WO2021202943A1

WO2021202943A1 - Treatment of phenylketonuria with aav and therapeutic formulations

Info

Publication number: WO2021202943A1
Application number: PCT/US2021/025486
Authority: WO
Inventors: Peter Colosi; Hassibullah Akeefe; Geoffrey BERGUIG; Rajeev MAHIMKAR; Haoling WENG; Zhonghua Gu; Marcus Wong; Kidisti Araya; Joyce Chou
Original assignee: Biomarin Pharmaceutical, Inc.
Priority date: 2020-04-03
Filing date: 2021-04-02
Publication date: 2021-10-07
Also published as: TW202144575A; AR122409A1

Abstract

Provided herein are pharmaceutical compositions and methods for treating phenylketonuria in a human subject.

Description

TREATMENT OF PHENYLKETONURIA WITH AAV AND THERAPEUTIC FORMULATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of U.S. Provisional Application No. 63/005,049, filed April 3, 2020, which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[002] This application incorporates by reference a Sequence Listing submitted with this application as a text file entitled “H808-466-228_SEQ_LISTING.txt,” was created on March 27, 2021, and is 191 bytes in size.

1. FIELD

[003] The invention relates to methods of treating phenylketonuria by normalizing levels of amino acids, neurotransmitters, and/or neurotransmitter metabolites by administering AAV virus particles to a subject having phenylketonuria, as well as therapeutic formulations comprising the same.

2. BACKGROUND

[004] Phenylketonuria (PKU) is an inborn error of amino acid metabolism that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for the metabolism of phenylalanine (Phe). Patients with PAH mutations that lead to PKU and hyperphenylalaninemia (HP A) display elevated levels of Phe, impaired neurophysiologic functioning, and reduced cognitive development. For patients with PKU, there is the potential for irreversible mental retardation unless Phe levels are maintained at low levels using dietary restrictions which can be leveraged in combination with pharmacotherapy. Current guidelines recommend patients maintain strict control of plasma Phe for life as uncontrolled blood Phe levels in PKU are associated with significant neuropsychological and neurocognitive issues, affecting patients beyond childhood into adolescence and adulthood. The neurological symptoms of PKU are caused by the abnormal production of a number of neurotransmitters, resulting from a loss of PAH which is required to convert Phe into the precursor metabolite required for the synthesis of various neurotransmitters.

[005] Current treatment for PKU includes the lifetime adherence to a diet that is low in Phe. This dietary therapy is difficult to maintain and does not always eliminate the damaging neurological effects that can be caused by elevated Phe levels. Less than ideal dietary control during pregnancy can lead to birth defects. In addition, it is very difficult for PKU/HPA patients to live a normal life while following the restricted diet, and dietary therapy can be associated with deficiencies of several nutrients, some of which are detrimental for brain development.

[006] Currently approved treatments for phenylketonuria include: PALYNZIQ® (pegvaliase-pqpz), a PEGylated version of the enzyme phenylalanine ammonia lyase that is administered once daily, and KUVAN® (sapropterin) or tetrahydrobiopterin (BH4), which is administered once daily to patients with HPA due to BH4-responsive PKU, typically in conjunction with a Phe-restricted diet. BH4 is the natural cofactor for the PAH enzyme, and can increase activity of the residual PAH enzyme to metabolize Phe into tyrosine. KUVAN® is described in US Patent Nos. 7,732,599, 8,003,126, 7,566,462, and 8,178,670, each of which is incorporated by reference in its entirety. PALYNZIQ® (pegvaliase-pqpz) is described in US Patent Nos. 7,531,341, 7,534,595, 7,537,923, 7,790,433, 7,560,263, 9,557,34. each of which is incorporated by reference in its entirety.

[007] A number of gene therapy approaches have been attempted in animal models, including approaches using adeno-associated virus (AAV) vector. For example, Ahmed et al. at American Society of Gene & Cell Therapy (ASGCT) Annual Meeting (May 17, 2018) reported that administration of AAVHSC15 packaging a human PAH transgene driven by a ubiquitously expressing promoter (not tissue-specific) normalized Phe levels to less than 150 mM in the PAHenu2 mouse model, while an optimized vector including a liver-specific promoter (HMI- 102) normalized serum Phe at ten-fold lower doses. Three human patients appear to have been treated with a gene therapy based on an AAVHSC15 vector containing a functional copy of the human PAH gene, in clinical trial NCT03952156, in which two participants at the low dose experienced no effect (no reduction in Phe levels), while a third patient at the mid dose was reported to experience some decline in Phe levels although not to therapeutic levels. [008] Int’l Pat. Pub. No. WO 2018/126112 (PCT/US2017/068897) reports administering AAV8 vector bearing a codon-optimized human PAH cDNA under the control of TBG, a hybrid promoter based on the human thyroid hormone-binding globulin promoter and microglobin/bikunin enhancer, to PAH knockout mice. There remains a need for treatment of human subjects with a one-time gene therapy that exhibits safe, durable and stable long term expression that is effective to reduce Phe levels in a clinically significant manner.

3. SUMMARY

[009] The present disclosure provides methods of treating phenylketonuria by normalizing levels of amino acids, neurotransmitters, and/or neurotransmitter metabolites by administering replication-deficient recombinant AAV (rAAV) particles to a subject having phenylketonuria, as well as pharmaceutical compositions comprising the same.

[0010] In one aspect, the disclosure provides a method of decreasing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver- specific transcription regulatory region.

[0011] In a related aspect, the disclosure provides a method of treating a human subject with phenylketonuria (PKU), comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region.

[0012] In a further related aspect, the disclosure provides compositions of the recombinant vector construct or AAV particle as described herein for use according to the disclosed methods. The disclosure also provides uses of a recombinant vector construct or AAV particle as described herein for the preparation of a medicament for treatment according to the methods described herein. [0013] The rAAV particle is preferably replication-deficient. The rAAV particle may comprise a recombinant vector construct, or the rAAV particle may be produced by methods comprising providing to a cell a recombinant vector construct, that comprises (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV3’ ITR, (b) a heterologous liver- specific transcription regulatory region, and (c) a nucleic acid sequence encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding the functional hPAH is operably linked to liver-specific expression control elements. The vector construct may include additional expression control elements, for example: a promoter and/or enhancer; an intron; optionally an exon from the same gene as the intron; and a polyadenylation (poly A) signal. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

[0014] IntT. Patent Pub. No. WO 2019/217513 (PCT/US2019/031252), incorporated herein by reference in its entirety, discloses codon-optimized PAH-encoding nucleic acid sequences, liver-specific transcription regulatory regions, enhancers, promoters, introns, polyadenylation signals, and other vector elements, AAV vectors and virus particles, and methods of treating phenylketonuria by normalizing levels of amino acids, neurotransmitters, and neurotransmitter metabolites in a subject having phenylketonuria.

[0015] In some embodiments, the nucleic acid encoding hPAH encodes a functional hPAH amino acid sequence at least 95% identical to SEQ ID NO: 2. Such a nucleic acid encoding functional hPAH may comprise a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 1. Alternatively, such a nucleic acid encoding functional hPAH may comprise a nucleotide sequence at least 80% identical to any one of SEQ ID NOs: 7-13. For example, the nucleic acid encoding functional hPAH is at least 80% identical to SEQ ID NO: 7.

[0016] In some embodiments, the liver-specific transcription regulatory region comprises a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer. In such embodiments, the nucleic acid encoding PAH is operably linked to the fragment of an hAAT promoter linked to the fragment of an HCR enhancer/ ApoE enhancer. In some embodiments, the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24. In some embodiments, the recombinant vector construct further comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6. Alternatively, in some embodiments, the liver-specific transcription regulatory region comprises a different type of promoter, and comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 25 or 26.

[0017] The recombinant vector construct may further comprise an intron, e.g. a native PAH intron or fragment thereof, a beta globin intron or fragment thereof, or an hAAT intron or fragment thereof, or a combination thereof. Portions of the bordering exon may need to be included to ensure proper splicing. In some embodiments, the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, SEQ ID NO: 27, SEQ ID NO: 29 or SEQ ID NO: 34.

[0018] The recombinant vector construct may further comprise a polyadenylation signal, e.g. a bovine growth hormone (bGH) or human growth hormone (hGH) polyadenylation signal. In some embodiments, the recombinant vector construct comprises a bGH polyadenylation signal. [0019] In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to any one of SEQ NOs: 15-23 or 52, and the AAV capsid is an AAV5 type capsid. [0020] In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to any one of SEQ NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

[0021] In specific embodiments, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises the nucleotide sequence of any one of SEQ NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

[0022] In any of the embodiments, the AAV capsid may comprise an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44 (AAV5). In some embodiments, the AAV capsid is an AAV capsid with liver tropism. In any of the embodiments, the AAV capsid with liver tropism may be an AAV capsid that excludes AAV8 and/or AAVHSC15. Preferably, the AAV capsid with liver tropism is an AAV5 type capsid.

[0023] In any of the embodiments, the subject has phenylketonuria (PKU), optionally classic PKU or severe PKU. In some embodiments, the subject has a plasma Phe level of 600 pmol/L or above prior to said administration. In some embodiments, the subject has a plasma Phe level of 1200 pmol/L or above prior to said administration. In some embodiments, the subject is 15 or more years old. In some embodiments, the subject is an adult. In some embodiments, the subject is a female, e.g. a nonpregnant female.

[0024] In some embodiments, the subject is not receiving pharmacotherapy to treat PKU. For example, the subject has not received pegvaliase at least 30 days prior to said administration, and/or the subject has not received sapropterin at least 7 days prior to said administration. In some embodiments, the subject has not received steroids at least 30 days prior to said administration. In some embodiments, the subject does not have clinically significant liver disease prior to said administration. In some embodiments, the subject does not have detectable anti-AAV capsid antibody in blood prior to said administration (e.g., is not AAV5 seropositive). [0025] In the methods of the disclosure, the rAAV particle is administered intravenously in a single administration. In some embodiments, the rAAV particle is administered at a dose ranging from about 1E13 to about 5E14 vector genomes per kilogram body weight of the subject (vg/kg), a dose of about 2E13 to about 2E14 (vg/kg), for example, a dose of about 1E13 vg/kg, or a dose of about 2E13 vg/kg, or a dose of about 6E13 vg/kg, or a dose of about 2E14 vg/kg. In one embodiment, the rAAV particle is administered at a dose of 2E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 3E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 4E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 5E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 6E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 7E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 8E13 vector genomes per kilogram body weight of the subject. In another embodiment, the rAAV particle is administered at a dose of 9E13 vector genomes per kilogram body weight of the subject.

[0026] The methods of the disclosure may further comprise administering to the subject a prophylactically effective amount of a corticosteroid or other systemic immunosuppressant to prevent hepatotoxicity, prior to detection of hepatotoxicity (e.g. as detected by ALT elevation above the upper limit of normal (ULN), or at least 2 times baseline ALT). This is also referred to herein as “prophylactic immunosuppression treatment.” In some embodiments, the prophylactically effective amount of corticosteroid or immunosuppressant is administered concurrent with administration of the rAAV particles of the invention. In other embodiments, the administration of the prophylactically effective amount of corticosteroid or immunosuppressant begins after administration of the rAAV particles, e.g. starting 3 to 10 weeks after administration of the rAAV particles, but prior to detection of hepatotoxicity. The corticosteroid or immunosuppressant may be administered for a prophylactic treatment time period, e.g., for a time period of at least about 3 to 13 weeks, and is preferably followed by a tapering period during which tapering amounts of the corticosteroid or immunosuppressant are administered, e.g. for a time period of about 2, 3 or 4 weeks. For example, the prophylactically effective amount of the corticosteroid is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day for a time period of at least about 3 to 13 weeks, followed by tapering amounts of the corticosteroid for a time period of about 2, 3 or 4 weeks. In some embodiments, the prophylactically effective amount of the corticosteroid is administered for a time period of about 13 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks. For example, a prednisone-equivalent dose of 40 mg/day is administered concurrent with said administration of rAAV particles for a time period of about 13 weeks, followed by tapering amounts of the prednisone equivalent for a time period of about 3 weeks (e.g. prednisone- equivalent dose of 30 mg/day, for one week, 20 mg/day for one week, and 10 mg/day for one week). Other prednisone equivalent corticosteroids may be used at appropriate doses, for example, dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, or budesonide. See description of prednisone equivalent doses in Liu et al. Allergy, Asthma & Clinical Immunology 9:30 (2013), herein incorporated by reference in its entirety. In some embodiments, budesonide is administered at a dose of 3 mg/day for a period of about 14 weeks followed by taper for a time period of about 3 weeks. Other systemic immunosuppressants that may be administered in prophylactically effective doses to prevent hepatotoxicity include (1) calcineurin inhibitors, e.g. tacrolimus or cyclosporine, (2) antiproliferative agents or IMDH inhibitors, e.g. mycophenolate, leflunomide or azathioprine, (3) mTOR inhibitors, e.g., sirolimus or everolimus. (4) janus kinase inhibitors, e.g. tofacitinib, or (5) immunosuppressant antibodies. For example, the immunosuppressant is tacrolimus or mycophenolate.

[0027] The methods of the disclosure may comprise administering to the subject a therapeutically effective amount of a corticosteroid or other systemic immunosuppressant to treat hepatotoxicity, upon detection of hepatotoxicity (e.g. as detected by ALT elevation above the upper limit of normal (ULN), or at least 2 times baseline ALT). This is also referred to herein as “therapeutic immunosuppression treatment.” The methods of the disclosure may further comprise the step of (a) determining a baseline level of a marker of hepatotoxicity in the blood of the subject prior to said administration, optionally about one month prior to said administration, and (b) determining a post-administration level of said marker for hepatotoxicity in the blood of the subject after said administration, optionally every week or more frequently. Such methods may further comprise the step of: (c) upon detection of hepatotoxicity by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a corticosteroid or other systemic immunosuppressant for a therapeutic treatment time period, e.g., for a time period of at least about 5 to about 8 weeks or longer (e.g. 5, 6, 7, or 8 weeks or longer), and is preferably followed by a tapering period during which tapering amounts of the corticosteroid or other immunosuppressant are administered, e.g., for a time period of about 2, 3 or 4 weeks. For example, the step (c) comprises, upon detection of hepatotoxicity by (i) a post-administration level of said marker of hepatotoxicity greater than the upper limit of normal (ULN), or (ii) a post-administration level of said marker of hepatotoxicity greater than or equal to twice the baseline level of said marker of hepatotoxicity, administering to the subject a therapeutically effective amount of a corticosteroid for a time period of at least about 5 to about 8 weeks, followed by tapering amounts of the corticosteroid for a time period of about 2, 3 or 4 weeks. In any of such embodiments, the marker of hepatotoxicity is ALT and/or AST, preferably ALT. In some embodiments, upon said detection, a prednisone-equivalent dose of 40 mg/day is administered for a time period of about 5 weeks, followed by tapering amounts of the prednisone equivalent for a time period of about 3 weeks (e.g. prednisone-equivalent dose of 30 mg/day, for one week, 20 mg/day for one week, and 10 mg/day for one week). Other systemic immunosuppressants that may be administered in effective doses upon detection of hepatotoxicity include (1) calcineurin inhibitors, e.g. tacrolimus or cyclosporine, (2) antiproliferative agents or IMDH inhibitors, e.g. mycophenolate, leflunomide or azathioprine, (3) mTOR inhibitors, e.g., sirolimus or everolimus. (4) janus kinase inhibitors, e.g. tofacitinib, or (5) immunosuppressant antibodies. For example, the immunosuppressant is tacrolimus or mycophenolate.

[0028] In certain related embodiments, the disclosure provides a composition of a recombinant vector construct or AAV particle as described herein for use for co-administration with the prophylactic administration of immunosuppressant (e.g., corticosteroids) and/or the therapeutic administration of immunosuppressant (e.g., corticosteroids) described herein. The disclosure also provides for use of a recombinant vector construct or AAV particle as described herein in preparation of a medicament for co-administration with the prophylactic administration of immunosuppressant and/or the therapeutic administration of immunosuppressant described herein. Similarly, the disclosure provides a composition of an immunosuppressant for the prevention and/or treatment of any hepatotoxicity associated with administration of the AAV particle according to the prophylactic administration of immunosuppressant and/or the therapeutic administration of immunosuppressant described herein. The disclosure also provides for use of an immunosuppressant in preparation of a medicament for the prevention and/or treatment of any hepatotoxicity associated with administration of the AAV particle according to the prophylactic administration of immunosuppressant and/or the therapeutic administration of immunosuppressant described herein. [0029] The methods may also further comprise the step of monitoring episome formation by steps comprising extracting DNA from liver cells of the subject and detecting circular vector genomes, optionally by PCR or southern blotting. The methods may also further comprise the step of monitoring AAV integration by steps comprising extracting chromosomes or DNA from liver cells of the subject and detecting AAV vector genomes, e.g. by PCR as described in Schnepp et al., ./. Virol., 79(23): 14793-14803 (2005) or by target enrichment sequencing (TES) as described in Gnirke et al., Nat Biotechnol. 27(2): 182-189 (2009).

[0030] The methods may also further comprise the step of measuring plasma Phe level of the subject every week, and optionally performing a Phe challenge test or Phe breath test (which measures Phe oxidation) on the subject.

[0031] The methods may also further comprise the step of measuring plasma level of one or more neurotransmitters or neurotransmitter metabolites of the subject every week. For example, the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5-hydroxyindoleacetic acid.

[0032] The methods of the disclosure may result in clinically significant lowering of plasma Phe levels (e.g. mean plasma Phe levels, or the mean of two consecutive plasma Phe levels) in the absence of concurrent pharmacotherapy. For example, the plasma Phe level of said subject is lowered to 360 pmol/L or less by 8 weeks after said administration, or 360 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy. For example, the plasma Phe level of said subject is between 120 and 360 pmol/L by 8 weeks after said administration, without concurrent pharmacotherapy. In some embodiments, the plasma Phe level of said subject is 120 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy, or 120 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy.

[0033] The methods of the disclosure may permit the subject to tolerate an increase in Phe intake from intact food sources. For example, the plasma Phe level of said subject is between 120 and 360 mihoI/L, or between 30 and 360 mihoI/L after said administration, and the subject tolerates an increase in Phe intake compared to a Phe restricted diet at baseline.

[0034] The methods of the disclosure may reduce plasma level of a neurotransmitter or neurotransmitter metabolite of the subject after said administration. For example, the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5-hydroxyindoleacetic acid. [0035] The methods of the disclosure may result in improved quality of life of said subject improves after said administration, optionally as measured by PKU-QOL or Q-LES-Q-SF questionnaire. The methods of the disclosure may result in improved neurocognitive symptoms or measures of the subject after said administration, optionally as measured by CANTAB. Preferably, the subject does not suffer from hypophenylalaninemia after said administration. [0036] In yet another aspect, the disclosure provides a pharmaceutical composition comprising rAAV particle at a concentration of at least 1E13 vg/ml, for example, about lE13vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, about 1E13 vg/ml. about 2E13 vg/ml, about 3E13 vg/ml, about 4E13 vg/ml, 5E13 vg/ml, about 6E13 vg/ml, about 7E13vg/ml, about 8E13 vg/ml, about 9E13 vg/ml or about 2E14 vg/ml, a buffering agent, an isotonicity agent, a cryopreservative agent and a surfactant which is stable during storage at about minus60°C or less for at least about 1 year, 1.5 years, or 2 years. In some embodiments, the surfactant is poloxamer at a concentration of less than 0.2% w/v, or less than 0.15% w/v, for example, about 0.1% w/v. In some embodiments, the cryopreservative agent is a sugar, for example, trehalose.

[0037] In some embodiments, the pharmaceutical composition is aqueous and comprises rAAV particle at a concentration of at least 1E13 vg/ml, sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 mM to about 165 mM, a cryopreservative agent that is a sugar, optionally trehalose, and a poloxamer at a concentration of less than 0.2% w/v. The sodium phosphate may comprise sodium phosphate, dibasic and sodium phosphate, monobasic. Optionally, the sugar is trehalose at a concentration of about 60 mM to about 80 mM. Optionally, the poloxamer is poloxamer 188 at a concentration of about 0.05% to 0.15% w/v.

[0038] In some embodiments, the sodium phosphate, monobasic is at a concentration that is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 mg/mL, and the sodium phosphate, dibasic, is at a concentration that is greater than 2.5 mg/ml and less than 3 mg/ml, optionally about 2.7 mg/ml. In some embodiments, the sodium chloride is at a concentration that is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. In some embodiments, the sugar is trehalose dihydrate at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml. In some embodiments, the poloxamer 188 is at a concentration less than 1.5 mg/ml, or about 1 mg/ml. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 2E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 3E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 4E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 5E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 7E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 8E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In certain embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 9E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188.

[0039] In a specific embodiment, the pharmaceutical composition comprises a recombinant AAV particle described herein. In specific emboidments, the recombinant AAV particle comprise a recombinant vector construct, wherein the recombinant vector construct comprises: (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV3’ ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid sequence encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding the functional hPAH is operably linked to liver-specific expression control elements. The vector construct may include additional expression control elements, for example: a promoter and/or enhancer; an intron; optionally an exon from the same gene as the intron; and a polyadenylation (poly A) signal. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

[0040] In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to any one of SEQ NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

[0041] In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 95% identical to any one of SEQ NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

[0042] In specific embodiments, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 18, and the AAV capsid is an AAV5 type capsid. In certain embodiments, the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. In some embodiments, the recombinant vector construct comprises the nucleotide sequence of any one of SEQ NOs: 15-23, and the AAV capsid is an AAV5 type capsid.

[0043] Preferably the pharmaceutical composition is a liquid aqueous solution, or lyophilized, and is for storage at freezing temperature. In any of these embodiments, the composition is for use in intravenous administration of rAAV particle to a patient with phenylketonuria.

[0044] Other embodiments will be evident to one skilled in the art upon reading the present specification.

4. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 A shows deamidation levels of AAV5 particles (percent deamidation) over time when stored at 4°C in buffer at pH 7.1, 7.5 or 7.9. Figure IB shows deamidation levels of AAV5 particles (percent deamidation) over time when stored at room temperature in buffer at pH 7.1, 7.5 or 7.9.

[0046] Figures 2A-2D show stability of AAV5 particles (percent change in the aggregation levels from the initial time point) formulated in either 2% mannitol or 2.8% trehalose under various storage conditions: <-60°C, 2-8°C , 25°C/60% RH and 37°C, respectively. Figure 2A: Change in aggregate over time for rAAV5 particles stored at <-60°C. Figure 2B: Change in aggregate over time for rAAV5 particles stored at 2-8°C. Figure 2C: Change in aggregate over time for rAAV5 particles stored at 25°C/60%RH. Figure 2D: Change in aggregate over time for rAAV5 particles stored at 37°C.

5. DETAILED DESCRIPTION

[0047] Definitions:

[0048] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes of the present disclosure, the following terms are defined below.

[0049] As used herein, in the context of gene delivery, the term "vector" or "gene delivery vector" may refer to a particle that functions as a gene delivery vehicle, and which comprises nucleic acid (i.e., the vector genome comprising any of the vector constructs described herein) packaged within, for example, an envelope or capsid. A gene delivery vector may be a viral gene delivery vector or a non-viral gene delivery vector. Alternatively, in some contexts, the term "vector" may be used to refer only to the vector genome or vector construct. Viral vectors suitable for use herein may be a parvovirus, an adenovirus, a retrovirus, a lentivirus or a herpes simplex virus. The parvovirus may be an adenovirus-associated virus (AAV).

