Generation of modified human PAH for severe PKU treated by liver-directed gene replacement therapy

Abstract

Provided herein are variant phenylalanine hydroxylase (PAH) polypeptides that are more stable and have greater activity than wild-type human PAH. Also provided are methods for treating phenylketonuria (PKU) and / or reducing phenylalanine levels in individuals in need thereof. Also provided herein are expression cassettes, vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions, and kits for expressing the variant PAH polypeptides in individuals in need thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 62 / 744,944, filed on October 12, 2018, which is incorporated herein by reference in its entirety.

[0003] ASCII Text File Sequence Listing Submission

[0004] The contents of the following submitted ASCII text file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: 159792016640SEQLIST.TXT, Record Date: October 16, 2019, Size: 33KB). Technical Field

[0005] The present disclosure relates to variant phenylalanine hydroxylase polypeptides.In some aspects, the present disclosure relates to compositions and methods for treating phenylketonuria using gene therapy. Background Art

[0006] Phenylketonuria (PKU) is a genetic defect in the liver enzyme phenylalanine hydroxylase (PAH), which catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine (Tyr). The disease is the most common inborn error of amino acid metabolism, with an overall prevalence of 1:10,000-1:15,000 in North America, and is detected through newborn screening programs in most developed countries. In the absence of any treatment, severe forms of PKU result in highly elevated blood Phe levels, which are neurotoxic and associated with severe mental retardation (Kochhar 2012, Ho 2014, Blau 2015). The affected protein, PAH, is a multidomain protein consisting of an N-terminal regulatory domain (1-117), a central catalytic domain (118-410), and a C-terminal tetramerization domain (411-452) (Flydal 2013). To date, more than 560 pathogenic mutations have been mapped to each domain, with the catalytic region being the most frequently affected site (Erlandsen 2003). The homomultimeric enzyme undergoes complex regulation by phosphorylation and allosteric activation through binding to the substrate Phe in the N-terminal domain, which fine-tunes PAH enzyme activity by altering the various conformational and multimeric states of the enzyme (Knappskog 1996, Jaffe 2013, Arturo 2016).

[0007] Current treatment for PKU is lifelong dietary restriction of Phe using a low-protein diet and liquid medication formulas (Kochhar 2012, Ho 2014, Blau 2015). Although effective, the poor taste of the medication and the severe restrictions on food choices make it difficult for children to adhere to the diet, and noncompliance with the diet steadily increases, and by the late teens, nearly 80% of patients have blood Phe levels above recommended levels (Waisbren 2007, Thomas 2017). There is also emerging evidence that despite good adherence to the Phe-restricted diet, many patients have deficits in various neurocognitive and neuropsychiatric functions and a high incidence of attention deficit hyperactivity disorder (ADHD). Although the reasons for this are unclear, potential explanations include brain amino acid imbalances, nutritional deficiencies of certain vitamins and trace elements, and fluctuations in blood Phe levels that are normally maintained stable by hepatic PAH activity (Cleary 2013, Gonzales 2016, Vogel 2017). Interestingly, treatment of mild PKU patients with a synthetic form of the cofactor tetrahydrobiopterin (BH4) (sapropterin dihydrochloride) has not only been shown to effectively reduce blood Phe levels, but has also been shown to improve neurological function, such as reducing ADHD symptoms (Burton 2015). This therapy increases residual PAH enzyme activity by acting as a pharmacological chaperone and may therefore partially correct the genetic defect by providing normal Phe-regulated PAH activity (Blau 2015). Another recently approved therapy consists of enzyme replacement therapy using a pegylated form of bacterial phenylalanine ammonia lyase (PAL) that metabolizes Phe to trans-cinnamic acid. This therapy significantly reduces blood Phe levels but appears to be less effective for neurological endpoints (Longo 2014). It is unclear whether this therapy, or any other therapy based primarily on reducing blood Phe levels, without correcting the function of PAH as a regulator of systemic Phe levels and a Tyr producer, will be able to address the cognitive and neuropsychiatric problems observed even in dietary compliant PKU patients.

[0008] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the invention

[0009] The present invention is based, at least in part, on the inventors' discovery of improved hPAH variants, and in particular variant-1 (V1), which contains four amino acid changes that result in improved protein stability and enzyme activity compared to endogenous hPAH. Delivery of cDNA encoding this hPAH-V1 variant to the liver using clinically relevant doses of rAAV improves PAH in the human PKU model enu2"We found that the novel variant of hPAH was more effective than unmodified hPAH in the treatment of various disease endpoints in mice and was more efficacious than unmodified hPAH. Therefore, rAAV vectors encoding this novel variant may provide a pathway for PKU gene therapy by allowing efficacy to be achieved with reduced vector doses."

[0010] In some aspects, the present invention provides variant phenylalanine hydroxylase (PAH) polypeptides comprising two amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250 and G256 of a wild-type human PAH polypeptide. In some aspects, the present invention provides variant phenylalanine hydroxylase (PAH) polypeptides comprising three amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250 and G256 of a wild-type human PAH polypeptide. In some aspects, the present invention provides variant phenylalanine hydroxylase (PAH) polypeptides comprising four amino acid substitutions, wherein the amino acid substitutions are located at sites M180, K199, S250 and G256 of a wild-type human PAH polypeptide. In some embodiments, the amino acid substitutions include one or more of M180T, K199P, S250P and G256A. In some embodiments, the amino acid substitutions include K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P. In some embodiments, the amino acid substitutions include M180T, K199P, S250P, and G256A. In some embodiments, the variant PAH polypeptide further comprises H264P, G272A, G272P, P275L, P279Q, G272P, and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the wild-type human PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant PAH polypeptide is a human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant PAH polypeptide further comprises one or more amino acid substitutions selected from G33A, G46A, G46P, G103A, G139A, G139P, G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, and G442A of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide further comprises one or more amino acid substitutions selected from P9G, G10V, G12S, K184R, K192R, S196A, Y206H, H220R, Q336E, E360D, I374C, N376E, N401T, I421V, I441V, S446H of the wild-type human PAH polypeptide, and the addition of S at position 453 of the wild-type human PAH polypeptide.In some embodiments, the variant PAH polypeptide further comprises one or more amino acid substitutions selected from F240W, A246P, G247A, Y268W, C284F, T323R, F327Y, E319P, I306(Y,F), K113P, G188A, F191Y, T193R, Y206H, G337P and N376P of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises one or more amino acid substitutions selected from G33A, G46A, G46P, G103A, G139A, G139P, G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, and G442A of a wild-type human PAH polypeptide.

[0011] In some aspects, the present invention provides variant PAH polypeptides, wherein the variant PAH polypeptide comprises one or more amino acid substitutions selected from P9G, G10V, G12S, K184R, K192R, S196A, Y206H, H220R, Q336E, E360D, I374C, N376E, N401T, I421V, I441V, S446H of a wild-type human PAH polypeptide, and an addition of S at position 453 of the wild-type human PAH polypeptide. In some aspects, the present invention provides a variant PAH polypeptide comprising one or more amino acid substitutions selected from F240W, A246P, G247A, Y268W, C284F, T323R, F327Y, E319P, I306(Y,F), K113P, G188A, F191Y, T193R, Y206H, G337P and N376P of a wild-type human PAH polypeptide.

[0012] In some embodiments, the variant PAH polypeptide comprises an N-terminal truncation. In some embodiments, the N-terminal truncation comprises a truncation of an N-terminal regulatory domain. In some embodiments, the N-terminal truncation comprises a truncation of amino acid residues 1-102 of a wild-type PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises a C-terminal truncation. In some embodiments, the C-terminal truncation comprises a truncation of a tetramerization domain. In some embodiments, the C-terminal truncation comprises a truncation of amino acid residues 429-452 of a wild-type PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises an amino acid sequence corresponding to amino acid residues 103-428 of a wild-type PAH polypeptide.

[0013] In some embodiments, the variant PAH polypeptide comprises one or more amino acid substitutions to eliminate a potential protease cleavage site. In some embodiments, the one or more amino acid substitutions that eliminate a potential protease cleavage site are located at positions 270-295 and / or 380-405 of the wild-type PAH polypeptide.

[0014] In some embodiments, the variant PAH polypeptide is fused to a liver targeting polypeptide. In some embodiments, the liver targeting polypeptide is HGF or a fragment thereof or a glycoprotein that binds to the hepatocyte asialoglycoprotein receptor. In some embodiments, the variant PAH polypeptide is pegylated and / or nitrosylated. In some embodiments, the variant PAH polypeptide comprises an I374C amino acid substitution, wherein the cys residue at position 374 is nitrosylated.

[0015] In some embodiments, the invention provides compositions comprising a variant PAH polypeptide as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0016] In some aspects, the invention provides isolated nucleic acids encoding variant PAH polypeptides as described herein. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is operably linked to a promoter. In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), mTTR482 promoter, mA1MB2-mTTR482 promoter, TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, LP1 promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; a cytomegalovirus enhancer / chicken β-actin / rabbit β-globulin promoter (CAG) promoter, an elongation factor 1-α promoter (EF1-α) promoter, a human β-glucuronidase promoter, a chicken β-actin (CBA) promoter, a modified chicken β-actin (CBA) promoter, or a promoter of SEQ ID NO:17, reverse transcription Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter and 13-actin promoter. In some embodiments, the promoter is LP1 promoter or mA1MB2-mTTR482 promoter. In some embodiments, the nucleic acid further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal or an HSV TK polyadenylation signal. In some embodiments, the nucleic acid further comprises an intron. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globulin hybrid intron. In some embodiments, the intron is a modified chicken β-actin (CBA) / rabbit β-globulin hybrid intron of SEQ ID NO:15. In some embodiments, the nucleic acid further comprises one or more ITRs. In some embodiments, the nucleic acid further comprises a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid is optimized to remove the ATG sequence. In some embodiments, the stuffer nucleic acid is the A1AT intron stuffer sequence of SEQ ID NO:16.

[0017] In some aspects, the present invention provides an isolated nucleic acid encoding a human PAH polypeptide, wherein the nucleic acid is codon optimized. In some embodiments, the nucleic acid sequence is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid is mRNA. In some aspects, the present invention provides a composition comprising a nucleic acid as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0018] In some aspects, the present invention provides a vector comprising an isolated nucleic acid as described herein. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the rAAV vector comprises a nucleic acid as described herein, and its flank is one or more AAV reverse terminal repeat (ITR) sequences. In some embodiments, the flank of the nucleic acid is two AAV ITRs. In some embodiments, AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, cattle AAV or mouse AAV serotype ITR. In some embodiments, AAV ITR is AAV2 ITR. In some embodiments, the rAAV vector comprises, from 5' to 3', an AAV2 ITR, a promoter, an intron, a nucleic acid encoding a PAH polypeptide, a stuffer nucleic acid, a polyadenylation signal, and an AAV2 ITR. In some embodiments, the promoter is an m1A1MB2-mTTR482 promoter or an LP1 promoter. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globulin hybrid intron. In some embodiments, the PAH polypeptide is a variant PAH polypeptide as described herein. In some embodiments, the nucleic acid encoding the PAH polypeptide is a codon-optimized nucleic acid. In some embodiments, the stuffer nucleic acid comprises a nucleic acid from an intron of the human α1 antitrypsin gene. In some embodiments, the intron of the human α1 antitrypsin gene has been mutated to remove the ATG sequence. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal. In some embodiments, the vector is a self-complementary vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding the complement of the PAH polypeptide, wherein the first nucleic acid sequence can form intrachain base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutant AAV ITR, wherein the mutant AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal melting sequence.

[0019] In some aspects, the present invention provides rAAV particles comprising rAAV vectors as described herein. In some embodiments, the AAV virus particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV2 V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, mouse AAV or rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV virus particles comprise engineered AAV capsids. In some embodiments, the engineered AAV capsids are DJ capsids or LK03 capsids. In some embodiments, the ITRs and capsids of the rAAV virions are derived from the same AAV serotype. In some embodiments, the ITRs and capsids of the rAAV virions are derived from different AAV serotypes. In some embodiments, the rAAV virions comprise an AAV8 capsid. In some embodiments, the rAAV virions comprise an AAV8 capsid, and wherein the vector comprises an AAV2 ITR.

[0020] In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAVDJ, AAV2 N587A, AAV2E548A, AAV2 N708A, AAV2 V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV1 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV2 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV3 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV4 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV5 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to an AAV6 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity to an AAV7 serotype capsid, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.In some embodiments, the AAV virus particles contain a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to the AAV8 serotype capsid. In some embodiments, the AAV virus particles contain a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to the AAV9 serotype capsid. In some embodiments, the AAV virus particles contain a capsid having at least 85% sequence identity, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity, to the AAV11 serotype capsid. In some embodiments, the AAV virion comprises a capsid having at least 85% sequence identity to an AAV12 serotype capsid, such as at least 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0021] In some embodiments, the AAV virion comprises a hybrid capsid. In some embodiments, the hybrid capsid is an AAV1 / AAV6, AAV2 / AAV6, AAV3 / AAV6, AAV4 / AAV6, AAV5 / AAV6, AAV7 / AAV6, AAV8 / AAV6, AAV9 / AAV6, AAV10 / AAV6, AAV11 / AAV6, AAV12 / AAV6, AAV1 / AAV8, AAV2 / AAV8, AAV3 / AAV8, AAV4 / AAV8, AAV5 / AAV8, AAV7 / AAV8, AAV9 / AAV8, AAV10 / AAV8, AAV11 / AAV8, AAV12 / AAV8, or AAV1 / AAV6 / AAV8 hybrid capsid. In some embodiments, the hybrid capsid is an AAV8 / AAV6 hybrid capsid. In some embodiments, the hybrid capsid is an AAV1 / AAV6 / AAV8 hybrid capsid.

[0022] In some embodiments, the AAV virion comprises a hybrid capsid. In some embodiments, the hybrid capsid is an AAV1 / AAV6, AAV2 / AAV6, AAV3 / AAV6, AAV4 / AAV6, AAV5 / AAV6, AAV7 / AAV6, AAV8 / AAV6, AAV9 / AAV6, AAV10 / AAV6, AAV11 / AAV6, AAV12 / AAV6, AAV1 / AAV8, AAV2 / AAV8, AAV3 / AAV8, AAV4 / AAV8, AAV5 / AAV8, AAV7 / AAV8, AAV9 / AAV8, AAV10 / AAV8, AAV11 / AAV8, AAV12 / AAV8, or AAV1 / AAV6 / AAV8 hybrid capsid. In some embodiments, the hybrid capsid is an AAV8 / AAV6 hybrid capsid. In some embodiments, the hybrid capsid is an AAV1 / AAV6 / AAV8 hybrid capsid.

[0023] In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of an AAV1 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of an AAV2 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of an AAV3 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of an AAV4 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of an AAV5 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of the AAV6 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of the AAV7 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of the AAV8 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of the AAV9 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98% or 99% sequence identity, to a portion of the AAV10 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98%, or 99% sequence identity, to a portion of an AAV12 capsid sequence. In some embodiments, the hybrid capsid comprises an amino acid sequence having at least 95% sequence identity, such as at least 96%, 97%, 98%, or 99% sequence identity to a portion of an AAV8 capsid sequence. In some embodiments, the portion comprises at least 100 amino acids, such as at least 150, 200, 250, 300, 350, or 400 amino acids.

[0024] In some aspects, the invention provides a composition comprising a rAAV particle as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier.

[0025] In some aspects, the invention provides a cell comprising an isolated nucleic acid as described herein. In some aspects, the invention provides a method of producing a variant PAH polypeptide, the method comprising culturing the above-mentioned cell under conditions that produce the variant PAH polypeptide. In some embodiments, the method further comprises the step of purifying the variant PAH polypeptide.

[0026] In some aspects, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a variant PAH polypeptide as described herein or a composition as described herein. In some aspects, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a nucleic acid encoding a variant PAH polypeptide as described herein or a nucleic acid as described herein. In some aspects, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a rAAV vector as described herein. In some aspects, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a rAAV particle as described herein. In some embodiments, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a composition as described herein. In some embodiments, the present invention provides a method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a cell as described herein. In some embodiments, the individual lacks PAH activity.

[0027] In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a variant PAH polypeptide as described herein. In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a nucleic acid encoding a variant PAH polypeptide as described herein or a nucleic acid as described herein. In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a rAAV vector as described herein. In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a rAAV particle as described herein. In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a composition as described herein. In some embodiments, the level of phenylalanine in the blood of an individual before treatment is increased compared to the level of phenylalanine in the blood of an equivalent matched control (peer-matched control) individual. In some aspects, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need, comprising administering to the individual a cell as described herein.

[0028] In some embodiments, the nucleic acid, rAAV vector, rAAV particle, composition, or cell is administered intravenously, intraarterially, intrahepatically, intraportally, intraperitoneally, or subcutaneously. In some embodiments, the administration is combined with another therapy. In some embodiments, the another therapy is treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia lyase (PAL) or pegylated PAL, or a phenylalanine restricted diet.

[0029] In some aspects, the invention provides methods for preparing a PAH polypeptide, comprising culturing a cell as described herein under conditions where the PAH polypeptide is produced. In some embodiments, the method further comprises purifying the PAH polypeptide.

[0030] In some aspects, the invention provides kits comprising a variant PAH polypeptide as described herein. In some embodiments, the invention provides kits comprising a nucleic acid as described herein, a rAAV vector as described herein, a rAAV particle as described herein, or a composition as described herein. In some embodiments, the kit further comprises instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, vial, and / or syringe.

[0031] In some aspects, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (pPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer. In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is on the 5' side of the mTTR promoter.

[0032] In some aspects, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element linked to a human α1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR). In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the 3' element is located on the 3' side of the transgene.

[0033] In some aspects, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (pPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer, and wherein the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element (3'AlbSMAR) linked to a human α1 antitrypsin scaffold / matrix attachment region (SMAR). In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is on the 5' side of the mTTR promoter. In some embodiments, the 3' element is located on the 3' side of the transgene.

[0034] In some embodiments, the expression cassette further comprises an intron. In some embodiments, the intron is a chicken β-actin / rabbit β-globin hybrid intron. In some embodiments, the expression cassette further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0035] In some embodiments, the transgene encodes a PAH polypeptide or a variant PAH polypeptide. In some embodiments, the invention provides a vector comprising an expression cassette as described herein. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector.

[0036] In some embodiments, the present invention provides a rAAV vector comprising an expression cassette as described herein, the flank of the expression cassette is one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the flank of the expression cassette is two AAV ITRs. In some embodiments, the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV or mouse AAV serotype ITR. In some embodiments, the AAV ITR is AAV2 ITR.

[0037] In some embodiments, the vector is a self-complementary vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding the complement of the PAH polypeptide, wherein the first nucleic acid sequence can form an intrachain base pair with the second nucleic acid sequence along most or all of its length. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutant AAV ITR, wherein the mutant AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal melting sequence.

[0038] In some embodiments, the present invention provides rAAV particles comprising rAAV vectors as described herein. In some embodiments, the AAV virus particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2N587A, AAV2E548A, AAV2 N708A, AAV2 V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV, mouse AAV or rAAV2 / HBoV1 serotype capsid. In some embodiments, the AAV virus particles comprise engineered AAV capsids. In some embodiments, the engineered AAV capsids are DJ capsids or LK03 capsids. In some embodiments, the ITRs and capsids of the rAAV virions are derived from the same AAV serotype. In some embodiments, the ITRs and capsids of the rAAV virions are derived from different AAV serotypes. In some embodiments, the invention provides a composition comprising rAAV particles as described herein. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A-Figure 1C Optimization of the liver promoter is shown. Figure 1A Schematic diagram of proviral plasmid constructs is shown. Construct mTTR482 (No. 1) was modified by adding various liver enhancer elements (Construct Nos. 2-6) or 3' stability elements (Construct Nos. 7-8). Constructs containing CBA promoter were used for comparison. All constructs contained flanking ITRs, hybrid introns and SEAP reporter. The constructs were then transfected into Huh7 cells ( Figure 1B ) or HepG2 cells ( Figure 1C ). Secreted SEAP activity levels were measured 72 hours after transfection and normalized by the level of b-galactosidase from the co-transfected LacZ plasmid. Each evaluation included n=3-4 / plasmid.

[0040] Figure 2A and Figure 2B Evaluation of liver promoters by in vivo SEAP production is shown. Figure 2A SEAP levels in normal C57BL / 6 mice are shown. Figure 2B PAH enu2 SEAP levels in mice. In both experiments, plasmid vectors were delivered by bolus injection and plasma SEAP activity was measured at different time points. Each treatment group contained n=3-6 animals.

[0041] Figure 3A-3D PAH enu2 Comparison of liver promoter performance in mice. rAAV8 vectors expressing mPAH from either sc-LP1 or ss-A1MB2-mTTR promoter (leader) were administered to male PAH mice at 4e10 (L) or 1e11 (M) VG / mouse. enu2 Mouse. Figure 3A Blood Phe levels are shown over a 56 day time course. Levels are mean values ​​of n=8 / group, except for n=2 naive animals. Figure 3B Shown are blood Phe levels at day 56. Shown are plasma Phe levels of animals treated with both constructs administered at 4e10 or 1e11 VG / mouse or untreated animals. Figure 3C Blood Tyr levels are shown. Levels were measured before vehicle administration (pre-bleeding) and 7 and 56 days after vehicle administration. Each value is the mean of n=8 / group. Figure 3D Shown are vector genomes in the liver at day 56. Vector copies were determined by qPCR and the average of n=5 / group is shown.

[0042] Figure 4A-4D Shown for PAH enu2 Analysis of mouse brains. PAH from untreated, ss-mA1MB2-mTTR (leader) construct (4e10 and 1e11 vg / mouse) treated, and sc-LP1 (1e11 vg / mouse) treated mice were sacrificed 56 days after treatment. enu2 Five animals of mice were perfused with PBS and brains were collected for analysis. Balb / C mice were used as wild-type controls (WT). Figure 4A Brain Phe levels are shown. Figure 4B Correlation of blood and brain Phe levels is shown. Figure 4C Brain dopamine levels are shown. Figure 4D Brain serotonin levels are shown. All values ​​represent the mean of n=5 / group (except untreated PAH enu2 Mice n = 2). Figures 3A-3C The research was conducted as described.

[0043] Figure 5A and Figure 5B Codon optimization of human PAH cDNA is shown. The cDNA encoding FLAG-tagged hPAH was cloned into the mTTR482-hPAH-BGHpA expression plasmid. The production of hPAH was tested in vitro and in vivo. Figure 5A FLAG-hPAH levels in Huh7 cells are shown. Plasmids were transfected and cell lysates were prepared 48 hours later. PAH protein levels in cell lysates were analyzed by Western blotting. Figure 5B FLAG-hPAH levels in liver of C57BL / 6 mice are shown. Plasmids were administered by hydrodynamic injection and livers were collected 24 hours later. FLAG-tagged hPAH levels in liver lysates were quantified using FLAG-ELISA and normalized by total protein. Values ​​represent the mean of n=4-5 animals / group. Abbreviations: C, negative control plasmid; GS, Genscript, GA, GeneArt, GS CpG, Genscript sequence with CpG removed; non, original hPAH DNA sequence; original Untreated animals.

[0044] Fig. 6A is a Western blot showing the expression of human phenylalanine hydroxylase (hPAH) and mouse phenylalanine hydroxylase (mPAH) under the control of different promoters (mTTR482, CBA and LP1). The expression plasmids were transfected into Huh7 cells and cell lysates were analyzed for FLAG-PAH levels two days later using an antibody against FLAG.

[0045] Figure 6B is a Western blot showing a comparison of the in vitro expression of full-length (FL) and double-truncated (DT) human and mouse PAH. Fig. 6A .

[0046] Figure 6C Comparison of hPAH and mPAH protein levels of both full-length (FL) and double-truncated (DT) forms. Quantification of FLAG-tagged proteins was performed by ELISA on Huh7 cell lysates after transfection with the expression plasmids.

[0047] Fig.6D The mRNA levels of hPAH and mPAH were compared by qPCR. Figure 6C The materials used were used for RNA analysis.

[0048] Fig. 6Eis a Western blot showing analysis of purified hPAH and mPAH (both produced in full length and double truncated forms). Proteins were purified by FLAG affinity column and run in SDS-PAGE gel followed by detection with anti-FLAG antibody.

[0049] Fig. 6F The efficacy of rAAV8 vectors encoding hPAH or mPAH in the PKU mouse model is shown. Both hPAH and mPAH were expressed from a self-complementary vector (sc-LP1) with the LP1 promoter. Efficacy was measured as a single IV injection into PAH enu2 The results were measured by the reduction of blood Phe levels in mice.

[0050] Figure 7A-7C The generation of a mouse / human hybrid PAH construct is shown. Fig. 7A A schematic diagram of the mouse / human hybrid PAH construct is shown. Regions derived from human (green) and mouse (grey) PAH are shown. Figure 7B Shown is the effect of replacing the N-terminal region of hPAH on protein expression levels. Figure 7C The effect of changes in the C-terminal region of hPAH on protein expression levels is shown. The experiment was performed by transfecting a plasmid with a CBA-PAH expression cassette into 293T cells. After 48 hours, cells were harvested for quantification of FLAG-PAH by FLAG ELISA.

[0051] Fig. 8A and Figure 8B In vitro screening of double-truncated hPAH (hPAH-DT) variants is shown. Fig. 8A The protein expression levels of the plasmid-expressed hPAH protein variants (No. 1 to No. 8) are shown. Figure 8B PAH activity levels of plasmid expressed hPAH protein variants are shown. All variants were transfected into 293 cells and expressed from plasmids with a CBA promoter. The results for the variant plasmids were compared to similar plasmids expressing human or mouse PAH.

[0052] Figure 9A-9C Characterization of hPAH-V1-DT derivatives for in vitro PAH production is shown. Fig. 9A The PAH activity levels of hPAH-V1-DT derivatives are shown. Fig. 9B PAH protein levels of hPAH-V1-DT derivatives are shown. Fig. 9C Specific PAH activities of hPAH-V1-DT derivatives are shown. Results are based on analysis of cell lysates from transfected 293 cells.

[0053] Figure 10A-10CShown are replicate analyses of hPAH-V1-DT derivatives for in vitro PAH production. Fig. 10A The PAH activity levels of selected hPAH-V1-DT derivatives are shown. Fig. 10B PAH protein levels of hPAH-V1-DT derivatives are shown. Fig. 10C The specific PAH activity of hPAH-V1-DT derivatives is shown. Fig. 8A and 8B Generated as described in . Fig. 10D PAH activities of three double mutant forms of variant-1 are shown. Fig. 8A and 8B Generated as described in .

[0054] Figure 11A-11C Shown is an in vitro comparison of full-length hPAH-V1 with mouse PAH (mPAH) and human PAH (hPAH). Fig.11A is Western blot analysis of protein expression. Fig. 11B Shown are PAH protein levels by FLAG-ELISA. Fig. 11C PAH activity levels are shown. Data were generated by transfecting an expression plasmid encoding the full-length PAH protein with the liver promoter A1MB2-mTTR482 into Huh7 cells followed by analysis of cell lysates. m indicates the marker lane.

[0055] Figure 12A-12C Shown in PAH enu2 Comparison of the efficacy of rAAV vectors expressing full-length hPAH-V1 in mice with that in mouse and human PAH. Fig. 12A Blood phenylalanine levels are shown. Fig. 12B Blood tyrosine levels are shown. Fig. 12C Blood phenylalanine metabolite levels are shown. P values ​​are indicated as: * for P < 0.05, ** for P < 0.01, and *** for P < 0.001. All treatment groups consisted of AAV8 vectors expressing FLAG-tagged PAH from a liver promoter. Vectors were injected at 3e11 (hPAH, hPAH-V1, and mPAH) or 1e12 vg / mouse (hPAH and hPAH-V1) by IV route on day 0. Initial PAH enu2 Mice and heterozygotes (HET) were used as negative and positive controls, respectively.

