[0278] In some embodiments of the foregoing method, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be any AAV transposase vector of the present disclosure.

[0279] Accordingly, the present disclosure provides a method for treating at least one metabolic liver disorder in a subject, the method comprising administering to the subject a) at least one therapeutically effective dose of at least one AAVpiggyBac transposon vector of the present disclosure and b) at least one therapeutically effective dose of at least one AAV transposase vector of the present disclosure.

[0280] Accordingly, the present disclosure provides a combination of at least one AAVpiggyBac transposon vector and at least one AAV transposase vector of the present disclosure for use in the treatment of at least one metabolic liver disorder in a subject, wherein the at least one AAVpiggyBac transposon vector is to be administered to a subject in at least one therapeutically effective dose, and the at least one AAV transposase vector is to be administered to a subject in at least one therapeutically effective dose.

[0281] Accordingly, this disclosure provides the use of a combination of at least one AAVpiggyBac transposon vector and at least one AAV transposase vector of this disclosure in the manufacture of a pharmaceutical product for the treatment of at least one metabolic disorder in a subject, wherein the at least one AAVpiggyBac transposon vector is for administration to a subject in at least one therapeutically effective dose, and the at least one AAV transposase vector is for administration to a subject in at least one therapeutically effective dose.

[0282] Metabolic liver disorders may include, but are not limited to, urea cycle disorders, N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinateuria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof. In some embodiments, metabolic liver dysfunction is ornithine transcarbamylase (OTC) deficiency.

[0283] In some embodiments of the above-described method, a composition comprising a nucleic acid molecule containing a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered simultaneously, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, a composition comprising a nucleic acid molecule containing a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered sequentially, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, a composition comprising a nucleic acid molecule containing a transposon and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be administered in close proximity in time, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein.

[0284] As used herein, the term “temporal proximity” means that the administration of one therapeutic composition (e.g., a composition containing a transposon) occurs within a time period prior to or after the administration of another therapeutic composition (e.g., a composition containing a transposase), and therefore 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, “temporal proximity” means that the administration of one therapeutic agent occurs within a time period prior to or after the administration of another therapeutic agent, and therefore there is a synergistic effect between one therapeutic agent and the other therapeutic agent. “Temporal proximity” can vary depending on a variety of factors, including, but is not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent is administered; the disease or condition to be treated or improved; the therapeutic outcome 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 through which the therapeutic agent is administered. In some embodiments, “temporal proximity” 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 doses of one therapeutic agent can be administered in temporal proximity to a single dose of another therapeutic agent. In some embodiments, temporal proximity can vary during a treatment cycle or in a dosing plan.

[0285] In some embodiments of the therapeutic methods of the present disclosure, administration of at least one composition and / or vector of the present disclosure to a subject may result in the expression of an exogenous protein (e.g., a therapeutic protein, a transposase, etc.) in at least one organ and / or tissue of the subject.

[0286] In some embodiments, administration of at least one composition and / or vector of the Disclosure results in the expression of exogenous proteins 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 cells in tissues and / or organs.

[0287] In some embodiments, administration of at least one composition and / or vector of the present disclosure results in the expression of exogenous proteins 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 of cells in a tissue and / or organ.

[0288] In some embodiments, administration of at least one composition and / or vector of the Disclosure results in the expression of an 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.

[0289] In some embodiments, administration of at least one composition and / or vector of the Disclosure results in the expression of an exogenous protein in a specific subset 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.

[0290] In some embodiments, administration of at least one composition and / or vector of the Disclosure results in the expression of exogenous proteins in tissues and / or organs for a period of 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.

[0291] In some embodiments, administration of at least one composition and / or vector of the Disclosure results in the expression of exogenous proteins in a specific subset of cells within a tissue and / or organ for a period of 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.

[0292] In some embodiments, the tissue and / or organ may be the liver. In some embodiments, a particular subset of cells, without being particularly limited, may include hepatocytes, hepatic astrocytes, Kupffer cells, hepatic sinusoidal endothelial cells, or any combination thereof.

[0293] Any method of the present disclosure may include administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal, or subject in need of such adjustment, treatment, or therapy. Such method may optionally further include concurrent or combination therapy for treating such disease or disorder, wherein the administration of any composition or pharmaceutical composition disclosed herein further includes concurrently and / or after administering at least one additional treatment for a urea cycle disorder.

[0294] Further treatments for urea cycle disorders may include, but are not limited to, dialysis, hemofiltration, calorie supplementation, hormone suppression, glucose infusion, insulin infusion, pharmacological removal of excess nitrogen, dextrose administration, fluid administration, Intralipid® administration, ammonia scavenger administration, arginine administration, sodium phenylacetate administration, sodium benzoate administration, Ammonul administration, phenylbutyrate administration, citrulline supplementation, arginine supplementation, or any combination thereof.

[0295] Exemplary Embodiments of the Present Disclosure

[0296] Embodiment 1. Adeno-associated virus (AAV) piggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) The first AAV reverse terminal repeat (ITR) sequence, b) The first piggyBacITR sequence, c) First insulator array, d) At least one promoter sequence, e) at least one transgene sequence, f) Poly-A sequence, g) Second insulator array, h) Second piggyBacITR sequence, and i) The second AAVITR sequence.

[0297] Embodiment 2. The AAVpiggyBac transposon polynucleotide of Embodiment 1, wherein the AAVpiggyBac transposon polynucleotide comprises DNA, cDNA, gDNA, RNA, mRNA, or any combination thereof.

[0298] Embodiment 3. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first and / or second AAVITR sequence comprises one nucleic acid sequence of sequence numbers 1-4, 93-94, 105-106, and 127.

[0299] Embodiment 4. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 3 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 4.

[0300] Embodiment 5. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence and / or the second piggyBacITR sequence comprises one nucleic acid sequence of sequence numbers 5-6, 86-90, 95-96, and 125.

[0301] Embodiment 6. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 5 and the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 6.

[0302] Embodiment 7. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first insulator sequence and / or the second insulator sequence comprises one of the nucleic acid sequences of SEQ ID NOs: 7-8, 77-80, and 91-92.

[0303] Embodiment 8. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 7 and the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 8.

[0304] Embodiment 9. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence is a liver-specific promoter.

[0305] Embodiment 10. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the liver-specific promoter is leukocyte-specific expression of a hybrid liver promoter (HLP), LP1 promoter, pp52(LSP1) long promoter, thyroxine-binding globulin (TBG) promoter, wTBG promoter, liver combinatorial bundle (HCB) promoter, 2xApoE-hAAT promoter, or leukocyte-specific expression of pp52(LSP1) and chimerichiontron promoter.

[0306] Embodiment 11. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises one of the nucleic acid sequences of SEQ ID NOs: 9-16, 69, 107, 126, and 132.

[0307] Embodiment 12. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide.

[0308] Embodiment 13. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the MUT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 17, 18, 121, or 122.

[0309] Embodiment 14. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the MUT1 polypeptide includes the nucleic acid sequence of SEQ ID NOs. 19, 20, or 111-120.

[0310] Embodiment 15. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide.

[0311] Embodiment 16. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the OTC polypeptide comprises the amino acid sequence of SEQ ID NO: 21 or 81.

[0312] Embodiment 17. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the OTC polypeptide includes the nucleic acid sequence of SEQ ID NOs. 22, 23, 82, and 83.

[0313] Embodiment 18. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises a nucleic acid sequence encoding an iCas9 polypeptide.

[0314] Embodiment 19. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the iCas9 polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or 84.

[0315] Embodiment 20. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the iCas9 polypeptide comprises the nucleic acid sequence of SEQ ID NO: 25 or 85.

[0316] Embodiment 21. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence is operably linked to at least one promoter sequence.

[0317] Embodiment 22. An AAVpiggyBac transposon polynucleotide according to any of the above embodiments, wherein the expression of at least one transgene sequence is controlled by at least one promoter sequence.

[0318] Embodiment 23. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the polyA sequence comprises one nucleic acid sequence of sequence numbers 26-27, 97, 108, 128, and 136.

[0319] Embodiment 24. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the AAVpiggyBac transposon polynucleotide further comprises at least a second transgene sequence.

[0320] Embodiment 25. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least a second transgene sequence comprises a nucleic acid sequence encoding an iCas9 polypeptide.

[0321] Embodiment 26. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the iCas9 polypeptide comprises the amino acid sequence of SEQ ID NO: 24 or 84.

[0322] Embodiment 27. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the iCas9 polypeptide includes the nucleic acid sequence of SEQ ID NO: 25 or 85.

[0323] Embodiment 28. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least a second transgene sequence comprises a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide.

[0324] Embodiment 29. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the MUT1 polypeptide comprises the amino acid sequence of SEQ ID NO: 17, 18, 121, or 122.

[0325] Embodiment 30. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the MUT1 polypeptide includes the nucleic acid sequence of SEQ ID NOs. 19, 20, or 111-120.

[0326] Embodiment 31. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least a second transgene sequence comprises a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide.

[0327] Embodiment 32. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the OTC polypeptide comprises the amino acid sequence of SEQ ID NO: 21 or 81.

