Non-viral DNA vectors and uses thereof for expressing phenylalanine hydroxylase (PAH) therapeutics

Abstract

To provide non-viral DNA vectors and uses thereof for expressing phenylalanine hydroxylase (PAH) therapeutics.SOLUTION: The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding PAH protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with the ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 817,771, filed March 13, 2019, and U.S. Provisional Application No. 62 / 857,514, filed June 5, 2019, the contents of each of which are incorporated by reference herein in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, as well as the sequences in Tables 1-15 herein, each of which is incorporated by reference in its entirety. The ASCII copy, created on March 10, 2020, is named 131698-05720_SL.txt and is 116,806 bytes in size.

[0003] The present invention relates to the field of gene therapy, including the production of non-viral vectors for expressing transgenes or isolated polynucleotides in subjects or cells.The present disclosure also relates to nucleic acid constructs, promoters, vectors, and host cells that contain polynucleotides, as well as methods for delivering exogenous DNA sequences to target cells, tissues, organs, or organisms.For example, the present disclosure provides a method for expressing phenylalanine hydroxylase (PAH) from cells using non-viral ceDNA vectors, for example, expressing PAH therapeutic proteins for the treatment of subjects with phenylketonuria (PKU).The methods and compositions can be used, for example, to treat disease by expressing PAH in cells or tissues of subjects in need of treatment. [Background technology]

[0004] Gene therapy aims to improve clinical outcomes for patients suffering from either genetic mutations or acquired diseases caused by abnormalities in gene expression profiles. Gene therapy includes the treatment or prevention of medical conditions resulting from defective genes or abnormal regulation or expression, e.g., under- or over-expression, which can lead to disorders, diseases, malignancies, etc. For example, diseases or disorders caused by defective genes can be treated, prevented, or improved by delivering repair genetic material to the patient, or by modifying or silencing the defective gene with repair genetic material to the patient, which results in therapeutic expression of genetic material in the patient.

[0005] The basis of gene therapy is to provide a transcription cassette with an active gene product (sometimes referred to as a transgene), which may result in, for example, a positive gain-of-function effect, a negative loss-of-function effect, or another outcome. Such outcomes may result from the expression of therapeutic proteins, such as antibodies, functional enzymes, or fusion proteins. Gene therapy may also be used to treat diseases or malignancies caused by other factors. Human monogenic disorders may be treated by the delivery and expression of normal genes into target cells. The delivery and expression of repair genes in target cells of patients may be carried out through many methods, including the use of engineered viruses and viral gene delivery vectors. Among the many virus-derived vectors available (e.g., recombinant retroviruses, recombinant lentiviruses, recombinant adenoviruses, etc.), recombinant adeno-associated viruses (rAAVs) have gained popularity as versatile vectors in gene therapy.

[0006] Adeno-associated viruses (AAV) belong to the Parvoviridae family and more specifically constitute the Dependoparvovirus genus. AAV-derived vectors (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for delivering genetic material because (i) they can infect (transduce) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons, (ii) they lack viral structural genes, thereby reducing host cell responses to viral infection, such as interferon-mediated responses, (iii) wild-type viruses are considered non-pathogenic in humans, (iv) in contrast to wild-type AAV, which can integrate into the host cell genome, replication-defective AAV vectors lack the rep gene and generally persist as episomes, thus limiting the risk of insertional mutagenesis or genotoxicity, and (v) compared to other vector systems, AAV vectors are generally considered to be relatively poor immunogens and therefore do not induce significant immune responses (see ii), thus obtaining sustained vector DNA and potentially long-term expression of therapeutic transgenes.

[0007] However, there are several significant deficiencies in using AAV particles as gene delivery vectors. One significant drawback associated with rAAV is its limited viral packaging capacity of approximately 4.5 kb of heterologous DNA (Dong et al., 1996; Athanasopoulos et al., 2004; Lai et al., 2010), and as a result, the use of AAV vectors is limited to protein coding capacities of less than 150,000 Da. A second drawback is that as a result of the prevalence of wild-type AAV infection in the population, rAAV gene therapy candidates need to be screened for the presence of neutralizing antibodies that would clear the vector from the patient. A third drawback concerns capsid immunogenicity, which prevents re-administration to patients who were not excluded from initial treatment. The immune system in the patient may respond to the vector effectively acting as a "booster" shot, priming the immune system to generate high titer anti-AAV antibodies that would prevent future treatment. Several recent reports have shown a relationship to immunogenicity in high-dose situations. Another notable drawback is the relatively slow onset of AAV-mediated gene expression, given that single-stranded AAV DNA must be converted to double-stranded DNA prior to heterologous gene expression.

[0008] Additionally, conventional AAV virions with capsids are produced by introducing a plasmid containing the AAV genome, rep gene, and cap gene (Grimm et al., 1998). However, such encapsidated AAV viral vectors have been found to inefficiently transduce certain cell and tissue types, and the capsids also induce immune responses.

[0009] Thus, the use of adeno-associated virus (AAV) vectors for gene therapy is limited due to a single administration to the patient (due to the patient immune response), the limited range of transgene genetic material suitable for delivery in AAV vectors due to minimal viral packaging capacity (approximately 4.5 kb), and the slow AAV-mediated gene expression.

[0010] Phenylketonuria (PKU) is a rare inherited inborn error of metabolism caused by mutations in the PAH gene. Phenylketonuria (PKU) is an inborn error of metabolism that results in decreased metabolism of the amino acid phenylalanine. Untreated PKU can lead to intellectual disability, seizures, behavioral problems, and mental disabilities. It can also lead to a musty odor and pale skin. Infants born to mothers with poorly treated PKU can have heart problems, small heads, and low birth weight. PKA is due to a mutation in the PAH gene, which results in low levels of phenylalanine hydroxylase (PAH), i.e., subjects with PKU have a mutation in PAH that causes a deficiency in its enzyme activity. PKU is autosomal recessive, meaning that both copies of the gene must be mutated for the condition to develop. There are two main types, classical PKU and variant PKU, depending on whether any enzyme function remains. People with one copy of the mutated PAH gene typically have no symptoms.

[0011] PAHs are enzymes normally expressed in the liver that are necessary to metabolize dietary phenylalanine to tyrosine, an amino acid involved in the production of neurotransmitters. PAHs catalyze the hydroxylation of phenylalanine to tyrosine. Defective PAH enzymes result in the accumulation of dietary phenylalanine to potentially toxic levels.

[0012] PKU can be caused by a single gene deficiency in the enzyme phenylalanine hydroxylase (PAH), which results in elevated serum Phe levels. PAH converts Phe to tyrosine in vertebrates. In the absence of PAH, the only other mechanisms for removing Phe are protein synthesis and a few degradative pathways that involve deamination and oxidative decarboxylation of the alanine side chain, which produce the characteristic phenyllactate and phenylacetate found in the urine of PKU patients. Unfortunately, a typical diet contains more Phe than could be removed in the absence of PAH. The resulting accumulation of Phe in PKU patients leads to several symptoms, including abnormal brain development and severe mental retardation. (Kaufman, Proc Nat'l Acad Sci USA 96:3160-3164, 1999).

[0013] The current standard of treatment is a very restrictive diet (restriction of phenylalanine (Phe)), but such dietary restrictions are difficult to maintain and are not always effective because they do not correct the underlying defect. Current treatment for PKU involves a diet low in foods containing phenylalanine and special supplements. The strict diet should be started as early as possible after birth and continued for at least 10 years, if not lifelong. The drug therapy sapropterin dihydrochloride may help some PKU patients. Left untreated, PKU can lead to progressive and severe neurological disorders. PKU is estimated to affect approximately 15,000 people in the United States, and there are no available treatments that address the genetic defect of PKU.

[0014] Despite tremendous advances in understanding the biochemistry, molecular biology, and genetics of PKU, little has been done to develop new treatments for the disorder. PKU has a large unmet need for disease-modifying therapies. First, current treatments are not disease-modifying, are effective only in a fraction of patients, and still require strict dietary restrictions, and noncompliance can lead to neurological damage. Second, there is no approved gene therapy for PKU, and AAV-based treatments cannot be used by 25%-40% of patients due to preexisting antibodies. AAV can only be administered once, resulting PAH levels may not be high enough to be effective or may be abnormal, and dose levels cannot be titrated.

[0015] Thus, there is a need in the art for technologies that allow for the expression of therapeutic PAH proteins in cells, tissues, or subjects for the treatment of PKU. Summary of the Invention [Means for solving the problem]

[0016] The technology described herein relates to methods and compositions for the treatment of phenylketonuria (PKU) by expression of the enzyme phenylalanine hydroxylase (PAH) from a capsid-free (e.g., non-viral) DNA vector with covalently closed ends (referred to herein as a "closed-end DNA vector" or "ceDNA vector"), the ceDNA vector comprising a PAH nucleic acid sequence or a codon-optimized version thereof. These ceDNA vectors can be used to produce PAH proteins for treatment, monitoring, and diagnosis. Application of ceDNA vectors expressing PAH to subjects for the treatment of PKU is useful to (i) provide disease-modifying levels of PAH enzyme, (ii) be minimally invasive in delivery, (iii) be reproducible and administered to be effective, (iv) have rapid onset of therapeutic effect, (v) result in sustained expression of the corrected PAH enzyme in the liver, (vi) restore phenylalanine metabolism of urea cycle function, and / or (vii) be titratable to achieve appropriate pharmacological levels of the defective enzyme.

[0017] Thus, the invention described herein relates to capsid-free (e.g., non-viral) DNA vectors with covalently closed ends (referred to herein as "closed-end DNA vectors" or "ceDNA vectors") containing a heterologous gene encoding a PAH to enable expression of a PAH therapeutic protein in a cell.

[0018] In one aspect, disclosed herein is a closed-end DNA (ceDNA) vector comprising at least one heterologous nucleotide sequence between adjacent inverted terminal repeats (ITRs), wherein the at least one heterologous nucleotide sequence encodes at least one PAH protein, and the at least one heterologous nucleotide sequence encoding at least one PAH protein is selected from sequences having at least 90% identity with any of the sequences in Table 1. In one embodiment, the ceDNA vector is a capsid-free vector. In one embodiment, the sequence in Table 1 is selected from the group consisting of SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, and SEQ ID NO:394.

[0019] In one embodiment, the heterologous nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 392. In one embodiment, the heterologous nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 84. In one embodiment, the heterologous nucleotide sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 394.

[0020] The ceDNA vectors for expression of PAH protein production described herein are non-encapsid-containing linear double-stranded DNA molecules formed from continuous strands of complementary DNA with covalently closed ends (linear, continuous, and non-encapsidated structures), and include a 5' inverted terminal repeat (ITR) sequence and a 3' ITR sequence, where the 5' ITR and the 3' ITR can have the same symmetrical three-dimensional configuration relative to each other (i.e., symmetric or substantially symmetric), or alternatively the 5' ITR and the 3' ITR can have a different three-dimensional configuration relative to each other (i.e., asymmetric ITR). Furthermore, the ITRs can be from the same or different serotypes. In some embodiments, the ceDNA vectors can include ITR sequences that have a symmetrical three-dimensional spatial configuration (i.e., they are the same or are mirror images of each other) such that their structures are the same shape in geometric space or have the same A, C-C' and B-B' loops in three-dimensional space. In some embodiments, one ITR may be derived from one AAV serotype and the other ITR may be derived from a different AAV serotype.

[0021] Thus, some embodiments of the technology described herein relate to ceDNA vectors for improved protein expression and / or production of the above-mentioned PAH proteins comprising ITR sequences flanking a heterologous nucleic acid sequence comprising any of the PAH nucleic acid sequences disclosed in Table 5, wherein the ITR sequences are selected from either (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., an asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration. The ceDNA vectors disclosed herein can be produced in eukaryotic cells and thus can be devoid of prokaryotic DNA modifications and bacterial endotoxin contamination in insect cells.

[0022] The methods and compositions described herein relate, in part, to the discovery of non-viral, capsid-free DNA vectors (ceDNA vectors) with covalently closed ends that can be used to express at least one PAH protein, or two or more PAH proteins, from cells, including, but not limited to, cells of the liver.

[0023] Thus, in one aspect, provided herein is a DNA vector (e.g., ceDNA vector) comprising at least one heterologous nucleic acid sequence encoding at least one transgene encoding a PAH protein operably linked to a promoter located between two different AAV inverted terminal repeats (ITRs), one of the ITRs comprising a functional AAV terminal resolution site and a Rep binding site, one of the ITRs comprising a deletion, insertion or substitution relative to the other ITR, the transgene encoding the PAH protein, the DNA having the presence of a characteristic band of linear, continuous DNA when digested with a restriction enzyme having a single recognition site on the DNA vector, compared to a linear, discontinuous DNA control when analyzed on a non-denaturing gel. Other aspects include the delivery of the PAH protein by expressing it in vivo from the ceDNA vector described herein, and further the treatment of phenylketonuria (PKU) using a ceDNA vector encoding a PAH. Cells comprising the ceDNA vector encoding a PAH described herein are also contemplated herein. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 85% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 90% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 95% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 96% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 97% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 98% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 99% identical to SEQ ID NO: 192. According to some embodiments, the ceDNA vector consists of SEQ ID NO: 192. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 85% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 90% identical to SEQ ID NO: 194.According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 95% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 96% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 97% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 98% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector comprises a nucleic acid sequence that is 99% identical to SEQ ID NO: 194. According to some embodiments, the ceDNA vector consists of SEQ ID NO: 194.

[0024] Aspects of the present invention relate to a method for producing a ceDNA vector useful for producing a PAH protein in a cell as described herein. In other embodiments, the ceDNA vector produced by the method provided herein. In one embodiment, a capsid-free (e.g., non-viral) DNA vector (ceDNA vector) for producing a PAH protein is obtained from a plasmid (referred to herein as a "ceDNA-plasmid") that contains a polynucleotide expression construct template that includes an initial 5' inverted terminal repeat (e.g., AAV ITR), a heterologous nucleic acid sequence, and a 3' ITR (e.g., AAV ITR) in this order, and the 5' ITR and the 3' ITR can be asymmetric or symmetric (e.g., WT-ITR or modified symmetric ITR) with respect to each other as defined herein.

[0025] The ceDNA vector for expression of the PAH protein disclosed herein can be obtained by several means that will be known to the skilled artisan upon reading this disclosure. For example, the polynucleotide expression construct template used to generate the ceDNA vector of the present invention can be a ceDNA-plasmid, a ceDNA-bacmid, and / or a ceDNA-baculovirus. In one embodiment, the ceDNA-plasmid comprises a restriction cloning site (e.g., SEQ ID NO: 123 and / or 124) operably located between the ITRs, and can insert, for example, an expression cassette comprising a promoter operably linked to a transgene, e.g., a nucleic acid encoding a PAH. In some embodiments, the ceDNA vector for expression of the PAH protein is produced from a polynucleotide template (e.g., ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus) comprising symmetric or asymmetric ITRs (modified or WT ITRs).

[0026] In permissive host cells, for example, in the presence of Rep, a polynucleotide template having at least two ITRs replicates to produce a ceDNA vector expressing a PAH protein. ceDNA vector production involves two steps: first, excision ("rescue") of the template from the template backbone (for example, ceDNA-plasmid, ceDNA-bacmid, ceDNA-baculovirus genome, etc.) via Rep protein, and second, Rep-mediated replication of the excised ceDNA vector. Rep proteins and Rep binding sites of various AAV serotypes are well known to those skilled in the art. Those skilled in the art will understand that Rep protein is selected from the serotype that binds and replicates a nucleic acid sequence based on at least one functional ITR. For example, if a replicable ITR is from AAV serotype 2, the corresponding Rep is from the AAV serotype that cooperates with that serotype, for example, AAV2 ITR cooperates with AAV2 or AAV4 Rep, but not with AAV5 Rep. Upon replication, the covalently closed-end ceDNA vectors continue to accumulate in permissive cells, and the ceDNA vectors preferably accumulate under standard replication conditions and in the presence of Rep proteins sufficiently stably over long periods of time, for example in amounts of at least 1 pg / cell, preferably at least 2 pg / cell, preferably at least 3 pg / cell, more preferably at least 4 pg / cell, and even more preferably at least 5 pg / cell.

[0027] Thus, one aspect of the present invention relates to a process for producing ceDNA vectors for expression of such PAH proteins, comprising: a) incubating a population of host cells (e.g., insect cells) harboring polynucleotide expression construct templates (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), where the host cells lack viral capsid coding sequences under conditions effective to induce production of ceDNA vectors in the host cells in the presence of Rep proteins, and for a sufficient time therefor; and b) harvesting and isolating the ceDNA vectors from the host cells. The presence of Rep proteins induces replication of vector polynucleotides with modified ITRs to produce ceDNA vectors for expression of PAH proteins in the host cells. However, viral particles (e.g., AAV virions) are not expressed. Thus, there is no virion-imposed size restriction.

[0028] The presence of a ceDNA vector useful for expressing a PAH protein isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on denaturing and non-denaturing gels to confirm the presence of a characteristic linear, continuous DNA band compared to linear, discontinuous DNA.

[0029] Also provided herein is a method for expressing a PAH protein having therapeutic use in a cell or a subject using a ceDNA vector. Such a PAH protein can be used for the treatment of phenylketonuria (PKU). Thus, provided herein is a method for the treatment of phenylketonuria (PKU), comprising administering a ceDNA vector encoding a therapeutic PAH protein to a subject in need thereof. According to some embodiments, the subject exhibits at least about 50% reduction in serum phenylalanine levels compared to the serum phenylalanine levels in the subject before administration. According to some embodiments, the subject exhibits at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% reduction in serum phenylalanine levels. According to some embodiments, the subject has a serum phenylalanine level of less than about 1500 uM after administration. According to some embodiments, the subject has a serum phenylalanine level of less than 1500, less than 1250, less than 1000, less than 750, less than 500, less than 400, less than 300, less than 250, less than 200, less than 100, less than 50 mM after administration. According to some embodiments, the subject exhibits at least about a 10% increase in PAH activity after administration compared to the level of PAH activity before administration. According to some embodiments, the subject exhibits at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% increase in PAH activity after administration compared to the level of PAH activity before administration.

[0030] In some embodiments, one aspect of the technology described herein relates to a non-viral capsid-free DNA vector (ceDNA vector) with covalently closed ends, the ceDNA vector comprising at least one heterologous nucleotide sequence operably positioned between two inverted terminal repeat sequences, where the ITR sequences can be asymmetric, or symmetric, or substantially symmetric, as these terms are defined herein, at least one of the ITRs comprises a functional terminal resolution site and a Rep binding site, and optionally the heterologous nucleic acid sequence encodes a transgene (e.g., a PAH protein), and the vector is not present in a viral capsid.

[0031] These and other aspects of the invention are described in further detail below. [Brief description of the drawings]

[0032]

[0013] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to the exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. The accompanying drawings, however, depict only typical embodiments of the present disclosure and therefore should not be considered as limiting in scope, since the present disclosure may admit of other equally effective embodiments.