[0056] Figure 13A-13C Quantification of rAAV vector genome and 3x-FLAG-PAH levels in the liver is shown. Fig.13A PAH protein levels are shown. Fig. 13B PAH activity levels are shown. Fig. 13C Vector genome copies in the liver are shown. Figures 12A-12C The experiments were performed as described, and livers were collected 69 days after vector administration. In each figure, data for 3e11 VG / mouse treatment groups (hPAH, hPAH-V1, and mPAH) and 1e12 vg / mouse treatment groups (hPAH, hPAH-V1) are shown. P value, *** is <0.001.

[0057] Figures 14A-14D Quantification of various amino acid levels in the brain is shown. Fig.14A Brain phenylalanine levels are shown. Fig. 14B Brain tyrosine levels are shown. Fig. 14C A correlation between brain and blood phenylalanine levels is shown. Fig.14D Brain tryptophan levels are shown. Figures 12A-12C The experiments were performed as described. Data shown are from the treatment group (n=5 / group) dosed with 3e11 VG / mouse (day 69). P value, *** is <0.001.

[0058] Figures 15A-15D Neurotransmitter levels in the brain are shown. Fig.15A Brain dopamine levels are shown. Fig. 15B Shown are brain levels of the dopamine metabolite DOPAC. Fig. 15C Brain serotonin levels are shown. Fig.15D Brain levels of the serotonin metabolite HIAA are shown. Figures 12A-12C The results were performed as described, and values ​​are from 3e11 VG / mouse cohort (day 69). P values, ** is P<0.01, and *** is P<0.001.

[0059] Figure 16A-16B Shown is a comparison of the efficacy of 3.8 and 4.6 kb A1MB2-mTTR-hPAV-V1 vector genomes. Fig.16A A schematic diagram of the vector is shown. Fig. 16B PAH enu2 Efficacy testing in mice. AAV8 vectors expressing hPAH-V1 from a liver promoter (leader) were administered once at 1e11 vg / mouse and efficacy was measured as blood Phe levels. These vectors were also compared to a 4.6 kb vector with hPAH-V1 and no N-terminal tag (tested at 1e11 and 3e11 vg / mouse).

[0060] Figure 17A-17C Shown is an in vivo comparison of hPAH-V1 and hPAH production in non-human primate livers following delivery with rAAV vectors. Fig.17A The levels of rAAV vector genomes in the liver of each individual animal are shown. Fig. 17BVector-derived mRNA levels in the liver and spleen of each treated animal are shown. The mRNA levels of each animal were normalized to vector genome copies. Fig. 17C Detection of FLAG-tagged PAH protein in liver homogenates is shown. Equal lysate loading was confirmed by detection of the housekeeping protein GAPDH. Experiments were performed by IV administration of 5e12 g / kg of rAAV vector expressing FLAG-tagged PAH or PAH-V1 from the A1MB2-mTTR promoter. Tissues were collected 2 weeks later for analysis of vector genome, vector-derived mRNA, and PAH protein in the liver. DETAILED DESCRIPTION

[0061] In some aspects, the invention described herein provides variants of human PAH (hPAH-V1) polypeptides comprising at least two or four amino acid changes that confer higher PAH protein and / or activity levels in vitro and in vivo compared to wild-type hPAH. In other aspects, the invention described herein provides improved variants of human PAH (hPAH-V1) comprising at least two, three or four amino acid changes that confer higher PAH protein and / or activity levels in vitro and in vivo compared to wild-type hPAH. In some aspects, the invention provides nucleic acids encoding the human PAH variants. In some aspects, the invention provides expression cassettes, recombinant adeno-associated virus (rAAV) vectors and viral particles, and pharmaceutical compositions comprising variant PAH polypeptides of the present disclosure. In other aspects, the invention provides methods for treating phenylketonuria (PKU); for example, by increasing PAH activity, increasing tyrosine and tryptophan transport to the brain, and normalizing brain neurotransmitter levels including dopamine and serotonin. In yet other aspects, the invention provides kits for treating PKU in an individual using the variant PAH of the present disclosure.

[0062] I. General Technology

[0063] Those skilled in the art are generally familiar with and routinely use routine methods using the techniques and procedures described or referenced herein, such as, for example, the widely used methods described in the following literature: Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012); Current Protocols in Molecular Biology (FM Ausubel, et al., ed., 2003); these series Methods in Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, ed., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, ed., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (edited by A.Doyle, JBGriffiths and DG Newell, J.Wiley and Sons, 1993-8); Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell, 1996); Gene TransferVectors for Mammalian Cells (JMMiller and MPCalos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (J. E. Coligan et al., 1991); Short Protocols in Molecular Biology (Ausubel et al., 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (VT DeVita et al., eds., JB Lippincott Company, 2011).

[0064] II. Definitions

[0065] As used herein, "vector" refers to a recombinant plasmid or virus comprising a nucleic acid to be delivered into a host cell in vitro or in vivo.

[0066] As used herein, the term "polynucleotide" or "nucleic acid" refers to a polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. Therefore, this term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA; genomic DNA; cDNA; DNA-RNA hybrids; or polymers including purine and pyrimidine bases or other natural, chemically modified or biochemically modified nucleotide bases, non-natural or derived nucleotide bases. The backbone of a polynucleotide may include sugars and phosphate groups (as typically found in RNA or DNA) or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide may include a polymer of synthetic subunits (such as aminophosphorylates), and may therefore be oligodeoxynucleoside phosphoramidates (P-NH2) or mixed phosphoramidate-phosphodiester oligomers. In addition, double-stranded polynucleotides may be obtained from chemically synthesized single-stranded polynucleotide products by synthesizing complementary chains and annealing these chains under appropriate conditions or by synthesizing complementary chains de novo using DNA polymerase with appropriate primers.

[0067] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers and polymers of amino acid residues. The definition encompasses both full-length proteins and fragments thereof. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In addition, for the purposes of this disclosure, "polypeptide" refers to a protein including modifications (such as deletions, additions and substitutions, which are generally conservative in nature) to the native sequence, as long as the protein maintains the desired activity. These modifications may be intentional (such as by site-directed mutagenesis), or may be accidental (such as by mutations of the host producing the protein or due to errors caused by PCR amplification).

[0068] "Recombinant viral vector" refers to a recombinant polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one, and in embodiments two, inverted terminal repeats (ITRs).

[0069] "Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more flanks, which are at least one, and in an embodiment, two AAV reverse terminal repeats (ITRs) of heterologous sequences (i.e., nucleic acid sequences not derived from AAV). When such rAAV vectors are present in a host cell that has been infected with a suitable helper virus (or the helper virus expresses a suitable auxiliary function) and is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins), the rAAV vector can be replicated and packaged in infectious virions. When the rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid for cloning or transfection), the rAAV vector can be referred to as a "pre-vector", which can be "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable auxiliary functions. The rAAV vector can be any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsulated in liposomes, and encapsidated in viral particles (particularly AAV particles). rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)".

[0070] "Heterologous" means an entity that is genotypically different from the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and may encode a heterologous polypeptide when expressed). Similarly, a cellular sequence (e.g., a gene or portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence relative to the vector.

[0071] The term "transgene" refers to a polynucleotide that is introduced into a cell and can be transcribed into RNA and optionally translated and / or expressed under appropriate conditions. In many aspects, it confers a desired characteristic to the cell into which it is introduced, or otherwise produces a desired therapeutic or diagnostic result.

[0072] "Chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from a chicken β-actin gene (e.g., Gallus gallus β-actin, represented by GenBank Entrez Gene ID 396526). As used herein, "chicken β-actin promoter" may refer to a promoter containing a cytomegalovirus (CMV) early enhancer element, a promoter and the first exon and intron of a chicken β-actin gene, and a splice acceptor of a rabbit β-globin gene, such as the sequence described in Miyazaki, J. et al. (1989) Gene 79 (2): 269-77. As used herein, the term "CAG promoter" may be used interchangeably. As used herein, the term "CMV early enhancer / chicken β-actin (CAG) promoter" may be used interchangeably.

[0073] The term "genomic particles (gp)", "genome equivalents" or "genomic copies" as used in relation to viral titer refers to the number of viral particles containing the recombinant AAV DNA genome, and is not related to infectivity or functionality. The number of genomic particles in a particular vector preparation can be measured by procedures such as described in the Examples herein or, for example, in the following literature: Clark et al. (1999) Hum. Gene Ther., 10: 1031-1039; Veldwijk et al. (2002) Mol. Ther., 6: 272-278.

[0074] The term "vector genome (vg)" as used herein may refer to one or more polynucleotides comprising a polynucleotide sequence of a set of vectors (e.g., viral vectors). The vector genome may be encapsidated in virions. Depending on the specific viral vector, the vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA or double-stranded RNA. The vector genome may include endogenous sequences associated with a specific viral vector and / or any heterologous sequences inserted into a specific viral vector by recombinant techniques. For example, a recombinant AAV vector genome may include at least one ITR sequence flanking a promoter, a stuffing fragment, a sequence of interest (e.g., RNAi), and a polyadenylation sequence. A complete vector genome may include a full set of polynucleotide sequences of a vector. In some embodiments, the nucleic acid titer of a viral vector may be measured in vg / mL. Methods suitable for measuring this titer are known in the art (e.g., quantitative PCR).

[0075] The terms "infectious unit (iu)", "infectious particles" or "replication units" as used in reference to viral titers refer to the number of infectious and replicative recombinant AAV vector particles as measured by the infectious center assay (also called the replication center assay), as described, for example, in McLaughlin et al. (1988) J. Virol., 62: 1963-1973.

[0076] The term "transducing unit (tu)" as used in reference to viral titer refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured in a functional assay, such as described in the Examples herein or, for example, in the following references: Xiao et al. (1997) Exp. Neurobiol., 144: 113-124; or Fisher et al. (1996) J. Virol., 70: 520-532 (LFU assay).

[0077] "Inverted terminal repeat" or "ITR" sequence is a term well known in the art and refers to relatively short sequences in opposite orientations found at the ends of a viral genome.

[0078] "AAV inverted terminal repeat (ITR)" sequence is a term well known in the art and is a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (designated A, A', B, B', C, C' and D regions) that allow intrastrand base pairing to occur within this portion of the ITR.

[0079] "Terminal melting sequence" or "trs" is a sequence in the D region of the AAV ITR that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal melting sequences are difficult to be cleaved by the AAV rep protein.

[0080] "AAV helper function" refers to a function that allows AAV to be replicated and packaged by a host cell. AAV helper function can be provided in any of a variety of forms, including but not limited to a helper virus or helper virus gene that assists in AAV replication and packaging. Other AAV helper functions are known in the art, such as genotoxic agents.

[0081] The "helper virus" of AAV refers to a virus that allows AAV (which is a defective parvovirus) to be replicated and packaged by host cells. Helper viruses provide "helper functions" that allow AAV to replicate. A variety of such helper viruses have been identified, including adenoviruses, herpes viruses, and poxviruses, such as vaccinia and baculoviruses. Adenoviruses cover a variety of different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Many adenoviruses of human, non-human mammalian and avian origin are known and can be obtained from depository institutions such as ATCC. Herpes family viruses that can also be obtained from depository institutions such as ATCC include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) and pseudorabies virus (PRV). Examples of adenovirus helper functions for replicating AAV include E1A function, E1B function, E2A function, VA function and E4orf6 function. Baculoviruses available from depository institutions include the Autographa californica nuclear polyhedrosis virus.

[0082] If the ratio of infectious AAV particles to infectious helper virus particles is at least about 10 2 :1; at least about 10 4 :1. At least about 10 6 :1; or at least about 10 8 :1 or more, then the preparation of rAAV is said to be "substantially free" of helper virus. In some embodiments, the preparation also does not contain an equivalent amount of helper virus protein (i.e., if the above-mentioned helper virus particle impurity is present in a disrupted form, the protein will be present due to this level of helper virus). Viral and / or cellular protein contamination can usually be observed on SDS gels as the presence of Coomassie-stained bands (e.g., the appearance of bands different from those corresponding to the AAV capsid proteins VP1, VP2 and VP3).

[0083] "Percentage (%) of sequence identity" with respect to a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in a reference polypeptide or nucleic acid sequence after the sequences are aligned and spaces are introduced (if necessary) to achieve the maximum percentage of sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment for the purpose of determining amino acid or nucleic acid sequence identity percentages can be achieved in a variety of ways within the technical scope of the art, such as using publicly available computer software programs, such as Current Protocols in Molecular Biology (Ausubel et al., ed., 1987), Supplement 30, Chapter 7.7.18, those described in Table 7.7.1, and including BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the sequence being compared. For purposes herein, the % amino acid sequence identity of a given amino acid sequence A to, with or against a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity to, with or against a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. For purposes herein, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which may alternatively be expressed as a given nucleic acid sequence C having or comprising a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 multiplied by a fraction W / Z, where W is the number of nucleotides scored as identical matches by a sequence alignment program in the program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that when the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not be equal to the % nucleic acid sequence identity of D to C.

[0084] An "isolated" molecule (eg, nucleic acid or protein) or cell means one that has been identified and separated and / or recovered from a component of its natural environment.

[0085] An "effective amount" is an amount sufficient to produce a beneficial or desired result, including a clinical result (e.g., improvement of symptoms, achievement of a clinical endpoint, etc.). An effective amount can be administered in one or more administrations. With respect to a disease state, an effective amount is an amount sufficient to improve, stabilize the disease, or delay the progression of the disease.

[0086] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0087] As used herein, "treatment" is an approach for obtaining a beneficial or desired clinical outcome. For purposes of this disclosure, beneficial or desired clinical outcomes include, but are not limited to, the following: relief of symptoms, reduction in extent of disease, stabilization of the disease state (e.g., not worsening), prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or alleviation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean prolonging survival compared to the expected survival if treatment is not received.

[0088] As used herein, the term "prophylactic treatment" refers to treatment in which an individual is known or suspected to have a disorder or is at risk for developing a disorder but does not yet exhibit symptoms of the disorder or exhibits minimal symptoms of the disorder. An individual undergoing prophylactic treatment may be treated prior to the onset of symptoms.

[0089] As used herein, "phenylalanine hydroxylase (PAH)" is an enzyme (EC 1.14.16.1) that catalyzes the hydroxylation of the aromatic side chain of phenylalanine to produce tyrosine. PAH is a monooxygenase that uses tetrahydrobiopterin (BH4, a pterin cofactor) and non-heme iron for catalysis. During the reaction, molecular oxygen is heterolytically cleaved, wherein one oxygen atom is successively incorporated into BH4 and the phenylalanine substrate. The hydroxylation of phenylalanine to tyrosine is the rate-limiting step in the catabolism of phenylalanine, and the lack of this enzyme activity leads to the autosomal recessive disorder phenylketonuria. PAH may also be referred to as PH, PKU or PKU1. PAH is a multidomain protein that consists of an N-terminal regulatory domain (1-117), a central catalytic domain (118-410) and a C-terminal tetramerization domain (411-452). Human PAH is provided in GenBank; for example, NM_000277, NM_00877, NM_001354304, NP_000268, NP_032803, NP_001341233, AAA60082.1, AAH26251.1, AAC51772.1, and AAL78816.1 (GI: 18765885). An example of human PAH is provided as SEQ ID NO:1.

[0090] As used herein, "phenylketonuria (PKU)" refers to a genetic defect in the liver enzyme phenylalanine hydroxylase (PAH). In the absence of any treatment, severe forms of PKU result in highly elevated blood Phe levels, which are neurotoxic and associated with severe mental retardation.

[0091] "mTTR promoter" refers to a polynucleotide sequence derived from the mouse transthyretin gene. Examples of mTTR promoters, mTTR482, are provided by Kyostio-Moore, (2016) and Nambiar (2017).

[0092] "Modified prothrombin enhancer (mPrT2)" refers to two copies of a polynucleotide sequence derived from the human prothrombin gene. Examples of mPrT2 enhancers are provided by (McEachern 2006, Jacobs 2008). An example of an mPrT2 sequence is provided by SEQ ID NO:7.

[0093] "Modified α1-microbikunin (mA1MB2)" refers to two copies of a polynucleotide sequence derived from the human α1-microbikunin / bikunin gene. An example of mA1MB2 is the enhancer of (McEachern 2006, Jacobs 2008). An example of a mA1MB2 sequence is provided by SEQ ID NO:8.

[0094] "Modified mouse albumin enhancer (mEalb)" refers to a polynucleotide sequence derived from the murine albumin gene. An example of an mEalb enhancer is provided by (Kramer 2003). An example of an mEalb sequence is provided by SEQ ID NO:9.

[0095] "Hepatitis B virus enhancer II (HEII)" refers to a polynucleotide sequence located upstream of the pre-C region promoter from the hepatitis B virus. An example of the hEII enhancer is provided by (Kramer 2003). An example of the HEII sequence is provided by SEQ ID NO:10.

[0096] "CRM8" refers to a cis-acting regulatory module derived from a polynucleotide sequence from the human Serpinal gene (Chuah 2014). An example of a CRM8 sequence is provided by SEQ ID NO:11.

[0097] "Alb 3" refers to the polynucleotide sequence on the 3' side of the coding region of the human albumin gene. Examples of Alb 3' elements are provided by Wooddell (2008). Examples of Alb 3' sequences are provided by SEQ ID NO: 12. "Alb3' / SMAR" refers to Alb3' attached to the scaffold / matrix attachment region of the human α1-antitrypsin gene (AF156542). Examples of Alb3' / SMAR sequences are provided by SEQ ID NO: 13.

[0098] References herein to "about" a value or parameter include (and describe) embodiments involving the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0099] As used herein, the singular forms "a," "an," and "the" include plural referents unless otherwise indicated.

[0100] It should be understood that aspects and embodiments of the disclosure described herein include "comprising," "consisting of," and / or "consisting essentially of" aspects and embodiments.

[0101] III. PAH variants

[0102] In some aspects, the present invention provides variant PAH polypeptides that, when expressed in an individual, confer higher PAH expression and / or activity levels. In some embodiments, the variant PAH polypeptide comprises at least two amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250, S251, H264, G256, G272, G275, P279, T323, and F327 of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises at least two amino acid substitutions at H264 and G275. In some embodiments, the variant PAH polypeptide comprises at least three amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises four amino acid substitutions at sites M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises five amino acid substitutions at M180, K199, S250, S251, G256, G272, and P279. In some embodiments, the variant PAH polypeptide comprises six amino acid substitutions at M180, K199, S250, G256, T323, and F327. In some embodiments, the variant PAH polypeptide comprises at least two amino acid substitutions of H264P and G275H. In some embodiments, the variant PAH polypeptide comprises at least three amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions including: K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P. In some embodiments, the variant PAH polypeptide further comprises amino acid substitutions H264P, G272A, G272P, P275L, P279Q, G272P and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the variant PAH polypeptide is any one of the variant PAH polypeptides listed in Tables 1-3. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions M180T, K199P, S250P and G256A, and comprises additional amino acid substitutions while at least approximately retaining the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the positions of the amino acid substitutions are based on a wild-type human PAH polypeptide; for example, a human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0103] In some embodiments, the variant PAH polypeptide is a human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant PAH polypeptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the variant PAH polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, a variant PAH polypeptide comprising an amino acid sequence that is at least about any of 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of the wild-type PAH of SEQ ID NO: 1.

[0104] In some embodiments, the variant PAH polypeptide is a truncated PAH polypeptide that retains phenylalanine hydroxylase activity. In some embodiments, the truncated PAH polypeptide comprises an N-terminal truncation. In some embodiments, the N-terminal truncation is a partial or complete truncation of the N-terminal regulatory domain. In some embodiments, the N-terminal truncation is a deletion of amino acid residue 1 to about amino acid residue 102 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the truncated PAH polypeptide comprises a C-terminal truncation. In some embodiments, the C-terminal truncation is a partial or complete truncation of a tetramerization domain. In some embodiments, the C-terminal truncation is a deletion of amino acid residues from about 429 to 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the variant PAH polypeptide comprises an N-terminal truncation and a C-terminal truncation. In some embodiments, the truncated PAH polypeptide comprises a partial or complete truncation of the N-terminal regulatory sequence and a partial or complete truncation of the tetramerization domain. In some embodiments, the truncated PAH polypeptide comprises a deletion of amino acid residue 1 to about amino acid residue 102 and about 429 to about 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the variant PAH polypeptide comprises an amino acid sequence corresponding to amino acid residues from about 102 to about 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the variant PAH polypeptide comprises an amino acid sequence corresponding to amino acid residues from 102 to 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the truncated PAH polypeptide further comprises four amino acid substitutions at positions M180, K199, S250, and G256 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the truncated PAH polypeptide comprises four amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the truncated variant PAH polypeptide further comprises any combination of amino acid substitutions listed in Tables 1 to 3. In some embodiments, the truncated PAH polypeptide has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of a wild-type PAH (e.g., a PAH polypeptide of SEQ ID NO: 1).

[0105] In some embodiments, the variant PAH polypeptide comprises one or more amino acid substitutions at one or more of the following positions: G33, G46, G103, G139, G148, G188, G218, G239, G247, G257, G272, G289, G307, G312, G332, G337, G344, G352, or G442. In some embodiments, the variant PAH polypeptide comprises one of the following amino acid substitutions: G33A, G46(A, P), G103A, G139(A, P), G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, or G442A. In some embodiments, the Variant No. 1 PAH polypeptide further comprises one or more amino acid substitutions at one or more of the following positions: G33, G46, G103, G139, G148, G188, G218, G239, G247, G257, G272, G289, G307, G312, G332, G337, G344, G352, or G442. In some embodiments, the variant number 1 PAH polypeptide comprises one or more of the following amino acid substitutions: G33A, G46(A, P), G103A, G139(A, P), G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, or G442A.

[0106] In some embodiments, the variant PAH polypeptide (e.g., variant number 1 PAH polypeptide) comprises one or more of the following amino acid substitutions: P9G, G10V, G12S, K184R, K192R, S196A, Y206H, H220R, Q336E, E360D, I374C, N376E, N401T, I421V, I441V, S446H. In some embodiments, the variant PAH polypeptide (e.g., variant number 1) further comprises a serine residue (Ser453) at the C-terminus of human PAH.

[0107] In some embodiments, the variant PAH polypeptide (e.g., variant number 1 PAH polypeptide) comprises one or more of the following amino acid substitutions: F240W, A246P, G247A, Y268W, C284F, T323R, F327Y, E319P, I306 (Y, F), K113P, G188A, F191Y, T193R, Y206H, G337P, N376P.

[0108] In some embodiments, the variant PAH polypeptide (eg, variant number 1 PAH polypeptide) comprises any amino acid substitution to eliminate a potential protease cleavage site. In some embodiments, the amino acid substitution to eliminate a potential protease cleavage site is located within PAH fragments 270-295 and 380-405.

[0109] In some embodiments, the variant PAH polypeptide (eg, variant number 1 PAH polypeptide) comprises a post-translational modification to enhance the stability of human PAH. In some embodiments, the variant PAH polypeptide comprises a post-translational modification such as PEGylation and nitrosation of Cys residues, particularly I374C, by an external nitrosation agent.

[0110] III. Nucleic Acids

[0111] In some aspects, the present invention provides nucleic acids encoding variant PAH polypeptides that, when expressed in an individual, confer a higher level of PAH activity. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising at least three amino acid substitutions, wherein the amino acid substitutions are located at sites selected from the group consisting of M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising four amino acid substitutions at sites M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising at least three amino acid substitutions selected from the group consisting of M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions including: K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide further comprising amino acid substitutions H264P, G272A, G272P, P275L, P279Q, G272P, and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the nucleic acid encodes a variant PAH polypeptide that is any one of the variant PAH polypeptides listed in Tables 1-3. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A, and comprising additional amino acid substitutions, while at least substantially retaining the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the positions of the amino acid substitutions encoded by the nucleic acid are based on a wild-type human PAH polypeptide; for example, a human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0112] In some embodiments, the nucleic acid encodes a variant human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid encodes an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 and has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about any of 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 and has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of the wild-type PAH of SEQ ID NO: 1.

[0113] In some embodiments, the nucleic acid encodes a variant PAH polypeptide that is a truncated PAH polypeptide that retains phenylalanine hydroxylase activity. In some embodiments, the nucleic acid encodes a truncated PAH polypeptide comprising an N-terminal truncation. In some embodiments, the nucleic acid encodes an N-terminal truncation that is a partial or complete truncation of the N-terminal regulatory domain. In some embodiments, the nucleic acid encodes an N-terminal truncation that is a deletion of amino acid residue 1 to about amino acid residue 102 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide that is a C-terminal truncation. In some embodiments, the nucleic acid encodes a C-terminal truncation that is a partial or complete truncation of a tetramerization domain. In some embodiments, the nucleic acid encodes a C-terminal truncation that is a deletion of amino acid residues from about 429 to 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide that comprises an N-terminal truncation and a C-terminal truncation. In some embodiments, the truncated PAH polypeptide comprises a partial or complete truncation of the N-terminal regulatory sequence and a partial or complete truncation of the tetramerization domain. In some embodiments, the truncated PAH polypeptide comprises a deletion of amino acid residue 1 to about amino acid residue 102 and about 429 to about 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from about 102 to about 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from 102 to 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide further comprising four amino acid substitutions at positions M180, K199, S250, and G256 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide comprising four amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a truncated variant PAH polypeptide further comprising any combination of the amino acid substitutions listed in Tables 1-3. In some embodiments, the nucleic acid encodes a truncated PAH polypeptide having at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of a wild-type PAH (e.g., a PAH polypeptide of SEQ ID NO: 1).

[0114] In some embodiments, the nucleic acid encoding the variant PAH polypeptide is operably linked to a promoter. In some embodiments, the promoter is selected from the group consisting of the cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, mouse transthyretin promoter (mTTR), mTTR482 promoter, mA1MB2-mTTR482 promoter, TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, LP1 promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globulin promoter (CAG) promoter, elongation factor 1-α promoter (EF1-α) promoter, human β-glucuronidase promoter, chicken β-actin (CBA) promoter, retroviral Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter and 13-actin promoter. In some embodiments, the promoter is the LP1 promoter or the mA1MB2-mTTR482 promoter.

[0115] In some embodiments, the nucleic acid further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA. In some embodiments, the polyadenylation signal is a synthetic polyadenylation signal, as described in Levitt, N et al. (1989), Genes Develop. 3: 1019-1025.

[0116] In some embodiments, the nucleic acid further comprises an intron. Various introns that can be used in the present invention are known to those skilled in the art, and include MVM intron, F IX truncated intron 1, β-globulin SD / immunoglobulin heavy chain SA, adenovirus SD / immunoglobulin SA, SV40 late SD / SA (19S / 16S) and hybrid adenovirus SD / IgGSA. (Wu et al. 2008, Kurachi et al., 1995, Choi et al. 2014, Wong et al., 1985, Yew et al. 1997, Huang and Gorman (1990). In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globulin hybrid intron. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globulin hybrid promoter and intron, wherein all ATG sites are removed to minimize erroneous translation start sites (SEQ ID NO: 15).

[0117] In some embodiments, the nucleic acid may include (one or more) filler nucleic acids. In some embodiments, the filler nucleic acid may include a sequence encoding a reporter polypeptide. As will be appreciated by those skilled in the art, the filler nucleic acid may be located in multiple regions within the nucleic acid and may consist of one continuous sequence within the nucleic acid (e.g., a single filler nucleic acid in a single position) or multiple sequences (e.g., more than one filler nucleic acid in more than one position (e.g., 2 positions, 3 positions, etc.)). In some embodiments, the filler nucleic acid may be located downstream of the nucleic acid sequence encoding the variant PAH polypeptide. In an embodiment, the filler nucleic acid may be located upstream of the nucleic acid sequence encoding the variant PAH polypeptide (e.g., between a promoter and a nucleic acid sequence encoding a variant PAH polypeptide). As will be appreciated by those skilled in the art, various nucleic acids may be used as filler nucleic acids. In some embodiments, the filler nucleic acid includes all or part of a human alpha-1-antitrypsin (AAT) filler sequence or a C16 P1 chromosome 16P1 clone (human C16) filler sequence. In some embodiments, the filler sequence includes all or part of a gene. For example, the filler sequence includes a portion of a human AAT sequence. It will be appreciated by those skilled in the art that different portions of a gene (e.g., a human AAT sequence) may be used as a filler fragment. For example, the filler fragment can be from the 5' end of the gene, the 3' end of the gene, the middle part of the gene, the non-coding part of the gene (e.g., intron), the coding region of the gene (e.g., exon), or a mixture of the non-coding part and the coding part of the gene. It will also be understood by those skilled in the art that all or part of the filler sequence can be used as a filler sequence. In some embodiments, the filler sequence is modified to remove the internal ATG codon. In some embodiments, the filler sequence comprises the nucleotide sequence of SEQ ID NO:16.