[0328] Embodiment 33. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the nucleic acid sequence encoding the OTC polypeptide includes the nucleic acid sequence of SEQ ID NOs. 22, 23, 82, and 83.

[0329] Embodiment 34. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the AAVpiggyBac transposon polynucleotide further comprises at least a second promoter sequence.

[0330] Embodiment 35. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least a second promoter sequence is located between at least one trans gene sequence and at least a second trans gene sequence.

[0331] Embodiment 36. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the AAVpiggyBac transposon polynucleotide further comprises at least one self-cleaving peptide sequence, the at least one self-cleaving peptide sequence being a nucleic acid sequence encoding a T2A peptide, a GSG-T2A peptide, an E2A peptide, a GSG-E2A peptide, an F2A peptide, a GSG-F2A peptide, a P2A peptide, or a GSG-P2A peptide.

[0332] Embodiment 37. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one self-cleaving peptide sequence is located between at least one transgene sequence and at least a second transgene sequence.

[0333] Embodiment 38. An AAVpiggyBac transposon polynucleotide according to any of the above embodiments, wherein the AAVpiggyBac transposon polynucleotide comprises at least two transgene sequences.

[0334] Embodiment 39. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least two transgene sequences are the same sequence.

[0335] Embodiment 40. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least two transgene sequences are different sequences.

[0336] Embodiment 41. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, further comprising at least one DNA spacer sequence.

[0337] Embodiment 42. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises one nucleic acid sequence of sequence numbers 103, 109, 129-131, and 137.

[0338] Embodiment 43. An AAVpiggyBac transposon polynucleotide according to any of the above embodiments, wherein the AAVpiggyBac transposon polynucleotide comprises at least two promoter sequences.

[0339] Embodiment 44. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least two promoter sequences are the same sequence.

[0340] Embodiment 45. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least two promoter sequences are different sequences.

[0341] Embodiment 46A. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) The first AAVITR sequence, b) The first piggyBacITR sequence, c) First insulator array, d) At least one promoter sequence, e) at least one transgene sequence, f) Poly-A sequence, g) Second insulator array, h) Second piggyBacITR sequence, i) at least one DNA spacer sequence, and j) The second AAVITR sequence.

[0342] Embodiment 46B. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 3.

[0343] Embodiment 46C. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0344] Embodiment 46D. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0345] Embodiment 46E. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 7.

[0346] Embodiment 46F. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 9.

[0347] Embodiment 46G. An AAVpiggyBac transposon polynucleotide from any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 126.

[0348] Embodiment 46H. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO: 22.

[0349] Embodiment 46I. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the polyA sequence comprises the nucleic acid sequence of SEQ ID NO: 97.

[0350] Embodiment 46J. An AAVpiggyBac transposon polynucleotide from any of the above embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 8.

[0351] Embodiment 46K. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 96.

[0352] Embodiment 46L. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of Sequence ID No. 129.

[0353] Embodiment 46M. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 4.

[0354] Embodiment 46N. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 126, e) At least one transgene sequence containing the nucleic acid sequence of sequence number 22, f) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, g) A second insulator sequence containing the nucleic acid sequence of sequence number 8, h) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, i) at least one DNA spacer sequence containing the nucleic acid sequence of sequence number 129, and j) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0355] Embodiment 47. Any one of Embodiments 46A to 46N, an AAVpiggyBac transposon polynucleotide, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 9.

[0356] Embodiment 48. One of the AAVpiggyBac transposon polynucleotides of Embodiments 46A to 46N, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 126.

[0357] Embodiment 49. One of the AAGpiggyBac transposon polynucleotides of Embodiments 46A to 46N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0358] Embodiment 50. One of the AAGpiggyBac transposon polynucleotides of Embodiments 46A to 46N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0359] Embodiment 51. One of the AAVpiggyBac transposon polynucleotides from Embodiments 46A to 50, wherein the AAVpiggyBac transposon polynucleotide contains the nucleic acid sequence of SEQ ID NO: 138.

[0360] Embodiment 52A. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) The first AAVITR sequence, b) The first piggyBacITR sequence, c) First insulator array, d) At least one promoter sequence, e) at least one transgene sequence, f) Poly-A sequence, g) Second insulator array, h) Second piggyBacITR sequence, i) at least one DNA spacer sequence, and j) The second AAVITR sequence.

[0361] Embodiment 52B. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 3.

[0362] Embodiment 52C. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0363] Embodiment 52D. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0364] Embodiment 52E. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 7.

[0365] Embodiment 52F. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 10.

[0366] Embodiment 52G. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 132.

[0367] Embodiment 52H. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO: 22.

[0368] Embodiment 52I. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the polyA sequence comprises the nucleic acid sequence of SEQ ID NO: 97.

[0369] Embodiment 52J. An AAVpiggyBac transposon polynucleotide from any of the above embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 8.

[0370] Embodiment 52K. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 96.

[0371] Embodiment 52L. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of Sequence ID No. 130.

[0372] Embodiment 52M. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 4.

[0373] Embodiment 52N. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of SEQ ID NO: 10 or SEQ ID NO: 132, e) At least one transgene sequence containing the nucleic acid sequence of sequence number 22, f) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, g) A second insulator sequence containing the nucleic acid sequence of sequence number 8, h) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, i) at least one DNA spacer sequence containing the nucleic acid sequence of Sequence ID No. 130, and j) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0374] Embodiment 53. An AAVpiggyBac transposon polynucleotide of any one of Embodiments 52A to 52N, wherein at least one promoter sequence contains the nucleic acid sequence of SEQ ID NO: 10.

[0375] Embodiment 54. An AAVpiggyBac transposon polynucleotide of any one of Embodiments 52A to 52N, wherein at least one promoter sequence contains the nucleic acid sequence of SEQ ID NO: 132.

[0376] Embodiment 55. One of the AAGpiggyBac transposon polynucleotides of Embodiments 52A to 52N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of Sequence ID No. 95.

[0377] Embodiment 56. One of the AAGpiggyBac transposon polynucleotides of Embodiments 52A to 52N, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0378] Embodiment 57. Any one of Embodiments 52A to 56, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 139.

[0379] Embodiment 58A. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) The first AAVITR sequence, b) The first piggyBacITR sequence, c) First insulator array, d) At least one promoter sequence, e) at least one transgene sequence, f) Poly-A sequence, g) Second insulator array, h) Second piggyBacITR sequence, i) at least one DNA spacer sequence, and j) The second AAVITR sequence.

[0380] Embodiment 58B. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 3.

[0381] Embodiment 58C. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0382] Embodiment 58D. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0383] Embodiment 58E. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the first insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 7.

[0384] Embodiment 58F. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 13.

[0385] Embodiment 58G. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transgene sequence comprises the nucleic acid sequence of SEQ ID NO: 22.

[0386] Embodiment 58H. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the polyA sequence comprises the nucleic acid sequence of SEQ ID NO: 97.

[0387] Embodiment 58I. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second insulator sequence comprises the nucleic acid sequence of SEQ ID NO: 8.

[0388] Embodiment 58J. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 96.

[0389] Embodiment 58K. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of Sequence ID No. 131.

[0390] Embodiment 58L. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 4.

[0391] Embodiment 58M. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of sequence number 13, e) At least one transgene sequence containing the nucleic acid sequence of sequence number 22, f) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, g) A second insulator sequence containing the nucleic acid sequence of sequence number 8, h) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, i) at least one DNA spacer sequence containing the nucleic acid sequence of sequence number 131, and j) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0392] Embodiment 59. One of the AAGpiggyBac transposon polynucleotides of Embodiments 58A to 58M, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of Sequence ID No. 95.

[0393] Embodiment 60. One of the AAGpiggyBac transposon polynucleotides of Embodiments 58A to 58M, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of Sequence ID No. 125.

[0394] Embodiment 61. Any one of the AAVpiggyBac transposon polynucleotides from Embodiments 58A to 60, wherein the AAVpiggyBac transposon polynucleotide contains the nucleic acid sequence of SEQ ID NO: 140.

[0395] Embodiment 62. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 126, e) A first transgene sequence containing the nucleic acid sequence of Sequence ID No. 22, f) A first self-cleaved peptide sequence containing the nucleic acid sequence of SEQ ID NO: 31, g) A second transgene sequence containing the nucleic acid sequence of sequence number 133, h) At least a second self-cleaved peptide sequence containing the nucleic acid sequence of Sequence ID No. 32, i) At least a third transgene sequence containing the nucleic acid sequence of sequence number 134, j) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, k) A second insulator sequence containing the nucleic acid sequence of sequence number 8, l) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, and m) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0396] Embodiment 63. The AAGpiggyBac transposon polynucleotide of Embodiment 62, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0397] Embodiment 64. The AAGpiggyBac transposon polynucleotide of Embodiment 62, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0398] Embodiment 65. The AAVpiggyBac transposon polynucleotide of Embodiment 62, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 9.

[0399] Embodiment 66. The AAVpiggyBac transposon polynucleotide of Embodiment 62, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 126.

[0400] Embodiment 67. Any one of the AAVpiggyBac transposon polynucleotides from Embodiments 62 to 66, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 141.

