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Key Points AAV delivery of ZFNs and corrective Donor vectors to adult mouse liver results in stable human factor IX levels, normalizing hemophilic clotting times.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disorder caused by deficiency of iduronate 2-sulfatase (IDS), leading to accumulation of glycosaminoglycans (GAGs) in tissues of affected individuals, progressive disease, and shortened lifespan. Currently available enzyme replacement therapy (ERT) requires lifelong infusions and does not provide neurologic benefit. We utilized a zinc finger nuclease (ZFN)-targeting system to mediate genome editing for insertion of the human IDS (hIDS) coding sequence into a "safe harbor" site, intron 1 of the albumin locus in hepatocytes of an MPS II mouse model. Three dose levels of recombinant AAV2/8 vectors encoding a pair of ZFNs and a hIDS cDNA donor were administered systemically in MPS II mice. Supraphysiological, vector dose-dependent levels of IDS enzyme were observed in the circulation and peripheral organs of ZFN+donor-treated mice. GAG contents were markedly reduced in tissues from all ZFN+donor-treated groups. Surprisingly, we also demonstrate that ZFN-mediated genome editing prevented the development of neurocognitive deficit in young MPS II mice (6-9 weeks old) treated at high vector dose levels. We conclude that this ZFN-based platform for expression of therapeutic proteins from the albumin locus is a promising approach for treatment of MPS II and other lysosomal diseases.

Mucopolysaccharidosis type I (MPS I) is a severe disease due to deficiency of the lysosomal hydrolase α-L-iduronidase (IDUA) and the subsequent accumulation of the glycosaminoglycans (GAG), leading to progressive, systemic disease and a shortened lifespan. Current treatment options consist of hematopoietic stem cell transplantation, which carries significant mortality and morbidity risk, and enzyme replacement therapy, which requires lifelong infusions of replacement enzyme; neither provides adequate therapy, even in combination. A novel in vivo genome-editing approach is described in the murine model of Hurler syndrome. A corrective copy of the IDUA gene is inserted at the albumin locus in hepatocytes, leading to sustained enzyme expression, secretion from the liver into circulation, and subsequent uptake systemically at levels sufficient for correction of metabolic disease (GAG substrate accumulation) and prevention of neurobehavioral deficits in MPS I mice. This study serves as a proof-of-concept for this platform-based approach that should be broadly applicable to the treatment of a wide array of monogenic diseases.

Our previous studies showed that the homeobox (Hox) D3 transcription factor induces expression of alphavbeta3 integrin and promotes endothelial cell (EC) migration and angiogenesis. Since binding of Hox 3 factors to target DNA is enhanced by the co-factor Pbx, we investigated whether Pbx1 is also required for angiogenesis. We observed that EC predominantly express the Pbx1b isoform. Nuclear extracts from angiogenic EC express higher levels of active Pbx1 and more effectively form complexes on Pbx1/Hox consensus DNA oligonucleotides as compared to nuclear extracts from quiescent EC. Introduction of anti-sense against Pbx1 impaired the formation of Pbx1/Hox complexes on target DNA consensus in nuclear extracts from angiogenic EC. Anti-sense against Pbx1 also impaired EC migration and blocked angiogenesis induced by bFGF in vivo. Furthermore, although the levels of Hox D3 were unchanged, expression of its target gene, beta3 integrin was reduced, consistent with impaired transcriptional activation by Hox D3. Together, these studies suggest that Pbx1 is required for pro-angiogenic Hox DNA binding and transcriptional activity in endothelial cells.

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