[0118] In some embodiments, the isolated nucleic acid encoding the human PAH polypeptide is codon optimized. In some embodiments, the isolated nucleic acid encoding the human PAH polypeptide is codon optimized for expression in specific cells (such as eukaryotic cells). Eukaryotic cells can be those of or derived from specific organisms, such as mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence with a codon that is more frequently or most commonly used in the genes of the host cell, while maintaining the native amino acid sequence. Various species show particular preferences for certain codons for specific amino acids. Codon usage tables are readily available, such as in the "Codon Usage Database", and these tables can be modified in a variety of ways (see, for example, Nakamura, Y. et al. (2000) Nucleic Acids Res. 28: 292). Computer algorithms are also available for codon optimization of specific sequences for expression in specific host cells, such as Gene Forge (Aptagen; Jacobus, Pennsylvania), DNA2.0, GeneArt (GA), or Genscript (GS), and the GS algorithm combined with reduction of CpG content. In some embodiments, the nucleic acid encoding the PAH polypeptide is codon optimized using the GA algorithm. In some embodiments, the nucleic acid sequence is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 14.

[0119] IV. Liver-specific expression cassette

[0120] In some aspects, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (mPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer. In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is on the 5' side of the mTTR promoter. In some embodiments, the transgene encodes a variant PAH polypeptide as described herein.

[0121] In some embodiments, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element linked to a human α1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR). In some embodiments, the mTTR promoter is an mTTR482 promoter. In some embodiments, the 3' element is located on the 3' side of the transgene. In some embodiments, the transgene encodes a variant PAH polypeptide as described herein.

[0122] In some embodiments, the present invention provides an expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (mPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer, and wherein the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element linked to a human α1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR). In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is on the 5' side of the mTTR promoter. In some embodiments, the 3' element is located on the 3' side of the transgene. In some embodiments, the transgene encodes a variant PAH polypeptide as described herein.

[0123] In some embodiments, the expression cassette further comprises an intron. In some embodiments, the intron is an MVM intron, F IX truncated intron 1, β-globulin SD / immunoglobulin heavy chain SA, adenovirus SD / immunoglobulin SA, SV40 late SD / SA (19S / 16S) or hybrid adenovirus SD / IgG SA. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globulin hybrid intron.

[0124] In some embodiments, the expression cassette further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, a SV40 polyadenylation signal, or HSV TK pA.

[0125] In some embodiments, the expression cassette is incorporated into a vector. In some embodiments, the expression cassette is incorporated into a viral vector. In some embodiments, the viral vector is an rAAV vector as described herein.

[0126] V. Vectors and Viral Particles

[0127] In certain aspects, the nucleic acid encoding the variant PAH polypeptide is contained in a vector. In some embodiments, the present invention contemplates the use of a recombinant viral genome to introduce a nucleic acid sequence encoding a variant PAH polypeptide for packaging into a viral particle, such as the viral particles described below. The recombinant viral genome may include any element that establishes expression of the variant PAH polypeptide, such as a promoter, ITR, ribosome binding element, terminator, enhancer, selection marker, intron, poly A signal and / or replication origin. Exemplary viral genomic elements and delivery methods for viral particles are described in more detail below.

[0128] Non-viral delivery systems

[0129] Conventional non-viral gene transfer methods can also be used to introduce nucleic acids into cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids compounded with delivery systems. For example, the carrier can be compounded with lipids (e.g., cations or neutral lipids), liposomes, polycations, nanoparticles, or agents that enhance nucleic acid cellular uptake. The carrier can be compounded with agents suitable for any delivery method described herein. In some embodiments, the nucleic acid comprises one or more viral ITRs (e.g., AAV ITRs).

[0130] Virus particles

[0131] In some embodiments, the vector comprising a nucleic acid encoding a variant PAH polypeptide is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector.

[0132] rAAV particles

[0133] In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the viral particle is a recombinant AAV particle comprising a nucleic acid comprising a nucleic acid encoding a variant PAH polypeptide flanked by one or two ITRs. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is flanked by two AAV ITRs.

[0134] In some embodiments, nucleic acids encoding variant PAH polypeptides of the present disclosure are operably linked to components, control sequences (including transcriptional start and stop sequences) in the direction of transcription, thereby forming an expression cassette. The 5' and 3' ends of the expression cassette are flanked by at least one functional AAV ITR sequence. "Functional AAV ITR sequence" means an ITR sequence function intended for rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7):3428-32; Passini et al., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference in their entirety. For the purpose of implementing some aspects of the present invention, the recombinant vector comprises at least all AAV sequences necessary for encapsidation and a physical structure for infection by rAAV. The AAV ITRs used in the vectors of the present invention need not have wild-type nucleotide sequences (e.g., as described in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may be altered by insertion, deletion or substitution of nucleotides, or the AAV ITRs may be derived from any of several AAV serotypes. Currently, more than 40 AAV serotypes are known, and new serotypes and variants of existing serotypes are still being identified. See Gao et al., PNAS, 2002, 99(18):11854-6; Gao et al., PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003, 77(12):6799-810.

[0135] The use of any AAV serotype is considered to be within the scope of the present invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including but not limited to, AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV or mouse AAV ITR, etc. In some embodiments, the nucleic acid in AAV comprises the ITR of AAV ITR, and the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV or mouse AAV, etc. In certain embodiments, AAV ITR is AAV2 ITR.

[0136] In some embodiments, the vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein. In some embodiments, the stuffer nucleic acid may be located 3' to the nucleic acid encoding the variant PAH polypeptide of the present disclosure.

[0137] In some aspects, the present invention provides virus particles comprising a recombinant self-complementary genome. In some embodiments, the vector is a self-complementary vector. AAV virus particles with self-complementary genomes and methods using self-complementary AAV genomes are described in the following documents: U.S. Patent Nos. 6,596,535; 7,125,717; 7,765,583; 7,785,888; 7,790,154; 7,846,729; 8,093,054; and 8,361,457; and Wang Z., et al., (2003) Gene Ther 10: 2105-2111, each of which is incorporated herein by reference in its entirety. rAAV comprising a self-complementary genome will rapidly form double-stranded DNA molecules with the aid of its partially complementary sequences (e.g., complementary coding strands and non-coding strands of transgenics). In some embodiments, the present invention provides AAV virions comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., a coding strand of a variant PAH polypeptide of the present invention) and a second heterologous polynucleotide sequence (e.g., a non-coding or antisense strand of a variant PAH polypeptide of the present disclosure), wherein the first heterologous polynucleotide sequence can form intrastrand base pairs with the second polynucleotide sequence along most or all of its length.

[0138] In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are connected by a sequence that promotes intrachain base pairing; for example, a hairpin DNA structure. Hairpin structures are known in the art, for example, in siRNA molecules. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are connected by a mutant ITR (e.g., right ITR). The mutant ITR comprises a deletion of the D region containing the terminal melting sequence. Therefore, when the AAV viral genome is replicated, the rep protein will not cut the viral genome at the mutant ITR, and therefore, the recombinant viral genome containing the following in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heterologous polynucleotide sequence including a regulatory sequence, a mutant AAV ITR, a second heterologous polynucleotide in the reverse direction of the first heterologous polynucleotide, and a third AAV ITR.

[0139] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are connected by a mutated ITR (e.g., right ITR). In some embodiments, the ITR comprises a polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA AAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 17). The mutated ITR comprises a deletion of the D region containing the terminal melting sequence. Therefore, when the AAV viral genome is replicated, the rep protein will not cut the viral genome at the mutated ITR, and therefore, the recombinant viral genome comprising the following in 5' to 3' order will be packaged in the viral capsid: AAV ITR, a first heterologous polynucleotide sequence including a regulatory sequence, a mutated AAV ITR, a second heterologous polynucleotide in the reverse direction of the first heterologous polynucleotide, and a third AAV ITR.

[0140] In some embodiments, the vector is encapsidated in a virion. In some embodiments, the virion is a recombinant AAV virion comprising a recombinant AAV vector. Different AAV serotypes are used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues (e.g., eye tissue). rAAV particles can contain viral proteins and viral nucleic acids of the same serotype or mixed serotypes. For example, in some embodiments, rAAV particles can contain AAV2 capsid proteins and at least one AAV2 ITR of the present invention, or they can contain AAV2 capsid proteins and at least one AAV1 ITR. Any combination of AAV serotypes for producing rAAV particles is provided herein, as if each combination has been clearly described herein. In some embodiments, the present invention provides rAAV particles comprising the AAV2 capsid of the present invention. In some embodiments, the present invention provides rAAV particles comprising the AAVrh8R capsid of the present invention.

[0141] In some embodiments, the rAAV particles comprise an AAV1 capsid, an AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, an AAV6 capsid (e.g., a wild-type AAV6 capsid, or a variant AAV6 capsid such as ShH10 as described in U.S. Pre-Grant Publication No. 2012 / 0164106), an AAV7 capsid, an AAV8 capsid, an AAVrh8 capsid, an AAVrh8R capsid, an AAV9 capsid (e.g., a wild-type AAV9 capsid, or a modified AAV9 capsid as described in U.S. Pre-Grant Publication No. 2013 / 0323226), an AAV10 capsid, an AAVrh10 capsid, an AAV11 capsid, an AAV12 capsid, a tyrosine capsid mutant, a heparin-binding capsid mutant, an AAV2R471A capsid, an AAV AAV2 / 2-7m8 capsid, an AAV DJ capsid (e.g., AAV-DJ / 8 capsid, AAV-DJ / 9 capsid, or any other capsid described in U.S. Pre-Grant Publication No. 2012 / 0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 capsid, or an AAV capsid described in U.S. Pat. No. 8,283,151 or International Publication No. WO / 2003 / 042397. In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the rAAV particle comprises an AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832. In further embodiments, the rAAV particle comprises a capsid protein of an AAV serotype from clade AF (Gao et al., J. Virol. 2004, 78(12):6381). In some embodiments, the rAAV particle comprises an AAV1 capsid protein or a mutant thereof. In other embodiments, the rAAV particle comprises an AAV2 capsid protein or a mutant thereof. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, the rAAV particles comprise an AAV serotype 1 (AAV1) capsid. In some embodiments, the rAAV particles comprise an AAV serotype 2 (AAV2) capsid. In some embodiments, the recombinant AAV virus particles comprise an AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsid.In some embodiments, AAV1, AAV2, AAV8, AAVrh8R, AAV9 and / or AAVrh10 capsids comprise tyrosine mutations or heparan binding mutations, such as described below. In some embodiments, the capsid is a liver-targeted capsid; for example, but not limited to, a LK03 capsid, a HSC15 capsid or a 17 capsid. In some embodiments, the capsid is an engineered AAV capsid (e.g., a shuffled capsid). Examples of engineered AAV capsids include, but are not limited to, DJ (Grimm D et al., J Virol. 2008, 82: 5887-911), LK03 (Lisowski L et al., Nature, 2014, 506: 382-6) and HSC15 and HSC17 (Smith LJ et al., Mol Ther, 2014 Sep; 22 (9): 1625-34).

[0142] It is known that the capsid of AAV (e.g., AAV2, AAV8, etc.) includes three capsid proteins: VP1, VP2, and VP3. These proteins contain a large number of overlapping amino acid sequences and unique N-terminal sequences. The AAV2 capsid includes 60 subunits arranged in icosahedral symmetry (Xie, Q., et al. (2002) Proc. Natl. Acad. Sci. USA 99 (16): 10405-10). It has been found that VP1, VP2, and VP3 exist in a ratio of 1:1:10.

[0143] In some embodiments, the rAAV particle comprises a) a rAAV capsid comprising a rAAV capsid protein comprising one or more amino acid substitutions at one or more positions that interact with heparan sulfate proteoglycans, and b) a rAAV vector comprising a heterologous nucleic acid and at least one AAV inverted terminal repeat sequence.

[0144] In some embodiments, the rAAV particle comprises one or more amino acid substitutions of the capsid protein that reduce or eliminate binding of the rAAV particle to heparan sulfate proteoglycans, and / or wherein the one or more amino acid substitutions are located at positions numbered based on VP1 numbering of AAV2 at 484, 487, 532, 585, or 588. As used herein, "based on VP1 numbering of AAV2" refers to amino acids of the capsid protein that correspond to the amino acids of VP1 of AAV2. For example, if one or more amino acid substitutions are at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 based on the numbering of VP1 of AAV2, then the one or more amino acid substitutions are at one or more amino acids of the capsid protein corresponding to amino acids 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 of VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 484, 487, 532, 585, or 588 of VP1 of AAV3 based on the numbering of VP1 of AAV2. In some embodiments, the one or more amino acid substitutions are at positions 485, 488, 528, 533, 586, or 589 based on VP1 numbering of AAVrh8R. In some embodiments, one or more amino acids at one or more positions corresponding to amino acids 585 and / or 588 (based on VP1 numbering of AAV2) are replaced with arginine residues (e.g., S586 and / or T589 of AAV1 or AAV6; S586 and / or A589 of AAV9; A586 and / or T589 of AAVrh8R; Q588 and / or T591 of AAV8; and Q588 and / or A591 of AAVrh10). In other embodiments, one or more amino acids (e.g., arginine or lysine) at one or more positions corresponding to amino acids 484, 487, 527 and / or 532 (based on VP1 numbering of AAV2) are replaced with one or more non-positively charged amino acids (e.g., alanine) (e.g., R485, R488, K528 and / or K533 of AAV1 or AAV6; RR485, R488, K528 and / or R533 of AAV9 or AAVrh8; and R487, R490, K530 and / or R535 of AAV8 or AAVrh10).

[0145] Production of AAV particles

[0146] Many methods are known in the art for producing rAAV vectors, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpes virus-AAV hybrids (Conway, JE et al., (1997) J. Virology 71(11):8780-8789) and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). rAAV production cultures for producing rAAV virions all require: 1) a suitable host cell; 2) a suitable helper virus function; 3) AAV rep and cap genes and gene products; 4) a nucleic acid (such as a therapeutic nucleic acid) flanked by at least one AAV ITR sequence (e.g., an AAV genome encoding a variant PAH polypeptide); and 5) a suitable culture medium and culture medium components that support rAAV production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human cell line, such as HeLa, A549, 293, or Perc.6 cells. In some embodiments, suitable helper virus functions are provided by wild-type or mutant adenoviruses (such as temperature-sensitive adenoviruses), herpes virus (HSV), baculovirus, or plasmid constructs that provide helper functions. In some embodiments, AAV rep and cap gene products can be from any AAV serotype. Usually, but not necessarily, the AAV rep gene product has the same serotype as the ITR of the rAAV vector genome, as long as the rep gene product can play a role in replicating and packaging the rAAV genome. Suitable culture media known in the art can be used to produce rAAV vectors. These culture media include, but are not limited to, culture media produced by Hyclone Laboratories and JRH, including modified Eagle's medium (MEM); Dulbecco's modified Eagle's medium (DMEM); custom formulations, such as those described in U.S. Pat. No. 6,566,118; and Sf-900 II SFM culture media as described in U.S. Pat. No. 6,723,551, each of which (particularly with respect to custom culture media formulations for producing recombinant AAV vectors) is incorporated herein by reference in its entirety. In some embodiments, AAV helper functions are provided by adenovirus or HSV. In some embodiments, the AAV helper functions are provided by a baculovirus and the host cell is an insect cell (eg, a Spodoptera frugiperda (Sf9) cell).

[0147] A method for producing rAAV particles is a triple transfection method. In short, a plasmid containing rep genes and capsid genes can be transfected (e.g., using a calcium phosphate method) into a cell line (e.g., HEK-293 cells) together with an auxiliary adenovirus plasmid, and the virus can be collected and optionally purified. Therefore, in some embodiments, rAAV particles are produced by triple transfection of nucleic acids encoding rAAV vectors, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus functions into host cells, wherein the transfection of nucleic acids into host cells produces host cells capable of producing rAAV particles.

[0148] In some embodiments, rAAV particles can be produced by a production cell line method (see Martin et al., (2013) Human Gene Therapy Methods 24:253-269; U.S. Patent Publication No. US2004 / 0224411; and Liu, XL et al. (1999) Gene Ther.6:293-299). In short, a cell line (e.g., HeLa, 293, A549 or Perc.6 cell line) can be stably transfected with a plasmid containing a rep gene, a capsid gene, and a vector genome containing a promoter-heterologous nucleic acid sequence (e.g., a variant PAH polypeptide). The cell line can be screened to select a lead clone for rAAV production, which can then be amplified to a production bioreactor and infected with a helper virus (e.g., adenovirus or HSV) to initiate rAAV production. The virus can then be harvested, the adenovirus can be inactivated (e.g., by heating) and / or removed, and the rAAV particles can be purified. Thus, in some embodiments, rAAV particles are produced by a production cell line comprising one or more of a nucleic acid encoding a rAAV vector, a nucleic acid encoding AAV rep and cap, and a nucleic acid encoding an AAV helper virus function. As described herein, the production cell line approach may be advantageous for producing rAAV particles with an overly large genome compared to the triple transfection approach.

[0149] In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are stably maintained in production cell lines. In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are introduced into cell lines on one or more plasmids to produce production cell lines. In some embodiments, AAV rep, AAVcap and rAAV genomes are introduced into cells on the same plasmid. In other embodiments, AAV rep, AAV cap and rAAV genomes are introduced into cells on different plasmids. In some embodiments, cell lines stably transfected with plasmids maintain plasmids in multiple passages (e.g., 5, 10, 20, 30, 40, 50 or more than 50 cell passages) of cell lines. For example, the one or more plasmids can be replicated when the cell replicates, or the one or more plasmids can be integrated into the cell genome. A variety of sequences that enable plasmids to replicate autonomously in cells (e.g., human cells) have been identified (see, e.g., Krysan, PJ et al. (1989) Mol. Cell Biol. 9: 1026-1033). In some embodiments, the one or more plasmids may contain a selection marker (e.g., an antibiotic resistance marker) that allows selection of cells that maintain the plasmid. Selection markers commonly used for mammalian cells include, but are not limited to, blasticidin, G418, hygromycin B, bleomycin, puromycin and derivatives thereof. Methods for introducing nucleic acids into cells are known in the art, and include, but are not limited to, viral transduction, cationic transfection (e.g., using cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for more details, see, e.g., Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem. 397: 3173-3178).

[0150] In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are stably integrated into the genome of the production cell line. In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are introduced into the cell line on one or more plasmids to produce the production cell line. In some embodiments, AAV rep, AAV cap and rAAV genomes are introduced into cells on the same plasmid. In other embodiments, AAV rep, AAV cap and rAAV genomes are introduced into cells on different plasmids. In some embodiments, the one or more plasmids may contain a selection marker (e.g., an antibiotic resistance marker) that allows selection of cells that maintain plasmids. Methods for stably integrating nucleic acids into a variety of host cell lines are known in the art. For example, repeated selection (e.g., by using a selection marker) can be used to select cells that have been integrated with nucleic acids containing a selection marker (and AAV cap and rep genes and / or rAAV genomes). In other embodiments, nucleic acids can be integrated into the cell line in a site-specific manner to produce a production cell line. Several site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O'Gorman, S. et al. (1991) Science 251:1351-1355), Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, AC et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0151] In some embodiments, the production cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the production cell line is derived from HeLa, 293, A549, or PERC. For example, the cell line is HeLa, 293, A549 or PERC. (Crucell) cell line or its derivatives.

[0152] In some embodiments, the production cell line is suitable for growth in suspension. As known in the art, anchorage-dependent cells cannot usually grow in suspension without a matrix (such as microcarrier beads). Making the cell line suitable for growth in suspension can include, for example, using a culture medium lacking calcium and magnesium ions to prevent agglomeration (and optionally a defoamer), using a culture container coated with a siliconized compound, growing the cell line in a rotary culture with a stirring paddle, and selecting the cells in the culture (rather than in a large mass or on the side of the container) at each passage. For further description, see, for example, ATCC FAQs (available at www.atcc.org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx) and the references cited therein.

[0153] In some aspects, a method for producing any rAAV particle disclosed herein is provided, the method comprising (a) culturing a host cell under conditions that produce rAAV particles, wherein the host cell comprises (i) one or more AAV packaging genes, wherein each of the AAV packaging genes encodes an AAV replication protein and / or encapsidation protein; (ii) an rAAV provector comprising a nucleic acid encoding a heterologous nucleic acid as described herein, flanked by at least one AAV ITR, and (iii) AAV helper functions; and (b) recovering the rAAV particles produced by the host cell. In some embodiments, the at least one AAV ITR is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV or mouse AAV serotype ITR, etc. For example, in some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh 10. In certain embodiments, the nucleic acid in the AAV comprises an AAV2 ITR. In some embodiments, the encapsidation protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimeric, bovine AAV or mouse AAV capsid rAAV2 / HBoV1 serotype capsid protein or a mutant thereof. In some embodiments, the encapsidation protein is an AAV8 capsid protein. In some embodiments, the rAAV particle comprises an AAV8 capsid and a recombinant genome comprising an AAV2 ITR, and a nucleic acid encoding a therapeutic transgene / nucleic acid (e.g., a nucleic acid encoding a variant PAH polypeptide).

[0154] Suitable rAAV production medium of the present invention can be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as known in the art, rAAV vectors can be produced under serum-free conditions, which can also be referred to as medium without animal-derived products. It will be appreciated by those of ordinary skill in the art that commercial or custom culture media designed to support rAAV vector production can also be supplemented with one or more cell culture components known in the art, including but not limited to glucose, vitamins, amino acids and / or growth factors, in order to increase the titer of rAAV in the production culture.

[0155] rAAV production cultures can be grown under a variety of conditions (over a wide range of temperatures, for varying lengths of time, etc.) appropriate to the particular host cells used. As is known in the art, rAAV production cultures include attachment-dependent cultures, which can be cultured in suitable attachment-dependent containers such as roller bottles, hollow fiber filters, microcarriers, and packed bed or fluidized bed bioreactors. rAAV vector production cultures can also include suspension-adapted host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as Wave bag systems.

[0156] The rAAV vector particles of the invention can be harvested from rAAV production cultures by lysing the host cells of the production culture or by harvesting spent culture medium from the production culture, provided that the cells are cultured under conditions known in the art to cause the release of rAAV particles from intact cells into the culture medium, as described more fully in U.S. Pat. No. 6,566, 118. Suitable methods for lysing cells are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0157] In another embodiment, the rAAV particles are purified. As used herein, the term "purified" includes preparations of rAAV particles that lack at least some other components that may also be present in the place where the rAAV particles naturally exist or are initially prepared. Thus, for example, the separated rAAV particles can be prepared by enriching them from a source mixture (such as a culture lysate or a production culture supernatant) using purification techniques. Enrichment can be measured in a variety of ways, such as according to the ratio of DNA enzyme-resistant particles (DRP) or genome copies (gc) present in the solution, or according to infectivity, or can be measured according to a second potential interfering substance (such as a contaminant, including a production culture contaminant or a process contaminant, including a helper virus, a culture medium component, etc.) present in the source mixture.

[0158] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtering through a series of depth filters, including, for example, a DOHC grade Millipore Millistak + HC Pod filter, an A1HC grade Millipore Millistak + HC Pod filter, and a 0.2 μm Filter Opticap XL10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques known in the art, such as centrifugation or filtering through any cellulose acetate filter of 0.2 μm or greater pore size known in the art.

[0159] In some embodiments, The rAAV production culture harvest is further processed to digest any high molecular weight DNA present in the production culture. Digestion was performed under standard conditions known in the art, including, for example, 1-2.5 units / ml of The concentration of 50% HCl is 0.1% HCl and the ... temperature ranges from ambient temperature to 37°C for a period of time ranging from 30 minutes to several hours.

[0160] rAAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating rAAV particles; capture of rAAV by apatite chromatography; heat inactivation of helper virus; capture of rAAV by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and capture of rAAV by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying rAAV particles are found, for example, in the following literature: Xiao et al., (1998) Journal of Virology 72: 2224-2232; U.S. Pat. Nos. 6,989,264 and 8,137,948; and WO 2010 / 148143.

[0161] VI. Treatment Methods

[0162] Certain aspects of the present disclosure relate to methods of treating phenylketonuria and / or reducing phenylalanine levels in individuals in need thereof. Phenylketonuria (PKU) is caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH), which results in elevated blood Phe levels that are toxic to the brain and subsequently lead to severe psychiatric disorders if untreated. Current treatments through dietary restriction are effective, but noncompliance in adolescents and adults is a major problem.

[0163] Efforts to treat PKU have been hampered by low human PAH activity in gene therapy vectors. The present approach is based, in part, on the discovery of variant PAH polypeptides with improved protein stability and enzymatic activity compared to endogenous human PAH polypeptides. Treatment of PKU by gene therapy using rAAV vectors encoding the variant PAH polypeptides described herein resulted in better reduction of blood and brain Phe levels compared to vectors encoding endogenous human PAH. Furthermore, while both hPAH and hPAH-V1 improved transport of Tyr and Trp to the brain, only variant PAH-treated animals had normalized brain neurotransmitter levels (including dopamine and serotonin), suggesting differential sensitivity to Phe-affected functions.

[0164] In some embodiments, the present invention provides a method of treating PKU, comprising administering to an individual in need thereof a therapeutically effective amount of a variant PAH polypeptide as described herein. In some embodiments, the variant PAH polypeptide comprises at least three amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises four amino acid substitutions at sites M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the variant PAH polypeptide comprises at least three amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions including: K199P, S250P and G256A; M180T, S250P and G256A; M180T, K199P and G256A; or M180T, K199P and S250P. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions M180T, K199P, S250P and G256A. In some embodiments, the variant PAH polypeptide further comprises amino acid substitutions H264P, G272A, G272P, P275L, P279Q, G272P and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the variant PAH polypeptide is any one of the variant PAH polypeptides listed in Tables 1-3. In some embodiments, the variant PAH polypeptide comprises amino acid substitutions M180T, K199P, S250P, and G256A, and comprises additional amino acid substitutions while at least approximately maintaining the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the positions of the amino acid substitutions are based on a wild-type human PAH polypeptide; for example, a human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant PAH polypeptide is a fusion polypeptide. In some embodiments, the variant PAH polypeptide is a fusion polypeptide fused to a tissue targeting peptide. In some embodiments, the variant PAH polypeptide is a fusion polypeptide fused to a liver targeting polypeptide. Examples of liver targeting polypeptides include, but are not limited to, fragments of human hepatocyte growth factor (Eavri and Lorberboum-Galkski, J Biol Chem 2007, 282: 23402-23409) or a glycoprotein that binds to the hepatocyte asialoglycoprotein receptor (Huang, et al., Bioconjugate Chemistry 2017, 28: 283-295).

[0165] In some embodiments, the present invention provides methods for treating PKU by administering an effective amount of a variant PAH polypeptide of the present disclosure. The variant PAH polypeptide may be administered to a specific tissue of interest, or may be administered systemically. In some embodiments, an effective amount of a variant PAH polypeptide may be administered parenterally. Parenteral administration routes may include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intracerebroventricular, intrahepatic, and the like. In some embodiments, an effective amount of a variant PAH polypeptide may be administered via one route of administration. In some embodiments, an effective amount of a variant PAH polypeptide may be administered via a combination of more than one route of administration. In some embodiments, an effective amount of a variant PAH polypeptide is administered to one location. In other embodiments, an effective amount of a variant PAH polypeptide may be administered to more than one location.