[0401] Embodiment 68. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of SEQ ID NO: 10 or SEQ ID NO: 132, e) A first transgene sequence containing the nucleic acid sequence of Sequence ID No. 22, f) A first self-cleaved peptide sequence containing the nucleic acid sequence of SEQ ID NO: 31, g) A second transgene sequence containing the nucleic acid sequence of sequence number 133, h) At least a second self-cleaved peptide sequence containing the nucleic acid sequence of Sequence ID No. 32, i) At least a third transgene sequence containing the nucleic acid sequence of sequence number 134, j) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, k) A second insulator sequence containing the nucleic acid sequence of sequence number 8, l) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, and m) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0402] Embodiment 69. The AAGpiggyBac transposon polynucleotide of Embodiment 68, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0403] Embodiment 70. The AAGpiggyBac transposon polynucleotide of Embodiment 68, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0404] Embodiment 71. The AAVpiggyBac transposon polynucleotide of Embodiment 68, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 10.

[0405] Embodiment 72. The AAVpiggyBac transposon polynucleotide of Embodiment 68, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 132.

[0406] Embodiment 73. An AAVpiggyBac transposon polynucleotide from any one of Embodiments 68 to 72, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 142.

[0407] Embodiment 74. AAVpiggyBac transposon polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 3, b) A first piggyBacITR sequence containing the nucleic acid sequence of SEQ ID NO: 95 or SEQ ID NO: 125, c) A first insulator sequence containing the nucleic acid sequence of sequence number 7, d) At least one promoter sequence containing the nucleic acid sequence of sequence number 13, e) A first transgene sequence containing the nucleic acid sequence of Sequence ID No. 22, f) At least one self-cleaved peptide sequence containing the nucleic acid sequence of SEQ ID NO: 135, g) At least a second transgene sequence containing the nucleic acid sequence of sequence number 134, h) A poly(A) sequence containing the nucleic acid sequence of sequence number 97, i) A second insulator sequence containing the nucleic acid sequence of sequence number 8, j) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, and k) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0408] Embodiment 75. The AAGpiggyBac transposon polynucleotide of Embodiment 74, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 95.

[0409] Embodiment 76. The AAGpiggyBac transposon polynucleotide of Embodiment 74, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of SEQ ID NO: 125.

[0410] Embodiment 77. An AAVpiggyBac transposon polynucleotide from any one of Embodiments 74-76, wherein the AAVpiggyBac transposon polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 143.

[0411] Embodiment 78. A vector comprising an AAVpiggyBac transposon polynucleotide of any of the embodiments described above.

[0412] Embodiment 79. A vector according to any of the above embodiments, wherein the vector is a viral vector.

[0413] Embodiment 80. A vector according to any of the above embodiments, wherein the viral vector is an adeno-associated virus (AAV) viral vector.

[0414] Embodiment 81. A vector according to any of the above embodiments, wherein the AAV virus vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 virus vector.

[0415] Embodiment 82. A vector according to any of the above embodiments, wherein the AAV virus vector is an AAV-KP-1 or AAV-NP59 virus vector, preferably an AAV-KP-1 virus vector.

[0416] Embodiment 83. A composition comprising a vector according to any of Embodiments 78 to 82.

[0417] Embodiment 84. An AAV transposase polynucleotide comprising a first AAVITR sequence, at least one promoter sequence, at least one transposase sequence, a polyA sequence, and a second AAVITR sequence in the 5' to 3' direction.

[0418] Embodiment 85. The AAV transposase polynucleotide of Embodiment 84, wherein the AAVpiggyBac transposon polynucleotide comprises DNA, cDNA, gDNA, RNA, mRNA, or any combination thereof.

[0419] Embodiment 86. An AAV transposase polynucleotide of any of the above embodiments, wherein the first and / or second AAVITR sequence comprises any of the nucleic acid sequences of SEQ ID NOs: 1-4, 93-94, 105-106, and 127.

[0420] Embodiment 87. An AAV transposase polynucleotide according to any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 1 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 2.

[0421] Embodiment 88. An AAV transposase polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 105 and the second AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 106.

[0422] Embodiment 89. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one promoter sequence is a liver-specific promoter.

[0423] Embodiment 90. An AAV transposase polynucleotide of any of the above embodiments, wherein the liver-specific promoter is leukocyte-specific expression of a hybrid liver promoter (HLP), an LP1 promoter, a pp52(LSP1) long promoter, a thyroxine-binding globulin (TBG) promoter, a wTBG promoter, a liver combinatorial bundle (HCB) promoter, a 2xApoE-hAAT promoter, or leukocyte-specific expression of pp52(LSP1) and a chimeric intron promoter.

[0424] Embodiment 91. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises one of the nucleic acid sequences of SEQ ID NOs: 9-16, 69, 107, 126, and 132.

[0425] Embodiment 92. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding a piggyBac®(PB) transposase polypeptide, a piggyBac-like(PBL) transposase polypeptide, or a SuperpiggyBac®(SPB) transposase polypeptide.

[0426] Embodiment 93. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding any of the amino acid sequences of SEQ ID NOs. 39-42, 47, and 49.

[0427] Embodiment 94. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one transposase sequence comprises the nucleic acid sequence of SEQ ID NO: 48 or 50.

[0428] Embodiment 95. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding Sleeping Beauty transposase polypeptide, hyperactive Sleeping Beauty (SB100X) transposase polypeptide, helitron transposase polypeptide, Tol2 transposase polypeptide, TcBuster transposase polypeptide, or mutant TcBuster transposase polypeptide.

[0429] Embodiment 96. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one transposase sequence comprises a nucleic acid sequence encoding any of the amino acid sequences of SEQ ID NOs. 51-60.

[0430] Embodiment 97. An AAV transposase polynucleotide according to any of the above embodiments, wherein the polyA sequence comprises the nucleic acid sequence of SEQ ID NOs. 26-27, 97, or 108.

[0431] Embodiment 98. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein at least one transposase sequence is operably linked to at least one promoter sequence.

[0432] Embodiment 99. An AAVpiggyBac transposon polynucleotide of any of the above embodiments, wherein the expression of at least one transposase sequence is controlled by at least one promoter sequence.

[0433] Embodiment 100. An AAV transposase polynucleotide of any of the above embodiments, wherein the AAV transposase polynucleotide further comprises at least one DNA spacer sequence.

[0434] Embodiment 101. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 103 or 109.

[0435] Embodiment 102A. AAV transposase polynucleotide comprising the following in the 5' to 3' direction: a) The first AAVITR sequence, b) at least one promoter sequence, c) At least one transposase sequence, d) Poly-A sequence, e) at least one DNA spacer sequence, and f) The second AAVITR sequence.

[0436] Embodiment 102B. An AAV transposase polynucleotide of any of the above embodiments, wherein the first AAVITR sequence comprises the nucleic acid sequence of SEQ ID NO: 127.

[0437] Embodiment 102C. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 9.

[0438] Embodiment 102D. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 126.

[0439] Embodiment 102E. An AAV transposase polynucleotide according to any of the above embodiments, wherein at least one transposase sequence comprises the nucleic acid sequence of SEQ ID NO: 48.

[0440] Embodiment 102F. An AAV transposase polynucleotide according to any of the above embodiments, wherein the polyA sequence comprises the nucleic acid sequence of SEQ ID NO: 136.

[0441] Embodiment 102G. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of Sequence ID No. 137.

[0442] Embodiment 102H. An AAV transposase polynucleotide of any of the above embodiments, wherein at least one DNA spacer sequence comprises the nucleic acid sequence of Sequence ID No. 4.

[0443] Embodiment 102I. AAV transposase polynucleotide comprising the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of sequence number 127, b) At least one promoter sequence containing the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 126, c) At least one transposase sequence containing the nucleic acid sequence of sequence number 48, d) A poly(A) sequence containing the nucleic acid sequence of sequence number 136, e) At least one DNA spacer sequence containing the nucleic acid sequence of sequence number 137, and f) A second AAVITR sequence containing the nucleic acid sequence of sequence number 4.

[0444] Embodiment 103. Any one of Embodiments 102A to 102I of an AAV transposase polynucleotide, wherein at least one promoter sequence comprises the nucleic acid sequence of Sequence ID No. 9.

[0445] Embodiment 104. One of the AAV transposase polynucleotides of Embodiments 102A to 102I, wherein at least one promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 126.

[0446] Embodiment 105. AAVpiggyBac transposase polynucleotide of Embodiments 102A-104, wherein at least one promoter sequence contains the nucleic acid sequence of SEQ ID NO: 144.

[0447] Embodiment 106. A vector comprising an AAV transposase polynucleotide of any of the embodiments described above.

[0448] Embodiment 107. A vector according to any of the above embodiments, wherein the vector is a viral vector.

[0449] Embodiment 108. A vector according to any of the above embodiments, wherein the viral vector is an adeno-associated virus (AAV) viral vector.

[0450] Embodiment 109. A vector according to any of the above embodiments, wherein the AAV virus vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 virus vector.

[0451] Embodiment 110. A vector according to any of the above embodiments, wherein the AAV virus vector is an AAV-KP-1 or AAV-NP59 AAV virus vector, and preferably the AAV virus vector is an AAV-KP-1 virus vector.

