In addition to correcting the hepatic metabolic defect, liver-targeted AAV8 therapy in presymptomatic AIP mice improved neuromotor function, as evidenced by their performance on the rotarod and footprint analysis (Figures 5a,b). Although this is encouraging, it should be noted that there is a distinct difference between the neuropathy that occurs in the AIP mice and human patients. In Temsirolimus CCI-779 the mice, the peripheral motor neuropathy develops chronically and progressively in the absence of ALA and PBG accumulation,8 whereas in humans, it typically occurs during an acute attack accompanied by elevated porphyrin precursors, and the symptoms remit once the attack resolves. The reason for this species difference is unknown, but presumably explains why AAV8 therapy only partially improved the neuromotor function in the AIP mice despite the metabolic abnormality in the liver being abolished.

In summary, these studies demonstrate that treatment with the AAV8 liver-targeted vector effectively transduced hepatic cells and provided rapid and prolonged HMB-synthase enzyme activity that continuously protected the AIP mice from the biochemical induction of acute attacks. Further, AAV8 therapy significantly improved neuromotor function in the AIP mice. These studies not only provide the rationale for the development of AAV8-mediated gene therapy for AIP patients with recurrent attacks, but further serve as a treatment model for the other hepatic porphyrias. Materials and Methods AAV2/8-HMBS vector construction and production.

The full-length murine HMB-synthase complementary DNA of the housekeeping isoform25 was subcloned into the DC-172 expression vector containing the liver-specific ��1-microglobulin enhancer and ��1-antityrpsin promoter, as previously described.22 The entire expression cassette was excised and cloned into the AAV2 previral plasmid pTR-UF12 (a gift from Michael Linden, Mount Sinai School of Medicine) and designated pTR172-HMBS. Plasmid DNA was purified using the QIAfilter plasmid Giga kit (Qiagen, Valencia, CA) and both inverted terminal repeat sites were confirmed by sequence analysis, following HgaI digestion. To produce rAAV2/8-HMBS, the pTR172-HMBS plasmid was cotransfected into HEK 293 cells with an adenovirus helper plasmid and a chimeric packaging construct that had the AAV2 rep gene fused to AAV8-derived cap genes. Following purification by column chromatography, rAAV2/8-HMBS was titered for DNase-resistant particles using a real-time TaqMan PCR assay with primers specific to the bovine growth hormone polyadenylation signal sequence. Animal studies. Animal procedures were reviewed and approved by the Mount Sinai Institutional Carfilzomib Animal Care and Use Committee.