[0166] In some embodiments, the present invention provides a method for treating PKU, comprising administering to an individual in need thereof a therapeutically effective amount of a nucleic acid (e.g., DNA or mRNA) encoding a variant PAH polypeptide as described herein. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising at least three amino acid substitutions, wherein the amino acid substitutions are located at sites selected from M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising four amino acid substitutions at sites M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising at least three amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions including: K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a variant PAH polypeptide further comprising amino acid substitutions H264P, G272A, G272P, P275L, P279Q, G272P, and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the nucleic acid encodes a variant PAH polypeptide that is any one of the variant PAH polypeptides listed in Tables 1-3. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A, and comprising additional amino acid substitutions, while at least substantially retaining the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the positions of the amino acid substitutions encoded by the nucleic acid are based on a wild-type human PAH polypeptide; for example, a human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0167] In some embodiments, the nucleic acid encodes a variant human PAH polypeptide. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid encodes an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 and has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence having at least about any of 80%, 85%, 90%, 95%, or 99% identity to the amino acid sequence of SEQ ID NO: 3 and having at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of the wild-type PAH of SEQ ID NO: 1. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is DNA. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is RNA (e.g., mRNA).

[0168] In some embodiments, the nucleic acid encodes a variant PAH polypeptide that is a truncated PAH polypeptide that retains phenylalanine hydroxylase activity. In some embodiments, the nucleic acid encodes a truncated PAH polypeptide comprising an N-terminal truncation. In some embodiments, the nucleic acid encodes an N-terminal truncation that is a partial or complete truncation of the N-terminal regulatory domain. In some embodiments, the nucleic acid encodes an N-terminal truncation that is a deletion of amino acid residue 1 to about amino acid residue 102 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide that is a C-terminal truncation. In some embodiments, the nucleic acid encodes a C-terminal truncation that is a partial or complete truncation of a tetramerization domain. In some embodiments, the nucleic acid encodes a C-terminal truncation that is a deletion of amino acid residues from about 429 to 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide that comprises an N-terminal truncation and a C-terminal truncation. In some embodiments, the truncated PAH polypeptide comprises a partial or complete truncation of the N-terminal regulatory sequence and a partial or complete truncation of the tetramerization domain. In some embodiments, the truncated PAH polypeptide comprises a deletion of amino acid residue 1 to about amino acid residue 102 and about 429 to about 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from about 102 to about 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from 102 to 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide further comprising four amino acid substitutions at positions M180, K199, S250, and G256 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encodes a truncated PAH polypeptide comprising four amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid encodes a truncated variant PAH polypeptide further comprising any combination of the amino acid substitutions listed in Tables 1-3. In some embodiments, the nucleic acid encodes a truncated PAH polypeptide having at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of wild-type PAH (e.g., the PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid encoding the variant PAH polypeptide encodes a fusion polypeptide fused to a tissue targeting peptide.In some embodiments, the variant PAH polypeptide is a fusion polypeptide fused to a liver targeting peptide. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is DNA. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is RNA (eg, mRNA).

[0169] In some embodiments, the present invention provides methods for treating PKU by administering an effective amount of a nucleic acid encoding a variant PAH polypeptide of the present disclosure. Nucleic acids encoding variant PAH polypeptides can be administered to a specific tissue of interest, or can be administered systemically. In some embodiments, an effective amount of nucleic acids encoding variant PAH polypeptides can be administered parenterally. Parenteral administration routes can include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, intrahepatic, and the like. In some embodiments, expression of variant PAH from tissues other than the liver may require the presence of cofactor BH4 (e.g., systemic delivery or co-expression from nucleic acid) Ding et al., Mol Ther 2008, 16: 673-681. In some embodiments, an effective amount of nucleic acids encoding variant PAH polypeptides can be administered by one administration route. In some embodiments, an effective amount of nucleic acids encoding variant PAH polypeptides can be administered by a combination of more than one administration route. In some embodiments, an effective amount of nucleic acids encoding variant PAH polypeptides can be administered to one location. In other embodiments, an effective amount of variant PAH polypeptides can be administered to more than one location. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is DNA. In some embodiments, the nucleic acid encoding the variant PAH polypeptide is RNA (eg, mRNA).

[0170] In some aspects of the invention, nucleic acids encoding variant PAH polypeptides are delivered to an individual via a viral vector. Viral vectors for gene therapy are known in the art. In some aspects, the invention provides methods for treating PKU by administering an effective amount of lentiviral particles encoding variant PAH polypeptides of the present disclosure. In some aspects, the invention provides methods for treating PKU by administering an effective amount of rAAV particles encoding variant PAH polypeptides of the present disclosure. rAAV can be administered to a specific tissue of interest, or can be administered systemically. In some embodiments, an effective amount of rAAV can be administered parenterally. Parenteral administration routes can include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, intrahepatic, and the like. In some embodiments, an effective amount of rAAV can be administered via one route of administration. In some embodiments, an effective amount of rAAV can be administered via a combination of more than one route of administration. In some embodiments, an effective amount of rAAV is administered to one location. In other embodiments, an effective amount of rAAV can be administered to more than one location.

[0171] According to the purpose of treatment, an effective amount of rAAV (in particle form in some embodiments) is administered. For example, when a low percentage of transduction can achieve the desired therapeutic effect, the goal of treatment is usually to reach or exceed this transduction level. In some cases, this transduction level can be achieved by transducing only about 1% to 5% of the target cells of the desired tissue type, at least about 20% of the cells of the desired tissue type in some embodiments, at least about 50% in some embodiments, at least about 80% in some embodiments, at least about 95% in some embodiments, and at least about 99% of the cells of the desired tissue type in some embodiments. The rAAV composition can be administered by one or more administrations during the same procedure or at intervals of days, weeks, months or years. One or more of any administration routes described herein can be used. In some embodiments, multiple vectors can be used to treat people.

[0172] Methods for identifying cells transduced by AAV virions are known in the art; for example, immunohistochemistry or the use of markers such as enhanced green fluorescent protein can be used to detect transduction by virions; for example, virions comprising an rAAV capsid having one or more amino acid substitutions.

[0173] In some embodiments, an effective amount of rAAV particles is administered to more than one location simultaneously or sequentially. In other embodiments, an effective amount of rAAV particles is administered to a single location more than once (e.g., repeatedly). In some embodiments, multiple injections of rAAV virions are spaced no more than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours apart.

[0174] In some embodiments, the present invention provides methods for treating a human suffering from PKU by administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding a variant PAH polypeptide of the disclosure. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0175] In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding a variant PAH polypeptide of the disclosure to treat PKU in an individual in need thereof. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about any of the following: 5×10 12 , 6×10 12 ,7×10 12 , 8×10 12 ,9×10 12 , 10×10 12 , 11×10 12, 15×10 12 , 20×10 12 , 25×10 12 ,30×10 12 or 50×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about any of the following: 5×10 12 Up to 6×10 12 , 6×10 12 Up to 7×10 12 ,7×10 12 Up to 8×10 12 , 8×10 12 Up to 9×10 12 ,9×10 12 Up to 10×10 12 , 10×10 12 Up to 11×10 12 , 11×10 12 Up to 15×10 12 , 15×10 12 Up to 20×10 12 , 20×10 12 Up to 25×10 12 , 25×10 12 Up to 30×10 12 ,30×10 12 Up to 50×10 12 or 50×10 12 Up to 100×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about any of the following: 5×10 12 Up to 10×10 12 , 10×10 12 Up to 25×10 12 or 25×10 12 Up to 50×10 12 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about any of the following: 5×10 9 , 6×10 9 ,7×10 9 , 8×10 9 ,9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 ,30×109 or 50×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about any of the following: 5×10 9 Up to 6×10 9 , 6×10 9 Up to 7×10 9 ,7×10 9 Up to 8×10 9 , 8×10 9 Up to 9×10 9 ,9×10 9 Up to 10×10 9 , 10×10 9 Up to 11×10 9 , 11×10 9 Up to 15×10 9 , 15×10 9 Up to 20×10 9 , 20×10 9 Up to 25×10 9 , 25×10 9 Up to 30×10 9 ,30×10 9 Up to 50×10 9 or 50×10 9 Up to 100×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about any of the following: 5×10 9 Up to 10×10 9 , 10×10 9 Up to 15×10 9 , 15×10 9 Up to 25×10 9 or 25×10 9 Up to 50×10 9 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about any of the following: 5×10 10 , 6×10 10 ,7×10 10 , 8×10 10 ,9×10 10 , 10×10 10 , 11×10 10 , 15×10 10 , 20×10 10 , 25×10 10 ,30×10 10 , 40×10 10 or 50×1010 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about any of the following: 5×10 10 Up to 6×10 10 , 6×10 10 Up to 7×10 10 ,7×10 10 Up to 8×10 10 , 8×10 10 Up to 9×10 10 ,9×10 10 Up to 10×10 10 , 10×10 10 Up to 11×10 10 , 11×10 10 Up to 15×10 10 , 15×10 10 Up to 20×10 10 , 20×10 10 Up to 25×10 10 , 25×10 10 Up to 30×10 10 ,30×10 10 Up to 40×10 10 , 40×10 10 Up to 50×10 10 or 50×10 10 Up to 100×10 10 In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about any of the following: 5×10 10 Up to 10×10 10 , 10×10 10 Up to 15×10 10 , 15×10 10 Up to 25×10 10 or 25×10 10 Up to 50×10 10 infectious units / mL.

[0176] In some embodiments, the dose of viral particles administered to a subject is at least about any of the following: 1×10 8 About 6×10 13 In some embodiments, the dose of viral particles administered to an individual is about any of the following: 1×10 8 About 6×10 13 genome copies / kg body weight.

[0177] In some embodiments, the total amount of viral particles administered to a subject is at least about any of the following: 1×10 9 About 1×10 14 In some embodiments, the total amount of viral particles administered to an individual is about any of the following: 1×10 9 About 1×10 14 genome copies.

[0178] The compositions of the invention (e.g., recombinant viral particles comprising a vector encoding a variant PAH polypeptide of the disclosure) can be used alone or in combination with one or more additional therapeutic agents for the treatment of PKU. The interval between sequential administrations can be calculated as at least (or, alternatively, less than) minutes, hours or days.

[0179] According to the purpose of treatment, an effective amount of rAAV (in particle form in some embodiments) is administered. For example, when a low percentage of transduction can achieve the desired therapeutic effect, the goal of treatment is usually to reach or exceed this transduction level. In some cases, this transduction level can be achieved by transducing only about 1% to 5% of the target cells, in some embodiments at least about 20% of the cells of the desired tissue type, in some embodiments at least about 50%, in some embodiments at least about 80%, in some embodiments at least about 95%, and in some embodiments at least about 99% of the cells of the desired tissue type. The rAAV composition can be administered by one or more administrations during the same procedure or at intervals of days, weeks, months or years. In some embodiments, multiple vectors can be used to treat mammals (e.g., humans).

[0180] In some embodiments, the rAAV compositions of the present disclosure can be used for administration to people. In some embodiments, the rAAV compositions of the present disclosure can be used for pediatric administration. It is not desirable to be bound by theory, because many symptoms of PKU are developmental in nature (e.g., severe mental disorders), so it may be particularly advantageous to treat PKU as early as possible. In some embodiments, an effective amount of rAAV (in particle form in some embodiments) is administered to patients whose age is less than one month, less than two months, less than three months, less than four months, less than five months, less than six months, less than seven months, less than eight months, less than nine months, less than ten months, less than eleven months, less than one year old, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, less than 18 months, less than 19 months, less than 20 months, less than 21 months, less than 22 months, less than two years old or less than three years old.

[0181] In some embodiments, the rAAV compositions of the present disclosure can be used for administration to young adults. In some embodiments, an effective amount of rAAV (in some embodiments, in particle form) is administered to a patient less than 12 years of age, less than 13 years of age, less than 14 years of age, less than 15 years of age, less than 16 years of age, less than 17 years of age, less than 18 years of age, less than 19 years of age, less than 20 years of age, less than 21 years of age, less than 22 years of age, less than 23 years of age, less than 24 years of age, or less than 25 years of age.

[0182] In some embodiments, the present invention provides a method of treating PKU, comprising administering to an individual in need thereof a therapeutically effective amount of a cell comprising a nucleic acid encoding a variant PAH polypeptide as described herein. In some embodiments, the cell comprises a nucleic acid encoding a variant PAH polypeptide comprising at least three amino acid substitutions, wherein the amino acid substitutions are located at a site selected from the group consisting of M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising four amino acid substitutions at sites M180, K199, S250, and G256 of a wild-type human PAH polypeptide. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising at least three amino acid substitutions selected from the group consisting of M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising amino acid substitutions including: K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide further comprising amino acid substitutions H264P, G272A, G272P, P275L, P279Q, G272P, and P275L, or T323R and F327T amino acid substitutions. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide that is any one of the variant PAH polypeptides listed in Tables 1-3. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising amino acid substitutions M180T, K199P, S250P, and G256A, and comprising additional amino acid substitutions, while at least substantially retaining the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the positions of the amino acid substitutions encoded by the nucleic acid in the cell are based on a wild-type human PAH polypeptide; for example, a human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0183] In some embodiments, the nucleic acid in the cell encodes a variant human PAH polypeptide. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 and has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of wild-type PAH. In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an amino acid sequence that is at least about any of 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3 and has at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of the wild-type PAH of SEQ ID NO: 1.

[0184] In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide that is a truncated PAH polypeptide that retains phenylalanine hydroxylase activity. In some embodiments, the nucleic acid in the cell encodes a truncated PAH polypeptide comprising an N-terminal truncation. In some embodiments, the nucleic acid in the cell encodes an N-terminal truncation that is a partial or complete truncation of the N-terminal regulatory domain. In some embodiments, the nucleic acid in the cell encodes an N-terminal truncation that is a deletion of amino acid residue 1 to about amino acid residue 102 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a truncated PAH polypeptide that is a C-terminal truncation. In some embodiments, the nucleic acid in the cell encodes a C-terminal truncation that is a partial or complete truncation of the tetramerization domain. In some embodiments, the nucleic acid in the cell encodes a C-terminal truncation that is a deletion of amino acid residues from about 429 to 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an N-terminal truncation and a C-terminal truncation. In some embodiments, the truncated PAH polypeptide comprises a partial or complete truncation of the N-terminal regulatory sequence and a partial or complete truncation of the tetramerization domain. In some embodiments, the truncated PAH polypeptide comprises a deletion of amino acid residue 1 to about amino acid residue 102 and about 429 to about 452 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from about 102 to about 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a variant PAH polypeptide comprising an amino acid sequence corresponding to amino acid residues from 102 to 428 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a truncated PAH polypeptide further comprising four amino acid substitutions at positions M180, K199, S250, and G256 of a wild-type human PAH polypeptide (e.g., a PAH polypeptide of SEQ ID NO: 1). In some embodiments, the nucleic acid in the cell encodes a truncated PAH polypeptide comprising four amino acid substitutions selected from M180T, K199P, S250P, and G256A. In some embodiments, the nucleic acid in the cell encodes a truncated variant PAH polypeptide further comprising any combination of the amino acid substitutions listed in Tables 1-3.In some embodiments, the nucleic acid in the cell encodes a truncated PAH polypeptide having at least about 25%, 50%, 75%, 100%, or greater than 100% of the phenylalanine hydroxylase activity of a wild-type PAH (eg, a PAH polypeptide of SEQ ID NO: 1).

[0185] In some embodiments, the present invention provides methods for treating PKU by administering an effective amount of cells comprising a nucleic acid encoding a variant PAH polypeptide of the present disclosure. Cells comprising a nucleic acid encoding a variant PAH polypeptide can be administered to a specific tissue of interest, or can be administered systemically. In some embodiments, an effective amount of cells comprising a nucleic acid encoding a variant PAH polypeptide can be administered parenterally. Parenteral administration routes can include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intracerebroventricular, intrahepatic, and the like. In some embodiments, the cells are encapsulated or in a device. In some embodiments, cells expressing PAH outside the liver may require exogenous addition or co-expression of the cofactor BH4. In some embodiments, the cells are encapsulated or in a device that also comprises BH4. In some embodiments, an effective amount of cells comprising a nucleic acid encoding a variant PAH polypeptide can be administered by one administration route. In some embodiments, an effective amount of a nucleic acid encoding a variant PAH polypeptide can be administered by a combination of more than one administration route. In some embodiments, an effective amount of a nucleic acid encoding a variant PAH polypeptide can be administered to one location. In other embodiments, an effective amount of a variant PAH polypeptide may be administered to more than one location.

[0186] In some embodiments, the cell comprising a nucleic acid encoding a variant PAH polypeptide is a hepatocyte, a muscle cell, a fibroblast, an endothelial cell, an epithelial cell, a blood cell, a bone marrow cell, a stem cell, or an induced pluripotent stem cell. In some embodiments, the cell further comprises exogenously added cofactor BH4 and / or coexpressed cofactor BH4.

[0187] In some embodiments, the cell is a cell line (e.g., a CHO cell line, a HeLa cell line, etc.). In some embodiments, the invention provides a method for producing a variant PAH polypeptide, comprising culturing a cell comprising a nucleic acid encoding a variant PAH polypeptide (e.g., an expression vector encoding a variant PAH polypeptide) under conditions that produce a variant PAH polypeptide. In some embodiments, the method for producing a variant PAH polypeptide further comprises one or more steps of purifying the variant PAH polypeptide.

[0188] VII. Kits or Products

[0189] The variant PAH polypeptides, nucleic acids, rAAV vectors, particles and / or pharmaceutical compositions described herein can be included, for example, in a kit or article of manufacture designed for use in one of the methods of the invention as described herein.

[0190] Typically, the system includes a cannula suitable for use in the methods of the invention, one or more syringes (eg, 1, 2, 3, 4, or more), and one or more fluids (eg, 1, 2, 3, 4, or more).

[0191] Syringe can be any suitable syringe, as long as it can be connected to the cannula for delivering fluid.In some embodiments, the system has a syringe.In some embodiments, the system has two syringes.In some embodiments, the system has three syringes.In some embodiments, the system has four or more syringes.The fluid suitable for the inventive method comprises fluid as described herein, for example, one or more fluids of one or more carriers as described herein, and one or more fluids comprising one or more therapeutic agents, each comprising an effective amount.

[0192] In some embodiments, the kit comprises a single fluid (e.g., a pharmaceutically acceptable fluid comprising an effective amount of a carrier). In some embodiments, the kit comprises 2 fluids. In some embodiments, the kit comprises 3 fluids. In some embodiments, the kit comprises 4 or more fluids. The fluid may include a diluent, a buffer, an excipient, or any other liquid described herein or known in the art suitable for delivering, diluting, stabilizing, buffering, or otherwise transporting a variant PAH polypeptide or rAAV vector composition of the present disclosure. In some embodiments, the kit comprises one or more buffers, such as an aqueous pH buffer solution. Examples of buffers may include, but are not limited to, phosphates, citrates, Tris, HEPES, and other organic acid buffers.

[0193] In some embodiments, the kit comprises a container. Suitable containers may include, for example, vials, bags, syringes, and bottles. The container may be made of one or more materials (such as glass, metal, or plastic). In some embodiments, the container is used to hold the rAAV composition of the present disclosure. In some embodiments, the container may also hold fluids and / or other therapeutic agents.

[0194] In some embodiments, the kit comprises an additional therapeutic agent and the rAAV composition of the present disclosure. In some embodiments, the rAAV composition and the additional therapeutic agent can be mixed. In some embodiments, the rAAV composition and the additional therapeutic agent can be kept separate. In some embodiments, the rAAV composition and the additional therapeutic agent can be in the same container. In some embodiments, the rAAV composition and the additional therapeutic agent can be in different containers. In some embodiments, the rAAV composition and the additional therapeutic agent can be administered simultaneously. In some embodiments, the rAAV composition and the additional therapeutic agent can be administered on the same day. In some embodiments, the rAAV composition can be administered within one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, two months, three months, four months, five months or six months of administering the additional therapeutic agent.

[0195] In some embodiments, the kit includes a therapeutic agent that temporarily suppresses the immune system before AAV administration. In some embodiments, the patient is briefly immunosuppressed shortly before and after the injection of the virus to suppress the response of T cells to AAV particles (e.g., see Ferreira et al., Hum. Gene Ther. 25: 180-188, 2014). In some embodiments, the kit also provides cyclosporine, mycophenolate mofetil and / or methylprednisolone.

[0196] The rAAV particles and / or compositions of the present invention can also be packaged into a kit including instructions for use. In some embodiments, the kit also includes a device for delivering (e.g., any type of parenteral administration described herein) rAAV particle compositions. In some embodiments, the instructions for use include instructions according to one of the methods described herein. In some embodiments, the instructions are printed on a label provided with the container (e.g., attached to the container). In some embodiments, the instructions for use include instructions for administering an effective amount of rAAV particles to an individual (e.g., a human), such as for treating PKU in an individual.

[0197] VIII. Exemplary Embodiments

[0198] Embodiment 1. A variant phenylalanine hydroxylase (PAH) polypeptide comprising two amino acid substitutions, wherein the amino acid substitutions are located at positions selected from the group consisting of M180, K199, S250 and G256 of a wild-type human PAH polypeptide.

[0199] Embodiment 2. A variant phenylalanine hydroxylase (PAH) polypeptide comprising three amino acid substitutions, wherein the amino acid substitutions are located at positions selected from the group consisting of M180, K199, S250 and G256 of a wild-type human PAH polypeptide.

[0200] Embodiment 3. A variant phenylalanine hydroxylase (PAH) polypeptide comprising four amino acid substitutions at positions M180, K199, S250 and G256 of a wild-type human PAH polypeptide.

[0201] Embodiment 4. The variant PAH polypeptide of any one of embodiments 1-3, wherein the amino acid substitutions comprise one or more of M180T, K199P, S250P, and G256A.

[0202] Embodiment 5. The variant PAH polypeptide of any one of embodiments 1-4, wherein the amino acid substitutions include K199P, S250P, and G256A; M180T, S250P, and G256A; M180T, K199P, and G256A; or M180T, K199P, and S250P.

[0203] Embodiment 6. The variant PAH polypeptide of any one of embodiments 1-5, wherein the amino acid substitutions include M180T, K199P, S250P, and G256A.

[0204] Embodiment 7. The variant PAH polypeptide of any one of embodiments 1-6, wherein the variant PAH polypeptide further comprises H264P, G272A, G272P, P275L, P279Q, G272P and P275L, or T323R and F327T amino acid substitutions.

[0205] Embodiment 8. The variant PAH polypeptide of any one of embodiments 1-7, wherein the wild-type human PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0206] Embodiment 9. The variant PAH polypeptide of any one of embodiments 1-8, wherein the variant PAH polypeptide is a human PAH polypeptide.

[0207] Embodiment 10. The variant PAH polypeptide of any one of embodiments 1-9, wherein the variant PAH polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence of SEQ ID NO:3.

[0208] Embodiment 11. The variant PAH polypeptide of any one of embodiments 1-6, wherein the variant PAH polypeptide comprises the amino acid sequence of SEQ ID NO:3.

[0209] Embodiment 12. The variant PAH of any one of embodiments 1-11, wherein the variant PAH polypeptide further comprises one or more amino acid substitutions selected from the group consisting of G33A, G46A, G46P, G103A, G139A, G139P, G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, and G442A of a wild-type human PAH polypeptide.

[0210] Embodiment 13. The variant PAH of any one of embodiments 1-12, wherein the variant PAH polypeptide further comprises one or more amino acid substitutions selected from the group consisting of P9G, G10V, G12S, K184R, K192R, S196A, Y206H, H220R, Q336E, E360D, I374C, N376E, N401T, I421V, I441V, S446H of the wild-type human PAH polypeptide, and the addition of S at position 453 of the wild-type human PAH polypeptide.

[0211] Embodiment 14. The variant PAH of any one of embodiments 1-13, wherein the variant PAH polypeptide further comprises one or more amino acid substitutions selected from the group consisting of F240W, A246P, G247A, Y268W, C284F, T323R, F327Y, E319P, I306(Y,F), K113P, G188A, F191Y, T193R, Y206H, G337P and N376P of a wild-type human PAH polypeptide.

[0212] Embodiment 15. A variant PAH polypeptide, wherein the variant PAH polypeptide comprises one or more amino acid substitutions selected from the group consisting of G33A, G46A, G46P, G103A, G139A, G139P, G148A, G188A, G218A, G239A, G247A, G257A, G272A, G289A, G307A, G312A, G332A, G337A, G344A, G352A, and G442A of a wild-type human PAH polypeptide.

[0213] Embodiment 16. A variant PAH polypeptide, wherein the variant PAH polypeptide comprises one or more amino acid substitutions selected from P9G, G10V, G12S, K184R, K192R, S196A, Y206H, H220R, Q336E, E360D, I374C, N376E, N401T, I421V, I441V, S446H of a wild-type human PAH polypeptide, and an addition of S at position 453 of the wild-type human PAH polypeptide.

[0214] Embodiment 17. A variant PAH polypeptide, wherein the variant PAH polypeptide comprises one or more amino acid substitutions selected from the group consisting of F240W, A246P, G247A, Y268W, C284F, T323R, F327Y, E319P, I306(Y,F), K113P, G188A, F191Y, T193R, Y206H, G337P and N376P of a wild-type human PAH polypeptide.

[0215] Embodiment 18. The variant PAH polypeptide of any one of embodiments 1-17, wherein the variant PAH polypeptide comprises an N-terminal truncation.

[0216] Embodiment 19. The variant PAH polypeptide of embodiment 18, wherein the N-terminal truncation comprises truncation of the N-terminal regulatory domain.

[0217] Embodiment 20. The variant PAH polypeptide of embodiment 18 or 19, wherein the N-terminal truncation comprises truncation of amino acid residues 1-102 of the wild-type PAH polypeptide.

[0218] Embodiment 21. The variant PAH polypeptide of any one of embodiments 1-20, wherein the variant PAH polypeptide comprises a C-terminal truncation.

[0219] Embodiment 22. The variant PAH polypeptide of embodiment 21, wherein the C-terminal truncation comprises a truncation of the tetramerization domain.

[0220] Embodiment 23. The variant PAH polypeptide of embodiment 21 or 22, wherein the C-terminal truncation comprises truncation of amino acid residues 429-452 of the wild-type PAH polypeptide.

[0221] Embodiment 24. The variant PAH polypeptide of any one of embodiments 1-23, wherein the variant PAH polypeptide comprises an amino acid sequence corresponding to amino acid residues 103-428 of a wild-type PAH polypeptide.

[0222] Embodiment 25. The variant PAH polypeptide of any one of embodiments 1-24, wherein the variant PAH polypeptide comprises one or more amino acid substitutions to eliminate potential protease cleavage sites.

[0223] Embodiment 26. The variant PAH polypeptide of embodiment 25, wherein the one or more amino acid substitutions that eliminate potential protease cleavage sites are located at positions 270-295 and / or 380-405 of the wild-type PAH polypeptide.

[0224] Embodiment 27. The variant PAH polypeptide of any one of embodiments 1-26, wherein the variant PAH polypeptide is fused to a liver targeting polypeptide.

[0225] Embodiment 28. The variant PAH polypeptide of embodiment 27, wherein the liver-targeted polypeptide is HGF or a fragment thereof or a glycoprotein that binds to the hepatocyte asialoglycoprotein receptor.

[0226] Embodiment 29. The variant PAH polypeptide of any one of embodiments 1-25, wherein the variant PAH polypeptide is pegylated and / or nitrosylated.

[0227] Embodiment 30. The variant PAH polypeptide of embodiment 26, wherein the variant PAH polypeptide comprises an I374C amino acid substitution, wherein the cys residue at position 374 is nitrosylated.

[0228] Embodiment 31. A composition comprising the variant PAH polypeptide of any one of embodiments 1-30.

[0229] Embodiment 32. The composition of embodiment 31, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0230] Embodiment 33. An isolated nucleic acid encoding the variant PAH polypeptide of any one of embodiments 1-30.

[0231] Embodiment 34. The isolated nucleic acid of embodiment 33, wherein the nucleic acid encoding the variant PAH polypeptide is operably linked to a promoter.

