Optimizing human α-galactosidase for treatment of Fabry disease

Abstract

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease. GLAv05 and GLAv09 were identified after screening more than 12,000 GLA variants through 8 rounds of directed evolution. Both GLAv05 and GLAv09 exhibit increased stability at both lysosomal and blood pH, stability to serum, and elevated enzyme activity in treated Fabry fibroblasts (19-fold) and GLA–/– podocytes (10-fold). GLAv05 and GLAv09 show improved pharmacokinetics in mouse and non-human primates. In a Fabry mouse model, the optimized variants showed prolonged half-lives in serum and relevant tissues, and a decrease of accumulated Gb3 in heart and kidney. To explore the possibility of diminishing the immunogenic potential of rhGLA, amino acid residues in sequences predicted to bind MHC II were targeted in late rounds of GLAv09 directed evolution. An MHC II-associated peptide proteomics assay confirmed a reduction in displayed peptides for GLAv09. Collectively, our findings highlight the promise of using directed evolution to generate enzyme variants for more effective treatment of lysosomal storage diseases.

Flow Cytometry-Based Assay to Detect Alpha Galactosidase Enzymatic Activity at the Cellular Level

“…AGAL activity may be influenced by several cellular environmental and metabolism factors, e.g., lysosomal stability, pH stability, and temperature stability. Also, AGAL activity shows cell-type specific variations [28]. The difference in cellular states during cell culturing may explain the higher variation observable in our in vitro experiments in wild-type Jurkat cells.…”

Engineering Synthetic and Recombinant Human Lysosomal b-Glucocerebrosidase for Enzyme Replacement Therapy for Gaucher Disease

“…These engineered enzymes have also been used as a drug-resistance gene to optimize lentiviral vectors for HIV and sickle disease gene therapy [42,43]. Additionally, engineered enzymes such as iduronate-2-sulfatase (IDS) have been developed for the treatment of Hunter syndrome-α lysosomal storage disease [44], and α-galactosidase (GLA) for the treatment of Fabry disease [45]. Furthermore, researchers have used synthetic biology technologies for programming, controlling transgene expression (35,36), and for synthesizing codon-optimized DNA constructs for gene therapy for several genetic diseases [46][47][48].…”