Human PAH expression cassette for treating PKUs with gene replacement therapy directed at the liver

Abstract

Provided herein is an expression cassette for expressing a transgene in liver cells, wherein the transgene encodes a PAH polypeptide. Also provided is a method for treating phenylketonuria (PKU) and / or reducing phenylalanine levels in an individual in need thereof. Further provided herein are vectors (e.g., rAAV vectors), viral particles, pharmaceutical compositions and kits for expressing a PAH polypeptide in an individual in need thereof.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 086,537, filed on 1 October 2020, and U.S. Provisional Application No. 63 / 121,797, filed on 4 December 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listings in ASCII text files. The contents of the following submission in ASCII text file are incorporated herein by reference in their entirety: Computer-Readable Sequence Listing (CRF) (filename: 159792017840SEQLIST.TXT, date recorded: September 22, 2021, size: 31,028 bytes).

[0003] The present invention relates to an expression cassette for expressing a phenylalanine hydroxylase polypeptide. In some embodiments, the present invention relates to compositions and methods for treating phenylketonuria using gene therapy. [Background technology]

[0004] Phenylketonuria (PKU) is a genetic deficiency of phenylalanine hydroxylase (PAH), a liver enzyme that catalyzes the hydroxylation of phenylalanine (Phe) to tyrosine (Tyr). This disorder is the most common congenital amino acid metabolism disorder in North America, with an overall incidence of 1:10–15,000, and is detected by newborn screening programs in most developed countries. If left untreated, severe forms of PKU result in neurotoxic and highly elevated blood Phe levels associated with intellectual disability (Non-Patent Literature 1, 2, and 3). The affected protein, PAH, is a multi-domain protein consisting of a regulatory N-terminal domain (1–117), a catalytic central domain (118–410), and a tetrameric domain at the C-terminal region (411–452) (Non-Patent Literature 4). To date, more than 560 disease-causing mutations have been mapped to each domain, with the catalytic region being the most frequently affected site (Non-Patent Literature 5). Homomultimeric enzymes undergo complex regulation via phosphorylation and allosteric activation when the substrate Phe binds to the N-terminal domain, which fine-tunes PAH enzyme activity by altering the enzyme's various structures and polymerization states (Non-Patent Literature 6, Non-Patent Literature 7, Non-Patent Literature 8).

[0005] Current treatment for PKU involves a lifelong pharmacokinetic (Phe) diet with a low-protein diet and liquid medical formulas (Non-Patent Literature 1, 2, 3). Despite its effectiveness, the unsatisfactory taste of medical foods and the strict limitations on food choices make dietary adherence difficult, and medication non-compliance gradually increases during childhood, with nearly 80% of patients having blood pharmacokinetic levels above recommended levels by late adolescence (Non-Patent Literature 9, 10). Furthermore, despite excellent adherence to the pharmacokinetic diet, there is emerging evidence that many patients experience various neurocognitive and neuropsychiatric deficits, in addition to a high incidence of attention deficit hyperactivity disorder (ADHD). The reasons for this are unclear, but possible explanations include amino acid imbalances in the brain, nutritional deficiencies in certain vitamins and trace elements, and fluctuations in blood pharmacokinetic levels that are normally maintained stably by hepatic PAH activity (Non-Patent Literature 11, 12, 13). Interestingly, treatment with a synthetic form of the cofactor tetrahydrobiopterin (BH4) (sapropterin dihydrochloride) in patients with milder forms of PKU has not only been shown to be effective in lowering blood Phe levels but has also demonstrated improvements in neurological outcomes, such as a reduction in ADHD symptoms (Non-Patent Literature 14). This therapy can provide partial modification of the genetic defect by increasing residual PAH enzyme activity by acting as a pharmacological chaperone, and thus by providing normal Phe with controlled PAH activity (Non-Patent Literature 3). Another therapy approved in recent years consists of enzyme substitution therapy using a pegylated form of bacterial phenylalanine ammonia lyase (PAL) that metabolizes Phe to trans-cinnamic acid. This therapy results in a significant reduction in blood Phe levels, but its effect on neurological endpoints does not appear to be as high (Non-Patent Literature 15). It remains unclear whether this or any other therapy, which is primarily based on lowering blood Phe levels without modifying PAH function as a regulator of systemic Phe levels and as a producer of Tyr, can address the cognitive and neuropsychiatric problems observed even in PKU patients who adhere to a diet.

[0006] Restoring Phe hydroxylase activity in the liver of PKU patients through gene transfer of the Pah gene is an attractive approach to treating the disease. If sufficient PAH expression can be restored, stable low blood Phe levels should be provided. Numerous studies have shown that Pah enu2 We have shown that rAAV-mediated delivery of cDNA encoding PAH to the mouse liver reduces blood Phe levels to within normal ranges and modifies behavior (Mochizuki 2004; Ding 2006; Harding 2006, Yagi 2011, Winn 2018). On average, one rAAV copy per cell or at least 10% of normal PAH activity in the liver is sufficient to correct defects in the liver (Hamman 2010, Yagi 2011, Viecelli 2014). Wild-type hepatocytes or heterozygous Pah enu2 / + Studies of hepatocyte regrowth in PKU mice using donor hepatocytes showed that 3–10% hepatocyte regrowth partially reduced blood Phe levels, and 10% hepatocyte regrowth completely corrected blood Phe levels (Hamman 2010). More recently, proof of this concept has been demonstrated in gene therapy trials delivering functional Pah gene copies to the liver, but this required relatively large vector doses (Chatterjee 2020). What is needed is an improved rAAV vector for efficient gene transfer to the liver, potent expression of hPAH in the liver, and subsequent correction of PKU pathology.

[0007] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Kochhar JSら, Drug Deliv Transl Res 2012, 2:223~237 [Non-licensed document 2] Ho G, Christodoulou J. Transl Pediatr 2014, 4:49~62 [Non-licensed document 3] Blau N, Longo N.Expert Opin Pharmacother 2015, 16:791~800

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Non-licensed literature 9

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[0009] This invention is at least in part based on the inventors' development of an expression cassette encoding phenylalanine hydroxylase (PAH). This expression cassette has enabled the driving of transgene expression in hepatocytes in human cell cultures, the liver of a PKU mouse model, and the liver of non-human primates. Furthermore, the wild-type human PAH polypeptide produced by the expression cassette was enzymatically active. Therefore, an rAAV vector having this expression cassette can pave the way for PKU gene therapy by delivering efficacy at reduced vector doses. [Means for solving the problem]

[0010] In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in liver cells, the expression cassette comprising a transgene operably linked to a promoter and an enhancer, the promoter comprising a mouse trans tyretin (mTTR) promoter, the enhancer comprising one or two modified prothrombin enhancers (pPrT2), one or two modified alpha-1-microbikunin (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HE11), or CRM8 enhancer, the transgene encoding a PAH polypeptide; and the AAV virus particles comprising an AAV-XL32 or AAV-XL32.1 capsid. In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is located at 5' relative to the mTTR promoter.

[0011] In some embodiments, the present invention provides recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in liver cells, the expression cassette comprises a promoter and a transgene operably ligated to a 3' element, the promoter comprising a mouse trans tyretin (mTTR) promoter, the 3' element being an albumin 3' element (3'Alb) or an albumin 3' element ligated to a human alpha-1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR), the transgene encoding a PAH polypeptide; and the AAV virus particles comprising an AAV-XL32 or AAV-XL32.1 capsid. In some embodiments, the mTTR promoter is an mTTR482 promoter. In some embodiments, the 3' element is located 3' relative to the transgene.

[0012] In some embodiments, the present invention relates to recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, an expression cassette for expressing a transgene in liver cells, wherein the expression cassette comprises a promoter and enhancers and a transgene operably ligated to the 3' element, the promoter comprising a mouse trans tyretin (mTTR) promoter, and the enhancers comprising one or two modified prothrombin enhancers (pPrT2) and one or two modified alpha-1-microbicinin enhancers (mA1 The rAAV particles are provided, comprising MB2), a modified mouse albumin enhancer (mEalb), a hepatitis B virus enhancer II (HE11), or a CRM8 enhancer; the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element (3'AlbSMAR) linked to a human alpha-1 antitrypsin scaffold / matrix attachment region (SMAR); the transgene encodes a PAH polypeptide; and the AAV virus particles comprise an AAV-XL32 or AAV-XL32.1 capsid. In some embodiments, the mTTR promoter is an mTTR482 promoter. In some embodiments, the enhancer is located 5' relative to the mTTR promoter. In some embodiments, the 3' element is located 3' relative to the transgene.

[0013] In some embodiments of the above-described aspects, the expression cassette further comprises an intron. In some embodiments, the intron is a chicken β-actin / rabbit β-globin hybrid intron. In some embodiments, the expression cassette further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0014] In some embodiments of the above-described model, the PAH polypeptide is a wild-type PAH polypeptide. In some embodiments, the PAH polypeptide is a human PAH polypeptide. In some embodiments, the PAH polypeptide contains the amino acid sequence of SEQ ID NO: 1. In some embodiments, the transgene is at least 80% identical to the nucleic acid sequence of SEQ ID NO: 2.

[0015] In some embodiments of the above-described aspects, the rAAV vector comprises an expression cassette containing one or more adjacent AAV reverse-terminal repeat (ITR) sequences. In some embodiments, the expression cassette described in any one of claims 1 to 18 contains adjacent ITRs of two AAVs. In some embodiments, the ITRs of AAVs are ITRs of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotypes. In some embodiments, the ITRs of AAVs are ITRs of AAV2. In some embodiments, the vector is a self-complementary vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding a complement of the PAH polypeptide, wherein the first nucleic acid sequence can form intrachain base pairs with the second nucleic acid sequence along most or all of its length. In some embodiments, the first and second nucleic acid sequences are linked by an ITR of a mutated AAV, the ITR of the mutated AAV includes a deletion in the D region and includes a mutation in the terminal decomposition sequence.

[0016] In some embodiments of the above-described set, the AAV capsid is the AAV-XL32 capsid. In some embodiments, the AAV-XL32 capsid comprises an AAV-XL32 capsid protein containing an amino acid sequence that is at least 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the AAV-XL32 capsid comprises VP1, VP2, and VP3, where VP1, VP2, and VP3 are encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV capsid is the AAV-XL32.1 capsid. In some embodiments, the AAV-XL32.1 capsid contains an amino acid sequence that is at least 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the AAV-XL32.1 capsid comprises VP1, VP2, and VP3, where VP1, VP2, and VP3 are encoded by the nucleic acid sequence of SEQ ID NO: 6.

[0017] In some embodiments, the present invention provides compositions comprising any of the rAAV particles described herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier.

[0018] In some embodiments, the present invention provides cells comprising any of the rAAV particles described herein. In some embodiments, the present invention provides a method for producing PAH polypeptides, comprising culturing the cells described herein under conditions for PAH polypeptide production. In some embodiments, the method further comprises a step of purifying the PAH polypeptides.

[0019] In some embodiments, the present invention provides a method for treating phenylketonuria in an individual requiring it, comprising administering rAAV particles as described herein to the individual. In some embodiments, the present invention provides a method for treating phenylketonuria in an individual requiring it, comprising administering a composition as described herein to the individual. In some embodiments, the present invention provides a method for treating phenylketonuria in an individual requiring it, comprising administering cells as described herein to the individual. In some embodiments, the individual lacks PAH activity.

[0020] In some embodiments, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need thereof, comprising administering rAAV particles as described herein to the individual. In some embodiments, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need thereof, comprising administering a composition as described herein to the individual. In some embodiments, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual in need thereof, comprising administering cells as described herein to the individual. In some embodiments, the level of phenylalanine in the blood of the individual before treatment is elevated compared to the level of phenylalanine in the blood of an equivalent corresponding control individual. In some embodiments, the rAAV particles, composition, or cells are administered intravenously, intra-arterially, intrahepatically, intra-portally, intraperitoneally, or subcutaneously. In some embodiments, the administration is combined with another therapy. In some embodiments, the other therapy is treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia lyase (PAL) or pegylated PAL, or a phenylalanine-restricted diet.

[0021] In some embodiments, the present invention provides a kit comprising any of the rAAV particles, compositions, or cells described herein. In some embodiments, the kit further comprises instructions for use; buffers and / or pharmaceutically acceptable excipients; and / or bottles, vials, and / or syringes. [Brief explanation of the drawing]