[0050] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are numerous serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228; and Bems, 1990, Virology, pp. 1743-1764, Raven Press, (New York); Gao et ah, 2011, Methods Mol. Biol. 807: 93-118; Ojala et ah, 2018, Mol. Ther. 26(1): 304-19. However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs).

[0051] As used herein, an "AAV vector construct" refers to nucleic acids, either single- stranded or double-stranded, having an AAV 5' inverted terminal repeat (ITR) sequence and an AAV 3' ITR flanking a protein-coding sequence (in one embodiment, a functional therapeutic protein-encoding sequence, e.g. hPAH-encoding sequence) operably linked to transcription regulatory elements (also called “expression control elements”) that are heterologous to protein encoding sequence and/or heterologous to the AAV viral genome, i.e., one or more promoters and/or enhancers and, optionally, a polyadenylation sequence and/or one or more introns inserted between exons of the protein-coding sequence. A single-stranded AAV vector refers to nucleic acids that are present in the genome of an AAV virus particle, and can be either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein. The size of such single- stranded nucleic acids is provided in bases. A double-stranded AAV vector refers to nucleic acids that are present in the DNA of plasmids, e.g., pUC19, or genome of a double-stranded virus, e.g., baculovirus, used to express or transfer the AAV vector nucleic acids. The size of such double-stranded nucleic acids in provided in base pairs (bp).

[0052] While AAV particles have been reported in the literature having AAV genomes of > 5.0 kb, in many of these cases the 5' or 3' ends of the encoded genes appear to be truncated (see Hirsch et al., Molec. Ther. 18:6-8, 2010, and Ghosh et al., Biotech. Genet. Engin. Rev. 24:165- 178, 2007). It has been shown, however, that overlapping homologous recombination occurs in AAV infected cells between nucleic acids having 5' end truncations and 3' end truncations so that a "complete" nucleic acid encoding the large protein is generated, thereby reconstructing a functional, full-length gene.

[0053] Oversized AAV vectors are randomly truncated at the 5' ends and lack a 5' AAV ITR. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5' AAV ITR and possibly portions of the 5' end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3' ITR and possibly portions of the 3' end of the target protein-coding gene. A functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the AAV PAH vectors and/or viral particles comprise at least one ITR.

[0054] The term "inverted terminal repeat (ITR)" as used herein refers to the art-recognized regions found at the 5' and 3' termini of the AAV genome which function in cis as origins of DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV-associated ITRs are disclosed by Yan et al., J. Virol. (2005) vol. 79, pp. 364-379 which is herein incorporated by reference in its entirety. ITR sequences that find use herein may be full length, wild-type AAV ITRs or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication. AAV ITRs useful in the recombinant AAV hPAH vectors of the embodiments provided herein may be derived from any known AAV serotype and, in certain embodiments, derived from the AAV2 serotype.

[0055] The term “control sequences” refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0056] A "transcription regulatory element" refers to nucleotide sequences of a gene involved in regulation of genetic transcription including a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression. The term "liver specific transcription regulatory element" or “liver-specific transcription regulatory region” refers to a regulatory element or region that produces preferred gene expression specifically in the liver tissue.

[0057] As used herein, the term “operably linked” is used to describe the connection between regulatory elements and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, for example, without limitation, constitutive or inducible promoters, tissue-specific regulatory elements, and enhancers. A gene or coding region is said to be “operably linked to” or “operatively linked to” or “operably associated with” the regulatory elements, meaning that the gene or coding region is controlled or influenced by the regulatory element. For instance, a promoter is operably linked to a coding sequence if the promoter effects transcription or expression of the coding sequence.

[0058] The term "isolated" when used in relation to a nucleic acid molecule of the present disclosure typically refers to a nucleic acid sequence that is identified and separated from at least one contaminant nucleic acid with which it is ordinarily associated in its natural source. Isolated nucleic acid may be present in a form or setting that is different from that in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells.

[0059] As used herein, the term “variant” refers to a polynucleotide (or polypeptide) having a sequence substantially similar to a reference polynucleotide (or polypeptide). Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide are known to the skilled artisan (see, e.g. , Sambrook et al. (1989)). In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and/or one or more internal sites in comparison to the reference polynucleotide. Similarities and/or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polynucleotide as determined by sequence alignment programs known by skilled artisans. In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and/or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot. Generally, a variant of a polypeptide, can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.

[0060] The term "identity," "homology" and grammatical variations thereof, mean that two or more referenced entities are the same, when they are "aligned" sequences. Thus, by way of example, when two polypeptide sequences are identical, they have the same amino acid sequence, at least within the referenced region or portion. Where two polynucleotide sequences are identical, they have the same polynucleotide sequence, at least within the referenced region or portion. The identity can be over a defined area (region or domain) of the sequence. An "area" or "region" of identity refers to a portion of two or more referenced entities that are the same. Thus, where two protein or nucleic acid sequences are identical over one or more sequence areas or regions they share identity within that region. An "aligned" sequence refers to multiple polynucleotide or protein (amino acid) sequences, often containing corrections for missing or additional bases or amino acids (gaps) as compared to a reference sequence. "Substantial homology" means that a molecule is structurally or functionally conserved such that it has or is predicted to have at least partial structure or function of one or more of the structures or functions (e.g., a biological function or activity) of the reference molecule, or relevant/corresponding region or portion of the reference molecule to which it shares homology. [0061] “Percent (%) nucleic acid sequence identity or homology” is defined as the percentage of nucleotides in a candidate sequence that are identical with a reference sequence after aligning the respective sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0062] “Percent (%) amino acid sequence identity or homology” with respect to the PAH amino acid sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a PAH polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0063] “Codon optimization” or “codon optimized” refers to changes made in the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non codon optimized sequence. It does not change the amino acid for which each codon encodes. [0064] As used herein, an “intron” is broadly defined as a sequence of nucleotides that is removable by RNA splicing. “RNA splicing” means the excision of introns from a pre-mRNA to form a mature mRNA. Introns may be upstream, downstream, or within the coding region of a gene. Insertion of an intron into a nucleotide sequence can be accomplished by any method known in the art. The only limitation of where the intron is inserted is in consideration of the packaging limitations of the AAV virus particles (about 5 kbp).

[0065] In certain embodiments, the recombinant AAV vector construct comprises (a) a nucleic acid comprising an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a liver-specific transcription regulatory region, (c) one or more introns, (d) a functional hPAH protein coding region, (e) a polyadenylation sequence, and (f) an AAV2 3' ITR (which may or may not be modified as known in the art).

[0066] Other embodiments provided herein are directed to vector constructs encoding a functional hPAH polypeptide, wherein the constructs comprise one or more of the individual elements of the above described constructs and combinations thereof, in one or more different orientation(s). Another embodiment provided herein is directed to the above described constructs in an opposite orientation. In another embodiment, provided are recombinant AAV virus particles comprising the herein described AAV vector constructs and their use for the treatment of PKU in subjects.

[0067] An "AAV virion" or "AAV viral particle" or "AAV vector particle" or "AAV virus" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated AAV vector construct as described herein. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as a "recombinant AAV vector particle", “AAV particle”, “recombinant AAV particle”, “rAAV particle”, or simply an "AAV vector". Production of AAV vector particles necessarily includes production of AAV vector genome, as such a vector genome is contained within an AAV vector particle. It is understood that reference to the polynucleotide AAV vector construct encapsulated within the vector particle, and replication thereof, refers to the AAV vector genome.

[0068] As used herein “therapeutic AAV virus” refers to an AAV virion, AAV viral particle, AAV vector particle, or AAV virus that comprises a heterologous polynucleotide that encodes a therapeutic protein such as the hPAH described herein. An "AAV vector construct" or “AAV vector genome” as used herein refers to a vector construct comprising a polynucleotide encoding a protein of interest (also called transgene) that are flanked by AAV terminal repeat sequences (ITRs) and operably linked to one or more expression control elements. Such AAV vector constructs can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. [0069] As used herein “therapeutic protein” refers to a polypeptide that has a biological activity that replaces or compensates for the loss or reduction of activity of an endogenous protein. For example, a functional PAH is a therapeutic protein for PKU.

[0070] “Neurotransmitter” as used herein refers to a chemical that is released from a nerve cell which thereby transmits an impulse from the nerve cell to another nerve, muscle, organ, or other tissue. A neurotransmitter is a messenger of neurologic information from one cell to another. In certain embodiments, neurotransmitters include phenethylamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, and serotonin. “Neurotransmitter metabolite” as used herein refers to the products following degradation of the neurotransmitters, one or two enzymatic steps downstream. Non-limiting examples of neurotransmitter metabolites include phenylacetic acid, phenylacetylglycine, phenylacetylglutamine, DOPAC, homovanillic acid, dihydroxyphenylethylene glycol (DOPEG), 3-methoxy-4-hydroxyphenylglycol (MHPG, MOPEG), indoleacetic acid and 5-hydroxyindoleacetic acid.

[0071] “Phenylketonuria (PKU)” as used herein refers to an inherited metabolic disease caused by a deficiency of the enzyme phenylalanine hydroxylase (PAH). This results in elevated, levels of phenylalanine (Phe) and reduced levels of neurotransmitters and neurotransmitter metabolites which can affect brain function, causing severe intellectual disability, behavioral abnormalities, cognitive impairments, psychiatric symptoms and delayed speech and seizures. [0072] “Treat” or “treatment” as used herein refers to therapeutic treatment which refers to a treatment administered to a subject who exhibits signs or symptoms of pathology, i.e., PKU, for the purpose of diminishing or eliminating those signs or symptoms or ameliorating their progression, severity or duration. The signs or symptoms can be biochemical, cellular, histological, functional, subjective or objective. Signs of PKU include elevated blood or plasma Phe levels, reduced blood or plasma neurotransmitter levels, e.g. Tyr levels, and neurocognitive symptoms.

[0073] “Neurocognitive symptoms” as used herein refers to specific neurological, behavioral, and cognitive symptoms associated with subjects having phenylketonuria. In particular, the loss of phenylalanine hydroxylase activity results in the inability of subjects having phenylketonuria from producing sufficient neurotransmitter levels. The inability to produce sufficient neurotransmitters directly results in a number of neurological, cognitive, and behavioral symptoms. In one embodiment, neurocognitive symptoms decreased IQ, attention deficits, and deficits in executive functions including strategic planning, inhibitory control, working memory, and cognitive flexibility.

[0074] “Therapeutically effective” as used herein means an amount effective to reduce the signs or symptoms of pathology. In the case of transient hepatotoxicity, it can refer to an amount of corticosteroid treatment effective to reduce markers of hepatotoxicity. In the case of PKU, it can refer to an amount effective to produce a clinically significant reduction in blood or plasma Phe levels. Although this will be the primary effect observed, other effects are observed that indicate effective reduction in the signs or symptoms of pathology, including increased Phe activity (e.g. as measured by increased rate of Phe oxidation on a Phe breath test), increased blood or plasma levels of neurotransmitters or neurotransmitter metabolites (e.g., increased Tyrosine (Tyr) levels), increased ability to tolerate an increase in dietary protein intake and/or a reduction in medical food intake (reduced need for Phe-reduced or Phe-free foods), decreased neurocognitive symptoms, decrease in symptoms of inattention and improvement in measures of executive function (e.g. improvements in ADHD-RS IV or CANTAB scores), improvements in health-related quality of life (e.g. improvements in PKU-QOL or Q-LES-Q-SF score, improvements in nutritional markers and/or normalization of fasting lipid panel. [0075] “Ameliorate” as used herein refers to the action of lessening the severity of symptoms, progression, or duration of a disease.

[0076] As used herein “stably treating” or “stable treatment” refers to using a therapeutic vector construct, AAV particle or cell administered to a subject where the subject stably expresses a therapeutic protein expressed by the vector construct, AAV particle or cell. Stably expressed therapeutic protein means that the protein is expressed for a clinically significant length of time.

[0077] “Clinically significant length of time” or “durability” as used herein with respect to PKU means expression at therapeutically effective levels for a length of time that has a meaningful impact on the plasma Phe levels and/or on other signs or symptoms of pathology. In certain embodiments clinically significant length of time is expression for at least six months, for at least eight months, for at least one year, for at least two years, for at least three years, for at least four years, for at least five years, for at least six years, for at least seven years, for at least eight years, for at least nine years, for at least ten years, or for the life of the subject.

[0078] As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.

[0079] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. “Animal” includes cold- and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles, and in particular, mammals. The term "avian" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys and pheasants. “Mammal,” as used herein, refers to an individual belonging to the class Mammalia and includes, but not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans. In some embodiments, the mammal is a human, including an infant, child, juvenile or adult human, e.g. a human of age less than 2 years old, age less than 5 years old, age 2 to less than 5 years old, age 5 to 9 years old, age 9 to less than 12 years old, age 5 to less than 12 years old, age 12 to less than 15 years old, or age 15 to 18 years old, or 15 years old or more, or 18 years old or more, or age less than 3 years old, age 3 to less than 6 years old, age less than 6 years old, age 6 to less than 9 years old, age 9 to less than 12 years old, age 6 to less than 12 years old, or age 12 to less than 18 years old, or 15 years old or more, or 18 years old or more. [0080] In general, a "pharmaceutically acceptable carrier" is one that is not toxic or unduly detrimental to cells. Exemplary pharmaceutically acceptable carriers include sterile, pyrogen-free water and sterile, pyrogen-free, phosphate buffered saline. Pharmaceutically acceptable carriers include physiologically acceptable carriers. The term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

5.1 RECOMBINANT AAV VECTOR CONSTRUCTS

[0081] The recombinant vector construct of the disclosure may be used to produce rAAV particles by methods described herein, comprising providing to a suitable host cell the recombinant vector construct, together with Rep and Cap genes. The recombinant vector construct may comprise (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) sequence and (ii) an AAV 3’ ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. Preferably, the nucleic acid encoding the functional hPAH is operably linked to liver-specific expression control elements. The vector construct may include additional expression control elements, for example: a promoter and/or enhancer; an intron; optionally an exon from the same gene as the intron; and a polyadenylation (poly A) signal. Such elements are further described herein. Preferably, the rAAV particles also comprise an AAV capsid with liver tropism, optionally an AAV5 type capsid.

[0082] In one embodiment, the vector construct comprises a nucleic acid encoding a functionally active hPAH protein. The hPAH encoding sequence may be wild-type, codon optimized, or a variant. One wild type hPAH gene has the nucleotide sequence of SEQ ID NO: 1. One wild type hPAH has the amino sequence of SEQ ID NO: 2.

[0083] The vector constructs described herein may comprise a nucleotide sequence that differs from wild type nucleotide sequence but still encodes a functional hPAH amino acid sequence at least 90%, 95% or 98% identical to amino acids of SEQ ID NO: 2. According to this aspect, the nucleotide sequence may have substantial homology, e.g. at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology, to SEQ ID NO: 1 as long as it encodes a functional hPAH at least 90% identical to amino acids of SEQ ID NO: 2. If the nucleic acid encodes a protein comprising a sequence having changes to any of the wild-type amino acids, the protein should still be a functional protein. A skilled person will appreciate that minor changes can be made to some of the amino acids of the protein without adversely affecting the function of the protein.

[0084]

[0085] Alternatively, the nucleotide sequence may have substantial homology, e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology, to any one of SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13, as long as the nucleotide sequence encodes functional hPAH at least 90%, 95% or 98% identical to amino acids of SEQ ID NO: 2, preferably a functional hPAH at least 95% identical to amino acids of SEQ ID NO: 2. The term substantial homology can be further defined with reference to a percent (%) homology. This is discussed in further detail elsewhere herein. In some embodiments, a vector construct comprises a nucleotide sequence encoding human PAH, wherein the nucleotide sequence is at least 90% identical to nucleotides 2894-4252 of SEQ ID NO:52. In certain embodiments, a vector construct comprises a nucleotide sequence encoding human PAH, wherein the nucleotide sequence is at least 95% identical to nucleotides 2894-4252 of SEQ ID NO:52. In some embodiments, a vector construct comprises a nucleotide sequence encoding human PAH, wherein the nucleotide sequence comprises nucleotides 2894- 4252 of SEQ ID NO:52.

[0086] As described herein, the nucleotide sequence encoding the hPAH protein can be modified to improve expression efficiency of the protein. For example, the nucleotide sequence can be codon optimized. This can be done in conjunction with manually reducing CpG di nucleotide content and removing any extra ORF in the sense and anti-sense direction. CpG di nucleotide content has been shown to activate TLR9 in dendritic cells leading to potential immune activation and CTL responses. A product in the AAV-vector genome may be delivered as ssDNA, thus reducing the CpG content. Reducing CpG content may reduce liver inflammation and ALT. As another example, one or more of the splice donors and/or splice acceptors in the nucleotide sequence of the protein of interest is modified to reduce the potential for extraneous splicing. [0087] Examples of liver specific regulatory elements suitable for inclusion in the heterologous liver-specific transcription regulatory regions include, but are not limited to, the mouse thyretin promoter (mTTR), the endogenous human factor VIII promoter (F8), human apolipoprotein E hepatic control region and active fragments thereof, human alpha- 1 -antitrypsin promoter (hAAT) and active fragments thereof, human alpha- 1 -microglobulin promoter and fragments thereof, human prothrombin promoter and active fragments thereof, human albumin minimal promoter, and mouse albumin promoter. Enhancers derived from liver-specific transcription factor binding sites are also contemplated, such as EBP, DBP, HNF1, HNF3,

HNF4, HNF6, and Enhl. The nucleic acid encoding hPAH is operably linked to any of such liver specific regulatory elements. In specific examples, the liver specific regulatory element is a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer. In one or more embodiments, the liver-specific transcription regulatory region is a synthetic promoter sequence comprising portions of a human alpha- 1- antitrypsin (hAAT) promoter, a hepatic control region (HCR) enhancer, and/or an apolipoprotein E (ApoE) enhancer. For example, the liver specific regulatory element comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24. In yet other examples, the liver specific regulatory element comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 25 or 26.

[0088] In some embodiments, the liver-specific transcription regulatory region comprises a shortened ApoE enhancer sequence (SEQ ID NO: 4) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto; a 186 base human alpha anti -trypsin (hAAT) proximal promoter (SEQ ID NO: 3) or a nucleotide sequence at least 80%, 85%, 90%, 95% or 98% identical thereto, including 42 bases of the 5' untranslated region (UTR); one or more enhancers selected from the group consisting of (i) a 34 base human ApoE/Cl enhancer, (ii) a 32 base human AAT promoter distal X region, and (iii) 80 additional bases of distal element of the human AAT proximal promoter; and a nucleic acid encoding human PAH. In another embodiment, the liver-specific transcription regulatory region comprises an a-microglobulin enhancer sequence and the 186 base human alpha anti -trypsin (AAT) proximal promoter.

[0089] In some embodiments, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6, which includes ApoE enhancer or HCR, a hAAT promoter, an LGI intron/hAAT intron, a hemoglobin intron, and a small portion of hemoglobin exon.

[0090] Specific examples of liver specific promoters include LP1, HLP, HCR-hAAT, ApoE- hAAT, LSP, TBG and TTR. These promoters are described in more detail in the following references: LP1 (human ApoE HCR core sequence (192 bp) with human AAT promoter (255 bp)): Nathwani A. etal. Blood. 2006 April 1; 107(7): 2653-2661; hybrid liver specific promoter (HLP) (human apolipoprotein E (ApoE) hepatic control region (HCR) fragment (34 bp) with modified human a -1 -antitrypsin (aAT) promoter (217 bp)): McIntosh J. etal. Blood. 2013 Apr 25; 121(17): 3335-3344; HCR-hAAT (ApoE-HCR (319 bp) with ApoE enhancer (1-4x154 bp) with human AAT promoter (408 bp) and including an Intron A (1.4 kbp) and 3’UTR (1.7 kbp)): Miao CH etal. Mol Ther. 2000; 1: 522-532; ApoE-hAAT: Okuyama T et al. Human Gene Therapy, 7, 637-645 (1996); LSP: Wang L etal. ProcNatl Acad Sci U S A. 1999 March 30; 96(7): 3906-3910, thyroxine binding globulin (TBG) promoter: Yan et al., Gene 506:289-294 (2012), and transthyretin (TTR) promoter: Costa et al., Mol. Cell. Biol. 8:81-90 (1988)

[0091] For example, De Simone et al. (EMBO Journal vol.6 no.9 pp.2759-2766, 1987) describes a number of promoters derived from human a- 1 -antitrypsin promoter. For example, it characterizes the cis- and trans-acting elements required for liver-specific activity within the human AAT promoter from -1200 to +44. The human AAT promoter in HLP consists of the distal X element (32 bp) and the proximal A and B elements (185 bp). Frain et al. (MOL CELL BIO, Mar. 1990, Vol. 10, No.3, p. 991-999) describes a number of promoters derived from human albumin promoter. For example, it characterizes promoter and enhancer elements within the human albumin gene from -1022 to -1.

[0092] Dang et al. (J BIOL CHEM, Vol. 270, No. 38, Issue of September 22, pp. 22577- 22585, 1995) describes the hepatic control region (HCR) of human apolipoprotein E gene (774 bp). Shachter et al. (J. Lipid Res. 1993. Vol.34: ppl699-1707) characterizes a liver-specific enhancer in the ApoE HCR (154 bp). These HCR fragments can be combined with other transcription regulatory elements such as the human AAT promoter or fragments thereof. Chow et al. (J Biol Chem. 1991 Oct 5;266(28): 18927-33) characterizes the human prothrombin enhancer from -940 to -860 (80 bp). Rouet et al. (Vol. 267, No . 29, Issue of October 15, PP. 20765-20773,1992; Nucleic Acids Res. 1995 Feb 11; 23(3): 395-404; and Biochemical Journal Sep 15, 1998, 334 (3) 577-584) characterize the sequence of the liver-specific human a-1- microglobulin/bikunin enhancer. U.S. Patent No. 7,323,324 also describes human AAT promoter, human a-microglobulin/bikunen enhancers, human albumin promoter, and human prothrombin enhancers.

[0093] In some embodiments, the promoter comprises multiple copies of one or more of the enhancers identified above. In some embodiments, the promoter constructs comprise one or more of the individual enhancer elements described above and combinations thereof, in one or more different orientation(s).

[0094] In some embodiments, the promoter is operably linked with a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked with a polynucleotide encoding the hPAH.

[0095] The size of the promoter can vary. Because of the limited packaging capacity of AAV, it is preferred to use a promoter that is small in size, but at the same time allows high level production of the protein(s) of interest in host cells. For example, in some embodiments the promoter is at most about 1.5 kb, at most about 1.4 kb, at most about 1.35 kb, at most about 1.3 kb, at most about 1.25 kb, at most about 1.2 kb, at most about 1.15 kb, at most about 1.1 kb, at most about 1.05 kb, at most about 1 kb, at most about 800 base pairs, at most about 600 base pairs, at most about 400 base pairs, at most about 200 base pairs, or at most about 100 base pairs. [0096] In some embodiments, a vector construct comprises a liver-specific transcription regulatory region, wherein the liver-specific regulatory region comprises a nucleotide sequence that is at least 90% identical to nucleotides 160 - 839 of SEQ ID NO:52. In certain embodiments, a vector construct comprises a liver-specific transcription regulatory region, wherein the liver-specific regulatory region comprises a nucleotide sequence that is at least 95% identical to nucleotides 160 - 839 of SEQ ID NO:52. In some embodiments, a vector construct comprises a liver-specific transcription regulatory region, wherein the liver-specific regulatory region comprises nucleotides 160 - 839 of SEQ ID NO:52.

