Lambert, JRA; (2018) Less is more: the efficacy of gene therapy to treat Fabry disease. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Fabry disease (FD) is caused by mutation in GLA that encodes lysosomal α-galactosidase-A (α-gal-A). Loss of α-gal-A leads to glycosphingolipid storage in cells. Symptoms are life-threatening and current treatment is often enzyme replacement with variable therapeutic benefit. Alternative therapies are required. The world’s first clinical gene therapy trial was recently approved using lentivirus to integrate GLA into target cells for long-term enzyme expression. This thesis examined the efficacy of a previously engineered lenti-vector which may inform design of future trials. Endogenous α-galA activity was characterised in human and murine tissues. The Jurkat lymphoblastic leukaemia cell-line exhibited low α-gal-A activity. Transduction of Jurkats resulted in dose-dependent increase of α-galA expression, without apparent toxicity. The enzyme produced by cells with 0.4 transgene copies per cell (vg/cell) had comparable kinetic properties to wild type. Increasing exposure to 1.8vg/cell resulted in an apparent increase in Michaelis Constant when compared to wild type. Therefore less virus dosage may be more therapeutically efficient. Increasing intracellular α-galA activity was accompanied by increased enzyme secretion and uptake of the extracellular enzyme into wild-type Jurkats, indicating cross-correction between cells. Previous research found deficits in mitochondrial function in FD. Here, inhibition of respiratory chain complex I appeared not to effect either lentivirus transduction efficiency or uptake of extracellular α-galA, but inhibited enzyme secretion. So FD may impede cross-correction. Extending the work into patient fibroblasts, this thesis found suggestion of reduced growth rate and impaired transduction efficiency. If correct these results may indicate the metabolic deficiencies in FD extend beyond complex I deficiency. In conclusion, lentivirus-mediated gene delivery is a promising therapeutic option for FD. However, excessive enzyme generation could result in a protein that has inferior kinetic properties, so dosage requires optimisation. Therapeutic strategies to support mitochondrial function may promote efficacy of treatment.

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