[0022] [Figure 1A] Figures 1A-1C show in vitro comparisons of PAH protein and activity levels of wild-type PAH and PAH variants. Four expression plasmids (n=2 / plasmid) were transfected into the human liver cell line Huh7 cells. After 72 hours, the cells were collected and lysates were prepared. Figure 1A shows PAH activity levels. The assay was performed for 30 minutes using 13C-Phe as the substrate. The amount of product (13C-Tyr) produced was measured by LC-MS / MS, and the value was normalized to total protein. Sample identity is shown on the x-axis, from left to right, including mock control, hPAH / G, hPAH-V1 / G, hPAH / E (wild-type hPAH), hPAH-V1 / E, and mouse PAH (mPAH). The y-axis shows the level of PAH activity (μM of 13C-Tyr per mg of protein). [Figure 1B] Figure 1B shows a Western blot indicating PAH protein levels. Cell lysates (15 μg / lane) were electrophoresed on an SDS-PAGE gel, transferred to the membrane, and then probed with anti-PAH antibodies reactive to both human and mouse PAH proteins. Sample identity is shown above the blot, from left to right, including hPAH-V1 / G, wild-type hPAH / E, hPAH-V1 / E, hPAH / G, and mPAH. [Figure 1C]Figure 1C shows the quantification of PAH protein levels as determined by Western blotting using AzureSpot software. Sample identity is shown on the x-axis, from left to right, including hPAH-V1 / G, wild-type hPAH / E, hPAH-V1 / E, hPAH / G, and mPAH. In Figures 1A-1C, "hPAH-V1 / G" represents human PAH variant 1 with the E183G amino acid substitution; "WT hPAH / E" represents wild-type human PAH with the E183 residue; "hPAH-V1 / G" represents human PAH variant 1 with the E183 residue; "hPAH / G" represents mutant human PAH with the E183G amino acid substitution; and "mPAH" represents FLAG-tagged mouse PAH used as a positive control. [Figure 2A] Figures 2A-2E show the results of experiments comparing AAV capsids for gene transfer into liver cells and organs in non-human primates (NHPs) and verifying the A1M2-mTTR promoter in NHPs. Figure 2A shows, from left to right, the EGFP protein levels in human liver cell lines (Huh7 cells) transduced with AAV vectors containing a CBA-EGFP expression cassette, using AAV8, AAV-DJ, AAV-LK03, AAV-XL14, and AAV-XL32 capsid vectors. [Figure 2B] Figure 2B shows the levels of vector genome / cells in NHP liver (dark gray) and spleen (light gray). Vector genome copies were measured by qPCR two weeks after IV delivery of 5e12vg / kg. VG copies in the liver and spleen of each animal 14 days post-vector administration are shown. The liver values ​​represent the mean of four samples collected from various locations. The spleen values ​​represent the mean of two adjacent samples collected from the central section of the spleen. [Figure 2C]Figure 2C shows the vector genome / cell levels in NHP kidney (dark gray), muscle (light gray), and heart (medium gray). The values ​​represent the average of two adjacent samples collected from each tissue. Capsid protein identity is shown along the x-axis, from left to right, including AAV8, AAV-DJ, AAV-LK03, AAV-XL14, or AAV-XL32. [Figure 2D] Figure 2D shows a summary of the in vivo distribution comparison of the five capsid vectors compared. The vector genome / cell level is shown on the x-axis from left to right, relative to the liver, spleen, muscle, heart, or kidney of NHP patients administered with the AAV8, AAV-DJ, AAV-LK03, AAV-XL14, or AAV-XL32 capsid vectors, shown on the y-axis. [Figure 2E] Figure 2E shows EGFP expression in three segments of the right medial lobe and one segment of the left medial lobe after administration of AAV8, AAV-DJ, AAV-LK03, AAV-XL14, or AAV-XL32 capsid vectors. [Figure 3-1]Figures 3A-3E show the results of experiments measuring the dose-response of XL32.1 / mA1MB2-mTTR482--EGFP for NHP liver gene transfer. Figure 3A shows the development of vector genomes in NHP livers as shown on the y axis, for vehicle-only controls as shown on the x axis, or in NHP administered doses of XL32.1 / mA1MB2-mTTR482- at 5e11, 2e12, 5e12, and 2e13 vg / kg. The lower x-axis panel shows the mean vector genome copies / cell (n=3 / administration cohort). M: Male NHP, F: Female NHP (significance: *, p<0.05; **, p<0.01). Figure 3B shows the levels of vector-derived transcripts in the liver of NHP patients, as shown on the y axis, for vehicle-only controls as shown on the x axis, or for NHP patients administered XL32.1 / mA1MB2-mTTR482-EGFP at doses of 5e11, 2e12, 5e12, and 2e13 vg / kg, as shown on the x axis. Figure 3C shows the levels of EGFP protein as shown on the y axis, for vehicle-only controls as shown on the x axis, or for NHP patients administered XL32.1 / mA1MB2-mTTR482-EGFP at doses of 5e11, 2e12, 5e12, and 2e13 vg / kg, as shown on the y axis. Figure 3D shows the correlation between the liver vector genome and vector-derived mRNA copies (high doses of 2e13 vg / kg were omitted in this analysis). Figure 3E shows the correlation between vector-derived mRNA copies and eGFP protein levels in the liver. [Figure 3-2] Continuation of Figure 3-1. [Figure 3-3] Continuation of Figure 3-2. [Figure 4A] Figures 4A-4C show the results of liver in situ hybridization analysis in a dose-response study of XL32.1 / mA1MB2-mTTR482-EGFP administered to NHP. Figure 4A shows a representative in situ hybridization image for detecting the vector in the liver. As an example, detection in animal #203 (2e12vg / kg group) (by qPCR, 3vg / cell) is shown on the right. [Figure 4B]Figure 4B shows the percentage of EGFP-vector DNA-positive cells in the liver detected by in-situ hybridization as shown on the y axis, in NHPs administered with vehicle-only controls as shown on the x axis, or with XL32.1 / mA1MB2-mTTR482-EGFP at doses of 5e11, 2e12, 5e12, and 2e13 vg / kg. [Figure 4C] Figure 4C shows the correlation between the average number of VG copies determined by qPCR (y-axis) and the percentage of VG-positive cells determined by in situ hybridization (x-axis). [Figure 5A] Figures 5A–5B show a comparison of the in vivo distribution of XL32 and XL32.1 capsid vectors in the NHP. Figure 5A shows the vector genome / cell levels in the liver and various other organs. The values ​​for the liver represent the average of three animals in each group (each animal was tested for one sample from the right and left medial lobes), while the values ​​for other organs are the average of three animals (one sample per animal). [Figure 5B] Figures 5A and 5B show a comparison of the in vivo distribution of XL32 and XL32.1 capsid vectors in NHP. Figure 5B shows the levels of mRNA-derived vectors in the liver. Each value represents the mean per treatment group (n=2 for each animal). [Figure 6]Figures 6A–6D illustrate the efficacy of XL32.1 / WT hPAH in Pah-KO mice. Figure 6A shows the time course of blood Phe levels. Figure 6B shows the blood Phe levels at day 36 (HOM mice are not included in the difference observation). Figure 6C shows the time course of blood Tyr levels. Figure 6D shows the blood Tyr levels at day 36. The vector (containing the mA1MB2-mTTR482 promoter) was administered via IV route to adult male homozygous Pah-KO mice at doses of 1e11, 3e11, and 1e12 vg / mouse. The vector-treated cohorts consisted of n=8–10 animals / groups, with HET mice (n=10) and C57BL66 mice (n=5). Abbreviations: HOM, homozygous Pah-KO mice; HET, heterozygous Pah-KO mice; C57BL / 6, wild-type mice. [Figure 7-1] Figures 7A–7E show the analysis of gene transfer and transduction by XL32.1 / WT hPAH in the liver of Pah-KO mice. Figure 7A shows the vector DNA copy in the liver. Figure 7B shows the vector DNA in all tested tissues (liver, spleen, muscle, kidney, and lung). Figure 7C shows the vector-derived mRNA levels in the liver. Figure 7D shows the correlation of the vector genome to the vector-derived mRNA levels per cell in the liver. Figure 7E shows the correlation of the vector genome to blood Phe levels in the liver. Each data point represents one animal. Normalization per cell is based on 5 pg of DNA / cell (vg / cell) and 30 pg / cell (mRNA / cell). Abbreviation: HOM, homozygous PAH-KO mouse. [Figure 7-2] Continuation of Figure 7-1. [Figure 8-1]Figures 8A–8C show the analysis of PAH activity and protein levels after delivery using XL32.1 / WT hPAH in the liver of Pah-KO mice. Figure 8A shows PAH activity in liver homogenates. Figure 8B shows the detection of PAH protein by Western blotting. Figure 8C shows the localization of PAH-positive cells by PAH immunohistochemistry. The vector-treated cohorts consisted of n=8–10 animals / groups, with HET, n=10 and C57BL, n=5. Three representative animals from each cohort were used for analysis for Western blotting. Their equal protein loading is indicated by β-actin probing. [Figure 8-2] Continuation of Figure 8-1. [Figure 9A] Figures 9A and 9B show the effects of XL32.1 / WT hPAH delivery to the liver on brain amino acid and neurotransmitter levels. Figure 9A shows brain Phe, Tyr, and Trp levels. [Figure 9B] Figures 9A and 9B show the effects of XL32.1 / WT hPAH delivery to the liver on brain amino acid and neurotransmitter levels. Figure 9B shows brain neurotransmitter levels of dopamine, norepinephrine, and serotonin. Each data point represents one animal. For brain amino acid analysis, the vector-treated cohorts included n=7–10 animals / groups, HET, n=9 and C57BL / 6, n=5. For brain neurotransmitter analysis, the vector-treated cohorts included n=7 animals / groups, HET, n=6 and C57BL / 6, n=4. Statistical analysis was performed by one-way ANOVA. [Figure 10A] Figures 10A and 10B show the behavioral analysis of Pah-KO mice after XL32.1 / WT hPAH delivery. Figure 10A shows images of nest quality, where each score ranges from 1 (poor quality) to 5 (high quality) of erosion. [Figure 10B]Figures 10A and 10B show behavioral analysis of Pah-KO mice after XL32.1 / WT hPAH delivery. Figure 10B shows animal scoring before and after rAAV-XL32.1 / WT hPAH treatment. Each data point represents one animal. The vector treatment cohorts included n=8–10 animals / groups, HET, n=10 and C57BL / 6, n=5, and statistical analysis was performed by one-way ANOVA. [Figure 11] Figures 11A–11C show the growth of animals in study 4 months after administration of AAVXL32.1 / WT hPAH to Pah-KO mice. Figure 11A shows that body weight was significantly different in Pah-KO mice (HOM and treatment cohorts) compared to pre-treatment HET and WT (C57BL / 6) mice. Figure 11B shows that body weight was significantly increased in the two higher-dose cohorts compared to untreated HOM mice 4 months after treatment with WT PAH. Figure 11C shows the increase in liver weight in the treatment group, which was similar to that of HET and WT mice. Significance was as follows, by one-way ANOVA and Tukey's multiple comparisons: *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001. Each data point represents one animal (n animals per group, HOM=9; low dose, n=6; medium dose, n=8; high dose, n=8; HET, n=8; and WT, n=8). Abbreviations: HOM, homozygous Pah-KO mouse; HET, heterozygous Pah-KO mouse; WT, wild-type C57BL / 6 mouse. Dose is vg / mouse. [Figure 12-1]Figures 12A–12C show plasma Phe levels during the study 4 months after administration of AAVXL32.1 / WT hPAH to Pah-KO mice. Figure 12A shows the mean blood Phe levels in each cohort over 120 days. Blood Phe levels in individual mice at 7 days (Figure 12B) and 120 days (Figure 12C) post-treatment. All treatment groups (dose vg / mouse) significantly reduced blood Phe levels compared to untreated HOM mice. At any time point, there were no significant differences between WT PAH treatment groups at 3e11 and 1e12 doses compared to HET and WT mice. The 1e11 dose showed variability and was not equivalent to HET / WT. The number of animals, statistical analysis, and abbreviations are shown in the legend of Figures 11A–11C. [Figure 12-2] Continuation of Figure 12-1. [Figure 13-1] Figures 13A–13E show vector DNA and mRNA in the liver of Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 13A shows vector DNA copies in the liver, and Figure 13B shows vector-derived mRNA levels in the liver. Both endpoints showed dose-responsive increases. A correlation of superior vector genome per cell in the liver to vector-derived mRNA levels (Figure 13C) and a correlation of vector genome in the liver to blood Phe levels (Figure 13D) were found. The latter indicated that normalization of blood Phe (100uM) required a minimum of 0.1VG / cell. Figure 13E shows a representative image of in-situ detection of vector DNA (red), and transcripts (green) are shown as H&E-stained fragments for each treatment cohort. Animal counts, statistical analyses, and abbreviations are as shown in Figures 11A–11C. [Figure 13-2] Continuation of Figure 13-1. [Figure 13-3] Continuation of Figure 13-2. [Figure 14-1]Figures 14A–14D show PAH activity and PAH protein detection in the liver of Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 14A shows PAH activity in liver homogenates. PAH enzyme activity in the 1e11 and 3e11 treatment cohorts was not significantly different from that in HET mice, but the 1e12 treatment resulted in significantly higher PAH activity than observed in normal mice. Figure 14B shows PAH protein production confirmed by PAH IHC in liver fragments, and the percentage of PAH-positive cells was quantified using HALO analysis. Figure 14C shows its correlation with vector DNA copies in the liver, showing that normalization of blood Phe (100 μM) required at least 20% PAH-positive cells. Figure 14D shows representative images of PAH IHC from all study cohorts. The percentage of PAH-positive cells in each image (# indicates the animal) is as follows: HOM (#4), 0%; 1e11 (#19), 38%; 3e11 (#21), 62%; 1e12 (#34), 72%; HET (#46), 94%; and WT (#56), 99%. [Figure 14-2] Continuation of Figure 14-1. [Figure 14-3] Continuation of Figure 14-2. [Figure 15A] Figures 15A and 15B show brain amino acid and neurotransmitter levels in Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 15A shows brain Phe, Tyr, and Trp levels. All treatment cohorts significantly reduced brain Phe levels, although variability was observed in the low (1e11vg / mouse) dose cohort. In this group, three animals with higher brain Phe levels correlated with higher blood Phe and lower hepatic gene transfer. [Figure 15B]Figures 15A and 15B show brain amino acid and neurotransmitter levels in Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 15B shows brain neurotransmitter levels of dopamine, norepinephrine, and serotonin. Here again, the three animals in the low-dose group with lower neurotransmitter levels represent the animals with higher blood Phe levels. The number of animals, statistical analysis, and abbreviations are as shown in the legend of Figures 11A–11C. [Figure 16-1] Figures 16A–16C show the effect of AAVXL32.1 / WT hPAH delivery to the liver on brain white matter content. White matter content was analyzed by quantifying corpus callosum volume using MRI. This was measured in live animals before treatment (Figure 16A) and 106 days after treatment (Figure 16B). Figure 16C shows the percentage change in corpus callosum volume for each animal. During the 4-month study period, all treated animals showed a significant increase, but no change was observed in HET and WT animals. Untreated Pah-KO mice (HOM) showed a slight decrease during this period. Figure 16D shows brain weight at the end of the study (120 days post-treatment). The cohort receiving only the 3e11 dose showed a significant increase in brain weight and did not reach the brain weight of normal animals. The number of animals, statistical analysis, and abbreviations are as shown in the legend of Figures 11A–11C. [Figure 16-2] Continuation of Figure 16-1. [Figure 17A] Figures 17A and 17B show the study-in-study behavioral analysis of Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 17A shows behavior as assessed by a nest-building assay that scored nest quality (from score 1 for no nest or low quality to score 5 for high quality). [Figure 17B]Figures 17A and 17B show the in-study behavioral analysis of Pah-KO mice 4 months after administration of AAVXL32.1 / WT hPAH. Figure 17B shows the results of nest-building assays performed before treatment and at 35 and 97 days after treatment. Improvement in nest-building scores was observed as early as day 35 and persisted until day 97 after treatment. The three animals in the low-dose group with lower scores represent animals with higher blood Phe levels. The number of animals, statistical analyses, and abbreviations are as shown in the legend of Figures 11A–11C. [Figure 18-1]Figures 18A–18G show a comparison of XL32.1 / WT hPAH vectors with various liver expression cassettes in vitro and in vivo. Figure 18A shows an illustration of the vectors. rAAVXL32.1 / mA1MB2-mTTR482-WT hPAH(A1MB2) represents the same vector used in the 4-month study. rAAVXL32.1 / LP1-HI2 contains the LP1 promoter with the same introns as used in rAAVXL32.1 / mA1MB2-mTTR482-WT hPAH. The rAAVXL32.1 / LP1-SI construct has the same promoter and introns as used in the hemophilia B clinical trial (Nathwani 2011). For in vitro analysis in human liver cell lines, each ITR containing the plasmid construct was transiently transfected into Huh7 cells in triplicates, and cell lysates were produced after 3 days. Figure 18B shows PAH protein levels in human cells in vitro. 10 μg of cell lysates were run in each lane, and PAH levels were analyzed by Western blotting using an anti-PAH antibody. Equal loading is shown with β-actin detection. Figure 18C shows the PAH activity assay in human cells in vitro. Phe to Tyr conversion was measured by a colorimetric assay and normalized to total protein measured by BCA. The data demonstrated that higher PAH protein and activity were generated by the A1MB2 construct in human liver cells. For in vivo analysis, each vector was administered IV to PAH-KO mice at 3e11vg / mouse and evaluated for 5 weeks. Figure 18D shows plasma Phe levels 36 days after vector delivery. Figure 18E shows the levels of vector-derived transcripts in the liver in each treatment cohort. Figure 18F shows liver PAH activity in each treatment cohort. Figure 18G shows PAH activity normalized by vector DNA in each animal. Due to the observed variability in vector DNA levels in the liver, PAH activity was normalized by vector DNA. Significance was determined by one-way ANOVA and Tukey's multiple comparisons as follows: *, p<0.05; **, p<0.01; ***, p<0.001; and ****, p<0.0001.Each data point represents one animal (n=10 in all groups, except for HOM, where n=4). [Figure 18-2] Continuation of Figure 18-1. [Figure 18-3] Continuation of Figure 18-2. [Modes for carrying out the invention]

[0023] In some embodiments, the present invention provides expression cassettes, recombinant adeno-associated virus (rAAV) vectors, and viral particles and pharmaceutical compositions, each comprising a transgene encoding a PAH polypeptide. In further embodiments, the present invention provides methods for treating phenylketonuria (PKU) by, for example, increasing PAH activity, increasing tyrosine and tryptophan transport to the brain, and normalizing brain neurotransmitter levels, including dopamine and serotonin. In further embodiments, the present invention provides methods for treating phenylketonuria (PKU), including, for example, increasing PAH activity, increasing tyrosine and tryptophan transport to the brain, and normalizing brain neurotransmitter levels, including dopamine and serotonin. In further additional embodiments, the present invention provides kits for treating PKU in an individual using the expression cassettes of the present disclosure.

[0024] General technology The techniques and procedures described or referenced herein are generally well understood and refer to, for example, Molecular Cloning: A Laboratory Manual (Sambrook et al., 4th edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012); Current Protocols in Molecular Biology (FMAusubel et al., eds., 2003); Methods in Enzymology series (Academic Press, Inc.); PCR 2: A Practical Approach (MJ MacPherson, B.D. Hames and GR. Taylor, eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane, eds., 1988); Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications (RI Freshney, 6th edition, J. Wiley and Sons, 2010); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, Academic Press, 1998); Introduction to Cell and Tissue Culture (JPMather and PERoberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, J. Wiley and Sons, 1993-98); Handbook of Experimental Immunology (DMWeir and C.C. Blackwell, 1996); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and MPCalos (ed.), 1987); PCR: The Polymerase Chain Reaction (Mullis et al., ed.), 1994; Current Protocols in Immunology (JEColigan et al., ed.), 1991; Short Protocols in Molecular Biology (Ausubel et al., J. Wiley and Sons, 2002); Immunobiology (CA Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, ed.), IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, eds.), Oxford University Press, 2000; Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JDCapra, eds.), Harwood Academic These methods are commonly adopted by those skilled in the art using conventional methodologies, such as those described in "Publishers, 1995" and "Cancer: Principles and Practice of Oncology" (edited by V.T. DeVita et al., JBLippincott Company, 2011).

[0025] definition As used herein, "vector" refers to a recombinant plasmid or virus containing nucleic acid intended to be delivered to a host cell, either in vitro or in vivo.

[0026] The terms “polynucleotide” or “nucleic acid,” as used herein, refer to polymeric forms of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. Therefore, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or other natural, chemically or biochemically modified, unnatural, or derivatized nucleotide bases. The backbone of a polynucleotide may contain sugars and phosphate groups (as typically found in RNA or DNA), or modified or substituted sugars or phosphate groups. Alternatively, the backbone of a polynucleotide may contain polymers of synthetic subunits such as phosphoramidates, and therefore may be oligodeoxynucleoside phosphoramidates (P-NH2) or hybrid phosphoramidate-phosphate diester oligomers. In addition, double-stranded polynucleotides can be obtained from chemically synthesized single-stranded polynucleotide products by either synthesizing a complementary strand and annealing the strand under appropriate conditions, or by de novo synthesizing the complementary strand with appropriate primers using DNA polymerase.

[0027] The terms “polypeptide” and “protein” are used synonymously and refer to polymers of amino acid residues, not limited to a minimum length. Such polymers of amino acid residues may contain native or non-native amino acid residues, and examples include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full-length proteins and their fragments are encompassed in this definition. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, and phosphorylation. Furthermore, for the purposes of this disclosure, “polypeptide” refers to proteins that include modifications such as deletions, additions, and substitutions (generally conserved in nature) of the native sequence, as long as the protein maintains the desired activity. These modifications may be planned, such as by site-directed mutagenesis, or they may be accidental, for example, accidental due to mutations in the host producing the protein or errors resulting from PCR amplification.

[0028] A "recombinant viral vector" refers to a recombinant polynucleotide vector containing one or more heterogeneous sequences (i.e., nucleic acid sequences that are not of viral origin). In the case of a recombinant AAV vector, the recombinant nucleic acid has at least one, in some embodiments, two, reverse terminal repeat sequences (ITRs) adjacent to each other.

[0029] A "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterogeneous sequences (i.e., nucleic acid sequences not of AAV origin) adjacent to at least one, in some embodiments, two AAV reverse-terminal repeat sequences (ITRs). Such rAAV vectors can be replicated and packaged into infectious viral particles when present in host cells infected with (or expressing) a suitable helper virus, and in host cells expressing AAV rep and cap gene products (i.e., AAV Re and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., into a chromosome, or into another vector, e.g., a plasmid used for cloning or transfection), the rAAV vector may also be referred to as a "pro-vector," which can be "rescued" by replication and capsidation in the presence of AAV packaging and suitable helper functions. rAAV vectors may be in any of many forms, including but not limited to those described above, complexed with lipids, encapsulated in liposomes, and viral particles, specifically capsidized within AAV particles, plasmids, linear artificial chromosomes, etc. rAAV vectors can be packaged in AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)."

[0030] "Heterogeneous" means that it is derived from an entity that is genetically distinct from the rest of the entity being compared to, introduced into, or incorporated into. For example, a polynucleotide introduced into a different cell type by genetic engineering is a heterogeneous polynucleotide (and may further encode a heterogeneous polypeptide if expressed). Similarly, a cellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterogeneous nucleotide sequence relative to the vector.

[0031] The term "transgene" refers to a polynucleotide that is introduced into a cell, transcribed into RNA, and can be optionally translated and / or expressed under appropriate conditions. In a manner, this may confer a desired characteristic to the cell into which it is introduced, or otherwise result in a desired therapeutic or diagnostic outcome.

[0032] The term "chicken β-actin (CBA) promoter" refers to a polynucleotide sequence derived from the chicken β-actin gene (e.g., chicken (Gallus gallus) beta-actin, represented by GenBank Entrez gene number 396526). As used herein, the term "chicken β-actin promoter" may also refer to a promoter containing the cytomegalovirus (CMV) early enhancer element, the promoter and first exon and intron of the chicken β-actin gene, and the splice acceptor of the rabbit β-globin gene, such as the sequence described in Miyazaki, J. et al. (1989) Gene 79(2):269-77. The term "CAG promoter" can be used synonymously as used herein. The term "CMV early enhancer / chicken β-actin (CAG) promoter" can be used synonymously as used herein.

[0033] The terms “genome particle (gp),” “genome equivalent,” or “genome copy,” when used in reference to a viral titer, refer to the number of virions containing the recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured, for example, in the examples herein, or by procedures such as those described, for example, Clark et al. (1999) Hum. Gene Ther., 10:1031-1039; Veldwijk et al. (2002) Mol. Ther., 6:272-278.

[0034] The term “vector genome (vg),” as used herein, may also refer to one or more polynucleotides comprising a set of polynucleotide sequences of a vector, such as a viral vector. A vector genome can be capsidized within a viral particle. Depending on the specific viral vector, a vector genome may comprise single-stranded DNA, double-stranded DNA, or single-stranded RNA, or double-stranded RNA. A vector genome may comprise endogenous sequences associated with a particular viral vector, and / or any heterologous sequences inserted into a particular viral vector via recombinant techniques. For example, a recombinant AAV vector genome may comprise at least one ITR sequence adjacent to the promoter, a stuffer, a sequence of interest (e.g., RNAi), and a polyadenylated sequence. A complete vector genome may comprise the complete set of polynucleotide sequences of the vector. In some embodiments, the titer of the nucleic acid of a viral vector can be measured in units of vg / mL. Preferred methods for measuring this titer are known in the art (e.g., quantitative PCR).

[0035] The terms “infectious unit (iu),” “infectious particle,” or “replication unit,” when used in reference to viral titers, refer to the number of infectious and replicable recombinant AAV vector particles as measured by an infectious center assay, also known as a replication center assay, as described, for example, by McLaughlin et al. (1988) J. Virol., 62:1963–1973.

[0036] When used in reference to a viral titer, the term “transduction unit (tu)” refers to the number of infectious recombinant AAV vector particles that result in the production of a functional transgene product, as measured by a functional assay, for example, the examples described herein, or, for example, Xiao et al. (1997) Exp. Neurobiol., 144:113-124; or Fisher et al. (1996) J. Virol., 70:520-532 (LFU assay).

[0037] "Reverse terminal repeats" or "ITR" sequences are well-understood terms in this field and refer to relatively short, reverse-direction sequences found at the ends of a viral genome.

[0038] An "AAV reverse terminal repeat (ITR)" sequence is a well-understood term in this field, referring to a sequence of approximately 145 nucleotides present at both ends of a natural single-stranded AAV genome. The outermost 125 nucleotides of an ITR may be located in one of two alternative directions, resulting in heterogeneity between different AAV genomes and between two ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter, self-complementary regions (named A, A', B, B', C, C', and D regions), which enable intra-strand base pairing within this portion of the ITR.

[0039] A "terminal degradation sequence" or "trs" is a sequence in the D region of the ITR of AAV that is cleaved by the AAV rep protein during viral DNA replication. Mutant terminal degradation sequences are refractory to cleavage by the AAV rep protein.

[0040] "AAV helper function" refers to a function that enables host cells to replicate and package AAV. AAV helper functions can be provided in any of numerous forms, such as helper viruses or helper viral genes, that facilitate AAV replication and packaging, but are not limited to these. Other AAV helper functions are known in the industry and include, for example, genotoxic substances.

[0041] A “helper virus” for AAV refers to a virus that enables host cells to replicate and package AAV (which is a deficient parvovirus). Helper viruses provide a “helper function” that enables AAV replication. Numerous such helper viruses have been identified, examples of which include adenoviruses, herpesviruses, and poxviruses, such as vaccinia and baculovirus. Adenoviruses encompass numerous different subgroups, but adenovirus type 5 (Ad5) of subgroup C is the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are publicly known and available from contract laboratories such as ATCC. Viruses of the herpes family are also available from contract laboratories such as ATCC, examples of which include, for example, herpes simplex virus (HSV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), and pseudorabies virus (PRV). Examples of adenovirus helper functions for AAV replication include E1A, E1B, E2A, VA, and E4orf6 functions. An example of a baculovirus available from contract laboratories is Autographa californica, a polyhedrosis virus.

[0042] The rAAV preparation has a ratio of infectious AAV particles to infectious helper virus particles of at least approximately 10 2 :1; at least about 10 4 :1, at least about 10 6 :1; or at least about 10 8A ratio of :1 or higher is said to be "substantially free" of helper viruses. In some embodiments, the preparation also does not contain an equivalent amount of helper virus protein (i.e., the protein that would be expected to be present as a result of such a level of helper virus if the helper virus particle impurities described above are present in a disintegrated form). Viral and / or cellular protein contamination can generally be observed as the presence of Coomassie staining bands in the SDS gel (e.g., the appearance of bands other than those corresponding to AAV capsid proteins VP1, VP2, and VP3).

[0043] The “sequence identity percentage (%)” of a reference polypeptide or nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to an amino acid residue or nucleotide in the reference polypeptide or nucleic acid sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum possible sequence identity percentage, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining amino acid or nucleic acid sequence identity percentage can be achieved in various ways within the capabilities of the art, for example, using publicly available computer software programs, such as those described in Current Protocols in Molecular Biology (Ausubel et al., 1987), Supp. 30, Section 7.7.18, Table 7.7.1, including BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A preferred alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve the best alignment over the entire length of the sequences being compared. For the purposes described herein, the amino acid sequence identity % of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can instead be said to be a given amino acid sequence A having or containing a specific amino acid sequence identity % to, with, or against a given amino acid sequence B) is calculated by multiplying the fraction X / Y by 100, where X is the number of amino acid residues that A and B have scored as identical in the alignment of that program by a sequence alignment program, and Y is the total number of amino acid residues in B. It is expected that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity % of A to B will not be equal to the amino acid sequence identity % of B to A.For the purposes described herein, the nucleic acid sequence identity % of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can instead be said to be a given nucleic acid sequence C having or containing a specific nucleic acid sequence identity % to, with, or against a given nucleic acid sequence D) is calculated by multiplying the fraction W / Z by 100, where W is the number of nucleotides scored by a sequence alignment program as identical matches in the alignment of C and D in that program, and Z is the total number of nucleotides in D. It is expected to be understood that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity % of C to D is not equal to the nucleic acid sequence identity % of D to C.