[0097] In some embodiments, the vector constructs comprise a nucleic acid sequence encoding functional hPAH that is operably linked to a heterologous liver-specific transcription regulatory region. The vector constructs may comprise other regulatory elements. In some embodiments, in the vector constructs described herein, the expression control elements include one or more of the following: a promoter and/or enhancer; an intron; and a polyadenylation (poly A) signal.

[0098] In some embodiments, the vector construct comprises at least one or both of a 5' inverted terminal repeat (ITR) of AAV and a 3 ' AAV ITR, a promoter, a nucleic acid encoding functional hPAH, and optionally a posttranscriptional regulatory element, where the promoter, the nucleic acid encoding functional hPAH and the posttranscription regulatory element are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR.

[0099] In certain embodiments, the recombinant AAV vector construct comprises a nucleic acid comprising (a) an AAV2 5' inverted terminal repeat (ITR) (which may or may not be modified as known in the art), (b) a liver-specific transcription regulatory region, (c) a functional hPAH coding region, (d) one or more introns including fragments of longer introns, (e) optionally an exon or fragment thereof, (f) a polyadenylation sequence, and (f) an AAV23' ITR (which may or may not be modified as known in the art).

[00100] Other embodiments provided herein are directed to vector constructs encoding a functional hPAH polypeptide, wherein the constructs comprise one or more of the individual elements of the above described constructs and combinations thereof, in one or more different orientation(s).

[00101] Various additional regulatory elements can be used in the vector constructs, for example enhancers to further increase expression level of the protein of interest in a host cell, a polyadenylation signal, a ribosome binding sequence, and/or a consensus splice acceptor or splice donor site. In some embodiments, the regulatory element can facilitate maintenance of the recombinant DNA molecule extrachromosomally in a host cell and/or improve vector potency (e.g. scaffold/matrix attachment regions (S/MARs)). Such regulatory elements are well known in the art.

[00102] The vector constructs disclosed herein may include regulatory elements such as a transcription initiation region and/or a transcriptional termination region. Examples of a transcription termination region include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, human growth hormone (hGH) poly(A), bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the posttranscriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence. In some embodiments, the transcriptional termination region is bGH poly(A) sequence.

[00103] In some embodiments, a vector construct comprises a poly(A) sequence, wherein the a poly(A) sequence comprises a nucleotide sequence that is at least 90% identical to nucleotides 4258 - 4484 of SEQ ID NO:52. In certain embodiments, a vector construct comprises a poly(A) sequence, wherein the a poly(A) sequence comprises a nucleotide sequence that is at least 95% identical to nucleotides 4258 - 4484 of SEQ ID NO:52. In some embodiments, a vector construct comprises a poly(A) sequence, wherein the a poly(A) sequence comprises nucleotides 4258 - 4484 of SEQ ID NO:52.

[00104] In some embodiments, the vector constructs can include additional transcription and translation initiation sequences, and/or additional transcription and translation terminators, which are known in the art.

[00105] In some embodiments, the vector comprises one or more introns. The introns may facilitate processing of the RNA transcript in mammalian host cells, increase expression of the protein of interest and/or optimize packaging of the vector into AAV particles. Inclusion of an intron element may enhance expression compared with expression in the absence of the intron element (see e.g. Kurachi et ah, 1995, J Biol Chem. 1995 Mar 10; 270(10):5276-81). AAV vectors typically accept inserts of DNA having a defined size range which is generally about 4 kb to about 5.2 kb, or slightly more. However, there is no minimum size for packaging and small vector genomes package very efficiently. Introns and intron fragments fulfill this requirement while also enhancing expression. Thus, the disclosure contemplates the inclusion of hPAH intron sequences in the AAV vector (e.g. portion of intron 2 of PAH), or other introns or other DNA sequences in place of portions of a hPAH intron. In some embodiments, the intron is the second hPAH intron or a 2116 bp truncated form of the hPAH second intron. Additionally, other 5' and 3' untranslated regions of nucleic acid may be used in place of those recited for hPAH. Non limiting examples of such an intron are a hemoglobin (b-globin) intron and/or hAAT (human alpha- 1 -antitrypsin) intron. In other embodiments, the intronic sequence is a composite hAAT/beta-globin intron. In some embodiments, the intron is a synthetic intron. [00106] In some embodiments, the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or SEQ ID NO: 27 or 29 or 34.

[00107] In some embodiments, a vector construct comprises an intron region, wherein the intron comprises a nucleotide sequence that is at least 90% identical to nucleotides 885 - 2828 of SEQ ID NO:52. In certain embodiments, a vector construct comprises an intron region, wherein the intron comprises a nucleotide sequence that is at least 95% identical to nucleotides 885 - 2828 of SEQ ID NO:52. In some embodiments, a vector construct comprises an intron region, wherein the intron comprises nucleotides 885 - 2828 of SEQ ID NO:52.

[00108] The location and size of the intron in the vector can vary. In some embodiments, the intron is located between the promoter and the sequence encoding the protein of interest. In some embodiments, the intron is located downstream of the sequence encoding the protein of interest. In some embodiments, the intron is located within the promoter. In some embodiments, the intron includes an enhancer element. In some embodiments, the intron is located within the sequence encoding the protein of interest, preferably between exons of the sequence encoding the protein of interest. In some embodiments, the intron may comprise all or a portion of a naturally occurring intron within the sequence encoding the protein of interest.

[00109] The vector constructs can incorporate sequences from the genome of any known organism. The sequences can be incorporated in their native form or can be modified in any way to obtain a desired activity. For example, the sequences can comprise insertions, deletions or substitutions.

[00110] In any of the embodiments herein, the rAAV particle comprises a recombinant vector construct comprising a nucleotide sequence at least 90% identical to any one of SEQ NOs: 15-23 or 52. Preferably the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18 or SEQ ID NO: 52, and the AAV capsid is an AAV5 type capsid. [00111] In some embodiments, a rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises one, two, three or all of the elements of SEQ ID NO: 52. In certain embodiments, a rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence that is is at least 90% identical to SEQ ID NO: 52. In some embodiments, a rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence that is is at least 95% identical to SEQ ID NO: 52. In certain embodiments, a rAAV particle comprises a recombinant vector construct, wherein the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO: 52. In specific embodiments, the rAAV particle comprises an AAV capsid which is an AAV5 type capsid.

[00112] The AAV vector constructs provided herein in single strand form are less than about 7.0 kb in length, or are less than 6.5 kb in length, or are less than 6.4 kb in length, or are less than

6.3 kb in length, or are less than 6.2 kb in length, or are less than 6.0 kb in length, or are less than

5.8 kb in length, or are less than 5.6 kb in length, or are less than 5.5 kb in length, or are less than

5.4 kb in length, or are less than 5.3 kb in length, or are less than 5.2 kb in length or are less than

5.0 kb in length, or are less than 4.8 kb in length, or are less than 4.6 kb in length, or are less than

4.5 kb in length, or are less than 4.4 kb in length, or are less than 4.3 kb in length, or are less than

4.2 kb in length, or are less than 4.1 kb in length, or are less than 4.0 kb in length, or are less than

3.9 kb in length, or are less than 3.8 kb in length, or are less than 3.7 kb in length, or are less than

3.6 kb in length, or are less than 3.5 kb in length, or are less than 3.4 kb in length, or are less than

3.3 kb in length, or are less than 3.2 kb in length, or are less than 3.1 kb in length, or are less than

3.0 kb in length. The AAV vector constructs provided herein in single strand form range from about 5.0 kb to about 6.5 kb in length, or range from about 4.8 kb to about 5.2 k in length, or 4.8 kb to 5.3 kb in length, or range from about 4.9 kb to about 5.5 kb in length, or about 4.8 kb to about 6.0 kb in length, or about 5.0 kb to 6.2 kb in length or about 5.1 kb to about 6.3 kb in length, or about 5.2 kb to about 6.4 kb in length, or about 5.5 kb to about 6.5 kb in length, or range from about 4.0 kb to about 5.0 kb in length, or range from about 3.8 kb to about 4.8 k in length, or 3.6 kb to 4.6 kb in length, or range from about 3.4 kb to about 4.4 kb in length, or range from about 3.2 kb to about 4.2 kb in length, or range from about 3.0 kb to 4.0 kb in length, or range from about 3.5 kb to about 4.0 kb in length, or range from about 3.0 kb to about 3.5 kb in length, or range from about 4 kb to about 4.5 kb in length

[00113] When AAV vectors are produced from oversized recombinant vector constructs, they may lack a portion of the 5' or 3’ ends of the recombinant vector construct. Because AAV is a single-stranded DNA virus, and packages either the sense or antisense strand, the sense strand in oversized AAV vectors lacks the 5' AAV ITR and possibly portions of the 5' end of the target protein-coding gene, and the antisense strand in oversized AAV vectors lacks the 3' ITR and possibly portions of the 3' end of the target protein-coding gene. A functional transgene is produced in oversized AAV vector infected cells by annealing of the sense and antisense truncated genomes within the target cell. Thus, in certain embodiments, the rAAV particles of the invention may comprise recombinant vector constructs that comprise at least one ITR, and a substantial portion of a nucleotide sequence encoding a functional hPAH, such as a fragment of any of SEQ ID NO: 7, 8, 9, 10, 11, 12, or 13 that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence. The rAAV particles of the invention may also comprise a substantial portion of any of any one of SEQ NOs: 15-23 or 52, e.g. a fragment that is greater than 50%, 60%, 70%, 80%, or 90% of the length of the nucleotide sequence set forth in any of SEQ ID NOs: 15-23 or 52.

[00114] Polynucleotides and polypeptides including modified forms can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques known to those of skill in the art (Sambrook et ah, Molecular Cloning: A Laboratory Manual, 2nd edition). Generation of the vector constructs can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

[00115] AAV vector constructs can be replicated and packaged into infectious AAV particles, preferably replication deficient AAV particles, when present in a host cell that has been transfected with a polynucleotide encoding and expressing rep and cap gene products.

5.2 RECOMBINANT AAV PARTICLES AND AAV CAPSIDS

[00116] Production of AAV particles requires AAV "rep" and "cap" genes, which are genes encoding replication and encapsidation proteins, respectively. AAV rep and cap genes have been found in all AAV serotypes examined to date, and are described herein and in the references cited. In wild-type AAV, the rep and cap genes are generally found adjacent to each other in the viral genome (i.e., they are "coupled" together as adjoining or overlapping transcriptional units), and they are generally conserved among AAV serotypes. AAV rep and cap genes are also indivi dually and collectively referred to as "AAV packaging genes." The AAV cap genes for use herein encode Cap proteins which are capable of packaging AAV vectors in the presence of rep and adeno helper function and are capable of binding target cellular receptors. In some embodiments, the AAV cap gene encodes a capsid protein having an amino acid sequence derived from a particular AAV serotype.

[00117] The AAV sequences employed for the production of AAV can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide a similar set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of AAV serotypes and a discussion of the genomic similarities. (See, e.g. , GenBank Accession number U89790; GenBank Accession number JO 1901 ; GenBank Accession number AF043303; GenBank Accession number AF085716; Chiorini et ak, J. Vir. (1997) vol. 71, pp. 6823-6833; Srivastava et ak, J. Vir. (1983) vol. 45, pp. 555-564; Chiorini et ak, J. Vir. (1999) vol. 73, pp. 1309-1319; Rutledge et ak, J. Vir. (1998) vol. 72, pp. 309-319; and Wu et ak, J. Vir. (2000) vol. 74, pp. 8635-8647).

[00118] The genomic organization of all known AAV serotypes is very similar. The genome of AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides (nt) in length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for the non- structural replication (Rep) proteins and the structural (VP) proteins. The VP proteins form the capsid. The assembly-activating protein (AAP) rapidly chaperones capsid assembly and prevents degradation of free capsid proteins (Grosse et ak, J. Virol. 91(20):e01198-17, 2017). The terminal 145 nt are self-complementary and are organized so that an energetically stable intramolecular duplex forming a T-shaped hairpin may be formed. These hairpin structures function as an origin for viral DNA replication, serving as primers for the cellular DNA polymerase complex. The Rep genes encode the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep 52 and Rep40 are transcribed from the pl9 promoter. The cap genes encode the VP proteins, VPl, VP2, and VP3. The cap genes are transcribed from the p40 promoter. The ITRs employed in the vectors of the present embodiment may correspond to the same serotype as the associated cap genes, or may differ. In one embodiment, the ITRs employed herein correspond to an AAV2 serotype and the cap genes correspond to an AAV5 serotype.

[00119] The AAV VP proteins are known to determine the cellular tropicity of the AAV virion. The VP protein-encoding sequences are significantly less conserved than Rep proteins and genes among different AAV serotypes. The ability of Rep and ITR sequences to cross complement corresponding sequences of other serotypes allows for the production of pseudotyped AAV particles comprising the capsid proteins of a serotype (e.g., AAV1, 5 or 8) and the Rep and/or ITR sequences of another AAV serotype (e.g., AAV2). Such pseudotyped rAAV particles are a part of the present disclosure.

[00120] The AAV particles described herein (and the encoding AAV vector genomes) may comprise any of the capsid proteins described in WO 2018/022608 or PCT/US19/32097, incorporated by reference herein in its entirety for its disclosure of human and simian AAV capsids and their properties such as transduction efficiency, tissue tropism, glycan-binding, and resistance to neutralization by IVIG, including but not limited to any of the capsids in the sequence listing and variants thereof, e.g. with chimeric swapped variable regions and/or glycan binding sequences and/or GH loop.

[00121] In one embodiment, the AAV ITR sequences for use in the context of the present disclosure are derived from AAV1, AAV2, AAV4 and/or AAV6. Likewise, the Rep (e.g., Rep78 and Rep52) coding sequences are in one embodiment derived from AAV1, AAV2, AAV4 and/or AAV6. The sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present disclosure may however be taken from any serotype, such as from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 or AAV 12, or from simian AAVs, including any of the capsid proteins described in WO 2018/022608 or PCT/US19/32097, or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries, or any capsid at least 90% identical to any of SEQ ID NOs: 35-51.

[00122] For example, the amino acid sequences of various capsids are published. See, e.g., [00123] AAVRh.l / hu.14 / AAV9 AAS99264.1 (SEQ ID NO: 35)

[00124] AAVRh.8 SEQ97 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 36)

[00125] AAVRh.lO SEQ81 of U.S. Pat. Pub. 2013/0045186 (SEQ ID NO: 37) [00126] AAVRh.74 SEQ 1 of Int’l. Pat. Pub. WO 2013/123503(SEQ ID NO: 38)

[00127] AAV1 AAB 95452.1 (SEQ ID NO: 39)

[00128] AAV2 YP 680426.1 (SEQ ID NO: 40)

[00129] AAV3 NP 043941.1 (SEQ ID NO: 41)

[00130] AAV3B AAB95452.1 (SEQ ID NO: 42)

[00131] AAV4 NP 044927.1 (SEQ ID NO: 43)

[00132] AAV5 YP 068409.1 (SEQ ID NO: 44)

[00133] AAV6 AAB95450.1 (SEQ ID NO: 45)

[00134] AAV7 YP 077178.1 (SEQ ID NO: 46)

[00135] AAV8 YP 077180.1 (SEQ ID NO: 47)

[00136] AAV 10 AAT46337.1 (SEQ ID NO: 48)

[00137] AAV11 AAT46339.1 (SEQ ID NO: 49)

[00138] AAV 12 ABI16639.1 (SEQ ID NO: 50)

[00139] AAV 13 ABZ10812.1 (SEQ ID NO: 51)

[00140] Wherein the the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51.

[00141] Preferably, the AAV capsid is an AAV capsid with liver tropism. In some instances, the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC15. Preferably, the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44. In specific embodiment, the AAV capsid comprises the sequence of SEQ ID NO: 44.

[00142] Modified "AAV" sequences also can be used in the context of the present disclosure, e.g. for the production of AAV gene therapy vectors. Such modified sequences e.g. sequences having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more nucleotide and/or amino acid sequence identity (e.g., a sequence having about 75-99% nucleotide sequence identity) to an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 ITR, Rep, or VP, can be used in place of wild- type AAV ITR, Rep, or VP sequences.

[00143] In some embodiments, a nucleic acid sequence encoding an AAV capsid protein is operably linked to expression control sequences for expression in a specific cell type, such as Sf9 or HEK cells. Techniques known to one skilled in the art for expressing foreign genes in insect host cells or mammalian host cells can be used to practice the embodiment. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith (1986) A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex.; Luckow (1991) In Prokop et ah, Cloning and Expression of Heterologous Genes in Insect Cells with Baculovirus Vectors' Recombinant DNA Technology and Applications, 97-152; King, L. A. and R. D. Possee (1992) The baculovirus expression system, Chapman and Hall, United Kingdom; O'Reilly, D. R., L. K. Miller, V. A. Luckow (1992) Baculovirus Expression Vectors:

A Laboratory Manual, New York; W.H. Freeman and Richardson, C. D. (1995) Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39; U.S. Pat. No. 4,745,051; US2003148506; and WO 03/074714, all of which are incorporated by reference in their entireties. A particularly suitable promoter for transcription of a nucleotide sequence encoding an AAV capsid protein is e.g. the polyhedron promoter. However, other promoters that are active in insect cells are known in the art, e.g. the plO, p35 or IE-1 promoters and further promoters described in the above references are also contemplated.

[00144] Use of insect cells for expression of heterologous proteins is well documented, as are methods of introducing nucleic acids, such as vectors, e.g., insect-cell compatible vectors, into such cells and methods of maintaining such cells in culture. (See, e.g., METHODS IN MOLECULAR BIOLOGY, ed. Richard, Humana Press, N J (1995); O'Reilly et ah, BACULOVIRUS EXPRESSION VECTORS, A LABORATORY MANUAL, Oxford Univ. Press (1994); Samulski et ah, J. Vir. (1989) vol. 63, pp.3822-3828; Kajigaya et ah, Proc. Nat'l. Acad. Sci. USA (1991) vol. 88, pp. 4646-4650; Ruffing et al., J. Vir. (1992) vol. 66, pp. 6922- 6930; Kirnbauer et al., Vir. (1996) vol. 219, pp. 37-44; Zhao et al., Vir. (2000) vol. 272, pp. 382- 393; and U.S. Pat. No. 6,204,059). In some embodiments, the nucleic acid construct encoding AAV in insect cells is an insect cell-compatible vector. An "insect cell-compatible vector" or "vector" as used herein refers to a nucleic acid molecule capable of productive transformation or transfection of an insect or insect cell. Exemplary biological vectors include plasmids, linear nucleic acid molecules, and recombinant viruses. Any vector can be employed as long as it is insect cell-compatible. The vector may integrate into the insect cells genome but the presence of the vector in the insect cell need not be permanent and transient episomal vectors are also included. The vectors can be introduced by any means known, for example by chemical treatment of the cells, electroporation, or infection. In some embodiments, the vector is a baculovirus, a viral vector, or a plasmid. In one embodiment, the vector is a baculovirus, i.e. the construct is a baculoviral vector. Baculoviral vectors and methods for their use are described in the above cited references on molecular engineering of insect cells.

5.3 METHODS FOR PRODUCING RECOMBINANT AAV PARTICLES

[00145] In another embodiment, provided are methods of producing recombinant adeno- associated virus (AAV) particles comprising any of the AAV vector constructs provided herein. The methods comprise the steps of culturing a cell that has been transfected with any of the AAV vector constructs provided herein (in association with various AAV cap and rep genes) and recovering recombinant therapeutic AAV particles from the transfected cell or supernatant of the transfected cell.

[00146] The cells useful for recombinant AAV production provided herein are any cell type susceptible to baculovirus infection, including insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

[00147] The present disclosure provides materials and methods for producing recombinant AAV particles in insect or mammalian cells that comprise any of the vector constructs described herein. In some embodiments, the vector construct further comprises a promoter and a restriction site downstream of the promoter to allow insertion of a polynucleotide encoding one or more proteins of interest, wherein the promoter and the restriction site are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a posttranscriptional regulatory element downstream of the restriction site and upstream of the 3' AAV ITR. In some embodiments, the vector construct further comprises a polynucleotide inserted at the restriction site and operably linked with the promoter, where the polynucleotide comprises the coding region of a protein of interest. As a skilled artisan will appreciate, any one of the AAV vector constructs disclosed in the present application can be used in methods to produce the recombinant AAV particle.

[00148] In some embodiments, the helper functions are provided by one or more helper plasmids or helper viruses comprising adenoviral or baculoviral helper genes. Non-limiting examples of the adenoviral or baculoviral helper genes include, but are not limited to, El A, E1B, E2A, E4 and VA, which can provide helper functions to AAV packaging.

[00149] Helper viruses of AAV are known in the art and include, for example, viruses from the family Adenoviridae and the family Herpes viridae. Examples of helper viruses of AAV include, but are not limited to, SAdV-13 helper virus and SAdV-13-like helper virus described in US Publication No. 20110201088 (the disclosure of which is incorporated herein by reference), and helper vectors pHELP (Applied Viromics). A skilled artisan will appreciate that any helper virus or helper plasmid of AAV that can provide adequate helper function to AAV can be used herein.

[00150] In some embodiments, the AAV cap genes are present in a plasmid. The plasmid can further comprise an AAV rep gene which may or may not correspond to the same serotype as the cap genes. The cap genes and/or rep gene from any AAV serotype described herein (including, but not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,

AAV1 1, AAV12, AAV13 and any variants thereof) can be used to produce the recombinant AAV. In some embodiments, the AAV cap genes encode a capsid from serotype 1, serotype 2, serotype 4, serotype 5, serotype 6, serotype 7, serotype 8, serotype 9, serotype 10, serotype 11, serotype 12, serotype 13 or a variant thereof.

[00151] In some embodiments, the insect or mammalian cell can be transfected with the helper plasmid or helper virus, the vector construct and the plasmid encoding the AAV cap genes; and the recombinant AAV virus can be collected at various time points after co transfection. For example, the recombinant AAV virus can be collected at about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, or a time between any of these two time points after the co-transfection.

[00152] Recombinant AAV particles can also be produced using any conventional methods known in the art suitable for producing infectious recombinant AAV. In some instances, a recombinant AAV can be produced by using an insect or mammalian cell that stably expresses some of the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising AAV rep and cap genes, and a selectable marker, such as a neomycin resistance gene, can be integrated into the genome of the cell. The insect or mammalian cell can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the viral vector construct comprising the 5' and 3' AAV ITR (and the nucleotide sequence encoding the heterologous protein, if desired). The advantages of this method are that the cells are selectable and are suitable for large-scale production of the recombinant AAV particle. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector construct containing the 5' and 3' AAV ITRs and the rep-cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the recombinant AAV.

[00153] Provided herein are methods for the production of a AAV particle, useful as a gene delivery vector, the method comprising the steps of:

(a) providing a cell permissive for AAV replication (e.g. an insect cell or mammalian cell) with one or more nucleic acid constructs comprising:

(i) a nucleic acid molecule (recombinant vector construct) provided herein that has at least one flanking AAV inverted terminal repeat nucleotide sequence;

(ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell;

(iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell;

(iv) and optionally AAP and MAAP contained in the VP2/3 mRNA

(b) culturing the cell defined in (a) under conditions permitting expression of the Rep and the capsid proteins; and, optionally (c) recovering the AAV particle, and optionally (d) purifying the AAV particle. For example, the recombinant vector construct of (i) comprises (1) at least one AAV ITR, (2) a heterologous liver-specific transcription regulatory region, and (3) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH).