[0033] [Figure 1A] Figure 1 shows an exemplary structure of a ceDNA vector for expressing a PAH protein disclosed herein, comprising asymmetric ITR. In this embodiment, the exemplary ceDNA vector comprises an expression cassette containing a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a PAH transgene can be inserted into the cloning site (R3 / R4) between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITR) - the wild-type AAV2 ITR upstream (5' end) of the expression cassette and the modified ITR downstream (3' end), and therefore the two ITRs flanking the expression cassette are asymmetric with respect to each other. [Figure 1B]1 shows an exemplary structure of a ceDNA vector for expressing PAH disclosed herein, which comprises an asymmetric ITR with an expression cassette comprising a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a PAH transgene can be inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) - a modified ITR upstream (5' end) of the expression cassette and a wild-type ITR downstream (3' end). [Figure 1C] 1 shows an exemplary structure of a ceDNA vector for the expression of PAH disclosed herein, which includes an asymmetric ITR with an expression cassette containing an enhancer / promoter, a PAH transgene, a post-transcriptional element (WPRE), and a polyA signal. The open reading frame (ORF) allows the insertion of the PAH transgene into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two inverted terminal repeats (ITRs) that are asymmetric with respect to each other, a modified ITR upstream (5' end) and a modified ITR downstream (3' end) of the expression cassette, where both the 5'ITR and the 3'ITR are modified ITRs, but with different modifications (i.e., not the same modifications). [Figure 1D] 1 shows an exemplary structure of a ceDNA vector for expressing PAH as disclosed herein, comprising a symmetric or substantially symmetric modified ITR as defined herein, with an expression cassette comprising a CAG promoter, a WPRE, and a BGHpA. An open reading frame (ORF) encoding a PAH transgene is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), with 5' and 3' modified ITRs being symmetric or substantially symmetric. [Figure 1E]1 shows an exemplary structure of a ceDNA vector for expressing a PAH disclosed herein, comprising a symmetric or substantially symmetric modified ITR as defined herein, with an expression cassette comprising an enhancer / promoter, a transgene, a post-transcriptional element (WPRE), and a polyA signal. The open reading frame (ORF) allows the insertion of a transgene (e.g., a PAH) into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two modified inverted terminal repeats (ITRs), with the 5' and 3' modified ITRs being symmetric or substantially symmetric. [Figure 1F] 1 shows an exemplary structure of a ceDNA vector for expressing PAH disclosed herein, comprising symmetric WT-ITR or substantially symmetric WT-ITR as defined herein, with an expression cassette comprising a CAG promoter, a WPRE, and BGHpA. An open reading frame (ORF) encoding a transgene (e.g., PAH) is inserted into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (WT-ITR), with 5'WT-ITR and 3'WT-ITR being symmetric or substantially symmetric. [Figure 1G] 1 shows an exemplary structure of a ceDNA vector for transgene production disclosed herein, comprising a symmetric or substantially symmetric modified ITR as defined herein, with an expression cassette comprising an enhancer / promoter, a transgene (e.g., PAH), a post-transcriptional element (WPRE), and a polyA signal. The open reading frame (ORF) allows the insertion of a transgene (e.g., PAH) into the cloning site between the CAG promoter and the WPRE. The expression cassette is flanked by two wild-type inverted terminal repeats (WT-ITR), with the 5'WT-ITR and 3'WT-ITR being symmetric or substantially symmetric. [Figure 2A]The T-shaped stem-loop structure of the wild-type left ITR of AAV2 (SEQ ID NO:52) is provided with the identification of the A-A', B-B', C-C' arms, two Rep binding sites (RBE and RBE'), and the terminal resolution site (TRS) is also shown. The RBE contains a series of four double tetramers that are believed to interact with either Rep 78 or Rep 68. In addition, the RBE' is also believed to interact with the Rep complex assembled on the wild-type or mutant ITR in the construct. The D and D' regions contain transcription factor binding sites and other conserved structures. [Figure 2B] Shown is the proposed Rep catalytic nicking and ligation activity in the wild-type left ITR (SEQ ID NO: 53), including the T-shaped stem-loop structure of the wild-type left ITR of AAV2, along with identification of the A-A' arm, the B-B' arm, the C-C' arm, two Rep binding sites (RBE and RBE'), and also the terminal resolution site (TRS), as well as the D and D' regions which contain several transcription factor binding sites and other conserved structures. [Figure 3A] 1 provides the primary structure (polynucleotide sequence) (left) and secondary structure (right) of the A-A' arm and the RBE-containing portions of the C-C' and B-B' arms of the wild-type left AAV2 ITR (SEQ ID NO:54). [Figure 3B] An exemplary mutant ITR (also referred to as modified ITR) sequence for the left ITR is shown. The primary structure (left) and predicted secondary structure (right) of the RBE portion of the A-A', C, and B-B' arms of an exemplary mutant left ITR (ITR-1, left) (SEQ ID NO: 113) are shown. [Figure 3C] The primary structure (left) and secondary structure (right) of the A-A' loop and the RBE-containing portions of the B-B' and C-C' arms of the wild-type right AAV2 ITR (sequence number 55) are shown. [Figure 3D]An exemplary right modified ITR is shown. The primary structure (left) and predicted secondary structure (right) of the A-A' arm and the RBE-containing portion of the B-B' and C arms of an exemplary mutant left ITR (ITR-1, right) (SEQ ID NO: 114) are shown. Any combination of left and right ITRs (e.g., AAV2 ITRs or other viral serotype or synthetic ITRs) can be used as taught herein. Each of the polynucleotide sequences in Figures 3A-3D refers to sequences used in the plasmid or bacmid / baculovirus genome used to produce the ceDNA described herein. The corresponding ceDNA secondary structure predicted from the ceDNA vector configuration and predicted Gibbs free energy values ​​in the plasmid or bacmid / baculovirus genome is also included in each of Figures 3A-3D. [Figure 4A] FIG. 4C is a schematic diagram showing the upstream process for generating baculovirus-infected insect cells (BIIC), which are useful for producing ceDNA vectors for expression of the PAHs disclosed herein, in the process described in the schematic diagram of FIG. 4B. [Figure 4B] FIG. 1 is a schematic diagram of an exemplary method for the production of ceDNA. [Figure 4C] 1 shows biochemical methods and processes for confirming the production of ceDNA vectors. [Figure 4D]FIG. 4B is a schematic illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left, either uncut or digested with a restriction endonuclease, and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel, showing multiple bands suggesting that in its duplex and uncut form, ceDNA exists at least in monomeric and dimeric states, appearing as a faster migrating, smaller monomer, and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears, corresponding to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel, since the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a banding distribution similar to that observed on a native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The rightmost schematic shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed bands are twice the size of those observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed in native conditions), depending on the context. Figure 4E shows DNA with a non-continuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. Figure 4E also shows ceDNA with a linear and continuous structure.The ceDNA vector can be cleaved by a restriction endonuclease to generate two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions, whereas in denaturing conditions the strands remain connected producing a single strand that migrates as 2 kb and 4 kb. [Figure 4E]FIG. 4B is a schematic illustrating a process for identifying the presence of ceDNA in DNA harvested from cell pellets obtained during the ceDNA production process. FIG. 4D shows schematic expected bands of an exemplary ceDNA on the left, either uncut or digested with a restriction endonuclease, and then subjected to electrophoresis on either a native or denaturing gel. The leftmost schematic is a native gel, showing multiple bands suggesting that in its duplex and uncut form, ceDNA exists at least in monomeric and dimeric states, appearing as a faster migrating, smaller monomer, and a slower migrating dimer that is twice the size of the monomer. The second schematic from the left shows that when ceDNA is cut with a restriction endonuclease, the original band disappears and a faster migrating (e.g., smaller) band appears, corresponding to the expected fragment size remaining after cleavage. Under denaturing conditions, the original duplex DNA is single stranded and migrates as a species twice as large as that observed on a native gel, since the complementary strand is covalently attached. Thus, in the second schematic from the right, digested ceDNA shows a banding distribution similar to that observed on a native gel, but the bands migrate as fragments twice the size of their native gel counterparts. The rightmost schematic shows that uncut ceDNA under denaturing conditions migrates as a single-stranded open circle, and thus the observed bands are twice the size of those observed under native conditions where the circle is not open. In this figure, "kb" is used to indicate the relative size of the nucleotide molecules based on the length of the nucleotide chain (e.g., for single-stranded molecules observed in denaturing conditions) or the number of base pairs (e.g., for double-stranded molecules observed in native conditions), depending on the context. Figure 4E shows DNA with a non-continuous structure. ceDNA can be cleaved by a restriction endonuclease with a single recognition site on the ceDNA vector, generating two DNA fragments with different sizes (1 kb and 2 kb) in both neutral and denaturing conditions. Figure 4E also shows ceDNA with a linear and continuous structure.The ceDNA vector can be cleaved by a restriction endonuclease to generate two DNA fragments that migrate as 1 kb and 2 kb in neutral conditions, whereas in denaturing conditions the strands remain connected producing a single strand that migrates as 2 kb and 4 kb. [Diagram 5] FIG. 1 is an illustrative diagram of an example denaturing gel run of ceDNA vectors with (+) or without (-) digestion with endonucleases (EcoRI for ceDNA constructs 1 and 2, BamH1 for ceDNA constructs 3 and 4, SpeI for ceDNA constructs 5 and 6, and XhoI for ceDNA vectors 7 and 8). Constructs 1-8 are described in Example 1 of International Application No. PCT / US18 / 49996, which is incorporated by reference in its entirety. The sizes of the bands highlighted with asterisks were determined and are indicated below the figure. [Figure 6] 1 shows the results of the experiment described in Example 7, specifically depicting IVIS images obtained from a mouse treated with LNP-PolyC control (mouse on the far left) and four mice treated with LNP-ceDNA-luciferase (all but the mouse on the far left). The four ceDNA-treated mice show significant fluorescence in the area containing the mouse's liver. [Figure 7] 1 depicts the results of the experiment described in Example 8. The dark spots (indicated by arrows) indicate the presence of protein resulting from the expressed ceDNA transgene and demonstrate the association of the administered LNP-ceDNA with hepatocytes. [Figure 8A]8A shows the results of the eye study described in Example 9. FIG. 8A shows representative IVIS images from the eyes of a rat injected with JetPEI®-ceDNA-luciferase (top left) and the uninjected eye of the same rat (top right), or the eyes of a rat injected with plasmid-luciferase DNA (bottom left) and the uninjected eye of the same rat (bottom right). FIG. 8B shows a graph of the average radiance observed in the treated eyes or the corresponding untreated eyes in each of the treatment groups. Rats treated with ceDNA demonstrated significant fluorescence (and thus expression of the luciferase transgene) over a 99-day period, in sharp contrast to rats treated with plasmid-luciferase, where minimal relative fluorescence (and thus expression of the luciferase transgene) was observed. [Figure 8B] 8A shows the results of the eye study described in Example 9. FIG. 8A shows representative IVIS images from the eyes of a rat injected with JetPEI®-ceDNA-luciferase (top left) and the uninjected eye of the same rat (top right), or the eyes of a rat injected with plasmid-luciferase DNA (bottom left) and the uninjected eye of the same rat (bottom right). FIG. 8B shows a graph of the average radiance observed in the treated eyes or the corresponding untreated eyes in each of the treatment groups. Rats treated with ceDNA demonstrated significant fluorescence (and thus expression of the luciferase transgene) over a 99-day period, in sharp contrast to rats treated with plasmid-luciferase, where minimal relative fluorescence (and thus expression of the luciferase transgene) was observed. [Figure 9A] Figure 9 shows the results of ceDNA persistence and re-dosing studies in Rag2 mice described in Example 10. Figure 9A shows a graph of the total flux over time observed in wild-type c57bl / 6 mice or Rag2 mice treated with LNP-ceDNA-Luc. Figure 9B provides a graph showing the effect of re-dosing on the expression levels of the luciferase transgene in Rag2 mice, resulting in a stable increase in expression observed after re-dosing (arrow indicates time of re-dosing). [Figure 9B]Figure 9 shows the results of ceDNA persistence and re-dosing studies in Rag2 mice described in Example 10. Figure 9A shows a graph of the total flux over time observed in wild-type c57bl / 6 mice or Rag2 mice treated with LNP-ceDNA-Luc. Figure 9B provides a graph showing the effect of re-dosing on the expression levels of the luciferase transgene in Rag2 mice, resulting in a stable increase in expression observed after re-dosing (arrow indicates time of re-dosing). [Figure 10] Provides data from a ceDNA luciferase expression study in treated mice described in Example 11, showing total flux in each group of mice over the duration of the study. High levels of unmethylated CpG correlated with a decrease in total flux observed in mice over time, while the use of a liver-specific promoter correlated with durable, stable expression of the transgene from the ceDNA vector for at least 77 days. [Figure 11] 1 is a graph showing the results of the experiment described in Example 12. Administration of each of the two ceDNA PAH constructs (ceDNA#1, ceDNA#2) by hydrodynamic delivery in PAHenu2 mice resulted in a significant reduction (approximately 75% reduction) in serum PHE levels relative to those seen in control (polyC) treated mice. [Figure 12]12 is a graph showing the results of the experiment described in Example 13. hPAH Codop2 refers to ceDNA containing codon-optimized version 2 (codop_v2) human PAH sequence linked to VandenDriessche (VD) promoter, hPAH Codop4 refers to ceDNA containing VD_promoter operably linked to codon-optimized and CpG-minimized human PAH version 4 (codop_CpGmin_v4), and ceDNA hPAH cDNA refers to unmodified human PAH cDNA tested for its effect on PHE correction in PAH-deficient PAHenu2 mice. FIG. 12 shows the time course of serum PHE levels (shown as PHE% corrected relative to control PAHenu2). Administration of ceDNA containing hPAH Codop2 and Codop4 resulted in a reduction in PHE serum levels, indicating sufficient PAH activity to correct blood phenylalanine levels in mice PKU. Correction was shown to be stable over the 15-day experiment. [Figure 13] 13 is a graph showing the results of the experiment described in Example 14. ceDNA containing hPAH codon-optimized version 2 (Codop2) was administered at low, medium, and high doses. FIG. 13 shows the time course of serum PHE levels (PHE) (μM). Administration of ceDNA hPAH Codop2 at low and medium doses resulted in a reduction in serum PHE in a dose-dependent manner. Notably, administration of ceDNA hPAH Codop2 at medium dose was significantly greater than administration of low dose. Correction was shown to be stable over the 15-day experiment. Serum PHE concentrations were not reduced in control animals (vehicle-KO). [Figure 14A] 14A-14C are graphs showing the results of the experiment described in Example 15. The effect of ceDNA Codop2 on individual animals on days 3 and 7 was examined. As shown in FIG. 14A, by day 3, administration of Codop2 resulted in a reduction in serum PHE levels, demonstrating sufficient PAH activity to correct blood phenylalanine levels in mice PKU as early as day 3. [Figure 14B]14B is a graph showing human PAH enzyme activity and resulting serum phenylalanine levels as measured 3 and 7 days after injection of ceDNA containing VD-hPAH Codop2. Ovals refer to non-responders collected on day 7 and correspond to the lack of PHE correction in FIG. 14A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Provided herein is a method for treating phenylketonuria (PKU) using a ceDNA vector comprising one or more nucleic acids encoding a PAH therapeutic protein or fragment thereof. Also provided herein is a ceDNA vector for expression of the PAH protein described herein, comprising one or more heterologous nucleic acids encoding the PAH protein. In some embodiments, expression of the PAH protein may include secretion of the therapeutic protein from the cell in which it is expressed, or alternatively, in some embodiments, the expressed PAH protein may act or function (e.g., exert its effect) within the cell in which it is expressed. In some embodiments, the ceDNA vector expresses the PAH protein in the liver, muscle (e.g., skeletal muscle) of a subject, or other body parts, which may act as a depot for production and secretion of the PAH therapeutic protein into many systemic compartments.

[0035] I. Definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those skilled in the art to which this disclosure belongs. It is to be understood that the present invention is not limited to the specific methodology, protocols, and reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp&Dohme Corp., 2011 (ISBN978-0-911910-19-3), Robert S.Porter et al. (eds.), Fields Virology, 6 thEdition,published by Lippincott Williams&Wilkins,Philadelphia,PA,USA(2013),Knipe,D.M.and Howley,P.M.(ed.),The Encyclopedia of Molecular Cell Biology and Molecular Medicine,published by Blackwell Science Ltd.,1999-2012(ISBN9783527600908)、およびRobert A.Meyers(ed.),Molecular Biology and Biotechnology:a Comprehensive Desk Reference,published by VCH Publishers,Inc.,1995(ISBN1-56081-569-8)、Immunology by Werner Luttmann,published by Elsevier,2006、Janeway‘s Immunobiology,Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor&Francis Limited,2014(ISBN0815345305,9780815345305)、Lewin’s Genes XI,published by Jones&Bartlett Publishers,2014(ISBN-1449659055)、Michael Richard Green and Joseph Sambrook,Molecular Cloning:A Laboratory Manual,4 th ed.,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,NY,USA(2012)(ISBN1936113414), Davis et al.,Basic Methods in Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(2012)(ISBN044460149X),Laboratory Methods in Enzymology:DNA,Jon Lorsch(ed.) Elsevier,2013(ISBN0124199542), Current Protocols in Molecular Biology(CPMB),Frederick M.Ausubel(ed.),John Wiley and Sons,2014(ISBN047150338X,9780471503385),Current Protocols in Protein Science(CPPS),John E.Coligan (ed.), John Wiley and Sons, Inc., 2005, and Current Protocols in Immunology(CPI)(John E.Coligan,ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of all of which are incorporated herein by reference in their entireties.

[0036] As used herein, the terms "administration", "administering" and variations thereof refer to the introduction of a composition or agent (e.g., a therapeutic nucleic acid or an immunosuppressant described herein) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. The introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. The introduction of a composition or agent into a subject is by electroporation. Administration includes self-administration and administration by another. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0037] As used herein, the phrases "nucleic acid therapy", "therapeutic nucleic acid" and "TNA" are used interchangeably and refer to any modality of therapy that uses nucleic acid as an active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutics and DNA-based therapeutics. Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vectors, closed-ended linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone (dbDNA™) DNA vectors, minimally immunologically defined gene expression (MIDGE) vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA").

[0038] As used herein, an "effective amount" or "therapeutically effective amount" of a therapeutic agent, such as a PAH therapeutic protein or fragment thereof, is an amount sufficient to produce a desired effect, for example, providing disease-modifying levels of PAH enzymes, resulting in sustained expression of repair PAH enzymes in the liver, restoring phenylalanine metabolism of urea cycle function, and / or achieving appropriate pharmacological levels of defective enzymes. Suitable assays for measuring expression of target genes or target sequences include, for example, testing protein or RNA levels using techniques known to those skilled in the art, such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays also known to those skilled in the art. However, dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, patient's condition, severity of the condition, route of administration, and the specific active agent used. Thus, dosage regimens can vary widely, but can be routinely determined by physicians using standard methods. Additionally, the terms "therapeutic amount", "therapeutically effective amount" and "pharmaceutical effective amount" include prophylactic or preventative amounts of the described compositions of the invention. In prophylactic or preventative applications of the described invention, the pharmaceutical composition or agent is administered to a patient susceptible to or otherwise at risk of a disease, disorder or condition, including the biochemical, histological and / or behavioral symptoms of the disease, disorder or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder or condition, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder or condition, according to some medical judgment. It is generally preferred to use the maximum dose, i.e., the highest safe dose. According to some embodiments, the disease, disorder, or condition is PKU. The terms "dose" and "administration" are used interchangeably herein.

[0039] As used herein, the term "therapeutic effect" refers to the outcome of treatment, which is deemed desirable and beneficial. Therapeutic effect can include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. Therapeutic effect can also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0040] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. It is within the ability of one of ordinary skill in the art to adjust the dose to achieve maximum efficacy based on the above and other well-known methods. See Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10, incorporated herein by reference. th General principles for determining therapeutic efficacy are summarized below and can be found in Chapter 1 of The American Clinical Trials Association (Academic Press, 2001).

[0041] Pharmacokinetic principles provide the basis for modifying dosing regimens to obtain the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where plasma concentrations of a drug can be measured and are related to the therapeutic window, additional guidance regarding dosage modifications is available.

[0042] As used herein, the terms "heterologous nucleotide sequence" and "transgene" are used interchangeably and refer to a nucleic acid of interest (other than a nucleic acid encoding a capsid polypeptide) that can be incorporated into, delivered and expressed by, the ceDNA vectors disclosed herein.

[0043] As used herein, the terms "expression cassette" and "transcription cassette" are used interchangeably and refer to a linear stretch of nucleic acid that includes a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not include capsid coding sequences, other vector sequences, or inverted terminal repeat regions. An expression cassette may additionally include one or more cis-acting sequences (e.g., promoters, enhancers, or repressors), one or more introns, and one or more post-transcriptional regulatory elements.

[0044] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. An "oligonucleotide" generally refers to a polynucleotide of about 5 to about 100 nucleotides of single-stranded or double-stranded DNA. However, for purposes of this disclosure, there is no upper limit to the length of an oligonucleotide. Oligonucleotides, also known as "oligomers" or "oligos," can be isolated from genes or chemically synthesized by methods known in the art. The terms "polynucleotide" and "nucleic acid" should be understood to include single-stranded (such as sense or antisense) and double-stranded polynucleotides, as applicable to the embodiments described. The DNA can be in the form of, for example, antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. The DNA can be in the form of minicircles, plasmids, bacmids, minigenes, ministring DNA (linear covalently closed DNA vectors), closed-end linear duplex DNA (CELiD or ceDNA), doggybone (dbDNA™) DNA, dumbbell-shaped DNA, minimally immunologically defined gene expression (MIDGE) vectors, viral vectors, or non-viral vectors. The RNA can be in the form of small interfering RNA (siRNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNAs), and peptide nucleic acids (PNAs). Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0045] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0046] "Base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogues, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0047] As used herein, the term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide), double-stranded RNA (i.e., double-stranded RNA such as siRNA, dicer substrate dsRNA, shRNA, aiRNA, or pre-miRNA), DNA-RNA hybrid (e.g., see PCT Publication No. WO2004 / 078941), or DNA-DNA hybrid (e.g., see PCT Publication No. WO2004 / 104199) that can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating degradation or inhibiting translation of mRNA complementary to the interfering RNA sequence) when the interfering RNA is present in the same cell as the target gene or sequence. Thus, interfering RNA refers to a single-stranded RNA that is complementary to a target mRNA sequence, or a double-stranded RNA formed by two complementary strands or a single self-complementary strand. Interfering RNA can have substantial or complete identity to target gene or sequence, or can contain mismatched regions (i.e., mismatch motifs). Interfering RNA sequence can correspond to full-length target gene or subsequence thereof. Preferably, interfering RNA molecule is chemically synthesized. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0048] Interfering RNA includes "small interfering RNA" or "siRNA", e.g., an interfering RNA that is about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA is The siRNA duplexes can be about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNAs can be about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length.) The siRNA duplexes can include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, the double-stranded polynucleotide molecule assembled from two separate strand molecules (one strand is sense strand and the other is complementary antisense strand), the double-stranded polynucleotide molecule assembled from single strand molecules (sense and antisense regions are linked by nucleic acid-based or non-nucleic acid-based linker), the double-stranded polynucleotide molecule with hairpin secondary structure with self-complementary sense and antisense regions, and the circular single-stranded polynucleotide molecule with stem with two or more loop structures and self-complementary sense and antisense regions (circular polynucleotide can be processed in vivo or in vitro to generate active double-stranded siRNA molecules).As used herein, the term "siRNA" includes RNA-RNA duplexes as well as DNA-RNA hybrids (see, for example, PCT Publication No. WO2004 / 078941, the entirety of which is incorporated herein by reference).

[0049] The term "nucleic acid construct" as used herein refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified to contain a segment of nucleic acid in a manner that would not otherwise occur in nature, or is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for the expression of the coding sequence of the present disclosure. An "expression cassette" comprises a DNA coding sequence operably linked to a promoter.

[0050] "Hybridizable" or "complementary" or "substantially complementary" means that a nucleic acid (e.g., RNA) contains a sequence of nucleotides that allows it to non-covalently bind, i.e., to "anneal" or "hybridize" to another nucleic acid in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength, forming Watson-Crick base pairs and / or G / U base pairs. As is known in the art, standard Watson-Crick base pairing includes adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that guanine (G) bases pair with uracil (U) for hybridization between two RNA molecules (e.g., dsRNA). For example, G / U base pairing is partially responsible for the degeneracy (i.e. redundancy) of the genetic code in the context of tRNA anticodon base pairing with codons in mRNA. In the context of this disclosure, the guanine (G) of the protein-binding segment (dsRNA duplex) of the subject DNA-targeting RNA molecule is considered to be complementary to the uracil (U), and vice versa. Thus, if a G / U base pair can be made at a given nucleotide position of the protein-binding segment (dsRNA duplex) of the subject DNA-targeting RNA molecule, that position is not considered to be non-complementary, but instead is considered to be complementary.

[0051] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, amino acids that are chemically or biochemically modified or derivatized, and polypeptides with modified peptide backbones.

[0052] A DNA sequence that "encodes" a particular PAH protein is a DNA nucleic acid sequence that is transcribed into a particular RNA and / or protein. The DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or the DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA targeting RNA, also called "non-coding" RNA or "ncRNA").

[0053] As used herein, the term "fusion protein" refers to a polypeptide that contains protein domains from at least two different proteins. For example, a fusion protein may contain (i) a PAH or a fragment thereof, and (ii) at least one non-GOI protein. Fusion proteins encompassed herein include, but are not limited to, an antibody, or an Fc or antigen-binding fragment of an antibody fused to an extracellular domain of a PAH protein, such as a receptor, ligand, enzyme, or peptide. The PAH protein or fragment thereof that is part of the fusion protein may be a monospecific antibody or a bispecific or multispecific antibody.

[0054] As used herein, the term "genomic safe harbor gene" or "safe harbor gene" refers to a gene or locus into which a nucleic acid sequence can be inserted such that the sequence can integrate and function in a predictable manner (e.g., express a protein of interest) without significantly adversely affecting endogenous gene activity or promoting cancer. In some embodiments, a safe harbor gene is also a locus or gene at which the inserted nucleic acid sequence can be expressed more efficiently and at higher levels than at a non-safe harbor site.

[0055] As used herein, the term "gene delivery" refers to the process by which foreign DNA is introduced into a host cell for gene therapy applications.

[0056] As used herein, the term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that includes at least one minimally necessary origin of replication and a region that includes a palindromic hairpin structure. Rep binding sequence ("RBS") (also referred to as RBE (Rep binding element)) and terminal resolution site ("TRS") together constitute the "minimally necessary origin of replication", and thus a TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given stretch of a polynucleotide sequence are typically referred to as "inverted terminal repeats" or "ITRs", respectively. In the context of viruses, ITRs mediate replication, viral packaging, integration, and proviral rescue. As unexpectedly found in the present invention herein, TRs that are not reverse complements over their entire length can still perform the traditional functions of ITRs, and thus the term ITR is used herein to refer to TRs in a ceDNA genome or ceDNA vector that can mediate the replication of ceDNA vectors. It will be understood by those skilled in the art that there can be three or more ITRs or asymmetric ITR pairs in the composite ceDNA vector configuration. ITR can be AAV ITR or non-AAV ITR, or can be derived from AAV ITR or non-AAV ITR. For example, ITR can be derived from Parvoviridae, which includes parvovirus and dependovirus (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, human parvovirus B-19), or the SV40 hairpin, which serves as the origin of SV40 replication, can be used as ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. Parvoviridae viruses are composed of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses capable of replication in vertebrate hosts, including but not limited to human, primate, bovine, canine, equine and ovine species.For convenience herein, an ITR located 5' to (upstream of) an expression cassette in a ceDNA vector is referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette in a ceDNA vector is referred to as the "3' ITR" or "right ITR."

[0057] "Wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other dependant virus, e.g., that retains Rep binding activity and Rep nicking ability. The nucleotide sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring canonical sequence due to degeneracy of the genetic code or drift, and thus WT-ITR sequences encompassed for use herein include WT-ITR sequences as a result of naturally occurring changes (e.g., replication errors) that occur during the production process.

[0058] As used herein, the term "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refers to a pair of WT-ITRs in a single ceDNA genome or ceDNA vector, both of which are wild-type ITRs with reverse-complementary sequences over their entire length. For example, an ITR can be considered to be a wild-type sequence even if it has one or more nucleotides that deviate from the naturally occurring canonical sequence, as long as the changes do not affect the sequence's properties and overall three-dimensional structure. In some embodiments, the deviating nucleotides represent conservative sequence changes. As a non-limiting example, the sequence has at least 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., as measured using BLAST with default settings) to the canonical sequence, and also has a symmetric three-dimensional spatial organization with respect to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT-ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (TRS) that pairs with the appropriate Rep protein. Optionally, other functions can be tested, including transgene expression under permissive conditions.

[0059] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutant ITR" are used interchangeably herein and refer to an ITR having a mutation in at least one or more nucleotides compared to the WT-ITR from the same serotype. The mutation may result in a change in one or more of the A, C, C', B, B' regions of the ITR, and may result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of the WT-ITR of the same serotype.

[0060] As used herein, the term "asymmetric ITR", also referred to as "asymmetric ITR pair", refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are not reverse complements over the entire length. As a non-limiting example, an asymmetric ITR pair does not have a symmetric three-dimensional spatial configuration with respect to their cognate ITRs, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair differs in overall geometric structure, i.e., the configuration of their A, C-C', and B-B' loops in three-dimensional space (e.g., compared to the cognate ITR, one ITR may have a shorter C-C' arm and / or a shorter B-B' arm). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. In one embodiment, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, and the two ITRs are modified ITRs that have different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm, or a short B-B' arm, etc.) such that they have a different three-dimensional spatial organization compared to their cognate asymmetric mod-ITR.

[0061] As used herein, the term "symmetric ITR" refers to a pair of ITRs in a single ceDNA genome or ceDNA vector that are wild-type or mutant (e.g., modified to wild-type) depend virus ITR sequences and are reverse-complementary over their entire length. In one non-limiting example, both ITRs are wild-type ITR sequences from AAV2. In another example, neither ITR is a wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may differ in sequence from the wild-type ITR due to nucleotide addition, deletion, substitution, truncation, or point mutation. For convenience herein, the ITR located 5' (upstream of) the expression cassette in the ceDNA vector is referred to as the "5'ITR" or "left ITR", and the ITR located 3' (downstream of) the expression cassette in the ceDNA vector is referred to as the "3'ITR" or "right ITR".

[0062] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a single ceDNA genome or ceDNA vector, both of which have reverse-complementary sequences over their entire length. For example, modified ITRs can be considered substantially symmetric even if there are some nucleotide sequences that deviate from the reverse-complementary sequence, as long as the changes do not affect the properties and overall shape. As a non-limiting example, the sequences have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity (measured using BLAST with default settings) to the canonical sequence, and also have a symmetric three-dimensional spatial organization with respect to their cognate modified ITR, such that their three-dimensional structures have the same shape in geometric space. In other words, substantially symmetric modified ITR pairs have the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair may have different reverse-complementary nucleotide sequences, but still have the same symmetrical three-dimensional spatial configuration. That is, both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., the 5'ITR) of a mod-ITR pair may be from one serotype, and the other ITR (e.g., the 3'ITR) may be from a different serotype, but both may have the same corresponding mutations (e.g., if the 5'ITR has a deletion in the C region, the cognate modified 3'ITR of the different serotype has a deletion in the corresponding position of the C' region), so that the modified ITR pair has the same symmetrical three-dimensional spatial configuration. In such an embodiment, each ITR of the modified ITR pair may be derived from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), such as a combination of AAV2 and AAV6, and the modification of one ITR is reflected in the corresponding position of the cognate ITR of the different serotype. In one embodiment, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs), so long as the nucleotide sequence differences between the ITRs do not affect the properties or overall shape, and they have substantially the same shape in three-dimensional space.As a non-limiting example, a mod-ITR has at least 95%, 96%, 97%, 98%, or 99% sequence identity to a canonical mod-ITR as determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and has a symmetric three-dimensional spatial organization such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric mod-ITR pair has the same A, C-C', and B-B' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in the geometric space of the cognate mod-ITR.

[0063] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is adjacent to A and C. Similarly for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous or immediately adjacent to the flanked sequence. In one embodiment, the term flanking refers to the terminal repeats at each end of a linear double-stranded ceDNA vector.

[0064] As used herein, the terms "treat", "treating" and / or "treatment" include suppressing, substantially inhibiting, slowing or reversing the progression of a condition, substantially improving the clinical symptoms of a condition, or substantially preventing the appearance of clinical symptoms, to obtain beneficial or desired clinical results. According to some embodiments, the condition is PKU. Treating further refers to achieving one or more of: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder. Beneficial or desired clinical results, such as pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder or condition from occurring in a subject who may have a predisposition to the disease, disorder or condition, but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the symptoms of the disease, disorder or condition, reducing the severity of the disease, disorder or condition, stabilizing (i.e., not worsening) the disease, disorder or condition, preventing the spread of the disease, disorder or condition, slowing or retarding the progression of the disease, disorder or condition, ameliorating or alleviating the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0065] As used herein, the terms "increase," "enhance," and "elevate" (and similar terms) generally refer to the act of directly or indirectly increasing a concentration, level, function, activity, or behavior relative to natural, expected or average, or relative to a control condition.