[0452] Embodiment 111. A composition comprising a vector according to any of Embodiments 102 to 110.

[0453] Embodiment 112. A composition comprising a vector from any of Embodiments 78 to 82 and a vector from any of Embodiments 102 to 110.

[0454] Embodiment 113. A method for treating at least one metabolic liver disorder (MLD) in a subject requiring such treatment, comprising administering to the subject at least one therapeutically effective amount of at least one polynucleotide, vector, or composition of any of the embodiments described above.

[0455] Embodiment 114. A method for treating at least one metabolic liver disorder (MLD) in a subject requiring the treatment, comprising administering to the subject the following: a) Any one, at least one therapeutically effective amount of any one of the AAVpiggyBac transposon polynucleotides of the embodiments described above, or of any one of the vectors and / or compositions of the embodiments described above comprising the AAVpiggyBac transposon polynucleotide, and b) Any one, at least one therapeutically effective amount of the AAVpiggyBac transposase polynucleotide of any one of the embodiments described above, or of any one vector and / or composition of the embodiments described above comprising the AAVpiggyBac transposase polynucleotide.

[0456] Embodiment 115. The method of Embodiment 114, wherein at least one MLD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

[0457] Embodiment 116. The method of Embodiment 115, wherein MLD is ornithine transcarbamylase (OTC) deficiency.

[0458] definition

[0459] Nucleic acid and polynucleotide molecules

[0460] The nucleic acid molecules and polynucleotide molecules of this disclosure may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including cDNA and genomic DNA obtained by cloning, synthetically, or in combination thereof. DNA may be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be a coding strand, also known as a sense strand, or a non-coding strand, also known as an antisense strand.

[0461] Construction of nucleic acid and polynucleotide molecules

[0462] The nucleic acids and polynucleotide molecules of this disclosure can be prepared using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, which are known in the art.

[0463] Nucleic acids and polynucleotide molecules may conveniently contain nucleotide sequences in addition to the polynucleotides of this disclosure. For example, a multicloning site containing one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Alternatively, a translatable sequence can be inserted to aid in the isolation of the translated polynucleotides of this disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the proteins of this disclosure. The nucleic acids of this disclosure, excluding the coding sequence, are optionally vectors, adapters, or linkers for cloning and / or expression of the polynucleotides of this disclosure.

[0464] Further sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, assist in the isolation of polynucleotides, or improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

[0465] Recombination methods for constructing nucleic acid and polynucleotide molecules

[0466] The nucleic acids and polynucleotide molecules of this disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methods known to those skilled in the art. In some embodiments, desired sequences in a cDNA or genomic DNA library are identified using oligonucleotide probes that selectively hybridize to the polynucleotides of this disclosure under strict conditions. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art.

[0467] Nucleic acid screening and isolation methods

[0468] cDNA or genomic libraries can be screened using the polynucleotide sequence-based probes of this disclosure. Using the probes, homologous genes from the same or different organisms can be isolated by hybridizing with genomic DNA or cDNA sequences. Those skilled in the art will understand that varying degrees of hybridization rigor can be used in assays, and that either the hybridization medium or the washing medium can be the rigor. As the hybridization conditions become more rigorous, a greater degree of complementarity between the probe and the target is required for double-strand formation to occur. The degree of rigor can be controlled by one or more of the following: temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the rigor of hybridization can be conveniently altered by changing the polarity of the reactant solution, for example, by manipulating the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding varies according to the rigor of the hybridization medium and / or washing medium. The degree of complementarity is optimally 100%, or 70-100%, or any range or value in between. However, it should be understood that slight sequence variations in the probe and primer can be compensated for by reducing the strictness of the hybridization medium and / or washing medium.

[0469] Methods for amplifying RNA or DNA are known in the art and can be used in accordance with this disclosure without excessive experimentation, based on the teachings and guidance presented herein.

[0470] Known methods for DNA or RNA amplification include, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (e.g., Mullis, et al. U.S. Patents No. 4,683,195, 4,683,202, 4,800,159, and 4,965,188; Tabor et al. No. 4,795,699 and 4,921,794; Innis No. 5,142,033; Wilson, et al. No. 5,122,464; Innis No. 5,091,310; Gyllensten, et al. No. 5,066,584; Gelfand, et al. No. 4,889,818; Silver, et al. This includes patents No. 4,994,370 by al., No. 4,766,067 by Biswas, and No. 4,656,134 by Ringold, as well as RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238, trade name NASBA, by Malek, et al.), the entire contents of which are incorporated herein by reference.

[0471] For example, polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences of this disclosure and the sequences of related genes directly from a genomic DNA or cDNA library. PCR and other in vitro amplification methods may also be useful, for example, to create nucleic acids to be used as probes for nucleic acid sequencing, for detecting the presence of a desired mRNA in a sample, or for other purposes, for cloning nucleic acid sequences encoding proteins to be expressed. Examples of techniques sufficient to direct those skilled in the art to in vitro amplification methods can be found in U.S. Patent 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). Furthermore, the yield of long PCR products can be improved by using, for example, the T4 gene 32 protein (Boehringer Mannheim).

[0472] Synthesis methods for constructing nucleic acids

[0473] The nucleic acids and polynucleotide molecules of this disclosure can also be prepared by direct chemical synthesis using known methods. Chemical synthesis typically produces single-stranded oligonucleotides, which can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization by DNA polymerase using the single strand as a template. Those skilled in the art will recognize that while the chemical synthesis of DNA may be limited to sequences of about 100 bases or more, longer sequences can be obtained by concatenating shorter sequences.

[0474] Recombinant expression cassette

[0475] The Disclosure further provides recombinant expression cassettes comprising nucleic acid or polynucleotide molecules of the Disclosure. Recombinant expression cassettes can be constructed using nucleic acid or polynucleotides of the Disclosure and introduced into at least one desired host cell. The recombinant expression cassette typically comprises polynucleotides of the Disclosure operably ligated to a transcription initiation regulatory sequence that directs the transcription of the polynucleotides in the intended host cell. Expression of nucleic acids of the Disclosure can be directed using both heterogeneous and non-heterogeneous (i.e., endogenous) promoters.

[0476] In some embodiments, isolated nucleic acids functioning as promoters, enhancers, or other elements can be introduced at appropriate locations (upstream, downstream, or within introns) of the non-heterogeneous forms of the polynucleotides of the Disclosure to upregulate or downregulate the expression of the polynucleotides of the Disclosure. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0477] Expression vectors and host cells

[0478] This disclosure also relates to vectors comprising isolated nucleic acids and polynucleotide molecules of this disclosure, host cells genetically engineered with recombinant vectors, and the production of at least polynucleotides by recombinant techniques well known in the art.

[0479] Polynucleotides can be selectively conjugated to vectors containing a selection marker for replication in a host. Generally, plasmid vectors are introduced into a precipitate, such as calcium phosphate precipitate, or into a complex with charged lipids. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and transduced into host cells.

[0480] The DNA insert should be operablely ligated to an appropriate promoter. The expression construct further includes sites for transcription initiation and transcription termination, and the transcription region includes a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct preferably includes a translation initiation codon and a appropriately positioned termination codon (e.g., UAA, UGA, or UAG) of the mRNA to be translated, with UAA and UAG being preferred for expression in mammalian or eukaryotic cells.

[0481] The expression vector preferably, but optionally, includes at least one select marker. Such markers include, for example, ampicillin, zeosin (Shbla 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 synthase (GS, U.S. Patent Nos. 5,122,464; 5,770,359; 5,827,739), blastosyl This includes din (bsd gene), resistance genes, and ampicillin, zeosin (Shbla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing Escherichia coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entirety). Suitable media and conditions for the host cells described above are known in the art. Suitable vectors will be readily apparent to those skilled in the art. Introduction of vector constructs into host cells can be carried out by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other known methods.

[0482] The expression vector preferably, but optionally, includes at least one selectable cell surface marker for isolating cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or groups of proteins that distinguish a cell or subset of cells from another defined subset of cells. Preferably, the selectable cell surface markers distinguish cells modified by the compositions or methods of the present disclosure from cells not modified by the compositions or methods of the present disclosure. Such cell surface markers include, but are not limited to, “designated cluster” or “classification determinant” proteins (often abbreviated as “CD”), e.g., truncated or full-length CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. Cell surface markers may further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8):1277-87).

[0483] The expression vector preferably, but optionally, 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 include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0484] Those skilled in the art are familiar with the numerous expression systems available for the expression of nucleic acids or polynucleotide molecules. Alternatively, the nucleic acids of the Disclosure may be expressed in host cells by being (manipulated) turned on in host cells containing endogenous DNA encoding the nucleic acids or polynucleotides of the Disclosure. Such methods are well known in the art and are described, for example, in U.S. Patents 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are incorporated herein by reference in their entirety.