[0044] The term "isolated" means that a molecule (e.g., nucleic acid or protein) or cell has been identified, separated, and / or recovered from its natural environment.

[0045] An "effective dose" is the amount sufficient to produce a beneficial or desirable outcome, including clinical outcomes (e.g., improvement of symptoms, achievement of clinical endpoints). An effective dose may be administered in one or more doses. In relation to a disease state, an effective dose is the amount sufficient to improve, stabilize, or delay the onset of the disease.

[0046] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0047] Where used herein, “treatment” is an approach to obtain a beneficial or desirable clinical outcome. For the purposes of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, a stabilized (e.g., non-exacerbating) disease state, prevention of disease spread (e.g., metastasis), delay or slowing of disease progression, improvement or temporary relief of disease state, and remission (whether partial or complete), whether detectable or undetectable. “Treatment” may also mean extending survival compared to the survival expected without treatment.

[0048] As used herein, the term "preventive treatment" refers to treatment given to an individual that is known to have, suspected to have, or at risk of having a disability, but is not exhibiting symptoms of the disability or exhibiting only minimal symptoms. Individuals receiving preventive treatment can be treated before symptoms develop.

[0049] Phenylalanine hydroxylase (PAH), as used herein, is an enzyme (EC1.14.16.1) that catalyzes the hydroxylation of the aromatic side chain of phenylalanine to produce tyrosine. PAH is a monooxygenase that uses tetrahydrobiopterin (BH4, pteridine cofactor) and non-heme iron for catalytic action. During the reaction, molecular oxygen is cleaved by heterolysis through the sequential incorporation of one oxygen atom into BH4 and the phenylalanine substrate. Hydroxylation of phenylalanine to tyrosine is the rate-limiting step in the catabolism of phenylalanine, and a deficiency in this enzyme activity causes phenylketonuria, an autosomal recessive disorder. PAH may also be referred to as PH, PKU, or PKU1. PAH is a multi-domain protein consisting of an N-terminal regulatory domain (1-117), a central catalytic domain (118-410), and a C-terminal tetramerizing domain (411-452). Human PAHs are available in GenBank, for example, GenBank:AAA60082.1, NCBI reference sequence:NP_000268.1 (protein), and NCBI reference sequence:NM_000277.3 (mRNA). An example of wild-type human PAH is provided as Sequence ID No. 1.

[0050] "Phenylketonuria (PKU)," as used herein, refers to a genetic deficiency of the liver enzyme phenylalanine hydroxylase (PAH). If left untreated, severe forms of PKU can lead to highly elevated blood Phe levels, which are neurotoxic and associated with severe intellectual disability.

[0051] The term "mTTR promoter" refers to a polynucleotide sequence derived from the mouse trans tyretin gene. An example of an mTTR promoter, mTTR482, is provided by Kyostio-Moore, (2016) and Nambiar (2017).

[0052] A "modified prothrombin enhancer (mPrT2)" refers to two copies of a polynucleotide sequence derived from the human prothrombin gene. Examples of mPrT2 enhancers are provided by (McEachern 2006, Jacobs 2008). An example of an mPrT2 sequence is provided by Sequence ID No. 7.

[0053] "Modified alpha-1-microbikunin (mA1MB2)" refers to two copies of a polynucleotide sequence derived from the human alpha-1-microglobulin / bikunin gene. An example of mA1MB2 is an enhancer by (McEachern 2006, Jacobs 2008). An example of the mA1MB2 sequence is provided by Sequence ID No. 8.

[0054] A "modified mouse albumin enhancer (mEalb)" refers to a polynucleotide sequence derived from the mouse albumin gene. An example of an mEalb enhancer is provided by (Kramer 2003). An example of an mEalb sequence is provided by Sequence ID No. 9.

[0055] "Hepatitis B virus enhancer II (HE11)" refers to a polynucleotide sequence derived from hepatitis B virus located upstream of the PreCore promoter. An example of an hEII enhancer is provided by (Kramer 2003). An example of an HEII sequence is provided by SEQ ID NO: 10.

[0056] "CRM8" refers to a cis-acting regulatory module derived from a polynucleotide sequence of the human Serpina1 gene (Chuah 2014). An example of a CRM8 sequence is provided by SEQ ID NO: 11.

[0057] "Alb3'" refers to the 3' polynucleotide sequence relative to the coding region of the human albumin gene. An example of an Alb3' element is provided by Wooddell (2008). An example of an Alb3' sequence is provided by Sequence ID No. 12. "Alb3' / SMAR" refers to Alb3' ligated to the scaffold / matrix attachment region of the human alpha-1 anti-trypsin gene (AF156542). An example of an Alb3' / SMAR sequence is provided by Sequence ID No. 13.

[0058] In this specification, any reference to a value or parameter "about" includes (and describes) embodiments directed to that value or parameter itself. For example, any statement referring to "about X" includes any statement of "X".

[0059] As used herein, the singular forms of the articles "a," "an," and "the" include plural subjects unless otherwise specified.

[0060] The aspects and embodiments of the disclosure described herein are understood to include aspects and embodiments that "include," "consist of," and / or "essentially consist of."

[0061] Liver-specific expression cassette In some embodiments, the present invention provides an expression cassette for expressing a transgene in hepatocytes, wherein the expression cassette comprises a transgene operably ligated to a promoter and an enhancer, the promoter comprising a mouse trans tiretin (mTTR) promoter, and the enhancer comprising one or two modified prothrombin enhancers (mPrT2), one or two modified alpha-1-microbicinin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HE11), or CRM8 enhancer. In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the promoter comprises an mTTR core promoter and an mTTR upstream enhancer. In some embodiments, the enhancer is 5' relative to the mTTR promoter. In some embodiments, the transgene encodes a PAH polypeptide as described herein.

[0062] In some embodiments, the present invention provides an expression cassette for expressing a transgene in liver cells, comprising a promoter and a transgene operably ligated to a 3' element, wherein the promoter comprises a mouse trans tiretin (mTTR) promoter and the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element ligated to a human α1 anti-trypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR). In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the 3' element is located 3' relative to the transgene. In some embodiments, the transgene encodes a PAH polypeptide as described herein.

[0063] In some embodiments, the present invention provides an expression cassette for expressing a transgene in hepatocytes, wherein the expression cassette comprises a promoter and an enhancer and a transgene operably ligated to a 3' element, the promoter comprising a mouse trans tyretin (mTTR) promoter, the enhancer comprising one or two modified prothrombin enhancers (mPrT2), one or two modified alpha-1-microbicinin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HE11), or a CRM8 enhancer, and the 3' element being an albumin 3' element (3'Alb) or an albumin 3' element ligated to a human alpha-1 antitrypsin scaffold / matrix attachment region (SMAR) (3'AlbSMAR). In some embodiments, the mTTR promoter is the mTTR482 promoter. In some embodiments, the enhancer is 5' relative to the mTTR promoter. In some embodiments, the 3' element is located 3' relative to the transgene. In some embodiments, the transgene encodes a PAH polypeptide described herein.

[0064] In some embodiments, the present invention provides an expression cassette for expressing a transgene in liver cells, wherein the transgene encodes a PAH polypeptide. In some embodiments, the PAH polypeptide is a wild-type PAH polypeptide. In some embodiments, the PAH polypeptide is a human PAH polypeptide. In some embodiments, the PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the PAH polypeptide is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the transgene is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the PAH polypeptide comprises the E183 residue. In some embodiments, the PAH polypeptide comprises a glutamic acid residue at amino acid residue number 183. In some embodiments, PAH polypeptides exhibit higher levels of PAH activity than PAH polypeptides containing the E183G amino acid substitution.

[0065] In some embodiments, the transgene encoding the PAH polypeptide is codon-optimized. In some embodiments, the transgene encoding the PAH polypeptide is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells may be cells of or derived from specific organisms, such organisms being, but not limited to, mammals, including humans, mice, rats, rabbits, dogs, or non-human primates. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by maintaining the native amino acid sequence while replacing at least one codon in the native sequence with a codon that is more frequently used or most frequently used in the host cell's gene. Different species exhibit specific biases for specific codons of specific amino acids. Codon frequency tables are readily available, for example, in "codon frequency databases," and these tables can be adapted in numerous ways (see, e.g., Nakamura, Y. et al. (2000) Nucleic Acids Res. 28:292). Computer algorithms are also available for codon-optimizing specific sequences for expression in specific host cells. Examples include Gene Forge (Aptagen; Jacobus, Pa.), DNA2.0, GeneArt (GA), or Genscript (GS), and the GS algorithm combined with CpG content reduction. In some embodiments, transgenes encoding PAH polypeptides have been codon-optimized using the GA algorithm.

[0066] In some embodiments, the expression cassette further comprises an intron. Various introns for use in the present invention are known to those skilled in the art, and examples include the MVM intron, F IX shortened intron 1, β-globin SD / immunoglobin heavy chain SA, adenovirus SD / immunoglobin SA, SV40 late SD / SA (19S / 16S), and hybrid adenovirus SD / IgG SA. (Wu et al., 2008; Kurachi et al., 1995; Choi et al., 2014; Wong et al., 1985; Yew et al., 1997; Huang and Gorman (1990). In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid promoter and intron in which all ATG sites have been removed to minimize the erroneous translation initiation site (SEQ ID NO: 15). In some embodiments, the intron is an MVM intron, a FIX shortened intron 1, β-globin SD / immunoglobin heavy chain SA, adenovirus SD / immunoglobin SA, SV40 late SD / SA (19S / 16S), or hybrid adenovirus SD / IgG SA. In some embodiments, the intron is a chicken β-actin (CBA) / rabbit β-globin hybrid intron.

[0067] In some embodiments, the expression cassette further includes a polyadenylation signal. In some embodiments, the polyadenylation signal is a bovine growth hormone polyadenylation signal, an SV40 polyadenylation signal, or an HSV TK pA. In some embodiments, the polyadenylation signal is a synthetic polyadenylation signal, such as those described by Levitt, N et al. (1989), Genes Develop. 3:1019-1025.

[0068] In some embodiments, the expression cassette includes a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may include a sequence encoding a reporter polypeptide. As expected to be recognized by those skilled in the art, the stuffer nucleic acid may be located in various regions within the nucleic acid, or may consist of a contiguous sequence within the nucleic acid (e.g., a single stuffer nucleic acid at a single location) or multiple sequences (e.g., one or more stuffer nucleic acids at more than one location (e.g., two locations, three locations, etc.)). In some embodiments, the stuffer nucleic acid may be located downstream of the transgene encoding the PAH polypeptide. In embodiments, the stuffer nucleic acid may be located upstream of the transgene encoding the PAH polypeptide (e.g., between the promoter and the transgene). Similarly, as expected to be recognized by those skilled in the art, various nucleic acids can be used as stuffer nucleic acids. In some embodiments, the stuffer nucleic acid includes all or part of a human alpha-1-anti-trypsin (AAT) stuffer sequence or a C16P1 chromosome 16 P1 clone (human C16) stuffer sequence. In some embodiments, the stuffer sequence includes all or part of a gene. For example, the stuffer sequence includes a portion of the human AAT sequence. Those skilled in the art are expected to recognize that different parts of a gene (e.g., the human AAT sequence) can be used as stuffer fragments. For example, a stuffer fragment may be from the 5' end of a gene, the 3' end of a gene, the middle of a gene, a non-coding portion of a gene (e.g., an intron), a coding region of a gene (e.g., an exon), or a mixture of non-coding and coding portions of a gene. Those skilled in the art are also expected to recognize that all or part of a stuffer sequence can be used as a stuffer sequence. In some embodiments, the stuffer sequence is modified to remove the internal ATG codon. In some embodiments, the stuffer sequence includes the nucleotide sequence of SEQ ID NO: 16.

[0069] In some embodiments, the expression cassette is incorporated into a vector. In some embodiments, the expression cassette is incorporated into a viral vector. In some embodiments, the vector contains the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the viral vector is the rAAV vector described herein.

[0070] Vectors and viral particles In certain embodiments, an expression cassette for expressing a PAH polypeptide (e.g., wild-type human PAH polypeptide) is contained within the vector. In some embodiments, the present invention intends to use a recombinant viral genome for introducing a nucleic acid sequence encoding a PAH polypeptide for packaging into viral particles, e.g., the viral particles described below. The recombinant viral genome may include any elements for establishing PAH polypeptide expression, e.g., promoters, ITRs, ribosome-binding elements, terminators, enhancers, selection markers, introns, poly(A) signals, and / or origins of replication. Exemplary viral genome elements and delivery methods for viral particles are described in more detail below.

[0071] Nonviral delivery systems Conventional non-viral gene delivery methods are also used to introduce nucleic acids into cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acids, and nucleic acids that are conjugated to the delivery system. For example, the vector may be conjugated to lipids (e.g., cationic or neutral lipids), liposomes, polycations, nanoparticles, or agents that enhance the cellular uptake of the nucleic acid. The vector may be conjugated to an agent suitable for any of the delivery methods described herein. In some embodiments, the nucleic acid may contain one or more viral ITRs (e.g., AAV ITRs).

[0072] Virus particles In some embodiments, the vector containing an expression cassette for expressing a PAH polypeptide (e.g., wild-type human PAH polypeptide) is a recombinant adeno-associated virus (rAAV) vector, a recombinant adenovirus vector, a recombinant lentivirus vector, or a recombinant herpes simplex virus (HSV) vector.

[0073] rAAV particles In some embodiments, the vector is a recombinant AAV (rAAV) vector. In some embodiments, the expression cassette for expressing a PAH polypeptide (e.g., wild-type human PAH polypeptide) has one or more AAV reverse-terminal repeat (ITR) sequences adjacent to each other. In some embodiments, the viral particle is a recombinant AAV particle containing an expression cassette for expressing a PAH polypeptide with one or two adjacent ITRs. In some embodiments, the expression cassette for expressing a PAH polypeptide has two adjacent AAV ITRs. In some embodiments, the vector contains the nucleic acid sequence of SEQ ID NO: 14.

[0074] In some embodiments, an expression cassette for expressing the PAH polypeptide of this disclosure is operably linked to a control sequence containing components of the transcription direction, transcription start and end sequences, thereby forming the expression cassette. The expression cassette is adjacent to at least one functional AAV ITR sequence at its 5' and 3' ends. "Functional AAV ITR sequence" means that the ITR sequence functions as intended for the rescue, replication, and packaging of AAV virions. See Davidson et al., PNAS, 2000, 97(7):34 pp. 28-32; Passini et al., J. Virol., 2003, 77(12):70 pp. 34-40; and Pechan et al., Gene Ther., 2009, 16:10-16, all of which are incorporated herein by reference as a whole. To carry out certain aspects of the present invention, the recombinant vector comprises at least all of the AAV sequence essential for encapsidation and the physical structure for infection by rAAV. The AAV ITR for use in the vector of the present invention does not need to have a wild-type nucleotide sequence (e.g., a sequence like the one described in Kotin, Hum. Gene Ther., 1994, 5:793-801), and may have modifications by nucleotide insertions, deletions, or substitutions, or the AAV ITR may be derived from any of several AAV serotypes. More than 40 AAV serotypes are currently known, and new serotypes and variants of existing serotypes continue to be identified. See Gao et al., PNAS, 2002, 99(18):11854-11866; Gao et al., PNAS, 2003, 100(10):6081-6066; and Bossis et al., J. Virol., 2003, 77(12):6799-810.

[0075] The use of any AAV serotype is considered to be within the scope of the present invention. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, for example, but not limited to, AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV, or mouse AAV ITR. In some embodiments, the nucleic acid in the AAV contains the ITR of the AAV. AAV ITRs such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV, or mouse AAV ITR. In certain embodiments, the AAV ITR is the AAV2 ITR.

[0076] In some embodiments, the vector may include a stuffer nucleic acid. In some embodiments, the stuffer nucleic acid may encode a green fluorescent protein. In some embodiments, the stuffer nucleic acid may be located at 3' relative to the expression cassette for expressing the PAH polypeptide of the Disclosure.

[0077] In some embodiments, the present invention provides viral particles containing a recombinant self-complementary genome. In some embodiments, the vector is a self-complementary vector. Methods of using AAV viral particles having a self-complementary genome and self-complementary AAV genomes are described in U.S. Patents 6,596,535, 7,125,717, 7,765,583, 7,785,888, 7,790,154, 7,846,729, 8,093,054, and 8,361,457, as well as Wang Z. et al., (2003) Gene Ther 10:2105-2111, each of which is incorporated herein by reference in whole. rAAV containing a self-complementary genome rapidly forms a double-stranded DNA molecule due to its partially complementary sequences (e.g., complementary coding and non-coding strands of the transgene). In some embodiments, the present invention provides an AAV virus particle comprising an AAV genome, wherein the rAAV genome comprises a first heterologous polynucleotide sequence (e.g., the coding strand of the PAH polypeptide of the present invention) and a second heterologous polynucleotide sequence (e.g., the non-coding strand or antisense strand of the PAH polypeptide of the present disclosure), wherein the first heterologous polynucleotide sequence can form intrachain base pairs with the second polynucleotide sequence along most or all of its length.

[0078] In some embodiments, a first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence are linked by a sequence that facilitates intrachain base pairing, such as a hairpin DNA structure. Hairpin structures are known in the art and are found, for example, in siRNA molecules. In some embodiments, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a mutant ITR (e.g., a right-handed ITR). The mutant ITR contains a deletion in the D region, which includes a terminal degradation sequence. As a result, when the AAV viral genome is replicated, the rep protein does not cleave the viral genome at the mutant ITR, so a recombinant viral genome containing the following in order from 5' to 3' is packaged into the viral capsid: AAV ITR, a first heterologous polynucleotide sequence containing a regulatory sequence, a mutant AAV ITR, a second heterologous polynucleotide oriented in the opposite direction to the first heterologous polynucleotide, and a third AAV ITR.

[0079] In some embodiments, the first heterologous nucleic acid sequence and the second heterologous nucleic acid sequence are linked by a mutant ITR (e.g., a right ITR). In some embodiments, the ITR contains the polynucleotide sequence 5'-CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCACGCCCGGGCTTTGCCCGGGCG-3' (SEQ ID NO: 17). The mutant ITR contains a deletion in the D region, which includes a terminal degradation sequence. As a result, when the AAV viral genome is replicated, the rep protein does not cleave the viral genome at the mutant ITR, so a recombinant viral genome containing the following in order from 5' to 3' is packaged into the viral capsid: AAV ITR, the first heterologous polynucleotide sequence including a regulatory sequence, the mutant AAV ITR, the second heterologous polynucleotide reversed relative to the first heterologous polynucleotide, and the third AAV ITR.

[0080] In some embodiments, the vector is capsid-formed within the viral particle. In some embodiments, the viral particle is a recombinant AAV viral particle containing a recombinant AAV vector. Different AAV serotypes are used to optimize the transduction of specific target cells or to target specific cell types within specific target tissues (e.g., liver tissue). rAAV particles may contain viral proteins and viral nucleic acids of the same or mixed serotypes. For example, in some embodiments, rAAV particles may contain the AAV2 capsid protein of the present invention and at least one AAV2 ITR, or the AAV2 capsid protein and at least one AAV1 ITR. Any combination of AAV serotypes for the preparation of rAAV particles is provided herein, as each combination is expressly described herein. In some embodiments, the present invention provides rAAV particles containing the AAV2 capsid of the present invention. In some embodiments, the present invention provides rAAV particles containing the AAVrh8R capsid of the present invention. In some embodiments, the present invention provides rAAV particles containing the manipulated AAV capsid of the present invention. In some embodiments, the present invention provides rAAV particles comprising the AAV-XL32 capsid of the present invention. In some embodiments, the present invention provides rAAV particles comprising the AAV-XL32.1 capsid of the present invention.