[0066] As used herein, the terms "minimize," "reduce," "lower," and / or "inhibit" (and similar terms) generally refer to the act of decreasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to natural, expected or average, or relative to a control condition.

[0067] As used herein, the term "ceDNA genome" refers to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments, the ceDNA genome is incorporated into a plasmid or virus genome as an intermolecular double-stranded polynucleotide of DNA.

[0068] As used herein, the term "ceDNA spacer region" refers to an intervening sequence that separates functional elements in a ceDNA vector or ceDNA genome. In some embodiments, the ceDNA spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, the ceDNA spacer region provides or increases the genetic stability of the ceDNA genome, for example, in a plasmid or baculovirus. In some embodiments, the ceDNA spacer region facilitates easy genetic manipulation of the ceDNA genome by providing a convenient location for cloning sites, etc. For example, in certain aspects, oligonucleotide "polylinkers" containing several restriction endonuclease sites, or non-open reading frame sequences designed to have no known protein (e.g., transcription factor) binding sites, can be positioned in the ceDNA genome to separate cis-acting elements, for example, inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element. Similarly, a spacer can be incorporated between the polyadenylation signal sequence and the 3' terminal resolution site.

[0069] As used herein, "Rep binding site", "Rep binding element", "RBE", and "RBS" are used interchangeably and refer to the binding site of a Rep protein (e.g., AAV Rep 78 or AAV Rep 68), which upon binding by the Rep protein allows the Rep protein to perform its site-specific endonuclease activity on a sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60). Any known RBS sequence may be used in embodiments of the invention, including other known AAV RBS sequences and other naturally known or synthetic RBS sequences. Without being bound by theory, it is believed that the nuclease domain of the Rep protein binds to the double-stranded nucleotide sequence GCTC, and thus the two known AAV Rep proteins bind directly to the double-stranded oligonucleotide, 5'-(GCGC)(GCTC)(GCTC)(GCTC)-3' (SEQ ID NO: 60), and stably assemble. In addition, the soluble aggregated conformers (i.e., an indefinite number of inter-associated Rep proteins) dissociate and bind to oligonucleotides containing Rep binding sites. Each Rep protein interacts with both the nitrogenous bases and the phosphodiester backbone on each strand. The interactions with the nitrogenous bases provide sequence specificity, while the interactions with the phosphodiester backbone are non- or low-sequence specific, stabilizing the protein-DNA complex.

[0070] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine that generates a 3'OH that serves as a substrate for DNA elongation via cellular DNA polymerases, such as DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex can participate in a coordinate ligation reaction. In some embodiments, the TRS minimally includes a non-base paired thymidine. In some embodiments, the nicking efficiency of the TRS can be controlled at least in part by its distance within the same molecule from the RBS. When the acceptor substrate is a complementary ITR, the resulting product is an intermolecular duplex. TRS sequences are known in the art and include, for example, the hexanucleotide sequence 5'-GGTTGA-3' (SEQ ID NO: 61) identified in AAV2. Any known TRS sequence can be used in embodiments of the invention, including other known AAV TRS sequences, other naturally known or synthetic TRS sequences such as AGTT (SEQ ID NO:62), GGTTGG (SEQ ID NO:63), AGTTGG (SEQ ID NO:64), AGTTGA (SEQ ID NO:65), and other motifs such as RRTTRR (SEQ ID NO:66).

[0071] As used herein, the term "ceDNA-plasmid" refers to a plasmid that contains a ceDNA genome as an intermolecular duplex.

[0072] As used herein, the term "ceDNA-bacmid" refers to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby acting as a shuttle vector for the baculovirus.

[0073] As used herein, the term "ceDNA-baculovirus" refers to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0074] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0075] As used herein, the term "ceDNA" refers to capsid-free closed-end linear double-stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. A detailed description of ceDNA is described in International Application PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for the production of ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Application Nos. PCT / US18 / 49996, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the production of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference.

[0076] As used herein, the term "closed-ended DNA vector" refers to a capsid-free DNA vector having at least one covalently closed end and at least a portion of the vector having an intramolecular double-stranded structure.

[0077] As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably and refer to a closed-end DNA vector that contains at least one terminal palindrome. In some embodiments, the ceDNA contains two covalently closed ends.

[0078] As used herein, the term "neDNA" or "nicked ceDNA" refers to closed-end DNA that has a nick or gap of 1-100 base pairs in the stem or spacer region 5' upstream of an open reading frame (e.g., the promoter and transgene to be expressed).

[0079] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interrupted portion of the synthetic DNA vector of the invention, creating a stretch of single-stranded DNA in the otherwise double-stranded ceDNA. A gap can be from 1 base pair to 100 base pairs in length for one strand of the double-stranded DNA. Exemplary gaps designed and created by the methods described herein, and synthetic vectors generated thereby, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp in length. Exemplary gaps in the present disclosure can be 1 bp to 10 bp in length, 1 to 20 bp in length, or 1 to 30 bp in length.

[0080] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferase causes cells to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0081] As used herein, the terms "sense" and "antisense" refer to the orientation of a structural element on a polynucleotide. The sense and antisense versions of an element are the reverse complements of each other.

[0082] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" refer to AAV vectors and methods for their synthetic production in an entirely cell-free environment.

[0083] As used herein, "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally produce a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed. For example, fluorescent proteins cause cells to fluoresce when excited with a particular wavelength of light, luciferase causes cells to catalyze a reaction that produces light, and enzymes such as β-galactosidase convert a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0084] As used herein, the term "effector protein" refers to a polypeptide that provides a detectable readout, for example, as a reporter polypeptide, or more appropriately, as a polypeptide that kills a cell, such as a toxin, or an agent that makes the cell susceptible to killing with a selected agent or its deletion. Effector proteins include any protein or peptide that directly targets or damages the DNA and / or RNA of a host cell. For example, effector proteins include, but are not limited to, restriction endonucleases (whether genomic or extrachromosomal) that target host cell DNA sequences, proteases that target polypeptides required for cell survival, DNA gyrase inhibitors, and ribonuclease-type toxins. In some embodiments, the expression of effector proteins controlled by the synthetic biological circuits described herein can participate as a factor in another synthetic biological circuit, thereby expanding the range and complexity of the responsiveness of the biological circuit system.

[0085] Transcriptional regulators refer to transcriptional activators and repressors that activate or repress the transcription of genes of interest, such as PAHs. Promoters are regions of nucleic acids that initiate the transcription of a particular gene. Transcriptional activators typically bind near transcriptional promoters and recruit RNA polymerase to directly initiate transcription. Repressors bind transcriptional promoters and sterically hinder transcription initiation by RNA polymerase. Other transcriptional regulators can serve as either activators or repressors, depending on where they bind and on cellular and environmental conditions. Non-limiting examples of transcriptional regulator classes include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine-zipper proteins.

[0086] As used herein, "repressor protein" or "inducer protein" refers to a protein that binds to a regulatory sequence element and represses or activates the transcription of the sequence operably linked to the regulatory sequence element, respectively.Preferred repressor and inducer proteins described herein are sensitive to the presence or absence of at least one input agent or environmental input.Preferred proteins described herein are modular in form, for example, comprising separable DNA binding and input agent binding or response elements or domains.

[0087] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0088] As used herein, an "input agent responsive domain" is a domain of a transcription factor that binds to a condition or input agent or otherwise responds to the condition or input agent in a manner that renders the linked DNA-binding fusion domain responsive to the presence of that condition or input. In one embodiment, the presence of the condition or input results in a conformational change in the input agent responsive domain or the protein to which it is fused, which modifies the transcriptional regulatory activity of the transcription factor.

[0089] The term "in vivo" refers to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the embodiments described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses carried out using live cells with intact membranes outside the body of a multicellular animal or plant, such as explants, cultured cells (including primary cells and cell lines), transformed cell lines, and extracted tissues or cells (including blood cells), among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may refer to introducing a programmable synthetic biological circuit into a non-cellular system, such as a cell or cell system-free medium, such as a cell extract.

[0090] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of the nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled. A promoter may also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. In some embodiments of the aspects described herein, a promoter may drive the expression of transcription factors that regulate the expression of the promoter itself. Within the promoter sequence will be found the transcription initiation site, as well as protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the ceDNA vectors disclosed herein. A promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' orientation) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0091] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50-1,500 base pairs) that binds one or more proteins (e.g., activator proteins or transcription factors) to increase transcriptional activation of a nucleic acid sequence. Enhancers can be located up to 1,000,000 base pairs upstream of the gene start site or downstream of the gene start site that they regulate. Enhancers can be located within intronic or exon regions of unrelated genes.

[0092] A promoter can be said to drive expression or drive transcription of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and regulates to control transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in an inverted orientation, whereby what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences are used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0093] A promoter may be one that is naturally associated with a gene or sequence, which can be obtained by isolating the 5' non-coding sequence located upstream of the coding segment and / or exon of a given gene or sequence. Such a promoter may be called "endogenous". Similarly, in some embodiments, an enhancer may be one that is naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.

[0094] In some embodiments, the coding nucleic acid segment is placed under the control of a "recombinant promoter" or a "heterologous promoter", both of which refer to a promoter that is not normally associated with an operably linked encoded nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and synthetic promoters or enhancers that are not "naturally occurring", i.e., may contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, for the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Additionally, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria, chloroplasts, etc., can be used as well.

[0095] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducer agent. As defined herein, an "inducer" or "inducer agent" can be endogenous or can be a compound or protein, usually exogenous, administered in such a manner that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducer agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of a certain agent, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter, and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0096] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolytic signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of the encoded polypeptide.

[0097] "Operably linked" refers to a juxtaposition in which the components described are in a relationship that allows them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. An "expression cassette" includes a heterologous DNA sequence that is operably linked to a promoter or other regulatory sequence sufficient to direct the transcription of a transgene in a ceDNA vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0098] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with the ceDNA vector according to the present invention is provided. Usually, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, such as domestic cats, canine species, such as dogs, foxes, wolves, avian species, such as chickens, emus, ostriches, and fish, such as trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, such as a primate or a human. The subject can be male or female. Additionally, the subject may be an infant or child. In some embodiments, the subject may be a neonatal or fetal subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals may be advantageously used as subjects that represent animal models of diseases and disorders. In addition, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African American, African European, Latin American, Middle Eastern, etc. In some embodiments, the subject may be a patient or other subject in a clinical setting. In some embodiments, the subject has already undergone treatment. In some embodiments, the subject is an embryo, fetus, neonatal, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, a human neonatal, a human infant, a human child, a human adolescent, or a human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo, hi some embodiments, the subject is a human embryo.

[0099] As used herein, the term "host cell" includes any cell type that is amenable to transformation, transfection, transduction, etc. with a nucleic acid construct or ceDNA expression vector of the present disclosure. By way of non-limiting example, host cells include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be any of a number of immortalized cell lines (e.g., HepG2 cells), induced pluripotent stem cells, or any of a number of immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be a cell in situ or in vivo in a tissue, organ, or organism.

[0100] The term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" may refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that is not normally found and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" may refer to a nucleic acid or polypeptide that is found in relatively small amounts and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to cause ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is natural to a biological system or cell.

[0101] The term "sequence identity" refers to the relatedness between two nucleotide sequences. For purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is measured using the Needleman-Wunsch algorithm (Needleman, 2000, supra), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. and Wunsch, 1970, supra). Optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle output labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides x 100) / length of alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0102] The term "homology" or "homology" as used herein is defined as the percentage of nucleotide residues that are identical to the nucleotide residues of the corresponding sequence on the target chromosome, after aligning the sequences as necessary and introducing gaps to achieve the maximum sequence identity percentage. Alignment for the purpose of determining nucleotide sequence homology percentage can be achieved in various ways that are within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of an arm of homology is considered "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the corresponding native or unedited nucleic acid sequence (e.g., a genomic sequence) of the host cell.

[0103] The term "heterologous" as used herein refers to a nucleotide or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence that encodes a chimeric polypeptide. A heterologous nucleic acid sequence may be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleotide sequence that encodes a fusion variant polypeptide.

[0104] A "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," can be attached to effect replication of the attached segment in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral in origin and / or final form, but for the purposes of this disclosure, "vector," as the term is used herein, generally refers to a ceDNA vector. The term "vector" encompasses any genetic element that can replicate and transfer a genetic sequence to a cell when associated with the appropriate control elements. In some embodiments, a vector can be an expression vector or a recombinant vector.

[0105] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence linked to a transcriptional regulatory sequence on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, so that it can be maintained in two organisms, for example, human cells for expression, and prokaryotic hosts for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions preceding and following the coding region, such as 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0106] "Recombinant vector" refers to a vector that contains a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vector described herein can be combined with other suitable compositions and therapies in some embodiments. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means to maintain the nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0107] The phrase "genetic disease" as used herein refers to a disease that is caused, directly or indirectly, partially or completely, by one or more abnormalities in the genome, especially conditions that are present from birth. The abnormality can be a mutation, insertion, or deletion. The abnormality can affect the coding sequence of the gene or its regulatory sequence. The genetic disease can be, but is not limited to, PKU, DMD, hemophilia, cystic fibrosis, Huntington's disease, familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, congenital hepatic porphyria, inherited disorders of hepatic metabolism, Lesch-Nyhan syndrome, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia telangiectasia, Bloom's syndrome, retinoblastoma, and Tay-Sachs disease.

[0108] As used herein, "inhibitory polynucleotide" refers to a DNA or RNA molecule that reduces or prevents the expression (transcription or translation) of a second (target) polynucleotide.Inhibitory polynucleotides include antisense polynucleotides, ribozymes, and external guide sequences.The term "inhibitory polynucleotide" also includes DNA and RNA molecules, such as RNAi that code for the actual inhibitory species, such as DNA molecules that code for ribozymes.

[0109] As used herein, "gene silencing" or "gene silenced" with respect to the activity of an RNAi molecule, eg, an siRNA or miRNA, refers to the reduction of the mRNA levels of a target gene in a cell.

[0110] As used herein, the term "RNAi" refers to any type of interfering RNA, including but not limited to siRNAi, shRNAi, endogenous microRNA, and artificial microRNA. For example, sequences previously identified as siRNAs are included, regardless of the mechanism of downstream processing of the RNA (i.e., siRNAs are believed to have a specific in vivo processing method that results in cleavage of mRNA, but such sequences can be incorporated into vectors in the context of adjacent sequences described herein). The term "RNAi" can include both gene silencing RNAi molecules and RNAi effector molecules that activate the expression of genes. By way of example only, in some embodiments, inhibitory or gene silencing RNAi agents are useful in the methods, kits and compositions disclosed herein, for example, to inhibit immune responses (e.g., innate immune responses).

[0111] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the method or composition, but are open to the inclusion of non-specified elements, whether essential or not.

[0112] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment. The use of "comprises" indicates inclusion rather than limitation.

[0113] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0114] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of the embodiment of the invention.

[0115] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that would be apparent to one of ordinary skill in the art upon reading this disclosure, and so forth. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with "for example."

[0116] Grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed herein to include the modified group, thus satisfying all Markush group descriptions used in the appended claims.

[0117] In some embodiments of any aspect, the disclosure described herein does not pertain to human cloning processes, processes for correcting the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, and processes for modifying the genetic identity of animals resulting from such processes.

[0118] Other terms are defined herein within the description of various aspects of the invention.

[0119] All patents and other publications cited throughout this application, including literature references, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of these documents.

[0120] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while steps or functions of a method are presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified as appropriate to provide further embodiments of the present disclosure using the compositions, functions, and concepts of the above references and applications. Furthermore, some changes can be made to the protein structure without affecting the type or amount of biological or chemical action, due to considerations of biological functional equivalence. These and other changes can be made to the present disclosure in light of the detailed description. All such modifications are intended to be within the scope of the appended claims.

[0121] Particular elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Additionally, although advantages associated with certain embodiments of the present disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily must exhibit such advantages, to be within the scope of the present disclosure.

[0122] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, and as such may vary. The terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0123] II. Expression of PAH proteins from closed-ended DNA (ceDNA) vectors The technology described herein is generally directed to the expression and / or production of PAH proteins in cells from non-viral DNA vectors, such as the ceDNA vectors described herein. ceDNA vectors for the expression of PAH proteins are described herein in the section entitled "ceDNA Vectors in General". In particular, ceDNA vectors for the expression of PAH proteins include a pair of ITRs (e.g., symmetric or asymmetric as described herein), and a nucleic acid encoding a PAH protein described herein, operably linked to a promoter or regulatory sequence between the ITR pair. A distinct advantage of ceDNA vectors for the expression of PAH proteins over traditional AAV vectors, and even lentiviral vectors, is that there is no size constraint on the heterologous nucleic acid sequence encoding the desired protein. Thus, even a full-length 6.8 kb PAH protein can be expressed from a single ceDNA vector. Thus, the ceDNA vectors described herein can be used to express therapeutic PAH proteins in subjects in need thereof, such as subjects with PKU.

[0124] As can be understood, the ceDNA vector technology described herein can be adapted to any level of complexity or can be used in a modular manner, where the expression of different components of PAH protein can be controlled in an independent manner.For example, the ceDNA vector technology designed herein can be as simple as using a single ceDNA vector to express a single heterologous gene sequence (e.g., PAH protein), or as complex as using multiple ceDNA vectors, each vector expressing multiple PAH proteins or related cofactors or accessory proteins, each independently controlled by different promoters.The following embodiments are specifically contemplated herein and can be adapted by those skilled in the art as needed.

[0125] In one embodiment, a single ceDNA vector can be used to express a single component of a PAH protein. Alternatively, a single ceDNA vector can be used to express multiple components (e.g., at least two) of a PAH protein under the control of a single promoter (such as a strong promoter), optionally using an IRES sequence to ensure proper expression of each of the components, e.g., cofactors or accessory proteins.

[0126] Also contemplated herein in another embodiment is a single ceDNA vector that contains at least two inserts (e.g., expressing heavy or light chains), with the expression of each insert being under the control of its own promoter.The promoter may include multiple copies of the same promoter, multiple different promoters, or any combination thereof.As those skilled in the art will appreciate, it is often desirable to express components of a PAH protein at different expression levels, and thus control the stoichiometry of the individual components expressed, to ensure efficient folding and combination of the PAH protein in cells.

[0127] Further variations of ceDNA vector technology can be envisioned by those skilled in the art or can be adapted from protein production methods using conventional vectors.

[0128] A. Nucleic acid Characterization and development of nucleic acid molecules of potential therapeutic use are provided herein. According to some embodiments, the nucleic acid of therapeutic use encodes a PAH protein. In some embodiments, the chemical modification of oligonucleotides for altered and improved in vivo properties (delivery, stability, life span, folding, target specificity) as well as their biological functions and mechanisms that directly correlate with therapeutic use are described, if necessary.

[0129] Exemplary therapeutic nucleic acids of the present disclosure that may be immunostimulatory and may require the use of the immunosuppressants disclosed herein can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), ribozymes, closed-ended double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone (dbDNA™) DNA, protelomeric closed-ended DNA, or dumbbell linear DNA), dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0130] siRNA or miRNA, which can downregulate the intracellular level of a specific protein through a process called RNA interference (RNAi), are also contemplated by the present invention as nucleic acid therapeutics.After siRNA or miRNA is introduced into the cytoplasm of host cells, these double-stranded RNA constructs can bind to a protein called RISC.The sense strand of siRNA or miRNA is removed by the RISC complex.When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand.RNAi is by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0131] Antisense oligonucleotides (ASOs) and ribozymes that inhibit mRNA translation into protein can be nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxynucleic acids have sequences complementary to the sequences of target protein mRNA, and can bind to mRNA by Watson-Crick base pairing. This binding prevents the translation of the target mRNA and / or induces RNaseH degradation of the mRNA transcript. As a result, antisense oligonucleotides have enhanced specificity of action (i.e., downregulation of specific disease-related proteins).

[0132] In any of the methods provided herein, the therapeutic nucleic acid can be a therapeutic RNA.The therapeutic RNA can be an inhibitor of mRNA translation, an agent of RNA interference (RNAi), a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), or an RNA that binds to mRNA transcript (ASO), a protein or other molecular ligand (aptamer).In any of the methods provided herein, the agent of RNAi can be a double-stranded RNA, a single-stranded RNA, a microRNA, a short interfering RNA, a small hairpin RNA, or a triplex-forming oligonucleotide.

[0133] According to some embodiments, the therapeutic nucleic acid is a closed-end double-stranded DNA, e.g., ceDNA. According to some embodiments, the expression and / or production of therapeutic protein in cells is derived from a non-viral DNA vector, e.g., ceDNA vector. The distinct advantage of ceDNA vectors over traditional AAV vectors, and even lentiviral vectors, for the expression of therapeutic proteins is that there is no size constraint on the heterologous nucleic acid sequence that codes for the desired protein. Thus, even large therapeutic proteins can be expressed from a single ceDNA vector. Thus, ceDNA vectors can be used to express therapeutic proteins in subjects in need thereof.

[0134] In general, the ceDNA vector for expressing a therapeutic protein disclosed herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. The ITR sequence is selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., an asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration.

[0135] In some embodiments, the transgene encoding the PAH protein can also encode a secretory sequence, which targets the PAH protein to the Golgi apparatus and the endoplasmic reticulum, where it is folded into the correct conformation by chaperone molecules as it passes through the ER and exits the cell. Exemplary secretory sequences include VH-02 (SEQ ID NO:88) and VK-A26 (SEQ ID NO:89) and Ig K These include, but are not limited to, a signal sequence (SEQ ID NO: 126), as well as a Gluc secretion signal (SEQ ID NO: 188), which enables the tagged protein to be secreted from the cytosol, and a TMD-ST secretion sequence (SEQ ID NO: 189), which targets the tagged protein to the Golgi apparatus.

[0136] Regulatory switches can also be used to fine-tune expression of a PAH protein, such that the PAH protein is expressed when needed (including, but not limited to, expression of the PAH protein at a desired expression level or amount), or alternatively, when there is the presence or absence of a particular signal, including a cell signaling event. For example, as described herein, expression of a PAH protein from a ceDNA vector can be turned on or off when a particular condition occurs, as described herein in the section entitled "Regulatory Switches."

[0137] For example, and for purposes of illustration only, the PAH protein can be used to stop an undesirable reaction, such as the production of too high levels of the PAH protein. The PAH gene may include a signal peptide marker to bring the PAH protein to the desired cells. However, in either situation, it may be desirable to regulate the expression of the PAH protein. ceDNA vectors are readily amenable to the use of regulatory switches.

[0138] A distinct advantage of ceDNA vectors over traditional AAV vectors, and even lentiviral vectors, is that there is no size constraint on the heterologous nucleic acid sequence encoding the PAH protein. Thus, full-length PAHs, as well as optionally any cofactors or evaluation proteins, can be expressed from a single ceDNA vector. Furthermore, depending on the atomic chemistry required, multiple segments of the same PAH protein can be expressed, the same or different promoters can be used, and regulatory switches can be used to fine-tune the expression of each region. For example, as shown in the examples, ceDNA vectors containing a dual promoter system can be used, whereby a different promoter is used for each domain of the PAH protein. The use of ceDNA plasmids to produce PAH proteins can include a unique combination of promoters for the expression of the domains of the PAH protein that results in the appropriate ratio of each domain for the formation of a functional PAH protein. Thus, in some embodiments, ceDNA vectors can be used to express different regions of the PAH protein separately (e.g., under the control of different promoters).

[0139] In another embodiment, the PAH protein expressed from the ceDNA vector further comprises additional functions, such as fluorescence, enzymatic activity, a secretion signal, or an immune cell activator.

[0140] In some embodiments, the ceDNA encoding the PAH protein can further include, for example, a linker domain. As used herein, a "linker domain" refers to an oligo- or polypeptide region of about 2-100 amino acids in length that links together any of the domains / regions of the PAH protein described herein. In some embodiments, the linker can include or consist of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to one another. Longer linkers can be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker can be cleavable or non-cleavable. Examples of cleavable linkers include 2A linkers (e.g., T2A), 2A-like linkers or functional equivalents thereof, and combinations thereof. The linker can be a linker region that is T2A from Thosea asigna virus.

[0141] For example, it is well within the ability of one of ordinary skill in the art to take a known and / or publicly available protein sequence, such as a PAH, and reverse engineer a cDNA sequence to encode such a protein. The cDNA can then be codon-optimized to match the intended host cell and inserted into a ceDNA vector as described herein.

[0142] B. ceDNA vectors expressing PAH proteins A ceDNA vector for expression of a PAH protein having one or more sequences encoding a desired PAH can include regulatory sequences such as a promoter, a secretion signal, a polyA region, and an enhancer. At a minimum, the ceDNA vector includes one or more heterologous sequences encoding a PAH protein.

[0143] To achieve highly efficient and accurate assembly of the PAH protein, in some embodiments it is specifically contemplated that the PAH protein contains an endoplasmic reticulum (ER) leader sequence that directs it to the ER where protein folding occurs, e.g., a sequence that targets the expressed protein to the ER for folding.

[0144] In some embodiments, a cellular or extracellular localization signal (e.g., a secretion signal, a nuclear localization signal, a mitochondrial localization signal, etc.) is included in the ceDNA vector to direct the secretion or desired subcellular localization of the PAH protein, thereby allowing the PAH protein to bind to an intracellular target (e.g., an intrabody) or an extracellular target.

[0145] In some embodiments, the ceDNA vectors for expression of PAH proteins described herein allow for the assembly and expression of any desired PAH protein in a modular manner. As used herein, the term "module" refers to an element in a ceDNA expression plasmid that can be easily removed from the construct. For example, the modular elements of a ceDNA production plasmid contain unique pairs of restriction sites flanking each element in the construct, allowing for exclusive manipulation of individual elements (see, e.g., Figures 1A-1G). Thus, the ceDNA vector platform can allow for the expression and assembly of any desired PAH protein configuration. In various embodiments, ceDNA plasmid vectors are provided herein that can reduce and / or minimize the amount of manipulation required to assemble a desired ceDNA vector encoding a PAH protein.

[0146] C. Exemplary PAH Proteins Expressed by ceDNA Vectors In particular, the ceDNA vectors for expression of the PAH proteins disclosed herein can encode, for example, but not limited to, PAH proteins, and variants and / or active fragments thereof, for use in treating, preventing, and / or ameliorating one or more symptoms of phenylketonuria (PKU). In one embodiment, the phenylketonuria (PKU) is human phenylketonuria (PKU).

