AAV2/6 Gene Therapy in a Murine Model of Fabry Disease Results in Supraphysiological Enzyme Activity and Effective Substrate Reduction

Abstract

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.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice

“…29 Unlike chaperone therapy, which is not effective for all GLA mutations, [20][21][22] the in vivo genome editing approach is applicable to patients with type 1 or type 2 disease. While the efficacy of AAV-mediated liver-directed gene therapy approaches has been previously demonstrated in the Fabry mice by us and others, [53][54][55][56] the genome-editing strategy described here leads to the permanent integration of the hGLA transgene and therefore permits treatment of children and/or adolescents with Fabry disease whose liver cells are still dividing as the liver grows. This is of significant importance, as it is well established that treatments for Fabry disease are more effective if initiated early, particularly before irreversible organ damage occurs.…”

“…Total DNA was isolated from liver, and ZFN and hGLA donor vector copy numbers were determined using previously described methods. 54 ZFN vector copy numbers were detected using forward primer 5 0 -GTGGAGGAGCTGCTGATCG-3 0 , reverse primer 5 0 -GA AGTTGATCTCGCCGTTGTTGA-3 0 and probe 5 0 -ATGATCAAAG CCGGCACCCTGACA-3 0 . These primers were designed to anneal to a sequence present in both the left and right ZFNs.…”

“…Gb3 and Lyso-Gb3 analyses in plasma and tissue samples Gb3 and Lyso-Gb3 concentrations were determined in tissues and plasma using liquid chromatography-mass spectrometry (LC-MS) at Brains-Online (Charles River Laboratories, Wilmington, MA, USA), as previously described. 54 LLOQs were as follows: Lyso-Gb3 = 1 nM for plasma, 0.05 nmol/g for heart and liver, and 0.1 nmol/g for kidney; Gb3 = 60 nM for plasma, 3 nmol/g for heart and liver, and 6 nmol/g for kidney.…”

Fabry Disease: Molecular Basis, Pathophysiology, Diagnostics and Potential Therapeutic Directions

“…Avobrio is also currently running a phase II clinical trial (NCT03454893) to study the efficacy and safety of a gene therapy (AVR-RD-01) for the treatment of classic FD patients. Recently, two new gene therapies (ST-920 and FLT190) making use of an AAV vector encoding human α-GalA cDNA with specific liver expression cassettes have been described to increase plasma and tissue α-GalA activities in an FD mouse model and are in phase I/II clinical trials (NCT04046224 and NCT04040049) [ 121 , 122 , 123 ]. Messenger RNA (mRNA) is also emerging as a new class of therapy for the treatment of rare monogenic disorders.…”

Fabry Cardiomyopathy: Current Treatment and Future Options

“…The current therapy for FD (Figure 2) is enzyme replacement therapy (ERT) or in amenable mutations Chaperone therapy. ERT is administered by lifelong biweekly infusion of recombinant enzyme [48,49], which is available as agalsidase alfa (Replagal ® , Shire Human Genetics Therapies AB, Stockholm, Sweden, since 2019 Takeda Pharma AG, 8152 Opfikon, Switzerland) and agalsidase beta (Fabrazyme ® , Sanofi Genzyme, Cambridge, MA, USA). After enzyme replacement, microvascular GL3 depositions were cleared in the kidneys, skin and the heart of most Fabry patients [49][50][51][52].…”