[0081] In some embodiments, the rAAV particles include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid (e.g., wild-type AAV6 capsid, or variant AAV6 capsid such as ShH10 described in U.S. Patent Application Publication 2012 / 0164106), AAV7 capsid, AAV8 capsid, AAVrh8 capsid, and AAVrh 8R capsid, AAV9 capsid (e.g., wild-type AAV9 capsid, or modified AAV9 capsid as described in U.S. Patent Application Publication No. 2013 / 0323226), AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid variant, heparin-binding capsid variant, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, AAV This includes DJ capsids (e.g., AAV-DJ / 8 capsid, AAV-DJ / 9 capsid, or any other capsid described in U.S. Patent Application Publication 2012 / 0066783), AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, rAAV2 / HBoV1 capsid, or AAV capsids described in U.S. Patent No. 8,283,151 or International Publication 2003 / 042397. In some embodiments, the AAV particles include AAV-XL32.1 capsid. In some embodiments, the AAV particles contain the AAV-XL32 capsid. In some embodiments, the AAV particles contain the AAV capsid described in International Publication No. 2019241324A1. In some embodiments, the mutant capsid protein retains the ability to form an AAV capsid. In some embodiments, the rAAV particles contain the AAV5 tyrosine mutant capsid (Zhong L. et al., (2008) Proc Natl Acad Sci USA 105(22):7827-7832). In further embodiments, the rAAV particles contain capsid proteins of AAV serotypes from clades A-F (Gao et al., J. Virol., 2004, 78(12):6381).In some embodiments, rAAV particles contain the AAV1 capsid protein or a variant thereof. In other embodiments, rAAV particles contain the AAV2 capsid protein or a variant thereof. In some embodiments, the AAV serotype is AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In some embodiments, rAAV particles contain the AAV serotype 1 (AAV1) capsid. In some embodiments, rAAV particles contain the AAV serotype 2 (AAV2) capsid. In some embodiments, recombinant AAV virus particles contain the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsids. In some embodiments, the AAV1, AAV2, AAV8, AAVrh8R, AAV9, and / or AAVrh10 capsids include, for example, tyrosine mutations or heparan-binding mutations as described below. In some embodiments, the capsid is a liver-targeted capsid, and is, for example, but not limited to, LK03 capsid, HSC15 capsid, 17 capsid, AAV-XL-32, or AAV-XL32.1 capsid. In some embodiments, the capsid is an engineered AAV capsid (e.g., a shuffled capsid). Examples of manipulated capsids include, but are not limited to, DJ (Grimm D et al., J Virol., 2008, 82:5887-911), LK03 (Lisowski L et al., Nature, 2014, 506:382-386), and HSC15 and HSC17 (Smith LJ et al., Mol Ther, September 2014; 22(9):1625-34).

[0082] The capsids of AAVs (e.g., AAV2, AAV8, etc.) are known to contain three capsid proteins: VP1, VP2, and VP3. These proteins contain a considerable amount of duplicated amino acid sequences and unique N-terminal sequences. The AAV2 capsid contains 60 subunits arranged by icosahedral symmetry (Xie, Q. et al. (2002) Proc. Natl. Acad. Sci. USA, 99(16):104 pp. 05-10). VP1, VP2, and VP3 are known to exist in a ratio of 1:1:10.

[0083] In some embodiments, the rAAV particles comprise a) an rAAV capsid protein having one or more amino acid substitutions at one or more positions that interact with a heparan sulfate proteoglycan, and b) an rAAV vector having heterogeneous nucleic acid and at least one AAV reverse terminal repeat.

[0084] In some embodiments, the rAAV particles include one or more amino acid substitutions in the capsid protein that reduce or eliminate the binding of the rAAV particles to the heparan sulfate proteoglycan, wherein the one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 in the numbering based on the VP1 numbering of AAV2. As used herein, “VP1 numbering of AAV2” means the listed amino acids of the capsid protein corresponding to the listed amino acids of the VP1 of AAV2. For example, if one or more amino acid substitutions are located at positions 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 in the numbering based on VP1 of AAV2, then one or more amino acid substitutions are located in the enumerated capsid protein amino acids corresponding to amino acids 347, 350, 390, 395, 448, 451, 484, 487, 527, 532, 585, and / or 588 of VP1 of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP1 of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 484, 487, 532, 585, or 588 of VP1 in AAV3, based on the VP1 numbering of AAV2. In some embodiments, one or more amino acid substitutions are located at positions 485, 488, 528, 533, 586, or 589 of AAVrh8R, based on the VP1 numbering. In some embodiments, one or more amino acids at the positions corresponding to amino acids 585 and / or 588 (based on the VP1 numbering of AAV2) are replaced by arginine residues (e.g., S586 and / or T589 for AAV1 or AAV6, S586 and / or A589 for AAV9, A586 and / or T589 for AAVrh8R, Q588 and / or T591 for AAV8, and Q588 and / or A591 for AAVrh10).In other embodiments, one or more amino acids (e.g., arginine or lysine) at the positions corresponding to amino acids 484, 487, 527 and / or 532 (numbering based on VP1 for AAV2) are replaced by positively uncharged amino acids such as alanine (e.g., R485, R488, K528 and / or K533 for AAV1 or AAV6; R485, R488, K528 and / or R533 for AAV9 or AAVrh8R; and R487, R490, K530 and / or R535 for AAV8 or AAVrh10).

[0085] XL32 and XL32.1 capsids and capsid proteins In some embodiments, the AAV particles include an engineered AAV capsid. In some embodiments, the engineered AAV capsid is an AAV-XL32 capsid. In some embodiments, the AAV-XL32 capsid includes an AAV-XL32 capsid protein. In some embodiments, the AAV-XL32 capsid includes an AAV-XL32 capsid protein having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the AAV-XL32 capsid includes a capsid protein encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid comprises a capsid protein encoded by a nucleic acid that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 4.

[0086] In some embodiments, the AAV particle contains an AAV-XL32 capsid. In some embodiments, the AAV-XL32 capsid contains VP1, VP2, and VP3, which are encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid contains VPX, which is encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid contains a capsid protein, which is encoded by an open reading frame within the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid contains 1, 2, 3, or 4 capsid proteins, which are encoded by an open reading frame within the nucleic acid sequence of SEQ ID NO: 4.

[0087] In some embodiments, the AAV particles contain the AAV-XL32 capsid protein. In some embodiments, the AAV-XL32 capsid protein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the AAV particles contain a capsid protein encoded by the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid contains a capsid protein encoded by a nucleic acid that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV-XL32 capsid protein is encoded by an open reading frame within the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the AAV particles contain 1, 2, 3, or 4 capsid proteins, which are encoded by an open reading frame within the nucleic acid sequence of Sequence ID No. 4. In some embodiments, the AAV virus particles contain the AAV capsid protein encoded by the nucleic acid sequence of Sequence ID No. 4. In some embodiments, the AAV particles contain VP1, VP2, and VP3, which are encoded by the nucleic acid sequence of Sequence ID No. 4. In some embodiments, the AAV particles contain VPX, which is encoded by the nucleic acid sequence of Sequence ID No. 4. In some embodiments, the AAV virus particles contain VP1, VP2, VP3, and VPX, which are encoded by the nucleic acid sequence of Sequence ID No. 4.

[0088] In some embodiments, the AAV particles include an engineered AAV capsid. In some embodiments, the engineered AAV capsid is an AAV-XL32.1 capsid. In some embodiments, the AAV-XL32.1 capsid includes an AAV-XL32.1 capsid protein. In some embodiments, the AAV-XL32.1 capsid includes an AAV-XL32.1 capsid protein having an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the AAV-XL32.1 capsid includes a capsid protein encoded by the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the AAV-XL32.1 capsid comprises a capsid protein encoded by a nucleic acid that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 6.

[0089] In some embodiments, the AAV particle contains an AAV-XL32.1 capsid. In some embodiments, the AAV-XL32.1 capsid contains VP1, VP2, and VP3, which are encoded by the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the AAV-XL32.1 capsid contains a capsid protein, which is encoded by an open reading frame within the nucleic acid sequence of SEQ ID NO: 6.

[0090] In some embodiments, the AAV particles contain the AAV-XL32.1 capsid protein. In some embodiments, the AAV-XL32.1 capsid protein contains an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 3. In some embodiments, the AAV particles contain a capsid protein encoded by the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the AAV-XL32.1 capsid contains a capsid protein encoded by a nucleic acid that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the AAV-XL32.1 capsid protein is encoded by an open reading frame within the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, AAV particles contain one, two, or three capsid proteins, which are encoded by an open reading frame within the nucleic acid sequence of Sequence ID No. 6. In some embodiments, AAV virus particles contain AAV capsid proteins encoded by the nucleic acid sequence of Sequence ID No. 6. In some embodiments, AAV particles contain VP1, VP2, and VP3, which are encoded by the nucleic acid sequence of Sequence ID No. 6. In some embodiments, AAV particles contain one, two, or three capsid proteins, which are encoded by an open reading frame within the nucleic acid sequence of Sequence ID No. 6.

[0091] In some embodiments, the AAV particles contain the AAV capsid described in International Publication No. 2019241324A1. In some embodiments, the AAV particles contain the AAV capsid protein described in International Publication No. 2019241324A1.

[0092] AAV particle production Numerous methods for producing rAAV vectors are known in the art, including transfection, stable cell line production, and infectious hybrid virus production systems, including adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J. Virology, 71(11):8780-8789) and baculovirus-AAV hybrids (Urabe, M. et al., (2002) Human Gene Therapy, 13(16):1935-1943; Kotin, R. (2011) Hum Mol Genet. 20(R1):R2-R6). All rAAV-producing cultures for the production of rAAV virus particles require 1) suitable host cells, 2) suitable helper virus function, 3) AAV rep and cap genes and gene products, 4) nucleic acids (such as therapeutic nucleic acids) adjacent to at least one AAV ITR sequence (e.g., an AAV genome encoding a PAH polypeptide), and 5) suitable culture media and culture components to support rAAV production. In some embodiments, suitable host cells are primate host cells. In some embodiments, suitable host cells are human-derived cell lines, e.g., HeLa, A549, 293, or Perc.6 cells. In some embodiments, suitable helper virus function is provided by wild-type or mutant adenovirus (e.g., temperature-sensitive adenovirus), herpesvirus (HSV), baculovirus, or plasmid constructs providing helper function. In some embodiments, the AAV rep and cap gene products may be derived from any AAV serotype. While not always the case, generally, the AAV rep gene product is a gene product of the same serotype as the ITR of the rAAV vector genome, insofar as the rep gene product can function in the replication and packaging of the rAAV genome. Suitable media known in the art are used for the production of the rAAV vector.These media include, but are not limited to, modified Eagle medium (MEM), Dulbecco's modified Eagle medium (DMEM), media manufactured by Hyclone Laboratories and JRH, custom preparations such as those described in U.S. Patent No. 6,566,118, and Sf-900II SFM medium described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference as a whole, particularly in relation to custom media preparations for use in the production of recombinant AAV vectors. In some embodiments, the AAV helper function is provided by an adenovirus or HSV. In some embodiments, the AAV helper function is provided by a baculovirus, and the host cell is an insect cell (e.g., Spodoptera frugiperda (Sf9) cell).

[0093] One method for producing rAAV particles is the triple transfection method. Briefly, a plasmid containing the rep gene and capsid gene is transfected into a cell line (e.g., HEK-293 cells) together with a helper adenovirus plasmid (e.g., using the calcium phosphate method), the virus is recovered, and optionally purified. Thus, in some embodiments, rAAV particles are produced by triple transfection into a host cell of nucleic acids encoding the rAAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus function, and the transfection of nucleic acids into a host cell generates a host cell capable of producing rAAV particles.

[0094] In some embodiments, rAAV particles are produced by a producer cell line method (Martin et al., (2013) Human Gene Therapy Methods, 24:253-269; U.S. Patent Application Publication No. 2004 / 0224411; and Liu, XL et al., (1999) Gene Ther. 6:293-299). Briefly, a cell line (e.g., HeLa, 293, A549, or Perc. 6 cell line) may be stably transfected with a plasmid containing a vector genome comprising a rep gene, a capsid gene, and a promoter-heterogeneous nucleic acid sequence (e.g., PAH polypeptide). The cell line may be screened to select a lead clone for rAAV production, then expanded into a production bioreactor, and infected with a helper virus (e.g., adenovirus or HSV) to initiate rAAV production. Subsequently, the viruses may be collected and the adenovirus may be inactivated (e.g., by heat) and / or removed to purify the rAAV particles. Thus, in some embodiments, the rAAV particles were produced using a producer cell line containing one or more nucleic acids from among those encoding the rAAV vector, nucleic acids encoding AAV rep and cap, and nucleic acids encoding AAV helper virus function. As described herein, the producer cell line method may be advantageous for producing rAAV particles with oversized genomes compared to the triple transfection method.

[0095] In some embodiments, the nucleic acids encoding the AAV rep and cap genes and / or the rAAV genome are stably maintained in the producer cell line. In some embodiments, the nucleic acids encoding the AAV rep and cap genes and / or the rAAV genome are introduced into the cell line in one or more plasmids to generate the producer cell line. In some embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell in the same plasmid. In other embodiments, the AAV rep, AAV cap, and rAAV genome are introduced into the cell in different plasmids. In some embodiments, the plasmid-transfected cell line maintains the plasmid over multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50, or more than 50 passages of the cell). For example, the plasmid is replicated when the cell replicates, or the plasmid is incorporated into the cell genome. Various sequences have been identified that enable plasmids to autonomously replicate within cells (e.g., human cells) (e.g., Krysan, PJ et al., (1989) Mol. Cell Biol. 9: pp. 1026-1033). In some embodiments, plasmids may contain selection markers (e.g., antibiotic resistance markers) that allow for the selection of cells that maintain the plasmid. Commonly used selection markers in mammalian cells include, but are not limited to, blasticidine, G418, hygromycin B, zeosin, puromycin, and their derivatives. Methods for introducing nucleic acids into cells are known in the art and include, but are not limited to, viral transduction, cationic transfection (using, for example, cationic polymers such as DEAE-dextran or cationic lipids such as lipofectamine), calcium phosphate transfection, microinjection, particulate guns, electroporation, and nanoparticle transfection (for details, see, for example, Kim, TK and Eberwine, JH (2010) Anal. Bioanal. Chem., 397: pp. 3173-3178).

[0096] In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are stably incorporated into a producer cell line. In some embodiments, nucleic acids encoding AAV rep and cap genes and / or rAAV genomes are introduced into a cell line in one or more plasmids to generate a producer cell line. In some embodiments, AAV rep, AAV cap, and rAAV genome are introduced into cells in the same plasmid. In other embodiments, AAV rep, AAV cap, and rAAV genome are introduced into cells in different plasmids. In some embodiments, the plasmid may contain a selection marker (e.g., an antibiotic resistance marker) that allows for the selection of cells that retain the plasmid. Methods for stably incorporating nucleic acids into various host cell lines are known in the art. For example, cells incorporating nucleic acids containing selection markers (as well as AAV cap and rep genes and / or rAAV genomes) are selected using repeated selection (e.g., through the use of a selection marker). In other embodiments, nucleic acids may be incorporated into a cell line in a site-specific manner to generate a producer cell line. Several site-directed recombination systems are known in the art, such as FLP / FRT (e.g., O'Gorman, S. et al., (1991), Science 251: pp. 1351-1355), Cre / loxP (e.g., Sauer, B. and Henderson, N. (1988), Proc. Natl. Acad. Sci. 85: pp. 5166-5170), and phi C31-att (e.g., Groth, AC et al., (2000), Proc. Natl. Acad. Sci. 97: pp. 5995-6000).

[0097] In some embodiments, the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line, e.g., Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from HeLa, 293, A549, or PERC.6® (Crucell) cells. For example, before introducing and / or stably maintaining / incorporating nucleic acids encoding AAV rep and cap genes and / or an oversized rAAV genome into the cell line to generate the producer cell line, the cell line is a HeLa, 293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof.

[0098] In some embodiments, producer cell lines are adapted for growth in suspension. As is well known in the art, anchor-dependent cells typically cannot grow in suspension without a substrate such as microcarrier beads. Adapting a cell line for growth in suspension may include, for example, growing the cell line in a spinner culture medium using a stirring paddle, using a medium lacking calcium and magnesium ions (and optionally an antifoaming agent) to prevent aggregation, using a culture vessel coated with a silicone compound, and selecting cells in the culture medium at each passage (not in large aggregates or on the sides of the vessel). For further explanation, see, for example, the ATCC Frequently Asked Questions document (available from www.atcc.org / Global / FAQs / 9 / 1 / Adapting%20a%20monolayer%20cell%20line%20to%20suspension-40.aspx) and the references cited therein.

[0099] In some embodiments, a method for producing any rAAV particles disclosed herein is provided, comprising the steps of (a) culturing host cells under conditions under which rAAV particles are produced, wherein the host cells include (i) one or more AAV package genes, each encoding an AAV replication and / or capsid-forming protein, (ii) an rAAV provector containing a nucleic acid encoding a heterologous nucleic acid as described herein adjacent to at least one AAV ITR, and (iii) an AAV helper function, and (b) recovering the rAAV particles produced by the host cells. In some embodiments, the at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, goat AAV, bovine AAV, or mouse AAV serotype ITR, etc. For example, in some embodiments, the AAV serotypes are AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, or AAVrh10. In certain embodiments, the nucleic acids in the AAV include AAV2 ITR. In some embodiments, the capsid-forming protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2 / 2-7m8, AAV DJ, AAV2 N587A, AAV2 E548A, AAV2N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV capsid, rAAV2 / HBoV1 serotype, AAV-XL32, or AAV-XL32.1 capsid protein or its variants. In some embodiments, the capsid-forming protein is the AAV8 capsid protein.In some embodiments, the rAAV particle comprises a recombinant genome including the AAV8 capsid and AAV2 ITR, as well as a nucleic acid encoding a therapeutic transgene / nucleic acid (e.g., an expression cassette for expressing a PAH polypeptide). In some embodiments, the capsidized protein is the AAV-XL32 capsid protein. In some embodiments, the capsidized protein is the AAV-XL32.1 capsid protein. In some embodiments, the capsidized protein comprises the amino acid sequence of SEQ ID NO: 3.

[0100] The preferred rAAV-producing culture medium of the present invention may be supplemented with serum or serum-derived recombinant protein at levels of 0.5% to 20% (v / v or w / v). Alternatively, as is well known in the art, rAAV vectors may be prepared under serum-free conditions, also referred to as a medium without animal-derived products. Those skilled in the art will understand that commercially available or custom media designed to support the preparation of rAAV vectors may also be supplemented with one or more cell culture components known in the art, such as, but not limited to, glucose, vitamins, amino acids, and / or growth factors, to increase the titer of rAAV in the resulting culture.

[0101] rAAV-producing cultures can be grown under a variety of conditions (wide temperature ranges, varying durations, etc.) suitable for the specific host cells being used. As is well known in the art, rAAV-producing cultures include adhesion-dependent cultures, such as those that can be cultured in suitable adhesion-dependent containers, such as roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluid-bed bioreactors. rAAV vector-producing cultures may also include suspension-compatible host cells, such as HeLa, 293, and SF-9 cells, which can be cultured in a variety of ways, including spinner flasks, stirred-tank bioreactors, and disposable systems such as WaveBag systems.

[0102] The rAAV vector particles of the present invention may be collected from rAAV-producing cultures by lysing the host cells of the producing culture or by collecting the used medium from the producing culture, provided that the cells are cultured under conditions known in the art to release the rAAV particles from intact cells into the culture medium, as further fully described in U.S. Patent No. 6,566,118. Suitable cell lysis methods are also known in the art and include, for example, multiple freeze / thaw cycles, sonication, microfluidization, and treatment with chemicals such as surfactants and / or proteases.

[0103] In further embodiments, rAAV particles are purified. As used herein, the term “purified” includes preparations of rAAV particles from which at least some of the other components present where the rAAV particles naturally exist or where they are first prepared have been removed. Thus, for example, isolated rAAV particles are prepared from a source mixture, such as a culture lysate or a productive culture supernatant, using concentration-purification techniques. Concentration can be measured in various ways, such as by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in the solution, or by infectivity, or concentration can be measured in relation to a second potentially interfering substance present in the source mixture, such as contaminants, productive culture contaminants, or process contaminants such as helper viruses or culture medium components.

[0104] In some embodiments, the rAAV-producing culture collection is clarified to remove host cell debris. In some embodiments, the producing culture collection is clarified by filtration through a series of depth filters, such as Grade DOHC Millipore Millistak+ HC Pod filters, Grade A1HC Millipore Millistak+ HC Pod filters, and 0.2 μm Filter Opticap XL10 Millipore Express SHC hydrophilic membrane filters. Clarification can also be achieved by various other standard techniques known in the art, such as centrifugation or filtration through cellulose acetate filters with pore sizes of 0.2 μm or larger known in the art.

[0105] In some embodiments, the rAAV-producing culture harvest is further treated with benzonase® to digest any high molecular weight DNA present in the producing culture. In some embodiments, benzonase® digestion is carried out under standard conditions known in the art, for example, at an ambient temperature range of ~37°C for 30 minutes to several hours with a final concentration of 1 to 2.5 units / ml of benzonase®.

[0106] rAAV particles are isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentration of rAAV particles; rAAV capture by apatite chromatography; thermal inactivation of helper viruses; rAAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and rAAV capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. These steps are used individually, in various combinations, or in different orders. In some embodiments, the method includes all steps in the order described below. Methods for purifying rAAV particles can be found, for example, in Xiao et al., (1998) Journal of Virology 72:2224-2232; U.S. Patents 6,989,264 and 8,137,948; and International Publication No. 2010 / 148143.

