Adeno-associated virus compositions for PAH gene transfer and methods of use thereof

#Phenylketonuria (PKU) is an autosomal recessive genetic disorder where the majority of cases are caused by mutations in the phenylalanine hydroxylase (PAH) gene. The PAH gene encodes a hepatic enzyme that catalyzes the hydroxylation of L-phenylalanine (Phe) to L-tyrosine (Tyr) upon multimerization. Reduction or loss of PAH activity leads to phenylalanine accumulation and its conversion into phenylpyruvate (also known as phenylketone). This abnormality in phenylalanine metabolism impairs neuronal maturation and the synthesis of myelin, resulting in mental retardation, seizures and other serious medical problems.

#Currently, there is no cure for PKU. The standard of care is diet management by minimizing foods that contain high amounts of phenylalanine. Dietary management from birth with a low phenylalanine formula largely prevents the development of the intellectual disability of the disorder. However, even on a low-phenylalanine diet, children still suffer from growth retardation, and adults often have osteoporosis and vitamin deficiencies. Moreover, adherence to life-long dietary treatment is difficult, particularly once children reach school age.

#New treatment strategies have recently emerged, including large neutral amino acid (LNAA) supplementation, cofactor tetrahydrobiopterin therapy, enzyme replacement therapy, and genetically modified probiotic therapy. However, these strategies suffer from shortcomings. The LNAA supplementation is suitable only for adults not adhering to a low Phe diet. The cofactor tetrahydrobiopterin can only be used in some mild forms of PKU. Enzyme replacement by administration of a substitute for PAH, e.g., phenylalanine ammonia-lyase (PAL), can lead to immune responses that reduce the efficacy and/or cause side effects. As to genetically modified probiotic therapy, the pathogenicity of PAL-expressing E. coli has been a concern.

#FIGS. 1A, 1B, 1C, 1D, and 1E are vector maps of the pHMI-hPAH-TC-004, pHMI-hPAH-TC-025, pHMI-hPAH-TC-010, pHMI-hPAH-TC-011, and pHMI-hPAH-TC-012 vectors, respectively.

#FIG. 2 is an image of Western blot showing the expression of human PAH from the pCOH-WT-PAH (“WT PAH”), pCOH—CO-PAH (“CO PAH pCOH”), and pHMI-CO-PAH (“CO PAH pHMI”) vectors. 5×105 HEK 293 cells were transfected with 1 μg of vector. Lysate of the cells was collected 48 hours after transfection. The expression of human PAH was detected by Western blotting with an anti-PAH antibody (Sigma HPA031642). The amount of GAPDH protein as detected by an anti-GAPDH antibody (Millipore MAB 374) was shown as a loading control.

#FIGS. 3A and 3B are graphs showing the Phe level in the serum of two pah−/− mice (“Mouse H1” and “Mouse H5”) each administered with 5×1013 vector genomes of the rAAV-CBA-mPAH vector packaged in an AAVHSC capsid per kg of body weight intravenously via the tail vein. Serum samples were collected in a time course. The Phe levels were measured with a BioAssay Systems ELISA kit EPHE-100 (FIG. 3A) or mass spectrometry (FIG. 3B).

#The instant disclosure provided adeno-associated virus (AAV) compositions that can restore PAH gene function in a cell. Also provide are packaging systems for making the adeno-associated virus compositions.

#As used herein, the term “replication-defective adeno-associated virus” refers to an AAV comprising a genome lacking Rep and Cap genes.

#As used herein, the term “PAH gene” refers to the phenylalanine hydroxylase gene. The human PAH gene is identified by Entrez Gene ID 5053. An exemplary nucleotide sequence of a PAH mRNA is provided as SEQ ID NO: 24. An exemplary amino acid sequence of a PAH polypeptide is provided as SEQ ID NO: 23.