Liver-directed gene targeting as a potential therapy for Fabry Disease

Saxena, Himanshi (2024). Liver-directed gene targeting as a potential therapy for Fabry Disease. PhD thesis The Open University.



Fabry disease (FD) is an X-linked inherited, lysosomal storage disorder caused by mutations in the Alpha Galactosidase-A (GLA) gene. This gene encodes for the GLA enzyme which is responsible for the catabolism of glycosphingolipids like globotriaosylceramide (Gb3). Accumulation of Gb3 in lysosomes results in systemic clinical manifestations and reduced lifespan. Enzyme replacement therapy (ERT) and chaperone therapy are the available treatments for FD however, noncurative and with limitations.

We developed a potential therapeutic approach based on the permanent genetic modification of hepatocytes to express the GLA enzyme by targeting the albumin locus in vivo. To model late-onset and early-onset FD, we treated juvenile (P30) and neonatal (P5) Fabry mice with an AAV8 donor vector containing mAlb homology arms and a codon-optimized version of the human GLA cDNA. This treatment was coupled with the AAV-mediated delivery of the CRISPR/SaCas9 platform to increase targeting efficiency. Treatment of juvenile Fabry mice (donor, 3.0E13 vg/kg; SaCas9, 6.0E12 vg/kg) resulted in elevated GLA enzyme activity which was stable till the termination of the experiment at 5 months of age, accompanied by a 70-80% reduction in Lyso-Gb3 accumulation in liver, kidneys and heart, compared to untreated mutant mice. To increase the safety of the procedure, concerns related to the use of programmable nucleases were avoided by applying a nuclease-free approach. Juvenile animals were treated only with the donor vector (3.0E13 vg/kg), coupled with the treatment with fludarabine, which enhances the gene targeting rate. This nuclease-free strategy resulted in increased plasma GLA activity compared to donor-only treated mice, accompanied by 80-95% of lyso-Gb3 clearance.

When we treated neonatal P5 Fabry mice with donor and SaCas9 AAVs, the treatment was significantly more efficient than in juvenile animals due to the increased targeting rate observed in proliferating hepatocytes present in a growing liver. In fact, we were able to completely clear lyso-Gb3 in plasma and in the different organs with the highest dose of 3.0E14 vg/kg of donor vector, while a dose of 3.0E13 vg/kg resulted in a reduction of 95-98% in plasma and target organs.

A dose escalation study with AAV-mediated episomal gene therapy was also done as a proof-of-principle in juvenile Faby KO mice using a strong liver-specific promoter and the human codon-optimized GLA transgene. Animals treated at 3.0E12 vg/kg and higher doses were able to reduce substrate accumulation by 98-100% in plasma and target tissues. Treatment with the lowest dose of the AAV vector (3.0E11vg/kg) resulted in the clearance of 85-95% lyso-Gb3 in the bloodstream and tissues proving the efficacy of the treatment for late-onset FD. ERT-treated animals were considered for comparative evaluation of the treatment.

This data is inclined towards a promising one-shot therapy using an AAV-based integrative gene-editing approach for early-onset and AAV-based episomal gene therapy for late-onset FD to revert the phenotype irrespective of the Fabry disease-causing mutation. To test the translational applicability of this integrative strategy, AAV-LK03 vectors containing human ALB homology arms have been tested in human liver cell lines and will be validated in primary cultures of human hepatocytes, and in humanized mice to generate consistent preclinical support.

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