[00154] Typically then, a method provided herein for producing a AAV gene delivery vector comprises: providing to a cell permissive for AAV replication (a) a nucleotide sequence encoding a template for producing vector genome, e.g. vector construct of the present disclosure (as described in detail herein); (b) nucleotide sequences sufficient for replication of the template to produce a vector genome (the first expression cassette defined above); (c) nucleotide sequences sufficient to package the vector genome into an AAV capsid (the second expression cassette defined above), under conditions sufficient for replication and packaging of the vector genome into the AAV capsid, whereby AAV particles comprising the vector genome encapsidated within the AAV capsid are produced in the cell.

[00155] Transient transfection of adherent HEK293 cells (Chahal et ah, J. Virol. Meth. 196: 163-73 (2014)) and transfection of Sf9 cells, using the baculovirus expression vector system (BEVS) (Mietzsch et ah, Hum. Gene Ther. 25: 212-22 (2014)), are two of the most commonly used methods to produce AAV vectors.

[00156] A method provided herein may comprise the step of affinity-purification of the (virions comprising the) recombinant parvoviral (rAAV) vector construct using an anti-AAV antibody, in one embodiment an immobilized antibody. In another embodiment, the anti-AAV antibody is a monoclonal antibody. One antibody for use herein is a single chain camelid antibody or a fragment thereof as e.g. obtainable from camels or llamas (see e.g. Muyldermans, 2001, Biotechnol. 74: 277-302). The antibody for affinity-purification of rAAV is an antibody that specifically binds an epitope on an AAV capsid protein, whereby in one embodiment the epitope is an epitope that is present on capsid protein of more than one AAV serotype. For example, the antibody may be raised or selected on the basis of specific binding to AAV5 capsid but at the same time also it may also specifically bind to AAV1, AAV2, AAV3, AAV6, AAV8 or AAV9 capsids.

[00157] The viral particles comprising the vector constructs described herein may be produced using any invertebrate cell type which allows for production of AAV or biologic products and which can be maintained in culture. For example, the insect cell line used can be from Spodoptera frugiperda, such as SF9, SF21, SF900+, drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyx mori cell lines, Trichoplusia ni cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines. In one embodiment, insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38.

[00158] Baculoviruses are enveloped DNA viruses of arthropods, two members of which are well known expression vectors for producing recombinant proteins in cell cultures.

Baculoviruses have circular double-stranded genomes (80-200 kbp) which can be engineered to allow the delivery of large genomic content to specific cells. The viruses used as a vector are generally Autographa califomica multicapsid nucleopolyhedrovirus (AcMNPV) or Bombyx mori nucleopolyhedrovirus (BmNPV) (Kato et ak, (2010), Applied Microbiology and Biotechnology, vol. 85, Issue 3, pp 459-470).

[00159] Baculoviruses are commonly used for the infection of insect cells for the expression of recombinant proteins. In particular, expression of heterologous genes in insects can be accomplished as described in for instance U.S. Pat. No. 4,745,051; EP 127,839; EP 155,476; Vlak et ak, (1988), Journal of General Virology, vol. 68, pp 765-776; Miller et ak, (1988),

Annual Review of Microbiology, vol. 42, pp 177-179; Carbonell et ak, (1998), Gene, vol. 73, Issue 2, pp 409-418; Maeda et ak, (1985), Nature, vol. 315, pp 592-594; Lebacq-Veheyden et ak, (1988), Molecular and Cellular Biology, vol. 8, no. 8, pp 3129-3135; Smith et ak, (1985), PNAS, vol. 82, pp 8404-8408; and Miyajima et ak, (1987), Gene, vol. 58, pp 273-281. Numerous baculovirus strains and variants and corresponding permissive insect host cells that can be used for protein production are described in Luckow et ak, (1988), Nature Biotechnology, vol. 6, pp 47-55; Maeda et ak, (1985), Nature, vol. 315, pp 592-594; and McKenna et ak, (1998), Journal of Invertebrate Pathology, vol. 71, Issue 1, pp 82-90.

[00160] In another embodiment, the methods provided herein are carried out with any mammalian cell type which allows for replication of AAV or production of biologic products, and which can be maintained in culture. In one embodiment, mammalian cells used can be HEK293, HeLa, CHO, NSO, SP2/0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE- 19, and MRC-5 cells.

[00161] The methods provided herein for producing rAAV particles produce a population of rAAV particles. In some embodiments, the population is enriched for particles comprising full length or nearly full length vector genomes by steps that reduce the number of empty capsids. [00162] The population of rAAV particles produced by the methods provided herein are used, for example, for administration in any of the methods of decreasing plasma Phe levels in human subjects described herein, and in any of the methods of treating PKU described herein. In certain embodiments, the rAAV particles are provided in the liquid formulation of Example 2. In certain embodiments, the formulation is suitable for intravenous administration for the method of treating PKU according to the protocol as described in Example 3 or Example 4.

5.4 PHARMACEUTICAL FORMULATIONS

[00163] In one embodiment, provided is a pharmaceutical composition comprising a nucleic acid or a vector provided herein and a pharmaceutically acceptable diluent, excipient, carrier and/or other medicinal agent, pharmaceutical agent or adjuvant, etc.

[00164] By "pharmaceutically acceptable" it is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example, in transfection of a cell ex vivo or in administering a viral particle or cell directly to a subject.

[00165] A carrier may be suitable for parenteral administration, which includes intravenous, intraperitoneal or intramuscular administration. Alternatively, the carrier may be suitable for sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein is contemplated. [00166] In other embodiments, provided herein are pharmaceutical compositions (i.e. formulations) of AAV particles useful for administration to subjects suffering from a genetic disorder to deliver gene encoding a protein of interest. In certain embodiments, the pharmaceutical formulations provided herein are liquid formulations that comprise recombinant AAV particles comprising any of the vector constructs disclosed herein. The concentration of recombinant AAV virions in the formulation may vary. In certain embodiments, the concentration of recombinant AAV particle in the formulation may range from 1 x 10¹³ to about 1 x 10¹⁴ vg/ml, for example, 6 x 10¹³ vg/ml. In certain embodiments, the concentration of recombinant AAV particle in the formulation is as described in Example 2.

[00167] In other embodiments, the AAV particle pharmaceutical formulation provided herein comprises one or more sterile pharmaceutically acceptable excipients to provide the formulation with advantageous properties for storage and/or administration to subjects for the treatment of the genetic disorder. In certain embodiments, the pharmaceutical formulations provided herein are capable of being stored at less than about -60°C (minus 60 degrees centigrade) for a period of at least 6 months, 1.5 years, or 2 years, with no appreciable change in stability. In addition, the pharmaceutical formulations provided herein are stable under suitable accelerated storage conditions. Example accelerated conditions include stored at about 40°C and about 75% humidity for a time period of, e.g., 6, 9, 12, 18 and/or 24 months, or at about 25°C and about 60% humidity for a time period of, e.g., 6, 9, 12, 18 and/or 24 months, or (for drug substances intended for storage in a freezer) at about -20°C for a time period of, e.g. 12 months. See, e.g., FDA Guidance for Industry: Stability Testing of New Drug Substances and Products, Nov 2003. [00168] In this regard, the term "stable" means that the recombinant AAV particle present in the formulation essentially retains its physical stability, chemical stability and/or biological activity during storage. In certain embodiments, the recombinant AAV particle present in the pharmaceutical formulation retains at least about 80% of its vg/ml (or at least about 80% of its infectious rAAV particles) in a human patient during storage for a determined period of time at - 65°C, in other embodiments at least about 85%, 90%, 95%, 98% or 99% of its vg/ml, or alternatively infectious rAAV particles, in a human subject.

[00169] In certain embodiments, the stability of a formulation described herein is assessed as described in Example 2. In certain embodiments, a formulation described herein is stable when stored at 2-8°C (e.g., 4°C) for at least 6 months (e.g., 6-9 months, 9-12 months, or 6-12 months) as assessed by an assay described in Example 2. In some embodiments, a formulation described herein is stable when stored at < minus 60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 12 months (e.g., 12-18 months, 12-24 months, 18-36 months, 24-48 months, 36-48 months, or 12-48 months) as assessed by an assay described in Example 2. In certain embodiments, a formulation described herein is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 24 months (e.g., 24-36 months, 24-48 months or 36-48 months) as assessed by an assay described in Example 2.

[00170] In some embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at 2- 8°C (e.g., 4°C) for at least 6 months (e.g., 6-9 months, 9-12 months, or 6-12 months) as assessed by measuring deamidation levels of the recombinant AAV particles (percent deamidation). In certain embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 12 months (e.g., 12-18 months, 12-24 months, 18-36 months, 24-48 months, 36-48 months, or 12-48 months) as assessed by measuring deamidation levels of the recombinant AAV particles (percent deamidation). In certain embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 24 months (e.g., 24-36 months, 24-48 months or 36-48 months) as assessed by measuring deamidation levels of the recombinant AAV particles (percent deamidation).

Standard techniques known to one of skill in the art may be used to measure deamidation.

[00171] In certain embodiments, the deamidation level of VPl protein at its N-terminus is quantified by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay accurately measures percent deamidation at the N-terminal region of AAV5 Viral Protein 1 (VPl), specifically at N50 and N56. Capsid particles in formulated bulk drug substance or drug product are denatured to dissociate viral proteins and digested to peptides prior to LC-MS analysis. The percent deamidation is calculated by measuring the intensity of deamidated peptide peak area relative to the sum of unmodified and deamidated peptide peak areas.

[00172] In some embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable for at least 6 months (e.g., 6-9 months, 9-12 months, or 6-12 months) at 2-8°C (e.g., 4°C) as assessed by measuring aggregation. In certain embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 12 months (e.g., 12-18 months, 12-24 months, 18-36 months, 24-48 months, 36- 48 months, or 12-48 months) as assessed by measuring aggregation. In certain embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 24 months (e.g., 24-36 months, 24-48 months, or 36-48 months) as assessed by measuring aggregation. Standard techniques known to one of skill in the art may be used to measure aggregation, such as capsid protein aggregation.

[00173] In certain embodiments, capsid protein aggregates are monitored by SEC-HPLC (Size Exclusion Chromatography High Performance Liquid Chromatography). The capsid protein particles are monitored by UV at 280 nm and elute according to size in order of trimer, dimer and monomer. Excipients and salts in the sample buffer such as poloxamer, elute after the monomer peak. Aggregate content is reported as % mul timer where % mul timer is the sum of the dimer and trimer peak area over the total peak area (of monomer, dimer and trimer). A reference is run with every assay to confirm assay performance.

[00174] In some embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at 2- 8°C (e.g., 4°C) for at least 6 months (e.g., 6-9 months, 9-12 months, or 6-12 months) as assessed by measuring deamidation levels of the recombinant AAV particles (percent deamidation) and aggregation. In certain embodiments, a formulation described herein, which comprises recombinant AAV particles (e.g, recombinant AAV5 particles) described herein, is stable when stored at < minus60°C (e.g., minus 60°C, minus 65 °C, minus 70 °C, minus 75 °C, or minus 80 °C) for at least 12 months (e.g., 12-18 months, 12-24 months, 18-36 months, 24-48 months, 36- 48 months, or 12-48 months) as assessed by measuring deamidation levels of the recombinant AAV particles (percent deamidation) and aggregation. Standard techniques known to one of skill in the art may be used to measure deamidation and aggregation. In certain embodiments, capsid protein aggregates are monitored by SEC-HPLC (Size Exclusion Chromatography High Performance Liquid Chromatography) and deamidation is measured by LC-MS, such as described herein.

[00175] In certain aspects, the formulation comprising recombinant AAV particle further comprises one or more buffering agents.

[00176] In another embodiment, the recombinant AAV particle formulation provided herein may comprise one or more isotonicity agents, such as sodium chloride. Other buffering agents and isotonicity agents known in the art are suitable and may be routinely employed for use in the formulations provided herein.

[00177] In another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more bulking agents, including cryoprotective agents. Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24).

[00178] In yet another embodiment, the recombinant AAV particle formulations provided herein may comprise one or more surfactants, which may be non-ionic surfactants. Exemplary surfactants include ionic surfactants, non-ionic surfactants, and combinations thereof. For example, the surfactant can be, without limitation, TWEEN 80 (also known as polysorbate 80, or its chemical name polyoxyethylene sorbitan monooleate), sodium dodecyl sulfate, sodium stearate, ammonium lauryl sulfate, TRITON AG 98 (Rhone-Poulenc), poloxamer 407, poloxamer 188 and the like, and combinations thereof.

[00179] The recombinant AAV particle formulations provided herein are stable and can be stored for extended periods of time without an unacceptable change in quality, potency, or purity. In one aspect, the formulation is stable at a temperature of about 5°C (e.g., 2°C to 8°C) for at least 1 month, for example, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -20°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -40°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of less than or equal to about -60°C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of minus 60 °C, minus 65 °C, minus 70°C, minus 75°C or minus 80 °C, or minus 60 °C to minus 80 °C for at least 6 months, for example, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more. In another embodiment, the formulation is stable at a temperature of minus 60 °C, minus 65 °C, minus 70°C, minus 75°C or minus 80 °C, or minus 60 °C to minus 80 °C for 6-12 months, 9-12 months, 12-18 months, 12-24 months, 18-24 months, 24-36 months, 36-48 months or 24-48 months.

[00180] Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to accommodate high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride are included in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. In certain embodiments, a nucleic acid or vector construct provided herein may be administered in a time or controlled release formulation, for example in a composition which includes a slow release polymer or other carriers that will protect the compound against rapid release, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may for example be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymners (PLG).

[00181] In yet another aspect, the disclosure provides a pharmaceutical composition comprising rAAV particle at a concentration of at least 1E13 vg/ml, for example, about lE13vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, about 1E13 vg/ml. about 2E13 vg/ml, about 6E13 vg/ml or about 2E14 vg/ml, a buffering agent, an isotonicity agent, a cryopreservative agent and a surfactant which is stable during storage at about -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years. In some embodiments, the surfactant is a poloxamer or alternatively a polysorbate at a concentration of less than 0.2% w/v, or less than 0.15% w/v, for example, about 0.1% w/v. In some embodiments, the cryopreservative agent is a sugar, for example, trehalose.

[00182] In some embodiments, the pharmaceutical composition is aqueous and comprises rAAV particle at a concentration of at least 1E13 vg/ml, for example, about lE13vg/ml to about 5E14 vg/ml, about 2E13 vg/ml to about 2E14 vg/ml, about 1E13 vg/ml. about 2E13 vg/ml, about 6E13 vg/ml or about 2E14 vg/ml, sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 mM to about 165 mM, a cryopreservative agent that is a sugar, optionally trehalose, and a poloxamer at a concentration of less than 0.2% w/v. The sodium phosphate may comprise sodium phosphate, dibasic and/or sodium phosphate, monobasic. Optionally, the sugar is trehalose at a concentration of about 60 mM to about 90mM, or about 60 mM to about 80 mM, or about 70 to about 90 mM, or about 70 to about 80 mM. Optionally, the poloxamer is poloxamer 188 at a concentration of about 0.05% to 0.15% w/v. In certain embodiments, the pharmaceutical composition comprises the formulation described in Example 2.

[00183] In some embodiments, the sodium phosphate is at a concentration of about 5 to about 15 mM. In some embodiments, the sodium chloride is at a concentration of about 100 to about 140 mM. In some embodiments, the sugar (e.g. trehalose) is at a concentration of about 60 to about 90 mM. In some embodiments, the poloxamer (e.g. poloxamer 188) is at a concentration of about 0.05% to about 0.15% w/v.

[00184] In some embodiments, the sodium phosphate is at a concentration of about 8 to about 12mM. In some embodiments, the sodium chloride is at a concentration of about 110 to about 130 mM. In some embodiments, the sugar (e.g. trehalose) is at a concentration of about 70 to about 80 mM. In some embodiments, the poloxamer (e.g. poloxamer 188) is at a concentration of about 0.08% to about 0.12% w/v, or about 0.1% w/v.

[00185] In some embodiments, the sodium phosphate, monobasic (dihydrate) is at a concentration that is greater than 0.1 mg/mL and less than 1 mg/mL, and the sodium phosphate, dibasic (dodecahydrate), is at a concentration that is greater than 0.5 mg/mL and less than 5 mg/mL. In some embodiments, the sodium chloride is at a concentration that is greater than 5 mg/mL and less than 10 mg/mL. In some embodiments, the sugar is trehalose (dihydrate) at a concentration of greater than 20 mg/ml to less than 40 mg/ml. In some embodiments, the poloxamer 188 is at a concentration of about 1.5 mg/ml or less, or about 1 mg/ml.

[00186] In some embodiments, the sodium phosphate, monobasic (dihydrate) is at a concentration that is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 to about 0.4 mg/mL, and the sodium phosphate, dibasic (dodecahydrate), is at a concentration that is greater than 2.5 mg/ml and less than 3 mg/ml, optionally about 2.7 mg/ml. In some embodiments, the sodium chloride is at a concentration that is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. In some embodiments, the sugar is trehalose (dihydrate) at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml. In some embodiments, the poloxamer 188 is at a concentration less than 1.5 mg/ml, or about 1 mg/ml.

[00187] In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 1E13 vg/ml to about 5E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 2E13 vg/ml to about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 1E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 2E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, the pharmaceutical composition comprises rAAV particle at a concentration of about 2E14 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. For example, a vial may comprise 8 mL aqueous solution, including 4.8E14 vg rAAV particles, 3.1 mg sodium dihydrogen phosphate, monobasic, dihydrate, 21.6 mg sodium phosphate, dibasic, dodecahydrate, 56.1 mg sodium chloride, 224 mg trehalose dihydrate and 8 mg poloxamer 188. [00188] In certain embodiments, provided herein is a pharmaceutical composition comprises rAAV particle at a unit dose of 480 x 10¹³ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, provided herein is a pharmaceutical composition comprises rAAV particle at a unit dose of 316 x 10¹³ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In certain embodiments, provided herein is a pharmaceutical composition comprises rAAV particle at a unit dose of 1.6 x 10¹⁶ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In some embodiments, provided herein is a pharmaceutical composition comprises rAAV particle at a unit dose of about 316 x 10¹³ vg to about 1.6 x 10¹⁶ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188. In certain embodiments, provided herein is a pharmaceutical composition comprises rAAV particle at a unit dose of about 250 x 10¹³ vg to about 2 x 10¹⁶ vg, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose (dihydrate), and 0.1% w/v poloxamer 188.

[00189] In a specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particle comprises an AAV capsid and a recombinant vector construct, wherein the recombinant AAV particle comprises a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region. In another specific embodiment, the recombinant AAV vector, and wherein the recombinant AAV vector comprises: (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV 3' ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. In one embodiment, the nucleic acid encoding functional hPAH encodes an amino acid sequence at least 95% identical to SEQ ID NO: 2. In another embodiment, the nucleic acid encoding functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 1 or 7- 13. In certain embodiments, the nucleic acid encoding PAH is operably linked to a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer. In some embodiments, the the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24, or alternatively at least 90% identical to any one of SEQ ID NOs: 25 or 26. In a specific embodiment, the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6. In certain embodiments, the recombinant vector construct further comprises an intron. In some embodiments, the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or 27 or 29 or 34. In certain embodiments, the recombinant vector construct further comprises a polyadenylation signal. In some embodiments, the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal. In a specific embodiment, the recombinant AAV particle comprises a recombinant vector construct at least 90% identical to any one of SEQ NOs: 15-23 or 52. In another specific embodiment, the recombinant AAV particle comprises a recombinant vector construct at least 95% identical to any one of SEQ NOs: 15-23 or 52. In another specific embodiment, the recombinant AAV particle comprises a recombinant vector construct comprising the nucleotide sequence of any one of SEQ NOs: 15-23 or 52. In another specific embodiment, the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35- 51. In another specific embodiment, the AAV capsid comprises an amino acid sequence at least 90% identical to any one of SEQ ID NOs: 35-51. In another specific embodiment, the AAV capsid comprises an amino acid sequence at least 95% identical to any one of SEQ ID NOs: 35- 51. In another specific embodiment, the AAV capsid is an AAV capsid with liver tropism. In certain embodiments, the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC15.

In some embodiments, the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44. In specific embodiments, the AAV capsid with liver tropism is an AAV5 type capsid, wherein the AAV5 type capsid comprises the sequence of SEQ ID NO: 44.

[00190] In a specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 18. In another specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 18. In another specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a recombinant vector construct comprising the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the AAV5 capsid is at least 85%, 90% or 95% identical to SEQ ID NO: 44. In specific embodiments, the AAV5 capsid comprises the sequence of SEQ ID NO: 44. [00191] In a specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO:52. In another specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises a nucleotide sequence at least 95% identical to SEQ ID NO:52. In another specific embodiment, a pharmaceutical composition described herein comprises recombinant AAV particle, wherein the recombinant AAV particles comprises an AAV5 capsid and a recombinant vector construct, wherein the recombinant vector construct comprises the nucleotide sequence of SEQ ID NO:52. In some embodiments, the AAV5 capsid is at least 85%, 90% or 95% identical to SEQ ID NO: 44. In specific embodiments, the AAV5 capsid comprises the sequence of SEQ ID NO: 44.

[00192] In a specific embodiment, a pharmaceutical composition described herein comprises a recombinant AAV particle described herein. In specific embodiments, a pharmaceutical composition described herein is for intravenous administration to a subject. In specific embodiments, a pharmaceutical composition described herein is for infusion into a subject. [00193] In specific embodiments, a pharmaceutical composition described herein is for use in a method described herein (e.g., Section 5, Example 3 or Example 4). In certain embodiments, a pharmaceutical composition described herein is for use in a method of decreasing plasma phenylalanine (Phe) levels in a human subject in need thereof. In some embodiments, pharmaceutical composition described herein is for use in a method of treating a human subject with phenylketonuria. See , e.g., Secton 5.5 and Example 3 and 4 for the subjects that may be administered a pharmaceutical composition described herein.

[00194] Preferably the pharmaceutical composition is a liquid aqueous solution, or lyophilized, and is for storage at freezing temperature. In any of these embodiments, the composition is for use in intravenous administration of rAAV particle to a patient with phenylketonuria.

[00195] In specific embodiments, the pharmaceutical composition comprises the formulation of Example 2.

5.5 METHODS OF TREATMENT

[00196] In any of the embodiments, the subject has phenylketonuria (PKU), optionally classic PKU or severe PKU. In some embodiments, the subject has a plasma Phe level of 600 pmol/L or above prior to said administration, or 700, 800, 900, 1000, or 1000 pmol/L or above. In some embodiments, the subject has a plasma Phe level of 1200 pmol/L or above prior to said administration. In certain embodiments, the subject to be treated will be treated according to the protocol described in Example 3 or Example 4.

[00197] In some embodiments, the subject is an infant less than 2 years old. In some embodiments, the subject is a human of the following ages, for example,

[00198] (1) 15 to 18, 12 to <15, 5-<12, or 0 (birth) to <5 years old

[00199] (2) 15 to 18, 12 to <15, 9 to <12, 5 to 9, 2 to <5, or 0 to <2 years old

[00200] (3) 12 to <18, 6 to <12, 0 to <6 years old

[00201] (4) 15 to <18, 12 to <15, 9 to <12, 6 to <9, 3 to <6, 0 to <3 years old.

