ZFN-Mediated In Vivo Genome Editing Corrects Murine Hurler Syndrome.

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.

Dose-Dependent Prevention of Metabolic and Neurologic Disease in Murine MPS II by ZFN-Mediated In Vivo Genome Editing.

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.

Robust ZFN-mediated genome editing in adult hemophilic mice.

Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.

A sensitive AAV transduction inhibition assay assists evaluation of critical factors for detection and concordance of pre-existing antibodies.

Pre-existing antibodies to viral capsids may have a negative impact on the efficacy and safety of adeno-associated virus (AAV)-based gene therapies. Total antibody (TAb) and/or cell-based transduction inhibition (TI) assays have been used to exclude seropositive individuals in clinical studies. Published AAV seroprevalence and patient enrollment criteria regarding antibody status lack comparability between assay formats, hindering a direct cross-study comparison. To identify critical factors impacting TI assay detection of AAV neutralizing antibodies (NAbs), we created a reporter construct expressing NanoLuc® luciferase (Nluc) that enabled a more sensitive and robust detection of AAV6 NAbs than using firefly luciferase. Assessment of additional factors including multiplicity of infection, cell lines, viral production, and capsid purity revealed the reporter is the major determinant of assay sensitivity impacting NAb detection. The Nluc reporter was further used to assess seroprevalence to AAV5, 8, and 9. Last, we compared AAV6 Nluc TI with two TAb assay formats. A higher correlation of Nluc TI was observed with direct binding (90%) than with the more sensitive bridging TAb assay (65%), suggesting both assay sensitivity and TAb formats contribute to AAV seropositivity concordance. Our results support a need to standardize assay formats to ensure proper assessment of pre-existing AAV immunity.