[0232] Embodiment 35. The isolated nucleic acid of embodiment 34, wherein the promoter is selected from the group consisting of cytomegalovirus (CMV) immediate early promoter, RSV LTR, MoMLV LTR, phosphoglycerate kinase-1 (PGK) promoter, simian virus 40 (SV40) promoter, CK6 promoter, transthyretin promoter (TTR), mTTR482 promoter, mA1MB2-mTTR482 promoter, TK promoter, tetracycline responsive promoter (TRE), HBV promoter, hAAT promoter, LSP promoter, LP1 promoter, chimeric liver-specific promoter (LSP), E2F promoter, telomerase (hTERT) promoter; cytomegalovirus enhancer / chicken β-actin / rabbit β-globulin promoter (CAG) promoter, elongation factor 1-α promoter (EF1-α) promoter, human β-glucuronidase promoter, chicken β-actin (CBA) promoter, modified chicken β-actin (CBA) promoter or SEQ ID NO:17, reverse transcription Rous sarcoma virus (RSV) LTR promoter, dihydrofolate reductase promoter and 13-actin promoter.

[0233] Embodiment 36. The isolated nucleic acid of embodiment 34 or 35, wherein the promoter is the LP1 promoter or the mA1MB2-mTTR482 promoter.

[0234] Embodiment 37. The isolated nucleic acid of any one of Embodiments 33-36, wherein the nucleic acid further comprises a polyadenylation signal.

[0235] Embodiment 38. The isolated nucleic acid of embodiment 37, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or HSV TK pA.

[0236] Embodiment 39. The isolated nucleic acid of any one of Embodiments 33-38, wherein the nucleic acid further comprises an intron.

[0237] Embodiment 40. The isolated nucleic acid of embodiment 39, wherein the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron.

[0238] Embodiment 41. The isolated nucleic acid of Embodiment 39, wherein the intron is a modified chicken β-actin (CBA) / rabbit β-globin hybrid intron of SEQ ID NO:15.

[0239] Embodiment 42. The isolated nucleic acid of any one of Embodiments 33-41, wherein the nucleic acid further comprises one or more ITRs.

[0240] Embodiment 43. The isolated nucleic acid of any one of Embodiments 33-42, wherein the nucleic acid further comprises a stuffer nucleic acid.

[0241] Embodiment 44. The isolated nucleic acid of embodiment 43, wherein the stuffer nucleic acid is optimized to remove the ATG sequence.

[0242] Embodiment 45. The isolated nucleic acid of Embodiment 44, wherein the stuffer nucleic acid is the A1AT intron stuffer sequence of SEQ ID NO:16.

[0243] Embodiment 46. An isolated nucleic acid encoding a human PAH polypeptide, wherein the nucleic acid is codon optimized.

[0244] Embodiment 47. The isolated nucleic acid of embodiment 46, wherein the nucleic acid sequence is at least 80% identical to the nucleic acid sequence of SEQ ID NO:14.

[0245] Embodiment 48. The isolated nucleic acid of Embodiment 46, wherein the nucleic acid comprises the nucleic acid sequence of SEQ ID NO:14.

[0246] Embodiment 49. The isolated nucleic acid of Embodiment 33, wherein the nucleic acid is mRNA.

[0247] Embodiment 50. A composition comprising the nucleic acid of any one of embodiments 33-49.

[0248] Embodiment 51. The composition of Embodiment 50, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0249] Embodiment 52. A vector comprising the nucleic acid of any one of embodiments 33-49.

[0250] Embodiment 53. The vector of Embodiment 52, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

[0251] Embodiment 54. A rAAV vector comprising the nucleic acid of any one of embodiments 33-41 or 43-49, wherein the nucleic acid is flanked by one or more AAV inverted terminal repeat (ITR) sequences.

[0252] Embodiment 55. The rAAV vector of embodiment 54, wherein the nucleic acid of any one of embodiments 33-48 is flanked by two AAV ITRs.

[0253] Embodiment 56. The rAAV vector of embodiment 54 or 55, wherein the AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR.

[0254] Embodiment 57. The rAAV vector of any one of Embodiments 54-56, wherein the AAV ITR is an AAV2 ITR.

[0255] Embodiment 58. The rAAV vector of Embodiment 57, wherein the rAAV vector comprises, from 5′ to 3′, AAV2 ITRs, a promoter, an intron, a nucleic acid encoding a PAH polypeptide, a stuffer nucleic acid, a polyadenylation signal, and AAV2 ITRs.

[0256] Embodiment 59. The rAAV vector of Embodiment 58, wherein the promoter is the mlA1MB2-mTTR482 promoter or the LP1 promoter.

[0257] Embodiment 60. The rAAV vector of embodiment 58 or 59, wherein the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron.

[0258] Embodiment 61. The rAAV vector of any one of embodiments 58-59, wherein the PAH polypeptide is a variant PAH polypeptide of any one of embodiments 1-30.

[0259] Embodiment 62. The rAAV vector of any one of embodiments 58-60, wherein the nucleic acid encoding the PAH polypeptide is the codon-optimized nucleic acid of any one of embodiments 46-48.

[0260] Embodiment 63. The rAAV vector of any one of Embodiments 58-62, wherein the stuffer nucleic acid comprises nucleic acid from an intron of the human α1 antitrypsin gene.

[0261] Embodiment 64. The rAAV vector of embodiment 63, wherein an intron of the human α1 antitrypsin gene has been mutated to remove the ATG sequence.

[0262] Embodiment 65. The rAAV vector of any one of Embodiments 58-64, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0263] Embodiment 66. The rAAV vector of any one of Embodiments 53-65, wherein the vector is a self-complementary vector.

[0264] Embodiment 67. The rAAV vector of embodiment 66, wherein the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding the complement of the PAH polypeptide, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0265] Embodiment 68. The rAAV vector of embodiment 67, wherein the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutant AAV ITR, wherein the mutant AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal melting sequence.

[0266] Embodiment 69. An rAAV particle comprising the rAAV vector of any one of embodiments 53-68.

[0267] Embodiment 70. The rAAV particle of embodiment 69, wherein the AAV virus particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsid.

[0268] Embodiment 71. The rAAV particle of Embodiment 69, wherein the AAV virion comprises an engineered AAV capsid.

[0269] Embodiment 72. The rAAV particle of Embodiment 71, wherein the engineered AAV capsid is a DJ capsid or a LK03 capsid.

[0270] Embodiment 73. The rAAV particle of embodiment 69 or 70, wherein the ITRs and capsid of the rAAV virion are derived from the same AAV serotype.

[0271] Embodiment 74. The rAAV particle of embodiment 69 or 70, wherein the ITRs and capsid of the rAAV virion are derived from different AAV serotypes.

[0272] Embodiment 75. The rAAV particle of any one of Embodiments 69-70 or 73-74, wherein the rAAV virion comprises an AAV8 capsid.

[0273] Embodiment 76. The rAAV particle of Embodiment 74, wherein the rAAV virion comprises an AAV8 capsid, and wherein the vector comprises an AAV2 ITR.

[0274] Embodiment 77. A composition comprising the rAAV particles of any one of embodiments 69-76.

[0275] Embodiment 78. The composition of Embodiment 77, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0276] Embodiment 79. A cell comprising the nucleic acid of any one of embodiments 33-49 or the vector of embodiments 52 or 53 or the rAAV vector of any one of embodiments 54-68.

[0277] Embodiment 80. A method of producing a variant PAH polypeptide, the method comprising culturing the cell of embodiment 79 under conditions such that the variant PAH polypeptide is produced.

[0278] Embodiment 81. The method of Embodiment 80, further comprising the step of purifying the variant PAH polypeptide.

[0279] Embodiment 82. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a variant PAH polypeptide of any one of embodiments 1-30 or a composition of embodiment 31 or 32.

[0280] Embodiment 83. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual a nucleic acid encoding a variant PAH polypeptide of any one of embodiments 1-30 or a nucleic acid of embodiment 33, 34 or 49.

[0281] Embodiment 84. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the rAAV vector of any one of Embodiments 53-68.

[0282] Embodiment 85. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the rAAV particle of any one of embodiments 69-76.

[0283] Embodiment 86. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the composition of embodiment 31, 32, 50, 51, 77 or 78.

[0284] Embodiment 87. A method for treating phenylketonuria in an individual in need thereof, comprising administering the cell of embodiment 79 to the individual.

[0285] Embodiment 88. The method of any one of Embodiments 82-87, wherein the subject lacks PAH activity.

[0286] Embodiment 89. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual a variant PAH polypeptide of any one of embodiments 1-30 or a composition of embodiment 31 or 32.

[0287] Embodiment 90. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual a nucleic acid encoding a variant PAH polypeptide of any one of embodiments 1-30 or a nucleic acid of embodiment 33, 34 or 49.

[0288] Embodiment 91. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual the rAAV vector of any one of embodiments 53-68.

[0289] Embodiment 92. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual the rAAV particle of any one of embodiments 69-76.

[0290] Embodiment 93. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual a composition of embodiment 31, 32, 50, 51, 77 or 78.

[0291] Embodiment 94. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering the cell of embodiment 79 to the individual.

[0292] Embodiment 95. The method of any one of embodiments 89-94, wherein the level of phenylalanine in the blood of the individual prior to treatment is elevated compared to the level of phenylalanine in the blood of an equivalent matched control individual.

[0293] Embodiment 96. The method of any one of embodiments 82-95, wherein the variant PAH polypeptide, nucleic acid, rAAV vector, rAAV particle, composition or cell is administered intravenously, intraarterially, intrahepatically, intraportally, intraperitoneally or subcutaneously.

[0294] Embodiment 97. The method of any one of Embodiments 82-96, wherein the administration is in combination with another therapy.

[0295] Embodiment 98. The method of embodiment 97, wherein the another therapy is treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia lyase (PAL) or pegylated PAL, or a phenylalanine restricted diet.

[0296] Embodiment 99. A method for producing a PAH polypeptide, comprising culturing the cell of embodiment 79 under conditions where the PAH polypeptide is produced.

[0297] Embodiment 100. The method of Embodiment 99, further comprising purifying the PAH polypeptide.

[0298] Embodiment 101. A kit comprising the variant PAH polypeptide of any one of embodiments 1-24.

[0299] Embodiment 102. A kit comprising the nucleic acid of any one of embodiments 33-46, the rAAV vector of any one of embodiments 53-68, the rAAV particle of any one of embodiments 69-76, or the composition of embodiment 77 or 78.

[0300] Embodiment 103. The kit of embodiment 101 or 102, wherein the kit further comprises instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, a vial and / or a syringe.

[0301] Embodiment 104. An expression cassette for expressing a transgene in hepatocytes, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (pPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer.

[0302] Embodiment 105. The expression cassette of embodiment 104, wherein the mTTR promoter is the mTTR482 promoter.

[0303] Embodiment 106. The expression cassette of embodiment 104 or 105, wherein the enhancer is on the 5' side of the mTTR promoter.

[0304] Embodiment 107. An expression cassette for expressing a transgene in a hepatocyte, wherein the expression cassette comprises a transgene operably linked to a promoter and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element linked to a human alpha 1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR).

[0305] Embodiment 108. The expression cassette of embodiment 107, wherein the mTTR promoter is the mTTR482 promoter.

[0306] Embodiment 109. The expression cassette of embodiment 107 or 108, wherein the 3' element is located on the 3' side of the transgene.

[0307] Embodiment 110. An expression cassette for expressing a transgene in hepatocytes, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer and a 3' element, wherein the promoter comprises a mouse transthyretin (mTTR) promoter, and the enhancer comprises one or two modified prothrombin enhancers (pPrT2), one or two modified α1-microbikunin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HEII), or a CRM8 enhancer, and wherein the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element linked to a human α1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR).

[0308] Embodiment 111. The expression cassette of embodiment 110, wherein the mTTR promoter is the mTTR482 promoter.

[0309] Embodiment 112. The expression cassette of embodiment 110 or 111, wherein the enhancer is on the 5' side of the mTTR promoter.

[0310] Embodiment 113. The expression cassette of any one of embodiments 110-112, wherein the 3' element is located on the 3' side of the transgene.

[0311] Embodiment 114. The expression cassette of any one of Embodiments 104-113, wherein the expression cassette further comprises an intron.

[0312] Embodiment 115. The expression cassette of embodiment 114, wherein the intron is a chicken β-actin / rabbit β-globin hybrid intron.

[0313] Embodiment 116. The expression cassette of any one of Embodiments 104-115, wherein the expression cassette further comprises a polyadenylation signal.

[0314] Embodiment 117. The expression cassette of embodiment 116, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0315] Embodiment 118. The expression cassette of any one of Embodiments 104-117, wherein the transgene encodes a PAH polypeptide or a variant PAH polypeptide.

[0316] Embodiment 119. The expression cassette of embodiment 118, wherein the variant PAH polypeptide is a variant PAH polypeptide of any one of embodiments 1-30.

[0317] Embodiment 120. A vector comprising the expression cassette of any one of embodiments 104-119.

[0318] Embodiment 121. The vector of Embodiment 120, wherein the vector is a recombinant adeno-associated virus (rAAV) vector.

[0319] Embodiment 122. An rAAV vector comprising the expression cassette of any one of embodiments 104-119, wherein the expression cassette is flanked by one or more AAV inverted terminal repeat (ITR) sequences.

[0320] Embodiment 123. The rAAV vector of embodiment 122, wherein the expression cassette of any one of embodiments 104-118 is flanked by two AAV ITRs.

[0321] Embodiment 124. The rAAV vector of embodiment 122 or 123, wherein the AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR.

[0322] Embodiment 125. The rAAV vector of any one of Embodiments 122-124, wherein the AAV ITR is AAV2 ITR.

[0323] Embodiment 126. The rAAV vector of any one of Embodiments 122-125, wherein the vector is a self-complementary vector.

[0324] Embodiment 127. The rAAV vector of embodiment 126, wherein the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding the complement of the PAH polypeptide, wherein the first nucleic acid sequence can form intrastrand base pairs with the second nucleic acid sequence along most or all of its length.

[0325] Embodiment 128. The rAAV vector of embodiment 127, wherein the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutant AAV ITR, wherein the mutant AAV ITR comprises a deletion of the D region and comprises a mutation of the terminal melting sequence.

[0326] Embodiment 129. An rAAV particle comprising the rAAV vector of any one of embodiments 122-127.

[0327] Embodiment 130. The rAAV particle of embodiment 129, wherein the AAV virus particle comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 serotype capsid.

[0328] Embodiment 131. The rAAV particle of Embodiment 130, wherein the AAV virion comprises an engineered AAV capsid.

[0329] Embodiment 132. The rAAV particle of Embodiment 131, wherein the engineered AAV capsid is a DJ capsid or a LK03 capsid.

[0330] Embodiment 133. The rAAV particle of embodiment 131 or 132, wherein the ITRs and capsid of the rAAV virion are derived from the same AAV serotype.

[0331] Embodiment 134. The rAAV particle of embodiment 131 or 132, wherein the ITRs and capsid of the rAAV virion are derived from different AAV serotypes.

[0332] Embodiment 135. A composition comprising the rAAV particle of any one of embodiments 129-134.

[0333] Embodiment 136. The composition of Embodiment 135, wherein the composition further comprises a pharmaceutically acceptable carrier.

[0334] Embodiment 137. A cell comprising the expression cassette of any one of embodiments 104-119 or the vector of any one of embodiments 120-128.

[0335] Embodiment 138. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the expression cassette of any one of embodiments 104-119.

[0336] Embodiment 139. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the rAAV vector of any one of embodiments 122-128.

[0337] Embodiment 140. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the rAAV particle of any one of embodiments 129-134.

[0338] Embodiment 141. A method for treating phenylketonuria in an individual in need thereof, comprising administering to the individual the composition of embodiment 135 or 136.

[0339] Embodiment 142. A method for treating phenylketonuria in an individual in need thereof, comprising administering the cell of embodiment 137 to the individual.

[0340] Embodiment 143. The method of any one of Embodiments 138-142, wherein the subject lacks PAH activity.

[0341] Embodiment 144. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual an expression cassette of any one of embodiments 104-119.

[0342] Embodiment 145. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual the rAAV vector of any one of embodiments 122-128.

[0343] Embodiment 146. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual the rAAV particle of any one of embodiments 129-134.

[0344] Embodiment 147. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering to the individual the composition of embodiment 135 or 136.

[0345] Embodiment 148. A method for reducing phenylalanine levels in the blood of an individual in need thereof, comprising administering the cell of embodiment 137 to the individual.

[0346] Embodiment 149. The method of any one of embodiments 144-148, wherein the level of phenylalanine in the blood of the individual prior to treatment is elevated compared to the level of phenylalanine in the blood of an equivalent matched control individual.

[0347] Embodiment 150. The method of any one of embodiments 138-149, wherein the nucleic acid, rAAV vector, rAAV particle, composition or cell is administered intravenously, intraarterially, intrahepatically, intraportally, intraperitoneally or subcutaneously.

[0348] Embodiment 151. The method of any one of Embodiments 138-150, wherein the administration is in combination with another therapy.

[0349] Embodiment 152. The method of embodiment 151, wherein the another therapy is treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia lyase (PAL) or pegylated PAL, or a phenylalanine restricted diet.

[0350] Embodiment 153. A kit comprising the expression cassette of any one of embodiments 104-119, the rAAV vector of any one of embodiments 122-128, the rAAV particle of any one of embodiments 129-134, or the composition of embodiments 135 or 136.

[0351] Embodiment 154. The kit of embodiment 153, wherein the kit further comprises instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, a vial and / or a syringe.

[0352] Example

[0353] The present invention will be more fully understood by reference to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It should be understood that the embodiments and embodiments described herein are for illustrative purposes only, and the various modifications or changes made according to them will be known to those skilled in the art, and should be included in the spirit and scope of the application and in the scope of the attached embodiments.

[0354] Example 1. Generation of liver-specific expression cassette

[0355] The following studies were performed to develop a robust expression construct that expressed the transgene in the liver of an individual.

[0356] Materials and Methods for Examples 1-3

[0357] Construction of PAH expression cassette and rAAV vector

[0358] To increase the strength of the liver promoter, the plasmid mTTR482-HI-hFVIII-BGHpA containing the mouse transthyretin (mTTR) promoter, the endogenous mTTR enhancer, and the bovine growth hormone (BGH) polyadenylation (pA) site was used for additional modifications (Kyostio-Moore 2016, Nambiar 2017). In this plasmid, the FVIII cDNA was replaced with a cDNA encoding secreted embryonic alkaline phosphatase (SEAP), and the existing intron was replaced with a 1069 bp chicken b-actin (CBA) / rabbit β-globin hybrid intron. Various liver enhancer sequences were cloned upstream of the mTTR482 enhancer. These include a modified prothrombin enhancer (mPrT2, two copies), a modified α1-microbikunin (mA1MB2, two copies) (McEachern 2006, Jacobs 2008), a modified mouse albumin enhancer (mEalb) (Kramer 2003), hepatitis B virus enhancer II (E11) (Kramer 2003), and CRM8 (Chuah 2014). In addition, two 3' elements were tested and cloned downstream of BGH pA, namely albumin (Alb 3') (Wooddell 2008) and albumin plus human A1AT1 SMAR element (AF156542) (Alb3' / SMAR) ( Figure 1A ). The modifications in some enhancers consisted of altering the hepatic nuclear factor (HNF) 3 and 4 binding sites to higher affinity sequences, similar to modifications previously made to mTTR482 (Nambiar 2017; Suppl 1).

[0359] To generate human or mouse PAH plasmid expression cassettes, the corresponding cDNA encoding full-length PAH (amino acids 1-452) was cloned into three expression plasmids. These plasmids contained liver-specific (LP1; Nathwani 2011), A1MB2-mTTR482, or CBA promoters, hybrid introns, and pA sequences (BGH or simian virus 40 [SV40] pA) (Nathwani 2011, Kyostio-Moore 2016). All expression plasmids contained flanking AAV2 ITRs. mTTR482 (Nambiar 2017) was used as the backbone to evaluate various cDNAs encoding hPAH. Four codon-optimized cDNAs were generated by DNA2.0, GeneArt (GA), or Genscript (GS) and the GS algorithm combined with a reduction in CpG content. In each expression plasmid, the PAH protein was tagged with an N-terminal 22-amino acid 3xFLAG peptide (MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 18)) for detection and quantification purposes.

[0360] The selected plasmids containing AAV2 ITR with liver promoter, hybrid intron, PAH cDNA and BGHpA were used for rAAV vector production. rAAV vectors with AAV8 serotype capsid were generated using triple transfection followed by CsCl2 purification (Univ. Massachusetts Gene Therapy Core). Vector batches were quantified by qPCR for BGHpA (Nambiar 2017).

[0361] In vitro cell culture

[0362] All tissue culture reagents were obtained from Irvine Scientific (Santa Ana, CA) or Invitrogen. For transient transfection, human 293 or human hepatoma cells (Huh7 or HepG2) (8 × 10 5 Cells were plated in 6-well culture dishes in Dulbecco's modified Eagle's medium (DMEM) with high glucose, 10% fetal bovine serum (FBS) and 10 ml / L Pen Strep (10 units / ml penicillin and 10 μg / ml streptomycin). Plasmids (2 μg) were transfected using Lipofectamine 2000 (Invitrogen). After 48 or 72 hours, cell lysates or culture medium were harvested for PAH analysis or SEAP activity, respectively.

[0363] Quantification of SEAP activity

[0364] Conditioned media or serum were collected and samples were heated at 65°C for 30 minutes to inactivate endogenous alkaline phosphatase. SEAP activity was measured using alkaline phosphatase (ALP)-SL reagent (Sekisui Diagnostics) and samples were incubated for 10 minutes to 2 hours before reading the absorbance at 405 nm. Human alkaline phosphatase HPAP was used as a standard (Calbiochem). SEAP activity was expressed as ug / ml by dividing the sample value by the conversion factor 0.103 mU / ng.

[0365] Quantification of PAH protein and activity

[0366] After 48 hours, whole cell lysates were produced by lysing cells in lysis buffer or RIPA buffer. In addition, in some experiments, ultrasonic treatment or shearing was used to enhance cell lysis. After thawing, the lysates were spun at 14,000g for 30 minutes before assay. Quantification of FLAG fusion protein levels was measured by FLAG ELISA (SE002-flag; ABSbio) according to the manufacturer's instructions and using kit standards or in-house purified 3xFLAG-mPAH-FL as protein standards. In vivo samples were normalized by total protein content measured by BCA protein assay kit (Pierce). The enzymatic activity of PAH proteins was measured as previously described by Yew et al. 2013, with some minor modifications. Western blotting for PAH detection was performed using anti-hPAH antibody (LS-C344145; LSBio) or anti-FLAG antibody (F1804; Sigma) using standard protocols.

[0367] In vivo studies

[0368] A group of BTBR-PAH was raised in Taconic enu2 / J mice (McDonald 1996). Homozygous (HOM) and heterozygous (HET) male mice were obtained at 8-12 weeks of age and were raised and maintained according to humane guidelines for animal care and use. For SEAP expression plasmid analysis, plasmids (10 μg / mouse) in sterile saline were administered by hydrodynamic large-volume injection into the tail vein of C57BL / 6 mice, with a minimum of n=8 per test article. Recombinant AAV8 vectors (6-10 animals / treatment) were administered via the tail vein via the intravenous route. In most studies, animals were killed by isoflurane anesthesia after 8-10 weeks. Whole blood was collected into EDTA collection tubes through the retro-orbital sinus, rotated and frozen until analysis. Some animals were perfused with PBS via the left ventricle before tissue collection. Liver samples were collected and frozen until analysis. For brain analysis, whole brains were harvested from the skull, weighed, sagittally cut, and frozen at -80°C until analysis.

[0369] Blood and tissue analysis

[0370] Plasma Phe and Tyr levels were analyzed by UHPLC-MS / MS using a Transcend II LX4 multiplex system equipped with a Dionex Ultimate 3000HPLC (Thermo Fisher, Waltham, Massachusetts, USA) coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, Massachusetts, USA). Standard solutions were prepared using L-Phe and L-Tyr (Sigma-Aldrich, St. Louis, Missouri, USA), and labeled L-Phe-13C9,15N and labeled L-Tyr-13C9,15N (Sigma-Aldrich) were used as internal standards. MS / MS detection was performed in positive ion mode. The analysis was performed using an Acquity BEH C18 column (1.7 μm, 2.1×30 mm) with a gradient separation consisting of holding at 98% mobile phase A (0.1% formic acid in water) for 10 seconds, followed by a gradient of 2%-45% mobile phase B (0.1% formic acid in acetonitrile) for 30 seconds, increasing to 75% B and holding for 15 seconds, washing at 75% B for 10 seconds, and re-equilibration at 98% A for 1 minute at a flow rate of 0.5 mL / min. MS / MS conversions were: 166.1 / 120.1 for Phe, 182.1 / 136.1 for Tyr, 176.1 / 129.1 for labeled Phe, and 192.1 / 145.1 for labeled Tyr.

[0371] For the quantitative of brain neurotransmitter, brain (Kankaanpaa2001) was processed according to the description of slight modification. In brief, with Bead Ruptor 24 (Omni) in ice-cold lysis buffer (1mM oxalic acid, 3mM cysteine, 0.1M acetic acid), at 4 ℃, half sagittal cut, PBS perfused brain homogenate 20 seconds with 4.85m / second. Sample was rotated 10 minutes with 14K, and supernatant was frozen subsequently. Then use Acquity UPLC (Waters Corporation, Milford City, Massachusetts, USA) coupled with API 5000 triple quadrupole mass spectrometer (AB SCIEX, Framingham, Massachusetts, USA), by UPLC-MS / MS quantitative brain homogenate (200mg / mL) L-DOPA, dopamine, homovanillic acid (HVA), serotonin, 5-hydroxytryptophan (5-HTP), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine level. In order to improve the stability of neurotransmitters, 300ng / mL cysteine ​​in 0.1% formic acid (FA) and acetonitrile was used as sample dilution buffer.Similar to the sample as described above, standard substance and standard solution of each analyte (Sigma-Aldrich) were prepared.MS / MS detection was carried out in positive ion mode (HVA was detected in negative ion mode).Acquity HSS C18 SB (1.7 μm, 2.1x100mm) was used for gradient separation, and the gradient separation was included in 98% mobile phase A (0.1% FA in water) and kept for 0.5 minute, followed by 2%-40% mobile phase B (0.1% FA in acetonitrile) gradient and kept for 3.5 minutes, increased to 95% B and kept for 0.1 minute, washed for 0.5 minute at 95% B, and rebalanced for 2.4 minutes at 98% A, with a flow rate of 0.5mL / minute. Positive ion MS / MS transitions were: 198 / 152 for L-DOPA, 177 / 160.1 for serotonin, 154 / 137.1 for dopamine, 192 / 146 for 5-HIAA, 170.1 / 107 for norepinephrine, and 221.1 / 201 for 5-HTP. Detection of HVA was performed in negative ion mode similar to the above method except that the initial hold phase of the gradient was eliminated. The MS / MS transition for HVA in negative ion mode was 181.1 / 137.1. The levels are expressed as μg / mL homogenate or uM. Brain Phe and Tyr levels were quantified as described in the previous section.

[0372] For liver samples, vector genomes, PAH activity, and protein levels were quantified. Copies of vector genomes were quantified by qPCR (Martin 2013). Quantification of FLAG-tagged proteins was performed as follows. Liver samples were homogenized in a tube with beads (15-340-153; Fisher Scientific) in 1× PBS or RIPA buffer containing 1× protease inhibitors at 5.65 rpm at 4°C for 20-30 seconds. The samples were spun at 14,000g for 30 minutes at 4°C. The supernatant was then used for PAH quantification by FLAG ELISA assay and normalized by total protein (BCA protein assay kit; Pierce). PAH protein activity in liver homogenates was performed as described above.