[00202] In some embodiments, the subject is a human aged [00203] (a) 12 to < 15 years.

[00204] (b) 5 to < 12 years; or 9 to < 12 years; or 5 to < 9 years

[00205] (c) 0 to < 5 years; or 2 to < 5 years or 0 to < 2 years).

[00206] In some embodiments, the subject is 15 or more years old, or 18 or more years old.

In some embodiments, the subject is an adult. In some embodiments, the subject is a male. In some embodiments, the subject is a female, e.g. a nonpregnant female.

[00207] In any of the embodiments, the subject has a mutation in an endogenous gene encoding PAH, optionally mutations F39L, L48S, I65T, R68S, A104D, SI IOC, D129G, E178G, VI 90 A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388M E390G, A395P, P407S, and Y414C.

[00208] In some embodiments, the subject is not receiving pharmacotherapy to treat PKU when the rAAV particles are administered. For example, the subject has not received pegvaliase at least 30 days prior to said administration and/ the subject has not received large neutral amino acids (LNAAs) at least 30 days prior to said administration, and/or the subject has not received sapropterin at least 7 days prior to said administration. In some embodiments, the subject has not received steroids at least 30 days prior to said administration.

[00209] In some embodiments, the subject does not have detectable anti- AAV capsid antibody in blood when the rAAV particles are administered (e.g., is not AAV5 seropositive). Anti-AAV neutralizing antibodies are undesirable because they may block cell transduction or otherwise reduce the overall efficiency of the treatment.

[00210] In certain embodiments, the subject does not have any detectable anti-AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject prior to administration of a rAAV particle described herein to the subject. In some embodiments, the subject does not have any detectable anti-AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject after administration of a rAAV particle described herein to the subject. In certain embodiments, the subject does not have any detectable anti- AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject prior to and after administration of a rAAV particle described herein to the subject.

[00211] In certain embodiments, the subject does not have any detectable anti -PAH antibody in blood sample from the subject prior to administration of a rAAV particle to the subject. In some embodiments, the subject does not have any detectable anti -PAH antibody in blood sample from the subject after to administration of a rAAV particle to the subject. In certain embodiments, the subject does not have any detectable anti -PAH antibody in blood sample from the subject prior to and after to administration of a rAAV particle to the subject.

[00212] In certain embodiments, the subject does not have any detectable anti-AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject prior to administration of a rAAV particle described herein to the subject and the subject does not have any detectable anti -PAH antibody in blood sample from the subject prior to administration of a rAAV particle to the subject. In some embodiments, the subject does not have any detectable anti-AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject after administration of a rAAV particle described herein to the subject and the subject does not have any detectable anti -PAH antibody in blood sample from the subject after to administration of a rAAV particle to the subject. In certain embodiments, the subject does not have any detectable anti-AAV capsid antibody (e.g., anti-AAV-5 capsid antibody) in a blood sample from the subject prior to and after administration of a rAAV particle described herein to the subject and the subject does not have any detectable anti -PAH antibody in blood sample from the subject prior to and after to administration of a rAAV particle to the subject.

[00213] In some embodiments, the subject does not have (1) evidence of an active infection or immunosuppressive disorder; (2) history of cancer; (3) substance use disorder, major depressive disorder, psychosis, or bipolar disorder; or (4) contraindication to corticosteroids. In some embodiments, the subject does not have prior infection with hepatitis B or C, or tuberculosis. In some embodiments, the subject does not have serum creatinine greater than or equal to 1.5 mg/dL.

[00214] In some embodiments, the subject does not have clinically significant liver disease prior to said administration. In some embodiments, the subject does not have a prior liver biopsy showing significant fibrosis of 3 or 4 as rated on a scale of 0-4. In some embodiments, the subject does not have an elevation in any of ALT (alanine transaminase), AST (aspartate aminotransferase), GGT (gamma-glutamyltransferase) or bilirubin to more than 1.25 times the upper limit of normal (ULN), or the international normalized ratio being equal to or greater than 1 2 [00215] In certain embodiments, the subject fulfills one, two, three, or more of the inclusion criteria in Example 3 or Example 4. In some embodiments, the subject does not meet one, two, three or more of the criteria listed under exclusion criteria in Example 3 or Example 4. In certain embodiments, the subject fulfills one, two, three, or more of the inclusion criteria in Example 3 or Example 4, and the subject does not meet one, two, three or more of the criteria listed under exclusion criteria in Example 3 or Example 4.

[00216] In some embodiments, the subject meets one, two, three or more of the criteria listed under exclusion criteria in Example 3 or Example 4. In certain embodiments, the subject fulfills one, two, three, or more of the inclusion criteria in Example 3 or Example 4, and the subject meets one, two, three or more of the criteria listed under exclusion criteria in Example 3 or Example 4.

[00217] Prior to infusion of the rAAV particles, subjects are evaluated for: (1) baseline physical examination; (2) baseline clinical laboratory tests, including (a) plasma Phe levels, (b) plasma tyrosine (Tyr) levels, and (c) liver enzyme tests, including ALT, AST, GGT and bilirubin; (d) and baseline AAV5 antibody detection; (3) baseline protein intake from intact food and from medical food; (4) measures of inattention and/or executive function, e.g., Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS IV) which is an investigator-rated inattention score), Cambridge Neuropsychological Test Automated Battery (CANTAB) scores (including Rapid Visual Processing, Stop Signal and Spatial Working Memory); (5) measures of health-related quality of life (HRQoL), e.g. Phenylketonuria Impact and Treatment Quality of Life Questionnaire (PKU-QOL) score or Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q-SF) score; (6) baseline levels of other parameters monitored during the study; and (7) PAH genotyping, if permitted. In certain embodiments, prior to rAAV particle infusion a subject is evaluated as described in Example 3 or Example 4.

[00218] In the methods of the disclosure, the rAAV particle is administered intravenously in a single administration. In some embodiments, the vector construct or recombinant AAV particle is administered by intravenous injection either as a single bolus or over a prolonged time period, which may be at least about 1, 5, 10, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 or 240 minutes, or more. [00219] In some embodiments, the rAAV particle is administered at a dose ranging from about 1E13 to about 5E14 vector genomes per kilogram body weight of the subject (vg/kg),

2E13 to about 2E14 (vg/kg), for example, a dose of about 1E13 vg/kg, or a dose of about 2E13 vg/kg, or a dose of about 6E13 vg/kg, or a dose of about 2E14 vg/kg. In certain embodiments, the rAAV particle is administered at a dose specified in Examples 3 and 4. In some embodiments, the subject administered the rAAV particle is about 80 kg. In other embodiments, the subject administered the rAAV particle is about 60 to 85 kg (e.g, 60, 65, 70, 75, 80 or 85 kg). [00220] In some embodiments, the rAAV particle is administered at a unit dose ranging from about 200E13 to about 2E16 vector genomes or 316E13 to about 1.6E16 vector genomes, for example, a unit dose of about 316E13 vg, or a unit dose of about 480E13 vg, or a unit dose of about 1.6E16 vg.

[00221] After infusion of the rAAV particles, the methods may further comprise the step of monitoring various parameters, e.g. measuring the parameters on a weekly basis. Measuring can alternatively occur every 1, 2, 3, 4, 5 or 6 days or every week or every two weeks or every three weeks or every month. Parameters may be monitored through Week 24, 48, 96 or longer. The methods may include measuring plasma Phe level of the subject. For example, plasma Phe levels are measured, and a reduction from baseline in mean plasma Phe levels at Week 8, 12 and 24 post-infusion is observed. Optionally the method includes performing a Phe challenge test or Phe breath test on the subject. An increase in Phe activity, as measured by increased rate of Phe oxidation on a Phe breath test, is observed.

[00222] The methods may also further comprise the step of measuring plasma level of one or more neurotransmitters or neurotransmitter metabolites of the subject. Tyrosine (Tyr) levels may be measured, and the Phe/Tyr ratio may be calculated. For example, the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5-hydroxyindoleacetic acid. : In one example, phenylacetylglutamine [PAG], homovanillic acid [HVA], 3-methoxy-4- hydroxyphenyl glycol [MOPEG], and 5-hydroxyindoleacetic acid [5HIAA] are monitored. [00223] The method may further comprise the step of monitoring the ability to tolerate an increase in dietary protein intake and/or a reduction in medical food intake (Phe-reduced or Phe- free foods). For example, after administration of the rAAV particles according to the methods described herein, subjects can consume at least 0.8 g/kg protein intake from intact food at Week 48 post-infusion while maintaining plasma Phe at less than or equal to 360 pmol/L, and/or subjects can consume no medical food. Such improvements in Phe intake may occur, e.g. by Week 24, 48, or 96.

[00224] The method may further comprise the step of monitoring symptoms of inattention and measures of executive function, e.g. as measured by ADHD-RS IV (investigator-rated inattention score), CANTAB scores (Rapid Visual Processing, Stop Signal and Spatial Working Memory), or health-related quality of life (HRQoL), e.g. as measured by PKU-QOL score or Q-LES-Q-SF score. A clinically significant improvement in any of these parameters is observed, e.g. by Week 24, 48, or 96.

[00225] Additional parameters include nutritional markers and/or fasting lipid panel. Several nutritional markers have been shown to be deficient in PKU patients and include: 25-hydroxy (OH) Vitamin D, methylmalonic acid (indicator of B-12 deficiency), serum ferritin (indicator of iron deficiency), selenium, and zinc. An improvement in any of these parameters is observed, e.g. by Week 24, 48 or 96.

[00226] The methods may also further comprise the step of monitoring episome formation by steps comprising extracting DNA from liver cells of the subject and detecting circular vector genomes, optionally by PCR or southern blotting.

[00227] After Week 24 post-infusion, subjects who achieve plasma Phe less than or equal to 360 pmol/L on two consecutive assessments at least one week apart are allowed to increase intact protein intake as follows:

[00228] (a) If the subject already has protein intake from intact sources that is greater than 2x the dietary reference intake DRI (greater than 1.6 g/kg), then the subject can maintain protein intake from intact sources and discontinue protein intake from medical food.

[00229] (b) If the subject has a protein intake from intact sources that is 0.5x to 2x DRI (0.4-

1.6 g/kg), then the subject can increase protein intake from intact sources by 10 g/day, and decrease protein intake from medical food by 10 g/day. [00230] (c) If the subject has a protein intake from intact sources that is less than 0.5x DRI

(less than 0.4 g/kg), then the subject can increase protein intake from intact sources by 20 g/day and decrease protein intake from medical food by 20 g/day.

[00231] After the dietary adjustment, if the subject has maintained plasma Phe less than or equal to 360 pmol/L for two weeks, further adjustment can be made according to a, b and c above. If the subject has not been able to maintain plasma Phe less than or equal to 360 pmol/L following dietary adjustment, the subject should reduce protein intake by a similar adjustment as the last increase.

[00232] If plasma Phe levels are less than 30 pmol/L and confirmed upon repeat plasma Phe measurement (performed within approximately 2 weeks), the dietician can consider the following adjustments: (a) If the subject is consuming less than 2x DRI (1.6 g/kg/day), the dietitian may instruct the subject to increase their intact protein by 20 grams/day and decrease their medical food protein by 20 grams/day, (b) If the subject is consuming 2x or more DRI, the dietitian may instruct the subject to increase their intact protein by 10 grams/day and decrease their medical food protein by 10 grams/day

[00233] The methods of the disclosure may result in clinically significant lowering of plasma Phe levels (e.g. mean plasma Phe levels, or the mean of two consecutive plasma Phe levels) in the absence of concurrent pharmacotherapy. For example, the plasma Phe level of said subject is lowered to 360 pmol/L or less by 8 weeks after said administration, or 360 pmol/L or less at 24, 48, or 96 weeks, or 2, 3 or 4 years after said administration, without concurrent pharmacotherapy. For example, the plasma Phe level of said subject is between 120 and 360 pmol/L by 8 weeks after said administration, without concurrent pharmacotherapy. In some embodiments, the plasma Phe level of said subject is 120 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy, or 120 pmol/L or less at 24, 48 or 96 weeks, or 2, 3 or 4 years after said administration, without concurrent pharmacotherapy.

[00234] The methods of the disclosure may permit the subject to tolerate an increase in Phe intake from intact food sources. For example, the plasma Phe level of said subject is between 120 and 360 pmol/L after said administration, and the subject tolerates an increase in Phe intake compared to a Phe restricted diet at baseline. In some embodiments, the subject may be permitted to increase intact protein consumed and decrease medical food protein, such as described in Example 3 or Example 4.

[00235] The methods of the disclosure may reduce plasma level of a neurotransmitter or neurotransmitter metabolite of the subject after said administration. For example, the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5-hydroxyindoleacetic acid. [00236] The methods of the disclosure may result in improved quality of life of said subject improves after said administration, optionally as measured by PKU-QOL or Q-LES-Q-SF questionnaire. The methods of the disclosure may result in improved neurocognitive symptoms or parameters (measures) of the subject after said administration. Preferably, the subject does not suffer from hypophenylalaninemia after said administration.

[00237] The methods of the disclosure provide administration of rAAV particles in a manner that is safe, e.g., no clinically significant treatment-emergent serious adverse events, no continuing incidences of hypophenylalaninemia (incidence of plasma Phe less than 30 pmol/L on 2 consecutive measurements), and no clinically significant changes in standard clinical laboratory values or markers of hepatotoxicity such as AST and/or ALT (or if changes occur, most are transient or resolve after treatment with systemic immunosuppressant). The methods may also provide a reduced immune response against the AAV capsid and/or PAH transgene.

The methods may also provide improved blood biodistribution, or reduced vector shedding in urine, stool, semen, or saliva.

[00238] In an aspect of the disclosure, hepatotoxicity, e.g. as detected through transient hepatic transaminase enzyme elevations, may be reduced or avoided by prophylactic immunosuppression treatment or therapeutic immunosuppression treatment. According to these aspects, in addition to administration of a therapeutically effective amount of AAV virus, the subject may be treated either prophylactically, therapeutically, or both with a corticosteroid or other immunosuppressant to prevent and/or treat any hepatotoxicity associated with administration of the AAV virus. Prophylactic immunosuppression treatment

[00239] The methods of disclosure may further comprise administering to the subject a prophylactically effective amount of a corticosteroid to prevent hepatotoxicity, prior to detection of hepatotoxicity (e.g. as detected by ALT elevation above the upper limit of normal (ULN), or at least 2 times baseline ALT). In some embodiments, the prophylactically effective amount of immunosuppressant (e.g. corticosteroid) is administered concurrent with administration of the rAAV particles of the invention. “Concurrent” as used herein means the same day, for example, or within one day or one week of (prior to or after) administration of the rAAV particles. In other embodiments, the administration of the prophylactically effective amount of immunosuppressant (e.g. corticosteroid) begins after administration of the rAAV particles, e.g. starting at 3, 4, 5, 6, 7, 8, 9 or 10 weeks after administration of the rAAV particles, but prior to detection of hepatotoxicity.

[00240] The corticosteroid or other immunosuppressant may be administered for a prophylactic treatment time period, e.g., for a time period of at least about 3 to 13 weeks (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks), and is preferably followed by tapering period during which tapering amounts of the corticosteroid or other immunosuppressant are administered, e.g., for a time period of about 2 to 4 weeks, or about 2, 3, or 4 weeks. For example, the prophylactically effective amount of the corticosteroid is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day for a time period of at least about 3 to 13 weeks (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13), followed by tapering amounts of the corticosteroid for a time period of about 2, 3 or 4 weeks. In some embodiments, the prophylactically effective amount of the corticosteroid is administered for a time period of about 13 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks. For example, a prednisone equivalent is administered at a prednisone-equivalent dose of 40 mg/day concurrent with said administration for a time period of about 13 weeks, followed by tapering amounts of the prednisone equivalent for a time period of about 3 weeks (e.g., prednisone-equivalent dose of 30 mg/day for a week, 20 mg/day for a week, and 10 mg/day for a week).

[00241] In some embodiments, the subject is administered a 16-week prophylactic corticosteroid course of a prednisone equivalent at a starting prednisone-equivalent dose of 40 mg/day, beginning on Day 1 a few hours pre-infusion of rAAV particles, for a time period of 13 weeks dosing at 40 mg/day, followed by a 3-week dose taper beginning at Week 14 (to a prednisone-equivalent dose of 30 mg/day for a week, 20 mg/day for a week, and 10 mg/day for a week). On the day of infusion, prophylactic corticosteroids should be administered at a minimum 3 hours before rAAV particle infusion. ALT and AST levels are monitored weekly. If there is ALT elevation to greater than upper limit of normal (ULN) or greater than 2x baseline ALT value, during the first 12 weeks, adjustments to corticosteroid dosing are based on clinical judgment, and liver enzymes may be monitored more frequently.

[00242] Therapeutic immunosuppression treatment

[00243] Administration of an AAV particle of the present disclosure may, in some cases, result in an observable degree of hepatotoxicity. Hepatotoxicity may be measured by a variety of well-known and routinely used techniques for example, measuring concentrations of certain liver-associated enzyme(s) (e.g., alanine transaminase, ALT) in the bloodstream of a subject both prior to AAV administration (i.e., baseline) and after AAV administration. An observable increase in ALT concentration after AAV administration (as compared to prior to administration) is indicative of drug-induced hepatotoxicity. The methods of the disclosure may comprise administering to the subject a therapeutically effective amount of a corticosteroid or other systemic immunosuppressant to treat hepatotoxicity, upon detection of hepatotoxicity.

[00244] Reactive immunosuppressant (e.g., corticosteroid) therapy may be initiated after the prophylactic regimen is completed, or in response to mild ALT elevations that meet pre-specified criteria, or based on clinical judgment. In some embodiments, it is initiated if ALT is greater than the ULN or greater than 2x baseline in two consecutive assessments within 72 hours, or 3x ULN in two consecutive assessments within 48 hours. In some embodiments, the reactive immunosuppressive (e.g. corticosteroid) regimen has a total duration of 8 weeks with 5 weeks of 40 mg/day prednisone-equivalent dosing, followed by a 3-week dose taper if ALT is both less than or equal to ULN and less than or equal to 2x baseline value. Liver enzymes are monitored weekly over 4 weeks in the period following discontinuation of reactive immunosuppression therapy, or more frequently if ALT values are above the ULN.

[00245] The methods of disclosure may further comprise the step of (a) determining a baseline level of a marker of hepatotoxicity in the blood of the subject prior to said administration, optionally about one month prior to said administration, and (b) determining a post-administration level of said marker for hepatotoxicity in the blood of the subject after said administration, optionally every week, or every 1, 2, 3, 4, 5, or 6 days.

[00246] Such methods may further comprise the step of: (c) upon detection of hepatotoxicity by biochemical or clinical signs, administering to the subject a therapeutically effective amount of an immunosuppressant (e.g., corticosteroid) for a therapeutic treatment time period, e.g., at least about 5 to about 8 weeks (e.g., 5, 6, 7 or 8 weeks), and is preferably followed by a tapering time period during which tapering amounts of the immunosuppressant (e.g. corticosteroid) are administered for a time period of about 2 to 4 weeks (e.g. 3 weeks). For example, the step (c) comprises, upon detection of hepatotoxicity by (i) a post-administration level of said marker of hepatotoxicity greater than the upper limit of normal (ULN), or (ii) a post-administration level of said marker of hepatotoxicity greater than or equal to twice the baseline level of said marker of hepatotoxicity, administering to the subject a therapeutically effective amount of a corticosteroid for a time period of at least about 5 to about 8 weeks or longer (e.g., 5, 6, 7 or 8 weeks or longer), followed by tapering amounts of the corticosteroid for a time period of about 2, 3 or 4 weeks. In any of such embodiments, the marker of hepatotoxicity is ALT and/or AST, preferably ALT. In some embodiments, upon said detection, a prednisone equivalent is administered at a prednisone-equivalent dose of 40 mg/day for a time period of about 5 weeks, followed by tapering amounts of the prednisone equivalent for a time period of about 3 weeks. [00247] "Prophylactic" corticosteroid or systemic immunosuppressant treatment refers to the administration of a corticosteroid or immunosuppressant to prevent hepatotoxicity and/or to prevent an increase in measured ALT levels in the subject. "Therapeutic" corticosteroid or immunosuppressant treatment refers to the administration of a corticosteroid or immunosuppressant to reduce hepatotoxicity caused by administration of an AAV virus and/or to reduce an elevated ALT concentration in the bloodstream of the subject caused by administration of an AAV virus. In certain embodiments, prophylactic or therapeutic corticosteroid treatment may comprise administration of a prednisone-equivalent dose of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or more mg/day, e.g. a prednisone-equivalent dose of between about 10 mg/day and about 60 mg/day of the corticosteroid to the subject. In certain embodiments, prophylactic or therapeutic corticosteroid treatment of a subject may occur over a continuous period of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 weeks, or more, followed by a period of administering tapering amounts. Corticosteroids that find use in the methods described herein include any known or routinely-employed corticosteroid including, for example, dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, budesonide and the like, at the equivalent doses for the same time periods.

[00248] Other systemic immunosuppressants that may be administered in prophylactically effective or therapeutically effective doses to prevent or reduce hepatotoxicity include (1) calcineurin inhibitors, e.g. tacrolimus or cyclosporine, (2) antiproliferative agents or IMDH inhibitors, e.g. mycophenolate, leflunomide or azathioprine, (3) mTOR inhibitors, e.g., sirolimus or everolimus. (4) janus kinase inhibitors, e.g. tofacitinib, or (5) immunosuppressant antibodies.

5.6 DETECTION OF ANTI-AAV ANTIBODIES

[00249] To maximize the likelihood of successful liver transduction with systemic AAV- mediated therapeutic gene transfer, prior to administration of an AAV particle in a therapeutic regimen to a human patient as described above, the prospective patient may be assessed for the presence of anti-AAV capsid antibodies or anti-AAV neutralizing antibodies that are capable of blocking cell transduction or otherwise reduce the overall efficiency of the therapeutic regimen. Such antibodies may be present in the serum of the prospective patient and may be directed against an AAV capsid of any serotype. In one embodiment, the serotype against which pre existing antibodies are directed is AAV5.

[00250] Methods to detect pre-existing AAV immunity are well known and routinely employed in the art and include cell-based in vitro transduction inhibition (TI) assays, in vivo (e.g., in mice) TI assays, and ELISA-based detection of total anti-capsid antibodies (TAb) (see, e.g.,Masat et ah, Discov. Med., vol. 15, pp. 379-389 and Boutin et ah, (2010) Hum. Gene Ther., vol. 21, pp. 704-712). TI assays may employ host cells into which an AAV-inducible reporter vector has been previously introduced. The reporter vector may comprise an inducible reporter gene such as GFP, etc. whose expression is induced upon transduction of the host cell by an AAV virus. Anti-AAV capsid antibodies present in human serum that are capable of preventing/reducing host cell transduction would thereby reduce overall expression of the reporter gene in the system. Therefore, such assays may be employed to detect the presence of anti-AAV capsid antibodies in human serum that are capable of preventing/reducing cell transduction by the therapeutic AAV particle.

[00251] The assays to detect anti-AAV capsid antibodies may employ solid-phase-bound AAV capsid as a "capture agent" over which human serum is passed, thereby allowing anti capsid antibodies present in the serum to bind to the solid-phase-bound capsid "capture agent". Once washed to remove non-specific binding, a "detection agent" may be employed to detect the presence of anti-capsid antibodies bound to the capture agent. The detection agent may be an antibody, an AAV capsid, or the like, and may be detectably-labeled to aid in detection and quantitation of bound anti-capsid antibody. In one embodiment, the detection agent is labeled with ruthenium or a ruthenium-complex that may be detected using electrochemiluminescence techniques and equipment.