[0485] Examples of cell cultures useful for producing nucleic acid and polynucleotide molecules, and specific parts or variants thereof, are known in the art, including bacteria, yeast, and mammalian cells. Mammalian cell lines are often in the form of a monolayer of cells, but mammalian cell suspensions or bioreactors can also be used. Numerous suitable host cell lines have been developed in the art, including COS-1 (e.g., ATCCCRL1650), COS-7 (e.g., ATCCCRL-1651), HEK293, BHK21 (e.g., ATCCCRL-10), CHO (e.g., ATCCCRL1610), and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hepG2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, etc., which are readily available, for example, from the American Type Culture Collection (Manassas, Va.) (www.atcc.org). Preferred host cells include lymphoid cells such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC registry number CRL-1580) and SP2 / 0-Ag14 cells (ATCC registry number CRL-1851). In a preferred embodiment, the recombinant cells are P3X63Ab8.653 or SP2 / 0-Ag14 cells.

[0486] Expression vectors for these cells may include, but are not limited to, one or more of the following regulatory sequences: promoters, e.g., late or early SV40 promoter, CMV promoter (US Patent No. 5,168,062; No. 5,385,839), HSVtk promoter, pgk (phosphoglycerin kinase) promoter, EF-1 alpha promoter (US Patent No. 5,266,491), or at least one human promoter; enhancers, and / or processing information sites, e.g., ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., SV40 large TAg poly-A addition site), and transcription stop sequences. See, for example, Ausubel et al., supra; Sambrook, et al. (see above). Other cells useful for the production of the nucleic acids or proteins of this disclosure are known and / or available from, for example, the Catalog of Cell Lines and Hybridomas of the American Type Culture Collection (www.atcc.org) or other known or commercially available sources.

[0487] When eukaryotic host cells are used, typically, polyadenylation or transcriptional terminator sequences are incorporated into the vector. An example of a terminator sequence is a polyadenylation sequence derived from the bovine growth hormone gene. Sequences for precise splicing of transcripts may 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)). Furthermore, as is known in the art, gene sequences that control replication in host cells can be incorporated into the vector.

[0488] This disclosure provides isolated or substantially purified polynucleotide or protein compositions. “Isolated” or “purified” polynucleotides or proteins, or their biologically active portions, substantially or essentially contain components that are normally associated with or interact with polynucleotides or proteins found in their natural environment. Therefore, isolated or purified polynucleotides or proteins, if produced by recombinant technology, substantially contain other cell material or culture medium, and if chemically synthesized, substantially contain chemical precursors or other chemicals. Optimally, “isolated” polynucleotides do not contain sequences (optimally, protein-coding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide originates. For example, in various embodiments, isolated polynucleotides may contain nucleotide sequences of approximately 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or less than 0.1kb that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide originates. Substantially cellular protein-free proteins include protein preparations having approximately 30%, 20%, 10%, 5%, or less than 1% (by dry weight) of contaminating proteins. When the proteins of this disclosure or their biologically active portions are produced by recombinant production, the culture medium is optimally less than approximately 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or chemicals other than the protein of interest.

[0489] This disclosure provides fragments and variants of disclosed DNA sequences and proteins encoded by these DNA sequences. The term “fragment” as used throughout this disclosure refers to a portion of a DNA sequence or a portion of an amino acid sequence, and therefore to a protein encoded thereby. A DNA sequence fragment containing a coding sequence may encode a protein fragment that retains the biological activity of a native protein and therefore may encode DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, DNA sequence fragments useful as hybridization probes generally do not encode proteins that retain biological activity or promoter activity. Therefore, DNA sequence fragments may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, to up to the full-length polynucleotides of this disclosure.

[0490] The nucleic acids or proteins of this disclosure can be constructed by a modular approach, which includes pre-assembling monomer units and / or repeating units in a target vector, which can then be assembled into a final target vector. The polypeptides of this disclosure may include the repeating monomers of this disclosure and can be constructed by a modular approach, which includes pre-assembling repeating units in a target vector, which can then be assembled into a final target vector. This disclosure provides polypeptides produced by this method, as well as nucleic acid sequences encoding these polypeptides. This disclosure provides host organisms and cells comprising nucleic acid sequences encoding polypeptides produced by this modular approach.

[0491] The term “contains” is intended to mean that compositions and methods include the listed elements but do not exclude other elements. “Essentially consisting of” as used to define compositions and methods shall mean excluding other elements that are essentially important to the combination when used for the intended purpose. Thus, compositions essentially consisting of the elements defined herein would not exclude trace contaminants or inert carriers. “Consists of” shall mean excluding more than trace amounts of other components and substantial method steps. The embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0492] 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 may include the splicing of mRNA in eukaryotic cells.

[0493] "Gene expression" refers to the conversion of information contained in a gene into a gene product. A gene product can be the direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA), or a protein produced by the translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins modified by processes such as methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0494] The "modulation" or "regulation" of gene expression refers to a change in gene activity. While not strictly limited, gene regulation includes gene activation and gene repression.

[0495] The term “operatively linked” or its equivalent (e.g., “linked operatively”) means that two or more molecules are positioned relative to each other so that they can interact and influence the function of one or both molecules or a combination thereof. In some embodiments, a transgene sequence or any other sequence is said to be operatively linked to a promoter sequence if the promoter sequence controls the expression of the transgene sequence or any other sequence. In some embodiments, a transposase sequence is said to be operatively linked to a promoter sequence if the promoter sequence controls the expression of a transposase sequence.

[0496] Non-covalently linked components and methods for constructing and using non-covalently linked components are disclosed. Various components can take on a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to enable transient interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional associations only under circumstances where such associations are required for desired activity, or primarily. The binding may be for a duration sufficient to enable the desired effect.

[0497] The terms “nucleic acid,” “oligonucleotide,” or “polynucleotide” refer to at least two nucleotides covalently linked to one another. A single-stranded description also defines the sequence of the complementary strand. Thus, a nucleic acid may also encompass the complementary strand of the described single-stranded structure. The nucleic acids of this disclosure also encompass substantially identical nucleic acids and their complements that retain the same structure or encode the same protein.

[0498] The nucleic acids of this disclosure may be single-stranded or double-stranded. The nucleic acids of this disclosure may contain double-stranded sequences even if the majority of the molecule is single-stranded. The nucleic acids of this disclosure may contain single-stranded sequences even if the majority of the molecule is double-stranded. The nucleic acids of this disclosure may include genomic DNA, cDNA, RNA, or hybrids thereof. The nucleic acids of this disclosure may include combinations of deoxyribonucleotides and ribonucleotides. The nucleic acids of this disclosure may include combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The nucleic acids of this disclosure may be synthesized to include non-natural amino acid modifications. The nucleic acids of this disclosure may be obtained by chemical synthesis or recombinant methods.

[0499] The nucleic acids of this disclosure may not exist in nature in their entirety or in any part thereof. The nucleic acids of this disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, which may make the entire nucleic acid sequence non-natural. The nucleic acids of this disclosure may contain one or more duplicate sequences, inverted sequences, or repeat sequences, which may result in sequences that do not exist in nature, which may make the entire nucleic acid sequence non-natural. The nucleic acids of this disclosure may contain modified nucleotides, artificial nucleotides, or synthetic nucleotides that do not exist in nature, which may make the entire nucleic acid sequence non-natural.

[0500] Given the redundancy of the genetic code, multiple nucleotide sequences can encode a particular protein. All such nucleotide sequences are intended herein.

[0501] As used throughout this disclosure, the term “operably linked” refers to the expression of a gene under the control of a spatially linked promoter. The promoter may be located 5' (upstream) or 3' (downstream) of the gene it controls. 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 originates. Variations in the distance between the promoter and the gene can be accommodated without loss of promoter function.

[0502] As used throughout this disclosure, the term “promoter” refers to a synthetic or naturally occurring molecule that can confer, activate, or enhance the expression of nucleic acids in a cell. A promoter may include one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter its spatial and / or temporal expression. A promoter may also include distal enhancer or repressor elements that can be located thousands of base pairs away from the transcription start site. Promoters may be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. A promoter may constitutively or differentially regulate the expression of a gene component depending on the cell, tissue, or organ in which the expression occurs, or depending on the growth stage in which the expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Typical 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, CMVIE promoter, EF-1 alpha promoter, CAG promoter, SV40 early promoter, or SV40 late promoter and CMVIE promoter.

[0503] As used throughout this disclosure, the term “substantially complementary” means that the first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the 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 exact hybridization conditions.

[0504] As used throughout this disclosure, the term “substantially identical” means that the first sequence and the second sequence are identical in terms of nucleic acids by at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%, if the first sequence is substantially complementary to the complement of the 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 if the first sequence is substantially complementary to the complement of the second sequence.

[0505] As used throughout this disclosure, the term “variant” means, when used to describe a nucleic acid, (i) a portion or fragment of a referenced nucleotide sequence; (ii) a complement or portion thereof of a referenced nucleotide sequence; (iii) a nucleic acid substantially identical to a referenced nucleic acid or its complement; or (iv) a nucleic acid that hybridizes under strict conditions to a referenced nucleic acid, its complement, or a sequence substantially identical thereto.

[0506] As used throughout this disclosure, the term “vector” refers to a nucleic acid sequence containing a replication origin. A vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. A vector may be a DNA vector or an RNA vector. A vector may also be a self-replicating extrachromosomal vector, preferably a DNA plasmid. A vector may contain amino acids and a combination of a DNA sequence, an RNA sequence, or both DNA and RNA sequences.