[0373] result

[0374] The hepatic mTTR482 promoter was previously described and contains the mouse transthyretin core promoter and its 5' enhancer element with modifications in the HNF-3 and HNF-4 binding sites (Nambiar 2016). The goal of this study was to further increase the promoter strength. Various liver-based enhancers upstream of this promoter or 3' stability elements downstream of BGH pA were added to the expression cassette, and secreted alkaline phosphatase (SEAP) levels were then measured in vitro as a reporter in two human liver lines ( Figure 1A Transient transfection of SEAP plasmids into Huh7 and HepG2 cells showed that the expression cassette with the mA1MB2 enhancer (No. 3) produced the highest SEAP levels in the culture medium and was approximately 2-fold higher than the parental mTTR482 construct in both cell lines ( Figure 1B , 1C In vivo evaluation of proviral plasmids by hydrodynamic injection in normal C57BL / 6 mice also showed the highest and most sustained SEAP expression with plasmids containing the mA1MB2 enhancer. SEAP levels were 2.8, 4.4, 4, and 3.6 times higher than those detected in animals treated with the parental mTTR482-SEAP plasmid on days 1, 7, 14, and 28, respectively ( Figure 2A ). In PAH enu2 mTTR482 proviral plasmids with and without the mA1MB2 enhancer were evaluated in mice ( Figure 2B). Plasmids containing the mA1MB2 enhancer consistently produced plasma SEAP levels that were 2-fold higher than those obtained with the parental mTTR482 construct. Although SEAP was stably secreted throughout the 28-day study, SEAP levels in PKU mice were approximately 10-fold lower than those obtained with these plasmids in C57Bl / 6 mice. Based on this data, the mA1MB2-mTTR482 promoter was selected as a candidate liver promoter.

[0375] Example 2. PAH enu2 Comparison of the optimized liver promoter and LP1 promoter in mice

[0376] To validate our optimized liver promoter in the PKU disease model, an AAV8 / mA1MB2-mPAH vector was generated and compared to an AAV8 / scLP1-mPAH vector comparable to the vector used by Yagi et al. (2011). Both vectors were administered intravenously at two different doses (4e10 and 1e11, VG / mouse), and blood Phe levels were measured. At both doses, the mA1MB2-mTTR482 vector was more effective in reducing blood Phe levels compared to the sc-LP1 vector ( Figure 3A , Figure 3B Both vectors increased blood Tyr levels at higher doses (1e11 vg / mouse); increases were detected already on day 7 (measured after the first administration) and remained elevated until day 56 (end of study) ( Figure 3C ). Although an increase in blood Tyr was also detected in animals treated with lower doses, the increase was not significantly different from the pre-treatment values ​​of the corresponding animals. Quantification of viral vector genomes in the liver showed that at day 56, the VG copies of the self-complementary vector increased by 2 to 3 times ( Figure 3D ).

[0377] Because brain Phe levels are the primary cause of pathology, Phe levels in the brain were measured. With the 1e11 dose, only the mA1MB2-mTTR482 construct provided low brain Phe levels similar to those of wild-type brain (HET) ( Figure 4A The reduction in brain Phe levels correlated closely with Phe levels measured in the blood ( Figure 4B ). Analysis of brain neurotransmitter levels showed that dopamine elevation was comparable between the two constructs ( Figure 4C Similar efficacy was observed with the mA1MB2-mTTR482 construct at a lower (4e10) dose. Brain serotonin quantification showed that treatment with the mA1MB2-mTTR482 construct produced significantly more serotonin compared to the sc-LP1 vector ( Figure 4D). In conclusion, the effective reduction of blood Phe levels with the mA1MB2-mTTR482 construct translated into a robust reduction of brain Phe levels and subsequently enhanced dopamine and serotonin synthesis in the brains of treated animals.

[0378] Example 3. Evaluation of codon-optimized hPAH cDNA for hPAH production

[0379] Previously published studies have shown that in PAH enu2 In mice, rAAV vectors encoding hPAH have poor efficacy. To test whether better produced hPAH could achieve enhanced efficacy, the effect of various codon usage was tested. To this end, four different codon-optimized cDNAs of hPAH were generated based on different algorithms. The resulting sequences were cloned downstream of the mTTR482 promoter and hPAH protein levels were evaluated in vitro and in vivo. Plasmid transfection into human Huh7 cells showed little difference (all within 2-fold) between the hPAH cDNAs ( Figure 5A When the expression plasmids were delivered into the liver of normal C57BL / 6 mice via hydrodynamic injection, much greater differences were observed between the constructs ( Figure 5B ). Specifically, the expression plasmid with hPAH cDNA generated with the GA algorithm resulted in 7-fold higher detection of FLAG-tagged protein in liver lysate compared to the plasmid with the endogenous, unmodified (non) sequence. Based on this, the human cDNA generated with the GA algorithm was used for subsequent studies.

[0380] Example 4: Comparison of human PAH and mouse PAH in vitro and in vivo

[0381] method

[0382] Plasmid vectors and recombinant AAV production.

[0383] The generation of liver-specific promoters (LP1 and mTTR482), hybrid introns, polyadenylation sites (bovine growth hormone [BGH] and simian virus 40 [SV40]) is known in the art and has been described in Nathwani (2012) and Nambiar (2017). Various plasmid vectors with liver-specific or chicken b-actin (CBA) promoters, hybrid introns, cDNA encoding full-length (FL) human (h) or mouse (m) PAH, and BGH polyA were constructed. Expression cassettes with double truncated forms (DT; amino acids 103-428) of mPAH and hPAH or with various hybrid constructs were also generated. Amino acid modifications in hPAH-DT and mPAH were evaluated in plasmid vectors with CBA promoter, hybrid intron, PAH-DT, and BGH polyA. The amino acid changes in the PAH-DT amino acid sequence variants (V) were generated by either synthesizing the altered DNA sequence or introducing the changes by site-directed mutagenesis (Genscript). For detection and quantification purposes, most constructs contained a sequence encoding the N-terminal 22-amino acid 3xFLAG-peptide (MDYKDHDG DYKDHDI DYKDDDDK (SEQ ID NO: 18)) upstream of the PAH cDNA.

[0384] The selected PAH plasmid vectors with liver-specific promoters (LP1 or mA1MB2-mTTR482), 1069 bp chicken β-actin (CBA) / rabbit β-globin mixed introns, hPAH cDNA (or variant) cDNA, and BGHpA were subcloned into the AAV2 ITR-containing plasmid. The final hPAH-V1 vector construct additionally contained a modified intron with the ATG removed and a 0.9 kb "stuffer" sequence from the α1 antitrypsin intron (reduced ATG) to increase the vector genome size to 4.6 kb. All rAAV vectors were produced using triple transfection with AAV8 serotype capsids followed by CsCl2 purification. Vector batches were quantified by qPCR for BGHpA (Martin 2013).

[0385] Purification of PAH proteins.

[0386] Transient transfection of Expi293F cells (Life Technologies) was performed using the Expi293F Expression System Kit (Life Technologies) in a serum-free suspension culture environment according to the manufacturer's instructions. The pCBA-PAH-BGHpA expression plasmid encoding hPAH, hPAH-V1, and mPAH tagged with FL or DT forms was used for transfection. The cells were spun down and whole cell lysates were generated in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% TritonX-100). Flag-tagged PAH proteins were purified using a FLAG affinity column (ANTI-FLAG M2 affinity gel; Sigma-Aldrich). Proteins were eluted using 0.1 M glycine-HCl (pH 3.5) or 100 ug / ml FLAG peptide (Sigma-Aldrich) in TBS. The proteins were resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl (some batches additionally contained 10% mannitol to improve stability, Nascimento 2010). The proteins were analyzed on SDS-PAGE 4%-12% Tris-BIS-MES gels and stained by Coomassie blue staining. The proteins were also analyzed by Western blotting using antibodies against FLAG peptide or PAH protein.

[0387] Quantification of PAH protein and activity levels.

[0388] HEK293T cells were transiently transfected with a plasmid containing a FLAG-tagged PAH expression cassette using Lipofectamine 2000 (Life Technologies). Whole cell lysates were generated after 48 h by lysing cells in lysis buffer or RIPA buffer (Pierce / ThermoFisher). Additionally, in some experiments, sonication or shearing was used to enhance cell lysis. After thawing, the lysates were spun at 14,000 g for 30 min prior to assay. Quantification of FLAG fusion protein levels was measured by FLAG ELISA (SE002-flag; ABSbio) according to the manufacturer's instructions and using the kit standard or in-house purified 3xFLAG-mPAH-FL as a protein standard. The enzymatic activity of PAH proteins was measured as previously described (Yew 2013) with some minor modifications. Western blotting for PAH detection was performed using anti-hPAH antibodies (LSBio) or anti-FLAG antibodies (Sigma) using standard protocols known in the art.

[0389] result

[0390] The production of human and mouse PAH proteins in Huh7 and 293 cells was evaluated in vitro. To quantify the production of hPAH and mPAH, all expressed proteins contained an N-terminal 3xFLAG tag. The data showed that the levels of hPAH were lower in whole Huh7 cell lysates compared to mPAH and were independent of the expression cassette used ( Fig. 6A Full-length (F1, 1-452) and double-truncated (DT) forms of hPAH showed lower production of hPAH compared to mPAH ( Figure 6B , Figure 6C ). The DT form is a shortened and constitutively active form of PAH consisting of amino acids 103 to 428 and therefore lacks the N-terminal regulatory domain and the C-terminal tetramerization domain of PAH.

[0391] Quantification of mRNA for each construct showed comparable message levels, suggesting that the difference in hPAH and mPAH protein levels is a post-transcriptional effect ( Fig.6D When hPAH and mPAH (FL and DT forms) were purified using a FLAG affinity column, human PAH (both FL and DT forms) showed a very different degradation pattern that was not observed for mouse PAH ( Fig. 6E ).

[0392] In PAH enu2 rAAV8 / sc-LP1 vectors encoding human and mPAH were tested in mice. Animals treated with the vector encoding mPAH showed normal blood Phe levels, whereas in mice treated with a comparable dose of the hPAH vector, little reduction in blood Phe levels was observed ( Fig. 6F The efficacy of the sc-LP1 vector encoding mPAH has been previously demonstrated (Yagi 2011).

[0393] Taken together, this data suggests that the endogenous form of hPAH is less potent than mPAH and that this correlates with the poor production / stability of hPAH protein observed in vitro.

[0394] Example 5: Generation of improved variants of human PAH

[0395] Hybrid hPAH constructs containing varying lengths of N-terminal or C-terminal sequences derived from mPAH were generated ( Fig. 7A These constructs were tested for in vitro production of FLAG-tagged proteins and showed that replacement of either the N-terminus or the C-terminus of hPAH with the corresponding mouse sequences did not increase protein levels ( Figure 7B , Figure 7C). Therefore, the region containing the catalytic core (103-448, DT) is responsible for the stability of mPAH.

[0396] To improve the stability of the hPAH core region, various modifications were introduced into the hPAH-DT backbone. Various amino acid modifications were introduced into the hPAH sequence using the hPAH-DT backbone. The amino acid changes were modeled based on the amino acid sequences of PAH in other species, including mice, chickens, and bacteria (Table 1). All constructs also contained a 3x-FLAG-tag in the flexible N-terminus. Human variants (V; V1-13) were expressed in vitro in 293 cells, and whole cell lysates were quantified for PAH protein and activity levels and compared with the DT forms of hPAH and mPAH ( Fig. 8A ). Protein quantification showed that some hPAH variants, such as V1, V5 (one additional change compared to V1), and V8 (two amino acid changes different from V1) had increased protein levels compared to unmodified hPAH. However, when these variants were tested for PAH enzyme activity, only V1 had significantly increased PAH activity ( Figure 8B ). V1 consists of four amino acid changes (M180T, K199P, S250P and G256A) located in three different parts of the catalytic domain. No changes were observed in construct V9-13 (data not shown).

[0397] Table 1. Description of human PAH variants

[0398]

[0399]

[0400] Additional derivatives of V1 were generated. The first group included constructs with only one amino acid change (V14-17, Table 1). PAH activity results showed that PAH levels and activities of all of these constructs were poor, comparable to unmodified hPAH, and thus indicated that none of these changes alone provided any improvement ( Figure 9A-9C , Figure 10A-10C ). The second set of constructs were derivatives of V1 in which one of the four amino acids was reduced to a human wild-type amino acid.

[0401] Table 2. Human PAH variant-1 derivatives

[0402]

[0403] It was then determined whether three amino acid changes were sufficient (V18-21). The best of these was V20 without the S250P change, which had approximately 67% of the activity of V1 ( Fig. 9AInterestingly, this variant, despite having lower total enzyme activity due to reduced PAH protein levels, had a 2-fold increase in specific activity compared to the parent V1 ( Fig. 9B , Fig. 9C ).

[0404] A set of double mutant derivatives of human PAH variant-1 was also tested. Unlike the single mutant series (V14-17), double mutants V29 and V30 achieved some improvement in activity, but not as much as V1 ( Fig. 10D ).

[0405] To further improve V1, additional changes were introduced, some of which were intended to increase specific activity (V22-28, Table 2). Although none of these changes increased total PAH activity, some modifications did increase PAH protein levels or their specific activity. For example, V23 had 2-fold more PAH protein than hPAH-V1.

[0406] "Reverse" mutagenesis of mPAH was performed by sequentially changing two or three residues at a time to those residues present in the region 103-428 in hPAH. Activity testing of five constructs spanning this region showed that none of these changes resulted in a reduction in mPAH production to the levels observed for hPAH (data not shown).

[0407] Table 3. Mouse PAH variants

[0408]

[0409]

[0410] An expression cassette was constructed encoding the best performing variant (V1) as a full-length protein expressed from the A1MB2-mTTR promoter. Following transfection of the plasmid into Huh7 cells, this construct was compared to comparable mouse PAH and hPAH expression plasmids for PAH protein production and activity. The data demonstrated that hPAH-V1 increased PAH protein production and activity by approximately 10-fold ( Figure 11A-11C ). All PAH proteins had comparable activity to protein ratios, indicating that the specific activities were roughly similar.

[0411] Taken together, the data demonstrate the feasibility of improving the PAH profile in humans through amino acid changes (eg, 2, 3, 4, 5, or 6 amino acid changes), all of which contribute to the observed improvements.

[0412] Example 6: Correction of hyperphenylalaninemia by PAH-V1

[0413] method

[0414] Blood and tissue analysis.

[0415] Brain and tissue analyses were performed as described in Examples 1-3.

[0416] Animal experiments.

[0417] Animal experiments were performed as described in Examples 1-3.

[0418] result

[0419] To test the efficacy of the modified hPAH in vivo, rAAV8 vectors encoding hPAH-V1 were generated, as well as similar vectors with either mouse or hPAH (both in FL form). enu2 Mouse models showed a rapid decrease in blood Phe levels and an increase in blood Tyr ( Fig. 12A , Fig. 12B Similar effects were observed for various Phe metabolites measured in blood ( Fig. 12C ). Overall, modified hPAH-V1 was more effective for all of these endpoints compared to endogenous hPAH. The increased efficacy of hPAH-V1 was directly correlated to increased PAH protein levels and activity in the liver ( Fig.13A , Fig. 13B All treatment groups had comparable vector genome copies in the liver ( Fig. 13C ).

[0420] Different brain endpoints were analyzed (performed in 3e11 VG / mouse treated groups). The data showed that compared with initial PAH enu2 Compared with mice, the Phe level in the brain of treated mice was significantly reduced, and the Tyr level was increased ( Fig.14A , Fig. 14B The changes in brain Phe levels were closely correlated with the reduction in blood Phe levels and also showed better control of Phe in the hPAH-V1 treated group ( Fig. 14C Because the amino acids Phe, Tyr, and Trp all use the same large neutral amino acid transporter (LAT1), similar improvements were observed in brain Trp levels ( Fig.14D ). Both hPAH and hPAH-V1 caused comparable increases in the levels of these amino acids in the brain.

[0421] Levels of the brain neurotransmitters dopamine and serotonin and their metabolites were measured in the brains of treated mice. The data showed that increases in these neurotransmitters were significantly higher in animals treated with hPAH-V1 compared to hPAH ( Figures 15A-15D ).

[0422] In summary, the improved properties of the hPAH-V1 protein translate into an enhanced ability to reduce blood and brain Phe levels and increase Tyr and neurotransmitter levels in the brain. As comparable vector genome levels were present in the liver, the data suggest that higher efficacy was obtained with vectors encoding hPAH-V1.

[0423] A 4.6 kb genome was generated for the mA1MB2-mTTR482 promoter / hPAH-V1 vector. This genome did not contain an "ATG" sequence in the intron sequence, except for the stuffer sequence introduced downstream of the BGH pA. Fig.16A ). When in PAH enu2 When this vector was tested in mice, significantly better efficacy was observed as measured by reduction in blood Phe levels when compared to the parental 3.8 kb vector ( Fig. 16B ). This experiment also included a 4.6 kb vector lacking the N-terminal FLAG tag for measuring PAH protein levels. The data showed that removal of this tag did not alter the efficacy of the vector genome. In summary, the modified hPAH-V1 confers increased potency relative to endogenous hPAH and, therefore, is expected to provide efficacy at lower vector doses compared to vectors encoding hPAH.

[0424] Example 7. Increased hPAH-V1 protein levels in NHP livers

[0425] Materials and methods:

[0426] Construction of recombinant AAV vector

[0427] Vector production of A1MB2-HI-hPAH and A1MB2-HI-hPAH-V1 was performed as described in Example 4, except that hybrid capsids were used.

[0428] Animal experiments

[0429] Male cynomolgus monkeys (Macaca fascicularis) aged 2 to 3 years (2-4 kg) received 10 ml of the test article by slow intravenous infusion (1 ml / min) into the saphenous vein. Treatment groups consisted of vehicle group (PBS, n=1), rAAV / hPAH (n=3), and rAAV / hPAH-V1 (n=3). Animals were humanely euthanized after two weeks, and various tissues were collected and frozen at -80°C until analysis.

[0430] Quantification of vector genome, mRNA, and PAH protein

[0431] Copies of the vector genome and vector-derived mRNA in the liver and spleen were quantified by qPCR using primers / probes against BGHpA as described in Examples 1-3. Levels of human PAH protein in liver homogenates were analyzed by Western blotting for the FLAG tag as described in Examples 1-3.

[0432] result:

[0433] To test the functionality of the A1MB2-mTTR promoter in NHP livers, mRNA and PAH production levels were evaluated at two times after vector delivery. On average, comparable levels of vector and vector-derived mRNA (normalized to vector genome copies) were detected in the livers of all vector-treated animals ( Fig.17A , Fig. 17B The liver specificity of expression was analyzed by comparing vector mRNA in liver and spleen (mRNA levels in both tissues were normalized to vector genome copies) and demonstrated that the transcript / VG ratio was higher in liver than in spleen ( Fig. 17B ). Transduction was also assessed by testing the detection of vector-derived FLAG-tagged PAH protein in the liver. All animals treated with vector encoding PAH-V1 demonstrated the presence of hPAH-V1 of the correct size in the liver. In contrast, little to no hPAH protein was detected in animals treated with vector encoding hPAH ( Fig. 17C ). Since both vector genome and mRNA levels were comparable in both treatment groups, these results suggest a better stability of the hPAH-V1 protein in the NHP liver. These observations are similar to studies performed in mice and in vitro studies with human hepatocyte lines, indicating the superiority of hPAH-V1 in all tested systems.

[0434] References

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[0450] Ledley FD,et al.,Biochem J 1990,267:399-406.

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[0464] Lisowski L,et al.,Nature,2014,506:382-6.

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[0466] Sequence Human phenylalanine hydroxylase (GenBank AAA60082.1 and AAC51772.1) - amino acid sequence MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQR

[0467] IEVLDNTQQLKILADSINSEIGILCSALQKIK(SEQ ID NO:1)

[0468] Human phenylalanine hydroxylase (GenBank AAH26251.1) - amino acid sequence

[0469] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPS

[0470] RLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGA

[0471] ELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEGKKTWGTVFKTLKSLYKTHACYEYNHIF

[0472] PLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPE

[0473] PDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSS

[0474] FGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQR

[0475] IEVLDNTQQLKILADSINSEIGILCSALQKIK(SEQ ID NO:2)

[0476] Human PAH-V1 amino acid sequence (M180T, K199P, S250P and G256A)

[0477] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPS

[0478] RLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGA

[0479] ELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYTEEEKKTWGTVFKTLKSLYPTHACYEYNHIF

[0480] PLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLPSRDFLAGLAFRVFHCTQYIRHGSKPMYTPE

[0481] PDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSS

[0482] FGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQR

[0483] IEVLDNTQQLKILADSINSEIGILCSALQKIK(SEQ ID NO:3)

[0484] Human PAH-V1 DNA sequence

[0485] ATGAGCACAGCCGTGCTGGAAAACCCCGGCCTGGGCAGAAAGCTGAGCGACTTCGGCCAGGAAACCAGCTACATCGAGGACAACTGCAACC

[0486] AGAACGGCGCCATCAGCCTGATCTTCAGCCTGAAAGAAGAAGTGGGCGCCCTGGCCAAGGTGCTGCGGCTGTTCGAGGAGAACGACGTGAA

[0487] CCTGACCCACATCGAGAGCCGGCCCAGCAGACTGAAGAAGGACGAGTACGAGTTCTTCACCCACCTGGACAAGCGGAGCCTGCCCGCCCTG

[0488] ACCAACATCATCAAGATCCTGCGGCACGACATCGGCGCCACCGTGCACGAGCTGAGCCGGGACAAGAAAAAGGACACCGTGCCCTGGTTCC

[0489] CCAGAACCATCCAGGAACTGGACAGATTCGCCAACCAGATCCTGTCCTACGGCGCCGAGCTGGATGCCGACCACCCTGGCTTCAAGGACCC

[0490] CGTGTACCGGGCCAGACGGAAGCAGTTCGCCGATATCGCCTACAACTACCGGCACGGCCAGCCCATCCCCAGAGTCGAGTACACCGAAGAG

[0491] GGCAAGAAAACCTGGGGCACCGTGTTCAAGACCCTGAAGTCCCTGTACCCCACCCACGCCTGCTACGAGTACAACCACATCTTCCCACTGC

[0492] TCGAAAAGTACTGCGGCTTCCACGAGGACAATATCCCTCAGCTGGAGGACGTGTCCCAGTTTCTGCAGACCTGCACCGGCTTCAGACTCAG

[0493] GCCTGTGGCCGGCCTGCTGCCCAGCAGAGATTTTCTGGCCGGACTGGCCTTCCGGGTGTTCCACTGCACCCAGTACATCAGACACGGCAGC

[0494] AAGCCCATGTACACCCCTGAGCCCGACATCTGCCACGAGCTGCTGGGACATGTGCCCCTGTTCAGCGACAGAAGCTTCGCCCAGTTCAGCC

[0495] AGGAAATCGGCCTGGCCTCTCTGGGCGCTCCCGACGAGTATATCGAGAAGCTGGCCACCATCTACTGGTTCACCGTGGAATTCGGCCTGTG

[0496] CAAGCAGGGCGACAGCATCAAGGCCTATGGCGCCGGACTCCTGTCCAGCTTCGGCGAGCTGCAGTACTGTCTGAGCGAGAAGCCCAAGCTG

[0497] CTGCCCCTGGAACTGGAAAAGACCGCCATCCAGAACTACACCGTGACCGAGTTCCAGCCCCTGTACTACGTGGCCGAGAGCTTCAACGACG

[0498] CCAAAGAAAAAGTGCGGAACTTCGCCGCCACCATCCCTCGGCCCTTCAGCGTCAGATACGACCCCTACACCCAGCGGATCGAGGTGCTGGACAACACAGCAGCTGAAAATCTGGCCGACTCCATCAACAGCGAGATCGGCATCCCTGTGCAGCGCCCTGCAGAAATTCAAGTGA(SEQ ID NO:4)

[0499] 3.8kb optimized vector genome sequence (ITR-A1MB2-mTTR-HI-hPAH V1-BGHpA-ITR)

[0500] GAGCTCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGCTTTGCCCGGGCGG

[0501] CCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTACGCGTGGCCCCAGGTTAATTTTTTAAAAAGC

[0502] AGTCAAAGGTCAAAGTGGCCCTTGGCAGCATTTACTCTCTCTATTTGACTTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGG

[0503] AGAAGAAATCAACATCCTGGACTTATCCTCTGGGCCTCTCCCCACCTTCGATGGCCCCAGGTTATTTTAAAGCAGTCAAAAGGTCAAA

[0504] GTGGCCCTTGGCAGCATTTACTCTCTCTATTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACA

[0505] TCCTGGACTTATCCTCTGGGCCTCTCCCCACCGATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATACTGTCGGGG

[0506] CAAAGGTCGGCAGTAGTTTTCCATCTTACTCAACATCCTCCCAGTGTACGTAGGATCCTGTCTGTCTGCACATTTCGTAGAGCGAGTGTTC

[0507] CGATACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGT

[0508] TTGGAGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCAC

[0509] AAGCTCCTGCTAGCAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCG

[0510] CGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTG

[0511] GCTGCGTGAAAGCCTTGAGGGGCTCCGGGAAGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGC

[0512] GCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCG

[0513] GGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGG

[0514] TGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACG

[0515] GGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCT

[0516] CGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAG

[0517] GGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGG

[0518] TGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC

[0519] CGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCA

[0520] TGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCATTTCGAAG

[0521] CCGCCACCATGAGCACAGCCGTGCTGGAAAACCCCGGCCTGGGCAGAAAGCTGAGCGACTTCGGCCAGGAAACCAGCTACATCGAGGACAA

[0522] CTGCAACCAGAACGGCGCCATCAGCCTGATCTTCAGCCTGAAAGAAGAAGTGGGCGCCCTGGCCAAGGTGCTGCGGCTGTTCGAGGAGAAC

[0523] GACGTGAACCTGACCCACATCGAGAGCCGGCCCAGCAGACTGAAGAAGGACGAGTACGAGTTCTTCACCCACCTGGACAAGCGGAGCCTGC

[0524] CCGCCCTGACCAACATCATCAAGATCCTGCGGCACGACATCGGCGCCACCGTGCACGAGCTGAGCCGGGACAAGAAAAAGGACACCGTGCC

[0525] CTGGTTCCCCAGAACCATCCAGGAACTGGACAGATTCGCCAACCAGATCCTGTCCTACGGCGCCGAGCTGGATGCCGACCACCCTGGCTTC

[0526] AAGGACCCCGTGTACCGGGCCAGACGGAAGCAGTTCGCCGATATCGCCTACAACTACCGGCACGGCCAGCCCATCCCCAGAGTCGAGTACA

[0527] CCGAAGAGGGCAAGAAAACCTGGGGCACCGTGTTCAAGACCCTGAAGTCCCTGTACCCCACCCACGCCTGCTACGAGTACAACCACATCTT

[0528] CCCACTGCTCGAAAAGTACTGCGGCTTCCACGAGGACAATATCCCTCAGCTGGAGGACGTGTCCCAGTTTCTGCAGACCTGCACCGGCTTC

[0529] AGACTCAGGCCTGTGGCCGGCCTGCTGCCCAGCAGAGATTTTCTGGCCGGACTGGCCTTCCGGGTGTTCCACTGCACCCAGTACATCAGAC

[0530] ACGGCAGCAAGCCCATGTACACCCCTGAGCCCGACATCTGCCACGAGCTGCTGGGACATGTGCCCCTGTTCAGCGACAGAAGCTTCGCCCA

[0531] GTTCAGCCAGGAAATCGGCCTGGCCTCTCTGGGCGCTCCCGACGAGTATATCGAGAAGCTGGCCACCATCTACTGGTTCACCGTGGAATTC

[0532] GGCCTGTGCAAGCAGGGCGACAGCATCAAGGCCTATGGCGCCGGACTCCTGTCCAGCTTCGGCGAGCTGCAGTACTGTCTGAGCGAGAAGC

[0533] CCAAGCTGCTGCCCCTGGAACTGGAAAAGACCGCCATCCAGAACTACACCGTGACCGAGTTCCAGCCCCTGTACTACGTGGCCGAGAGCTT

[0534] CAACGACGCCAAAGAAAAAGTGCGGAACTTCGCCGCCACCATCCCTCGGCCCTTCAGCGTCAGATACGACCCCTACACCCAGCGGATCGAG

[0535] GTGCTGGACAACACACAGCAGCTGAAAATTCTGGCCGACTCCATCAACAGCGAGATCGGCATCCTGTGCAGCGCCCTGCAGAAAATCAAGT

[0536] GACCTAGGTGATCAAGATCTGCTAGCTTAATTAACCCGGGACTAGTGCGGCCGCTCGAGACTAGTCTGTGCCTTCTAGTTGCCAGCCATCT

[0537] GTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT

[0538] GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGA

[0539] TGCGGTGGGCTCTATGGTACCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCC(SEQ ID NO:5)