[00252] The same above-described methodology may be employed to assess and detect the generation of an anti-AAV capsid immune response in a patient previously treated with a therapeutic AAV virus of interest. As such, not only may these techniques be employed to assess the presence of anti-AAV capsid antibodies prior to treatment with a therapeutic AAV virus, they may also be employed to assess and measure the induction of an immune response against the administered therapeutic AAV virus after administration. As such, contemplated herein are methods that combine techniques for detecting anti-AAV capsid antibodies in human serum and administration of a therapeutic AAV virus for the treatment of Fabry Disease, wherein the techniques for detecting anti-AAV capsid antibodies in human serum may be performed either prior to or after administration of the therapeutic AAV virus.

5.7 KITS

[00253] In certain embodiments, provided herein are containers comprising a pharmaceutical formulation described herein (e.g., a formulation described in Example 4). The container may be any type that is typically used to store recombinant AAV particle formulations, such as a vial. In certain embodiments, the pharmaceutical composition is a lyophilized formulation. In other embodiments, the pharmaceutical composition is a liquid formulation. In some embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 5-25 ml (e.g., 5 ml, 10 ml, 15 ml, 20 ml, or 25 ml) of a pharmaceutical formulation described herein. In certain embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 10-50 ml (e.g., 10 ml, 20 ml, 30 ml, 40 ml or 50 ml) of a pharmaceutical formulation described herein. In some embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 50- 100 ml of a pharmaceutical formulation described herein. In certain embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 100-500 ml of a pharmaceutical formulation described herein. In some embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 500-1000 ml of a pharmaceutical formulation described herein. In certain embodiments, provided herein is a container (e.g., an infusion bag or vial) comprising 250-500 ml of a pharmaceutical formulation described herein. In certain embodiments, the concentration of the recombinant AAV particle is about 1 x 10¹³ vg/ml to about 4 x 10¹⁴ vg/ml (e.g., 2 x 10¹³ vg/ml, 6 x 10¹³ vg/ml or 2 x 10¹⁴ vg/ml). In some embodiments, the concentration of the recombinant AAV particle is sufficient to administer a dose of 2 x 10¹³ vg/ml, 6 x 10¹³ vg/ml or 2 x 10¹⁴ vg/ml to a subject (e.g., a subject 60-85 kg, such as 60 kg, 65 kg, 70 kg, 75 kg, 80 kg, or 85 kg).

[00254] In some embodiments, a container comprising a pharmaceutical formulation described herein is accompanied by instructions or a product insert, which describes, e.g., the methods of administration, dose and use of the pharmaceutical formulation. For example, the instructions or product insert may describe the rate of infusion of the pharmaceutical formulation, the criteria for administration of the pharmaceutical formulation to a subject, and/or one, two or more of things to evaluate post-infusion, such as described herein (e.g, in Example 3 or Example 4).

[00255] In certain embodiments, provided herein is a kit comprising a pharmaceutical formulation described herein in a container (e.g., an infusion bag or vial). In some embodiments, the kit further comprises instructions or a product insert, which describes, e.g., the methods of administration, dose and use of the pharmaceutical formulation. The kit may further comprise one, two or more of the following: a syringe, IV pole, IV tubing, dressing, tape, antiseptic solution.

[00256] Other aspects and advantages of the present disclosure will be understood upon consideration of the following illustrative examples. 6. EXAMPLES

6.1 EXAMPLE 1: ADMINISTRATION OF AAV PARTICLES TO NON-HUMAN PRIMATES

[00257] Cynomolgus monkeys were administered vehicle or rAAV particles comprising a recombinant vector construct described herein and an AAV type capsid, at doses up to 4E14 vector genomes per kg body weight (vg/kg). Measures of safety were monitored, including weekly physicals, body weight measurements, monitoring for anti-AAV5 antibody response, anti-PAH antibody response, and liver enzyme levels such as ALT and AST. The primates were monitored for adverse clinical signs, and all major organs assessed for pathology.

6.2 EXAMPLE 2: PHARMACEUTICAL FORMULATION

[00258] The rAAV particles comprising an AAV5 type capsid and a recombinant vector construct described herein (one of SEQ ID NOS: 15-23 or 52) are provided in a liquid formulation suitable as a physiologically compatible IV solution for intravenous administration, that is stable for long periods of time, e.g. 1 or 2 years, while frozen at < 60°C (at about minus 60°C or less). The liquid formulation is also stable for a time period of, e.g., at least 6 or 12 months under appropriate accelerated storage conditions.

[00259] The deamidation level of VPl protein at its N-terminus were quantified by Liquid Chromatography-Mass Spectrometry (LC-MS). The assay accurately measures percent deamidation at the N-terminal region of AAV5 Viral Protein 1 (VPl), specifically atN50 and N56. Capsid particles in formulated bulk drug substance or drug product were denatured to dissociate viral proteins and digested to peptides prior to LC-MS analysis. The percent deamidation was calculated by measuring the intensity of deamidated peptide peak area relative to the sum of unmodified and deamidated peptide peak areas.

[00260] Accelerated and stressed stability studies evaluated the impact of buffer pH on deamidation levels of rAAV5 particles at pH ranging from 7 to 8 (at pH 7.1, 7.5 and 7.9).

Samples were stored at 4°C for 30 days or at room temperature (RT) for 15 days. Results shown in Figures 1 A and IB indicated elevated deamidation levels at increasing pH and greater storage temperature. Although there is greater thermal stability at acidic pH compared to basic pH conditions, a formulation that produces a final formulation near physiological pH (pH 7.4) is preferable. For a pharmaceutical formulation administered without diluent, the preferred pH of the pharmaceutical formulation was selected to be pH 7.2.

[00261] Sodium phosphate buffer was selected to maintain the target pH (7.2) of the solution. A 10 mM sodium phosphate concentration was demonstrated to be sufficient to maintain the pH at long-term and accelerated stability testing conditions. The pH stability of the formulation was evaluated under three different storage conditions: long term (<-60°C), accelerated (2-8°C), and stressed (25°C/60% RH). For all tested conditions, there were no significant changes of pH over time.

[00262] Sodium chloride within certain concentration ranges maintains capsid colloidal stability and solution clarity. In the absence of NaCl, the rAAV particles may precipitate out of solution. An aqueous solution containing at least 50 mM NaCl is necessary to reduce the overall haziness of the rAAV particle solution and maintain solubility of the rAAV particles. Increasing NaCl concentrations from 50 to 100 mM improved stability, while NaCl concentrations from 100 mM to 165 mM showed comparable results. A concentration of 120 mM NaCl within that range was selected to maintain the stabilizing effect while maintaining an isotonic solution. Thermal stability as a function of NaCl concentration was investigated and analytical results are provided in Table 1. The increased in the onset temperature corresponding to increase in NaCl concentration further affirms the sodium chloride has significant impact of AAV stability.

[00263] Table 1 Thermal Stability Screening of AAV as function of varying NaCl concentration

[00264] Various bulking agents that are cryopreservative or cryoprotectant agents, were tested for their ability to maintain stability of the liquid formulation under freezing temperature conditions. Comparison of sugars such as trehalose with polyols such as mannitol showed that trehalose was superior at maintaining stability. 74 mM trehalose dihydrate was determined to be the minimal amount of trehalose cryoprotectant in the presence of 120 mM NaCl. The resulting solution Tg' of the final formulation was approximately -52°C and will minimize the effects of minor temperature excursions that can occur at long-term storage conditions. The concentration of 74 mM trehalose (2.8% trehalose) was selected to achieve the stabilizing effect while maintaining an isotonic solution. A comparative study of rAAV particles of the invention formulated in either 2% mannitol or 2.8% trehalose was conducted under various storage conditions: <-60°C, 2-8°C , 25°C/60% RH and 37°C.

[00265] The capsid protein aggregates were monitored by SEC-HPLC (Size Exclusion Chromatography High Performance Liquid Chromatography). The capsid protein particles were monitored by UV at 280 nm and elute according to size in order of trimer, dimer and monomer. Excipients and salts in the sample buffer such as poloxamer, elute after the monomer peak. Aggregate content is reported as % mul timer where % mul timer is the sum of the dimer and trimer peak area over the total peak area (of monomer, dimer and trimer). A reference was run with every assay to confirm assay performance.

[00266] Results are depicted in Figures 2A-2D, respectively, as the percent change in the aggregation levels from the initial time point. The stability profiles for both formulations was comparable at <-60°C and 2-8°C (Figures 2A and 2B), with no significant change in the aggregate levels for the study duration. However, under elevated storage conditions of 25°C/60% RH and 37°C, AAV shows superior stability when formulated using trehalose instead of mannitol (Figures 2C and 2D).

[00267] Surfactants reduce adsorption of rAAV particles to contact surfaces, and thus reduce precipitation and increase formulation stability. While 0.2% w/v poloxamer, e.g. poloxamer 188, was previously determined to be desirable for other rAAV particle formulations, the rAAV particles of the invention containing nucleic acid encoding functional PAH have been shown to be stable when lesser amounts are used. Varying levels of poloxamer concentrations (0, 0.05%, 0.1% and 0.2% (w/v) were analyzed at a concentration of using 8E13 vg/mL rAAV5 particles. The adsorptive properties of the poloxamer were observed to mitigate adsorptive losses. As little as 0.05% poloxamer shows retention of the monomer and prevents the loss detected with absence of poloxamer. A concentration of poloxamer 188 at 0.1% w/v was suitable to maintain the stability of the liquid formulation under all tested conditions.

[00268] The formulation was able to maintain stability of a relatively high AAV particle concentration of 6E13 vg/mL (6 x 10¹³ vector genomes per mL). The final aqueous formulation of rAAV particles at a concentration of 6E13 vg/mL, with 10 mM sodium phosphate (2.47 mM or -0.4 mg/ml sodium dihydrogen phosphate, monobasic, dihydrate and 7.53 mM or 2.7 mg/ml sodium phosphate, dibasic, dodecahydrate), 120 mM or -7 mg/ml sodium chloride, 74 mM or 28 mg/ml trehalose dihydrate, and 0.1% w/v or 1 mg/ml poloxamer 188, resulted in a final solution that was clear to slightly opalescent, colorless to pale yellow, and essentially free of particles while maintaining overall product stability at the intended use and storage conditions. Testing showed that the formulation is expected to be stable for up to 2 years at about -60°C (minus 60) or less. The average osmolality of the drug product is 325 mOsm/kg, which is slightly greater than the approximately 290 mOsm/L value of human serum but considerably lower than 450 mOsm/L, which is stated to carry the lowest risk of phlebitis when administered through a peripheral vein. The slightly hypertonic nature of the product is not a concern.

[00269] The 6E13 vg/mL concentration of rAAV particles enables clinical dosing to occur with a reasonable volume of liquid. For example, a dose of 2E13 vg/kg (2 x 10¹³ vector genomes per kg body weight of subject) may be administered to a 70 kg patient with 23.3 mL of liquid, while a dose of 6E13 vg/kg (6 x 10¹³ vector genomes per kg body weight of subject) may be administered to a 70 kg patient with 70 mL of liquid.

6.3 EXAMPLE 3: ADMINISTRATION OF AAV PARTICLES TO HUMAN SUBJECTS

[00270] Human subjects are administered rAAV particles comprising an AAV5 type capsid and a recombinant vector construct described herein (one of SEQ ID NOS: 15-23 or 52), at doses of 2E13, 6E13, or 2E14 vector genomes per kg body weight (vg/kg), to assess the efficacy, safety and tolerability of the rAAV particles. Additional dose levels, not exceeding 2E14 vg/kg, may be studied.

[00271] The objective is to demonstrate a clinically meaningful reduction in plasma Phe in subjects with PKU after a single intravenous administration of the rAAV articles. Subjects with baseline mean plasma Phe level greater than 600 pmol/L are administered the rAAV particles at the desired dose, in a single intravenous infusion, and are followed for 5 years to evaluate durability of the response. A proportion of subjects in at least one dose cohort will achieve a clinically significant reduction in plasma Phe (for example, the subjects may achieve plasma Phe less than or equal to 360 pmol/L, or even normalization of Phe at less than or equal to 120 pmol/L) by Week 8, 24 or 48 post-infusion. A durable response will last at least 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years or 5 years or longer.

[00272] Additional subjects with more severe PKU that have baseline mean plasma Phe level of greater than 1200 pmol/L are administered the rAAV particles and will achieve a clinically significant reduction in plasma Phe (for example, the subjects may achieve plasma Phe less than or equal to 600 pmol/L, or less than or equal to 360 pmol/L, or less than or equal to 120 pmol/L) by Week 8, 24 or 48 post-infusion. The neurotoxicity of elevated Phe is a direct effect of excess Phe. Metabolic control of Phe levels has been shown to be correlated with higher executive functioning and better cognitive performance.

[00273] Further, a proportion of subjects in at least one dose cohort will exhibit an improvement in measures of inattention and/or executive function. A proportion of subjects in at least one dose cohort will achieve an improvement in health-related quality of life. For example, the improvements may be achieved by Week 24, Week 32, Week 48, Week 96, or later. [00274] In addition, a proportion of subjects in at least one dose cohort will achieve an increase in dietary protein intake from intact food (and a concomitant decrease in dietary protein intake from medical food) after administration of the rAAV particle, at Week 48 or later after administration of the rAAV particle. Improvements may be seen earlier, e.g. at Week 24 or Week 32, or later, e.g. Week 96 or later. A proportion of subjects in at least one dose cohort will be able to consume at least 0.8 g/kg protein intake from intact food at Week 48 or later after administration of the rAAV particle, while maintaining mean plasma Phe at less than or equal to 360 pmol/L. A proportion of subjects in at least one dose cohort will be able to discontinue medical food after administration of the rAAV particle.

[00275] Moreover, the administration of the rAAV particles will be demonstrated to be safe for most patients, e.g., low incidence of treatment-emergent serious adverse events, low incidence of plasma hypophenylalaninemia (Phe levels less than 30 pmol/L), and either low incidence of hepatic transaminase elevation or transient elevations that resolve after corticosteroid therapy.

Inclusion and exclusion criteria

[00276] The clinical study inclusion criteria include the following: (1) age 15 years or older, or 18 years or older; (2) diagnosis of phenylketonuria (PKU) and an average of two plasma Phe levels greater than 600 pmol/L prior to the administration of rAAV particles; (3) not currently receiving pharmacotherapy to treat PKU (e.g., last dose of pegvaliase or large neutral amino acids (LNAAs) at least 30 days prior to the rAAV particle administration, or last dose of sapropterin at least 7 days prior to the rAAV particle administration. Additional criteria include willingness to abstain from alcohol, herbal and natural remedies, dietary supplements, and hepatotoxic medications from screening through at least 52 weeks post-administration of rAAV particle.

[00277] The clinical study exclusion criteria include the following: (1) evidence of an active infection or immunosuppressive disorder; (2) history of cancer; (3) substance use disorder, major depressive disorder, psychosis, or bipolar disorder; (4) contraindication to corticosteroids; (5) detectable antibodies to AAV5 capsid (i.e. seropositivity); (6) prior liver biopsy showing significant fibrosis of 3 or 4 as rated on a scale of 0-4; (7) clinically significant liver disease including an elevation in any of ALT (alanine transaminase), AST (aspartate aminotransferase), GGT (gamma-glutamyltransferase) or bilirubin to more than 1.25 times the upper limit of normal (ULN), or the international normalized ratio being equal to or greater than 1.2; (8) prior infection with hepatitis B or C, or tuberculosis; (9) serum creatinine greater than or equal to 1.5 mg/dL. Monitoring of safety and efficacy

[00278] Prior to infusion of the rAAV particles, subjects are evaluated for: (1) baseline physical examination; (2) baseline clinical laboratory tests, including (a) plasma Phe levels, (b) plasma tyrosine (Tyr) levels, and (c) liver enzyme tests, including ALT, AST, GGT and bilirubin; (d) and baseline AAV5 antibody detection; (3) baseline protein intake from intact food and from medical food; (4) measures of inattention and/or executive function, e.g., Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS IV) which is an investigator-rated inattention score), Cambridge Neuropsychological Test Automated Battery (CANTAB) scores (including Rapid Visual Processing, Stop Signal and Spatial Working Memory); (5) measures of health-related quality of life (HRQoL), e.g. Phenylketonuria Impact and Treatment Quality of Life Questionnaire (PKU-QOL) score or Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q-SF) score; (6) baseline levels of other parameters monitored during the study; and (7) PAH genotyping, if permitted.

[00279] After infusion of the rAAV particles, parameters that are monitored through Week 24, 48, 96 and longer include: (1) weekly plasma Phe levels, detecting a change from baseline in mean plasma Phe levels at Week 8, 12 and 24 post-infusion; (2) weekly neurotransmitter or neurotransmitter metabolite levels, e.g. plasma Tyr levels, Phe/Tyr ratio; (3) after Week 24, ability to tolerate an increase in dietary protein intake and/or a reduction in medical food intake (Phe-reduced or Phe-free foods), for example, a proportion of subjects can consume at least 0.8 g/kg protein intake from intact food at Week 48 post-infusion while maintaining plasma Phe at less than or equal to 360 pmol/L, and/or consume no medical food at Week 48 post-infusion; (4) symptoms of inattention and measures of executive function, e.g. as measured by ADHD-RS IV (investigator-rated inattention score), CANTAB scores (Rapid Visual Processing, Stop Signal and Spatial Working Memory), (5) health-related quality of life (HRQoL), e.g. as measured by PKU-QOL score or Q-LES-Q-SF score. A clinically significant improvement in any of these parameters is observed. [00280] Additional parameters include: neurotransmitter metabolite levels (e.g., phenylacetylglutamine [PAG], homovanillic acid [HVA], 3-methoxy-4-hydroxyphenyl glycol [MOPEG], and 5-hydroxyindoleacetic acid [5HIAA]); rate of Phe oxidation; nutritional markers; fasting lipid panel. A clinically significant improvement in any of these parameters is observed. [00281] PAH catalyzes the conversion of Phe to tyrosine, so untreated PKU results in lower than normal tyrosine concentrations in the blood. Several nutritional markers have been shown to be deficient in PKU patients and include: 25-hydroxy (OH) Vitamin D, methylmalonic acid (indicator of B-12 deficiency), serum ferritin (indicator of iron deficiency), selenium, and zinc. [00282] The rate of Phe oxidation, as measured using the Phe breath test, is used as a measure of PAH activity. The fasting Phe breath test allows for the quantitative evaluation of Phe metabolism by the appearance of ¹³C02 in the breath following oral administration of a phenylalanine isotope (non-radioactive tracer, L-[l-¹³C]-phenylalanine). The cumulative recovery of ¹³CC>2 over the 120-minute period is an endpoint to assess change in rate of Phe oxidation from baseline over the course of the study.

[00283] The administration of the rAAV particles is safe, e.g., no clinically significant treatment-emergent serious adverse events, no continuing incidences of hypophenylalaninemia (incidence of plasma Phe less than 30 pmol/L on 2 consecutive measurements), and no clinically significant changes in standard clinical laboratory values or markers of hepatotoxicity such as AST and/or ALT (or if changes occur, most are transient or resolve after treatment with systemic immunosuppressant). Immune response against the AAV capsid and PAH transgene is monitored, as is blood biodistribution, and urine, stool, semen, and saliva vector shedding. Change in Phe-restricted diet

[00284] Before Week 24 post-infusion of the rAAV particles, subjects are instructed to maintain consistent dietary intake, e.g., dietary protein intake from intact foods changes less than 25% from baseline and the medical food protein intake changes less than 25% from baseline. After Week 24 post-infusion, subjects who achieve plasma Phe less than or equal to 360 pmol/L on two consecutive assessments at least one week apart are allowed to increase intact protein intake as follows: [00285] (a) If the subject already has protein intake from intact sources that is greater than 2x the dietary reference intake DRI (greater than 1.6 g/kg), then the subject can maintain protein intake from intact sources and discontinue protein intake from medical food.

[00286] (b) If the subject has a protein intake from intact sources that is 0.5x to 2x DRI (0.4-

1.6 g/kg), then the subject can increase protein intake from intact sources by 10 g/day, and decrease protein intake from medical food by 10 g/day.

[00287] (c) If the subject has a protein intake from intact sources that is less than 0.5x DRI

[00288] After the dietary adjustment, if the subject has maintained plasma Phe less than or equal to 360 pmol/L for two weeks, further adjustment can be made according to a, b and c above. If the subject has not been able to maintain plasma Phe less than or equal to 360 pmol/L following dietary adjustment, the subject should reduce protein intake by a similar adjustment as the last increase.

[00289] If plasma Phe levels are less than 30 pmol/L and confirmed upon repeat plasma Phe measurement (performed within approximately 2 weeks), the dietician can consider the following adjustments: (a) If the subject is consuming less than 2x DRI (1.6 g/kg/day), the dietitian may instruct the subject to increase their intact protein by 20 grams/day and decrease their medical food protein by 20 grams/day, (b) If the subject is consuming 2x or more DRI, the dietitian may instruct the subject to increase their intact protein by 10 grams/day and decrease their medical food protein by 10 grams/day.

[00290] If dietary adjustments are insufficient or not feasible to achieve target plasma Phe levels, pharmacotherapy with oral therapies may be considered following Week 24 assessments, and pharmacotherapy with injectables may be considered following Week 48 assessments, per standard of care.

[00291] Prophylactic corticosteroid therapy

[00292] Transient hepatic transaminase enzyme elevations may be reduced or avoided by prophylactic corticosteroid therapy. A 16-week prophylactic corticosteroid course is administered with a prednisone-equivalent starting dose of 40 mg/day, beginning on Day 1 pre infusion, for a time period of 13 weeks dosing at 40 mg/day, followed by a 3-week dose taper beginning at Week 14 (to a prednisone-equivalent dose of 30 mg/day for a week, 20 mg/day for a week, and 10 mg/day for a week). On the day of infusion, prophylactic corticosteroids should be administered at a minimum 3 hours before rAAV particle infusion. ALT and AST levels are monitored weekly. If there is ALT elevation to greater than upper limit of normal (ULN) or greater than 2x baseline ALT value, during the first 12 weeks, adjustments to corticosteroid dosing are based on clinical judgment, and liver enzymes may be monitored more frequently. [00293] Reactive corticosteroid therapy for transient hepatic enzyme elevations

[00294] Reactive corticosteroid therapy may be initiated after the prophylactic regimen is completed, in response to mild ALT elevations that meet pre-specified criteria, or based on clinical judgment. It may be initiated if ALT is greater than the ULN or greater than 2x baseline in two consecutive assessments within 72 hours, or 3x ULN in two consecutive assessments within 48 hours. The recommended reactive CS regimen has a total duration of 8 weeks with 5 weeks of 40 mg/day prednisone-equivalent dosing, followed by a 3-week dose taper if ALT is both less than or equal to ULN and less than or equal to 2x baseline value. Liver enzymes are monitored weekly over 4 weeks in the period following discontinuation of reactive corticosteroid therapy, or more frequently if ALT values are above the ULN.

[00295] Reactive corticosteroids are not administered if elevations in ALT are clearly not related to the therapeutic intervention with rAAV particles (e.g., elevated ALT with concurrent increase in creatine phosphokinase (CPK) due to intensive exercise, or viral hepatitis).