[0107] Treatment method Certain aspects of this disclosure relate to methods for treating phenylketonuria and / or reducing phenylalanine levels in individuals requiring such treatment. In some embodiments, the present invention provides a method for treating PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of this disclosure. In some embodiments, the PAH polypeptide is a wild-type PAH polypeptide. The expression cassette for expressing the PAH polypeptide may be administered to a tissue of particular interest or systemically. In some embodiments, the expression cassette for expressing an effective amount of the PAH polypeptide may be administered parenterally. Parenteral administration routes include, but are not limited to, intravenous, intraperitoneal, intraosseous, intraarterial, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, and intrahepatic. In some embodiments, expression of the PAH polypeptide from tissues beyond the liver may require the presence of cofactor BH4 (e.g., delivered systemically or co-expressed from nucleic acids). Ding et al., Mol Ther 2008, 16:673-681. In some embodiments, the expression cassette for expressing an effective amount of PAH polypeptide may be administered via a single administration route. In some embodiments, the expression cassette for expressing an effective amount of PAH polypeptide may be administered via a combination of more than one administration routes. In some embodiments, the expression cassette for expressing an effective amount of PAH polypeptide is administered at one site. In other embodiments, the expression cassette for expressing an effective amount of PAH polypeptide may be administered at more than one site. In some embodiments, the expression cassette for expressing PAH polypeptide is DNA. In some embodiments, the expression cassette for expressing PAH polypeptide is RNA (e.g., mRNA).

[0108] In some embodiments, the present invention provides a method for reducing phenylalanine levels in individuals having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the level of phenylalanine in individuals having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced to a level found in individuals without PKU. In some embodiments, the level of phenylalanine in individuals having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0109] In some embodiments, the present invention provides a method for reducing the level of phenylalanine in the blood of an individual having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the level of phenylalanine in the blood of an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced to the level found in the blood of an individual without PKU. In some embodiments, the level of phenylalanine in the blood of an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0110] In some embodiments, the present invention provides a method for reducing phenylalanine levels in the brains of individuals having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the level of phenylalanine in the brains of individuals having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced to a level found in individuals without PKU. In some embodiments, the level of phenylalanine in the brains of individuals having PKU after administration of the expression cassette for expressing the PAH polypeptide is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more.

[0111] In some embodiments, the present invention provides a method for increasing neurotransmitter levels in the brain of an individual having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the neurotransmitter is one or more of dopamine, norepinephrine, or serotonin. In some embodiments, the neurotransmitter levels in the brain of an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide are increased to levels found in an individual without PKU.

[0112] In some embodiments, the present invention provides a method for increasing tyrosine and / or tryptophan levels in an individual having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the levels of tyrosine and / or tryptophan in an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide increase to levels found in an individual without PKU.

[0113] In some embodiments, the present invention provides a method for increasing tyrosine and / or tryptophan levels in the blood of an individual having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the levels of tyrosine and / or tryptophan in the blood of an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide are increased to levels found in an individual without PKU.

[0114] In some embodiments, the present invention provides a method for increasing tyrosine and / or tryptophan levels in the brain of an individual having PKU by administering an expression cassette for expressing an effective amount of the PAH polypeptide of the present disclosure. In some embodiments, the levels of tyrosine and / or tryptophan in the brain of an individual having PKU after administration of the expression cassette for expressing the PAH polypeptide are increased to levels found in an individual without PKU.

[0115] In some embodiments of the present invention, an expression cassette for expressing a PAH polypeptide (e.g., wild-type human PAH polypeptide) is delivered to an individual by a viral vector. Viral vectors for gene therapy are known in the art. In some embodiments, the present invention provides a method for treating PKU by administering an effective amount of lentiviral particles encoding the PAH polypeptide of the present disclosure. In some embodiments, the present invention provides a method for treating PKU by administering an effective amount of rAAV particles encoding the PAH polypeptide of the present disclosure. rAAV may be administered to a specific tissue of interest or systemically. In some embodiments, an effective amount of rAAV may be administered parenterally. Parenteral administration routes include, but are not limited to, intravenous, intraperitoneal, intraosseous, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, and intrahepatic. In some embodiments, an effective amount of rAAV may be administered via a single administration route. In some embodiments, an effective amount of rAAV may be administered via a combination of two or more administration routes. In some embodiments, an effective amount of rAAV is administered to a single site. In other embodiments, an effective dose of rAAV may be administered to two or more locations.

[0116] An effective amount of rAAV (in some embodiments, in the form of particles) is administered according to the purpose of the treatment. For example, if the desired therapeutic effect can be achieved with a low percentage of transduction, the purpose of the treatment is generally to meet or exceed this transduction level. In some cases, this transduction level is achieved by transducing only about 1-5% of target cells of the desired tissue type, at least about 20% in some embodiments, at least about 50% in some embodiments, at least about 80% in some embodiments, at least about 95% in some embodiments, and at least about 99% in some embodiments. The rAAV composition is administered in one or more doses during the same treatment or at intervals of several days, weeks, months, or years. One or more of the administration routes described herein may be used. In some embodiments, multiple vectors may be used to treat humans.

[0117] Methods for identifying cells transduced by AAV virus particles are known in the art, and for example, transduction of viral particles, e.g., viral particles containing an rAAV capsid with one or more amino acid substitutions, can be detected using immunohistochemistry or markers such as enhanced green fluorescent protein.

[0118] In some embodiments, an effective dose of rAAV particles is administered simultaneously or consecutively to two or more locations. In other embodiments, an effective dose of rAAV particles is administered two or more times to one location (e.g., repeated). In some embodiments, multiple injections of rAAV virus particles are spaced at intervals of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 9 hours, 12 hours, or 24 hours or less.

[0119] In some embodiments, the present invention provides a method of treating a human having PKU by administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding a PAH polypeptide of the present disclosure. In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients.

[0120] In some embodiments, the method comprises administering an effective amount of a pharmaceutical composition comprising a recombinant viral vector encoding a PAH polypeptide of the present disclosure to an individual in need of treatment for PKU to treat PKU. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5×10 12 、6×10 12 、7×10 12 、8×10 12 、9×10 12 、10×10 12 、11×10 12 、15×10 12 、20×10 12 、25×10 12 、30×10 12 、or 50×10 12 genome copies / mL. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is about 5×10 12 ~6×10 12 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​12 , or 50 x 10 12 ~100×10 12 It is either genome copies / mL. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is approximately 5 × 10⁻¹⁶. 12 ~10×10 12 , 10×10 12 ~25×10 12 , or 25×10 12 ~50×10 12 It is either genome copies / mL. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5 × 10⁶ 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 15×10 9 , 20×10 9 , 25×10 9 , 30×10 9 , or 50 x 10 9 It is either transduction units / mL. In some embodiments, the viral titer of the virus particles (e.g., rAAV particles) is approximately 5 × 10⁻¹⁶. 9 ~6×10 9 , 6×10 9 ~7×10 9 , 7×10 9 ~8×10 9 , 8×10 9 ~9×10 9 , 9×10 9 ~10×10 9 , 10×10 9 ~11×10 9 , 11×10 9 ~15×10 9 , 15×10 9 ~20×10 9 , 20×10 9 ~25×10 9 , 25×10 9 ~30×10 9 , 30×10 9 ~50×10 9 , or 50 x 10 9~100×10 9 Either in transducing units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is about 5×10 9 ~10×10 9 、10×10 9 ~15×10 9 、15×10 9 ~25×10 9 、or 25×10 9 ~50×10 9 Either in transducing units / mL. In some embodiments, the viral titer of viral particles (e.g., rAAV particles) is at least about 5×10 10 、6×10 10 、7×10 10 、8×10 10 、9×10 10 、10×10 10 、11×10 10 、15×10 10 、20×10 10 、25×10 10 、30×10 10 、40×10<00001​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​10 , 40×10 10 ~50×10 10 or 50 x 10 10 ~100×10 10 It is either infectious units / mL. In some embodiments, the viral titer of the viral particles (e.g., rAAV particles) is at least about 5 × 10⁶ 10 ~10×10 10 , 10×10 10 ~15×10 10 , 15×10 10 ~25×10 10 , or 25×10 10 ~50×10 10 It is either infectious units / mL or one of the above. In some embodiments, the viral particles are rAAV particles. In some embodiments, the rAAV particles contain an XL32 capsid. In some embodiments, the rAAV particles contain an XL32.1 capsid.

[0121] In some embodiments, the dose of viral particles administered to an individual is at least about 1 × 10⁻⁶ 8 ~Approx. 6×10 13 The value is genome copies / kg body weight. In some embodiments, the dose of viral particles administered to an individual is approximately 1 × 10⁻⁶. 8 ~Approx. 6×10 13 The value is genome copies / kg body weight. In some embodiments, the dose of viral particles administered to an individual is approximately 1 × 10⁻⁶. 10 , 2×10 10 , 3 x 10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1 x 10 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12, 2×10 12 , 13×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , or 1 × 10 13 It is either genome copies / kg body weight.

[0122] In some embodiments, the total amount of viral particles administered to an individual is at least about 1 × 10⁻⁶ 9 ~Approx. 1×10 14 It is a genome copy. In some embodiments, the total amount of viral particles administered to an individual is approximately 1 × 10⁻⁶. 9 ~Approx. 1×10 14 It is a genome copy. In some embodiments, the total amount of viral particles administered to an individual is approximately 1 × 10⁻⁶. 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , 1 x 10 12 , 2×10 12 , 3 x 10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , 9×10 12 , 1 x 10 13 , 2×10 13 , about 13×10 13 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 , 9×10 13 , or 1 × 10 14 It is one of the genome copies.

[0123] The compositions of the present invention (e.g., recombinant viral particles comprising a vector encoding the PAH polypeptide of this disclosure) can be used alone or in combination with one or more additional therapeutic agents for treating PKU. The interval between consecutive doses may be at least a few minutes, hours, or days (or alternatively, less than a few minutes, hours, or days).

[0124] An effective amount of rAAV (in some embodiments, in the form of particles) is administered according to the purpose of the treatment. For example, if the desired therapeutic effect can be achieved with a low percentage of transduction, the purpose of the treatment is generally to meet or exceed this transduction level. In some cases, this transduction level is achieved by transducing only about 1-5% of target cells, at least about 20% of cells of the desired tissue type in some embodiments, at least about 50% in some embodiments, at least about 80% in some embodiments, at least about 95% in some embodiments, and at least about 99% of cells of the desired tissue type in some embodiments. The rAAV composition is administered in one or more doses during the same treatment or at intervals of several days, weeks, months, or years. In some embodiments, multiple vectors may be used to treat mammals (e.g., humans).

[0125] In some embodiments, the rAAV compositions of this disclosure are used for administration to humans. In some embodiments, the rAAV compositions of this disclosure are used for administration to children. While we do not wish to be bound by theory, since many of the symptoms of PKU are inherently developmental (e.g., severe mental disorders), it may be particularly advantageous to treat PKU as early in life as possible. In some embodiments, an effective dose of rAAV (in some embodiments, in the form of particles) is administered to patients under 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 2 years, or 3 years of age.

[0126] In some embodiments, the rAAV compositions of this disclosure are used for administration to young adults. In some embodiments, an effective dose of rAAV (in some embodiments, rAAV in particle form) is administered to patients under 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 years of age.

[0127] In some embodiments, the present invention provides a method for treating PKU by administering cells containing an expression cassette for expressing an effective amount of the PAH polypeptide of this disclosure (e.g., wild-type human PAH polypeptide). The cells containing the expression cassette for expressing the PAH polypeptide may be administered to a specific tissue of interest or systemically. In some embodiments, an effective amount of cells containing the expression cassette for expressing the PAH polypeptide may be administered parenterally. Parenteral administration routes include, but are not limited to, intravenous, intraperitoneal, intraosseous, intracerebral, intramuscular, intrathecal, subcutaneous, intraventricular, and intrahepatic. In some embodiments, the cells are encapsulated or contained within a device. In some embodiments, extrahepatic PAH-expressing cells may require the exogenously added or co-expressed cofactor BH4. In some embodiments, the cells are further encapsulated containing BH4 or contained within a device further containing BH4. In some embodiments, an effective amount of cells containing the expression cassette for expressing the PAH polypeptide may be administered via a single administration route. In some embodiments, an effective amount of the expression cassette for expressing the PAH polypeptide may be administered via a combination of two or more administration routes. In some embodiments, an effective amount of the expression cassette for expressing the PAH polypeptide is administered at one site. In other embodiments, an effective amount of the expression cassette for expressing the PAH polypeptide may be administered at two or more sites.

[0128] In some embodiments, the cells containing the expression cassette for expressing the PAH polypeptide are hepatocytes, muscle cells, fibroblasts, endothelial cells, epithelial cells, hematopoietic cells, myeloid cells, stem cells, or induced pluripotent stem cells. In some embodiments, the cells further include exogenously added cofactor BH4 and / or co-expressed cofactor BH4.

[0129] In some embodiments, the cells are cell lines (e.g., CHO cell lines, HeLa cell lines, etc.). In some embodiments, the present invention provides a method for producing PAH polypeptides (e.g., wild-type human PAH polypeptides), comprising culturing cells containing an expression cassette encoding a PAH polypeptide under conditions for PAH polypeptide production. In some embodiments, the method for producing PAH polypeptides further comprises one or more steps of purifying the PAH polypeptides.

[0130] Kit or manufactured article The expression cassettes described herein (e.g., expression cassettes for expressing PAH polypeptides, e.g., wild-type human PAH polypeptides), rAAV vectors, particles, and / or pharmaceutical compositions are included, for example, in kits or manufactured articles designed for use in one of the methods of the present invention described herein.

[0131] Generally, the system includes a cannula, one or more syringes (e.g., one, two, three, four, or more), and one or more fluids (e.g., one, two, three, four, or more) suitable for use in the method of the present invention.

[0132] The syringe can be any suitable syringe, as long as it is connectable to a cannula for fluid delivery. In some embodiments, the system has one syringe. In some embodiments, the system has two syringes. In some embodiments, the system has three syringes. In some embodiments, the system has four or more syringes. Suitable fluids for use in the method of the present invention include the fluids described herein, for example, one or more fluids each containing an effective amount of one or more vectors described herein, and one or more fluids containing one or more therapeutic agents.

[0133] In some embodiments, the kit comprises a single fluid (e.g., a pharmaceutically acceptable fluid containing an effective amount of the vector). In some embodiments, the kit comprises two fluids. In some embodiments, the kit comprises three fluids. In some embodiments, the kit comprises four or more fluids. The fluids may include any diluents, buffers, excipients, or other liquids described herein or known in the art that are suitable for delivering, diluting, stabilizing, buffering, or otherwise transporting expression cassettes for expressing the PAH polypeptide or rAAV vector composition of this disclosure. In some embodiments, the kit comprises one or more buffers, e.g., pH-buffered aqueous solutions. Examples of buffers include, but are not limited to, phosphoric acid, citrate, Tris, HEPES, and other organic acid buffers.

[0134] In some embodiments, the kit includes a container. Suitable containers include, for example, vials, bags, syringes, and bottles. The container is made from one or more materials such as glass, metal, or plastic. In some embodiments, the container is used to hold the rAAV composition of the Disclosure. In some embodiments, the container may hold a fluid and / or other therapeutic agent.

[0135] In some embodiments, the kit includes an additional therapeutic agent along with the rAAV composition of the Disclosure. In some embodiments, the rAAV composition and the additional therapeutic agent are mixed. In some embodiments, the rAAV composition and the additional therapeutic agent are kept separately. In some embodiments, the rAAV composition and the additional therapeutic agent are in the same container. In some embodiments, the rAAV composition and the additional therapeutic agent are in different containers. In some embodiments, the rAAV composition and the additional therapeutic agent are administered simultaneously. In some embodiments, the rAAV composition and the additional therapeutic agent are administered on the same day. In some embodiments, the rAAV composition is administered within 1, 2, 3, 4, 5, 6, 7 days, 2 weeks, 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months of the administration of the additional therapeutic agent.

[0136] In some embodiments, the kit includes a therapeutic agent for transiently suppressing the immune system before AAV administration. In some embodiments, the patient is transiently immunosuppressed immediately before and after viral injection to inhibit the T-cell response to AAV particles (see, e.g., Ferreira et al., Hum. Gene Ther. 25: pp. 180-188, 2014). In some embodiments, the kit further provides cyclosporine, mycophenolate mofetil, and / or methylprednisolone.

[0137] The rAAV particles and / or compositions of the present invention may be further packaged in a kit including instructions for use. In some embodiments, the kit further includes a device for delivery of the rAAV particle composition (e.g., parenteral administration of any kind described herein). In some embodiments, the instructions for use include instructions relating to one of the methods described herein. In some embodiments, the instructions are printed on a label provided with the container (e.g., affixed to the container). In some embodiments, the instructions for use include instructions for administering an effective amount of rAAV particles to an individual (e.g., a human) to treat PKU in the individual.

[0138] Exemplary Embodiments Embodiment 1. Recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in liver cells, the expression cassette comprising a transgene operably linked to a promoter and an enhancer, the promoter comprising a mouse trans tyretin (mTTR) promoter, the enhancer comprising one or two modified prothrombin enhancers (pPrT2), one or two modified alpha-1-microbicinin enhancers (mA1MB2), a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HE11), or CRM8 enhancer, the transgene encoding a PAH polypeptide; the AAV virus particle comprising an AAV-XL32 or AAV-XL32.1 capsid.

[0139] Embodiment 2. The rAAV particle according to Embodiment 1, wherein the mTTR promoter is the mTTR482 promoter.

[0140] Embodiment 3. The enhancer is located at 5' relative to the mTTR promoter in the rAAV particle according to Embodiment 1 or 2.

[0141] Embodiment 4. Recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in liver cells, the expression cassette comprising a promoter and a transgene operably ligated to a 3' element, the promoter comprising a mouse trans tyretin (mTTR) promoter, the 3' element being an albumin 3' element (3'Alb) or an albumin 3' element (3'AlbSMAR) ligated to a human alpha-1 antitrypsin scaffold / matrix attachment region (SMAR), the transgene encoding a PAH polypeptide; and the rAAV particles comprising an AAV-XL32 or AAV-XL32.1 capsid.

[0142] Embodiment 5. The rAAV particle according to Embodiment 4, wherein the mTTR promoter is the mTTR482 promoter.

[0143] Embodiment 6.3' element is located at 3' relative to the transgene, as described in Embodiment 4 or 5 of the rAAV particle.

[0144] Embodiment 7. Recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector and an expression cassette for expressing a transgene in liver cells, wherein the expression cassette comprises a promoter and enhancers and a transgene operably ligated to the 3' element, the promoter comprising a mouse trans tyretin (mTTR) promoter, and the enhancers comprising one or two modified prothrombin enhancers (pPrT2) and one or two modified alpha-1-microbicinin enhancers (mA1MB). 2) comprising a modified mouse albumin enhancer (mEalb), hepatitis B virus enhancer II (HE11), or CRM8 enhancer; the 3' element is an albumin 3' element (3'Alb) or an albumin 3' element (3'AlbSMAR) linked to a human alpha-1 antitrypsin scaffold / matrix attachment region (SMAR); the transgene encodes a PAH polypeptide; and the rAAV particles comprise an AAV-XL32 or AAV-XL32.1 capsid.

[0145] Embodiment 8. The rAAV particle according to Embodiment 7, wherein the mTTR promoter is the mTTR482 promoter.

[0146] Embodiment 9. The enhancer is located at 5' relative to the mTTR promoter in the rAAV particle according to Embodiment 7 or 8.

[0147] Embodiment 10.3' element is an rAAV particle according to any one of Embodiments 7 to 9, wherein the 3' element is positioned at 3' relative to the transgene.

[0148] Embodiment 11. The expression cassette further comprises an intron, the rAAV particle according to any one of Embodiments 1 to 10.

[0149] Embodiment 12. The rAAV particle according to Embodiment 11, wherein the intron is a chicken β-actin / rabbit β-globin hybrid intron.

[0150] Embodiment 13. An rAAV particle according to any one of Embodiments 1 to 12, wherein the expression cassette further comprises a polyadenylation signal.

[0151] Embodiment 14. The rAAV particle according to Embodiment 13, wherein the polyadenylation signal is a bovine growth hormone polyadenylation signal.

[0152] Embodiment 15. The rAAV particle according to any one of Embodiments 1 to 14, wherein the PAH polypeptide is a wild-type PAH polypeptide.

[0153] Embodiment 16. The rAAV particle according to any one of Embodiments 1 to 15, wherein the PAH polypeptide is a human PAH polypeptide.

[0154] Embodiment 17. The rAAV particle according to any one of Embodiments 1 to 16, wherein the PAH polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

[0155] Embodiment 18. An rAAV particle according to any one of Embodiments 1 to 17, wherein the introduced gene is at least 80% identical to the nucleic acid sequence of Sequence ID No. 2.

[0156] Embodiment 19. The rAAV vector comprises an expression cassette adjacent to one or more AAV reverse terminal repeat (ITR) sequences, as described in any one of Embodiments 1 to 18.

[0157] Embodiment 20. The expression cassette according to any one of Embodiments 1 to 18 is adjacent to the ITRs of two AAVs, wherein the rAAV particles are as described in Embodiment 19.

[0158] Embodiment 21. The rAAV particle according to Embodiment 19 or 20, wherein the ITR of AAV is the ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype.

