RescueModel: Mouse (Mus musculus)

GeneAlteration: PAH_enu2 mice were purchased and bred to obtain the homozygotic mice for this study. Original Generation Method: Mutations were induced in mice germline cells at a rate near 10^-3 per locus via an alkylating agent called ethylnitrosourea. Inbred BTBR males were treated with this agent and bred to normal BTBR females. Three hundred and fifty progeny were screened for mutations that did not complement the previous model PAH_enu1 and if present bred to a previous homozygote and tested for hyperphenylalaninemia. A PHE clearance test was performed in questionable cases. For those that tested positive the carrier parents were mated to a normal BTBR partner to establish a cogenic mutant line. Western blot confirmed reduced to absent CRM in liver.

RescuePhenotype: HP:0007513, HP:0004923, HP:0005982, HP:0100610 (Generalized hypopigmentation, Hyperphenylalaninemia, Reduced phenylalanine hydroxylase activity, Maternal hyperphenylalaninemia)

RescueMethod: The human PAH expression vector plasmid pAAV2-EF-hPAH-WPRE was created by subcloning a full-length human PAH cDNA into a preexisitng parent vector by substitution with eGFP cDNA. Alos included is an EF promoter, woodchuck hepatitis virus post-transcriptional element, and bovine growth hormone poly (A) signal. The rep2/cap2 plasmid or rep2/cap8 plasmid was used to package the vector and produce the final rAAV2/2-hPAH and rAAV2/8-hPAH vectors by triple plasmid transfection. Five-to-six week old PAH_enu2 mice were anesthetized with ketamine-xylazine and infused with viral particles of rAAV2/2-hPAH or rAAV2/8-hPAH through the hepatic portal vein or tail vein.

RescueEvidence: PAH activity in the livers of treated mice six weeks after treatment recovered to 58.43±5.73% and 89.25±8.79% of the wild-type activity in female and male PAH_enu2 mice respectively compared to <1% WT in untreated mice. Plasma phenylalanine concentration in female PAH_enu2 mice infused with rAAV2/8-hPAH vectors decreased markedly to 110±34.5 µM at two weeks from 2,891±336.2 µM and showed continued significant phenylalanine clearance for six month after rAAV2/8-hPAH infusion. Male PAH_enu2 showed a similar reduction, though they started at a lower base concentration of 1,800.6±233.7 µM. The fur color of treated mice also changed from greyish to black from one week after delivery with sustained fur color changes over the entire six month period. Investiation of effects on pregnant mice revealed a complete recovery of fetus length and body weight compared to the untreated mice (1.746±0.203 cm and 774±144 mg for untreated VS 2.081±0.269 cm and 916±67 mg for treated). Spontaneous abortion rate also normalized to 4.55±5.89% from 30.95±14.27%. The rAAV-hPAH genome was not found in the placentae or fetuses nor did the genome of fetuses affect the development parameters.

ModelVariant: NM_000277.3(PAH):c.788T>C (p.Phe263Ser)

ClinVar: 619150 https://www.ncbi.nlm.nih.gov/clinvar/variation/619150/