[0507] As used throughout this disclosure, the term “variant” means, when used to describe a peptide or polypeptide, a peptide or polypeptide having a different amino acid sequence due to an amino acid insertion, deletion, or conserved substitution, but retaining at least one biological activity. A variant may also mean a protein having an amino acid sequence that is substantially identical to a referenced protein having an amino acid sequence that retains at least one biological activity.

[0508] Conservative amino acid substitutions, i.e., substitution of an amino acid with a different amino acid having similar properties (e.g., hydrophilicity, degree and distribution of charged regions), are typically recognized in the art as resulting in minor changes. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. (Kyte et al., J. Mol. Biol. 157: 105-132 (1982)). The hydropathic index of amino acids is based on consideration of their hydrophobicity and charge. Amino acids with similar hydropathic indices can be substituted while retaining protein function. In one embodiment, amino acids with hydropathic indices of ±2 are substituted. The hydrophilicity of amino acids can also be used to identify substitutions that result in proteins that retain biological function. Considering the hydrophilicity of amino acids in relation to a peptide allows for the calculation of the peptide's maximum local mean hydrophilicity, which is a useful measure reported to correlate well with antigenicity and immunogenicity. (See U.S. Patent No. 4,554,101, which is incorporated herein by reference in its entirety.)

[0509] Substitutions of amino acids with similar hydrophilicity values ​​can result in peptides that retain biological activity, such as immunogenicity. Substitutions can be made with amino acids having hydrophilicity values ​​within ±2 of each other. Both the hydrophobicity index and hydrophilicity of an amino acid are influenced by its specific side chain. Consistent with this observation, it is understood that amino acid substitutions suitable for biological function depend on the relative similarity of the amino acids, particularly their side chains, as revealed by their hydrophobicity, hydrophilicity, charge, size, and other properties.

[0510] 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 include conservative substitutions introduced by modifications of the polynucleotides encoding the polypeptides of this disclosure. Amino acids can be classified by their physical properties and their contribution to the secondary and tertiary structures of proteins. A conservative substitution is the substitution of one amino acid with another amino acid having similar properties. Examples of conservative substitutions are shown in Table A.

[0511] [Table 1]

[0512] Alternatively, conserved amino acids can be grouped as shown in Table B, as described by Lehninger (Biochemistry, Second Edition; Worth Publishers, Inc. NY, NY (1975), pp. 71-77).

[0513] [Table 2]

[0514] Alternatively, exemplary conservative substitutions are shown in Table C.

[0515] [Table 3]

[0516] It should be understood that the polypeptides of this disclosure are intended to include polypeptides having one or more insertions, deletions, or substitutions of amino acid residues, or any combination thereof, as well as modifications other than insertions, deletions, or substitutions of amino acid residues. The polypeptides or nucleic acids of this disclosure may contain one or more conservative substitutions.

[0517] As used throughout this disclosure, the term “two or more” of the aforementioned amino acid substitutions means two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, four, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more of the listed amino acid substitutions. The term “two or more” may mean two, three, four, or five of the listed amino acid substitutions.

[0518] The polypeptides and proteins of this disclosure may have sequences, or any part thereof, that do not exist in nature. The polypeptides and proteins of this disclosure may contain one or more mutations, substitutions, deletions, or insertions that do not exist in nature, making the entire amino acid sequence non-natural. The polypeptides and proteins of this disclosure may contain one or more replicated, inverted, or repeatable sequences, the resulting sequences that do not exist in nature, making the entire amino acid sequence non-natural. The polypeptides and proteins of this disclosure may contain modified, artificial, or synthetic amino acids that do not exist in nature, making the entire amino acid sequence non-natural.

[0519] As used throughout this disclosure, “sequence identity” can be determined using default parameters with a standalone executable BLAST engine program (bl2seq) for blasting two sequences, which can be retrieved from the FTP site of the National Center for Biotechnology Information (NCBI) (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; the entire text is incorporated herein by reference). As used in the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “identical” refers to a specific percentage of residues that are the same across a particular region of each sequence. The percentage can be obtained by optimally aligning the two sequences, comparing the two sequences in a given region, determining the number of positions where identical residues exist in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the given region, and multiplying the result by 100 to obtain the percentage of sequence identity. If the two sequences have different lengths, or if alignment generates one or more staggered 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. Identity can be determined manually or using computer sequencing algorithms such as BLAST or BLAST 2.0.

[0520] As used throughout this disclosure, the term “endogenous” refers to nucleic acids or protein sequences that are naturally associated with the target gene or the host cell into which it is introduced.

[0521] As used throughout this disclosure, the term “exogenous” means nucleic acids or protein sequences that are not naturally associated with the target gene or the host cell into which it is introduced, and includes naturally occurring nucleic acids, such as multiple copies of a DNA sequence that are not naturally occurring, or naturally occurring nucleic acid sequences located at genomic locations that are not naturally occurring.

[0522] This disclosure provides a method for introducing a polynucleotide construct containing a DNA sequence into a host cell. “Introducing” means presenting the polynucleotide construct to the cell so that it can access the interior of the host cell. The method of this disclosure does not depend on a specific method for introducing the polynucleotide construct into the host cell, but only on the fact that the polynucleotide construct reaches the interior of a single host cell. Methods for introducing polynucleotide constructs into bacteria, plants, fungi, and animals are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0523] As used herein, the term “subject” is interchangeable with the term “subject requiring it,” both referring to a subject who has a disease or is at high risk of developing a disease. “Subject” includes mammals. Mammals may be, for example, humans or suitable non-human mammals, such as primates, mice, rats, dogs, cats, cattle, horses, goats, camels, sheep, or pigs. The subject may also be a bird or poultry. In one embodiment, the mammal is a human.

[0524] As used herein, the terms “treating” or “to treat” describe the management and care of a patient aimed at combating a disease, condition, or disorder, and include the administration of the compounds of the Disclosure, or pharmaceutically acceptable salts, polymorphs, or solvates thereof, to alleviate the symptoms or complications of the disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term “to treat” may also include the treatment of in vitro cell or animal models.

[0525] Example 1 - In vivo expression of a transgene mediated by the viral vector of the present disclosure

[0526] In the following non-limiting embodiments, mice were treated with the viral vectors of the present disclosure, and the expression of transgenes contained in the viral vectors was tracked.

[0527] Newborn mice were divided into four different treatment groups.

[0528] The mice in treatment group #1 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a dose of 3.3 × 10⁻¹⁴. 13 The vector genome (vg) was administered at a dose of 1 kg.

[0529] The mice in treatment group #2 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 8, in a dose of 3.3 × 10⁻¹⁴. 13 The drug was administered at a dose of vg / kg.

[0530] The mice in treatment group #3 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a dose of 3.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and an AAV transposase vector containing AAV transposase polynucleotides was administered in a dose of 1.1 × 10⁻¹⁶. 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding the SPB transposase.

[0531] The mice in treatment group #4 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 8, in a dose of 3.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and an AAV transposase vector containing AAV transposase polynucleotides was administered in a dose of 1.1 × 10⁻¹⁶. 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding the SPB transposase.

[0532] Next, bioluminescence (BLI) signals were measured in mice for 35 days after viral vector administration to determine the expression of the transgene encoded by the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in Figure 10. In Figure 10, treatment group #1 is called HLP-OTC, treatment group #2 is called TBG-OTC, treatment group #3 is called HLP-OTC+SPB, and treatment group #4 is called TBG-OTC+SPB.

[0533] As shown in Figure 10, mice in treatment groups #3 and #4 showed increased BLI levels over 35 days. While we do not wish to be bound by theory, these results suggest that co-administration of the AAVpiggyBac transposon vector and the AAV transposase vector integrates the trans gene of the AAVpiggyBac transposon vector into the host genome, resulting in increased and sustained expression of the trans gene. Furthermore, increased trans gene expression was observed in treatment group #4, which was administered an AAVpiggyBac transposon vector containing a TBG promoter. Again, while we do not wish to be bound by theory, these results suggest that the use of a TBG promoter can provide increased trans gene expression that occurs shortly after administration. Such activity is particularly advantageous in clinical settings where early-onset patients are being treated.

[0534] Example 2 - In vivo expression of transgenes mediated by different concentrations of the viral vector of the present disclosure

[0535] In the following non-limiting embodiments, mice were treated with various concentrations of the viral vectors of this disclosure, and the expression of transgenes contained in the viral vectors was tracked.

[0536] In this study, mice were administered one of the following: a) Only AAVpiggyBac transposon vectors containing increasing concentrations of AAVpiggyBac transposon polynucleotides as shown in Figure 8, or b) AAV transposase vector containing AAV transposase polynucleotides, along with increasing concentrations of AAVpiggyBac transposon vectors containing AAVpiggyBac transposon polynucleotides as shown in Figure 8, where the AAV transposase polynucleotides contained a transposase sequence encoding the SPB transposase.