[0159] Embodiment 22. The rAAV particle according to any one of Embodiments 19 to 21, wherein the ITR of AAV is the ITR of AAV2.

[0160] Embodiment 23. The vector is a self-complementary vector, the rAAV particle according to any one of Embodiments 19 to 22.

[0161] Embodiment 24. The rAAV particle according to Embodiment 23, wherein the vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding a complement of the PAH polypeptide, the first nucleic acid sequence being able to form intrachain base pairs with the second nucleic acid sequence along most or all of its length.

[0162] Embodiment 25. The rAAV particle according to Embodiment 24, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by an ITR of a mutated AAV, the ITR of the mutated AAV comprising a deletion in the D region and a mutation in the terminal degradation sequence.

[0163] Embodiment 26. rAAV particles comprising an rAAV vector, wherein the rAAV vector comprises, from 5' to 3', an AAV2 ITR, a modified alpha-1 microbicin enhancer (mA1MB2), a mouse trans tyretin (mTTR) promoter, a chicken β-actin / rabbit β-globin hybrid intron, a codon-optimized human PAH gene, a bovine growth hormone polyadenylation signal, a stuffer fragment derived from an alpha-1 anti-trypsin gene, and an AAV2 ITR, the rAAV particles.

[0164] Embodiment 27. The rAAV particle according to any one of Embodiments 1 to 26, wherein the AAV capsid is the AAV-XL32 capsid.

[0165] Embodiment 28. The rAAV particle according to Embodiment 27, wherein the AAV-XL32 capsid comprises an AAV-XL32 capsid protein having an amino acid sequence at least 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.

[0166] Embodiment 29. The rAAV particle according to Embodiment 28, wherein the AAV-XL32 capsid comprises VP1, VP2, and VP3, where VP1, VP2, and VP3 are encoded by the nucleic acid sequence of Sequence ID No. 4.

[0167] Embodiment 30. The rAAV particle according to any one of Embodiments 1 to 26, wherein the AAV capsid is the AAV-XL32.1 capsid.

[0168] Embodiment 31. The rAAV particle according to Embodiment 30, wherein the AAV-XL32.1 capsid contains an amino acid sequence that is at least 90%, 95%, 99%, or 100% identical to SEQ ID NO: 3.

[0169] Embodiment 32. The rAAV particle according to Embodiment 30, wherein the AAV-XL32.1 capsid comprises VP1, VP2, and VP3, where VP1, VP2, and VP3 are encoded by the nucleic acid sequence of Sequence ID No. 6.

[0170] Embodiment 33. A composition comprising rAAV particles as described in any one of Embodiments 1 to 32.

[0171] Embodiment 34. The composition according to Embodiment 33, further comprising a pharmaceutically acceptable carrier.

[0172] Embodiment 35. A cell containing rAAV particles as described in any one of Embodiments 1 to 32.

[0173] Embodiment 36. A method for producing a PAH polypeptide, comprising culturing the cells described in Embodiment 35 under conditions for producing the PAH polypeptide.

[0174] Embodiment 37. The method according to Embodiment 36, further comprising a step of purifying the PAH polypeptide.

[0175] Embodiment 38. A method for treating phenylketonuria in an individual who needs it, comprising administering the rAAV particles described in any one of Embodiments 1 to 37 to the individual.

[0176] Embodiment 39. A method for treating phenylketonuria in an individual who needs it, comprising administering the composition described in Embodiment 33 or 34 to the individual.

[0177] Embodiment 40. A method for treating phenylketonuria in an individual who needs it, comprising administering the cells described in Embodiment 35 to the individual.

[0178] Embodiment 41. The method according to any one of Embodiments 38 to 40, wherein the individual lacks PAH activity.

[0179] Embodiment 42. A method for reducing the level of phenylalanine in the blood of an individual who needs it, comprising administering the rAAV particles described in any one of Embodiments 1 to 32 to the individual.

[0180] Embodiment 43. A method for reducing the level of phenylalanine in the blood of an individual who needs it, comprising administering the composition described in Embodiment 33 or 34 to the individual.

[0181] Embodiment 44. A method for reducing the level of phenylalanine in the blood of an individual who needs it, comprising administering the cells described in Embodiment 35 to the individual.

[0182] Embodiment 45. The method according to any one of Embodiments 42 to 44, wherein the level of phenylalanine in the blood of an individual before treatment is elevated as compared to the level of phenylalanine in the blood of an equivalent corresponding control individual.

[0183] Embodiment 46. The method according to any one of Embodiments 38 to 45, wherein the rAAV particles, composition or cells are administered intravenously, intraarterially, intrahepatically, intraportally, intraperitoneally, or subcutaneously.

[0184] Embodiment 47. The method according to any one of Embodiments 38 to 46, wherein the administration is combined with another therapy.

[0185] Embodiment 48. The method according to Embodiment 47, wherein the another therapy is treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia-lyase (PAL) or pegylated PAL, or a phenylalanine-restricted diet.

[0186] Embodiment 49. A kit comprising the rAAV particles according to any one of Embodiments 1 to 32, the composition according to Embodiment 33 or 34, or the cells according to Embodiment 35.

[0187] Embodiment 50. The kit according to Embodiment 49, further comprising instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, vial and / or syringe.

Examples

[0188] The present invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the present invention. It is to be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes may be suggested to those skilled in the art in light of the examples and embodiments described herein, and are to be included within the spirit and scope of the present application and the scope of the appended embodiments.

Examples

[0189] In vitro evaluation of PAH coding sequences The following examples describe the generation of vectors encoding human PAH. Specifically, wild-type and variant PAH alleles with and without the E183G amino acid substitution were cloned into vectors, and PAH expression and activity were measured in liver cells.

[0190] material and method PAH code sequence Human PAH cDNA encoding wild-type or variant 1 PAH with or without the E183G amino acid substitution was tested as summarized in Table 1 below. The "variant 1" allele of PAH has the M180T, K199P, S250P, and G256A amino acid substitutions as described in International Publication No. 2020077250A1, incorporated herein by reference.

[0191] We optimized the codon frequency of PAH cDNA using GeneArt (GA) codon optimization.

[0192] [Table 1]

[0193] Generation of plasmid vectors and recombinant AAVs The PAH coding sequence described in International Publication No. 2020077250A1, incorporated herein by reference, was expressed.

[0194] Specifically, modifications were introduced to the plasmid mTTR482-HI-hFVIII-BGHpA, which contains a mouse trans tyretin (mTTR) promoter, an endogenous mTTR enhancer, and a bovine growth hormone (BGH) polyadenylation (pA) site, in order to increase liver promoter strength (Kyostio-Moore 2016, Nambiar 2017). In this plasmid, the FVIII cDNA was replaced with cDNA encoding secreted placental alkaline phosphatase (SEAP), and the existing intron was replaced with a 1069 bp chicken β-actin (CBA) / rabbit β-globin hybrid intron. Two copies of the modified alpha-1-microbicin enhancer (mA1MB2) (McEachern 2006, Jacobs 2008) were cloned upstream of the mTTR482 enhancer to generate the mA1MB2-mTTR482 promoter.

[0195] We used a hybrid intron (also known as HI2) made from a chicken beta-actin / rabbit beta-globin intron that was modified to eliminate five existing ATG sequences to reduce incorrect translation initiation.

[0196] All vectors contained bovine growth hormone polyadenylated sites (BGHpA).

[0197] Finally, a filler sequence ("stuffer") consisting of the alpha anti-trypsin gene intron sequence (SerpinA1=A1AT)NG_008290.1;nt13638~17363) on chromosome 14 was inserted between the BGHpA and ITR sites to reduce the total vector genome size to 4.6kb. Seven ATG sites in the stuffer sequence were modified to TTG to remove potential translation initiation sites.

[0198] A plasmid containing multiple AAV2 ITRs, having a liver promoter, a hybrid intron, PAH cDNA, and BGHpA, was used for rAAV vector production. rAAV vectors having the AAVXL32 serotype capsid were generated using a triple transfection method followed by CsCl purification (SabTech) or by column purification (Sanofi Vector Core). Vector lots were quantified by qPCR against BGHpA (Nambiar 2017).

[0199] In vitro culture All tissue culture reagents were obtained from Irvine Scientific (Santa Ana, CA) or Invitrogen. For transient transfection, human 293 or human liver cancer cells (Huh7 or HepG2) (8×10 5 cells / well) were seeded in 6-well dishes in Dulbecco's Modified Eagle Medium (DMEM) containing high glucose, 10% fetal bovine serum (FBS), and 10 mL Pen Strep (10 units / ml penicillin and 10 μg / ml streptomycin). Plasmid (2 μg) was transfected with Lipofectamine 2000 (Invitrogen). Cell lysates or culture media were collected 48 hours or 72 hours later, respectively, for PAH analysis or SEAP activity.

[0200] Activity assay and protein detection To measure PAH activity, whole cell lysates were generated 48 hours later by lysing cells in lysis buffer or RIPA buffer. Additionally, in some experiments, sonication or shearing was used to enhance cell lysis. Once thawed, the lysates were spun at 14,000 g for 30 minutes prior to assay. The enzymatic activity of PAH protein was measured as previously described by Yew et al., 2013, with some minor modifications. Activity was also measured using 13C-labeled Phe as previously described (Heintz 2012).

[0201] Western blotting for PAH detection was performed using a standard protocol with an anti-hPAH antibody (LS-C344145; LSBio). In vivo samples were normalized by total protein content measured by a BCA protein assay kit (Pierce). FLAG-PAH protein levels were quantified by FLAG-ELISA (SE002-flag; ABSbio) according to the manufacturer's instructions, using either the kit standard or in-house purified 3× FLAG-mPAH-FL as the protein standard.

[0202] result Four vectors encoding PAH were generated under the control of mA1MB2-mTTR482. Specifically, hPAH / 183G, hPAH-V1 / G, hPAH-V1 / E, and wild-type PAH ("hPAH / E") were expressed and tested for PAH activity (Figure 1A) and protein levels (Figures 1B, 1C) in Huh7 cells.

[0203] Wild-type PAH demonstrated the highest level of PAH activity, as shown in Figure 1A. For the hPAH / G mutant coding sequence, the addition of the four amino acid substitutions of variant 1 improved activity and protein production tenfold. Incorporating the four amino acid substitutions of variant 1 into the wild-type PAH coding sequence did not increase PAH activity, but resulted in a twofold increase in PAH protein levels (Figures 1B, 1C).

[0204] Based on these results, we selected wild-type PAH as the transgene for in vivo efficacy studies in Pah-KO mice. [Examples]

[0205] Evaluation of capsids, lead liver promoters, and dose-response in non-human primate livers. The following examples describe experiments evaluating the ability of various AAV capsid proteins to transduce liver cells. Furthermore, the ability of the mA1M2-mTTR482 promoter to promote transgene expression in non-human primates (NHPs) was tested, and dose-response experiments were performed to evaluate the administration of the XL32.1 / mA1MB2-mTTR482-EGFP vector to NHPs.

[0206] material and method AAV Capsid Protein Various AAV capsid proteins were tested for their ability to transduce Huh7 cells and non-human primate (NHP) livers. Specifically, XL32, LK03 (Lisowski L et al., Nature, 2014, 506:382-386), DJ (Grimm D et al., J Virol 2008, 82:5887-5911), AAV8, and XL14 capsid proteins were tested in Huh7 cells and NHP in the initial experiments (Figures 2A-2D). Subsequently, XL32.1 capsid protein was used in dose-response experiments in NHP (Figures 3A-3B, 4A-4C).

[0207] As described in International Publication No. 2019241324A1, XL32 and XL32.1 are hybrid capsids generated from an AAV capsid gene shuffle library consisting of capsid genes for AAV serotypes 1, 2, 3B, 4, 6, 7, 8, and 9. XL32 was selected from the library for its enrichment in mouse liver. In addition to the typical VP1, VP2, and VP3 protein products, XL32 also produced a fourth protein product (called "VPX") which is thought to be produced due to a weak non-ATG start codon in the XL32 coding sequence. Specifically, XL32 had a C-to-G mutation at nucleotide 219, counting from the VP1 start codon. XL32.1 was obtained from XL32 by site-directed mutagenesis to reverse the C-to-G mutation back to the original C and match the wild-type AAV7 and AAV8 sequences. According to International Publication No. 2019241324A1, XL32.1 showed no apparent difference in vector yield and infectivity. Table 2 below provides the amino acid sequences of XL32 and XL32.1.

[0208] [Table 2]

[0209] Research on non-human primates (NHPs) All studies used 2-3 year old (3-4 kg) male cynomolgus monkeys (Macaca fascicularis, of Asian origin). Animals were screened for neutralizing antibodies to the vector capsid prior to vector administration. Selected animals were administered the vector by slow intravenous infusion (1 ml / min) into the saphenous vein using an infusion pump. Blood samples were collected at various time points. At necropsy, samples were collected from the liver and several other organs for analysis of the in vivo distribution of the vector.

[0210] For vector detection, total tissue DNA was isolated from each tissue using 1.4 mm ceramic beads and Omni Ruptor-24, followed by proteinase K digestion and phenol / chloroform extraction. DNA was precipitated with isopropyl alcohol, spun, and resuspended in Tris-EDTA. DNA was quantified, and the level of vector-derived DNA was measured by qPCR using primers specific to the BGHpA sequence present in each VG (Kyostio-Moore et al., 2016). The level of VG was expressed as copy number per cell (using 5 pg of dsDNA per diploid cell genome).

[0211] Homogenates for total EGFP protein were prepared using an Omni bead disruptor in ELISA kit extraction buffer PRT (Abcam GFP ELISA kit; ab171581) supplemented with a protease inhibitor (Roche). After centrifugation, the supernatant was collected, diluted with kit extraction buffer PRT, and EGFP protein levels were quantified by ELISA according to the manufacturer's instructions. All values ​​were expressed as ng of EGFP / mg of total protein. Total protein was measured by BCA assay.

[0212] To quantify vector-derived transcripts, liver and spleen homogenates were generated using an Omni bead lithograph in 2 ml tubes containing 1.4 mm ceramic beads and 1 mL of Trizol (Thermofisher, A33250). Chloroform was added and mixed, and the aqueous phase was then transferred to a Promega Z3100 column using the SV total RNA kit. DNase treatment was performed on the column, and after washing the column, the RNA was eluted in water. Total RNA was quantified using Nanodrop 8000. cDNA was generated using a High Capacity cDNA RT kit (Thermofisher, 4368814) with random primers. The level of vector-derived mRNA was measured by qPCR using primers specific to the BGHpA sequence. Transcript levels were expressed as copy number per cell or per μg of RNA (using 5 pg of dsDNA per diploid cell genome).

[0213] Immunohistochemistry and in-situ hybridization analyses for liver were performed using 4 μM fragments cut from neutral-buffered formalin-fixed, paraffin-embedded blocks. All steps for automated fluorescence in-situ hybridization (RNAscope) and IHC were performed using a Leica Bond RX instrument (Leica Bios, Systems Inc., Buffalo Grove, IL). A sequential double-staining mode was used, starting with the RNAscope 2.5 LS Multiplex Fluorescent assay (Advanced Cell Diagnostics, Newark, CA), followed by IHC. In short, unbaked paraffin sections were baked at 60°C for 30 minutes, the paraffin was removed at 60°C, and then pre-treated at 42°C for 2 hours with Bond ER solution 2, protease, and hydrogen peroxide for target search before hybridization with the negative probe DapB (catalog no. 320878, Advanced Cell Diagnostics, Newark, CA), the positive probe cynomolgus monkey (M. fascicularis) PPIB (catalog no. 320908), or the target probe eGFP (catalog no. 400288, Advanced Cell Diagnostics, Newark, CA). The pre-amplifier and amplifier were connected in series, and the slides were incubated with OPAL690 (catalog no. FP1497001KT, Akoya Biosciences, Marlborough, MA) diluted 1:1500. After applying an HRP blocker, the fragments were subjected to target detection using Bond ER solution 1 before the IHC portion of the automated protocol.Slides were blocked with Antibody Diluent / Block (Akoya Biosciences, Marlborough, MA) and incubated with rabbit IgG isotype control (catalog no. AB105-C, R&D Systems, Minneapolis, MN) or rabbit anti-GFP (catalog no. A11122, Invitrogen / Thermofisher, Waltham, MA) used at 1 μg / mL for 30 minutes at room temperature. Secondary antibody, anti-rabbit polymer HRP (catalog no. PV6119, Leica Biosystems Inc., Buffalo Grove, IL), was applied, and detection was achieved with 1:150 dilution OPAL570 (catalog no. FP1488001KT, Akoya Biosciences, Marlborough, MA). Cells were counterstained with Spectral DAPI (Akoya Biosciences, Marlborough, MA). 20x images were acquired by Zeiss AxioScan Z1. 20x magnified images were imported into HALO (Indica labs) image analysis software and analyzed using the FISH-IF v1.1.3 imaging module.

[0214] Comparison of XL32 and XL32.1 capsid proteins Vectors expressing hPAH-V1 / G using the mA1M2-mTTR482 promoter were packaged in either XL32 or XL32.1 capsids and administered to NHP. 5e12vg / kg of vector was administered via IV delivery, and the vector genome copy number per cell in the liver and various other organs was measured by qPCR two weeks after administration (Figure 5A). Furthermore, the level of vector-derived mRNA in the liver was measured (Figure 5B).

[0215] result Early AAV capsid research To select an AAV capsid for PAH gene transfer that is expected to offer superior translatability for human gene transfer, five AAV capsids were generated, each containing a CBA-EGFP vector genome. The vectors showed varying levels of EGFP detection in Huh7 cells in vitro (Figure 2A). The AAV vectors were then delivered to NHP via an intravenous route, followed by collection of liver and various organs two weeks later. In the liver samples, VG was measured in the right medial lobe in three different regions, showing comparable levels among the samples, indicating a uniform distribution within the lobe (data not shown). Interlobar distribution was also evaluated by quantifying VG in the right and left medial lobes, demonstrating similar VG copies in both lobes (data not shown). Figure 2B shows the average of these four samples in the liver of each animal. The greatest vector gene transfer was observed with rAAV-XL32, followed by AAV-LK03, AAV8, and rAAV-XL14, with rAAV-DJ showing the lowest, and the difference between the highest and lowest ranked capsid vectors was approximately 22-fold. VG levels were comparable in individual animals within each capsid treatment group. VG copies were also measured in the spleen (Figure 2B) as well as in muscle, kidney, and heart (Figure 2C). In other tissues, only very small amounts of vector genome were observed with XL32. In summary, the XL32 capsid vector delivered by systemic administration provided robust hepatic uptake, but only very small amounts of vector were detected with this capsid in other organs examined (Figure 2D). Figure 2E shows intra- and inter-lobar EGFP expression.

[0216] NHP dose-response study In the second NHP study, the dose-response of liver gene translocation with XL32.1 / mA1MB2-mTTR482-EGFP was evaluated. The vector was delivered via the IV route, and tissue was collected 16 days later. Regarding liver analysis, two liver samples (collected from the right and left medial lobes) showed comparable VG levels (data not shown). The dose cohorts at 5e11, 2e12, 5e12, and 2e13 vg / kg had, on average, 0.9, 3.5, 8.8, and 7.3 (M and F) and 19.0 VG / cell, respectively (Figure 3A). Thus, dose response was demonstrated at all doses except the maximum dose. Within the two maximum doses, there was inter-animal variability that may be due to the immune response to the transgene. In the 5e12 vg / kg cohort, comparable vector genomes were detected in male and female animals (Figure 3A).

[0217] Vector promoter function was evaluated by measuring the level of vector-derived eGFP mRNA in liver samples. Overall, there was a trend of increasing transcription levels per cell with increasing dose (Figure 3B). The maximum dose tested (2e13vg / kg) resulted in low mRNA levels, possibly due to an immune response to the eGFP protein (corresponding to lower VG detection).

[0218] Vector transduction was evaluated by measuring eGFP protein levels in the liver. The data did not show a dose-response in eGFP protein levels; high eGFP levels were detected even at two lower doses, but the maximum dose resulted in the lowest eGFP level (Figure 3C). Nevertheless, a good correlation was observed between liver VG and vector-derived mRNA copies (r²=0.57) (Figure 3D). A good correlation was also observed between mRNA copies and eGFP protein levels (r²=0.57). 2 A value of 0.85 was observed (Figure 3E). This may suggest that not all vector genomes necessarily possess transcriptional activity, but once transcription occurs, eGFP protein production is directly proportional to the transcription level.

[0219] To understand the percentage of hepatocytes that incorporated the vector, vector uptake and localization in the liver were evaluated by hybridization using an eGFP probe. This probe is expected to detect both vector DNA and mRNA. Figure 4A shows an example of an in-situ hybridization image. Vector-positive hepatocytes were quantified to estimate the percentage of vector-positive cells in the liver (Figure 4B). The data demonstrated an increase in vector-positive cells with increasing vector dose. In the livers of both male and female animals, approximately 50–80% of liver cells were positive at a dose of 5e12vg / kg. A good correlation was observed between the percentage of positive cells in the liver and the mean number of VG copies (Figure 4C).