6.4 EXAMPLE 4: ADMINISTRATION OF AAV PARTICLES TO HUMAN SUBJECTS

[00296] Human subjects are administered rAAV particles comprising an AAV5 type capsid and a recombinant vector construct described herein (one of SEQ ID NOS: 15-23 or 52), at doses of 2E13, 6E13, or < 2E14 vector genomes per kg body weight (vg/kg), to assess the efficacy, safety and tolerability of the rAAV particles. Additional dose levels, not exceeding 2E14 vg/kg, may be studied.

[00297] The objective is to demonstrate a clinically meaningful reduction in plasma Phe in subjects with PKU after a single intravenous administration of the rAAV articles. Subjects with baseline mean plasma Phe level greater than 600 pmol/L are administered the rAAV particles at the desired dose, in a single intravenous infusion, and are followed for 5 years to evaluate durability of the response. A proportion of subjects in at least one dose cohort will achieve a clinically significant reduction in plasma Phe (for example, the subjects may achieve plasma Phe less than or equal to 360 pmol/L, or even normalization of Phe at less than or equal to 120 pmol/L) by Week 8, 24 or 48 post-infusion. A durable response will last at least 6 months, 1 year, 1.5 years, 2 years, 3 years, 4 years or 5 years or longer.

[00298] Additional subjects with more severe PKU that have baseline mean plasma Phe level of greater than 1200 pmol/L are administered the rAAV particles and will achieve a clinically significant reduction in plasma Phe (for example, the subjects may achieve plasma Phe less than or equal to 600 pmol/L, or less than or equal to 360 pmol/L, or less than or equal to 120 pmol/L) by Week 8, 24 or 48 post-infusion. The neurotoxicity of elevated Phe is a direct effect of excess Phe. Metabolic control of Phe levels has been shown to be correlated with higher executive functioning and better cognitive performance.

[00299] Further, a proportion of subjects in at least one dose cohort will exhibit an improvement in measures of inattention and/or executive function. For example, a proportion of subjects in at least one dose cohort will see an improvement from baseline post-infusion as assessed by ADHD-RS IV and/or CANTAB (including Rapid Visual Processing, Stop Signal, and Spatial Memory). A proportion of subjects in at least one dose cohort will achieve an improvement in health-related quality of life. For example, a proportion of subjects in at leaset one dose cohort will achieve an improvement from baseline post-infusion as assessed by PKU- QOL score and/or Q-LES-Q-SF score. Such improvements may be achieved by Week 24, Week 32, Week 48, Week 96, or later.

[00300] In addition, a proportion of subjects in at least one dose cohort will achieve an increase in dietary protein intake from intact food (and a concomitant decrease in dietary protein intake from medical food) after administration of the rAAV particle, at Week 48 or later after administration of the rAAV particle. Improvements may be seen earlier, e.g. at Week 24 or Week 32, or later, e.g. Week 96 or later. A proportion of subjects in at least one dose cohort will be able to consume at least 0.8 g/kg protein intake from intact food at Week 48 or later after administration of the rAAV particle, while maintaining mean plasma Phe at less than or equal to 360 mihoI/L. A proportion of subjects in at least one dose cohort will be able to discontinue medical food after administration of the rAAV particle.

[00301] Moreover, the administration of the rAAV particles will be demonstrated to be safe for most patients, e.g., low incidence of treatment-emergent serious adverse events, low incidence of plasma hypophenylalaninemia (Phe levels less than 30 pmol/L), and either low incidence of hepatic transaminase elevation or transient elevations that resolve after corticosteroid (CS) therapy.

[00302] Inclusion and exclusion cri/eriaJhe clinical study inclusion criteria include the following: (1) age 15 years or older, or 18 years or older; (2) diagnosis of phenylketonuria (PKU), which is a condition characterized by PAH deficiency, and an average of two plasma Phe levels greater than 600 pmol/L prior to the administration of rAAV particles; (3) never received pharmacotherapy to treat PKU or if previously on pharmacotherapy to treat PKU, pharmacotherapy must have been discontinued due to lack of tolerability or inability to achieve target efficacy; subject should not discontinue effective treatment to enroll in the clinical study; subjects previously on pharmacotherapy must have discontinued prior to rAAV particles administration (e.g., last dose of pegvaliase or large neutral amino acids (LNAAs) at least 30 days prior to the rAAV particle administration, or last dose of sapropterin at least 7 days prior to the rAAV particle administration). Additional criteria include (i)willingness to abstain from alcohol, herbal and natural remedies, dietary supplements, and hepatotoxic medications prior to rAAV particle administration through at least 52 weeks post-administration of rAAV particle;

(ii) willingness to avoid semen or blood donation until 3 consecutive semen or blood samples are below the limit for vector detection; (iii) willingness to avoid oocyte donation; (iv) willingness to abstain from organ donation and tissue donation; (v) willingness of males to use an method of effective of contraception for at least 12 weeks post-rAAV particle administration; (vi) negative serum pregnancy at day -28 and -7 for females; (vii) willingness of females to use an method of effective of contraception throughout the study; and (viii) up to date vaccination.

[00303] The clinical study exclusion criteria include the following: (1) subjects with primary BH4 deficiency or other forms of BH4 metabolism deficiency; (2) evidence of an active infection (including SARS-CoV-2) or immunosuppressive disorder, including HIV; (3) history of malignancy within 5 years of rAAV particle administration, or any hepatic malignancy; (4) substance use disorder or major depressive disorder 1 year prior to rAAV particle administration; (5) any history of psychosis or bipolar disorder; (6) contraindication to corticosteroids or a history of a condition that would worsen with corticosteroid therapy; (7) detectable antibodies to AAV5 capsid (i.e. seropositivity) prior to rAAV particle administration; (8) grade 3 or 4 fibrosis as assessed by either Fibroscan or Enhanced Liver Fibrosis (ELF) test score greater than 9.8 prior to rAAV particle administration; (9) prior liver biopsy showing significant fibrosis of 3 or 4 as rated on a scale of 0-4 on the Batts-Ludwig (Batts et al., The American journal of surgical pathology. DEC 1995; 19(12): 1409-17) or METAVIR (Bedossa and Poynard, Hepatology. 1996 Aug;24(2):289-93), or an equivalent grade of fibrosis if an alternative scale is used; (10) prior treatment with gene therapy; . (10) clinically significant liver disease as assessed by ultrasound prior to rAAV particle administration; (11) significant liver dysfunction prior to rAAV particle administration as defined by an elevation in any of ALT (alanine transaminase), AST (aspartate aminotransferase), GGT (gamma-glutamyltransferase) or bilirubin to more than 1.25 times the upper limit of normal (ULN), or the international normalized ratio being equal to or greater than 1.2; (11) prior infection with hepatitis B or C as assessed by, e.g., a serological assay or PCR;

(12) active tuberculosis or untreated latent tuberculosis infection as determined by positive QuantiFERON prior to rAAV particle adminsitration; (13) use of systemic agents, including corticosteroids within 30 days prior to rAAV particle administration; (14) use of systemic immunosuppressive agents within 30 days prior to rAAV particle administration; (15) immunization with live or live-attenuated vaccines within 30 days prior to rAAV particle administration, or other vaccines within 14 days prior to rAAV particle administration; (16) diagnosis of glaucoma prior to rAAV particle administration;; (17) serum creatinine greater than or equal to 1.5 mg/dL prior to rAAV particle administration; (18) hemoglobin A1C>8.0% or glucose >250 mg/dL; and (19) significant thrombocytopenia (e.g., platelet count <100 x 10⁹ /L) prior to rAAV particle adminsitration.

[00304] Monitoring of safety and efficacy

[00305] Prior to infusion of the rAAV particles, subjects are evaluated for: (1) baseline physical examination; (2) baseline clinical laboratory tests, including (a) plasma Phe levels, (b) plasma tyrosine (Tyr) levels, and (c) liver enzyme tests, including ALT, AST, GGT and bilirubin; (d) and baseline AAV5 antibody detection; (3) baseline protein intake from intact food and from medical food; (4) measures of inattention and/or executive function, e.g., Attention Deficit Hyperactivity Disorder Rating Scale (ADHD-RS IV) which is an investigator-rated inattention score), Cambridge Neuropsychological Test Automated Battery (CANTAB) scores (including Rapid Visual Processing, Stop Signal and Spatial Working Memory); (5) measures of health-related quality of life (HRQoL), e.g. Phenylketonuria Impact and Treatment Quality of Life Questionnaire (PKU-QOL) score or Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q-SF) score; (6) baseline levels of other parameters monitored during the study; and (7) PAH genotyping, if permitted.

[00306] Immunogenicity of rAAV particle is monitored routinely in the clinic and will include assessment of anti-AAV5 capsid and anti -PAH total binding antibodies as well as PAH- and capsid-specific cellular immunity. Immunogenicity is also monitored in the event of infusion related reactions occurring any time after administration of rAAV particle or in case of ALT elevations above a certain threshold (ALT > 3x upper limit of normal).

[00307] After infusion of the rAAV particles, parameters that are monitored through Week 24, 48, 96 and longer include: (1) weekly plasma Phe levels, detecting a change from baseline in mean plasma Phe levels at Week 8, 12 and 24 post-infusion; (2) weekly neurotransmitter or neurotransmitter metabolite levels, e.g. plasma Tyr levels, Phe/Tyr ratio; (3) after Week 24, ability to tolerate an increase in dietary protein intake and/or a reduction in medical food intake (Phe-reduced or Phe-free foods), for example, a proportion of subjects can consume at least 0.8 g/kg protein intake from intact food at Week 48 post-infusion while maintaining plasma Phe at less than or equal to 360 pmol/L, and/or consume no medical food at Week 48 post-infusion; (4) symptoms of inattention and measures of executive function, e.g. as measured by ADHD-RS IV (investigator-rated inattention score), CANTAB scores (Rapid Visual Processing, Stop Signal and Spatial Working Memory), (5) health-related quality of life (HRQoL), e.g. as measured by PKU-QOL score or Q-LES-Q-SF score. A clinically significant improvement in any of these parameters is observed.

[00308] Additional parameters include: neurotransmitter metabolite levels (e.g., phenylacetylglutamine [PAG], homovanillic acid [HVA], 3-methoxy-4-hydroxyphenyl glycol [MOPEG], and 5-hydroxyindoleacetic acid [5HIAA]); rate of Phe oxidation; nutritional markers; fasting lipid panel. A clinically significant improvement in any of these parameters is observed. Optional post-treatment liver biopsy(s) will be obtained starting at Week 20.

[00309] PAH catalyzes the conversion of Phe to tyrosine, so untreated PKU results in lower than normal tyrosine concentrations in the blood. Several nutritional markers have been shown to be deficient in PKU patients and include: 25-hydroxy (OH) Vitamin D, methylmalonic acid (indicator of B-12 deficiency), serum ferritin (indicator of iron deficiency), selenium, and zinc. [00310] The rate of Phe oxidation, as measured using the Phe breath test, is used as a measure of PAH activity. The fasting Phe breath test allows for the quantitative evaluation of Phe metabolism by the appearance of ¹³C02 in the breath following oral administration of a phenylalanine isotope (non-radioactive tracer, L-[l-¹³C]-phenylalanine). The cumulative recovery of ¹³CC>2 over the 120-minute period is an endpoint to assess change in rate of Phe oxidation from baseline over the course of the study. Subjects fast for at least 2.5 hours prior to any plasma Phe measurement, subjects fast for at least 4 hours prior to liver ultrasound and Fibroscan, and subjects fast at least 8 hours for any ¹³C-phenylalanine breath test (PBT).

[00311] The administration of the rAAV particles is safe, e.g., no clinically significant treatment-emergent serious adverse events, no continuing incidences of hypophenylalaninemia (incidence of plasma Phe less than 30 pmol/L on 2 consecutive measurements), and no clinically significant changes in standard clinical laboratory values or markers of hepatotoxicity such as AST and/or ALT (or if changes occur, most are transient or resolve after treatment with systemic immunosuppressant). Immune response against the AAV capsid and PAH transgene is monitored, as is blood biodistribution, and urine, stool, semen, and saliva vector shedding. [00312] Change in Phe-restricted diet

[00313] Before Week 24 post-infusion of the rAAV particles, subjects are instructed to maintain consistent dietary intake, e.g., dietary protein intake from intact foods changes less than 25% from baseline and the medical food protein intake changes less than 25% from baseline, unless: (1) plasma Phe levels are levels are < 30 pmol/L at any time on the study; (2) plasma Phe < 360 pmol/L on the two most recent consecutive assessments prior to week 24; or (3) as instructed by the study investigator or dietician to support adequate nutrition. After Week 24 post-infusion, subjects who achieve plasma Phe less than or equal to 360 m mol/L on two consecutive assessments at least one week apart are allowed to increase intact protein intake as follows:

(a) If the subject already has protein intake from intact sources that is greater than 2x the dietary reference intake DRI (greater than 1.6 g/kg), then the subject can maintain protein intake from intact sources and discontinue protein intake from medical food.

(b) If the subject has a protein intake from intact sources that is 0.5x to 2x DRI (0.4- 1.6 g/kg), then the subject can increase protein intake from intact sources by 10 g/day, and decrease protein intake from medical food by 10 g/day.

(c) If the subject has a protein intake from intact sources that is less than 0.5x DRI (less than 0.4 g/kg), then the subject can increase protein intake from intact sources by 20 g/day and decrease protein intake from medical food by 20 g/day.

[00314] After the dietary adjustment, if the subject has maintained plasma Phe less than or equal to 360 pmol/L for two weeks, further adjustment can be made according to a, b and c above. If the subject has not been able to maintain plasma Phe less than or equal to 360 pmol/L following dietary adjustment, the subject should reduce protein intake by a similar adjustment as the last increase.

[00315] If plasma Phe levels are less than 30 pmol/L and confirmed upon repeat plasma Phe measurement (performed within approximately 2 weeks), modifications to the subject’s diet should be made. If the subject is consuming less than 2x DRI (1.6 g/kg/day), the dietitian may instruct the subject to increase their intact protein by 20 grams/day and decrease their medical food protein by 20 grams/day, (b) If the subject is consuming 2x or more DRI, the dietitian may instruct the subject to increase their intact protein by 10 grams/day and decrease their medical food protein by 10 grams/day. Further increases in protein intake as per above are allowed if plasma Phe levels are < 30 pmol/L in order to achieve 2 x DRI guidelines (1.6 g/kg/day) of intact protein intake.

[00316] If dietary adjustments are insufficient or not feasible to achieve target plasma Phe levels, pharmacotherapy with oral therapies may be considered following Week 24 assessments, and pharmacotherapy with injectables may be considered following Week 48 assessments, per standard of care.

[00317] Prophylactic corticosteroid therapy [00318] Transient hepatic transaminase enzyme elevations may be reduced or avoided by prophylactic corticosteroid therapy. A 16-week prophylactic corticosteroid course is administered with a prednisone-equivalent starting dose of 40 mg/day, beginning on Day 1 pre infusion, for a time period of 13 weeks dosing at 40 mg/day, followed by a 3-week dose taper beginning at Week 14 (to a prednisone-equivalent dose of 30 mg/day for a week, 20 mg/day for a week, and 10 mg/day for a week). On the day of infusion, prophylactic corticosteroids should be administered at a minimum 3 hours before rAAV particle infusion. ALT and AST levels are monitored weekly. If there is ALT elevation to greater than upper limit of normal (ULN) or greater than 2x baseline ALT value, during the first 12 weeks, adjustments to corticosteroid dosing are based on clinical judgment, and liver enzymes may be monitored more frequently. [00319] After completion of prophylactic corticosteroid course, subjects undergo review of emergent adverse effects, brief physical examination (for in clinic visits), vital signs and clinical laboratory panel biweekly for 4 weeks and every 4 weeks thereafter for a total of 24 weeks. After this period the above-mentioned assessments are performed during at home visits are performed by a home healthcare professional. Subjects are checked for adverse effects related to corticosteroid- and hypothalamic-pituitary-adrenal (HPA)-axis suppression during corticosteroid treatment period and post-corticosteroid period respectively, for prompt identification of emergent related adverse effects.

[00320] Reactive corticosteroid therapy for transient hepatic enzyme elevations [00321] Reactive corticosteroid therapy may be initiated after the prophylactic regimen is completed, in response to mild ALT elevations that meet pre-specified criteria, or based on clinical judgment. It may be initiated if ALT is greater than the ULN or greater than 2x baseline in two consecutive assessments within 72 hours, or 3x ULN in two consecutive assessments within 48 hours. The recommended reactive CS regimen has a total duration of 8 weeks with 5 weeks of 40 mg/day prednisone-equivalent dosing, followed by a 3-week dose taper if ALT is both less than or equal to ULN and less than or equal to 2x baseline value. Liver enzymes are monitored weekly over 4 weeks in the period following discontinuation of reactive corticosteroid therapy, or more frequently if ALT values are above the ULN. [00322] Reactive corticosteroids are not administered if elevations in ALT are clearly not related to the therapeutic intervention with rAAV particles (e.g., elevated ALT with concurrent increase in creatine phosphokinase (CPK) due to intensive exercise, or viral hepatitis).

[00323] During reactive CS treatment, subjects undergo review of emergent adverse effects, brief physical examination (for in clinic visits), vital signs and clinical laboratory test panel every 4 weeks, either at in clinic visit or via home healthcare professional visits (at scheduled or unscheduled visits). Subjects are checked for adverse effects related to CS treatment, for evaluation of emergent adverse effects.

[00324] After completion of reactive CS, subjects undergo review of emergent adverse effects, brief physical examination (for in clinic visits), vital signs and clinical laboratory panel biweekly for 4 weeks and every 4 weeks thereafter for a total of 24 weeks. After this period, assessments are performed as scheduled. Assessments occurring during at home visits will be performed by a home healthcare professional. Subjects will be checked for adverse effects related to HPA-axis suppression for evaluation of subjects during post-corticosteroid, for prompt identification of emergent related adverse effects.

[00325] The following medications are prohibited starting 30 days before the start of rAAV particle administration and through the end of the study: Pegvaliase (except when permitted for rescue therapy); large neutral amino acids (LNAAs) (except when permitted for rescue therapy); and systemic immunosuppressive agents, including CS (beyond what is permitted per protocol to mitigate the risk of transaminase elevation). Systemic immunosuppressive agents (e.g., CS- sparing agents such as inosine monophosphate dehydrogenase inhibitors and calcineurin inhibitors) may be used should corticosteroids administration be clinically deemed to be ineffective, not tolerated, and/or contraindicated.

