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Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920., Graphical Abstract

Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

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., In vivo genome editing following a single injection of ZFN and IDUA donor encoding AAV8 resulted in metabolic correction and neurological benefit in a murine model of MPS I. These results enable a currently open clinical trial to evaluate targeted genome engineering for MPS I in humans.

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., AAV-mediated in vivo delivery of ZFN and IDS donor resulted in site-specific gene insertion and dose-dependent IDS expression in a mouse model of MPS II. These results support a currently open clinical trial, the first ever in vivo human genome editing study to be conducted.

Fabry disease (FD), an X-linked lysosomal storage disease, is caused by mutations in the GLA gene encoding α-galactosidase A (α-GalA). FD is characterized by progressive systemic accumulation of the enzyme’s substrates, globotriaosylceramide (Gb3) and lyso-Gb3, leading to renal, cardiac and/or cerebrovascular disease and culminating in premature demise. FD is treated by enzyme replacement therapy (ERT), however the short enzyme half-life necessitates life-long biweekly infusions. A more effective and long-lasting treatment would benefit FD patients. An AAV-mediated liver-targeted gene therapy was evaluated in a mouse model (GLAKO) that lacks α-GalA activity and accumulates high levels of Gb3/lyso-Gb3 in plasma and tissues. This strategy employs an episomal AAV (serotype 2/6) vector encoding human GLA cDNA (hGLA) driven by a liver-specific promoter. One-time administration of increasing amounts of AAV-hGLA cDNA generated using a clinical scale manufacturing process resulted in supraphysiological expression of plasma α-GalA (over 300-fold of WT) by study day 15, was well tolerated, and was stable for 3 months post-injection. Dose-dependent increases in α-GalA activities were achieved in liver, heart and kidney with a corresponding reduction of Gb3/lyso-Gb3. This initial vector was compared to an improved cDNA vector in a 1-month study using two different AAV doses in wild type C57BL/6 mice. The improved cDNA vector produced on average 7-fold higher levels of plasma α-GalA activity at study day 28 than mice administered the same dose of the initial cDNA. The high levels of α-GalA activity seen in these studies, along with the concomitant marked reduction in the accumulated Gb3/lyso-Gb3 in key tissues of the GLAKO mouse model, provide “proof-of-concept” for AAV-mediated targeting of hepatocytes to express therapeutic levels of human α-GalA. The clinical scale manufacturing process developed for these studies will enable rapid development and production in 2019 of clinical-grade material featuring the improved cDNA construct.

Mucopolysaccharidosis type I (MPS I) is characterized by progressive neurodegeneration, and premature death (

Hunter syndrome (Mucopolysaccharidosis Type II, MPS II) is a rare X-linked lysosomal disorder caused by lack of functional iduronate-2 sulfatase (IDS) enzyme and subsequent accumulation of glycosaminoglycans (GAG) in affected individuals. Manifestations include skeleton dysplasia, splenohepatomegaly, cardiopulmonary obstruction, and shortened life expectancy. In severe cases there is also neurologic impairment. Enzyme replacement therapy (ERT) is currently the only FDA-approved treatment to manage disease progression; however, ERT does not affect neurological aspects of the disease and requires that patients receive long and costly infusions of replacement factor on a frequent basis. We have developed a zinc-finger nuclease (ZFN) approach to insert the human IDS (hIDS) coding sequence into the albumin locus using AAV2/8 vectors. In this study IDS-deficient MPS II mice (n= 8-13 per group, age 7-8 weeks) were treated by intravenous infusion of a mixture of ZFN-encoding AAV vectors along with an AAV vector encoding the hIDS partial cDNA flanked by albumin sequence homology arms at three different vector doses. Wild-type littermates, untreated MPS II mice, and MPS II animals infused only with the hIDS donor vector (without ZFN-encoding vectors) were included as controls. Successful insertion of the hIDS coding sequence will result in hIDS expression regulated by the endogenous albumin promoter. Plasma and tissue IDS activities as well as urine and tissue GAG contents were monitored throughout the study to evaluate the effectiveness of the treatment. Sufficient animals were maintained for neurobehavioral testing at four-months post-injection to determine whether the treatment is neurologically beneficial. We found that IDS activities in the plasma of the treated groups were 10- to 100-fold higher than wild-type and stably expressed through the entire study duration in a dose-dependent fashion, while only very low levels of IDS activity were found in the animals infused with hIDS donor vector alone. At 4 weeks post-treatment IDS activities in peripheral tissues ranged from 1% to 200% wild-type in a dose-dependent fashion, while in the hIDS donor-only group enzyme activity was not detected in any tissue except liver (10% that of wild-type). We observed up to 2% of the wild-type IDS activity in the brains of animals administered the complete set of AAV vectors, while no IDS activity was observed in the brains of animals infused with the IDS donor vector alone. Urine GAGs were reduced in all of the ZFN + Donor treatment groups regardless of the vector dose. Tissue GAGs in the treatment groups were also decreased, but GAG content in the brain was not different from untreated MPS II litter mates at the initial analysis conducted four weeks post-treatment. No tissue GAG reduction was observed in animals infused with hIDS donor vector alone. Before conclusion of this study, animals from all six groups will be tested in the Barnes Maze as neurobehavioral assessment to determine the neurological effect of targeted hIDS expression in the liver. These results together with hIDS expression and GAG level data from final necropsy tissues will be presented. These results demonstrate that ZFN can be effectively used to mediate in vivo insertion of the hIDS coding sequence into the albumin locus with resultant stable and high-level hIDS enzyme expression and metabolic correction in MPS II.