[0147] (i) PAH therapeutic proteins and fragments thereof Essentially, any version of PAH therapeutic protein or fragment thereof (e.g., functional fragment) can be encoded by ceDNA vector and expressed in and from ceDNA vector as described herein.Those skilled in the art will understand that PAH therapeutic protein includes all splice variants and orthologs of PAH protein.PAH therapeutic protein includes intact molecule as well as fragment thereof (e.g., functional).

[0148] A distinct advantage of ceDNA vectors over traditional AAV and even lentiviral vectors is that there is no size constraint on the heterologous nucleic acid sequence encoding the desired protein, therefore, multiple full-length PAH therapeutic proteins can be expressed from a single ceDNA vector.

[0149] PAH Proteins and Genes: The PAH gene is located on chromosome 12, band 12q22-q24.2. As of 2000, approximately 400 disease-causing mutations had been discovered in the PAH gene. Phenylalanine hydroxylase (PAH) may also be referred to as phenylalanine 4-monooxygenase, phenylalanine-4-hydroxylase, Phe-4-monooxygenase, EC 1.14.16.1, EC 1.14.16, PKU1, PKU, or PH.

[0150] The protein sequence of PAH is as follows: Homo sapiens PAH enzyme translation (450 amino acids), accession number NM_000277.3 MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKK KDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLED VSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQK (SEQ ID NO: 195)

[0151] PAH is mainly expressed in the liver, with moderate expression in the kidney and gallbladder. Low levels of PAH expression can also be detected in the prostate and adrenal gland. During fetal development, PAH can be expressed in the adrenal gland, heart, intestine, lung, and stomach. Thus, the ceDNA vector expressing PAH can be administered to any one or more tissues selected from the liver, kidney, gallbladder, prostate, and adrenal gland. In some embodiments, when the ceDNA vector expressing PAH is administered to an infant or administered to a subject in utero, the ceDNA vector expressing PAH can be administered to any one or more tissues selected from the liver, adrenal gland, heart, intestine, lung, and stomach.

[0152] Expression of a PAH therapeutic protein or fragment thereof from a ceDNA vector can be achieved both spatially and temporally using one or more inducible or repressible promoters, including the regulatory switches described herein, as known in the art or as described herein.

[0153] In one embodiment, the PAH therapeutic protein is a "therapeutic protein variant," which refers to a PAH therapeutic protein that has an altered amino acid sequence, composition, or structure compared to the corresponding native PAH therapeutic protein. In one embodiment, the PAH is a functional version (e.g., wild type). It may also be useful to express mutant versions of the PAH protein, such as point or deletion mutations that lead to phenylketonuria (PKU), for example, to evaluate animal models of disease and / or drugs for phenylketonuria (PKU). Delivery of mutant or modified PAH proteins into cells or animal model systems can be performed to generate disease models. Such cells or animal models can be used for research and / or drug screening. PAH therapeutic proteins expressed from ceDNA vectors may further include sequences / moieties that confer additional functions, such as fluorescence, enzymatic activity, or secretion signals. In one embodiment, the PAH therapeutic protein variant includes a non-native tag sequence (e.g., an immunotag) for identification to enable it to be distinguished from the endogenous PAH therapeutic protein in the recipient host cell.

[0154] For example, it is well within the ability of one of ordinary skill in the art to take a known and / or publicly available protein sequence of a PAH therapeutic protein and reverse engineer a cDNA sequence to encode such a protein. The cDNA can then be codon optimized to match the intended host cell and inserted into a ceDNA vector as described herein.

[0155] In one embodiment, the PAH therapeutic protein coding sequence may be derived from an existing host cell or cell line, for example, by reverse transcribing mRNA obtained from the host and amplifying the sequence using PCR.

[0156] (ii) ceDNA vector expressing PAH therapeutic protein A ceDNA vector having one or more sequences encoding a desired PAH therapeutic protein can include regulatory sequences such as a promoter (see, e.g., Table 1), a secretion signal, a polyA region, and an enhancer. At a minimum, the ceDNA vector includes one or more heterologous sequences encoding a PAH therapeutic protein or a functional fragment thereof. Exemplary cassette insertions for generating a ceDNA vector encoding a PAH therapeutic protein are shown in Figures 1A-1G. In one embodiment, the ceDNA vector includes a PAH sequence listed in Table 1 herein.

[0157] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

[0158] In one embodiment, the ceDNA vector comprises a PAH sequence listed in Table 1 herein. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 90% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 91% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 92% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 93% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 94% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 95% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 96% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 97% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 98% identity to a PAH sequence listed in Table 1. In one embodiment, the ceDNA vector comprises a PAH sequence having at least 99% identity to a PAH sequence listed in Table 1.

[0159] In one embodiment, the PAH sequence has at least 90% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 91% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 92% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 93% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 94% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 95% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394.In one embodiment, the PAH sequence has at least 96% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 97% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 98% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence has at least 99% identity to SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence comprises SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394. In one embodiment, the PAH sequence consists of SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393, or SEQ ID NO:394.

[0160] In one embodiment, the PAH sequence has a sequence having at least 85% identity to SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence having at least 90% identity to SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence having at least 95% identity to SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence having at least 97% identity to SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence having at least 99% identity to SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a SEQ ID NO:. In one embodiment, the PAH sequence has a sequence comprising SEQ ID NO: 382. In one embodiment, the PAH sequence has a sequence consisting of SEQ ID NO: 382.

[0161] In one embodiment, the PAH sequence has a sequence having at least 85% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence having at least 90% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence having at least 95% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence having at least 97% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence having at least 99% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence comprising SEQ ID NO: 384. In one embodiment, the PAH sequence has a sequence consisting of SEQ ID NO: 384.

[0162] In one embodiment, the PAH sequence has a sequence having at least 85% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence having at least 90% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence having at least 95% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence having at least 97% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence having at least 99% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence having at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence comprising SEQ ID NO: 394. In one embodiment, the PAH sequence has a sequence consisting of SEQ ID NO: 394.

[0163] (iii) PAH therapeutic proteins and uses thereof for the treatment of PKU The ceDNA vectors described herein can be used to deliver therapeutic PAH proteins for the treatment of PKU, which is associated with inappropriate expression of and / or mutations within the PAH protein.

[0164] The ceDNA vectors described herein can be used to express any desired PAH therapeutic protein. Exemplary therapeutic PAH therapeutic proteins include, but are not limited to, any of the PAH proteins expressed by the sequences shown in Table 1 herein.

[0165] In one embodiment, the expressed PAH therapeutic protein is functional for the treatment of phenylketonuria (PKU). In some embodiments, the PAH therapeutic protein does not provoke an immune system response.

[0166] In another embodiment, a ceDNA vector encoding a PAH therapeutic protein or a fragment thereof (e.g., functional fragment) can be used to generate a chimeric protein.Therefore, it is specifically contemplated herein that a ceDNA vector expressing a chimeric protein can be administered to any one or more tissues selected from, for example, liver, kidney, gallbladder, prostate, and adrenal gland.In some embodiments, when a ceDNA vector expressing a PAH is administered to an infant or administered to a subject in utero, the ceDNA vector expressing a PAH can be administered to any one or more tissues selected from liver, adrenal gland, heart, intestine, lung, and stomach, or to its liver stem cell precursor for in vivo or ex vivo treatment of phenylketonuria (PKU).

[0167] PKU: PKU is a rare inherited inborn error of metabolism caused by a mutation in the PAH gene. PAH is an enzyme normally expressed in the liver, and is necessary to metabolize dietary phenylalanine into tyrosine, an amino acid involved in the production of neurotransmitters. PKU results from a mutation in PAH, resulting in a deficiency in its enzymatic activity. Thus, a ceDNA vector expressing a PAH protein can express PAH in the liver or other tissues, including retinal cells such as photoreceptors and / or RPE cells. In some embodiments, the ceDNA vector expresses at least one PAH protein in both photoreceptors and RPE cells.

[0168] PAH is normally expressed endogenously in both PR and RPE cell types.It has also been reported that low-level PAH expression in RPE may also be necessary for normal retinal function.Therefore, to prevent retinal degeneration, low-level or high-level expression of PAH protein by ceDNA vector in PR and also optionally RPE cells may sometimes be required.This level of expression can be fine-tuned by promoter and / or regulatory switch as described herein.

[0169] Thus, in some embodiments, ceDNA vectors are used to express PAH protein, a 6.8kb protein, from endogenous promoters (about 1kb), restoring normal retinoid processing in both photoreceptors and RPE. In some embodiments, ceDNA vectors expressing PAH protein are administered via suprachoroidal or intravitreal routes to treat larger areas of the retina. In some embodiments, ceDNA vectors are administered via any one or more of subretinal, suprachoroidal, or intravitreal injections.

[0170] The method includes administering to a subject an effective amount of a composition comprising a ceDNA vector encoding a PAH therapeutic protein or a fragment thereof (e.g., a functional fragment), as described herein. As will be understood by those skilled in the art, the term "effective amount" refers to the amount of the administered ceDNA composition that results in expression of the protein in a "therapeutically effective amount" for the treatment of a disease or disorder.

[0171] The dosage range of a composition comprising a ceDNA vector encoding a PAH therapeutic protein or a fragment thereof (e.g., a functional fragment) depends on the potency (e.g., promoter efficiency) and includes an amount sufficient to produce the desired effect, e.g., expression of the desired PAH therapeutic protein, for the treatment of phenylketonuria (PKU). The dosage should not be so large as to cause unacceptable adverse side effects. In general, the dosage will vary depending on the specific characteristics of the ceDNA vector, the expression efficiency, and the age, condition, and sex of the patient. The dosage can be determined by one skilled in the art and can also be adjusted by the individual physician in case of complications, unlike conventional AAV vectors, because ceDNA vectors do not contain immune-activating capsid proteins that prevent repeated administration.

[0172] The administration of the ceDNA composition described herein can be repeated for a limited period of time. In some embodiments, the dose is given periodically or by pulse administration. In a preferred embodiment, the dose listed above is administered over several months. The duration of treatment depends on the subject's clinical progress and responsiveness to treatment. Booster treatment over time is contemplated. Furthermore, the level of expression can be titrated as the subject grows.

[0173] The PAH therapeutic protein may be expressed in a subject for at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 12 months / year, at least 2 years, at least 5 years, at least 10 years, at least 15 years, at least 20 years, at least 30 years, at least 40 years, at least 50 years, or more. Long-term expression can be achieved by repeated administration of the ceDNA vectors described herein at predetermined or desired intervals.

[0174] As used herein, the term "therapeutically effective amount" refers to an amount of expressed PAH therapeutic protein or functional fragment thereof that is sufficient to cause a statistically significant measurable change in the expression of a disease biomarker or a reduction in a given disease symptom (see "Measurement of efficacy" below). Such effective amounts can be evaluated in clinical trials as well as animal studies for a given ceDNA composition.

[0175] The exact amount of ceDNA vector that needs to be administered depends on the practitioner's judgment and is specific to each individual. Suitable regimes for administration also vary, but are typified by an initial administration followed by repeated doses at one or more intervals by subsequent injections or other administrations. Alternatively, continuous intravenous infusion sufficient to maintain blood levels within the range specified for in vivo treatment is contemplated, particularly for the treatment of acute diseases / disorders.

[0176] The agents useful in the methods and compositions described herein can be administered locally, intravenously (by bolus or continuous infusion), intracellularly, intratissue, orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and can be delivered by peristaltic means or other means known to those skilled in the art, if necessary.The agents can be administered systemically, if necessary.They can also be administered intrauterinely.

[0177] The efficacy of a given treatment for phenylketonuria (PKU) can be determined by a skilled clinician. However, if any one or all of the signs or symptoms of the disease or disorder are altered in a beneficial manner, or other clinically acceptable symptoms or markers of the disease are improved or ameliorated by at least 10% after treatment with, for example, a ceDNA vector encoding PAH as described herein, or a functional fragment thereof, the treatment is considered to be an "effective treatment," as that term is used herein. Efficacy can also be measured by stabilization of the disease, or failure of the individual to deteriorate, as assessed by the need for medical intervention (i.e., progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including: (1) inhibiting the disease, e.g., halting or slowing the progression of the disease or disorder, or (2) palliating the disease, e.g., causing a reduction in symptoms, and (3) preventing or reducing the likelihood of development of the disease, such as liver or kidney failure, or preventing secondary diseases / disorders associated with the disease (e.g., hand deformities due to rheumatoid arthritis, or cancer metastasis). An effective amount for treating a disease means an amount that, when administered to a mammal in need thereof, is sufficient to provide effective treatment for the disease, as that term is defined herein.

[0178] The effectiveness of a drug can be determined by assessing physical indicators specific to phenylketonuria (PKU). Standard methods for analyzing PKU indicators are known in the art.

[0179] In some embodiments, the ceDNA vectors for expression of the PAH proteins disclosed herein can also encode cofactors or other polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding, e.g., siRNA, shRNA, microRNA, antisense counterparts (e.g., antagoMiR) that can be used in combination with the PAH protein expressed from ceDNA). Additionally, expression cassettes containing sequences encoding PAH proteins can also include exogenous sequences encoding reporter proteins used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others well known in the art.

[0180] In one embodiment, the ceDNA vector comprises a nucleic acid sequence for expressing a therapeutic PAH protein that is functional for the treatment of PKU. In a preferred embodiment, the therapeutic PAH protein does not provoke an immune system response unless one is desired.

[0181] III. General ceDNA Vectors for Use in the Production of PAH Therapeutic Proteins The embodiments of the present invention are based on methods and compositions comprising closed-end linear double-stranded (ceDNA) vectors capable of expressing PAH transgenes. In some embodiments, the transgene is a sequence encoding a PAH protein. The ceDNA vectors for expressing PAH proteins described herein are not limited by size, thereby allowing, for example, the expression of all components required for the expression of a transgene from a single vector. The ceDNA vectors for expressing PAH proteins are preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), for example, at 37° C. for 1 hour or more.

[0182] In general, the ceDNA vector for expressing the PAH protein disclosed herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. The ITR sequence is selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., an asymmetric modified ITR), (ii) two modified ITRs (e.g., an asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration.

[0183] Methods and compositions comprising ceDNA vectors for the production of PAH proteins are included herein, and may further comprise delivery systems, such as, but not limited to, liposomal nanoparticle delivery systems.Non-limiting exemplary liposomal nanoparticle systems are included for use are disclosed herein.In some embodiments, the present disclosure provides lipid nanoparticles comprising ceDNA and ionized lipids.For example, lipid nanoparticle formulations made and loaded with ceDNA obtained by the process are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein by reference in its entirety.

[0184] The ceDNA vector for expressing PAH protein disclosed herein does not have the packaging constraint imposed by the limited space in the viral capsid.The ceDNA vector represents a versatile eukaryotically produced alternative to the prokaryotically produced plasmid DNA vector, as opposed to the enclosed AAV genome.This allows the insertion of control elements, such as the regulatory switches disclosed herein, large transgenes, multiple transgenes, etc.

[0185] 1A-1E show schematic diagrams of non-limiting exemplary ceDNA vectors for expression of PAH proteins, or sequences of corresponding ceDNA plasmids. The ceDNA vectors for expression of PAH proteins do not contain capsids and can be obtained from plasmids encoding the first ITR, an expression cassette containing a transgene, and the second ITR in this order. The expression cassette can include one or more regulatory sequences that allow and / or control the expression of the transgene, for example, the expression cassette can include one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA) in this order.

[0186] The expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue and cell type specific promoters, and enhancers. In some embodiments, the ITRs may act as promoters of transgenes, such as PAH proteins. In some embodiments, the ceDNA vector may include additional components for regulating the expression of transgenes, such as a regulatory switch for controlling and regulating the expression of PAH proteins, as described in the section entitled "Regulatory Switches" herein, and may include a regulatory switch, which is a kill switch that allows for controlled cell death of cells containing the ceDNA vector, if desired.

[0187] The expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range of about 4000-10,000 nucleotides, or 10,000-50,000 nucleotides, or more than 50,000 nucleotides. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-50,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-75,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-10,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 1000-10,000 nucleotides in length. In some embodiments, the expression cassette may comprise a transgene that is in the range of 500-5,000 nucleotides in length. ceDNA vectors do not have the size limitations of encapsidated AAV vectors, and therefore allow the delivery of large expression cassettes to result in efficient transgene expression. In some embodiments, ceDNA vectors lack prokaryotic cell-specific methylation.

[0188] The ceDNA expression cassette may contain, for example, an expressible exogenous sequence (e.g., an open reading frame) or transgene that encodes a protein that is absent, inactive, or insufficiently active in the recipient subject, or a gene that encodes a protein with a desired biological or therapeutic effect. The transgene may encode a gene product that can function to correct the expression of a defective gene or transcript. In principle, the expression cassette may contain any gene that encodes a protein, polypeptide, or RNA that is reduced or absent due to a mutation, or that provides a therapeutic effect if overexpression is considered within the scope of this disclosure.

[0189] The expression cassette may include any transgene (e.g., encoding a PAH protein), for example, a PAH protein useful for treating PKU in a subject, i.e., a therapeutic PAH protein. The ceDNA vector can be used alone or in combination with exogenous genes and nucleotide sequences, including polypeptide-encoding or non-encoding nucleic acids (e.g., RNAi, miR, etc.), and viral sequences in the subject's genome, such as HIV viral sequences, to deliver and express any PAH protein of interest to a subject. Preferably, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary use) or immunogenic polypeptides. In certain embodiments, the ceDNA vectors are useful for expressing any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, or RNAs (encoding or non-encoding, e.g., siRNAs, shRNAs, microRNAs, and their antisense counterparts (e.g., antagoMiRs)), antibodies, fusion proteins, or any combination thereof.

[0190] The expression cassette may also encode a polypeptide, a sense or antisense oligonucleotide, or an RNA (coding or non-coding, e.g., siRNA, shRNA, microRNA, and their antisense counterparts (e.g., antagoMiR)). The expression cassette may also include an exogenous sequence encoding a reporter protein used for experimental or diagnostic purposes, such as β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, and others known in the art.

[0191] The sequences provided in the expression cassette, the expression construct of the ceDNA vector for the expression of the PAH proteins described herein, can be codon-optimized for the target host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are more frequently or most frequently used in the genes of a vertebrate of interest, for enhanced expression in the cells of the vertebrate of interest, such as a mouse or human. Various species exhibit a particular bias for certain codons of certain amino acids. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another public database. In some embodiments, the nucleic acid encoding the PAH protein is optimized for human expression and / or is a human PAH or a functional fragment thereof, as known in the art.

[0192] As disclosed herein, the transgene expressed by the ceDNA vector for expression of the PAH protein encodes the PAH protein. There are many structural features of the ceDNA vector for expression of the PAH protein that are different from the plasmid-based expression vector. The ceDNA vector may have one or more of the following characteristics: lack of original (i.e., uninserted) bacterial DNA, lack of prokaryotic replication origin, self-contained (i.e., does not require any sequence other than the two ITRs containing Rep binding and terminal resolution sites (RBS and TRS) and no exogenous sequence between the ITRs), presence of ITR sequences that form hairpins, and absence of bacterial-type DNA methylation, or indeed any other methylation that would be considered abnormal by a mammalian host. In general, the vector preferably does not contain any prokaryotic DNA, although it is contemplated that some prokaryotic DNA may be inserted as exogenous sequence, as a non-limiting example in a promoter or enhancer region. Another important feature that distinguishes ceDNA vectors from plasmid expression vectors is that ceDNA vectors are single-stranded linear DNA with closed ends, whereas plasmids are always double-stranded DNA.

[0193] The ceDNA vectors for expression of PAH proteins produced by the methods provided herein preferably have a linear, continuous structure rather than a discontinuous structure as determined by a restriction enzyme digestion assay ( FIG. 4D ). A linear, continuous structure is believed to be more stable against attack by cellular endonucleases while at the same time being less likely to recombine and cause mutagenesis. Thus, a linear, continuous structure ceDNA vector is a preferred embodiment. A continuous, linear, single-stranded intramolecular duplex ceDNA vector may be covalently linked at the termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. Complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, whereas, conversely, a ceDNA vector has complementary strands but is a single DNA molecule and thus remains a single molecule even when denatured. In some embodiments, the ceDNA vectors described herein, unlike plasmids, can be produced without DNA base methylation of prokaryotic cell types. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular linear vs. circular) and the methods used to produce and purify these different objects (see below), as well as in terms of their DNA methylation, which in the case of ceDNA-plasmids is of prokaryotic cell type and in the case of ceDNA vectors is of eukaryotic cell type.

[0194] There are several advantages of using ceDNA vectors for the expression of the PAH proteins described herein over plasmid-based expression vectors, including but not limited to the following: 1) Plasmids contain bacterial DNA sequences and are subject to prokaryotic cell-specific methylation, e.g., 6-methyladenosine and 5-methylcytosine methylation, whereas capsid-free AAV vector sequences are of eukaryotic origin and are not subject to prokaryotic cell-specific methylation, and as a result, capsid-free AAV vectors are less likely to induce inflammation and immune responses compared to plasmids. 2) Plasmids require the presence of resistance genes during the production process, whereas ceDNA vectors do not. 3) Circular plasmids are not delivered to the nucleus upon introduction into cells and require overloading to bypass degradation by cellular nucleases, whereas ceDNA vectors contain viral cis elements, i.e., ITRs, that confer resistance to nucleases and can be designed to be targeted and delivered to the nucleus. We hypothesize that the minimal defining elements essential for ITR function are a Rep binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and a terminal resolution site (TRS, 5'-AGTTGG-3' (SEQ ID NO: 64) for AAV2), plus a variable palindromic sequence that allows hairpin formation. 4) ceDNA vectors do not have the over-representation of CpG dinucleotides often found in prokaryotic cell-derived plasmids that reportedly bind members of the Toll-like family of receptors and elicit T cell-mediated immune responses. In contrast, transduction with the capsid-free AAV vectors disclosed herein can use a variety of delivery reagents and efficiently target cells and tissue types that are difficult to transduce with conventional AAV virions.

[0195] IV. Inverted terminal repeat (ITR) As disclosed herein, the ceDNA vector for expressing PAH protein comprises a transgene or heterologous nucleic acid sequence located between two inverted terminal repeat (ITR) sequences, and the ITR sequence can be an asymmetric ITR pair or a symmetric or substantially symmetric ITR pair, as these terms are defined herein. The ceDNA vector disclosed herein can comprise an ITR sequence selected from any of the following: (i) at least one WT ITR and at least one modified AAV inverted terminal repeat (mod-ITR) (e.g., asymmetric modified ITR), (ii) two modified ITRs (e.g., asymmetric modified ITR) in which the mod-ITR pair has a different three-dimensional spatial configuration relative to each other, or (iii) a symmetric or substantially symmetric WT-WT ITR pair in which each WT-ITR has the same three-dimensional spatial configuration, or (iv) a symmetric or substantially symmetric modified ITR pair in which each mod-ITR has the same three-dimensional spatial configuration, and the method of the present disclosure can further comprise a delivery system, such as, but not limited to, a liposomal nanoparticle delivery system.

[0196] In some embodiments, the ITR sequences may be derived from viruses of the Parvoviridae family, which includes two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. The Parvovirinae subfamily (referred to as parvoviruses) includes the Dependoviruses, whose members require co-infection with a helper virus, such as an adenovirus or a herpesvirus, for productive infection under most conditions. The Dependovirus family includes the adeno-associated viruses (AAVs), which normally infect humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), as well as related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). Parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, "Parvoviridae: The Viruses and Their Replication," Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).

[0197] ITRs are illustrated in the specification, and an example herein is the AAV2 WT-ITR, however, one of skill in the art will recognize that ITRs from any known parvovirus, e.g., dependovirus, e.g., AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes, e.g., NCBI: NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261), chimeric ITRs, or ITRs from any synthetic AAV, as described above. In some embodiments, AAV can infect warm-blooded animals, such as avian (AAAV), bovine (BAAV), canine, equine, and ovine adeno-associated viruses. In some embodiments, the ITRs are derived from B19 parvovirus (GenBank Accession No. NC 000883), minute virus of mice (MVM) (GenBank Accession No. NC 001510), goose parvovirus (GenBank Accession No. NC 001701), snake parvovirus 1 (GenBank Accession No. NC 006148). In some embodiments, the 5'WT-ITR is derived from one serotype and the 3'WT-ITR is derived from a different serotype, as discussed herein.

[0198] The skilled artisan knows that ITR sequences have the general structure of a double-stranded Holliday junction, typically a T-shaped or Y-shaped hairpin structure (see, for example, Figures 2A and 3A), with each WT-ITR formed by two palindromic arms or loops (B-B' and C-C') embedded in a larger palindromic arm (A-A'), and a single-stranded D sequence (the order of these palindromic sequences defines the flip or flop orientation of the ITR). See, for example, the structural analysis and sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6) and described in Grimm et al., J. Virology, 2006; 80(1); 426-439, Yan et al., J. Virology, 2005; 364-379, Duan et al., Virology 1999; 261; 8-14. Based on the exemplary AAV2 ITR sequences provided herein, one skilled in the art can easily determine the WT-ITR sequence from any AAV serotype for use in a ceDNA vector or ceDNA-plasmid. See, for example, the sequence comparison of ITRs from different AAV serotypes (AAV1-AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006; 80(1); 426-439. This shows the percent identity of the left ITR of AAV2 to the left ITRs from other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%).

[0199] A. Symmetric ITR Pair In some embodiments, a ceDNA vector for expression of a PAH protein described herein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other, i.e., the ceDNA vector may comprise ITR sequences having a symmetrical three-dimensional spatial configuration, whereby their structures are the same shape in geometric space or have the same A, C-C', B-B' loops in three-dimensional space. In such embodiments, the symmetrical ITR pair or the substantially symmetrical ITR pair may be a modified ITR (e.g., mod-ITR) that is not a wild-type ITR. A mod-ITR pair may have the same sequence that has one or more modifications from the wild-type ITR and is the reverse complement (inversion) of each other. In alternative embodiments, the modified ITR pair is substantially symmetric as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetric three-dimensional shapes.