7. EMBODIMENTS

1. A method of decreasing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region. A method of treating a human subject with phenylketonuria, comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region. The method of embodiment 1 or 2 wherein the recombinant vector construct of the rAAV particle comprises (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV 3' ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs. The method of embodiment 3 wherein the nucleic acid encoding functional hPAH encodes an amino acid sequence at least 95% identical to SEQ ID NO: 2. The method of any of embodiments 1-4 wherein the nucleic acid encoding functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 1 or 7-13. The method of any of embodiments 1-5 wherein the nucleic acid encoding PAH is operably linked to a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer. The method of any of embodiments 1-5 wherein the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24, or alternatively at least 90% identical to any one of SEQ ID NOs: 25 or 26. The method of any of embodiments 1-5 wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6. The method of any of embodiments 1-8 wherein the recombinant vector construct further comprises an intron. The method of embodiment 9 wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or 27 or 29 or 34. The method of any of embodiments 1-10 wherein the recombinant vector construct further comprises a polyadenylation signal. The method of embodiment 11 wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal. The method of any of embodiments 1-12 wherein the rAAV particle comprises a recombinant vector construct at least 90% identical to any one of SEQ NOs: 15-23 or 52. The method of any of embodiments 1-13 wherein the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51. The method of any of embodiments 1-13 wherein the AAV capsid is an AAV capsid with liver tropism. The method of embodiment 15 wherein the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC15. The method of embodiment 15 wherein the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44. The method of any of the preceding embodiments wherein the subject has phenylketonuria (PKU). The method of any of the preceding embodiments wherein the subject has classic PKU or severe PKU. The method of any of the preceding embodiments wherein the subject has a plasma Phe level of 600 pmol/L or above prior to said administration. The method of any of the preceding embodiments wherein the subject has a plasma Phe level of 1200 pmol/L or above prior to said administration. The method of any of the preceding embodiments wherein the subject is 15 or more years old. The method of any of the preceding embodiments wherein the subject is an adult. The method of any of the preceding embodiments wherein the subject is a female. The method of any of the preceding embodiments wherein the subject has a mutation in an endogenous gene encoding PAH, optionally mutations F39L, L48S, I65T, R68S, A104D, SI IOC, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T, K320N, A373T, V388ME390G, A395P, P407S, and Y414C. The method of any of the preceding embodiments wherein the subject is a nonpregnant female. The method of any of the preceding embodiments wherein the subject is not receiving pharmacotherapy to treat PKU. The method of embodiment 27 wherein the subject has not received pegvaliase at least 30 days prior to said administration. The method of embodiment 27 wherein the subject has not received large neutral amino acids at least 30 days prior to said administration. The method of embodiment 27 wherein the subject has not received sapropterin at least 7 days prior to said administration. The method of any of the preceding embodiments wherein the subject has not received steroids at least 30 days prior to said administration. The method of any of the preceding embodiments wherein the subject does not have detectable anti-AAV capsid antibody in blood prior to said administration. The method of any of the preceding embodiments wherein the subject does not have clinically significant liver disease prior to said administration. The method of any of the preceding embodiments wherein wherein the rAAV particle is administered intravenously in a single administration. The method of any of the preceding embodiments wherein wherein the rAAV particle is administered at a dose ranging from about 2E13 to about 2E14 vector genomes per kilogram body weight of the subject (vg/kg). The method of embodiment 35 wherein the rAAV particle is administered at a dose of about 2E13 vg/kg. The method of embodiment 35 wherein the rAAV particle is administered at a dose of about 6E13 vg/kg. The method of embodiment 35 wherein the rAAV particle is administered at a dose of about 2E14 vg/kg. The method of any of the preceding embodiments further comprising administering to the subject a prophylactic immunosuppression treatment. The method of embodiment 39 wherein the prophylactic immunosuppression treatment comprises administering a prophylactically effective amount of a systemic immunosuppressant to prevent hepatotoxicity, optionally concurrent with said administration of rAAV particles. The method of embodiment 40 wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, or budesonide. The method of embodiment 40 wherein the systemic immunosuppressant is a corticosteroid and the prophylactically effective amount is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day, optionally for a time period of at least about 13 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks. The method of any of the preceding embodiments further comprising the step of (a) determining a baseline level of a marker of hepatotoxicity in the blood of the subject prior to said administration, optionally about one month prior to said administration, and (b) determining a post-administration level of said marker for hepatotoxicity in the blood of the subject after said administration every week. The method of embodiment 43 further comprising administering to the subject a therapeutic immunosuppression treatment. The method of embodiment 44 wherein the therapeutic immunosuppression treatment comprises the step of: (c) upon detection of hepatotoxicity by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a systemic immunosuppressant to reduce hepatotoxicity. The method of embodiment 44 wherein detection of hepatotoxicity is by (i) a post administration level of said marker of hepatotoxicity greater than the upper limit of normal (ULN), or (ii) a post-administration level of said marker of hepatotoxicity greater than or equal to twice the baseline level of said marker of hepatotoxicity. The method of any of embodiments 44-46 wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, or budesonide. The method of any of embodiments 44-46 wherein the systemic immunosuppressant is a corticosteroid and the therapeutically effective amount is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day, optionally for a time period of at least about 5 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks. The method of any of embodiments 43-48 wherein the marker of hepatotoxicity is ALT and/or AST. The method of any of embodiments 43-48 wherein the marker of hepatotoxicity is ALT. The method of any of the preceding embodiments further comprising monitoring episome formation by steps comprising extracting DNA from liver cells of the subject and detecting circular vector genomes, optionally by PCR or southern blotting. The method of any of the preceding embodiments further comprising the step of measuring plasma Phe level of the subject every week. The method of any of the preceding embodiments further comprising administering a Phe breath test. The method of any of the preceding embodiments further comprising the step of measuring plasma level of one or more neurotransmitters or neurotransmitter metabolites of the subject every week. The method of embodiment 54 wherein the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5- hydroxyindoleacetic acid. The method of any of the preceding embodiments wherein the plasma Phe level of said subject is 360 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy . The method of any of the preceding embodiments wherein the plasma Phe level of said subject is 360 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy. The method of any of the preceding embodiments wherein the plasma Phe level of said subject is between 120 and 360 pmol/L by 8 weeks after said administration, without concurrent pharmacotherapy. The method of any of the preceding embodiments wherein the plasma Phe level of said subject is 120 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy . The method of any of the preceding embodiments wherein the plasma Phe level of said subject is 120 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy. The method of any of the preceding embodiments wherein the subject tolerates an increase in Phe intake compared to a Phe restricted diet at baseline. The method of any of the preceding embodiments wherein blood level of a neurotransmitter or neurotransmitter metabolite of the subject is reduced after said administration. The method of embodiment 62 wherein the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5- hydroxyindoleacetic acid. The method of any of the preceding embodiments wherein the quality of life of said subject improves after said administration, optionally as measured by PKU-QOL or Q- LES-Q-SF questionnaire. The method of any of the preceding embodiments wherein one or more neurocognitive symptoms or measures of the subject improves after said administration. The method of any of the preceding embodiments wherein the subject does not suffer from ongoing hypophenylalaninemia after said administration. A pharmaceutical composition comprising rAAV particle at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, a buffering agent, an isotonicity agent, a cryopreservative agent and a surfactant which is stable during storage at about -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years. The pharmaceutical composition of embodiment 67 wherein the surfactant is at a concentration of less than 0.2% w/v, or less than 0.15% w/v. The pharmaceutical composition of embodiment 67 wherein the surfactant is at a concentration of about 0.1% w/v. The pharmaceutical composition of any of embodiments 67-69 wherein the cryopreservative agent is a sugar. The pharmaceutical composition of any of embodiments 67-69 wherein the cryopreservative agent is trehalose. A pharmaceutical composition comprising rAAV particle at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml which comprises sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 mM to about 165 mM, a cryopreservative agent that is a sugar, optionally trehalose, and a poloxamer or polysorbate at a concentration of less than 0.2% w/v. The pharmaceutical composition of embodiment 72 which comprises sodium phosphate, dibasic and sodium phosphate, monobasic. The pharmaceutical composition of any of embodiments 72-73 wherein the sugar is trehalose at a concentration of about 60 mM to about 80 mM. The pharmaceutical composition of any of embodiments 72-74 wherein the poloxamer is poloxamer 188 at a concentration of about 0.05% to 0.15% w/v. The pharmaceutical composition of embodiment 72 which comprises sodium phosphate, sodium chloride, trehalose and poloxamer 188. The pharmaceutical composition of embodiment 72 or 76 which comprises sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 to about 140 mM, a sugar at a concentration of about 60 to about 90 mM, and a poloxamer at a concentration of about 0.05% to about 0.15% w/v. The pharmaceutical composition of embodiment 72 or 76 which comprises sodium phosphate at a concentration of about 8 to about 12mM, sodium chloride at a concentration of about 110 to about 130 mM, a sugar at a concentration of about 70 to about 80 mM, and a poloxamer at a concentration of about 0.08% to about 0.12% w/v. The pharmaceutical composition of any of embodiments 72 or 76 wherein the sodium phosphate, monobasic is at a concentration that is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 mg/mL, and the sodium phosphate, dibasic, is at a concentration that is greater than 2.5 mg/ml and less than 3 mg/ml, optionally about 2.7 mg/mL. The pharmaceutical composition of any of embodiments 72 or 76 or 79 wherein the sodium chloride is at a concentration that is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml. The pharmaceutical composition of any of embodiments 72 or 76 or 79-80 wherein the sugar is trehalose dihydrate at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml. The pharmaceutical composition of any of embodiments 72 or 76 or 79-81 wherein the poloxamer 188 is at a concentration less than 1.5 mg/ml, or about 1 mg/ml. The pharmaceutical composition of any of embodiments 72-82 wherein the rAAV particle is at a concentration of about 6E13 vg/ml. A pharmaceutical composition which comprises rAAV particle at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188. The pharmaceutical composition of any of embodiments 72-84 which is liquid. The pharmaceutical composition of any of embodiments 72-84 which is lyophilized. The pharmaceutical composition of any of embodiments 72-84 for use in intravenous administration of rAAV particle to a patient with phenylketonuria. A method of producing an rAAV particle comprising (a) providing a cell permissive for AAV replication with one or more nucleic acid constructs comprising: (i) a recombinant vector construct comprising (1) at least one AAV ITR, (2) a heterologous liver-specific transcription regulatory region, and (3) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), (ii) a nucleotide sequence encoding one or more AAV Rep proteins which is operably linked to a promoter that is capable of driving expression of the Rep protein(s) in the cell; and (iii) a nucleotide sequence encoding one or more AAV capsid proteins which is operably linked to a promoter that is capable of driving expression of the capsid protein(s) in the cell; (b) culturing the cell under conditions permitting expression of the Rep and the capsid proteins; and optionally (c) recovering the AAV particle. The method of embodiment 88 wherein the cell is an insect cell. The method of embodiment 88 wherein the cell is a mammalian cell. The method of any of embodiments 88-90 wherein the nucleic acid encoding functional hPAH encodes an amino acid sequence at least 95% identical to SEQ ID NO: 2. The method of any of embodiments 88-90 wherein the nucleic acid encoding functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 1 or 7-13. The method of any of embodiments 88-90 wherein the nucleic acid encoding PAH is operably linked to a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer. The method of any of embodiments 88-93 wherein the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24, or alternatively at least 90% identical to any one of SEQ ID NOs: 25 or 26.

95. The method of any of embodiments 88-93 wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6.

96. The method of any of embodiments 88-95 wherein the recombinant vector construct further comprises an intron.

97. The method of embodiment 96 wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or 27 or 29 or 34.

98. The method of any of embodiments 88-97 wherein the recombinant vector construct further comprises a polyadenylation signal.

99. The method of embodiment 98 wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal.

100. The method of any of embodiments 88-90 wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to any one of SEQ NOs: 15-23 or 52.

101. The method of any of embodiments 88-100 wherein the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51.

102. The method of any of embodiments 88-100 wherein the AAV capsid is an AAV capsid with liver tropism.

103. The method of embodiment 102 wherein the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC15.

104. The method of embodiment 102 wherein the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44.

105. A population of rAAV particles produced by the method of any one of embodiments 88- 104, optionally enriched for particles comprising full length or nearly full length vector genomes by steps that reduce the number of empty capsids.

106. A method of decreasing plasma Phe levels in a human subject in need thereof comprising administering the population of rAAV particles of embodiment 105.

107. A method of treating PKU in a human subject in need thereof comprising administering the population of rAAV particles of embodiment 105. . SELECT SEQUENCES

[00326] The embodiments described herein are intended to be merely exemplary, and those skilled in the art will recognize, or will be able to ascertain using no more than routine experimentation, numerous equivalents of specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the disclosure.

[00327] All of the patents, patent applications and publications referred to herein are incorporated by reference herein in their entireties. Citation or identification of any reference in this application is not an admission that such reference is available as prior art to this application. The full scope of the disclosure is better understood with reference to the appended claims.

Claims

1. A pharmaceutical composition comprising rAAV particle at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml which comprises sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 mM to about 165 mM, a cryopreservative agent that is a sugar, optionally trehalose, and a poloxamer or polysorbate at a concentration of less than 0.2% w/v.

2. The pharmaceutical composition of claim 1 which comprises sodium phosphate, dibasic and sodium phosphate, monobasic.

3. The pharmaceutical composition of claim 1 or 2, wherein the sugar is trehalose at a concentration of about 60 mM to about 80 mM.

4. The pharmaceutical composition of any one of claims 1-3, wherein the poloxamer is poloxamer 188 at a concentration of about 0.05% to 0.15% w/v.

5. The pharmaceutical composition of claim 1, which comprises sodium phosphate, sodium chloride, trehalose and poloxamer 188.

6. The pharmaceutical composition of claim 1 or 5, which comprises sodium phosphate at a concentration of about 5 to about 15 mM, sodium chloride at a concentration of about 100 to about 140 mM, a sugar at a concentration of about 60 to about 90 mM, and a poloxamer at a concentration of about 0.05% to about 0.15% w/v.

7. The pharmaceutical composition of claim 1 or 5, which comprises sodium phosphate at a concentration of about 8 to about 12mM, sodium chloride at a concentration of about 110 to about 130 mM, a sugar at a concentration of about 70 to about 80 mM, and a poloxamer at a concentration of about 0.08% to about 0.12% w/v.

8. The pharmaceutical composition of claim 1 or 5, wherein the sodium phosphate, monobasic is at a concentration that is greater than 0.1 mg/mL and less than 0.5 mg/mL, optionally about 0.3 mg/mL, and the sodium phosphate, dibasic, is at a concentration that is greater than 2.5 mg/ml and less than 3 mg/ml, optionally about 2.7 mg/mL.

9. The pharmaceutical composition of claim 1, 5 or 8, wherein the sodium chloride is at a concentration that is greater than 5 mg/ml and less than 8 mg/ml, optionally about 7 mg/ml.

10. The pharmaceutical composition of claim 1, 5, 8 or 9, wherein the sugar is trehalose dihydrate at a concentration of greater than 20 mg/ml to less than 40 mg/ml, or about 25 mg/ml to about 35 mg/ml, or about 28 mg/ml.

11. The pharmaceutical composition of any one of claims 1, 5 or 8-10, wherein the poloxamer 188 is at a concentration less than 1.5 mg/ml, or about 1 mg/ml.

12. The pharmaceutical composition of any one of claims 1-11 wherein the rAAV particle is at a concentration of about 6E13 vg/ml.

13. A pharmaceutical composition comprising recombinant AAV (rAAV) particle at a concentration of at least 1E13 vg/ml to about 1E14 vg/ml, a buffering agent, an isotonicity agent, a cryopreservative agent and a surfactant which is stable during storage at about -60°C (minus sixty degrees centigrade) or less for at least about 1 year, 1.5 years, or 2 years.

14. The pharmaceutical composition of claim 13, wherein the surfactant is at a concentration of less than 0.2% w/v, or less than 0.15% w/v.

15. The pharmaceutical composition of claim 13, wherein the surfactant is at a concentration of about 0.1% w/v.

16. The pharmaceutical composition of any one of claims 13-15, wherein the cryopreservative agent is a sugar.

17. The pharmaceutical composition of any one of claims 1-15, wherein the cryopreservative agent is trehalose.

18. A pharmaceutical composition which comprises rAAV particle at a concentration of about 6E13 vg/ml, 10 mM sodium phosphate, 120 mM sodium chloride, 74 mM trehalose dihydrate, and 0.1% w/v poloxamer 188.

19. The pharmaceutical composition of any one of claims 6-18 which is liquid.

20. The pharmaceutical composition of any of claims 6-18 which is lyophilized.

21. The pharmaceutical composition of any of claims 6-18 for use in intravenous administration of rAAV particle to a patient with phenylketonuria.

22. The pharmaceutical composition of any one of claims 1-21, wherein the rAAV particle comprises an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region.

23. The pharmaceutical composition of claim 22, wherein the recombinant AAV vector, and wherein the recombinant AAV vector comprises: (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV 3' ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs.

24. The pharmaceutical composition of claim 22 or 23, wherein the nucleic acid encoding functional hPAH encodes an amino acid sequence at least 95% identical to SEQ ID NO: 2

25. The pharmaceutical composition of any one of claims 22 or 23, wherein the nucleic acid encoding functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 1 or 7-13.

26. The pharmaceutical composition of any one of claims 22-26, wherein the nucleic acid encoding PAH is operably linked to a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer.

27. The pharmaceutical composition of any one of claims 22-26, wherein the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24, or alternatively at least 90% identical to any one of SEQ ID NOs: 25 or 26.

28. The pharmaceutical composition of any one of claims 22-27, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO:

6

29. The pharmaceutical composition of any one of any of claims 22-26, wherein the recombinant vector construct further comprises an intron.

30. The pharmaceutical composition of claim 29, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or 27 or 29 or 34.

31. The pharmaceutical composition of any of claims 22-29, wherein the recombinant vector construct further comprises a polyadenylation signal.

32. The pharmaceutical composition of claim 31, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal.

33. The pharmaceutical composition of any of claims 22-32, wherein the rAAV particle comprises a recombinant vector construct at least 90% identical to any one of SEQ NOs: 15-23 or 52.

34. The pharmaceutical composition of any of claims 22-33, wherein the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35- 51.

35. The pharmaceutical composition of any one of claims 1-34, wherein the AAV capsid is an AAV capsid with liver tropism.

36. The pharmaceutical composition of claim 35, wherein the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC15.

37. The pharmaceutical composition of claim 35, wherein the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44.

38. A method of decreasing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 22-37.

39. The method of claim 38, wherein the subject has phenylketonuria (PKU).

40. A method of treating a human subject with phenylketonuria (PKU), comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 22-37.

41. The method of claim 39 or 40, wherein the subject has classic PKU or severe PKU.

42. The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose of 2E13 vector genomes per kilogram of the subject.

43. The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose of 6E13 vector genomes per kilogram of the subject.

44. The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose of 2E14 vector genomes per kilogram of the subject.

45. The method of any one of claims 38 to 41, wherein the therapeutically effective amount is a dose ranging from about 2E13 to about 9E13 vector genomes per kilogram of the subject.

46. A method of decreasing plasma phenylalanine (Phe) levels in a human subject in need thereof, comprising administering to the subject a dose ranging from about 2E13 to about 2E14 of a recombinant adeno-associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region.

47. The method of claim 45, wherein the subject has phenylketonuria (PKU).

48. A method of treating a human subject with phenylketonuria, comprising administering to the subject a dose ranging from about 2E13 to about 2E14 of a recombinant adeno- associated virus (rAAV) particle comprising an AAV capsid, and a recombinant vector construct comprising a nucleic acid encoding a functional phenylalanine hydroxylase (PAH) and optionally a heterologous liver-specific transcription regulatory region.

49. The method of claim 47 or 48, wherein the subject has classic PKU or severe PKU.

50. The method of any one of claims 46 to 49, wherein the recombinant vector construct of the rAAV particle comprises (a) one or both of (i) an AAV 5' inverted terminal repeat (ITR) and (ii) an AAV 3' ITR, (b) a heterologous liver-specific transcription regulatory region, and (c) a nucleic acid encoding a functional human phenylalanine hydroxylase (hPAH), optionally wherein the AAV ITRs are AAV2 ITRs.

51. The method of claim 50, wherein the nucleic acid encoding functional hPAH encodes an amino acid sequence at least 95% identical to SEQ ID NO: 2.

52. The method of any one of claims 46-50, wherein the nucleic acid encoding functional hPAH comprises a nucleotide sequence at least 90% identical to SEQ ID NOs: 1 or 7-13.

53. The method of any one of claims 46-52, wherein the nucleic acid encoding PAH is operably linked to a promoter comprising a fragment of an hAAT promoter and a fragment of an HCR enhancer/ ApoE enhancer.

54. The method of any one of claims 46-53, wherein the liver-specific transcription regulatory region comprises a nucleotide sequence at least 90% identical to any one of SEQ ID NOs: 3, 4 or 24, or alternatively at least 90% identical to any one of SEQ ID NOs: 25 or 26.

55. The method of any one of claims 46-54, wherein the recombinant vector construct comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 6.

56. The method of any of claims 46-53 wherein the recombinant vector construct further comprises an intron.

57. The method of claim 56, wherein the intron comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 14, or 27 or 29 or 34.

58. The method of any one of claims 46-57, wherein the recombinant vector construct further comprises a polyadenylation signal.

59. The method of claim 58, wherein the recombinant vector construct comprises a bovine growth hormone (bGH) polyadenylation signal.

60. The method of any one of claims 46-59, wherein the rAAV particle comprises a recombinant vector construct at least 90% identical to any one of SEQ NOs: 15-23 or 52.

61. The method of any of claims 46-59, wherein the AAV capsid comprises an amino acid sequence at least 85% identical to any one of SEQ ID NOs: 35-51.

62. The method of any of claims 46-61, wherein the AAV capsid is an AAV capsid with liver tropism.

63. The method of claim 62, wherein the AAV capsid with liver tropism excludes AAV8 and/or AAVHSC 15.

64. The method of claim 62, wherein the AAV capsid with liver tropism is an AAV5 type capsid, optionally at least 85%, 90% or 95% identical to SEQ ID NO: 44.

65. The method of any one of the claims 38-64, wherein the subject has a plasma Phe level of 600 pmol/L or above prior to said administration.

66. The method of any one of the claims 38-64, wherein the subject has a plasma Phe level of 1200 pmol/L or above prior to said administration.

67. The method of any one of the claims 38-66, wherein the subject is 15 or more years old.

68. The method of any one of the claims 38-66, wherein the subject is an adult.

69. The method of any one of claims 38-68, wherein the subject is a female.

70. The method of any one of claims 38-69, wherein the subject has a mutation in an endogenous gene encoding PAH, optionally mutations F39L, L48S, I65T, R68S, A104D, SI IOC, D129G, E178G, V190A, P211T, R241C, R261Q, A300S, L308F, A313T,

K320N, A373T, V388M E390G, A395P, P407S, and Y414C.

71. The method of any one of claims 38-70, wherein the subject is a nonpregnant female.

72. The method of any one of claims 38-71, wherein the subject is not receiving pharmacotherapy to treat PKU.

73. The method of claim 72, wherein the subject has not received pegvaliase at least 30 days prior to said administration.

74. The method of claim 72, wherein the subject has not received large neutral amino acids at least 30 days prior to said administration.

75. The method of claim 72, wherein the subject has not received sapropterin at least 7 days prior to said administration.

76. The method of any one of claims 38-75, wherein the subject has not received steroids at least 30 days prior to said administration.

77. The method of any one of claims 38-76, wherein the subject does not have detectable anti-AAV capsid antibody in blood prior to said administration.

78. The method of any one of claims 38-77, wherein the subject does not have clinically significant liver disease prior to said administration.

79. The method of any one of claims 38-78, wherein wherein the rAAV particle is administered intravenously in a single administration.

80. The method of any one of claims 38-79, further comprising administering to the subject a prophylactic immunosuppression treatment.

81. The method of claim 80, wherein the prophylactic immunosuppression treatment comprises administering a prophylactically effective amount of a systemic immunosuppressant to prevent hepatotoxicity, optionally concurrent with said administration of rAAV particles.

82. The method of claim 81, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, or budesonide.

83. The method of claim 81, wherein the systemic immunosuppressant is a corticosteroid and the prophylactically effective amount is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day, optionally for a time period of at least about 13 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks.

84. The method of any one of claims 38-83, further comprising the step of (a) determining a baseline level of a marker of hepatotoxicity in the blood of the subject prior to said administration, optionally about one month prior to said administration, and (b) determining a post-administration level of said marker for hepatotoxicity in the blood of the subject after said administration every week.

85. The method of claim 84, further comprising administering to the subject a therapeutic immunosuppression treatment.

86. The method of claim 85, wherein the therapeutic immunosuppression treatment comprises the step of: (c) upon detection of hepatotoxicity by biochemical or clinical signs, administering to the subject a therapeutically effective amount of a systemic immunosuppressant to reduce hepatotoxicity.

87. The method of claim 86, wherein detection of hepatotoxicity is by (i) a post administration level of said marker of hepatotoxicity greater than the upper limit of normal (ULN), or (ii) a post-administration level of said marker of hepatotoxicity greater than or equal to twice the baseline level of said marker of hepatotoxicity.

88. The method of any one of claims 85-87, wherein the systemic immunosuppressant is a corticosteroid, optionally dexamethasone, prednisone, prednisolone, fludrocortisone, hydrocortisone, or budesonide.

89. The method of any one of claims 85-87, wherein the systemic immunosuppressant is a corticosteroid and the therapeutically effective amount is a prednisone-equivalent dose of from 10 mg/day to 40 mg/day, optionally for a time period of at least about 5 weeks, followed by tapering amounts of the corticosteroid for a time period of about 3 weeks.

90. The method of any one of claims 84-87, wherein the marker of hepatotoxicity is ALT and/or AST.

91. The method of any one of claims 84-87, wherein the marker of hepatotoxicity is ALT.

92. The method of any one of claims 38-91, further comprising monitoring episome formation by steps comprising extracting DNA from liver cells of the subject and detecting circular vector genomes, optionally by PCR or southern blotting.

93. The method of any one of claims 38-92, further comprising the step of measuring plasma Phe level of the subject every week.

94. The method of any one of claims 38-93, further comprising administering a Phe breath test.

95. The method of any one of claims 38-94, further comprising the step of measuring plasma level of one or more neurotransmitters or neurotransmitter metabolites of the subject every week.

96. The method of claim 95, wherein the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5-hydroxyindoleacetic acid.

97. The method of any one of claims 38-96, wherein the plasma Phe level of said subject is 360 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy .

98. The method of any one of claims 38-97, wherein the plasma Phe level of said subject is 360 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy .

99. The method of any one of claims 38-98, wherein the plasma Phe level of said subject is between 120 and 360 pmol/L by 8 weeks after said administration, without concurrent pharmacotherapy .

100. The method of any one of claims 38-98, wherein the plasma Phe level of said subject is 120 pmol/L or less by 8 weeks after said administration, without concurrent pharmacotherapy .

101. The method of any one of claims 38-100, wherein the plasma Phe level of said subject is 120 pmol/L or less at 2, 3 or 4 years after said administration, without concurrent pharmacotherapy.

102. The method of any one of claims 38-101, wherein the subject tolerates an increase in Phe intake compared to a Phe restricted diet at baseline.

103. The method of any one of claims 38-102, wherein blood level of a neurotransmitter or neurotransmitter metabolite of the subject is reduced after said administration.

104. The method of claim 103, wherein the one or more neurotransmitters or neurotransmitter metabolites is phenethylamine, phenylethanolamine, tyramine, dopamine, norepinephrine, epinephrine, tryptamine, hydroxytryptamine, phenylacetic acid, phenylacetylglutamine, mandelic acid, hydroxyphenylacetic acid, DOPAC, homovanillic acid, DOMA, MOPEG, vanillylmandelic acid, indoleactic acid, or 5- hydroxyindoleacetic acid.

105. The method of any one claims 38-104, wherein the quality of life of said subject improves after said administration, optionally as measured by PKU-QOL or Q-LES-Q-SF questionnaire.

106. The method of any one of claims 38-105, wherein one or more neurocognitive symptoms or measures of the subject improves after said administration.

107. The method of any one of claims 38-106, wherein the subject does not suffer from ongoing hypophenylalaninemia after said administration.

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