[0537] On day 21 after administration of the viral vector, BLI was measured in mice to assess the expression of the transgene encoded by the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in Figure 11. As shown in Figure 11, when both the AAVpiggyBac transposon vector and the AAV transposase vector were administered simultaneously, higher levels of transgene expression were achieved at lower doses. While we do not wish to be bound by theory, these results suggest that simultaneous administration of the AAVpiggyBac transposon vector and the AAV transposase vector integrates the transgene of the AAVpiggyBac transposon vector into the host genome, resulting in increased and sustained transgene expression. This is particularly advantageous in a clinical setting because it can reduce the total dose of AAV that needs to be administered to the subject and helps avoid the negative side effects usually associated with AAV vector administration.

[0538] Example 3 - Otc using the viral vector of the present disclosure spf-ash Mouse processing

[0539] The following are non-limiting embodiments, Otc spf-ash Mice were treated with the viral vector of this disclosure.

[0540] As will be understood by those skilled in the art, Otc spf-ashMice are a widely used model of urea cycle disorders, including OTC deficiency and chronic hyperammonemia. Mice have a mutation (c.386G>A;p.R129H) at the last nucleotide of exon 4 of the Otc gene, affecting the 5' splice site and resulting in partial use of a 48bp hidden splice site into an adjacent intron.

[0541] Newborn OTC spf-ash The mice were divided into two different treatment groups.

[0542] Mice in treatment group #1 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a quantity of 3.3 × 10⁻¹⁴. 13 The vector genome (vg) / kg was administered, and the AAV transposase vector containing the AAV transposase polynucleotide shown in Figure 4 was administered in a dose of 3.3 × 10⁻⁶. 13 The mice in treatment group #1 received AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio, resulting in a total AAV dose of 6.6 × 10⁶. 13 The samples were treated with vg / kg. The AAV transposase polynucleotide contained a transgene sequence encoding human OTC, which allowed for differentiation from endogenous mouse OTC.

[0543] In treatment group #2, mice were injected with an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a quantity of 3.3 × 10⁻¹⁴ units. 13 The vector genome (vg) / kg was administered, and the AAV transposase vector containing the AAV transposase polynucleotide shown in Figure 4 was administered in a dose of 1.7 × 10⁻⁶. 13 The mice in treatment group #2 received AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio, resulting in a total AAV dose of 5 × 10⁻¹⁴ 13The samples were treated with vg / kg. The AAV transposase polynucleotide contained a transgene sequence encoding human OTC, which allowed for differentiation from endogenous mouse OTC.

[0544] After administration of the viral vector, BLI was measured in mice to assess the expression of the transgene encoded by the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in Figure 12. As shown in Figure 12, high levels of transgene expression were measured in both treatment groups.

[0545] After administration of viral vectors, the amount of unintegrated vector copies per diploid genome was measured for both AAVpiggyBAC transposon vectors and AAV transposase vectors. The results of this analysis are shown in Figure 13. As shown in Figure 13, the amount of unintegrated AAV transposase vectors decreased with the aging of the mice. Furthermore, even on day 7, the amount of unintegrated AAV transposase vectors was lower compared to the amount of unintegrated AAVpiggyBac transposon vectors.

[0546] After administration of the viral vector, the number of copies of the non-integrated AAVpiggyBac transposon vector per diploid genome and the number of copies of the integrated AAVpiggyBac transposon vector were measured. The results of this analysis are shown in Figure 14. As shown in Figure 14, the integrated AAVpiggyBac transposon vector was detected 21 days after treatment. Furthermore, integration was more consistent in treatment group #1 (2:1 OTC:SPB) compared to treatment group #2 (2:2 OTC:SPB). While we do not wish to be constrained by theory, the results in Figure 14, particularly the integration of the transposon vector, indicate successful in vivo transposition of the transposon.

[0547] The number of integrated sites was also assayed by LM-PCR on days 21 and 43. Briefly, 2 μg of genomic DNA was isolated from mouse liver tissue and randomly sheared by sonication. A unique molecular identifier (UMI) was ligated to the resulting ends. Two PCR amplifications were performed. The final PCR product was sequenced using Illumina paired-end sequencing. The integrated sites were determined by confirming two breakpoints. The results of this PCR analysis are shown in Tables 1 and 2. While we do not wish to be constrained by theory, the results shown in Tables 1 and 2 indicate successful transposon transposition and integration in vivo.

[0548] [Table 4]

[0549] [Table 5]

[0550] After administration of a viral vector, the levels of human OTC mRNA and SPB mRNA were measured in mice relative to the level of mouse OTC mRNA. The results of this analysis are shown in Figure 15. As shown in Figure 15, mice treated with the viral vector expressed large amounts of human OTC mRNA. Furthermore, the level of SPB mRNA decreased with age. While we do not wish to be bound by theory, this decrease in SPB mRNA may be advantageous in a clinical setting to avoid off-target translocations after initial treatment. Correlation analysis between human OTC mRNA and SPB mRNA and the total vector copy number per diploid genome was also performed. The results of this analysis are shown in Figure 16. As shown in Figure 16, the mRNA levels of human OTC and SPB correlated with the corresponding vector copy number.

[0551] Liver samples were collected from mice 21 days after treatment, and GFP expression was analyzed. Hepatocytes in samples collected from both treatment group #1 and treatment group #2 showed strong GFP expression.

[0552] Example 4 - Treatment of an inducible hyperammonemia mouse model with the viral vector of the present disclosure

[0553] The following are non-limiting embodiments, Otc spf-ash Mice were treated with the viral vector of this disclosure, and a model of induced hyperammonemia was created using shRNA.

[0554] Newborn OTC spf-ash The mice were divided into two different treatment groups.

[0555] Mice in treatment group #1 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a quantity of 3.3 × 10⁻¹⁴. 13 The AAV transposase vector containing the AAV transposase polynucleotide shown in Figure 4 was administered at a dose of vector genome (vg) / kg, and 3.3 × 10⁻⁶. 13 The mice in treatment group #1 received AAVpiggyBac transposon vector and AAV transposase vector in a 1:1 dose ratio, resulting in a total AAV dose of 6.6 × 10⁶. 13 The samples were treated with vg / kg. The AAV transposase polynucleotide contained a transgene sequence encoding human OTC, which allowed for differentiation from endogenous mouse OTC.

[0556] In treatment group #2, mice were injected with an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a quantity of 3.3 × 10⁻¹⁴ units. 13 The vector genome (vg) / kg was administered, and the AAV transposase vector containing the AAV transposase polynucleotide shown in Figure 4 was administered in a dose of 1.7 × 10⁻⁶. 13 The mice in treatment group #2 received AAVpiggyBac transposon vector and AAV transposase vector in a 2:1 dose ratio, resulting in a total AAV dose of 5 × 10⁻¹⁴ 13The samples were treated with vg / kg. The AAV transposase polynucleotide contained a transgene sequence encoding human OTC, which allowed for differentiation from endogenous mouse OTC.

[0557] On day 38 after processing, each subset of the processing group was one of the following: a) Leave it unattended b) Administer a dose of shRNA targeting mouse OTC.

[0558] For control and comparison, OTCs of similar age that were not treated with a viral vector were used. spf-ash Mice were also administered a dose of shRNA targeting mouse OTC.

[0559] Figure 17 shows the survival rates of mice in treatment group #1, either left untreated (2:2 OTC:SPB) or further administered with a dose of shRNA targeting mouse OTC (2:2 OTC:SPB+shRNA). Figure 17 also shows the survival rates of OTC mice of similar age that were not treated with a viral vector and were administered a dose of shRNA targeting mouse OTC. spf-ash This shows the survival rate of the mice. Figure 18 shows the ammonia concentration in the plasma of the aforementioned mouse group. As shown in Figures 17 and 18, the adverse effects of shRNA administration are delayed in mice treated with the viral vector.

[0560] Example 5 - In vivo expression of a trans gene operably linked to a different promoter sequence in the AAVpiggyBac transposon vector of the present disclosure.

[0561] In the following non-limiting examples, mice were treated with the viral vectors of this disclosure, which contained transgenes operably linked to either the HLP promoter, LP1 promoter, or TBG promoter sequence. The expression of the transgenes contained in the viral vectors was tracked to determine the efficiency with which each promoter could drive transgene expression in vivo, particularly in the liver.

[0562] Neonatal wild-type mice, adult wild-type mice, and neonatal Otc spf-ash The mice were divided into 12 different treatment groups.

[0563] Treatment groups #1 through #6 included neonatal wild-type mice.

[0564] The mice in treatment group #1 were given 5 × 10⁶ AAVpiggyBac transposon vectors containing the AAVpiggyBac transposon polynucleotides shown in Figure 1. 13 The drug was administered at a dose of vg / kg.

[0565] The mice in treatment group #2 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in a dose of 3.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 1.7 × 10⁻⁶ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0566] The mice in treatment group #3 were given 5 × 10⁶ AAVpiggyBac transposon vectors containing the AAVpiggyBac transposon polynucleotides shown in Figure 8. 13 The drug was administered at a dose of vg / kg.

[0567] The mice in treatment group #4 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 8, in a dose of 3.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 1.7 × 10⁻⁶ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0568] The mice in treatment group #5 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 9, in a 95 × 10⁶ dose. 13 The drug was administered at a dose of vg / kg.