[0200] (i) Wild-type ITR In some embodiments, the symmetric or substantially symmetric ITR is wild type (WT-ITR) as described herein. That is, both ITRs have wild type sequences, but are not necessarily WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR can be derived from one AAV serotype, and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetric as defined herein, that is, they can have one or more conservative nucleotide modifications while maintaining a symmetric three-dimensional spatial configuration.

[0201] Thus, as disclosed herein, a ceDNA vector comprises a transgene or heterologous nucleic acid sequence located between two adjacent wild-type inverted terminal repeat (WT-ITR) sequences, which are reverse-complementary (inverted) to each other, or alternatively are substantially symmetrical to each other. That is, the WT-ITR pair has a symmetrical three-dimensional spatial configuration. In some embodiments, the wild-type ITR sequence (e.g., AAV WT-ITR) comprises a functional Rep binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3' for AAV2, SEQ ID NO: 60) and a functional terminal resolution site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 62).

[0202] In one aspect, a ceDNA vector for expression of a PAH protein can be obtained from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two WT inverted terminal repeats (WT-ITRs) (e.g., AAV WT-ITRs). That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs of the same AAV serotype. That is, in some embodiments, one WT-ITR can be derived from one AAV serotype and the other WT-ITR can be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they can have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration. In some embodiments, the 5'WT-ITR is derived from one AAV serotype and the 3'WT-ITR is derived from the same or different AAV serotype. In some embodiments, the 5'WT-ITR and the 3'WT-ITR are mirror images of each other, i.e., they are symmetrical. In some embodiments, the 5'WT-ITR and the 3'WT-ITR are from the same AAV serotype.

[0203] WT ITR is well known. In one embodiment, the two ITRs are from the same AAV2 serotype. In certain embodiments, WT from other serotypes can be used. There are several serotypes that are homologous, such as AAV2, AAV4, AAV6, AAV8. In one embodiment, closely homologous ITRs (e.g., ITRs with similar loop structures) can be used. In another embodiment, more diverse AAV WT ITRs can be used, such as AAV2 and AAV5, and in yet another embodiment, an ITR that is substantially WT can be used, i.e., it has the basic loop structure of WT, but has some conservative nucleotide changes that do not change or affect the properties. When using WT-ITR from the same virus serotype, one or more regulatory sequences can be further used. In certain embodiments, the regulatory sequence is a regulatory switch that allows the activity of ceDNA, such as the expression of the encoded PAH protein, to be adjusted.

[0204] In some embodiments, one aspect of the technology described herein relates to a ceDNA vector for expression of a PAH protein, the ceDNA vector comprises at least one heterologous nucleotide sequence encoding a PAH protein operably positioned between two wild-type inverted terminal repeats (WT-ITRs), the WT-ITRs may be from the same serotype, different serotypes, or may be substantially symmetrical with respect to each other (i.e., have a symmetrical three-dimensional spatial organization such that their structures are the same shape in geometric space, or have the same A, C-C', and B-B' loops in three-dimensional space). In some embodiments, the symmetric WT-ITRs comprise a functional terminal resolution site and a Rep binding site. In some embodiments, the heterologous nucleic acid sequence encodes a transgene, and the vector is not in a viral capsid.

[0205] In some embodiments, the WT-ITRs are the same but are reverse complements of each other. For example, the sequence AACG of the 5'ITR can be CGTT (i.e., the reverse complement) of the 3'ITR of the corresponding site. In one example, the 5'WT-ITR sense strand comprises a sequence of ATCGATCG and the corresponding 3'WT-ITR sense strand comprises CGATCGAT (i.e., the reverse complement of ATCGATCG). In some embodiments, the WT-ITR ceDNA further comprises a terminal resolution site and a replication protein binding site (RPS) (sometimes referred to as a replication protein binding site), e.g., a Rep binding site.

[0206] Exemplary WT-ITR sequences for use in ceDNA vectors for expression of PAH proteins containing WT-ITRs are shown in Table 3 herein, which shows the WT-ITR pairs (5'WT-ITR and 3'WT-ITR).

[0207] As an illustrative example, the present disclosure provides a ceDNA vector for expression of a PAH protein comprising a promoter, with or without a regulatory switch, operably linked to a transgene (e.g., a heterologous nucleic acid sequence), wherein the ceDNA lacks a capsid protein, (a) is produced from a ceDNA-plasmid (see, e.g., Figures 1F-1G) encoding WT-ITRs, each WT-ITR having the same number of intramolecularly duplexed base pairs in its hairpin secondary configuration (preferably excluding any AAA or TTT terminal loop deletions in this configuration compared to these reference sequences), and (b) is identified as ceDNA using the assay for identification of ceDNA by agarose gel electrophoresis under native and denaturing conditions of Example 1.

[0208] In some embodiments, adjacent WT-ITRs are substantially symmetrical to each other.In this embodiment, 5'WT-ITR can be derived from one serotype of AAV, and 3'WT-ITR can be derived from a different serotype of AAV, so that WT-ITRs are not identical reverse complements.For example, 5'WT-ITR can be derived from AAV2, and 3'WT-ITR can be derived from a different serotype (e.g., AAV1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12). In some embodiments, the WT-ITRs may be selected from two different parvoviruses selected from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. In some embodiments, such a combination of WT ITRs is a combination of WT-ITRs from AAV2 and AAV6. In one embodiment, the substantially symmetric WT-ITRs are at least 90% identical, at least 95% identical, at least 96%...97%...98%...99%...99.5%, and all points in between, when one is inverted relative to the other ITR, and have the same symmetric three-dimensional spatial configuration. In some embodiments, the WT-ITR pair is substantially symmetric because they have a symmetric three-dimensional spatial organization, e.g., have the same three-dimensional organization of the A, C-C', B-B', and D arms. In one embodiment, a substantially symmetric WT-ITR pair is inverted with respect to the other and is at least 95% identical to each other, at least 96%...97%...98%...99%...99.5%, and all points in between, where one WT-ITR retains a Rep binding site (RBS) and terminal resolution site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60).In some embodiments, the substantially symmetric WT-ITR pair is inverted relative to each other and is at least 95% identical to each other, at least 96%...97%...98%...99%...99.5%, and all points in between, where one WT-ITR retains a Rep binding site (RBS) and terminal resolution site (trs) of 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. Homology can be determined by standard means well known in the art, such as BLAST (Basic Local Alignment Search Tool), BLASTN with default settings.

[0209] In some embodiments, the structural element of the ITR can be any structural element involved in the functional interaction of the ITR with a large Rep protein (e.g., Rep 78 or Rep 68). In certain embodiments, the structural element provides selectivity for the interaction of the ITR with a large Rep protein, i.e., determines, at least in part, which Rep protein functionally interacts with the ITR. In other embodiments, the structural element physically interacts with the large Rep protein when the Rep protein is bound to the ITR. Each structural element can be, for example, the secondary structure of the ITR, the nucleotide sequence of the ITR, the space between two or more elements, or any combination of the above. In one embodiment, the structural element is selected from the group consisting of A and A' arms, B and B' arms, C and C' arms, D arms, Rep binding sites (RBE) and RBE' (i.e., complementary RBE sequences), and terminal resolution sites (trs).

[0210] By way of example only, Table 2 shows exemplary combinations of WT-ITR.

[0211] Table 2: Exemplary combinations of WT-ITRs from the same or different serotypes, or different parvoviruses. The order shown does not indicate the ITR positions, e.g., "AAV1, AAV2" specifies that the ceDNA may contain WT-AAV1 ITRs at the 5' position and WT-AAV2 ITRs at the 3' position, or vice versa, WT-AAV2 ITRs at the 5' position and WT-AAV1 ITRs at the 3' position. Abbreviations: AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12), AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genomes (e.g., from NCBI: NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261), ITRs from warm-blooded animals (avian AAV (AAAV), bovine AAV (BAAV), canine, equine, and ovine AAV), ITRs from B19 parvovirus (GenBank accession number: NC000883), minute virus from mice (MVM) (GenBank accession number NC001510), goose: goose parvovirus (GenBank accession number: NC 001701), snake: snake parvovirus 1 (GenBank accession number NC 006148). [Table 2-1] [Table 2-2] [Table 2-3]

[0212] By way of example only, Table 3 shows the sequences of exemplary WT-ITRs from several different AAV serotypes. [Table 3]

[0213] In some embodiments, the nucleotide sequence of the WT-ITR sequence may be modified (e.g., by modifying 1, 2, 3, 4 or 5 or more nucleotides, or any range therein), whereby the modification is a substitution of a complementary nucleotide, e.g., G for C and vice versa, T for A and vice versa.

[0214] In certain embodiments of the invention, the ceDNA vector for expression of a PAH protein does not have a WT-ITR consisting of a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14. In alternative embodiments of the invention, when the ceDNA vector has a WT-ITR comprising a nucleotide sequence selected from any of SEQ ID NOs: 1, 2, 5-14, the adjacent ITR is also WT and the ceDNA comprises a regulatory switch, e.g., as disclosed herein and in International Application No. PCT / US18 / 49996 (see, e.g., Table 11 of PCT / US18 / 49996, which is incorporated herein by reference in its entirety). In some embodiments, the ceDNA vector for expression of a PAH protein comprises a regulatory switch as disclosed herein and a selected WT-ITR having a nucleotide sequence selected from any of the group consisting of SEQ ID NOs: 1, 2, 5-14.

[0215] The ceDNA vector for expression of PAH proteins described herein may comprise a WT-ITR structure that retains an operable RBE, trs, and RBE' portion. Figures 2A and 2B, using the wild-type ITR for illustrative purposes, show one possible mechanism for the operation of the trs site within the wild-type ITR structure portion of the ceDNA vector. In some embodiments, the ceDNA vector for expression of PAH proteins comprises one or more functional WT-ITR polynucleotide sequences that include a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one WT-ITR is functional. In an alternative embodiment where the ceDNA vector for expression of PAH proteins comprises two WT-ITRs that are substantially symmetrical to each other, at least one WT-ITR is functional and at least one WT-ITR is non-functional.

[0216] B. Common modified ITRs (mod-ITRs) of ceDNA vectors containing asymmetric or symmetric ITR pairs As discussed herein, the ceDNA vector for expression of a PAH protein may contain a symmetric ITR pair or an asymmetric ITR pair. In either case, one or both ITRs may be modified ITRs, the difference being that in the first case (i.e., symmetric mod-ITR), the mod-ITRs have the same three-dimensional spatial organization (i.e., have the same A-A', C-C', and B-B' arm organization), whereas in the second case (i.e., asymmetric mod-ITR), the mod-ITRs have a different three-dimensional spatial organization (i.e., have a different organization of A-A', C-C', and B-B' arms).

[0217] In some embodiments, the modified ITR is an ITR that is modified by deletion, insertion, and / or substitution compared to a wild-type ITR sequence (e.g., AAV ITR). In some embodiments, at least one of the ITRs of the ceDNA vector comprises a functional Rep binding site (RBS, e.g., 5'-GCGCGCTCGCTCGCTC-3', SEQ ID NO: 60 for AAV2) and a functional terminal resolution site (TRS, e.g., 5'-AGTT-3', SEQ ID NO: 62). In one embodiment, at least one of the ITRs is a non-functional ITR. In one embodiment, each of the different or modified ITRs is not a wild-type ITR from a different serotype.

[0218] Although certain alterations and mutations of ITRs are described in detail herein, in the context of ITRs, "altered" or "mutated" or "modified" refers to nucleotides that have been inserted, deleted, and / or substituted relative to the wild-type, reference, or original ITR sequence. An altered or mutant ITR may be an engineered ITR. As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polypeptide is considered to be "engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been engineered by the hand of man and is different from the aspect it occurs in nature.

[0219] In some embodiments, the mod-ITR can be synthetic. In one embodiment, the synthetic ITR is based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITR does not contain AAV base sequences. In yet another embodiment, the synthetic ITR preserves the ITR structure described above, but has little or no AAV source sequence. In some aspects, the synthetic ITR can preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, is not recognized by wild-type Rep, but only by mutant Rep.

[0220] Those skilled in the art can determine the corresponding sequence in other serotypes by known means. For example, determine whether there is a change in the A, A', B, B', C, C', or D region, and determine the corresponding region in another serotype. BLAST® (Basic Local Alignment Search Tool) or other homology alignment programs can be used in default settings to determine the corresponding sequence. The present invention further provides a collection and a plurality of ceDNA vectors that include mod-ITRs from different AAV serotype combinations. That is, one mod-ITR can be from one AAV serotype and another mod-ITR can be from a different serotype. Without being bound by theory, in one embodiment, one ITR may be derived from or based on the AAV2 ITR sequence and the other ITR of the ceDNA vector may be derived from or based on any one or more ITR sequences of AAV serotype 1 (AAV1), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV11), or AAV serotype 12 (AAV12).

[0221] Any parvovirus ITR can be used for modification as ITR or as base ITR. Preferably, the parvovirus is a dependovirus. More preferably, it is AAV. The serotype selected can be based on the tissue tropism of the serotype. AAV2 has a wide tissue tropism, AAV1 preferentially targets neurons and skeletal muscle, and AAV5 targets neurons, retinal pigment epithelium, and photoreceptors. AAV6 preferentially targets skeletal muscle and lung. AAV8 preferentially targets liver, skeletal muscle, heart, and pancreatic tissue. AAV9 preferentially targets liver, skeletal, and lung tissue. In one embodiment, the modified ITR is based on AAV2 ITR.

[0222] More specifically, the ability of a structural element to functionally interact with a particular large Rep protein can be altered by modifying the structural element. For example, the nucleotide sequence of the structural element can be modified compared to the wild-type sequence of the ITR. In one embodiment, the structural elements of the ITR (e.g., A arm, A' arm, B arm, B' arm, C arm, C' arm, D arm, RBE, RBE', and trs) can be removed and replaced with wild-type structural elements from a different parvovirus. For example, the replacement structure can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, snake parvovirus (e.g., royal python parvovirus), bovine parvovirus, caprine parvovirus, avian parvovirus, canine parvovirus, equine parvovirus, shrimp parvovirus, porcine parvovirus, or insect AAV. For example, the ITRs can be AAV2 ITRs and the A or A' arms or RBE can be replaced with structural elements from AAV5. In another embodiment, the ITRs can be AAV5 ITRs and the C or C' arms, RBE, and trs can be replaced with structural elements from AAV2. In another embodiment, the AAV ITRs can be AAV5 ITRs and the B and B' arms are replaced with the AAV2 ITR B and B' arms.

[0223] By way of example only, Table 4 shows exemplary modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide in a region of a modified ITR, where X indicates a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleic acid in that section relative to the corresponding wild-type ITR. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any of the C and / or C' and / or B and / or B' regions retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. For example, if the modification results in either a single-arm ITR (e.g., a single C-C' arm, or a single B-B' arm), or a modified C-B' arm or C'-B arm, or a two-arm ITR with at least one truncated arm (e.g., a truncated C-C' arm and / or a truncated B-B' arm), at least the single arm, or at least one of the arms of the two-arm ITR (one arm may be truncated), retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In some embodiments, the truncated C-C' arm and / or the truncated B-B' arm have three consecutive T nucleotides (i.e., TTT) in the terminal loop.

[0224] Table 4: Exemplary combinations of modifications (e.g., deletions, insertions, and / or substitutions) of at least one nucleotide for different B-B' and C-C' regions or arms of the ITR (X indicates a nucleotide modification, e.g., addition, deletion, or substitution of at least one nucleotide in the region). [Table 4]

[0225] In some embodiments, a mod-ITR for use in a ceDNA vector for expression of a PAH protein comprises an asymmetric ITR pair or a symmetric mod-ITR pair as disclosed herein and may comprise any one of the combinations of modifications shown in Table 4, as well as a modification of at least one nucleotide in any one or more of the regions selected from A' and C, C and C', C' and B, B and B', and B' and A. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the C or C' or B or B' regions still preserves the terminal loop of the stem loop. In some embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' retains three consecutive T nucleotides (i.e., TTT) in at least one terminal loop. In alternative embodiments, any modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide between C and C' and / or B and B' retains three consecutive A nucleotides (i.e., AAA) in at least one terminal loop. In some embodiments, a modified ITR for use herein may include any one of the combinations of modifications shown in Table 4, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in any one or more of the regions selected from A', A, and / or D. For example, in some embodiments, a modified ITR for use herein may include any one of the combinations of modifications shown in Table 4, as well as at least one modification (e.g., deletion, insertion, and / or substitution) in the A region. In some embodiments, a modified ITR for use herein may include any one of the combinations of modifications shown in Table 4, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A' region.In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 4, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the A and / or A' region. In some embodiments, modified ITRs for use herein may include any one of the combinations of modifications shown in Table 4, as well as a modification (e.g., deletion, insertion, and / or substitution) of at least one nucleotide in the D region.

[0226] In one embodiment, the nucleotide sequence of the structural element can be modified (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein) to produce a modified structural element. In one embodiment, specific modifications to the ITRs are exemplified herein (e.g., SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187) or shown in Figures 7A-7B of PCT / US2018 / 064242, filed December 6, 2018 (e.g., SEQ ID NOs: 97-98, 101-103, 105-108, 111-112, 117-134, 545-54 of PCT / US2018 / 064242). In some embodiments, the ITRs may be modified (e.g., by modifying 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides, or any range therein). In other embodiments, the ITR can have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to one of the modified ITRs of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or to the RBE-containing sections of the A-A' arm and the C-C' and B-B' arms as set forth in SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187, or in Tables 2-9 of International Application No. PCT / US18 / 49996 (i.e., SEQ ID NOs: 110-112, 115-190, 200-468), which are incorporated by reference in their entireties.

[0227] In some embodiments, the modified ITR may include, for example, removal or deletion of all of a particular arm, e.g., all or part of the A-A' arm, or all or part of the B-B' arm, or all or part of the C-C' arm, or alternatively, removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs that form the stem of a loop (see, e.g., ITR-21 in Figure 7A of PCT / US2018 / 064242, filed December 6, 2018, which is incorporated by reference in its entirety herein), so long as there is still a final loop capping the stem (e.g., a single arm). In some embodiments, the modified ITR may include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. In some embodiments, the modified ITR may include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm (see, e.g., ITR-1 in Figure 3B or ITR-45 in Figure 7A of PCT / US2018 / 064242, filed December 6, 2018, which is incorporated by reference herein in its entirety). In some embodiments, the modified ITR may include removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the C-C' arm and removal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or more base pairs from the B-B' arm. Any combination of base pair removal is envisioned, e.g., 6 base pairs in the C-C' arm and 2 base pairs in the B-B' arm may be removed. As an illustrative example, Figure 3B shows an exemplary modified ITR having at least seven base pairs deleted from each of the C and C' portions, a substitution of a nucleotide in the loop between the C and C' regions, and a deletion of at least one base pair from each of the B and B' regions, such that the modified ITR comprises two arms with at least one arm (e.g., C-C') being truncated. In some embodiments, the modified ITR also comprises a deletion of at least one base pair from each of the B and B' regions, such that the B-B' arm is also truncated relative to the WT ITR.

[0228] In some embodiments, the modified ITR may have a 1-50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotide deletion relative to the full-length wild-type ITR sequence. In some embodiments, the modified ITR may have a 1-30 nucleotide deletion relative to the full-length WT ITR sequence. In some embodiments, the modified ITR has a 2-20 nucleotide deletion relative to the full-length wild-type ITR sequence.

[0229] In some embodiments, the modified ITRs do not contain any nucleotide deletions in the RBE-containing portions of the A or A' regions so as not to interfere with DNA replication (e.g., binding to the RBE by Rep proteins or nicking at the terminal resolution site). In some embodiments, modified ITRs encompassed for use herein have one or more deletions in the B, B', C, and / or C regions as described herein.

[0230] In some embodiments, a ceDNA vector for expression of a PAH protein comprising a symmetric or asymmetric ITR pair comprises a regulatory switch as disclosed herein and at least one selected modified ITR having a nucleotide sequence selected from any of the group consisting of SEQ ID NOs: 3, 4, 15-47, 101-116, or 165-187.

[0231] In another embodiment, the structure of the structural element may be modified. For example, the structural element may have a change in the height of the stem and / or the number of nucleotides in the loop. For example, the height of the stem may be about 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides or more, or any range therein. In one embodiment, the height of the stem may be about 5 nucleotides to about 9 nucleotides and may functionally interact with Rep. In another embodiment, the height of the stem may be about 7 nucleotides and may functionally interact with Rep. In another example, the loop may have 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or more, or any range therein.

[0232] In another embodiment, the number of GAGY binding sites or GAGY-related binding sites in an RBE or extended RBE can be increased or decreased. In one example, an RBE or extended RBE can contain 1, 2, 3, 4, 5, 6 or more GAGY binding sites, or any range therein. Each GAGY binding site can independently be the exact GAGY sequence or a sequence similar to GAGY, so long as the sequence is sufficient to bind to the Rep protein.

[0233] In another embodiment, the spacing between two elements (such as, but not limited to, an RBE and a hairpin) can be altered (e.g., increased or decreased) to alter functional interactions with a large Rep protein. For example, the spacing can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides or more, or any range therein.

[0234] The ceDNA vector for expression of PAH proteins described herein may contain an ITR structure that is modified with respect to the wild-type AAV2 ITR structure disclosed herein, but still retains an operable RBE, trs, and RBE' portion. Figures 2A and 2B show one possible mechanism for the manipulation of the trs site in the wild-type ITR structure of a ceDNA vector for expression of PAH proteins. In some embodiments, the ceDNA vector for expression of PAH proteins contains one or more functional ITR polynucleotide sequences that include a Rep-binding site (RBS, 5'-GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60) for AAV2) and a terminal resolution site (TRS, 5'-AGTT (SEQ ID NO: 62)). In some embodiments, at least one ITR (wild-type or modified ITR) is functional. In alternative embodiments where the ceDNA vector for expression of PAH proteins contains two modified ITRs that are different or asymmetric from each other, at least one modified ITR is functional and at least one modified ITR is non-functional.

[0235] In some embodiments, the modified ITR (e.g., left or right ITR) of the ceDNA vector for expression of the PAH proteins described herein has a modification in the loop arm, cleavage arm, or spacer. Exemplary sequences of ITRs having modifications in the loop arm, cleavage arm, or spacer are listed in Table 2 (i.e., SEQ ID NOs: 135-190, 200-233), Table 3 (e.g., SEQ ID NOs: 234-263), Table 4 (e.g., SEQ ID NOs: 264-293), Table 5 (e.g., SEQ ID NOs: 294-318 herein), Table 6 (e.g., SEQ ID NOs: 319-468), and Tables 7-9 (e.g., SEQ ID NOs: 101-110, 111-112, 115-134) or Table 10A or 10B (e.g., SEQ ID NOs: 9, 100, 469-483, 484-499) of International Application No. PCT / US18 / 49996, which are incorporated herein by reference in their entirety.

[0236] In some embodiments, the modified ITRs for use in ceDNA vectors for expression of PAH proteins comprising asymmetric ITR pairs or symmetric mod-ITR pairs are selected from any of those set forth in Tables 2, 3, 4, 5, 6, 7, 8, 9, and 10A-10B of International Application No. PCT / US18 / 49996, the entireties of which are incorporated herein by reference, or a combination thereof.

[0237] Additional exemplary modified ITRs for use in ceDNA vectors for expression of PAH proteins, including asymmetric or symmetric mod-ITR pairs in each of the above classes, are shown in Tables 5A and 5B. The predicted secondary structures of the right-modified ITRs in Table 5A are shown in Figure 7A of International Application No. PCT / US2018 / 064242, filed December 6, 2018, and the predicted secondary structures of the left-modified ITRs in Table 5B are shown in Figure 7B of International Application No. PCT / US2018 / 064242, filed December 6, 2018, which is incorporated herein by reference in its entirety.

[0238] Tables 5A and 5B show exemplary right- and left-modified ITRs.

[0239] Table 5A: Exemplary modified right ITRs. These exemplary modified right ITRs can include an RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), a spacer ACTGAGGC (SEQ ID NO: 69), a spacer complement GCCTCAGT (SEQ ID NO: 70), and an RBE' (i.e., a complement to the RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 5A]

[0240] Table 5B: Exemplary Modified Left ITRs These exemplary modified left ITRs can include the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE complement (RBE') GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 5B]

[0241] In one embodiment, the ceDNA vector for expression of a PAH protein comprises, in the 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette as described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with each other, i.e., they have different three-dimensional spatial configurations with each other. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutant or modified ITR, or vice versa, where the first ITR can be a mutant or modified ITR and the second ITR can be a wild-type ITR. In some embodiments, the first ITR and the second ITR are both mod-ITRs, but have different sequences or have different modifications, and thus are not the same modified ITRs, but have different three-dimensional spatial configurations. In other words, ceDNA vectors using asymmetric ITRs contain ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR, or alternatively, the asymmetric ITRs may have different sequences and different three-dimensional shapes relative to each other if they have a modified asymmetric ITR pair. Exemplary asymmetric ITRs for use in generating ceDNA-plasmids in ceDNA vectors for expression of PAH proteins are shown in Tables 5A and 5B.

[0242] In an alternative embodiment, the ceDNA vector for the expression of PAH proteins comprises two symmetric mod-ITRs. That is, both ITRs have the same sequence but are the reverse complements of each other. In some embodiments, the symmetric mod-ITR pair comprises at least one or any combination of deletion, insertion, or substitution compared to the wild-type ITR sequence from the same AAV serotype. The additions, deletions, or substitutions of the symmetric ITRs are the same but are the reverse complements of each other. For example, the insertion of three nucleotides into the C region of the 5'ITR is reflected in the insertion of three reverse complementary nucleotides into the corresponding section of the C' region of the 3'ITR. For illustrative purposes only, if the addition is AACG in the 5'ITR, the addition is CGTT in the 3'ITR at the corresponding site. For example, if the 5'ITR sense strand is ATCGATCG, with the addition of AACG between the G and A, resulting in the sequence ATCGAACGATCG (SEQ ID NO: 51). The corresponding 3'ITR sense strand is CGATCGAT (the reverse complement of ATCGATCG), with the addition of CGTT (i.e., the reverse complement of AACG) between the T and C, resulting in the sequence CGATCGTTCGAT (SEQ ID NO:49) (the reverse complement of ATCGAACGATCG) (SEQ ID NO:51).