[0220] Comparison of XL32 and XL32.1 capsid proteins Experiments were conducted to compare the in vivo distribution of XL32 and XL32.1 capsid vectors in NHP. As shown in Figures 5A–5B, XL32 and XL32.1 yielded comparable levels of viral genome and vector-derived mRNA in the liver. [Examples]

[0221] Efficacy of XL32.1 / mA1MB2-mTTR482-WT PAH in vivo in Pah-KO mice The following examples describe studies on the efficacy of XL32.1 / mA1MB2-mTTR482-WT PAH in vivo in Pah-KO mice.

[0222] material and method Efficacy study in PAH-KO mice Homozygous (HOM) and heterozygous (HET) Pah-KO male mice were obtained at 8–12 weeks of age and housed and maintained in accordance with humane guidelines for animal care and use. All animal procedures were approved by the Sanofi Animal Ethics Committee (IACUC).

[0223] Recombinant AAVXL32.1 vector was administered intravenously via the tail vein (6–10 animals / procedure). Animals were sacrificed under isoflurane anesthesia. Whole blood was collected from the posterior orbital sinus into an EDTA collection tube, spun, and cryopreserved until analysis. Tissue collection was performed in some animals after perfusing the left ventricle with PBS. Liver samples were collected and frozen until analysis. For brain analysis, the whole brain was collected from the skull, weighed, sagittally segmented, and frozen at -80°C until analysis.

[0224] Blood and tissue analysis Plasma Phe and Tyr levels were analyzed by UHPLC-MS / MS. Brain was processed as described in Kankaanpaa 2001, with minor modifications. For liver samples, vector genome, PAH activity, and protein levels were quantified. Copies of the vector genome were quantified by qPCR (Martin 2013). PAH protein activity in liver homogenates was normalized by total protein as described above (BCA protein assay kit; Pierce).

[0225] result The XL32.1 / mA1MB2-mTTR482-WT PAH was tested for efficacy in a PKU mouse model. A Pah-KO model with no PAH protein production was used. The vector was administered intravenously to adult mice, and efficacy was evaluated 35 days or 4 months after treatment.

[0226] Levels of Phe and Tyr were measured in the blood and brain. Dopamine and serotonin levels were measured in the brain. Finally, behavior was evaluated, for example, by a nest-building assay.

[0227] Four months after administration, a thorough analysis of neuropathology and white matter changes was performed. These were evaluated by in vivo MRI and further by endpoint white matter staining. [Examples]

[0228] Short-term in vivo efficacy of XL32.1 / mA1MB2-mTTR482-WT hPAH in PAH-KO mice. The XL32.1 / mA1MB2-mTTR482-WT hPAH vector (also known as XL32.1 / WT hPAH) was evaluated in vivo in a PKU mouse model for 5 weeks. Following Pah gene transfer into the liver, a dose-dependent reduction in blood Phe levels and an increase in blood Tyr were observed. This correlated with dose-dependent detection of the vector genome, vector-derived mRNA, and PAH activity in the liver of treated mice. Lower Phe levels were associated with increased amino acid transport to the brain and increased levels of various neurotransmitters. Evaluation of PAH-positive cell levels in the liver demonstrated detection of pericentral PAH-positive hepatocytes, along with increasing intensity correlated with vector dose. In summary, our study demonstrates that the optimized rAAVXL32.1 vector encoding WT hPAH corrected PKU-related pathology in a PAH-KO mouse model, thus supporting its use for the treatment of human PKU.

[0229] material and method Vector generation. An XL32.1 capsid vector (mA1MB2-mTTR482-HI2) encoding WT hPAH expressed from an optimized liver promoter and introns was produced by triple transfection as described above. The vector was purified by affinity column followed by a CsCl gradient. The vector was titrated by qPCR, and PAH production and function were tested in vitro prior to animal experiments.

[0230] Efficacy studies in PAH-KO mice. Colonies of Pah-KO mice generated in a C57BL / 6 background were maintained at Jackson Laboratory (Singh et al., 2020; submitted). Some studies also used WT C57BL / 6 mice as normal controls. Homozygous (HOM) and heterozygous (HET) male mice were obtained at 8–12 weeks of age and housed and maintained according to humane guidelines for animal care and use. The vector was administered intravenously via the tail vein (6–10 animals / treatment). Animals were sacrificed under isoflurane anesthesia. Whole blood was collected from the posterior orbital sinus into an EDTA collection tube, spun, and cryopreserved until analysis. Tissue was collected from some animals after perfusing the left ventricle with PBS. Liver samples were collected and frozen until analysis. For brain analysis, the entire brain was collected from the skull, weighed, sagittally sliced, and frozen at -80°C until analysis.

[0231] Blood and tissue analyses. Plasma Phe and Tyr levels were analyzed by UHPLC-MS / MS as described in Singh et al., 2021. Brain was processed for the quantification of brain neurotransmitters as described in Kankaanpaa 2001;Singh 2020, with minor modifications. Copies of the vector genome in various tissues were quantified by qPCR (Martin 2013). PAH protein activity and detection of PAH protein in liver homogenates were performed as previously described (Heintz 2012, Nambiar 2017) and normalized by total protein (BCA protein assay kit; Pierce).

[0232] Animal behavior assay. The nest-building assay was performed as previously published, with minor modifications (Deacon 2006). Animals were transferred to clean, individual cages and supplied with 3.0 gm + / - 0.02 sq. oz cotton (Nestlet; cabfm00088, Ancare). The following day, all unused nest-building materials were weighed, and the quality of the nests was scored by two individuals based on the following rating scale: 1 - Nestlet untouched (more than 90% untouched), 2 - Nestlet partially torn (50-90% untouched), 3 - Most of the nestlet shredded, but the nest location is often not identifiable (less than 50% untouched), 4 - Identifiable but flat nest (more than 90% of the nestlet torn), 5 - Perfect nest with craters and high walls (more than 90% of the nestlet torn).

[0233] Plotting and statistical analysis. All data was graphed using GraphPad Prism (version 8.0.2) or Excel (Microsoft). Statistical analysis was performed using Student's t-test in Excel or one-way ANOVA in GraphPad Prism.

[0234] result Correction of blood Phe and Tyr after XL32 / WT hPAH gene transfer into Pah-KO mice. The efficacy of the XL32 / WT PAH vector after IV delivery was tested using a Pah-KO model with no PAH protein production, and efficacy was evaluated for 36 days. This treatment reduced blood Phe levels to levels comparable to HET and WT mice in a dose-dependent manner (Figures 6A and 6B). The treatment also increased blood Tyr levels, and normal blood Tyr levels were achieved at all vector doses tested (Figures 6C and 6D).

[0235] The livers of treated mice were evaluated for gene transfer efficiency and hepatic transduction. In the liver, vector DNA was detected in a dose-dependent manner at 1e11, 3e11, and 1e12 vg / mice, resulting in average levels of 0.2, 1.3, and 6.8 vg / cell, respectively (Figure 7A). The vector was predominantly detected in the liver, with levels measured in other tissues tested (spleen, heart, muscle, kidney, and lung) being less than one-tenth (Figure 7B). Dose-dependent detection of vector DNA was translated into a dose-dependent increase in vector-derived PAH mRNA in the liver (Figures 7C and 7D). Correlation of vg / cell copies with blood Phe levels demonstrated that a minimum of 0.1 vg / cell was required for normalization of blood Phe (Figure 7E). The functionality of the PAH protein was tested by measuring PAH activity in the liver. Low doses (1 e11 vg / mouse) induced PAH activity comparable to that in het mice, while two higher doses exceeded the activity measured in het livers (Figure 8A). Similar dose-response patterns in PAH protein levels were also observed by Western blotting of liver homogenates (Figure 8B). To understand the location of transduced hepatocytes, treated livers were also evaluated by immunohistochemistry (IHC). Increased PAH staining was detected with increasing dose, and the transduction pattern was primarily pericentral (Figure 8C).

[0236] Effects of XL32.1 / WT hPAH on brain amino acid and neurotransmitter levels. Brain amino acid levels of Phe, Tyr, and Trp were measured. The data demonstrated normalization of brain Phe levels after treatment, as levels were comparable to those measured in Het and WT mice. The reduction in blood Phe also increased the transport of these amino acids to the brain, as Tyr and Trp share the same amino acid transporter (Figure 9A).

[0237] The neurotransmitters dopamine and serotonin are known to be reduced in the brains of PKU patients. Therefore, we quantified the levels of these neurotransmitters in the brains of vector-treated mice. Treatment with the rAAVXL32.1 / WT hPAH vector resulted in normalization of dopamine, norepinephrine, and serotonin to levels observed in Het and WT mice (Figure 9B).

[0238] Behavioral analysis of Pah-KO mice. Nest-building behavior assays were performed to evaluate the effects of biochemical changes in the brain on animal behavior (Figures 10A and 10B). This assay measures the ability of mice to build nests, which were then scored based on the amount of nest-building material used and the overall quality of the nest (Figure 10A). Before treatment, all untreated PKU mice had significantly lower nest scores compared to those created by Het mice (Figure 10B). However, 36 days after treatment, scores significantly improved in treated mice (Figure 10B). No difference in nest-building scores was observed between Het and WT mice.

[0239] summary Our data demonstrate that an rAAV vector consisting of the XL32.1 capsid and expressing WT hPAH from an optimized liver promoter was able to correct multiple PKU-related pathologies in a mouse model of human PKU five weeks after treatment. Systemic delivery of this vector resulted in a dose-dependent increase in vector, vector-derived mRNA, and PAH activity in the liver of Pah-KO mice. This was associated with an increase in Tyr levels, in addition to a decrease in Phe levels in the blood and subsequently in the brain, at all doses tested. The reduction in Phe in both the blood and brain also normalized the levels of the neurotransmitters dopamine and serotonin in the brain. These biochemical changes correlated with improved behavior in the mice. The lowest dose used in this study, 5 e12 vg / kg (1 e11 vg / mouse), provided a vector with an average of 0.2 vg / cell, which resulted in comparable liver PAH activity as measured in Het mice. [Examples]

[0240] Long-term in vivo efficacy of XL32.1 / mA1MB2-mTTR482-WT hPAH in PAH-KO mice. material and method Vector generation. The XL32.1 capsid vector encoding WT hPAH contained a liver-specific expression cassette with a modified A1MB2 enhancer (two copies of alpha-1-microglobulin), a modified mouse trans tyretin core promoter and distal enhancer (mTTR482), hybrid intron 2 (HI2, an intron consisting of a chicken beta-actin / rabbit beta-globin hybrid intron), and a bovine growth hormone (BGH) polyadenylation site (BGH) (Nambiar 2017) (the complete genome name is ITR- / mA1M2-mTTR482-HI2-WT hPAH-BGHpA-Stuffer-ITR, also known as XL32.1 / WT hPAH). Two additional vectors with the LP1 liver promoter were also constructed; one vector contained either a hybrid intron 2 (HI2) (identical to the one used in the A1MB2-mTTR482 construct) or a short intron (SI, Nathwani 2012). The size of all constructs was adjusted to the size of the wild-type AAV genome by adding a stuffer sequence (A1AT intron sequence). Plasmids containing all ITRs were tested for PAH protein production and activity in vitro by transient transfection, PAH Western blotting, and activity testing in the human liver cell line Huh7 cells as previously described (Singh 2021). All XL32.1 capsid vectors were produced by triple transfection as described above. Vectors for 4-month efficacy studies were purified by affinity column, followed by a CsCl gradient. Vectors for 1-month and 4-month efficacy studies were purified by affinity column, followed by a CsCl gradient. All vector lots were quantified by qPCR against BGHpA (Nambiar 2017).

[0241] Efficacy study in PAH-KO mice over 4 months. The efficacy study was performed as described in Example 4. All animals underwent tissue collection after perfusion of the left ventricle with PBS.

[0242] Blood and tissue analysis. Blood and tissue analysis was performed as described in Example 4. Copies of vector DNA in liver were quantified by qPCR in liver (Nambiar 2017). PAH protein activity and detection of PAH protein in liver homogenates were performed as previously described (Heintz 2012, Nambiar 2017) and normalized by total protein (BCA protein assay kit; Pierce). Formalin-fixed paraffin-embedded livers were used for PAH IHC as described in Singh 2021. For digital quantification of IHC-positive cell percentage, IHC slides were analyzed using VISIOPHARM image analysis software (version 2020.08), and regions of interest (ROI) in whole liver slide images were measured. PAH staining intensity was measured by drawing a 3 μm boundary from the nucleus, and each cell was classified as either PAH-positive or PAH-negative. Formalin-fixed, paraffin-embedded liver fragments were also used for in-situ hybridization in selected animals using the BaseScope® double-strand reagent kit in manual mode, according to the protocol of Advanced Cell Diagnostics, Inc. (ACD). ISH staining of the entire fragment was analyzed using the HALO ISH Image Analysis Module (v4.1). The quantified endpoint was the percentage of vector DNA and mRNA-positive cells.

[0243] Brain imaging. As described in Singh 2021, brain white matter content was analyzed at various time points in living animals. The region of interest surrounding the brain and visible corpus callosum structures were plotted across coronal sections, and the volume of the corpus callosum was calculated.

[0244] Animal behavior assay. Animal behavior was evaluated as described in Example 4.

[0245] Plotting and statistical analysis. All data was graphed using GraphPad Prism (version 8.0.2) or Excel (Microsoft). Statistical analysis was performed using one-way ANOVA in GraphPad Prism with Tukey's multiple comparisons.

[0246] result Effects of XL32.1 / WT PAH gene transfer on health status, blood Phe levels, and hepatic PAH correction 4 months after treatment. The efficacy of the XL32.1 / WT PAH vector was tested after IV delivery using a Pah-KO model with no PAH protein production, and efficacy was evaluated for 4 months. The vector doses tested were 1e11, 3e11, and 1e12vg / mouse, which translate to approximately 5e12, 2e12, and 5e13vg / kg, respectively. Animal body weight was assessed throughout the study to monitor growth during the 4-month study. Baseline body weight was established by weighing animals 8 days prior to administration, and then weighed again at the end of the study, 120 days after vector delivery (Figures 11A, 11B). All Pah-KO mice treated with XL32.1 / WT PAH increased body weight, on average by 130–145%. The treatment also increased liver weight in PAH-KO mice, and body weight reached that of HET and WT mice in all treatment groups (Figure 11C). Therefore, PAH expression in the liver resulted in significant growth and improved health status in the treated mice.

[0247] Delivery of the XL32.1 / WT hPAH vector to the liver resulted in reduced blood Phe levels (Figures 12A-12C). The data demonstrated a rapid reduction in blood Phe levels in the treatment cohort, with Phe levels at day 7 being 337±123 μM (1 e11vg / mouse), 94±14 μM (3 e11vg / mouse), and 70±11 μM (1 e12vg / mouse), compared to naive Pah-KO mice (2742±70 μM). These were comparable to Phe levels in HET and WT mice (84±9 and 62±5 μM, respectively) (Figure 12B). On day 120, the blood Phe level in untreated Pah-KO mice was 2612±71 μM, while the mean Phe levels in the treated cohort were 1237±483 μM (1e11vg / mouse), 158±32 μM (3e11vg / mouse), and 64±5 μM (1e12vg / mouse). Phe levels in the two higher-dose cohorts were not significantly different from those in HET (110±13 μM) and WT (82±3 μM) mice (Figure 12C). Therefore, the intermediate and high-dose vector cohorts (3e11 and 1e12vg / mouse) resulted in sustained normalization of blood Phe levels. The lowest dose (1e11vg / mouse) showed variability, with 3 out of 6 mice exhibiting normal blood Phe levels. The remaining three animals initially had reduced blood Phe levels, but the effect was not sustained.

[0248] The livers of treated mice were evaluated for gene transfer efficiency and hepatic transduction. Vector DNA was detected in the liver in a dose-dependent manner, and the 1e11, 3e11, and 1e12 vg / mouse cohorts yielded, on average, the following vg / cell levels: 0.042 vg / cell ± 0.02, 0.321 vg / cell ± 0.111, and 3.40 vg / cell ± 0.49, respectively (Figure 13A). Quantification of vector-derived mRNA in the liver demonstrated, on average, 2.1e6 ± 1.0e6, 9.0e6 ± 2.0e6, and 3.7e7 ± 0.6e7 mRNA copies / μg RNA in each dose cohort, respectively (Figure 13B). This represents an approximately four-fold increase in expression at each dose. Excellent correlation with vector DNA and mRNA levels was observed (R 2 =0.90) (Figure 13C). The correlation with blood Phe levels at vg copies / cell demonstrated that a minimum of 0.1 vg / cell was required for blood Phe normalization (Figure 13D). Vector DNA analysis in the low-dose cohort revealed that the three animals whose blood Phe was normalized contained 0.1 vg / cell, while the remaining three animals whose Phe levels increased had levels below 0.1 vg / cell over the long term. Vector uptake and gene expression were also confirmed by in situ hybridization of vector DNA. Figure 13E shows vector-derived transcripts and representative images.

[0249] The functionality of PAH expressed from the vector was tested by measuring PAH activity in the liver. PAH activity was measured by quantifying Phe conversion to Tyr using an MS-based assay. PAH activity in three treatment cohorts (low to high dose) was 11.5±7.9 μM (n=6), 42.8±12.4 μM (n=8), and 168.2±21.4 μM (n=8). 13The protein was C-Tyr / mg (Figure 14A). For comparison, PAH activity in HET and WT animals was 63.7±5.9 μM (n=6) and 111.8±9.9 μM (n=6), respectively, while PAH activity was not detected in untreated Pah-KO mice (HOM, n=7). The assay demonstrated three animals in the low-dose cohort that lacked detectable PAH activity, and these same animals had very low vector DNA and mRNA copies. PAH activity showed a good correlation with vector transcription levels (R 2 =0.88 (not shown).

[0250] To understand the level of PAH-positive cells in the liver, liver fragments were evaluated by IHC using anti-PAH antibodies. The mean PAH-positive cell content in the liver of each treatment cohort was 0.4±0.1% for group 1 (HOM), 21.0±8.6% for group 2 (low), 42.2±4.0% for group 3 (moderate), 52.8±4.6% for group 4 (high), 93.6±1.7% for group 5 (HET), and 97.4±0.4% (WT) for group 6 (WT) (Figure 14B). The data showed that approximately 20% PAH-positive liver was required for normalization of blood Phe levels (Figure 14C). The 20% PAH-positive liver condition is consistent with the published liver regrowth results by Hamman et al. (2011), which demonstrated that a minimum of 10% wild-type or heterozygous hepatocytes are required in hepatocyte transplantation experiments to normalize blood Phe levels in Pahenu2 mice. Representative images of PAH IHC showed increased staining intensity with increasing dose of the vector encoding WT PAH (Figure 14D). PAH staining exhibited a heterogeneous staining pattern in the treated liver, with highly positive cell clusters dispersed among negative cells. This contrasted with the staining observed in HET and WT livers, which showed uniform staining intensity across liver fragments (Figure 14D).

[0251] The effects of XL32.1 / WT PAH on brain amino acid, neurotransmitter levels, and white matter content. High blood Phe levels can cause neurotoxicity due to increased Phe uptake into the brain. High blood Phe levels may also reduce the uptake of other large neutral amino acids (Tyr, Trp) into the brain, as they utilize the same amino acid transporter (LAT1). Our data demonstrated that low doses reduced brain Phe levels in each treatment cohort, showing a variable efficacy (Figure 15A). Mean brain Phe levels were 167±4 μM for group 1 (HOM), 94±27 μM for group 2 (low), 28±3 μM for group 3 (medium), 26±1 μM for group 4 (high), 30±3 μM for group 5 (HET), and 30±1 μM for group 6 (WT). All groups except the low-dose WT PAH vector cohort normalized brain Phe levels to levels equivalent to those of HET and WT mice. The reduction in blood Phe also improved Tyr levels in the brain: Group 1 (HOM), 13±1 μM; Group 2 (low), 16±1 μM; Group 3 (moderate), 16±1 μM; Group 4 (high), 19±1 μM; Group 5 (HET), 20±2 μM; and Group 6 (WT), 20±1 μM. Trp transport to the brain also improved; mean Trp levels in each treatment group were: Group 1 (HOM), 4.6±0.2 μM; Group 2 (low), 5.7±0.5 μM; Group 3 (moderate), 5.4±0.3 μM; Group 4 (high), 6.5±0.3 μM; Group 5 (HET), 6.3±0.5 μM; and Group 6 (WT), 6.4±0.3 μM. However, significant treatment effects on brain Tyr and Trp levels were observed only at the maximum dose.

