PCR was performed on an iQ5 Multicolor Real-Time PCR (Bio-Rad), using the iQ5 Standard Edition Software, version 2.0. Genomic DNA (10 ng) was subjected to a two-step PCR amplification under the following conditions: 1 cycle 95°C × 3 minutes, followed by 45 cycles of 95°C × 15 seconds and 68°C × 40 seconds. Primer sequences: hAAT forward: 5′-TCCTGGGTCAACTGGGCATC-3′; hAAT reverse: 5′-CAGGGGTGCCTCCTCTGTGA-3′; Gapdh forward: 5′-CCACCCCAGCAAGGACACTG-3′; Gapdh reverse 5′-GCTCCCTAGGCCCCTCCTGT-3′. RAD001 Dilutions of hAAT plasmid into mouse genomic DNA were used to generate copy number standards. Results were normalized to Gapdh expression. In Fah5981SB mice, a single point mutation (GA transversion) at the terminal nucleotide

of Fah exon 8 leads to mis-splicing and exon-8 deletion from the messenger RNA (mRNA). Several important criteria derived from the literature18 were considered for the design of

the gene repair vector to correct the Fah5981SB point mutation (Fig. 1A). First, the vector should not contain elements needed for driving gene expression such as promoters, enhancers, or cDNA expression cassettes. Second, the fidelity and length of homology should be maximized with the packaging capacity of AAV (4.7 kb) check details being the limit. Third, the position of the nucleotide targeted for repair should be at the center of the homology. A 4.5-kb PCR product homologous to murine Fah was cloned into an AAV plasmid backbone and verified by DNA sequencing. Recombinant AAV-Fah of serotypes 2 and 8 were produced and administered to Fah5981SB mice as neonates or adults. Correction of the point mutation by homologous recombination (Fig. 1B) leads to normal Fah gene and protein expression. The evaluation of homologous recombination as a strategy for gene repair has traditionally relied on detecting alterations in reporter sequences rather than correcting

a disease phenotype. Given the selective advantage of FAH+ hepatocytes in the HTI liver, Fah5981SB mice can be used to study the clinical significance of AAV-mediated gene repair by homologous recombination. Four d3 Fah5981SB neonates were intravenously injected with 1 × 1011 vg of AAV2-Fah and kept on NTBC until weaning, followed by NTBC withdrawal to select for corrected Amino acid hepatocytes. Two control groups were injected with isotonic NaCl solution. Control group I (n = 3) did not receive a course of NTBC post-weaning, continued to lose weight and died. Control group II (n = 2) did receive one course of NTBC post-weaning but failed to maintain a healthy weight and died. AAV-treated mice began to stabilize in weight at 8 weeks after treatment, suggesting the onset of sufficient liver function. At age 11 weeks, a two-thirds partial hepatectomy was performed to induce liver regeneration and subsequent episomal AAV loss. Continued clinical improvement following partial hepatectomy strongly suggested stable gene repair at the Fah locus.