[0243] In an alternative embodiment, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair may have different sequences but have corresponding or the same symmetrical three-dimensional shapes. For example, one modified ITR may be derived from one serotype, and the other modified ITR may be derived from a different serotype, but they have the same mutation (e.g., nucleotide insertion, deletion, or substitution) in the same region. In other words, for illustrative purposes only, the 5'mod-ITR may be derived from AAV2 and have a deletion in the C region, and the 3'mod-ITR may be derived from AAV5 and have a corresponding deletion in the C' region. Provided that the 5'mod-ITR and the 3'mod-ITR have the same or symmetrical three-dimensional spatial configuration, they are encompassed in the use of modified ITR pair herein.

[0244] In some embodiments, a substantially symmetric mod-ITR pair has the same A, C-C' and B-B' loops in three-dimensional space. For example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR has a corresponding deletion of the C-C' loop and has a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in the geometric space of the cognate mod-ITR. By way of example only, substantially symmetric ITRs can have a symmetric spatial organization such that their structures are the same shape in geometric space. This can occur, for example, when a GC pair is modified, for example, to a CG pair or vice versa, or when an AT pair is modified to a TA pair or vice versa. Thus, using the above illustrative examples of a modified 5'ITR as ATCGAACGATCG (SEQ ID NO:51) and a modified 3'ITR as CGATCGTTCGAT (SEQ ID NO:49) (i.e., the reverse complement of ATCGAACGATCG (SEQ ID NO:51)), if, for example, the 5'ITR had a sequence of ATCGAACCATCG (SEQ ID NO:50) (with the addition of G modified to C), these modified ITRs would still be symmetrical and a substantially symmetrical 3'ITR would have a sequence of CGATCGTTCGAT (SEQ ID NO:49) without the addition of T to the corresponding a. In some embodiments, such modified ITRs are substantially symmetrical because the modified ITR pair has symmetric stereochemistry.

[0245] Table 6 shows exemplary symmetrically modified ITR pairs (i.e., left-modified ITR and symmetrically right-modified ITR) for use in ceDNA vectors for expression of PAH proteins. The bold (red) portions of the sequences identify the partial ITR sequences (i.e., the sequences of the A-A', C-C', and B-B' loops). These exemplary modified ITRs can include the RBE GCGCGCTCGCTCGCTC-3' (SEQ ID NO: 60), the spacer ACTGAGGC (SEQ ID NO: 69), the spacer complement GCCTCAGT (SEQ ID NO: 70), and the RBE' (i.e., the complement to the RBE) GAGCGAGCGAGCGCGC (SEQ ID NO: 71). [Table 6-1] [Table 6-2] [Table 6-3]

[0246] In some embodiments, a ceDNA vector for expression of a PAH protein comprising an asymmetric ITR pair may comprise an ITR having an ITR sequence or ITR subsequence set forth in any one or more of Tables 5A-5B herein, or a modification corresponding to any of the modifications in the sequences shown in Figures 7A-7B of International Application No. PCT / US2018 / 064242, filed December 6, 2018, which is incorporated herein in its entirety, or disclosed in Tables 2, 3, 4, 5, 6, 7, 8, 9, or 10A-10B of International Application No. PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety.

[0247] V. Exemplary ceDNA Vectors As described above, the present disclosure relates to recombinant ceDNA expression vectors and ceDNA vectors encoding PAH proteins comprising any one of the above-mentioned asymmetric ITR pairs, symmetric ITR pairs, or substantially symmetric ITR pairs. In certain embodiments, the present disclosure relates to recombinant ceDNA vectors for expressing PAH proteins having adjacent ITR sequences and transgenes, the ITR sequences being asymmetric, symmetric, or substantially symmetric with respect to each other as defined herein, and the ceDNA further comprises a nucleotide sequence of interest (e.g., an expression cassette comprising the nucleic acid of the transgene) located between the adjacent ITRs, and the nucleic acid molecule lacks a viral capsid protein coding sequence.

[0248] The ceDNA expression vector for expressing a PAH protein may be any ceDNA vector that can be conveniently subjected to recombinant DNA procedures, including the nucleotide sequence described herein, if at least one ITR is modified. The ceDNA vector for expressing a PAH protein of the present disclosure is compatible with the host cell into which the ceDNA vector is introduced. In certain embodiments, the ceDNA vector may be linear. In certain embodiments, the ceDNA vector may exist as an extrachromosomal entity. In certain embodiments, the ceDNA vector of the present disclosure may include elements that allow the integration of the donor sequence into the genome of the host cell. As used herein, "transgene" and "heterologous nucleotide sequence" are synonymous and encode a PAH protein, as described herein.

[0249] Referring now to Figures 1A-1G, there is shown a schematic diagram of the functional components of two non-limiting plasmids useful for making a ceDNA vector for expression of a PAH protein. Figures 1A, 1B, 1D, 1F show the construct of a ceDNA vector for expression of a PAH protein or the sequence of the corresponding ceDNA plasmid. The ceDNA vector can be obtained from a plasmid that does not contain a capsid and that encodes in this order a first ITR, an expressible transgene cassette, and a second ITR, where the first and second ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. The ceDNA vector for expression of a PAH protein can be obtained from a plasmid that does not contain a capsid and that encodes in this order a first ITR, an expressible transgene (protein or nucleic acid), and a second ITR, where the first and second ITR sequences are asymmetric, symmetric, or substantially symmetric with respect to each other, as defined herein. In some embodiments, the expressible transgene cassette optionally includes an enhancer / promoter, one or more homology arms, a donor sequence, a post-transcriptional regulatory element (e.g., a WPRE, e.g., SEQ ID NO:67), and a polyadenylation and termination signal (e.g., a BGH polyA, e.g., SEQ ID NO:68).

[0250] Figure 5 is a gel confirming the production of ceDNA from multiple ceDNA plasmid constructs using the methods described in the Examples. The ceDNA is confirmed by the characteristic banding pattern in the gel, as discussed above with respect to Figure 4A and in the Examples.

[0251] A. Regulatory Elements The ceDNA vector for expression of the PAH protein described herein, including asymmetric or symmetric ITR pairs as defined herein, may further include a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. Exemplary promoters are listed in International Application No. PCT / US2020 / 021328, e.g., Table 7, which is incorporated herein by reference in its entirety. Exemplary enhancers are listed in International Application No. PCT / US2020 / 021328, e.g., Table 8, which is incorporated herein by reference in its entirety. In some embodiments, the ITRs may act as a promoter of a transgene, e.g., a PAH protein. In some embodiments, the ceDNA vector for expression of the PAH protein described herein includes additional components for regulating the expression of the transgene, e.g., a regulatory switch as described herein, to regulate the expression of the transgene, or a kill switch that may kill a cell containing the ceDNA vector encoding the PAH protein. Regulatory elements, including regulatory switches, that may be used in the present invention are discussed more fully in International Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0252] In an embodiment, the second nucleotide sequence comprises a regulatory sequence and a nucleotide sequence encoding a nuclease. In certain embodiments, the gene regulatory sequence is operably linked to the nucleotide sequence encoding the nuclease. In certain embodiments, the regulatory sequence is suitable for controlling the expression of the nuclease in the host cell. In certain embodiments, the regulatory sequence comprises a suitable promoter sequence capable of directing the transcription of a gene operably linked to a promoter sequence, such as the nucleotide sequence encoding the nuclease of the present disclosure. In certain embodiments, the second nucleotide sequence comprises an intron sequence linked to the 5' end of the nucleotide sequence encoding the nuclease. In certain embodiments, an enhancer sequence is provided upstream of the promoter to increase the efficacy of the promoter. In certain embodiments, the regulatory sequence comprises an enhancer and a promoter, the second nucleotide sequence comprises an intron sequence upstream of the nucleotide sequence encoding the nuclease, the intron comprises one or more nuclease cleavage sites, and the promoter is operably linked to the nucleotide sequence encoding the nuclease.

[0253] ceDNA vectors for expression of PAH proteins produced synthetically or using cell-based production methods as described in the Examples herein may further include certain combinations of cis-regulatory elements such as the WHP post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) and BGH polyA (SEQ ID NO: 68). Suitable expression cassettes for use in expression constructs are not limited by packaging constraints imposed by the viral capsid.

[0254] (i) Promoter: One of skill in the art will understand that promoters used in the ceDNA vectors for expression of the PAH proteins disclosed herein should be appropriately tailored to the particular sequence that they promote.

[0255] The expression cassette of the ceDNA vector for the expression of PAH proteins may contain a promoter that can affect cell specificity as well as the overall expression level. In the case of transgene expression, for example, expression of PAH proteins, they may contain a highly active virus-derived immediate early promoter. The expression cassette may contain a tissue-specific eukaryotic promoter to restrict transgene expression to a specific cell type and reduce toxic effects and immune reactions resulting from unregulated ectopic expression. In some embodiments, the expression cassette may contain a promoter or synthetic regulatory element such as the CAG promoter (SEQ ID NO: 72). The CAG promoter contains (i) a cytomegalovirus (CMV) early enhancer element, (ii) a promoter, the first exon and first intron of the chicken β-actin gene, and (iii) a splice acceptor of the rabbit β-globin gene. Alternatively, the expression cassette may include an alpha-1-antitrypsin (AAT) promoter (SEQ ID NO: 73 or SEQ ID NO: 74), a liver-specific (LP1) promoter (SEQ ID NO: 75 or SEQ ID NO: 76), or a human elongation factor-1 alpha (EF1a) promoter (e.g., SEQ ID NO: 77 or SEQ ID NO: 78). In some embodiments, the expression cassette includes one or more constitutive promoters, such as a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with an RSV enhancer), or a cytomegalovirus (CMV) immediate early promoter (optionally with a CMV enhancer, e.g., SEQ ID NO: 79). Alternatively, an inducible promoter, a native promoter of the transgene, a tissue-specific promoter, or various promoters known in the art can be used. According to some embodiments, the promoter is any promoter or promoter sequence set forth in International Application No. PCT / US2020 / 021328, filed March 6, 2020, which is incorporated herein by reference in its entirety.

[0256] According to some embodiments, the promoter is a VandenDriessche (VD) promoter. According to some embodiments, the VD promoter comprises SEQ ID NO: 191, as shown below. CCGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGATACTCTAATCTCCCTAGGCAAGGTTCATATTTGTGTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGCTCCTG (SEQ ID NO: 191) According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 85% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 90% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 95% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 96% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 97% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 98% identical to SEQ ID NO: 191. According to some embodiments, the promoter comprises a nucleic acid sequence that is at least about 99% identical to SEQ ID NO: 191. According to some embodiments, the promoter consists of the nucleic acid sequence of SEQ ID NO: 191.

[0257] Suitable promoters may be derived from viruses and therefore may be referred to as viral promoters, or they may be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter, such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 micronucleus promoter (U6, e.g., SEQ ID NO: 80) (Miyagishi et al., J. Clin. Imaging, 2002, 143:1311-1323, 2002). al., Nature Biotechnology 20,497-500(2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep.1;31(17)), human H1 promoter (H1) (e.g., SEQ ID NO:81 or SEQ ID NO:155), CAG promoter, human alpha1-antitypsin (HAAT) promoter (e.g., SEQ ID NO:82), and the like. In certain embodiments, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In certain embodiments, the DNA containing the nuclease cleavage sites is foreign to the promoter DNA.

[0258] In one embodiment, the promoter used is the native promoter of the gene that codes for therapeutic protein.The promoter and other regulatory sequences of each gene that codes for therapeutic protein are known and characterized.The promoter region used can further include one or more additional regulatory sequences (e.g., native), such as enhancers (e.g., SEQ ID NO:79 and SEQ ID NO:83), including SV40 enhancer (SEQ ID NO:126).

[0259] In some embodiments, the promoter may be a promoter from a human gene, such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue-specific promoter, such as a liver-specific promoter, such as human alpha 1-antitrypsin (HAAT). In one embodiment, delivery to the liver may be achieved using endogenous ApoE-specific targeting of the composition comprising the ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0260] Non-limiting examples of suitable promoters for use in accordance with the present invention include, for example, the CAG promoter (SEQ ID NO: 72), the HAAT promoter (SEQ ID NO: 82), the human EF1-alpha promoter (SEQ ID NO: 77), or the EF1a promoter (SEQ ID NO: 78), the IE2 promoter (e.g., SEQ ID NO: 84), and any of the rat EF1-alpha promoter (SEQ ID NO: 85), the mEF1 promoter (SEQ ID NO: 59), or the 1E1 promoter fragment (SEQ ID NO: 125).

[0261] (ii) Enhancer In some embodiments, the ceDNA expressing the PAH comprises one or more enhancers. In some embodiments, the enhancer sequence is located 5' of the promoter sequence. In some embodiments, the enhancer sequence is located 3' of the promoter sequence. According to some embodiments, the enhancer is any enhancer or enhancer sequence shown in International Application No. PCT / US2020 / 021328, filed March 6, 2020, which is incorporated herein by reference in its entirety.

[0262] (iii) Exemplary 5'UTR and Intron Sequences In some embodiments, the ceDNA vector comprises an intron sequence located 3' of the 5'UTR sequence and / or the 5'ITR sequence.In some embodiments, the 5'UTR is located 5' of the sequence encoding the transgene, for example, the PAH protein.Exemplary 5'UTR sequences are listed in International Application No. PCT / US2020 / 021328, for example, Table 9A, which is incorporated herein by reference in its entirety.

[0263] (iv) 3'UTR sequence In some embodiments, the ceDNA vector comprises a 3'UTR sequence located 5' of the 3'ITR sequence.In some embodiments, the 3'UTR is located 3' of the sequence encoding the transgene, for example, the PAH protein.Exemplary 3'UTR sequences are listed in International Application No. PCT / US2020 / 021328, for example, Table 9B, which is incorporated herein by reference in its entirety.

[0264] (v) polyadenylation sequence A sequence encoding a polyadenylation sequence may be included in the ceDNA vector for expression of a PAH protein to stabilize the mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment, the ceDNA vector does not include a polyadenylation sequence. In other embodiments, the ceDNA vector for expression of a PAH protein includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments, the polyadenylation sequence includes about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0265] The expression cassette can include any polyadenylation sequence known in the art or a variation thereof. In some embodiments, the polyadenylation (polyA) sequence is selected from any of those listed in International Application No. PCT / US2020 / 021328, for example, Table 10, which is incorporated herein by reference in its entirety. Other polyA sequences commonly known in the art can also be used, including, but not limited to, naturally occurring sequences isolated from bovine BGHpA (e.g., SEQ ID NO: 68) or viral SV40pA (e.g., SEQ ID NO: 86), or synthetic sequences (e.g., SEQ ID NO: 87). Some expression cassettes can also include an SV40 late polyA signal upstream enhancer (USE) sequence. In some embodiments, the USE sequence can be used in combination with SV40pA or a heterologous polyA signal. The polyA sequence is located 3' of the transgene encoding the PAH protein.

[0266] The expression cassette may also include post-transcriptional elements to increase expression of the transgene. In some embodiments, the Woodchuck Hepatitis Virus (WHP) post-transcriptional regulatory element (WPRE) (e.g., SEQ ID NO: 67) is used to increase expression of the transgene. Other post-transcriptional processing elements can be used, such as the post-transcriptional elements from the thymidine kinase gene of Herpes Simplex Virus or Hepatitis B Virus (HBV). Secretory sequences can be linked to the transgene, such as the VH-02 and VK-A26 sequences, e.g., SEQ ID NO: 88 and SEQ ID NO: 89.

[0267] (vi) Nuclear localization sequence In some embodiments, the ceDNA vector for expression of PAH protein comprises one or more nuclear localization sequences (NLS), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In some embodiments, the one or more NLS is located at or near the amino terminus, at or near the carboxy terminus, or a combination thereof (e.g., one or more NLS at the amino terminus and / or one or more NLS at the carboxy terminus). When two or more NLSs are present, each can be selected independently from the other NLSs, such that a single NLS is present in two or more copies and / or in combination with one or more other NLSs present in one or more copies. Non-limiting examples of NLSs are shown in Table 7. [Table 7]

[0268] B. Additional Components of ceDNA Vectors The ceDNA vector for expression of the PAH proteins of the present disclosure may include nucleotides encoding other components for gene expression. For example, to select a specific gene targeting event, a protective shRNA can be embedded in a microRNA and inserted into a recombinant ceDNA vector designed for site-specific integration into a highly active locus, such as the albumin locus. Such an embodiment may provide a system for in vivo selection and expansion of gene-modified hepatocytes in any genetic background, such as described in Nygaard et al., A universal system to select gene-modified hepatocytes in vivo, Gene Therapy, June 8, 2016. The ceDNA vector of the present disclosure may include one or more selectable markers that allow for the selection of transformed, transfected, transduced, etc. cells. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, NeoR, etc. In certain embodiments, a positive selection marker is incorporated into the donor sequence, such as NeoR. A negative selection marker can be incorporated downstream of the donor sequence, for example, a nucleic acid sequence encoding the negative selection marker, HSV-tk, can be incorporated into the nucleic acid construct downstream of the donor sequence.

[0269] C. Adjustment switch A molecular regulatory switch is one that responds to a signal to produce a measurable change in state. Such regulatory switches can be usefully combined with the ceDNA vectors for expression of PAH proteins described herein to control the output of expression of the PAH protein from the ceDNA vector. In some embodiments, the ceDNA vectors for expression of PAH proteins include regulatory switches that serve to fine-tune the expression of the PAH protein. For example, they can serve as a biological containment function for the ceDNA vector. In some embodiments, the switch is an "on / off" switch designed to start or stop (i.e., shut down) the expression of the PAH protein in the ceDNA vector in a controllable and regulatable manner. In some embodiments, the switch can include a "kill switch" that can instruct a cell containing the ceDNA vector to undergo programmed cell death once the switch is activated. Exemplary regulatory switches included for use in ceDNA vectors for expression of PAH proteins can be used to regulate expression of transgenes and are discussed more fully in International Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0270] (i) Binary Adjustment Switch In some embodiments, the ceDNA vector for expression of PAH protein includes a regulatory switch that can help to controllably adjust the expression of PAH protein. For example, the expression cassette located between the ITRs of the ceDNA vector can additionally include a regulatory region, such as a promoter, a cis-element, a repressor, an enhancer, etc., operably linked to the nucleic acid sequence encoding the PAH protein, which is regulated by one or more cofactors or exogenous agents. By way of example only, the regulatory region can be regulated by a small molecule switch or an inducible or repressible promoter. A non-limiting example of an inducible promoter is a hormone-inducible or metal-inducible promoter. Other exemplary inducible promoter / enhancer elements include, but are not limited to, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

[0271] (ii) Small molecule regulatory switches A variety of art-known small molecule-based regulatory switches are known in the art and can be combined with ceDNA vectors for expression of the PAH proteins disclosed herein to form ceDNA vectors controlled by regulatory switches. In some embodiments, the regulatory switch is an orthogonal ligand / nuclear receptor pair, such as retinoid receptor mutant / LG335 and GRQCIMFI (Taylor, et al. al. BMC Biotechnology 10 (2010): 15), engineered steroid receptors, such as modified progesterone receptors with C-terminal truncations that cannot bind progesterone but bind RU486 (mifepristone) (U.S. Pat. No. 5,364,791), the ecdysone receptor from Drosophila and their ecdysteroid ligands (Saez, et al., PNAS, 97(26) (2000), 14512-14517), or Sando R 3 rd;Nat Methods. 2013,10(11):1085-8. In some embodiments, the regulatory switch for controlling the transgene expressed by the ceDNA vector is a prodrug activation switch, such as those disclosed in U.S. Patent Nos. 8,771,679 and 6,339,070, which are incorporated herein by reference in their entirety.

[0272] (iii) "Passcode" adjustment switch In some embodiments, the regulatory switch may be a "passcode switch" or "passcode circuit." A passcode switch allows fine tuning of the control of expression of a transgene from a ceDNA vector when certain conditions occur. That is, a combination of conditions must be present for expression and / or repression of the transgene to occur. For example, at least conditions A and B must occur for expression of the transgene to occur. A passcode regulatory switch may be any number of conditions, for example, at least 2, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7 or more conditions are present for expression of the transgene to occur. In some embodiments, at least 2 conditions (e.g., A, B conditions) must occur, and in some embodiments, at least 3 conditions must occur (e.g., A, B, and C or A, B, and D). By way of example only, conditions A, B, and C must be present for gene expression from a ceDNA with a passcode "ABC" regulatory switch to occur. Conditions A, B, and C may be as follows: Condition A is the presence of a pathology or disease, Condition B is a hormonal response, and Condition C is a response to expression of the transgene. For example, if the transgene edits a defective EPO gene, condition A is the presence of chronic kidney disease (CKD), condition B occurs when the subject has hypoxia in the kidney, and condition C is defective erythropoietin-producing cell (EPC) mobilization in the kidney or, alternatively, defective HIF-2 activation. Once oxygen levels increase or reach desired EPO levels, the transgene is turned off again and turned back on until the three conditions occur.

[0273] In some embodiments, the passcode regulatory switches or "passcode circuits" included for use in ceDNA vectors include hybrid transcription factors (TFs) to expand the range and complexity of environmental signals used to define biological containment conditions. In contrast to deadman switches, which induce cell death in the presence of predefined conditions, the "passcode circuits" allow cell survival or transgene expression in the presence of specific "passcodes" and can be easily reprogrammed to allow transgene expression and / or cell survival only when a given environmental condition or passcode is present.

[0274] Any and all combinations of the regulatory switches disclosed herein, such as small molecule switches, nucleic acid-based switches, small molecule-nucleic acid hybrid switches, post-transcriptional transgene regulatory switches, post-translational regulatory radiation-controlled switches, hypoxia-mediated switches, and other regulatory switches known to those skilled in the art disclosed herein, can be used in the passcode regulatory switches disclosed herein. Regulatory switches encompassed for use are also discussed in the review article Kis et al., JR Soc Interface. 12:20141000 (2015) and summarized in Table 1 of Kis. In some embodiments, the regulatory switches for use in the passcode system can be selected from any or combinations of the switches disclosed in Table 11 of International Patent Application No. PCT / US18 / 49996, which is incorporated herein by reference in its entirety.

[0275] (iv) a nucleic acid-based regulatory switch for controlling transgene expression In some embodiments, the regulatory switch for controlling the expression of PAH protein by ceDNA is based on a nucleic acid-based control mechanism.Exemplary nucleic acid control mechanisms are known in the art and are envisaged for use.For example, such mechanisms include riboswitches, such as those disclosed in US2009 / 0305253, US2008 / 0269258, US2017 / 0204477, WO2018 / 026762A1, US Patent No. 9,222,093, and European Patent Application No. 288071, as well as those disclosed in Villa JK Such as those disclosed in the review by Friedman et al., Microbiol Spectr. 2018 May;6(3). Also included are metabolite-responsive transcriptional biosensors, such as those disclosed in WO2018 / 075486 and WO2017 / 147585. Other mechanisms known in the art that are envisioned for use include silencing of transgenes by siRNA or RNAi molecules (e.g., miR, shRNA). For example, a ceDNA vector may contain a regulatory switch that encodes an RNAi molecule that is complementary to a portion of a transgene expressed by the ceDNA vector. A transgene (e.g., a PAH protein) is silenced by a complementary RNAi molecule if such an RNAi is expressed even when expressed by a ceDNA vector, and a transgene (e.g., a PAH protein) is not silenced by RNAi if the RNAi is not expressed when the transgene is expressed by a ceDNA vector.

[0276] In some embodiments, the regulatory switch is a tissue-specific self-inactivating regulatory switch, e.g., as disclosed in US2002 / 0022018, whereby the regulatory switch purposefully switches off the transgene (e.g., a PAH protein) at sites where transgene expression would otherwise be detrimental. In some embodiments, the regulatory switch is a recombinase reversible gene expression system, e.g., as disclosed in US2014 / 0127162 and U.S. Patent No. 8,324,436.

[0277] (v) Post-transcriptional and post-translational regulatory switches. In some embodiments, the regulatory switch for controlling the expression of the PAH protein by the ceDNA vector is a post-transcriptional modification system. For example, such regulatory switches can be used as described in US2018 / 0119156, GB2011 / 07768, WO2001 / 064956A3, EP2707487, and Beilstein et al., ACS Synth.Biol., 2015,4(5),pp 526-534, Zhong ... It may be an aptazyme riboswitch that is sensitive to tetracycline or theophylline, as disclosed in et al., Elife. 2016 Nov 2;5.pii:e18858. In some embodiments, it is envisioned that one skilled in the art can code both the transgene and an inhibitory siRNA that contains a ligand-sensitive (off-switch) aptamer, the end result of which is a ligand-sensitive on-switch.

[0278] (vi) Other exemplary adjustable switches Any known regulatory switch can be used in the ceDNA vector to control expression of the PAH protein by the ceDNA vector, including those triggered by environmental changes. Additional examples include Suzuki et al., Scientific Reports 8;10051(2018), genetic code expansion and non-physiological amino acids, radiation- or ultrasound-regulated on / off switches (see, e.g., Scott S et al., Gene Ther. 2000 Jul;7(13):1121-5, U.S. Pat. Nos. 5,612,318, 5,571,797, 5,770,581, 5,817,636, and WO1999 / 025385A1). In some embodiments, the regulatory switch is controlled by an implantable system, e.g., U.S. Pat. No. 7,840,263, US2007 / 0190028A1, and gene expression is controlled by one or more forms of energy, including electromagnetic energy, that activate a promoter operably linked to a transgene in a ceDNA vector.

[0279] In some embodiments, the regulatory switch envisioned for use in the ceDNA vector is a hypoxia-mediated or stress-activated switch, such as those disclosed in WO1999 / 060142A2, U.S. Patent Nos. 5,834,306, 6,218,179, 6,709,858, US2015 / 0322410, Greco et al., (2004) Targeted Cancer Therapies 9, S368, which are incorporated herein by reference in their entirety, as well as FROG, TOAD, and NRSE elements, and conditionally inducible silence elements, including, for example, hypoxia response element (HRE), inflammatory response element (IRE), and shear stress activation element (SSAE), which are disclosed in U.S. Patent No. 9,394,526, which are incorporated herein by reference in their entirety. Such an embodiment is useful for turning on the expression of a transgene from a ceDNA vector after ischemia or in ischemic tissue and / or tumor.