[0540] 4.6kb optimized vector genome sequence (ITR-A1MB2-mTTR-HI[noATGs]-hPAH V1-BGHpA-filler Nucleic acid-ITR)(4561bp)

[0541] TTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAG

[0542] TGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTACCGCGTGGCCCCAGGTTAATTTTTAAAAAGCAGTC

[0543] AAAGGTCAAAGTGGCCCTTGGCAGCATTTACTCTCTCTATTGACTTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAA

[0544] GAAATCAACATCCTGGACTTATCCTCTGGGCCTCTCCCCACCTTCGATGGCCCCAGGTTAATTTTTAAAAAGCAGTCAAAGGTCAAAGTGG

[0545] CCCTTGGCAGCATTTACTCTCTCTATTGACTTTGGTTAATAATCTCAGGAGCACAAACATTCCTGGAGGCAGGAGAAGAAATCAACATCCT

[0546] GGACTTATCCTCTGGGCCTCTCCCCACCGATATCTACCTGCTGATCGCCCGGCCCCTGTTCAAACATGTCCTAATACTCTGTCGGGGCAAA

[0547] GGTCGGCAGTAGTTTTCCATCTTACTCAACATCCTCCCAGTGTACGTAGGATCCTGTCTGTCTGCACATTTCGTAGAGCGAGTGTTCCGAT

[0548] ACTCTAATCTCCCGGGGCAAAGGTCGTATTGACTTAGGTTACTTATTCTCCTTTTGTTGACTAAGTCAATAATCAGAATCAGCAGGTTTGG

[0549] AGTCAGCTTGGCAGGGATCAGCAGCCTGGGTTGGAAGGAGGGGGTATAAAAGCCCCTTCACCAGGAGAAGCCGTCACACAGATCCACAAGC

[0550] TCCTGCTAGCCAATTGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGAC

[0551] CGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTATTGACGGCTTGTTTCTTTTCTG

[0552] TGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAAGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGA

[0553] GCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAG

[0554] CGGGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGG

[0555] GGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTA

[0556] CGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGG

[0557] CTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTTTGGTAATCGTGCGAG

[0558] AGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGC

[0559] GGTGCGGCGCCGGCAGGAAGGAATTGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG

[0560] TCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAAC

[0561] CTTGTTCTTGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCATTTCGA

[0562] AGCCGCCACCATGAGCACAGCCGTGCTGGAAAACCCCGGCCTGGGCAGAAAGCTGAGCGACTTCGGCCAGGAAACCAGCTACATCGAGGAC

[0563] AACTGCAACCAGAACGGCGCCATCAGCCTGATCTTCAGCCTGAAAGAAGAAGTGGGCGCCCTGGCCAAGGTGCTGCGGCTGTTCGAGGAGA

[0564] ACGACGTGAACCTGACCCACATCGAGAGCCGGCCCAGCAGACTGAAGAAGGACGAGTACGAGTTCTTCACCCACCTGGACAAGCGGAGCCT

[0565] GCCCGCCCTGACCAACATCATCAAGATCCTGCGGCACGACATCGGCGCCACCGTGCACGAGCTGAGCCGGGACAAGAAAAAGGACACCGTG

[0566] CCCTGGTTCCCCAGAACCATCCAGGAACTGGACAGATTCGCCAACCAGATCCTGTCCTACGGCGCCGAGCTGGATGCCGACCACCCTGGCT

[0567] TCAAGGACCCCGTGTACCGGGCCAGACGGAAGCAGTTCGCCGATATCGCCTACAACTACCGGCACGGCCAGCCCATCCCCAGAGTCGAGTA

[0568] CACCGAAGAGGGCAAGAAAACCTGGGGCACCGTGTTCAAGACCCTGAAGTCCCTGTACCCCACCCACGCCTGCTACGAGTACAACCACATC

[0569] TTCCCACTGCTCGAAAAGTACTGCGGCTTCCACGAGGACAATATCCCTCAGCTGGAGGACGTGTCCCAGTTTCTGCAGACCTGCACCGGCT

[0570] TCAGACTCAGGCCTGTGGCCGGCCTGCTGCCCAGCAGAGATTTTCTGGCCGGACTGGCCTTCCGGGTGTTCCACTGCACCCAGTACATCAG

[0571] ACACGGCAGCAAGCCCATGTACACCCCTGAGCCCGACATCTGCCACGAGCTGCTGGGACATGTGCCCCTGTTCAGCGACAGAAGCTTCGCC

[0572] CAGTTCAGCCAGGAAATCGGCCTGGCCTCTCTGGGCGCTCCCGACGAGTATATCGAGAAGCTGGCCACCATCTACTGGTTCACCGTGGAAT

[0573] TCGGCCTGTGCAAGCAGGGCGACAGCATCAAGGCCTATGGCGCCGGACTCCTGTCCAGCTTCGGCGAGCTGCAGTACTGTCTGAGCGAGAA

[0574] GCCCAAGCTGCTGCCCCTGGAACTGGAAAAGACCGCCATCCAGAACTACACCGTGACCGAGTTCCAGCCCCTGTACTACGTGGCCGAGAGC

[0575] TTCAACGACGCCAAAGAAAAAGTGCGGAACTTCGCCGCCACCATCCCTCGGCCCTTCAGCGTCAGATACGACCCCTACACCCAGCGGATCG

[0576] AGGTGCTGGACAACACACAGCAGCTGAAAATTCTGGCCGACTCCATCAACAGCGAGATCGGCATCCTGTGCAGCGCCCTGCAGAAAATCAA

[0577] GTGAACTAGTCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTG

[0578] TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGG

[0579] GGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATACCGGTCCAGGGGTGAGTGAAGGTTTGGAAGAGTGTAGCA

[0580] GAATAAGAAACCATGAGTCCCCTCCCTGAGAAGCCCTGAGCCCCCTTGACGACACACATCCCTCGAGGCTCAGCTTCATCATCTGTAAAAG

[0581] GTGCTGAAACTGACCATCCAAGCTGCCGAAAAAGATTGTGTGGGGATAATTCAAAACTAGAGGAAGATGCAGAATTTCTACATCGTGGCGA

[0582] TGTCAGGCTAAGAGTTGCCATCGTGGCTGTCCATCGATTTTATTGGAATCATATGTTTATTTGAGGGTGTCTTGGATATTACAAATAAATT

[0583] GTTGGAGCATCAGGCATATTTGGTAATTCTGTCTAAGGCTCCCTGCCCCTTGTTAATTGGCAGCTCAGTTATTCATCCAGGGCAAACATTC

[0584] TGCTTACTATTCCTGAGAGCTTTCCTCATCCTCTAGATTGGCAGGGGAATTGCAGTTGCCTGAGCAGCCTCCCCTCTGCCATACCAACAGA

[0585] GCTTCACCATCGAGGCTTGCAGAGTGGACAGGGGCCTCAGGGACCCCTGATCCCAGCTTTCTCATTGGACAGAAGGAGGAGACTGGGGCTG

[0586] GAGAGGGACCTGGGCCCCACTAAGGCCACAGCAGAGCCAGGACTTTAGCTGTGCTGACTGCAGCCTGGCTTGCCTCCACTGCCCTCCTTT

[0587] GCCTCAAGAGCAAGGGAGCCTCAGAGTGGAGGAAGCAGCCCCTGGCCTTGCCTCCCACCTCCCCTCCCCTTTGCTGTTTTCCTGGGACAGT

[0588] GGGAGCTGGCTTAGATTGCCCTGGGGCCCCCAGGACCCTGGCATTTTAACCCCTCAGGGGCAGGAAGGCAGCCTGAGATACAGAAGAGTCC

[0589] ATCACCTGCTGTATGCCACACACCATCCCAGTCGACATTTAAATTAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC

[0590] TCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAA (SEQ ID NO: 6)

[0591] 5' and 3' sequences used in the liver expression cassette

[0592] Modified PrT2 enhancer sequence

[0593]

[0594] Underlined, hepatocyte nuclear factor binding site; bold, modifications introduced to generate higher affinity binding site, italics, repeated sequences

[0595] Modified A1MB2 enhancer

[0596]

[0597] Underlined, hepatocyte nuclear factor binding site; bold, modifications introduced to generate higher affinity binding site, italics, repeated sequences

[0598] Modified Ealb sequence

[0599]

[0600] Underlined, hepatocyte nuclear factor binding site; bold, modifications introduced to generate higher affinity binding site, italics, repeated sequences

[0601] HEII enhancer

[0602] CCATCAGATCCTGCCCAAGGTCTTACATAAGA GGACTCTTGGAC TCCCAGCAATGTCAACGACCGACCTTGAGGCCTACTTCAAAGACTGT GTGTTTAAGGACTGGGAGGAGCTGGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTG(SEQ ID NO:10)

[0603] CRM8 enhancer

[0604] GGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCAC (SEQ ID NO: 11)

[0605] 3'Alb stabilizing element

[0606] CTCAATTGGATGACACTAGTCATCACATTTAAAAGCATCTCAGGTAACTATATTTTGAATTTTTTAAAAAAGTAACTATAATAGTTATTAT

[0607] TAAAATAGCAAAGATTGACCATTTCCAAGAGCCATATAGACCAGCACCGACCACTATTCTAAACTATTTATGTATGTAAATATTAGCTTTT

[0608] AAAATTCTCAAAATAGTTGCTGAGTTGGGAACCACTATTATTTCTATCGATTCAGCAGCCGTAAGTCTAGGACAGGCTTAAATTGTTTTCA

[0609] CTGGTGTAAATTGCAGAAAGATGATCTAAGTAATTTGGCATTTATTTTAATAGGTTTGAAAAACACATGCCATTTTACAAATAAGACTTAT

[0610] ATTTGTCCTTTTGTTTTTCAGCCTACCATGAATAAGAAAGAAAATGAAGATCAAAAGCTTATTCTCTGTTTTTCTTTTTCGTTGGT

[0611] GTAAAGCCAACACCCTGTCTAAAAAACATAAATTTCTTTAATTTTTGCCTCTTTTCTCTGTGCTTCAATTAAAAAATGGAAAGAAT

[0612] CTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGAAAATCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCT

[0613] TATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAAATAATTAATACTCTTCTAAGTTATGGATTATAAACATTCAA

[0614] AATAATATTTTGACATTATGATAATCTGAATAAAAGAACAAAAACCATGGTATAGGTAAGGAATAAAACATGGCTTTTACCTTAGAAAAAACAATTCTAAAATTCATGGAATCAAAAAGAGCCTGCAGGTACCCT(SEQID NO:12)

[0615] 3'alb and SMAR stability elements

[0616] CTCAATTGGATGACACTAGTCATCACATTTAAAAGCATCTCAGGTAACTATATTGAATTTTTTAAAAGTAACTAATAGTTATTAT

[0617] TAAAATAGCAAAGATTGACCATTCCAAGAGCCATATAGACCAGCACCGACCACTATTCTAAACTATTTATGTATGTAAATTAGCTTTT

[0618] AAAATTCTCAAAATAGTTGCTGAGTTGGGAACCACTATTATTTCTATCTACTGTTTTAATTAAAATTATCTCTAAGGCATGTGAACTGGCT

[0619] GTCTTGGTTTTCATCTGTACTTCATCTGCTACCTCTGTGACCTGAAACATATTTATAATTCCATTAAGCTGTGCATATGATAGATTTATCA

[0620] TATGTATTTTCCTTAAAGGATTTTTGTAAGAACTAATTGAATTGATACCTGTAAAGTCTTTATCACACTACCCAATAAAATAATAAATCTCT

[0621] TTGTTCAGCTCTCTGTTTCTATAAATATGTACCAGTTTTATTGTTTTTAGTGGTAGTGATTTTATTCTCTTTCTATATATATACACACACA

[0622] TGTGTGCATTCATAAATATATACAATTTTTATGAATAAAATTATTAGCAATCAATATTGAAAACCACTGATTTTTTGTTTATGTGAGCAA

[0623] ACAGCAGATTAAAAGGAATTCCTGCAGATTCAGCAGCCGTAAGTCTAGGACAGGCTTAAATTGTTTTCACTGGTGTAAATTGCAGAAAGAT

[0624] GATTTAAGTAATTTGGCATTTATTTTAATAGGTTTGAAAAACACATGCCATTTTACAAATAAGACTTATATTTGTCCTTTTGTTTTTCAGC

[0625] CTACCATGAGAATAAGAGAAAGAAAATGAAGATCAAAAGCTTATTCATCTGTTTTTCTTTTTCGTTGGTGTAAAGCCAACACCCTGTCTAAAAAAACATAAATTTCTTTAATCATTTTGCCTCTTTTCTCTGTGCTTCAATTAATAAAAAATGGAAAGAATCTAATAGAGTGGTACAGCACTGTTATTTTTCAAAGATGTGTTGCTATCCTGGAAAATTCTGTAGGTTCTGTGGAAGTTCCAGTGTTCTCTCTTATTCCACTTCGGTAGAGGATTTCTAGTTTCTTGTGGGCTAATTAAATAAATCATTAATACTCTTCTAAGTTATGGATTATAAACATTCAAAATAATATTTTGACATTATGATAATTCTGAATAAAAGAACAAAAACCATGGTATAGGTAAGGAATATAAAACATGGCTTTTACCTTAGAAAAAACAATTCTAAAATTCATATGGAATCAAAAAAGAGCCTGCAGGTACCCT(SEQ ID NO:13)

[0626] Codon-optimized human PAH cDNA

[0627] ATGAGCACAGCCGTGCTGGAAAACCCCGGCCTGGGCAGAAAGCTGAGCGACTTCGGCCAGGAAACCAGCTACATCGAGGACAACTGCAACC

[0628] AGAACGGCGCCATCAGCCTGATCTTCAGCCTGAAAGAAGAAGTGGGCGCCCTGGCCAAGGTGCTGCGGCTGTTCGAAGAGAACGACGTGAA

[0629] CCTGACCCACATCGAGAGCCGGCCCAGCAGACTGAAGAAGGACGAGTACGAGTTCTTCACCCACCTGGACAAGCGGAGCCTGCCCGCCCTG

[0630] ACCAACATCATCAAGATCCTGCGGCACGACATCGGCGCCACCGTGCACGAGCTGAGCCGGGACAAGAAAAAGGACACCGTGCCCTGGTTCC

[0631] CCAGAACCATCCAGGAACTGGACAGATTCGCCAACCAGATCCTGTCCTACGGCGCCGAGCTGGATGCCGACCACCCTGGCTTCAAGGACCC

[0632] CGTGTACCGGGCCAGACGGAAGCAGTTCGCCGATATCGCCTACAACTACCGGCACGGCCAGCCCATCCCCAGAGTCGAGTACATGGAAGAG

[0633] GGCAAGAAAACCTGGGGCACCGTGTTCAAGACCCTGAAGTCCCTGTACAAGACCCACGCCTGCTACGAGTACAACCACATCTTCCCACTGC

[0634] TCGAGAAGTACTGCGGCTTCCACGAGGACAATATCCCTCAGCTCGAGGACGTGTCCCAGTTTCTGCAGACCTGCACCGGCTTCAGACTCAG

[0635] GCCTGTGGCCGGCCTGCTGAGCAGCAGAGATTTTCTGGGCGGACTGGCCTTCCGGGTGTTCCACTGCACCCAGTACATCAGACACGGCAGC

[0636] AAGCCCATGTACACCCCTGAGCCCGACATCTGCCACGAGCTGCTGGGACATGTGCCCCTGTTCAGCGACAGAAGCTTCGCCCAGTTCAGCC

[0637] AGGAAATCGGCCTGGCCTCTCTGGGCGCTCCCGACGAGTATATCGAGAAGCTGGCCACCATCTACTGGTTCACCGTGGAATTCGGCCTGTG

[0638] CAAGCAGGGCGACAGCATCAAGGCCTATGGCGCCGGACTCCTGTCCAGCTTCGGCGAGCTGCAGTACTGTCTGAGCGAGAAGCCCAAGCTG

[0639] CTGCCCCTGGAACTGGAAAAGACCGCCATCCAGAACTACACCGTGACCGAGTTCCAGCCCCTGTACTACGTGGCCGAGAGCTTCAACGACG

[0640] CCAAAGAAAAAGTGCGGAACTTCGCCGCCACCATCCCTCGGCCCTTCACGTCAGATACGACCCCTACACCCAGCGGATCGAGGTGCTGGACAACACACAGCAGCTGAAAATTCTGGCCGACTCCATCAACAGCGAGATCGGCATCCTGTGCAGCGCCCTGCAGAAAATCAAGTGA (SEQ ID NO: 14)

[0641] Modified chicken β-actin (CBA) / rabbit β-globin hybrid promoter / intron

[0642]

[0643] Bold = changed to T to eliminate G, A, A, A, A of ATG (5 changes) 0.9 kb A1AT intron stuffer sequence

[0644]