[0252] Levels of the neurotransmitters dopamine and serotonin are reduced in the brains of PKU patients. As an explanation, both Phe toxicity and substrate deficiencies (Tyr, Trp) in the synthesis of these neurotransmitters have been proposed. Here, data demonstrate that PAH gene delivery to the liver improved both dopamine and serotonin levels in the brain (Figure 15B). Treatment with 3e11 and 1e12 / mouse doses normalized neurotransmitter levels to levels comparable to those in HET and WT mice, although variability was observed in the low-dose cohort. The variability in the low-dose cohort (1e11vg / mouse) correlated with its gene transfer efficiency and subsequent Phe regulation (3 effective animals and 3 ineffective animals). Correlation analysis of dopamine and serotonin levels with brain Phe or their substrate levels (Tyr for dopamine and Trp for serotonin) showed that a reduction in Phe resulted in a greater improvement than their amino acid substrate levels. Therefore, dopamine production showed a moderate correlation with brain Tyr levels (R 2 (=0.3887), showed a better correlation with reduced Phe levels in the brain (R 2 (=0.5057). Similarly, serotonin production did not show much modification to the increase in Trp levels in the brain (R 2 (=0.1938), showed a close correlation with a decrease in brain Phe levels (R 2 (=0.6418).

[0253] In vivo MRI studies were performed to assess changes in brain white matter and evaluate the efficacy of the treatment on brain health. 3D volumetric MRI segmentation and measurements were applied to quantify the characteristic MRI appearance of the corpus callosum. MRI analysis showed that MRI corpus callosum volume was significantly lower in Pah-KO mice compared to HET and WT mice, while all Pah-KO cohorts were comparable at baseline assessment (Figure 16A). 106 days after treatment in Pah-KO mice, corpus callosum volume increased in all dose cohorts, particularly when compared to baseline in each individual animal as a percentage (Figures 16B, 16C). However, at the 106-day time point, none of the treatment cohorts corrected corpus callosum volume to normal levels; brain weight was not normalized in any of the treatment cohorts (Figure 16D).

[0254] Behavioral analysis of Pah-KO mice. PKU patients suffer from a higher frequency of anxiety, depression, and motor tremor. These problems were measured using a behavioral assay that assesses the ability of mice to build nests (Deacon et al., 2006). Normal animals tear apart the stuffing and assemble it into a round, raised nest, while animals with depression are expected to use little to no of this material. Figure 17A shows the scoring of nest building used. Before treatment, the scores were 1.7±0.4 for group 1 (HOM), 1.4±0.2 for group 2 (low), 1.7±0.4 for group 3 (moderate), 1.7±0.4 for group 4 (high), 5.0±0.0 for group 5 (HET), and 5.0±0.0 for group 6 (WT), with no significant differences between the untreated Pah-KO mouse cohort (Figure 17B). 35 days after treatment, nest-building scores significantly improved among treated Pah-KO mice, with group mean scores of 2.1±0.6 for Group 1 (HOM), 3.8±0.5 for Group 2 (low), 4.6±0.2 for Group 3 (medium), 3.9±0.4 for Group 4 (high), 5.0±0.0 for Group 5 (HET), and 4.9±0.1 for Group 6 (WT). There was no significant difference in scores between treated Pah-KO mice and HET and WT mice. On day 97, group mean scores were similar to those on day 35, as follows: 1.7±0.3 for Group 1 (HOM), 2.5±0.6 for Group 2 (low), 4.1±0.3 for Group 3 (medium), 4.0±0.3 for Group 4 (high), 5.0±0.0 for Group 5 (HET), and 4.9±0.1 for Group 6 (WT). With the exception of the low-dose treatment group, all groups showed significant improvement compared to untreated Pah-KO mice. Similarly, with the exception of the low-dose cohort, all groups showed no significant difference compared to HET or WT mice.

[0255] summary Gene therapy by transferring a functional Pah gene into the liver to correct defective PAH activity in the liver of PKU patients is an attractive strategy for providing long-term Phe control in PKU patients. Our data demonstrate that an rAAV vector (mA1MB2-mTTR482-HI2) consisting of an XL32.1 capsid and expressing WT hPAH from an optimized liver expression cassette was able to correct multiple PKU-related pathologies in Pah-KO mice, a model of human PKU, over a 4-month study period. Systemic delivery of this vector resulted in dose-dependent increases in vector DNA, vector-derived mRNA, PAH protein, and PAH activity in the liver of Pah-KO mice. All three vector doses tested (approximately 5e11, 2e13, and 5e13vg / kg) reduced blood Phe levels, although variability was observed in the lowest dose cohort. Modification of blood Phe levels to various research endpoints, therapeutic utility over a 4-month study period, and normalization of blood Phe levels using a minimum of 0.1 vector DNA / cell, 3 × 10⁶ 6The study demonstrated the need for maintenance of mRNA / μg RNA and 20% PAH-positive liver. The 20% PAH-positive liver condition is similar to the results of hepatic regrowth published by Hamman et al. (16), which demonstrated that transplantation of a minimum of 10% wild-type or heterozygous hepatocytes was required to normalize blood Phe levels in Pahenu2 mice. The embodiments of the present invention also demonstrated sustained improvements in brain health over 4 months, including normalization of amino acid transport, neurotransmitter dopamine and serotonin levels, and increased corpus callosum volume. These biochemical changes correlated with improved behavior in the mice; efficacy was already observed 35 days after treatment and was maintained until a later time point (day 97). Interestingly, three animals in the low-dose group without sustained Phe control demonstrated inferior values ​​in all measured endpoints, suggesting a correlation between blood Phe and disease pathology. Furthermore, normalization of blood Phe increased the body weight of the treated animals, highlighting the significant impact of hyperphenylalanemia on the overall growth and metabolism of PKU animals. This growth was reflected in increased liver weight to levels in WT and HET mice, suggesting hepatocyte proliferation. Nevertheless, the medium and high-vector cohorts maintained sufficient vector DNA to provide efficacy until the end of the study. In addition, the low-dose 1e11vg / mouse (5×10) 12 The variability observed in the vg / kg dose cohort allowed us to define the threshold level for gene transfer required in situations that could lead to loss of the vector genome, such as liver injury or proliferation.

[0256] In summary, the embodiments of the present invention demonstrated that rAAVXL32.1 / WT hPAH gene transfer can reduce blood Phe levels, resulting in sustained improvements in growth, increased brain white matter, brain amino acid content and neurotransmitter levels, and overall improved behavior. Comparison of the lead genome (mA1MB2-mTTR482-HI2) in vivo with a liver expression cassette (LP1-SI, Nathwani 2011) already used in clinical practice showed that the lead candidate exhibited higher levels of transcriptional and enzymatic activity in mouse liver. Therefore, the combination of higher expression levels of the lead candidate and superior gene transfer efficiency with the XL32.1 capsid allows for the treatment of PKU patients in clinical practice at potentially lower vector doses, providing improved therapeutic safety. Taken together, the embodiments of the present invention support the use of this vector for the treatment of PKU by enabling treatment at clinically effective, feasible, and safe AAV vector doses. [Examples]

[0257] Comparison of liver expression elements in vivo The XL32.1 vector containing mA1MB2-mTTR482-HI2, used in the 4-month study described above, was evaluated against the liver expression cassette (LP1-SI) (Nathwani 2011) used in the hemophilia B clinical trial. An intermediate construct with an LP1 promoter containing an HI2 intron was also evaluated (Figure 18A). Testing of the ITR-containing plasmid construct in the human liver cell line Huh7 cells by transient transfection showed higher PAH protein and activity from the A1MB2 construct compared to the construct with the LP1 promoter (Figures 18B, 18C). Three expression cassettes packaged in XL32.1 capsids were administered to Pah-KO mice for 5 weeks at equivalent doses (3e11vg / mouse) and evaluated. All rAAV vectors used for expression cassette comparison were purified by CsCl gradient. All vectors significantly reduced blood Phe levels compared to untreated Pah-KO mice (HOM), and Phe levels were similar to those observed in HET mice (Figure 18D). Close-up analysis showed differences in vector expression levels. Quantification of mRNA levels in the liver showed a tendency for higher expression from vectors with the mA1MB2-mTTR482 promoter compared to LP1-SI, and when transcription levels were normalized, approximately three times higher mRNA levels per VG were observed using the mA1MB2-mTTR482 promoter. The mean normalized RNA levels (mRNA / VG) were 133.9±19.5 for A1MB2, 95.5±11.0 for LP1-HI2, and 43.0±4.9 for LP1-SI (Figure 18E). Similarly, hepatic PAH enzyme activity was three times higher in animals treated with mA1MB2-mTTR482-HI2 compared to the LP1-SI construct. The mean hepatic PAH activity (μM Tyr / mg protein) in the study cohort was 157.4±25.7 for A1MB2, 42.9±10.2 for LP1-HI2, 54.9±13.4 for LP1-SI, and 67.8±10.9 for HET (Figure 18F). When PAH activity was normalized to VG copies, this difference was six-fold (Figure 18G).When activity was normalized to mRNA copies, a smaller difference (2x) was observed, suggesting that the increase in expression from vector DNA, rather than PAH production per mRNA, was the main reason for the difference (not shown). Therefore, the data demonstrate that in mouse liver, expression from mA1MB2-mTTR482-HI2 was stronger than expression from the LP1-SI expression cassette.

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Viecelli HM, Harbottle RP, Wong SP, Schlegel A, Chuah MK, VandenDriessche T, Harding CO, Thony B. Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology 2014, 60:1035-1043. Chatterjee, S., Sivanandam, V, Wong, Jr, K. K. AAV and Hematopoietic Stem Cells: The Potential of AAVHSCs in Genetic Medicines. Human Gene Ther 2020, 31: 542-552. Sabatino, D. E., Lange, A M., Altynova, E. S., Sarkar R., Zhou, S., Merricks, E. P., Franck, H. G., Nicols, T. C., Arruda, V. R.,Kazazian Jr, H. H. Efficacy and safety of long-term prophylaxis in severe hemophilia A dogs following liver gene therapy using AAV vectors. Mol Ther 2011, 19: 442-9. Singh K, Cornell CS, Jackson R, Kabiri M, Phipps M, Desai M, et al. CRISPR / Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria. Scientific Reports 202111: 7254.. Kankaanpaa, A., Meririnne, E., Ariniemi, K. & Seppala, T. 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Liver-specific transcriptional modules identified by genome-wide in silico analysis enable efficient gene therapy in mice and non-human primates. Mol Ther 2014, 9:1605-1613. Wooddell CI, Reppen T, Wolff JA, Herweijer H. Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery. J Gene Med 2008, 10:551-563. Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC, Chowdary P, Riddell A, Jaquilmac A, Harrington C, O’Beirne J, Rustagi P, Ng CYC, Kay MA, Zhou J, Spence Y, Morton CL, Allay J, Coleman J, Sleep S, Cunningham JM, Srivastava D, Basner-Tschakarjan E, Mingozzi F, High KA, Gray JT, Reiss U, Nienhuis A, Davidoff AM. Jiang J, Larimore K, Gupta S, Gu Z, Northrup H; PRISM investigators. Mol Genet Metab. Adeno-associated virus vector-mediated gene transfer in hemophilia B. NEJM 2011, 365:2357-2365. 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[0259] Sequence Human phenylalanine hydroxylase (GenBank AAA60082.1 / NP_000268.1 protein / NM_000277.3 mRNA; WT PAH amino acid sequence containing E183) MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDVNLTHIESRPS RLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQELDRFANQILSYGA ELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTVFKTLKSLYKTHACYEYNHIF PLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFLGGLAFRVFHCTQYIRHGSKPMYTPE PDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSS FGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQR IEVLDNTQQLKILADSINSEIGILCSALQKIK(Sequence No. 1) Human WT PAH coding sequence XL32 and XL32.1 capsid amino acid sequences (Sequence ID 3) XL32 capsid DNA sequence XL32.1 capsid DNA sequence

[0260] Modified PrT2 enhancer sequence [ka] Underlined section: Hepatic nuclear factor binding site; Bold: Modification introduced to a higher affinity binding site; Italic: Repeating sequence

[0261] Modified A1MB2 enhancer [ka] Underlined section: Hepatic nuclear factor binding site; Bold: Modification introduced to a higher affinity binding site; Italic: Repeating sequence

[0262] Modified Ealb sequence [ka] Underlined section: Hepatic nuclear factor binding site; Bold: Modification introduced to a higher affinity binding site; Italic: Repeating sequence

[0263] HEII Enhancer CCATCAGATCCTGCCCAAGGTCTTACATAAGA GGACTCTTGGAC TCCCAGCAATGTCAACGACCGACCTTGAGGCCTACTTCAAAGACTGTGTGTTTAAGGACTGGGAGGAGCTGGGGGAGGAGATTAGGTTAAAGGTCTTTGTATTAGGAGGCTG (SEQ ID NO: 10) CRM8 Enhancer GGGGAGGCTGCTGGTGAATATTAACCAAGGTCACCCCAGTTATCGGAGGAGCAAACAGGGGCTAAGTCCAC (SEQ ID NO: 11) 3'Alb Stability Element (Sequence ID 12) 3'alb and SMAR stability elements

[0264] ITR-mA1MB2-mTTR482-HI2-WT hPAH / E-BGHpA-Stuffer-ITR sequence;The code sequence for WT PAH is underlined. ATGAGCACAGCCGTGCTGGAAAACCCCGGCCTGGGCAGAAAGCTGAGCGACTTCGGCCAGGAAACCAGCTACATCGAGGACAACTGCAACCAGAACGGCGCCATCAGCCTGATCTTCAGCCTGAAAGAAGAAGTGGGCGCCCTGGCCAAGGTGCTGCGGCTGTTCGAGGAGAACGACGTGAACCTGACCCACATCGAGAGCCGGCCCAGCAGACTGAAGAAGGACGAGTACGAGTTCTTCACCCACCTGGACAAGCGGAGCCTGCCCGCCCTGACCAACATCATCAAGATCCTGCGGCACGACATCGGCGCCACCGTGCACGAGCTGAGCCGGGACAAGAAAAAGGACACCGTGCCCTGGTTCCCCAGAACCATCCAGGAACTGGACAGATTCGCCAACCAGATCCTGTCCTACGGCGCCGAGCTGGATGCCGACCACCCTGGCTTCAAGGACCCCGTGTACCGGGCCAGACGGAAGCAGTTCGCCGATATCGCCTACAACTACCGGCACGGCCAGCCCATCCCCAGAGTCGAGTACATGGAAGAGGAGAAGAAAACCTGGGGCACCGTGTTCAAGACCCTGAAGTCCCTGTACAAGACCCACGCCTGCTACGAGTACAACCACATCTTCCCACTGCTCGAAAAGTACTGCGGCTTCCACGAGGACAATATCCCTCAGCTGGAGGACGTGTCCCAGTTTCTGCAGACCTGCACCGGCTTCAGACTCAGGCCTGTGGCCGGCCTGCTGAGCAGCAGAGATTTTCTGGGCGGACTGGCCTTCCGGGTGTTCCACTGCACCCAGTACATCAGACACGGCAGCAAGCCCATGTACACCCCTGAGCCCGACATCTGCCACGAGCTGCTGGGACATGTGCCCCTGTTCAGCGACAGAAGCTTCGCCCAGTTCAGCCAGGAAATCGGCCTGGCCTCTCTGGGCGCTCCCGACGAGTATATCGAGAAGCTGGCCACCATCTACTGGTTCACCGTGGAATTCGGCCTGTGCAAGCAGGGCGACAGCATCAAGGCCTATGGCGCCGGACTCCTGTCCAGCTTCGGCGAGCTGCAGTACTGTCTGAGCGAGAAGCCCAAGCTGCTGCCCCTGGAACTGGAAAAGACCGCCATCCAGAACTACACCGTGACCGAGTTCCAGCCCCTGTACTACGTGGCCGAGAGCTTCAACGACGCCAAAGAAAAAGTGCGGAACTTCGCCGCCACCATCCCTCGGCCCTTCAGCGTCAGATACGACCCCTACACCCAGCGGATCGAGGTGCTGGACAACACACAGCAGCTGAAAATTCTGGCCGACTCCATCAACAGCGAGATCGGCATCCTGTGCAGCGCCCTGCAGAAAATCAAGTGA

[0265] Modified chicken β-actin (CBA) / rabbit β-globin hybrid / intron (HI2) [ka]

[0266] 0.9kb A1AT intron stuffer arrangement [ka]

Claims

1. Recombinant adeno-associated virus (rAAV) particles comprising an rAAV vector, wherein the rAAV vector comprises an expression cassette for expressing a transgene in liver cells, the expression cassette comprising a transgene operably ligated to a promoter and an enhancer, the promoter comprising a mouse trans tyretin (mTTR) promoter, the enhancer comprising one or two modified alpha-1-microbicinol enhancers (mA1MB2), and the transgene encoding a PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1; The AAV virus particle contains an AAV-XL32 or AAV-XL32.1 capsid, which has an amino acid sequence 100% identical to SEQ ID NO:

3. mA1MB2 contains the sequence of sequence number 8, The aforementioned rAAV particle.

2. The rAAV particle according to claim 1, wherein the mTTR promoter is the mTTR482 promoter.

3. The expression cassette is a. Introns that are introns, which may be chicken β-actin / rabbit β-globin hybrid introns; and / or b. Polyadenylation signals that are, in some cases, bovine growth hormone polyadenylation signals; The rAAV particle according to claim 1 or 2, further comprising:

4. The rAAV particle according to any one of claims 1 to 3, wherein the introduced gene is at least 80% identical to the nucleic acid sequence of Sequence ID No.

2.

5. The rAAV vector contains an expression cassette with one or more AAV reverse terminal repeat (ITR) sequences adjacent to each other, and may include The expression cassette has two adjacent ITRs of AAV; The ITRs of AAV are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITRs; The ITR of AAV is the ITR of AAV2; and / or The vector is a self-complementary vector, and in some cases, The vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding a complement of the first nucleic acid sequence, wherein the first nucleic acid sequence can form intrachain base pairs with the second nucleic acid sequence along most or all of its length, and optionally, The first nucleic acid sequence and the second nucleic acid sequence are linked by the ITR of the mutated AAV, the ITR of the mutated AAV containing a deletion in the D region and a mutation in the terminal decomposition sequence. rAAV particles according to any one of claims 1 to 4.

6. The rAAV particle according to claim 1, comprising, from 5' to 3', an AAV2 ITR, a modified alpha-1-microbicin enhancer (mA1MB2), a mouse trans-tyretin (mTTR) promoter, a chicken β-actin / rabbit β-globin hybrid intron, a codon-optimized human PAH gene encoding a PAH polypeptide comprising the amino acid sequence of SEQ ID NO: 1, a bovine growth hormone polyadenylation signal, a stuffer fragment derived from the alpha-1-anti-trypsin gene, and an AAV2 ITR.

7. The rAAV particle according to claim 6, wherein the chicken β-actin / rabbit β-globin hybrid intron comprises the sequence of SEQ ID NO:

15.

8. The rAAV particle according to claim 6, wherein the stuffer fragment derived from the alpha-1-anti-trypsin gene comprises the sequence of SEQ ID NO:

16.

9. The rAAV particle according to claim 6, comprising the nucleic acid sequence of Sequence ID No. 2 of the codon-optimized human PAH gene.

10. The rAAV particle according to claim 6, wherein the rAAV vector comprises the nucleic acid sequence of sequence number 14.

11. The rAAV vector comprises a first nucleic acid sequence encoding a PAH polypeptide and a second nucleic acid sequence encoding a complement of the first nucleic acid sequence, wherein the first nucleic acid sequence can form intrachain base pairs with the second nucleic acid sequence along most or all of its length. The rAAV particle according to claim 1, wherein the first nucleic acid sequence and the second nucleic acid sequence are linked by an ITR of mutated AAV, the ITR of mutated AAV comprising a deletion of the D region and a mutation of the terminal degradation sequence.

12. The rAAV particle according to any one of claims 1 to 11, wherein the AAV-XL32 capsid comprises VP1, VP2, and VP3, wherein VP1, VP2, and VP3 are encoded by the nucleic acid sequence of Sequence ID No. 4, or the AAV-XL32.1 capsid comprises VP1, VP2, and VP3, wherein VP1, VP2, and VP3 are encoded by the nucleic acid sequence of Sequence ID No.

6.

13. A composition comprising rAAV particles according to any one of claims 1 to 12, further comprising a pharmaceutically acceptable carrier, in some cases.

14. A cell containing rAAV particles according to any one of claims 1 to 12.

15. A method for producing a PAH polypeptide, comprising culturing the cells described in claim 14 under conditions for producing a PAH polypeptide, and optionally further comprising a step of purifying the PAH polypeptide.

16. The composition according to claim 13, for use in treating phenylketonuria in individuals that require it and which may lack PAH activity.

17. The composition according to claim 13, for use in reducing the level of phenylalanine in the blood of an individual in need, where, in some cases, the level of phenylalanine in the blood of the individual before treatment is elevated compared to the level of phenylalanine in the blood of an equivalent, corresponding control individual.

18. The composition is administered intravenously, intra-arterially, intrahepatically, intra-portally, intraperitoneally, or subcutaneously; and / or, The administration is combined with other therapies, which may include treatment with tetrahydrobiopterin, treatment with phenylalanine ammonia lyase (PAL) or pegylated PAL, or a phenylalanine-restricted diet. A composition for use according to claim 16 or 17.

19. A kit comprising, depending on the circumstances, instructions for use; a buffer and / or a pharmaceutically acceptable excipient; and / or a bottle, vial and / or syringe, comprising rAAV particles according to any one of claims 1 to 12, the composition according to claim 13, or the cells according to claim 14.

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