[0280] (vii) Kill Switch Other embodiments described herein relate to ceDNA vectors for expression of PAH proteins as described herein that include a kill switch. The kill switch disclosed herein can cause cells containing the ceDNA vector to be killed or undergo programmed cell death as a means of permanently removing the introduced ceDNA vector from the subject's system. It will be understood by those skilled in the art that the use of a kill switch in a ceDNA vector for expression of a PAH protein is usually coupled with targeting of the ceDNA vector to a limited number of cells that the subject can tolerately lose, or to a cell type (e.g., cancer cells) where apoptosis is desired. In all aspects, the "kill switch" disclosed herein is designed to provide rapid and robust cell killing of cells containing the ceDNA vector in the absence of an input survival signal or other specified condition. In other words, the kill switch encoded by the ceDNA vector for expression of a PAH protein as described herein can restrict cell survival of cells containing the ceDNA vector to an environment defined by a particular input signal. Such a kill switch serves as a biological containment function when it is desirable to remove the ceDNA vector for expression of a PAH protein in a subject, or to ensure that the encoded PAH protein is not expressed.

[0281] Other kill switches known to those of skill in the art are encompassed for use in the ceDNA vectors for expression of the PAH proteins disclosed herein, e.g., those disclosed in US2010 / 0175141, US2013 / 0009799, US2011 / 0172826, US2013 / 0109568, as well as the kill switches disclosed in Jusiak et al, Reviews in Cell Biology and molecular Medicine;2014;1-56, Kobayashi et al., PNAS,2004;101;8419-9, Marchisio et al., Int. Journal of Biochem and Cell Biol.,2011;43;310-319, and Reinshagen et al., Science Translational Medicine,2018,11, the contents of all of which are incorporated by reference in their entireties.

[0282] Thus, in some embodiments, a ceDNA vector for expression of a PAH protein can include a kill switch nucleic acid construct that includes a nucleic acid encoding an effector toxin or a reporter protein, and the expression of the effector toxin (e.g., death protein) or reporter protein is controlled by a predetermined condition. For example, the predetermined condition can be the presence of an environmental substance, such as an exogenous substance, without which the cell will by default express the effector toxin (e.g., death protein) and be killed. In an alternative embodiment, the predetermined condition is the presence of two or more environmental substances, for example, the cell will only survive if two or more required exogenous substances are provided, and in the absence of either, the cell containing the ceDNA vector will be killed.

[0283] In some embodiments, the ceDNA vector for expression of a PAH protein is modified to incorporate a kill switch that destroys cells containing the ceDNA vector, effectively terminating the in vivo expression of the transgene expressed by the ceDNA vector (e.g., expression of a PAH protein). Specifically, the ceDNA vector is further engineered to express a switch protein that does not function in mammalian cells under normal physiological conditions. Only upon administration of a drug or environmental condition that specifically targets this switch protein will the cells expressing the switch protein be destroyed, thereby terminating the expression of the therapeutic protein or peptide. For example, it has been reported that cells expressing HSV-thymidine kinase can be killed upon administration of drugs such as ganciclovir and cytosine deaminase. See, for example, Dey and Evans, Suicide Gene Therapy by Herpes Simplex Virus-1 Thymidine Kinase (HSV-TK), in Targets in Gene Therapy, edited by You (2011), and Beltinger et al. See, et al., Proc. Natl. Acad. Sci. USA 96(15):8699-8704 (1999). In some embodiments, the ceDNA vector can include a siRNA kill switch called DISE (death by excision induced survival gene) (Murmann et al., Oncotarget. 2017;8:84643-84658. Induction of DISE in ovarian cancer cells in vivo).

[0284] VI. Detailed Methods for the Production of ceDNA Vectors A. General production Certain methods of producing ceDNA vectors for expression of PAH proteins, including asymmetric or symmetric ITR pairs as defined herein, are described in Section IV of International Application No. PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety. In some embodiments, ceDNA vectors for expression of PAH proteins disclosed herein may be produced using insect cells, as described herein. In alternative embodiments, ceDNA vectors for expression of PAH proteins disclosed herein may be produced synthetically, and in some embodiments, cell-free, as disclosed in International Application No. PCT / US19 / 14122, filed January 18, 2019, which is incorporated herein by reference in its entirety.

[0285] As described herein, in one embodiment, a ceDNA vector for expression of a PAH protein can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., ceDNA-plasmid, ceDNA-bacmid, and / or ceDNA-baculovirus), where the host cells lack viral capsid coding sequences under conditions effective to induce production of the ceDNA vector in the host cells in the presence of Rep proteins, and for a sufficient time therefor; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of Rep proteins induces replication of the vector polynucleotide with modified ITRs to produce the ceDNA vector in the host cells. However, no viral particles (e.g., AAV virions) are expressed. Thus, there is no size restriction, such as that naturally imposed in AAV or other virus-based vectors.

[0286] The presence of a ceDNA vector isolated from a host cell may be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0287] In yet another aspect, the present invention provides the use of host cell lines that stably integrate a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, for example as described in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. If the host cell line is a mammalian cell line, for example, HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpes virus, may be used to introduce Rep protein into the cell, allowing excision and amplification of ceDNA in the presence of Rep and a helper virus.

[0288] In one embodiment, the host cells used to generate ceDNA vectors for expression of PAH proteins as described herein are insect cells, and a baculovirus is used to deliver both the polynucleotides encoding the Rep proteins and the non-viral DNA vector polynucleotide expression construct template of ceDNA, e.g., as described in Figures 4A-4C and in Example 1. In some embodiments, the host cells are engineered to express the Rep proteins.

[0289] The ceDNA vector is then harvested and isolated from the host cell. The time for harvesting the ceDNA vector described herein from the cell can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. In one embodiment, the cells are grown under sufficient conditions and harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector, but before the majority of the cells begin to die due to the toxicity of the baculovirus. The DNA-vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free plasmid kit. Other methods developed for plasmid isolation can also be adapted for DNA-vectors. In general, any nucleic acid purification method can be employed.

[0290] DNA vector can be purified by any means known to those skilled in the art for DNA purification.In one embodiment, ceDNA vector is purified as DNA molecule.In another embodiment, ceDNA vector is purified as exosome or microparticle.

[0291] The presence of a ceDNA vector for expression of a PAH protein can be confirmed by digesting the vector DNA isolated from the cells with a restriction enzyme that has a single recognition site on the DNA vector and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of characteristic linear and continuous DNA compared to linear and discontinuous DNA. Figures 4C and 4D show one embodiment for identifying the presence of a closed-ended ceDNA vector produced by the processes herein.

[0292] B. ceDNA plasmid ceDNA-plasmid is a plasmid used for the later production of ceDNA vector for expression of PAH protein. In some embodiments, ceDNA-plasmid can be constructed using known techniques to provide at least (1) modified 5'ITR sequence, (2) expression cassette containing cis-regulatory elements, such as promoter, inducible promoter, regulatory switch, enhancer, etc., and (3) modified 3'ITR sequence (3'ITR sequence is asymmetric with respect to 5'ITR sequence) as operably linked components in the transcription direction. In some embodiments, the expression cassette flanked by ITRs contains cloning sites for introducing exogenous sequences. The expression cassette replaces the rep and cap coding regions of the AAV genome.

[0293] In one aspect, the ceDNA vector for expression of a PAH protein is derived from a plasmid, referred to herein as a "ceDNA-plasmid," that encodes, in this order, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), an expression cassette comprising a transgene, and a mutant or modified AAV ITR, said ceDNA-plasmid lacking an AAV capsid protein coding sequence. In an alternative embodiment, the ceDNA-plasmid encodes, in this order, a first (or 5') modified or mutant AAV ITR, an expression cassette comprising a transgene, and a second (or 3') modified AAV ITR, said ceDNA-plasmid lacking an AAV capsid protein coding sequence, and the 5' and 3' ITRs are symmetrical with respect to each other. In an alternative embodiment, a ceDNA-plasmid encodes, in that order, a first (or 5') modified or mutated AAV ITR, an expression cassette containing a transgene, and a second (or 3') mutated or modified AAV ITR, wherein the ceDNA-plasmid lacks the AAV capsid protein coding sequence, and the 5' and 3' modified ITRs have the same modifications (i.e., they are reverse complementary or symmetrical to each other).

[0294] In further embodiments, the ceDNA-plasmid system lacks viral capsid protein coding sequences (i.e., lacks not only AAV capsid genes, but also capsid genes of other viruses). Additionally, in certain embodiments, the ceDNA-plasmid also lacks AAV Rep protein coding sequences. Thus, in preferred embodiments, the ceDNA-plasmid lacks functional AAV cap and AAV rep genes (GG-3' for AAV2) as well as variable palindrome sequences that allow hairpin formation.

[0295] The ceDNA-plasmid of the present invention can be generated using the native nucleotide sequence of any AAV serotype genome known in the art. In one embodiment, the ceDNA-plasmid backbone is derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8 genome. See, for example, NCBI: NC 002077, NC 001401, NC001729, NC001829, NC006152, NC 006260, NC 006261, Kotin and Smith, The Springer Index of Viruses available at URLs maintained by Springer (www web address: oesys.springer.de / viruses / database / mkchapter.asp?virID=42.04) (Note - references to URLs or databases refer to the contents of the URLs or databases as of the effective filing date of this application). In a particular embodiment, the ceDNA-plasmid backbone is derived from the AAV2 genome. In another particular embodiment, the ceDNA-plasmid backbone is a synthetic backbone engineered to include 5' and 3' ITRs from one of these AAV genomes.

[0296] The ceDNA-plasmid may optionally contain a selectable or selectable marker for use in establishing a ceDNA vector producing cell line. In one embodiment, the selectable marker may be inserted downstream (i.e., 3') of the 3'ITR sequence. In another embodiment, the selectable marker may be inserted upstream (i.e., 5') of the 5'ITR sequence. Suitable selectable markers include, for example, those that confer drug resistance. The selectable marker may be, for example, the Blasticidin S resistance gene, kanamycin, geneticin, etc. In a preferred embodiment, the drug selectable marker is the Blasticidin S resistance gene.

[0297] An exemplary ceDNA (e.g., rAAV0) vector for expression of a PAH protein is produced from a rAAV plasmid. A method for producing a rAAV vector can include: (a) providing a host cell with a rAAV plasmid as described above, where both the host cell and the plasmid lack a capsid protein-encoding gene; (b) culturing the host cell under conditions that allow the production of a ceDNA genome; and (c) harvesting the cell and isolating the produced AAV genome from the cell.

[0298] C. Exemplary Methods for Producing ceDNA Vectors from ceDNA Plasmids Also provided herein are methods for generating capsid-free ceDNA vectors for expression of PAH proteins, particularly methods that have yields high enough to provide sufficient vector for in vivo experiments.

[0299] In some embodiments, the method for producing a ceDNA vector for expression of a PAH protein includes the steps of: (1) introducing a nucleic acid construct comprising an expression cassette and two symmetric ITR sequences into a host cell (e.g., Sf9 cell); (2) optionally establishing a clonal cell line, e.g., by using a selection marker present on a plasmid; (3) introducing a Rep-encoding gene into said insect cell (either by transfection or infection with a baculovirus carrying said gene); and (4) harvesting the cells and purifying the ceDNA vector. The nucleic acid construct comprising the expression cassette and two ITR sequences described above for the production of a ceDNA vector can be in the form of a ceDNA-plasmid, or a bacmid or baculovirus produced with a ceDNA plasmid as described below. The nucleic acid construct can be introduced into a host cell by transfection, viral transduction, stable integration, or other methods known in the art.

[0300] D. Cell line The host cell line used in the production of ceDNA vector for expression of PAH protein can include insect cell lines derived from Spodoptera frugiperda (Sf9, Sf21, etc.) or Trichoplusia ni cells, or other eukaryotic cell lines including other invertebrate, vertebrate, or mammalian cells. Other cell lines known to those skilled in the art can also be used, such as HEK293, Huh-7, HeLa, HepG2, HeplA, 911, CHO, COS, MeWo, NIH3T3, A549, HT1 180, monocytes, and mature and immature dendritic cells. The host cell line can be transfected for stable expression of ceDNA-plasmid for high yield ceDNA vector production.

[0301] The ceDNA-plasmid can be introduced into Sf9 cells by transient transfection using reagents (e.g., liposomes, calcium phosphate) or physical means (e.g., electroporation) known in the art. Alternatively, stable Sf9 cell lines that stably integrate the ceDNA-plasmid into their genome can be established. Such stable cell lines can be established by incorporating a selection marker into the above ceDNA-plasmid. If the ceDNA-plasmid used to transfect the cell line contains a selection marker such as an antibiotic, the cells that are transfected with the ceDNA-plasmid and have integrated the ceDNA-plasmid DNA into their genome can be selected by adding the antibiotic to the cell growth medium. Resistant clones of cells can then be isolated and propagated by single cell dilution or colony transfer techniques.

[0302] Isolation and purification of E. ceDNA vector Examples of processes for obtaining and isolating ceDNA vectors are described in Figures 4A-4E and in the specific examples below. ceDNA-vectors for expression of the PAH proteins disclosed herein can be obtained from producer cells expressing AAV Rep proteins and further transformed with ceDNA-plasmids, ceDNA-bacmids, or ceDNA-baculoviruses. Plasmids useful for producing ceDNA vectors include plasmids encoding PAH proteins or plasmids encoding one or more REP proteins.

[0303] In one embodiment, the polynucleotide encodes an AAV Rep protein (Rep 78 or 68) delivered to a producer cell in a plasmid (Rep-plasmid), bacmid (Rep-bacmid), or baculovirus (Rep-baculovirus). The Rep-plasmid, Rep-bacmid, and Rep-baculovirus can be generated by the methods described above.

[0304] Methods for producing ceDNA vectors for expression of PAH proteins are described herein. The expression constructs used to generate ceDNA vectors for expression of PAH proteins described herein can be plasmids (e.g., ceDNA-plasmids), bacmids (e.g., ceDNA-bacmids), and / or baculoviruses (e.g., ceDNA-baculoviruses). By way of example only, ceDNA vectors can be generated from cells co-infected with ceDNA-baculoviruses and Rep-baculoviruses. The Rep protein produced from the Rep-baculovirus replicates the ceDNA-baculovirus to generate ceDNA vectors. Alternatively, ceDNA vectors for expression of PAH proteins can be generated from cells stably transfected with constructs comprising sequences encoding AAV Rep proteins (Rep78 / 52) delivered in Rep-plasmids, Rep-bacmids, or Rep-baculoviruses. The ceDNA-baculoviruses can be transiently transfected into cells and replicated by the Rep protein to generate ceDNA vectors.

[0305] The bacmid (e.g., ceDNA-bacmid) can be transfected into permissive insect cells, such as Sf9, Sf21, Tni (Trichoplusia ni) cells, High Five cells, and the like, to generate ceDNA-baculovirus, which is a recombinant baculovirus that contains sequences that include symmetric ITRs and an expression cassette. The ceDNA-baculovirus can be reinfected into insect cells to obtain the next generation of recombinant baculovirus. Optionally, this step can be repeated one or more times to produce larger amounts of recombinant baculovirus.

[0306] The time for harvesting and collecting the ceDNA vector for expression of the PAH protein described herein from the cells can be selected and optimized to achieve high yield production of the ceDNA vector. For example, the harvest time can be selected taking into consideration cell viability, cell morphology, cell growth, etc. Usually, the cells can be harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector (e.g., ceDNA vector), but before a portion of the cells begin to die due to viral toxicity. The ceDNA vector can be isolated from Sf9 cells using a plasmid purification kit, such as the Qiagen ENDO-FREE PLASMID® kit. Other methods developed for plasmid isolation can also be adapted for ceDNA vectors. In general, any art-known nucleic acid purification method can be employed, as well as commercially available DNA extraction kits.

[0307] Alternatively, purification can be implemented by subjecting the cell pellet to an alkaline lysis process, centrifuging the resulting lysate, and carrying out chromatographic separation.As one non-limiting example, this process can be carried out by loading the supernatant onto an ion exchange column (e.g., SARTOBIND Q®) that retains nucleic acid, then eluting (e.g., with 1.2M NaCl solution), and carrying out further chromatographic purification on a gel filtration column (e.g., 6 Fast Flow GE).The capsid-free AAV vector is then recovered, for example, by precipitation.

[0308] In some embodiments, the ceDNA vector for expression of PAH protein can also be purified in the form of exosomes or microparticles. It is known in the art that many cell types release not only soluble proteins but also complex protein / nucleic acid cargoes through shedding of membrane microvesicles (Cocucci et al, 2009, EP10306226.1, which are incorporated herein by reference in their entirety). Such vesicles include microvesicles (also called microparticles) and exosomes (also called nanovesicles), both of which contain proteins and RNA as cargo. Microvesicles are generated from direct budding of plasma membrane, and exosomes are released into the extracellular environment upon fusion of multivesicular endosomes with plasma membrane. Thus, microvesicles and / or exosomes containing ceDNA vectors can be isolated from cells transduced with ceDNA plasmids, or bacmids or baculoviruses generated with ceDNA plasmids.

[0309] Microvesicles can be isolated by subjecting the culture medium to filtration or ultracentrifugation at 20,000xg and the exosomes at 100,000xg. The optimal duration of ultracentrifugation can be determined experimentally and will depend on the particular cell type from which the vesicles are isolated. Preferably, the culture medium is first cleared by low speed centrifugation (e.g., 2000xg for 5-20 minutes) and subjected to spin concentration, for example, using AMICON® spin columns (Millipore, Watford, UK). Microvesicles and exosomes can be further purified via FACS or MACS by using specific antibodies that recognize specific surface antigens present on the microvesicles and exosomes. Other microvesicles and exosome purification methods include, but are not limited to, immunoprecipitation, affinity chromatography, filtration, and magnetic beads coated with specific antibodies or aptamers. During purification, the vesicles are washed, for example, with phosphate buffered saline. One advantage of using microvesicles or exosomes to deliver ceDNA-containing vesicles is that these vesicles can be targeted to various cell types by including on them membrane proteins that are recognized by specific receptors on each cell type. (See also EP10306226)

[0310] Another aspect of the invention herein relates to a method for purifying ceDNA vector from host cell lines that stably integrate ceDNA constructs into their own genome.In one embodiment, ceDNA vector is purified as DNA molecule.In another embodiment, ceDNA vector is purified as exosome or microparticle.

[0311] Figure 5 of International Application No. PCT / US18 / 49996 shows a gel confirming the production of ceDNA from multiple ceDNA-plasmid constructs using the methods described in the Examples. The ceDNA is confirmed by a characteristic banding pattern in the gel, as discussed with respect to Figure 4D in the Examples.

[0312] VII. Pharmaceutical Compositions In another embodiment, a pharmaceutical composition is provided, comprising a ceDNA vector for expression of a PAH protein as described herein, and a pharma- ceutically acceptable carrier or diluent.

[0313] The ceDNA vector for expressing the PAH protein disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to cells, tissues, or organs of the subject.Typically, the pharmaceutical composition comprises the ceDNA vector disclosed herein and a pharmaceutically acceptable carrier.For example, the ceDNA vector for expressing the PAH protein described herein can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration).Passive tissue transduction via high pressure intravenous or intraarterial infusion, as well as intracellular injection such as intranuclear microinjection or intracytoplasmic injection, are also contemplated.The pharmaceutical composition for therapeutic purposes can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high ceDNA vector concentration. Sterile injection solution can be prepared by incorporating the required amount of ceDNA vector compound in a suitable buffer with one or a combination of the above-listed components, and then filter sterilization containing ceDNA vector, and can be formulated to deliver the transgene in nucleic acid to recipient cells, resulting in the therapeutic expression of transgene or donor sequence therein.This composition can also include a pharmaceutically acceptable carrier.

[0314] Pharmaceutically active compositions containing ceDNA vectors for expression of PAH proteins can be formulated to deliver transgenes for a variety of purposes to cells, e.g., cells of a subject.

[0315] Pharmaceutical compositions for therapeutic purposes must typically be sterile and stable under the conditions of manufacture and storage.Compositions can be formulated as solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high ceDNA vector concentration.Sterile injectable solution can be prepared by incorporating the required amount of ceDNA vector compound in a suitable buffer with one or a combination of the above-listed components as necessary, and sterilizing by filtration.

[0316] The ceDNA vectors for expression of the PAH proteins disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-thecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intratissue (e.g., intramuscular, intracardiac), intrahepatic, intrarenal, intracerebral), intrathecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrainterstitial, intracameral, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high pressure intravenous or intra-arterial infusion is also contemplated, as well as intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0317] In some embodiments, the methods provided herein include delivering one or more ceDNA vectors for expression of the PAH proteins disclosed herein to a host cell. Also provided herein are cells produced by such methods, and organisms (such as animals, plants, or fungi) that contain or are produced from such cells. Methods for delivery of nucleic acids can include lipofection, nucleofection, microinjection, biolistics, liposomes, immunoliposomes, polycations or lipids:nucleic acid conjugates, naked DNA, and drug-enhanced uptake with DNA. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, which are incorporated herein by reference in their entirety, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Delivery can be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0318] Various techniques and methods for delivering nucleic acid to cells are known in the art.For example, nucleic acid, such as ceDNA for expressing PAH protein, can be formulated in lipid nanoparticle (LNP), lipidoid, liposome, lipid nanoparticle, lipoplex, or core-shell nanoparticle.Typically, LNP is composed of nucleic acid (e.g., ceDNA) molecule, one or more ionized or cationic lipids (or their salts), one or more non-ionic or neutral lipids (e.g., phospholipids), molecules that prevent aggregation (e.g., PEG or PEG-lipid conjugate), and optionally sterol (e.g., cholesterol).

[0319] Another method for delivering nucleic acid, such as ceDNA for expression of PAH protein, to cells is by complexing the nucleic acid with a ligand that is internalized by the cell.For example, the ligand can bind to a receptor on the cell surface and be internalized via endocytosis.The ligand can be covalently linked to the nucleotide in the nucleic acid.Exemplary conjugates for delivering nucleic acid into cells are described, for example, in WO2015 / 006740, WO2014 / 025805, WO2012 / 037254, WO2009 / 082606, WO2009 / 073809, WO2009 / 018332, WO2006 / 112872, WO2004 / 090108, WO2004 / 091515, and WO2017 / 177326, the entire contents of which are incorporated herein by reference in their entirety.

[0320] Nucleic acids such as ceDNA for expressing PAH proteins can also be delivered to cells by transfection. Useful transfection methods include, but are not limited to, lipid-mediated transfection, cationic polymer-mediated transfection, or calcium phosphate precipitation. Transfection reagents are well known in the art and include TurboFect™ Transfection Reagent (Thermo Fisher Scientific®), Pro-Ject Reagent (Thermo Fisher Scientific®), TRANSPASS™ P Protein Transfection Reagent (New England Biolabs®), CHARIOT™ Protein Delivery Reagent (Active Motif), PROTEOJUICE™ Protein Transfection Reagent (EMD Millipore®), 293fectin, LIPOFECTAMINE™ 2000, LIPOFECTAMINE™ 3000 (Thermo Fisher Scientific®), LIPOFECTAMINE™ (Thermo Fisher Scientific®), LIPOFECTIN™ (Thermo Fisher Scientific®), DMRIE-C, CELLFECTIN™ (Thermo Fisher Scientific®), OLIGOFECTAMINE™ (Thermo Fisher Scientific®), IL-10 ... Scientific®), LIPOFECTACE™, FUGENE™ (Roche®, Basel, Switzerland), FUGENE™ HD (Roche®), TRANSFECTAM™ (Transfectam, Promega®, Madison, Wis.), TFX-10™ (Promega®), TFX-20™ (Promega®), TFX-50™ (Promega), TRANSFECTIN™ (BioRad®, Hercules, Calif.), SILENTFECT™ (Bio-Rad®), Effectene™ (Qiagen®, Valencia, Calif.), DC-chol (Avanti Polar Lipids), GENEPORTER™ (Gene Therapy Systems®, San Diego, Calif.), DHARMAFECT 1™ (Dharmacon, Lafayette, Colo.), DHARMAFECT 2™ (Dharmacon), DHARMAFECT 3™ (Dharmacon), DHARMAFECT 4™ (Dharmacon), ESCORT™ III (Sigma, St. Louis, Mo.), and ESCORT™ IV (Sigma Chemical Co.). Nucleic acids such as ceDNA can also be delivered to cells via microfluidics methods known to those of skill in the art.

[0321] The ceDNA vector for expressing PAH protein described herein can also be directly administered to organisms for in vivo cell transduction.Administration can be by any of the routes that are usually used to bring molecules into contact with blood or tissue cells, including but not limited to injection, infusion, topical application, and electroporation.Suitable methods for administering such nucleic acids are available and well known by those skilled in the art, and more than one route can be used to administer a particular composition, although a particular route can often provide a more immediate and effective response than another route.

[0322] Methods for introducing ceDNA vectors into nucleic acid vectors for expression of the PAH proteins disclosed herein can be delivered, for example, to hematopoietic stem cells, for example, by the methods described in U.S. Pat. No. 5,928,638, the entirety of which is incorporated herein by reference.

[0323] The ceDNA vector for expression of the PAH protein according to the present invention can be added to liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids. Exemplary liposomes and liposomal formulations, including but not limited to polyethylene glycol (PEG) functional group containing compounds, are disclosed in International Application Nos. PCT / US2018 / 050042, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, the entire contents of each of which are incorporated herein by reference, see, for example, the section entitled "Pharmaceutical Formulations."

[0324] The ceDNA vector can be delivered in vitro or in vivo using various delivery methods or modifications thereof known in the art. For example, in some embodiments, the ceDNA vector for expression of PAH proteins is delivered by creating a temporary penetration in the cell membrane by mechanical, electrical, ultrasonic, hydrodynamic, or laser-based energy, thereby facilitating DNA entry into the targeted cells. For example, the ceDNA vector can be delivered by temporarily disrupting the cell membrane by squeezing the cells through a size-restricted channel or by other means known in the art. In some cases, the ceDNA vector alone is injected as naked DNA directly into any one of the liver, kidney, gallbladder, prostate, adrenal gland, heart, intestine, lung, and any one or more tissues selected from the liver, skin, thyroid, cardiac muscle, or skeletal muscle. In some cases, the ceDNA vector is delivered by a gene gun. Gold or tungsten spherical particles (1-3 μm diameter) coated with capsid-free AAV vectors can be accelerated to high speed by pressurized gas and penetrate into the target tissue cells.