[0569] The mice in treatment group #6 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 9, in a dose of 3.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 1.7 × 10⁻⁶ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0570] Next, for 42 days after viral vector administration, the bioluminescence (BLI) signal in the liver of mice in treatment groups #1 to #6 was measured to determine the expression of the transgene encoded by the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in Figure 19. In Figure 19, treatment group #1 is called HLP-OTC, treatment group #2 is called HLP-OTC+SPB, treatment group #3 is called TBG-OTC, treatment group #4 is called TBG-OTC+SPB, treatment group #5 is called LP1-OTC+SPB, and treatment group #6 is called LP1-OTC+SPB. As shown in Figure 19, the TBG promoter most efficiently drove transgene expression, followed by the LP1 promoter, and then the HLP promoter. Increased and sustained expression was observed in treatment groups #2, #4, and #6. While we do not wish to be constrained by theory, these results indicate that co-administration of the AAVpiggyBac transposon vector and the AAV transposase vector leads to the integration of the AAVpiggyBac transposon vector's transgene into the host genome, resulting in increased and sustained transgene expression. The transposon vector integration observed in Figure 19 demonstrates successful in vivo transposon transposition.

[0571] The levels of human OTC mRNA and SPB mRNA relative to mouse OTC mRNA levels were also measured in mice from treatment groups #1 to #6. The results of this analysis are shown in Figure 20 (human OTC mRNA) and Figure 21 (SPB mRNA). Similar to the results shown in Figure 19, the results in Figures 20 and 21 indicate that the TBG promoter yields the highest levels of transgene mRNA, followed by the LP1 promoter, and then the HLP promoter.

[0572] On day 21 after viral vector administration, the amount of human OTC protein relative to the amount of mouse OTC protein was also measured. The results of this analysis are shown in Figure 22. Similar to the results shown in Figures 19-21, the results shown in Figure 22 indicate that the TBG promoter yielded the highest levels of human OTC protein, followed by the LP1 promoter, and then the HLP promoter.

[0573] Furthermore, hepatocytes from mice in treatment group #1 and treatment group #2 were also analyzed by immunohistochemistry and stained for GFP. Briefly, liver tissue was collected on day 21, fixed with 10% neutral buffered formalin, embedded in paraffin, and then stained. The results of immunohistochemistry are shown in Figure 27, which shows that higher levels of GFP were observed in treatment group #2. While we do not wish to be bound by theory, these results suggest that co-administration of the AAVpiggyBac transposon vector and the AAV transposase vector integrates the trans gene of the AAVpiggyBac transposon vector into the host genome, resulting in increased and sustained expression of the trans gene.

[0574] Furthermore, the number of integrated sites in the genome of the treated mice was assayed by LM-PCR. Briefly, 2 μg of genomic DNA was isolated from mouse liver tissue and randomly sheared by sonication. Unique molecular identifiers (UMIs) were ligated to the resulting ends. Two PCR amplifications were performed. The final PCR products were sequenced using Illumina paired-end sequencing. The total number of unique integrated sites was determined by a one-sided breakpoint with two or more UMIs. The results of this analysis are shown in Table 3.

[0575] [Table 6]

[0576] Treatment groups #7 through #12 included adult wild-type mice.

[0577] The mice in treatment group #7 were given 2 × 10⁶ AAVpiggyBac transposon vectors containing the AAVpiggyBac transposon polynucleotides shown in Figure 1. 13 The drug was administered at a dose of vg / kg.

[0578] The mice in treatment group #8 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 1, in an amount of 1.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 0.7 × 10⁻¹⁴ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0579] The mice in treatment group #9 were given 2 × 10⁶ AAVpiggyBac transposon vectors containing the AAVpiggyBac transposon polynucleotides shown in Figure 8. 13 The drug was administered at a dose of vg / kg.

[0580] The mice in treatment group #10 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 8, in an amount of 1.3 × 10⁶. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 0.7 × 10⁻¹⁴ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0581] The mice in treatment group #11 were given 2 × 10⁶ AAVpiggyBac transposon vectors containing the AAVpiggyBac transposon polynucleotides shown in Figure 9. 13 The drug was administered at a dose of vg / kg.

[0582] The mice in treatment group #12 were given an AAVpiggyBac transposon vector containing the AAVpiggyBac transposon polynucleotide shown in Figure 9, in an amount of 1.3 × 10⁻¹⁴. 13 At a dose of vg / kg, and with an AAV transposase vector containing AAV transposase polynucleotides, 0.7 × 10⁻¹⁴ 13 The drug was administered at a dose of vg / kg. The AAV transposase polynucleotide contained a transposase sequence encoding an SPB transposase operably ligated to the HLP promoter.

[0583] Next, at 7 and 14 days after viral vector administration, the bioluminescence (BLI) signal in the liver of mice from treatment groups #7 to #12 was measured to determine the expression of the transgene encoded by the AAVpiggyBac transposon polynucleotide. The results of this analysis are shown in Figure 23. In Figure 23, treatment group #7 is referred to as HLP-OTC, treatment group #8 as HLP-OTC+SPB, treatment group #9 as TBG-OTC, treatment group #10 as TBG-OTC+SPB, treatment group #11 as LP1-OTC+SPB, and treatment group #12 as LP1-OTC+SPB. As shown in Figure 23, similar strengths of transgene expression were observed for each promoter.

[0584] The levels of human OTC mRNA and SPB mRNA relative to mouse OTC mRNA levels were also measured in mice from treatment groups #7 to #12 on day 14 after viral vector administration. The results of this analysis are shown in Figure 24 (human OTC mRNA) and Figure 25 (SPB mRNA). As shown in Figures 24 and 25, similar levels of human OTC were observed for the HLP and LP1 promoters, with the strongest expression observed for the BG promoter.

[0585] Fourteen days after viral vector administration, the amount of human OTC protein relative to the amount of mouse OTC protein was also measured. The results of this analysis are shown in Figure 26. Similar to the results shown in Figures 19-21, the results shown in Figure 22 indicate that the TBG promoter yielded the highest levels of human OTC protein, followed by the LP1 promoter, and then the HLP promoter.

[0586] equivalent The foregoing description is provided for illustrative purposes only and is not intended to limit the disclosure to the exact form disclosed. Details of one or more embodiments of the disclosure are described in the accompanying description above. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the disclosure, but preferred methods and materials are described herein. Other features, purposes, and advantages of the disclosure will be apparent from this description and the claims. In this specification and the accompanying claims, singular nouns include plural nouns unless the context explicitly indicates otherwise. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the disclosure belongs. All patents and publications cited herein are incorporated by reference.

Claims

1. Adeno-associated virus (AAV) piggyBac transposon polynucleotides, including the following in the 5' to 3' direction: a) A first AAVITR sequence containing the nucleic acid sequence of SEQ ID NO: 93, b) A first piggyBacITR sequence containing the nucleic acid sequence of sequence number 125, c) A first insulator sequence containing the nucleic acid sequence of Sequence ID No. 7, d) At least one promoter sequence containing the nucleic acid sequence of sequence number 132, e) At least one transgene sequence containing the nucleic acid sequence of Sequence ID No. 22, f) A polyA sequence containing the nucleic acid sequence of sequence number 97, g) A second insulator sequence containing the nucleic acid sequence of Sequence ID No. 8, h) A second piggyBacITR sequence containing the nucleic acid sequence of sequence number 96, i) At least one DNA spacer sequence containing the nucleic acid sequence of Sequence ID No. 130, and j) A second AAVITR sequence containing the nucleic acid sequence of sequence number 94.

2. The AAVpiggyBac transposon polynucleotide according to claim 1, comprising a nucleic acid sequence that is 97% identical to the nucleic acid sequence shown in Sequence ID No.

139.

3. The AAVpiggyBac transposon polynucleotide according to claim 2, comprising a nucleic acid sequence that is 97% identical to nucleic acid residues 12-4782 of sequence number 139.

4. The AAVpiggyBac transposon polynucleotide according to claim 1, wherein the first piggyBacITR sequence comprises the nucleic acid sequence of sequence number 95.

5. A vector comprising the AAVpiggyBac transposon polynucleotide as described in claim 1.

6. The vector according to claim 5, wherein the vector is a viral vector, and the viral vector is an AAV virus vector.

7. The vector according to claim 6, wherein the AAV virus vector is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 virus vector.

8. The vector according to claim 6, wherein the AAV virus vector is an AAV8 virus vector.

9. The vector according to claim 6, wherein the AAV virus vector is an AAV9 virus vector.

10. The vector according to claim 6, wherein the AAV virus vector is an AAV-KP-1 or AAV-NP59 virus vector.

11. The vector according to claim 6, wherein the AAV virus vector is an AAV-KP-1 virus vector.

12. A composition comprising the vector described in claim 5.

13. A composition for treating at least one metabolic liver disorder (MLD) in a subject, comprising at least one therapeutically effective amount of the polynucleotide described in claim 1.

14. The composition according to claim 13, wherein the at least one MLD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinateuria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

15. The composition according to claim 14, wherein the MLD is OTC deficiency.

16. A composition for treating at least one metabolic liver disorder (MLD) in a subject, comprising at least one therapeutically effective amount of the vector described in claim 5.

17. The composition according to claim 16, wherein the at least one MLD is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinateuria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

18. The composition according to claim 17, wherein the MLD is OTC deficiency.

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