[0645] Bold = 7 bases changed to eliminate ATG; all A to T. Sequence Listing <110> New Company <120> Generation of modified human PAH for severe PKU treated by liver-directed gene replacement therapy <130> 15979-20166.00 <140> Not yet assigned <141> Submit together <150> US 62 / 744,944 <151> 2018-10-12 <160> 18 <170> FastSEQ for Windows version 4.0 <210> 1 <211> 452 <212> PRT <213> Homo sapiens <400> 1 Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu Ser 1 5 10 15 Asp Phe Gly Gln Glu Thr Ser Tyr Ile Glu Asp Asn Cys Asn Gln Asn 20 25 30 Gly Ala Ile Ser Leu Ile Phe Ser Leu Lys Glu Glu Val Gly Ala Leu 35 40 45 Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val Asn Leu Thr His 50 55 60 Ile Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp Glu Tyr Glu Phe Phe 65 70 75 80 Thr His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Ile Lys 85 90 95 Ile Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg Asp 100 105 110 Lys Lys Lys Asp Thr Val Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu 115 120 125 Asp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala 130 135 140 Asp His Pro Gly Phe Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln 145 150 155 160 Phe Ala Asp Ile Ala Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg 165 170 175 Val Glu Tyr Met Glu Glu Glu Lys Lys Thr Trp Gly Thr Val Phe Lys 180 185 190 Thr Leu Lys Ser Leu Tyr Lys Thr His Ala Cys Tyr Glu Tyr Asn His 195 200 205 Ile Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp Asn Ile 210 215 220 Pro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr Gly Phe 225 230 235 240 Arg Leu Arg Pro Val Ala Gly Leu Leu Ser Ser Arg Asp Phe Leu Gly 245 250 255 Gly Leu Ala Phe Arg Val Phe His Cys Thr Gln Tyr Ile Arg His Gly 260 265 270 Ser Lys Pro Met Tyr Thr Pro Glu Pro Asp Ile Cys His Glu Leu Leu 275 280 285 Gly His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln 290 295 300 Glu Ile Gly Leu Ala Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu Lys 305 310 315 320 Leu Ala Thr Ile Tyr Trp Phe Thr Val Glu Phe Gly Leu Cys Lys Gln 325 330 335 Gly Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly 340 345 350 Glu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lys Leu Leu Pro Leu Glu 355 360 365 Leu Glu Lys Thr Ala Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro 370 375 380 Leu Tyr Tyr Val Ala Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg 385 390 395 400 Asn Phe Ala Ala Thr Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro 405 410 415 Tyr Thr Gln Arg Ile Glu Val Leu Asp Asn Thr Gln Gln Leu Lys Ile 420 425 430 Leu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala Leu 435 440 445 Gln Lys Ile Lys 450 <210> 2 <211> 452 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu Ser 1 5 10 15 Asp Phe Gly Gln Glu Thr Ser Tyr Ile Glu Asp Asn Cys Asn Gln Asn 20 25 30 Gly Ala Ile Ser Leu Ile Phe Ser Leu Lys Glu Glu Val Gly Ala Leu 35 40 45 Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val Asn Leu Thr His 50 55 60 Ile Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp Glu Tyr Glu Phe Phe 65 70 75 80 Thr His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Ile Lys 85 90 95 Ile Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg Asp 100 105 110 Lys Lys Lys Asp Thr Val Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu 115 120 125 Asp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala 130 135 140 Asp His Pro Gly Phe Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln 145 150 155 160 Phe Ala Asp Ile Ala Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg 165 170 175 Val Glu Tyr Met Glu Glu Gly Lys Lys Thr Trp Gly Thr Val Phe Lys 180 185 190 Thr Leu Lys Ser Leu Tyr Lys Thr His Ala Cys Tyr Glu Tyr Asn His 195 200 205 Ile Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp Asn Ile 210 215 220 Pro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr Gly Phe 225 230 235 240 Arg Leu Arg Pro Val Ala Gly Leu Leu Ser Ser Arg Asp Phe Leu Gly 245 250 255 Gly Leu Ala Phe Arg Val Phe His Cys Thr Gln Tyr Ile Arg His Gly 260 265 270 Ser Lys Pro Met Tyr Thr Pro Glu Pro Asp Ile Cys His Glu Leu Leu 275 280 285 Gly His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln 290 295 300 Glu Ile Gly Leu Ala Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu Lys 305 310 315 320 Leu Ala Thr Ile Tyr Trp Phe Thr Val Glu Phe Gly Leu Cys Lys Gln 325 330 335 Gly Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly 340 345 350 Glu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lys Leu Leu Pro Leu Glu 355 360 365 Leu Glu Lys Thr Ala Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro 370 375 380 Leu Tyr Tyr Val Ala Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg 385 390 395 400 Asn Phe Ala Ala Thr Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro 405 410 415 Tyr Thr Gln Arg Ile Glu Val Leu Asp Asn Thr Gln Gln Leu Lys Ile 420 425 430 Leu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala Leu 435 440 445 Gln Lys Ile Lys 450 <210> 3 <211> 452 <212> PRT <213> Homo sapiens <400> 3 Met Ser Thr Ala Val Leu Glu Asn Pro Gly Leu Gly Arg Lys Leu Ser 1 5 10 15 Asp Phe Gly Gln Glu Thr Ser Tyr Ile Glu Asp Asn Cys Asn Gln Asn 20 25 30 Gly Ala Ile Ser Leu Ile Phe Ser Leu Lys Glu Glu Val Gly Ala Leu 35 40 45 Ala Lys Val Leu Arg Leu Phe Glu Glu Asn Asp Val Asn Leu Thr His 50 55 60 Ile Glu Ser Arg Pro Ser Arg Leu Lys Lys Asp Glu Tyr Glu Phe Phe 65 70 75 80 Thr His Leu Asp Lys Arg Ser Leu Pro Ala Leu Thr Asn Ile Ile Lys 85 90 95 Ile Leu Arg His Asp Ile Gly Ala Thr Val His Glu Leu Ser Arg Asp 100 105 110 Lys Lys Lys Asp Thr Val Pro Trp Phe Pro Arg Thr Ile Gln Glu Leu 115 120 125 Asp Arg Phe Ala Asn Gln Ile Leu Ser Tyr Gly Ala Glu Leu Asp Ala 130 135 140 Asp His Pro Gly Phe Lys Asp Pro Val Tyr Arg Ala Arg Arg Lys Gln 145 150 155 160 Phe Ala Asp Ile Ala Tyr Asn Tyr Arg His Gly Gln Pro Ile Pro Arg 165 170 175 Val Glu Tyr Thr Glu Glu Glu Lys Lys Thr Trp Gly Thr Val Phe Lys 180 185 190 Thr Leu Lys Ser Leu Tyr Pro Thr His Ala Cys Tyr Glu Tyr Asn His 195 200 205 Ile Phe Pro Leu Leu Glu Lys Tyr Cys Gly Phe His Glu Asp Asn Ile 210 215 220 Pro Gln Leu Glu Asp Val Ser Gln Phe Leu Gln Thr Cys Thr Gly Phe 225 230 235 240 Arg Leu Arg Pro Val Ala Gly Leu Leu Pro Ser Arg Asp Phe Leu Ala 245 250 255 Gly Leu Ala Phe Arg Val Phe His Cys Thr Gln Tyr Ile Arg His Gly 260 265 270 Ser Lys Pro Met Tyr Thr Pro Glu Pro Asp Ile Cys His Glu Leu Leu 275 280 285 Gly His Val Pro Leu Phe Ser Asp Arg Ser Phe Ala Gln Phe Ser Gln 290 295 300 Glu Ile Gly Leu Ala Ser Leu Gly Ala Pro Asp Glu Tyr Ile Glu Lys 305 310 315 320 Leu Ala Thr Ile Tyr Trp Phe Thr Val Glu Phe Gly Leu Cys Lys Gln 325 330 335 Gly Asp Ser Ile Lys Ala Tyr Gly Ala Gly Leu Leu Ser Ser Phe Gly 340 345 350 Glu Leu Gln Tyr Cys Leu Ser Glu Lys Pro Lys Leu Leu Pro Leu Glu 355 360 365 Leu Glu Lys Thr Ala Ile Gln Asn Tyr Thr Val Thr Glu Phe Gln Pro 370 375 380 Leu Tyr Tyr Val Ala Glu Ser Phe Asn Asp Ala Lys Glu Lys Val Arg 385 390 395 400 Asn Phe Ala Ala Thr Ile Pro Arg Pro Phe Ser Val Arg Tyr Asp Pro 405 410 415 Tyr Thr Gln Arg Ile Glu Val Leu Asp Asn Thr Gln Gln Leu Lys Ile 420 425 430 Leu Ala Asp Ser Ile Asn Ser Glu Ile Gly Ile Leu Cys Ser Ala Leu 435 440 445 Gln Lys Ile Lys 450 <210> 4 <211> 1359 <212> DNA <213> Homo sapiens <400> 4 atgagcacag ccgtgctgga aaaccccggc ctgggcagaa agctgagcga cttcggccag 60 gaaaccagct acatcgagga caacgcaac cagaacggcg ccatcagcct gatcttcagc 120 ctgaaagaag aagtgggcgc cctggccaag gtgctgcggc tgttcgagga gaacgacgtg 180 aacctgaccc acatcgagag ccggcccagc agactgaaga aggacgagta cgagttcttc 240 acccacctgg acaagcggag cctgcccgcc ctgaccaca tcatcagat cctgcggcac 300 gatacggcg ccaccgtgca cgagctgagc cgggacaaga aaaggacac cgtgccctgg 360 ttccccagaa ccatccagga actggacaga ttcgccacc agatcctgtc ctacggcgcc 420 gagctggatg ccgaccaccc tggctcaag gaccccgtgt accggccag acggaagcag 480 ttcgccgata tcgcctacaa ctaccggcac ggccagccca tcccagagt cgagtacacc 540 gaagagggca agaaaacctg gggcaccgtg ttcagaccc tgaagtccct gtaccccacc 600 cacgcctgct acgagtacaa ccacatctc ccactgctcg aaagtactg cggcttccac 660 gaggacaata tccctcagct ggaggacgtg tcccagtttc tgcagacctg caccggcttc 720 agactcaggc ctgtggccgg cctgctgccc agcagagatt ttctggccgg actggccttc 780 cgggtgttcc actgcaccca gtacatcaga cacggcagca agcccatgta cacccctgag 840 cccgacatct gccacgagct gctgggacat gtgcccctgt tcagcgacag aagcttcgcc 900 cagttcagcc aggaaatcgg cctggcctct ctgggcgctc ccgacgagta tatcgagaag 960 ctggccacca tctactggtt caccgtggaa ttcggcctgt gcaagcaggg cgacagcatc 1020 aaggcctatg gcgccggact cctgtccagc ttcggcgagc tgcagtactg tctgagcgag 1080 aagcccaagc tgctgcccct ggaactggaa aagaccgcca tccagaacta caccgtgacc 1140 gagttccagc ccctgtacta cgtggccgag agcttcaacg acgccaaaga aaaagtgcgg 1200 aacttcgccg ccaccatccc tcggcccttc agcgtcagat acgaccccta cacccagcgg 1260 atcgaggtgc tggacaacac acagcagctg aaaattctgg ccgactccat caacagcgag 1320 atcggcatcc tgtgcagcgc cctgcagaaa atcaagtga 1359 <210> 5 <211> 3713 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 5 gagctcttgg ccactccctc tctgcgcgct cgctcgctca ctgaggccgg gcgaccaaag 60 gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga gcgagcgagc gcgcagagag 120 ggagtggcca actccatcac taggggttcc tacgcgtggc cccaggttaa tttttaaaaa 180 gcagtcaaag gtcaaagtgg cccttggcag catttactct ctctattgac tttggttaat 240 aatctcagga gcacaaacat tcctggaggc aggagaagaa atcaacatcc tggacttatc 300 ctctgggcct ctccccacct tcgatggccc caggttaatt tttaaaaagc agtcaaaggt 360 caaagtggcc cttggcagca tttactctct ctattgactt tggttaataa tctcaggagc 420 acaaacattc ctggaggcag gagaagaaat caacatcctg gacttatcct ctgggcctct 480 ccccaccgat atctacctgc tgatcgcccg gcccctgttc aaacatgtcc taatactctg 540 tcggggcaaa ggtcggcagt agttttccat cttactcaac atcctcccag tgtacgtagg 600 atcctgtctg tctgcacatt tcgtagagcg agtgttccga tactctaatc tcccggggca 660 aaggtcgtat tgacttaggt tacttattct ccttttgttg actaagtcaa taatcagaat 720 cagcaggttt ggagtcagct tggcagggat cagcagcctg ggttggaagg agggggtata 780 aaagcccctt caccaggaga agccgtcaca cagatccaca agctcctgct agcagtcgct 840 gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 900 tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 960 aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga aagccttgag 1020 gggctccggg aaggcccttt gtgcgggggg agcggctcgg ggggtgcgtg cgtgtgtgtg 1080 tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg ctgcgggcgc 1140 ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcggggccgg gggcggtgcc 1200 ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt gtgcgtgggg 1260 gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca cccccctccc 1320 cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg tggcgcgggg 1380 ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc ggggccgcct 1440 cgggccgggg agggctcggg ggaggggcgc ggcggccccc ggagcgccgg cggctgtcga 1500 ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc gcagggactt 1560 cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac cccctctagc 1620 gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga gggccttcgt 1680 gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc gcggggggac 1740 ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt gaccggcggc 1800 tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct cctgggcaac 1860 gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tcatttcgaa gccgccacca 1920 tgagcacagc cgtgctggaa aaccccggcc tgggcagaaa gctgagcgac ttcggccagg 1980 aaaccagcta catcgaggac aactgcaacc agaacggcgc catcagcctg atcttcagcc 2040 tgaaaaga agtgggcgcc ctggccaagg tgctgcggct gttcgaggag aacgacgtga 2100 acctgaccca catcgagagc cggcccagca gactgaagaga gacgagtac gagttcttca 2160 cccacctgga caagcggagc ctgcccgccc tgaccaacat catcaagatc ctgcggcacg 2220 acatcggcgc caccgtgcac gagctgagcc gggacaagaa aaaggacacc gtgccctggt 2280 tccccagaac catccaggaa ctggacagat tcgccaacca gatcctgtcc tacggcgccg 2340 agctggatgc cgaccaccct ggcttcaagg accccgtgta ccgggccaga cggaagcagt 2400 tcgccgatat cgcctacaac taccggcacg gccagcccat ccccagagtc gagtacaccg 2460 aagagggcaa gaaaacctgg ggcaccgtgt tcaagaccct gaagtccctg taccccaccc 2520 acgcctgcta cgagtacaac cacatcttcc cactgctcga aaagtactgc ggcttccacg 2580 aggacaatat ccctcagctg gaggacgtgt cccagtttct gcagacctgc accggcttca 2640 gactcaggcc tgtggccggc ctgctgccca gcagagattt tctggccgga ctggccttcc 2700 gggtgttcca ctgcacccag tacatcagac acggcagcaa gcccatgtac acccctgagc 2760 ccgacatctg ccacgagctg ctgggacatg tgcccctgtt cagcgacaga agcttcgccc 2820 agttcagcca ggaaatcggc ctggcctctc tgggcgctcc cgacgagtat atcgagaagc 2880 tggccaccat ctactggttc accgtggaat tcggcctgtg caagcagggc gacagcatca 2940 aggcctatgg cgccggactc ctgtccagct tcggcgagct gcagtactgt ctgagcgaga 3000 agcccaagct gctgcccctg gaactggaaa agaccgccat ccagaactac accgtgaccg 3060 agttccagcc cctgtactac gtggccgaga gcttcaacga cgccaaagaa aaagtgcgga 3120 acttcgccgc caccatccct cggcccttca gcgtcagata cgacccctac acccagcgga 3180 tcgaggtgct ggacaacaca cagcagctga aaattctggc cgactccatc aacagcgaga 3240 tcggcatcct gtgcagcgcc ctgcagaaaa tcaagtgacc taggtgatca agatctgcta 3300 gcttaattaa cccgggacta gtgcggccgc tcgagactag tctgtgcctt ctagttgcca 3360 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 3420 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 3480 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca 3540 tgctggggat gcggtgggct ctatggtacc aggaacccct agtgatggag ttggccactc 3600 cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg 3660 gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag agagggagtg gcc 3713 <210> 6 <211> 4561 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 6 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgcccgggc aaagcccggg 60 cgtcgggcga cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg 120 gccaactcca tcactagggg ttccttaccg cgtggcccca ggttaatttt taaaaagcag 180 tcaaaggtca aagtggccct tggcagcatt tactctctct attgactttg gttaataatc 240 tcaggagcac aaacattcct ggaggcagga gaagaaatca acatcctgga cttatcctct 300 gggcctctcc ccaccttcga tggccccagg ttaattttta aaaagcagtc aaaggtcaaa 360 gtggcccttg gcagcattta ctctctctat tgactttggt fatherctc aggagcacaa 420 acattcctgg aggcaggaga agaaatcaac atcctggact tatcctctgg gcctctcccc accgatatct acctgctgat cgcccggccc ctgttcaaac atgtcctaat actctgtcgg 540 ggcaaaggtc ggcagtagtt ttccatctta ctcaacatcc tcccagtgta cgtaggatcc 600 tgtctgtctg cacatttcgt agagcgagtg ttccgatact ctaatctccc ggggcaaagg 660 720. tcgtattgac ttaggttact tattctcctt ttgttgacta agtcaataat cagaatcagc aggtttggag tcagcttggc aggtcagc agcctgggtt ggaggaggg ggtataaaag 780 ccccttcacc aggagaagcc gtcacacaga tccacaagct cctgctagcc aattgagtcg 840 ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc 900 tctgactgac cgcgttactc ccacaggtga gcggggcggga cggcccttct cctccggggct gtaattagcg cttggtttat tgacggcttg tttctttct gtggctgcgt gaaagccttg aggggctccg ggaaggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg 1080 tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc 1140 gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcggggcc gggggcggtg 1200 ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg 1260 gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc 1320 cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg 1380 ggctcgccgt gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc 1440 ctcgggccgg ggagggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc 1500 gaggcgcggc gagccgcagc cattgccttt tttggtaatc gtgcgagagg gcgcagggac 1560 ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta 1620 gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga attgggcggg gagggccttc 1680 gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg 1740 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg 1800 gctctagagc ctctgctaac cttgttcttg ccttcttctt tttcctacag ctcctgggca 1860 acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcatttcg aagccgccac 1920 catgagcaca gccgtgctgg aaaaccccgg cctgggcaga aagctgagcg acttcggcca 1980 ggaaaccagc tacatcgagg acaactgcaa ccagaacggc gccatcagcc tgatcttcag 2040 cctgaaagaa gaagtgggcg ccctggccaa ggtgctgcgg ctgttcgagg agaacgacgt 2100 gaacctgacc cacatcgaga gccggcccag cagactgaag aaggacgagt acgagttctt 2160 cacccacctg gacaagcgga gcctgcccgc cctgaccaac atcatcaaga tcctgcggca 2220 cgacatcggc gccaccgtgc acgagctgag ccgggacaag aaaaaggaca ccgtgccctg 2280 gttccccaga accatccagg aactggacag attcgccaac cagatcctgt cctacggcgc 2340 cgagctggat gccgaccacc ctggcttcaa ggaccccgtg taccgggcca gacggaagca 2400 gttcgccgat atcgcctaca actaccggca cggccagccc atccccagag tcgagtacac 2460 cgaagagggc aagaaaacct ggggcaccgt gttcaagacc ctgaagtccc tgtaccccac 2520 ccacgcctgc tacgagtaca accacatctt cccactgctc gaaaagtact gcggcttcca 2580 cgaggacaat atccctcagc tggaggacgt gtcccagttt ctgcagacct gcaccggctt 2640 cagactcagg cctgtggccg gcctgctgcc cagcagagat tttctggccg gactggcctt 2700 ccgggtgttc cactgcaccc agtacatcag acacggcagc aagcccatgt acacccctga 2760 gcccgacatc tgccacgagc tgctgggaca tgtgcccctg ttcagcgaca gaagcttcgc 2820 ccagttcagc caggaaatcg gcctggcctc tctgggcgct cccgacgagt atatcgagaa 2880 gctggccacc atctactggt tcaccgtgga attcggcctg tgcaagcagg gcgacagcat 2940 caaggcctat ggcgccggac tcctgtccag cttcggcgag ctgcagtact gtctgagcga 3000 gaagcccaag ctgctgcccc tggaactgga aaagaccgcc atccagaact acaccgtgac 3060 cgagttccag cccctgtact acgtggccga gagcttcaac gacgccaaag aaaaagtgcg 3120 gaacttcgcc gccaccatcc ctcggccctt cagcgtcaga tacgacccct acacccagcg 3180 gatcgaggtg ctggacaaca cacagcagct gaaaattctg gccgactcca tcaacagcga 3240 gatcggcatc ctgtgcagcg ccctgcagaa aatcaagtga actagtctgt gccttctagt 3300 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 3360 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 3420 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 3480 aggcatgctg gggatgcggt gggctctata ccggtccagg ggtgagtgaa ggtttggaag 3540 agtgtagcag aataagaaac catgagtccc ctccctgaga agccctgagc ccccttgacg 3600 acacacatcc ctcgaggctc agcttcatca tctgtaaaag gtgctgaaac tgaccatcca 3660 agctgccgaa aaagattgtg tggggataat tcaaaactag aggaagatgc agaatttcta 3720 catcgtggcg atgtcaggct aagagttgcc atcgtggctg tccatcgatt ttattggaat 3780 catatgttta tttgagggtg tcttggatat tacaaataaa ttgttggagc atcaggcata 3840 tttggtaatt ctgtctaagg ctccctgccc cttgttaatt ggcagctcag ttattcatcc 3900 agggcaaaca ttctgcttac tattcctgag agctttcctc atcctctaga ttggcagggg 3960 aattgcagtt gcctgagcag cctcccctct gccataccaa cagagcttca ccatcgaggc 4020 ttgcagagtg gacaggggcc tcagggaccc ctgatcccag ctttctcatt ggacagaagg 4080 aggagactgg ggctggagag ggacctgggc ccccactaag gccacagcag agccaggact 4140 ttagctgtgc tgactgcagc ctggcttgcc tccactgccc tcctttgcct caagagcaag 4200 ggagcctcag agtggaggaa gcagcccctg gccttgcctc ccacctcccc tcccctttgc 4260 tgttttcctg ggacagtggg agctggctta gattgccctg gggcccccag gaccctggca 4320 ttttaacccc tcaggggcag gaaggcagcc tgagatacag aagagtccat cacctgctgt 4380 atgccacaca ccatccccac agtcgacatt taaattagga acccctagtg atggagttgg 4440 ccactccctc tctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 4500 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 4560 a 4561 <210> 7 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 7 gcgagaactt gtgcctcccc gtgttcctga cctttgaccc tctgtcctac ttagactaat 60 attgactttg ggtactgcaa acaggaaatg ggggagggat tcgatgcgag aacttgtgcc 120 tccccgtgtt cctgaccttt gaccctctgt cctacttaga ctaatattga ctttgggtac 180 tgcaaacagg aaatggggga ggga 204 <210> 8 <211> 330 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 8 ggccccaggt taatttttaa aaagcagtca aaggtcaaag tggcccttgg cagcatttac 60 tctctctatt gactttggtt aataatctca ggagcacaaa cattcctgga ggcaggagaa 120 gaaatcaaca tcctggactt atcctctggg cctctcccca ccttcgatgg ccccaggtta 180 atttttaaaa agcagtcaaa ggtcaaagtg gcccttggca gcatttactc tctctattga 240 ctttggttaa taatctcagg agcacaaaca ttcctggagg caggagaaga aatcaacatc 300 ctggacttat cctctgggcc tctccccacc 330 <210> 9 <211> 377 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 9 gttcctagat tacattacac attctgcaag catagcacag gtcaaagttc aactttaatt 60 actttcattt tcttgtatcc tcacagccta gaaaataacc tgcgttacag catccactca 120 gtatcccttg agcatgaggt gacactactt aacataggga cgagatggta ctttgtgtct 180 cctgctctgt cagcagggca ctgtacttgc tgataccagg gaatattgat ttgtaaatac 240 catcattccg aacgtgtttg ccttggccag ttttccatgt acatgcagaa agaagtttgg 300 gactgatcaa tacagtcctc tgcctttaaa gcaataggaa aaggccaact tgtctacgtt 360 tagtatgtgg ctgtaga 377 <210> 10 <211> 156 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 10 ccatcagatc ctgcccaagg tcttacataa gaggactctt ggactcccag caatgtcaac 60 gaccgacctt gaggcctact tcaaagactg tgtgtttaag gactgggagg agctggggga 120 ggagattagg ttaaaggtct ttgtattagg aggctg 156 <210> 11 <211> 71 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 11 ggggaggctg ctggtgaata ttaaccaagg tcaccccagt tatcggagga gcaaacaggg 60 gctaagtcca c 71 <210> 12 <211> 869 <212> DNA <213> Artificial sequence <220> <223> Synthetic construct <400> 12 ctcaattgga tgacactagt catcacattt aaaagcatct caggtaacta tattttgaat 60 tttttaaaaa agtaactata atagttatta ttaaaatagc aaagattgac catttccaag 120 agccatatag accagcaccg accactattc taaactattt atgtatgtaa atattagctt 180 ttaaaattct caaaatagtt gctgagttgg gaaccactat tatttctatc gattcagcag 240 ccgtaagtct aggacaggct taaattgttt tcactggtgt aaattgcaga aagatgatct 300 aagtaatttg gcatttattt taataggttt gaaaaacaca tgccatttta caaataagac 360 ttatatttgt ccttttgttt ttcagcctac catgagaata agagaaagaa aatgaagatc 420 aaaagcttat tcatctgttt ttctttttcg ttggtgtaaa gccaacaccc tgtctaaaaa 480 acataaattt ctttaatcat tttgcctctt ttctctgtgc ttcaattaat aaaaaatgga 540 aagaatctaa tagagtggta cagcactgtt atttttcaaa gatgtgttgc tatcctgaaa 600 attctgtagg ttctgtggaa gttccagtgt tctctcttat tccacttcgg tagaggattt 660 ctagtttctt gtgggctaat taaataaatc attaatactc ttctaagtta tggattataa 720 acattcaaaa taatattttg acattatgat aattctgaat aaaagaacaa aaaccatggt 780 ataggtaagg aatataaaac atggctttta ccttagaaaa aacaattcta aaattcatat 840 ggaatcaaaa aagagcctgc aggtaccct 869 <210> 13 <211> 1302 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 13 ctcaattgga tgacactagt catcacattt aaaagcatct caggtaacta tattttgaat 60 tttttaaaaa agtaactata atagttatta ttaaaatagc aaagattgac catttccaag 120 agccatatag accagcaccg accactattc taaactattt atgtatgtaa atattagctt 180 ttaaaattct caaaatagtt gctgagttgg gaaccactat tatttctatc tactgtttta 240 attaaaatta tctctaaggc atgtgaactg gctgtcttgg ttttcatctg tacttcatct 300 gctacctctg tgacctgaaa catatttata attccattaa gctgtgcata tgatagattt 360 atcatatgta ttttccttaa aggatttttg taagaactaa ttgaattgat acctgtaaag 420 tctttatcac actacccaat aaataaa tctctttgtt cagctctctg tttctataaa 480 tatgtaccag ttttatgtt tttagtggta gtgattttat tctctttcta tatatataca 540 cacacatgtg tgcattcata aatatataca atttttatga ataaaaatt attagcaatc 600 aatattgaaa accactgatt tttgtttatg tgagcaaa gcagattaaa aggaattcct 660 gcagattcag cagccgtaag tctaggacag gcttaaattg ttttcactgg tgtaaattgc 720 agaagatga tctaagtaat ttggcattta ttttaatagg tttgaaaaac acatgccatt 780 ttacaataa gacttatatt tgtccttttg tttttcagcc taccatgaga atagaagaa 840 gaaaatgaag atcaaaagct tattcatctg tttttctttt tcgttggtgt aaagccaaca 900 ccctgtctaa aaaacataaa tttctttaat cattttgcct cttttctctg tgcttcaatt 960 aataaaaaat ggaaagaatc taatagagtg gtacagcact gttatttttc aaagatgtgt 1020 tgctatcctg aaaattctgt aggttctgtg gaagttccag tgttctctct tattccactt 1080 cggtagagga tttctagttt cttgtgggct aattaaataa atcattaata ctcttctaag 1140 ttatggatta taaacattca aaataatatt ttgacattat gataattctg aataaaagaa 1200 caaaaaccat ggtataggta aggaatataa aacatggctt ttaccttaga aaaaacaatt 1260 ctaaaattca tatggaatca aaaaagagcc tgcaggtacc ct 1302 <210> 14 <211> 1359 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 14 atgagcacag ccgtgctgga aaaccccggc ctgggcagaa agctgagcga cttcggccag 60 gaaaccagct acatcgagga caactgcaac cagaacggcg ccatcagcct gatcttcagc 120 ctgaaagaag aagtgggcgc cctggccaag gtgctgcggc tgttcgaaga gaacgacgtg 180 aacctgaccc acatcgagag ccggcccagc agactgaaga aggacgagta cgagttcttc 240 acccacctgg acaagcggag cctgcccgcc ctgaccaaca tcatcaagat cctgcggcac 300 gacatcggcg ccaccgtgca cgagctgagc cgggacaaga aaaaggacac cgtgccctgg 360 ttccccagaa ccatccagga actggacaga ttcgccaacc agatcctgtc ctacggcgcc 420 gagctggatg ccgaccaccc tggcttcaag gaccccgtgt accgggccag acggaagcag 480 ttcgccgata tcgcctacaa ctaccggcac ggccagccca tccccagagt cgagtacatg 540 gaagagggca agaaaacctg gggcaccgtg ttcaagaccc tgaagtccct gtacaagacc 600 cacgcctgct acgagtacaa ccacatcttc ccactgctcg agaagtactg cggcttccac 660 gaggacaata tccctcagct cgaggacgtg tcccagtttc tgcagacctg caccggcttc 720 agactcaggc ctgtggccgg cctgctgagc agcagagatt ttctgggcgg actggccttc 780 cgggtgttcc actgcaccca gtacatcaga cacggcagca agcccatgta cacccctgag 840 cccgacatct gccacgagct gctgggacat gtgcccctgt tcagcgacag aagcttcgcc 900 cagttcagcc aggaaatcgg cctggcctct ctgggcgctc ccgacgagta tatcgagaag 960 ctggccacca tctactggtt caccgtggaa ttcggcctgt gcaagcaggg cgacagcatc 1020 aaggcctatg gcgccggact cctgtccagc ttcggcgagc tgcagtactg tctgagcgag 1080 aagcccaagc tgctgcccct ggaactggaa aagaccgcca tccagaacta caccgtgacc 1140 gagttccagc ccctgtacta cgtggccgag agcttcaacg acgccaaaga aaaagtgcgg 1200 aacttcgccg ccaccatccc tcggcccttc agcgtcagat acgaccccta cacccagcgg 1260 atcgaggtgc tggacaacac acagcagctg aaaattctgg ccgactccat caacagcgag 1320 atcggcatcc tgtgcagcgc cctgcagaaa atcaagtga 1359 <210> 15 <211> 1069 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 15 agtcgctgcg cgctgccttc gccccgtgcc ccgctccgcc gccgcctcgc gccgcccgcc 60 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc 120 gggctgtaat tagcgcttgg tttattgacg gcttgtttct tttctgtggc tgcgtgaaag 180 ccttgagggg ctccgggaag gccctttgtg cgggggagc ggctcgggg gtgcgtgcgt 240 gtgtgtgtgc gtggggagcg ccgcgtgcgg ctccgcgctg cccggcggct gtgagcgctg 300 cgggcgcggc gcggggcttt gtgcgctccg cagtgtgcgc gaggggagcg gggccgggg 360 cggtgccccg cggtgcgggg ggggctgcga ggggaaaaa gggctcgtgc ggggtgtgtg 420 cgtgggggg tgagcagggg gtgtgggcgc gtcggtcggg ctgcaacccc ccctgcaccc 480 ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgta cggggcgtgg 540 cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 600 gccgcctcgg gccgggggagg gctcggggga ggggcgcggc ggccccccgga gcgccggcgg 660 ctgtcgaggc gcggcgagcc gcagccattg ccttttttgg taatcgtgcg agagggcgca 720 gggacttcct ttgtcccaaa tctgtgcgga gccgaaatct gggaggcgcc gccgcacccc 780 ctctagcggg cgcggggcga agcggtgcgg cgccggcagg aaaggaattgg gcggggaggg 840 ccttcgtgcg tcgccgcgcc gccgtcccct tctccctctc cagcctcggg gctgtccgcg 900 gggggacggc tgccttcggg ggggacgggg cagggcgggg ttcggcttct ggcgtgtgac 960 cggcggctct agagcctctg ctaaccttgt tcttgccttc ttctttttcc tacagctcct 1020 gggcaacgtg ctggttattg tgctgtctca tcattttggc aaagaattc 1069 <210> 16 <211> 901 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 16 ccaggggtga gtgaaggttt ggaagagtgt agcagaataa gaaaccatga gtcccctccc 60 tgagaagccc tgagccccct tgacgacaca catccctcga ggctcagctt catcatctgt 120 aaaaggtgct gaaactgacc atccaagctg ccgaaaaaga ttgtgtgggg ataattcaaa 180 actagaggaa gatgcagaat ttctacatcg tggcgatgtc aggctaagag ttgccatcgt 240 ggctgtccat cgattttatt ggaatcatat gtttatttga gggtgtcttg gatattacaa 300 ataaattgtt ggagcatcag gcatatttgg taattctgtc taaggctccc tgccccttgt 360 taattggcag ctcagttatt catccagggc aaacattctg cttactattc ctgagagctt 420 tcctcatcct ctagattggc aggggaattg cagttgcctg agcagcctcc cctctgccat 480 accaacagag cttcaccatc gaggcttgca gagtggacag gggcctcagg gacccctgat 540 cccagctttc tcattggaca gaaggaggag actggggctg gagagggacc tgggccccca 600 ctaaggccac agcagagcca ggactttagc tgtgctgact gcagcctggc ttgcctccac 660 tgccctcctt tgcctcaaga gcaagggagc ctcagagtgg aggaagcagc ccctggcctt 720 gcctcccacc tcccctcccc tttgctgttt tcctgggaca gtgggagctg gcttagattg 780 ccctggggcc cccaggaccc tggcatttta acccctcagg ggcaggaagg cagcctgaga 840 tacagaagag tccatcacct gctgtatgcc acacaccatc cccacagtcg acatttaaat 900 t 901 <210> 17 <211> 78 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Construct <400> 17 cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccacgc 60 ccgggctttg cccgggcg 78 <210> 18 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 18 Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp 1 5 10 15 Tyr Lys Asp Asp Asp Asp Lys 20

Claims

1. A recombinant adeno-associated virus (rAAV) particle comprising a rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in hepatocytes, wherein the expression cassette comprises a transgene operably linked to a promoter and an enhancer, wherein the expression cassette comprises the enhancer, the promoter and the transgene from 5' to 3', wherein the promoter comprises a mouse transthyretin (mTTR) promoter, wherein the mTTR promoter is an mTTR482 promoter, and the enhancer comprises one or two modified α1-microbikunin enhancers (mA1MB2), wherein the mA1MB2 comprises the sequence of SEQ ID NO: 8, and wherein the transgene encodes a phenylalanine hydroxylase (PAH) polypeptide.

2. The rAAV particle of claim 1, wherein the enhancer is on the 5' side of the mTTR promoter.

3. The rAAV particle of claim 1, wherein the 3' element is located on the 3' side of the transgene.

4. The rAAV particle of claim 1, wherein the expression cassette further comprises an intron.

5. The rAAV particle of claim 4, wherein the intron is a chicken β-actin / rabbit β-globulin hybrid intron, wherein the chicken β-actin / rabbit β-globulin hybrid intron comprises the sequence of SEQ ID NO:

15.

6. The rAAV particle of claim 1, wherein the expression cassette further comprises a polyadenylation signal.

7. The rAAV particle of claim 6, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

8. The rAAV particle of claim 1, wherein the PAH polypeptide is a wild-type human PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

9. The rAAV particle of claim 1, wherein the expression cassette is flanked by one or more AAV inverted terminal repeat sequences.

10. The rAAV particle of claim 9, wherein the expression cassette is flanked by two AAV ITRs.

11. The rAAV particle of claim 10, wherein the AAV ITR is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR.

12. The rAAV particle of claim 11, wherein the AAV ITR is an AAV2 ITR.

13. The rAAV particle of claim 1, wherein the vector is a self-complementary vector.

14. The rAAV particle of claim 1, wherein the vector comprises a first nucleic acid sequence encoding the PAH polypeptide and a second nucleic acid sequence encoding the complement of the PAH polypeptide, wherein the first nucleic acid sequence forms intrastrand base pairs with the second nucleic acid sequence along most or all of their lengths.

15. The rAAV particle of claim 14, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by a mutated AAV ITR, wherein the mutated AAV ITR comprises a deletion of the D region and comprises a mutation of a terminal melting sequence.

16. The rAAV particle of claim 1, wherein the rAAV particle comprises an engineered AAV capsid.

17. The rAAV particle of claim 16, wherein the engineered AAV capsid is a DJ capsid or a LK03 capsid.

18. The rAAV particle of claim 17, wherein the ITRs and capsid of the rAAV particle are derived from the same AAV serotype.

19. The rAAV particle of claim 17, wherein the ITRs and capsid of the rAAV particle are derived from different AAV serotypes.

20. The rAAV particle of claim 1, wherein the rAAV vector comprises, from 5′ to 3′, AAV2 ITRs, a modified α1-microbikunin enhancer (mA1MB2), a mouse transthyretin (mTTR) promoter, a chicken β-actin / rabbit β-globulin hybrid intron, a codon-optimized human PAH gene, a bovine growth hormone polyadenylation signal, a stuffer fragment derived from an intron of the α1 antitrypsin gene, and the AAV2 ITRs.

21. The rAAV particle of claim 20, wherein the stuffer fragment derived from an intron of the α1 antitrypsin gene comprises the sequence of SEQ ID NO:

16.

22. The rAAV particle of claim 20, wherein the codon-optimized human PAH gene comprises the sequence of SEQ ID NO:

14.

23. A composition comprising the rAAV particles of any one of claims 1-22.

24. The composition of claim 23, wherein the composition further comprises a pharmaceutically acceptable carrier.

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