[0325] Specifically contemplated herein are compositions comprising a ceDNA vector and a pharma- ceutically acceptable carrier for expression of a PAH protein. In some embodiments, the ceDNA vector is formulated in a lipid delivery system, e.g., liposomes, as described herein. In some embodiments, such compositions are administered by any route desired by a skilled practitioner. The compositions may be administered to a subject by different routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via intrabuccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or combinations thereof. For veterinary use, the compositions may be administered in a suitably acceptable formulation according to normal veterinary practice. A veterinarian can readily determine the most appropriate dosing regimen and route of administration for a particular animal. The compositions may be administered by conventional syringes, needleless injection devices, "microprojectile bombardment guns," or other physical methods, such as electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0326] In some cases, ceDNA vectors for expression of PAH proteins are delivered by hydrodynamic injection, a simple and highly efficient method for the direct intracellular delivery of any water-soluble compound and particle to skeletal muscles throughout the viscera and limbs.

[0327] In some cases, ceDNA vectors for the expression of PAH proteins are delivered by ultrasound, by creating nanoscopic pores in the membrane, facilitating the intracellular delivery of DNA particles to cells of internal organs or tumors, so the size and concentration of plasmid DNA play a major role in the efficiency of this system. In some cases, ceDNA vectors are delivered by magnetofection, by using a magnetic field to concentrate the particles containing nucleic acids in the target cells.

[0328] In some cases, chemical delivery systems can be used by using nanomer complexes, including, for example, cationic liposomes / micelles or polycationic nanomer particles that belong to cationic polymers, compressing negatively charged nucleic acids. The cationic lipids used in the delivery method include, but are not limited to, monovalent cationic lipids, polyvalent cationic lipids, guanidine-containing compounds, cholesterol derivative compounds, cationic polymers (e.g., poly(ethyleneimine), poly-L-lysine, protamine, other cationic polymers), and lipid-polymer hybrids.

[0329] A. Exosomes: In some embodiments, the ceDNA vectors for expression of the PAH proteins disclosed herein are delivered by being packaged into exosomes. Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the cell that produced the exosomes, and exhibit membrane proteins from the parent cell on the surface. Exosomes are produced by various cell types, including epithelial cells, B and T lymphocytes, mast cells (MC), as well as dendritic cells (DC). In some embodiments, exosomes with diameters of 10 nm to 1 μm, 20 nm to 500 nm, 30 nm to 250 nm, 50 nm to 100 nm are envisioned for use. Exosomes can be isolated for delivery to target cells, either by using their donor cells or by introducing specific nucleic acids into them. Various approaches known in the art can be used to produce exosomes containing the capsid-free AAV vectors of the invention.

[0330] A. Microparticles / Nanoparticles In some embodiments, the ceDNA vector for expression of the PAH protein disclosed herein is delivered by lipid nanoparticles. Generally, lipid nanoparticles include ionized amino lipids (e.g., heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA, phosphatidylcholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), cholesterol, and coating lipids (polyethylene glycol-dimyristol glycerol, PEG-DMG), as disclosed, for example, by Tam et al. (2013). Advances in Lipid Nanoparticles for siRNA delivery. Pharmaceuticals 5(3):498-507.

[0331] In some embodiments, the lipid nanoparticles have an average diameter of about 10 to about 1000 nm. In some embodiments, the lipid nanoparticles have a diameter less than 300 nm. In some embodiments, the lipid nanoparticles have a diameter of about 10 to about 300 nm. In some embodiments, the lipid nanoparticles have a diameter less than 200 nm. In some embodiments, the lipid nanoparticles have a diameter of about 25 to about 200 nm. In some embodiments, the lipid nanoparticle preparation (e.g., a composition comprising a plurality of lipid nanoparticles) has a size distribution, with an average size (e.g., diameter) of about 70 nm to about 200 nm, and more typically, the average size is about 100 nm or less.

[0332] Various lipid nanoparticles known in the art can be used to deliver the ceDNA vector for expressing the PAH protein disclosed herein.For example, various delivery methods using lipid nanoparticles are described in US Patent No. 9,404,127, No. 9,006,417 and No. 9,518,272.

[0333] In some embodiments, the ceDNA vector for expressing the PAH protein disclosed herein is delivered by gold nanoparticles.Generally, the nucleic acid can be covalently bound to the gold nanoparticles or non-covalently bound to the gold nanoparticles (e.g., bound by charge-charge interactions), as described, for example, by Ding et al.(2014).Gold Nanoparticles for Nucleic Acid Delivery.Mol.Ther.22(6);1075-1083.In some embodiments, the gold nanoparticle-nucleic acid conjugate is produced using the method described, for example, in U.S. Patent No. 6,812,334.

[0334] B. Conjugates In some embodiments, the ceDNA vector for expression of the PAH protein disclosed herein is conjugated (e.g., covalently linked to an agent that increases cellular uptake). An "agent that increases cellular uptake" is a molecule that promotes the transport of nucleic acids through lipid membranes. For example, the nucleic acid may be conjugated to a lipophilic compound (e.g., cholesterol, tocopherol, etc.), a cell penetrating peptide (CPP) (e.g., penetratin, TAT, Syn1B, etc.), and a polyamine (e.g., spermine). Further examples of agents that increase cellular uptake are disclosed, for example, in Winkler (2013). Oligonucleotide conjugates for therapeutic applications. Ther. Deliv. 4(7); 791-809.

[0335] In some embodiments, the ceDNA vector for expression of the PAH protein disclosed herein is conjugated to a polymer (e.g., a polymer molecule) or a folate molecule (e.g., a folic acid molecule). In general, delivery of nucleic acids conjugated to a polymer is known in the art, for example, as described in WO2000 / 34343 and WO2008 / 022309. In some embodiments, the ceDNA vector for expression of the PAH protein disclosed herein is conjugated to a poly(amide) polymer, for example, as described by U.S. Patent No. 8,987,377. In some embodiments, the nucleic acid described by the present disclosure is conjugated to a folic acid molecule, for example, as described in U.S. Patent No. 8,507,455.

[0336] In some embodiments, the ceDNA vectors for expression of the PAH proteins disclosed herein are conjugated to carbohydrates, for example, as described in U.S. Pat. No. 8,450,467.

[0337] C. Nanocapsules Alternatively, nanocapsule formulations of ceDNA vectors for expressing PAH proteins disclosed herein can be used.Nanocapsules can generally capture substances in a stable and reproducible manner.To avoid side effects caused by intracellular polymer overload, such fine particles (approximately 0.1 μm in size) should be designed using polymers so that they can be degraded in vivo.Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are contemplated for use.

[0338] D. Liposomes The ceDNA vector for expressing the PAH protein according to the present invention can be added to liposomes for delivery to cells or target organs in a subject. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0339] The formation and use of liposomes are generally known to those skilled in the art. Liposomes with improved serum stability and circulation half-life have been developed (US Pat. No. 5,741,516). In addition, various methods of liposomes and liposome-like preparations as potential drug carriers have been described (US Pat. Nos. 5,567,434, 5,552,157, 5,565,213, 5,738,868, and 5,795,587, which are incorporated herein by reference in their entirety).

[0340] E. Exemplary Liposome and Lipid Nanoparticle (LNP) Compositions The ceDNA vector for expression of the PAH protein according to the present invention can be added to liposomes for delivery to cells, for example, cells that require expression of the transgene. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic agent delivery in the context of formulation development. They act by fusing with cell membranes and rearranging their lipid structure to deliver drugs or active pharmaceutical ingredients (APIs). Liposome compositions for such delivery are composed of compounds with phospholipids, particularly phosphatidylcholine, although these compositions may also contain other lipids.

[0341] Lipid nanoparticles (LNPs) containing ceDNA vectors are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and International Application No. PCT / US2018 / 064242, filed December 6, 2018, which are incorporated herein in their entireties, and are contemplated for use in the methods and compositions for ceDNA vectors for expression of PAH proteins disclosed herein.

[0342] In some embodiments, the present disclosure provides liposomal formulations that include one or more compounds with polyethylene glycol (PEG) functional groups (so-called "PEGylated compounds"), which can reduce immunogenicity / antigenicity, provide hydrophilic and hydrophobic properties to the compounds, and reduce dosing frequency. Alternatively, the liposomal formulations simply include polyethylene glycol (PEG) polymers as additional components. In such embodiments, the molecular weight of the PEG or PEG functional group can be from 62 Da to about 5,000 Da.

[0343] In some embodiments, the present disclosure provides liposomal formulations that will deliver APIs with extended or controlled release profiles over a period of hours to weeks. In some related embodiments, the liposomal formulations may include aqueous chambers bounded by lipid bilayers. In other related embodiments, the liposomal formulations encapsulate APIs with components that undergo physical transitions at elevated temperatures that release the API over a period of hours to weeks.

[0344] In some embodiments, the liposomal formulation comprises sphingomyelin and one or more lipids disclosed herein. In some embodiments, the liposomal formulation comprises an Optisome.

[0345] In some aspects, the present disclosure provides a lipid composition comprising N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, (distearoyl-sn-glycero-phosphoethanolamine), MPEG (methoxypolyethylene glycol) conjugated lipids, HSPC (hydrogenated soy phosphatidylcholine), PEG (polyethylene glycol), DSPE (distearoyl-sn-glycero-phosphoethanolamine), DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), DPPG (dipalmitoylphosphatidylglycerol), EPC (egg phosphatidylcholine), DOPS ... phatidylserine), POPC (palmitoyloleoylphosphatidylcholine), SM (sphingomyelin), MPEG (methoxypolyethylene glycol), DMPC (dimyristoylphosphatidylcholine), DMPG (dimyristoylphosphatidylglycerol), DSPG (distearoylphosphatidylglycerol), DEPC (dierucoylphosphatidylcholine), DOPE (dioleoyl-sn-glycero-phosphoethanolamine), cholesteryl sulfate (CS), dipalmitoylphosphatidylglycerol (DPPG), DOPC (dioleoyl-sn-glycero-phosphatidylcholine), or any combination thereof.

[0346] In some embodiments, the present disclosure provides a liposomal formulation comprising a phospholipid, cholesterol, and a PEGylated lipid in a molar ratio of 56:38:5. In some embodiments, the total lipid content of the liposomal formulation is 2-16 mg / mL. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, a lipid containing an ethanolamine functional group, and a PEGylated lipid in a molar ratio of 3:0.015:2, respectively. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group, cholesterol, and a PEGylated lipid. In some embodiments, the present disclosure provides a liposomal formulation comprising a lipid containing a phosphatidylcholine functional group and cholesterol. In some embodiments, the PEGylated lipid is PEG-2000-DSPE. In some embodiments, the present disclosure provides a liposome formulation comprising DPPG, soy PC, an MPEG-DSPE lipid conjugate, and cholesterol.

[0347] In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids containing phosphatidylcholine functional groups and one or more lipids containing ethanolamine functional groups.In some embodiments, the present disclosure provides a liposomal formulation comprising one or more lipids containing phosphatidylcholine functional groups, lipids containing ethanolamine functional groups, and a sterol, such as cholesterol.In some embodiments, the liposomal formulation comprises DOPC / DEPC and DOPE.

[0348] In some embodiments, the present disclosure provides liposomal formulations further comprising one or more pharmaceutical excipients, such as sucrose and / or glycine.

[0349] In some embodiments, the present disclosure provides liposomal formulations that are either unilamellar or multilamellar structures. In some embodiments, the present disclosure provides liposomal formulations that include multivesicular particles and / or foam-based particles. In some embodiments, the present disclosure provides liposomal formulations that are larger in size relative to typical nanoparticles, about 150-250 nm in size. In some embodiments, the liposomal formulation is a lyophilized powder.

[0350] In some embodiments, the present disclosure provides liposomal formulations made and loaded with ceDNA vectors disclosed or described herein by adding a weak base to a mixture having isolated ceDNA outside the liposome. This addition increases the pH outside the liposome to approximately 7.3, sending the API into the liposome. In some embodiments, the present disclosure provides liposomal formulations having an acidic pH inside the liposome. In such cases, the inside of the liposome can be pH 4-6.9, more preferably pH 6.5. In other embodiments, the present disclosure provides liposomal formulations made by using intraliposomal drug stabilization techniques. In such cases, a polymeric or non-polymeric highly charged anion and an intraliposomal entrapment agent, such as polyphosphate or sucrose octasulfate, are utilized.

[0351] In some embodiments, the present disclosure provides lipid nanoparticles comprising ceDNA and ionized lipids. For example, lipid nanoparticle formulations made and loaded with ceDNA obtained by the process are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, and are incorporated herein. This can be achieved by high energy mixing of ethanol lipids with aqueous ceDNA at low pH, which protonates ionized lipids and provides favorable energetics for ceDNA / lipid association and nucleation of particles. Particles can be further stabilized by aqueous dilution and removal of organic solvent. Particles can be concentrated to a desired level.

[0352] Generally, lipid particles are prepared with a total lipid to ceDNA (mass or weight) ratio of about 10:1 to 30:1. In some embodiments, the lipid to ceDNA ratio (mass / mass ratio, w / w ratio) can be in the range of about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, or about 6:1 to about 9:1. The amount of lipid and ceDNA can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or more. Generally, the total lipid content of the lipid particle formulation can be in the range of about 5 mg / mL to about 30 mg / mL.

[0353] Ionized lipids are typically used to condense nucleic acid cargo, such as ceDNA, at low pH and to drive membrane association and fusogenicity.Generally, ionized lipids are lipids that contain at least one amino group that is positively charged or protonated under acidic conditions, such as pH 6.5 or less.Ionized lipids are also referred to herein as cationic lipids.

[0354] Exemplary ionizable lipids are described in International PCT Patent Publication Nos. WO2015 / 095340, WO2015 / 199952, WO2018 / 011633, WO2017 / 049245, WO2015 / 061467, WO2012 / 040184, WO2012 / 000104, WO2015 / 074085, WO2016 / 081029, WO2017 / 004143, WO2017 / 075531, WO2017 / 117528, WO2011 / 022460, WO2013 / 148541, WO 2013 / 116126, WO2011 / 153120, WO2012 / 044638, WO2012 / 054365, WO2011 / 090965, WO2013 / 016058, WO2012 / 162210, WO2008 / 042973 , No. WO2010 / 129709, No. WO2010 / 144740, No. WO2012 / 099755, No. WO2013 / 049328, No. WO2013 / 086322, No. WO2013 / 086373, No. WO2011 / 071860, No. WO2009 / 132 No. 131, No. WO2010 / 048536, No. WO2010 / 088537, No. WO2010 / 054401, No. WO2010 / 054406, No. WO2010 / 054405, No. WO2010 / 054384, No. WO2012 / 016184, No. WO2009 / 086558, WO2010 / 042877, WO2011 / 000106, WO2011 / 000107, WO2005 / 120152, WO2011 / 141705, WO2013 / 126803, WO2006 / 007712, WO Nos. 2011 / 038160, WO2005 / 121348, WO2011 / 066651, WO2009 / 127060, WO2011 / 141704, WO2006 / 069782, WO2012 / 031043, WO2013 / 006825, WO2013 / 033563, WO2013 / 089151, WO2017 / 099823, WO2015 / 095346, and WO2013 / 086354, and U.S. Patent Publication Nos. 2016 / 0311759, 2015 / 0376115,No. 2016 / 0151284, No. 2017 / 0210697, No. 2015 / 0140070, No. 2013 / 0178541, No. 2013 / 0303587, No. 2015 / 0141678, No. 2015 / 0239926, 2016 / 0376224, 2017 / 0119904, 2012 / 0149894, 2015 / 0057373, 2013 / 0090372, 2013 / 02745 No. 23, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 0023673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, No. 2 No. 015 / 0203446, No. 2018 / 0005363, No. 2014 / 0308304, No. 2013 / 0338210, No. 2012 / 0101148, No. 2012 / 0027796, No. 2012 / 00 No. 58144, No. 2013 / 0323269, No. 2011 / 0117125, No. 2011 / 0256175, No. 2012 / 0202871, No. 2011 / 0076335, No. 2006 / 0083780 No. 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, 201 Nos. 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of all of which are incorporated herein by reference in their entireties.

[0355] In some embodiments, the ionizable lipid is MC3(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoic acid (DLin-MC3-DMA or MC3), having the following structure: [ka]

[0356] The lipid DLin-MC3-DMA is described in Jayaraman et al., Angew. Chem. Int. Ed Engl. (2012), 51(34):8529-8533, the contents of which are incorporated herein by reference in their entirety.

[0357] In some embodiments, the ionizable lipid is the lipid ATX-002, described in WO2015 / 074085, the contents of which are incorporated herein by reference in their entirety.

[0358] In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (compound 32), described in WO2012 / 040184, the contents of which are incorporated herein by reference in their entirety.

[0359] In some embodiments, the ionizable lipid is compound 6 or compound 22, described in WO2015 / 199952, the contents of which are incorporated herein by reference in their entirety.

[0360] Without limitation, the ionized lipids may comprise 20-90% (mol) of the total lipids present in the lipid nanoparticles. For example, the ionized lipid molar content may be 20-70% (mol), 30-60% (mol), or 40-50% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments, the ionized lipids comprise about 50 mol% to about 90 mol% of the total lipids present in the lipid nanoparticles.

[0361] In some embodiments, lipid nanoparticles can further comprise non-cationic lipid.Non-ionic lipid includes amphipathic lipid, neutral lipid, and anionic lipid.Thus, non-cationic lipid can be neutral uncharged, zwitterionic, or anionic lipid.Non-cationic lipid is typically used to enhance membrane fusogenicity.

[0362] Exemplary non-cationic lipids contemplated for use in the methods and compositions disclosed herein are described in International Application Nos. PCT / US2018 / 050042, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018. Exemplary non-cationic lipids are described in International Application Publication No. WO2017 / 099823 and U.S. Patent Publication No. 2018 / 0028664, the contents of both of which are incorporated herein by reference in their entireties.

[0363] The non-cationic lipid may comprise 0-30% (mol) of the total lipid present in the lipid nanoparticle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid nanoparticle. In various embodiments, the molar ratio of ionized lipid to neutral lipid ranges from about 2:1 to about 8:1. 【0...

Claims

1. A nucleotide sequence encoding a phenylalanine hydroxylase (PAH) protein, the nucleotide sequence comprising a sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393 and SEQ ID NO:

394.

2. The nucleotide sequence of claim 1, comprising a sequence having at least 95% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393 and SEQ ID NO:

394.

3. The nucleotide sequence of claim 1, comprising a sequence having at least 97% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393 and SEQ ID NO:

394.

4. The nucleotide sequence of claim 1, comprising a sequence having at least 98% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393 and SEQ ID NO:

394.

5. The nucleotide sequence of claim 1, comprising a sequence having at least 99% identity to a sequence selected from the group consisting of SEQ ID NO:382, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:384, SEQ ID NO:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:390, SEQ ID NO:391, SEQ ID NO:392, SEQ ID NO:393 and SEQ ID NO:

394.

6. A nucleotide sequence described in any one of claims 1 to 5, wherein the nucleotide sequence comprises SEQ ID NO:

382.

7. A nucleotide sequence described in any one of claims 1 to 6, wherein the nucleotide sequence consists of SEQ ID NO:

382.

8. A capsid-free closed-end DNA (ceDNA) vector comprising at least one nucleotide sequence described in any one of claims 1 to 7 positioned between adjacent inverted terminal repeats (ITRs).

9. The ceDNA vector of claim 8, wherein the ceDNA vector comprises a promoter linked to the at least one nucleotide sequence encoding at least one PAH protein.

10. The ceDNA vector, (i) an enhancer; (ii) 5′ UTR and / or intron sequences; (iii) a 3′ UTR; and / or (iv) at least one polyA sequence 10. The ceDNA vector of claim 8 or claim 9, further comprising:

11. At least one of the adjacent ITRs contains a functional terminal resolution site (TRS) and a Rep binding site; one or both of the flanking ITRs are derived from a virus selected from the group consisting of parvovirus, dependovirus, and adeno-associated virus (AAV); the adjacent ITRs are symmetric; the adjacent ITRs are substantially symmetric; the adjacent ITRs are asymmetric; one of the ITRs is a wild-type ITR or both of the flanking ITRs are wild-type ITRs; one of the flanking ITRs is not a wild-type ITR or both of the flanking ITRs are not wild-type ITRs; the flanking ITRs are from different viral serotypes; The adjacent ITRs are: 【Table 2-4】 【Table 2-5】 【Table 2-6】 from a viral serotype pair selected from the group consisting of the viral serotype pairs shown in at least one of the flanking ITRs is altered from the wild-type AAV ITR sequence by deletions, additions, and / or substitutions that affect the overall three-dimensional conformation of the ITR; one or both of the flanking ITRs are derived from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12; and / or The ceDNA vector according to any one of claims 8 to 10, wherein one or both of the flanking ITRs comprises a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO:2, SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:12, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:9, SEQ ID NO:14, and SEQ ID NOs:15 to 48.

12. A ceDNA vector described in any one of claims 8 to 11, wherein one or both of the ITRs are synthetic.

13. One or both of the adjacent ITRs are modified by deletion, insertion, and / or substitution in at least one of the ITR regions selected from the group consisting of A, A', B, B', C, C', D, and D' regions; one or both of the flanking ITRs are modified by deletion, insertion, and / or substitution, resulting in the loss of all or part of the stem-loop structure formed by the B and B' regions; one or both of the flanking ITRs are modified by deletions, insertions, and / or substitutions that result in the loss of all or part of the stem-loop structure formed by the C and C' regions; one or both of the flanking ITRs are modified by deletion, insertion, and / or substitution resulting in the loss of all or a portion of the stem-loop structure formed by the B and B' regions and / or all or a portion of the stem-loop structure formed by the C and C' regions; one or both of the flanking ITRs contains a single stem-loop structure in a region that in a wild-type ITR contains a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions; one or both of the flanking ITRs comprises a single stem and two loops in a region which in the wild-type ITR comprises a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions; one or both of the flanking ITRs comprises a single stem and a single loop in a region that in the wild-type ITR comprises a first stem-loop structure formed by the B and B' regions and a second stem-loop structure formed by the C and C' regions; and / or 13. The ceDNA vector according to any one of claims 8 to 12, wherein both flanking ITRs are modified in such a manner as to result in overall three-dimensional symmetry when said flanking ITRs are inverted relative to each other.

14. A ceDNA vector described in any one of claims 8 to 13, wherein the ceDNA vector comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 382, ​​or wherein the at least one nucleotide sequence comprises a sequence having at least 96% identity to SEQ ID NO: 382, ​​or wherein the at least one nucleotide sequence comprises a sequence having at least 97% identity to SEQ ID NO:

382.

15. A ceDNA vector described in any one of claims 8 to 14, wherein the ceDNA vector comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:192, SEQ ID NO:193, or SEQ ID NO:

194.

16. The ceDNA vector described in any one of claims 8 to 15, wherein the ceDNA vector comprises at least one regulatory switch selected from the group consisting of a binary regulatory switch, a small molecule regulatory switch, a passcode regulatory switch, a nucleic acid-based regulatory switch, a post-transcriptional regulatory switch, a radiation-controlled or ultrasound-controlled regulatory switch, a hypoxia-mediated regulatory switch, an inflammatory response regulatory switch, a shear-activated regulatory switch, and a kill switch.

17. A pharmaceutical composition for use in expressing a PAH protein in a cell, comprising contacting the cell with a nucleotide sequence described in any one of claims 1 to 7 or a ceDNA vector described in any one of claims 8 to 16.

18. The pharmaceutical composition of claim 17, wherein the cells are selected from the group consisting of hepatocytes, photoreceptor cells and RPE cells.

19. The pharmaceutical composition described in claim 17 or 18, wherein the cells are in vitro or in vivo.

20. A pharmaceutical composition described in any one of claims 17 to 19, wherein at least one nucleotide sequence is codon optimized for expression in the cell.

21. A pharmaceutical composition for use in treating a subject having phenylketonuria (PKU), comprising administering to the subject a nucleotide sequence described in any one of claims 1 to 7 or a ceDNA vector described in any one of claims 8 to 15.

22. A pharmaceutical composition described in any one of claims 20 to 21, characterized in that the nucleotide sequence or the ceDNA vector is administered to a cell selected from the group consisting of a hepatocyte, a photoreceptor cell, and an RPE cell.

23. The pharmaceutical composition of any one of claims 20 to 22, wherein the ceDNA vector expresses the PAH protein in cells selected from the group consisting of hepatocytes, photoreceptor cells, and RPE cells.

24. The pharmaceutical composition of any one of claims 20 to 23, wherein the ceDNA vector is administered by one or more routes selected from the group consisting of intravenous injection, intramuscular injection, infusion, subretinal injection, suprachoroidal injection, and intravitreal injection.

25. The subject exhibits at least a 50% decrease in serum phenylalanine levels compared to the level of serum phenylalanine in the subject prior to administration; the subject has a serum phenylalanine level of less than 1500 μM after administration; and / or 24. The pharmaceutical composition of any one of claims 20 to 23, wherein the subject exhibits at least a 10% increase in PAH activity after administration compared to the level of PAH activity before administration.

26. A cell comprising a nucleotide sequence described in any one of claims 1 to 7 or a ceDNA vector described in any one of claims 8 to 16.

27. The cell of claim 26, wherein the cell is selected from the group consisting of a hepatocyte, a photoreceptor cell, and an RPE cell.

28. A composition comprising a nucleotide sequence described in any one of claims 1 to 7 or a ceDNA vector described in any one of claims 8 to 16 and a lipid.

29. The composition of claim 28, wherein the lipid is a lipid nanoparticle (LNP).

30. A kit comprising a nucleotide sequence described in any one of claims 1 to 7 or a ceDNA vector described in any one of claims 8 to 16, a pharmaceutical composition described in claim 26, a cell described in claim 26 or claim 27, or a composition described in claim 28 or 29.

31. A composition for use in treating a subject having phenylketonuria (PKU), comprising a nucleotide sequence according to any one of claims 1 to 7, or a ceDNA vector according to any one of claims 8 to 16, or a cell according to claim 26 or claim 27, or a composition according to claim 28 or 29.

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