Base editing of Galactose-1-Phosphate Uridylyl Transferase as a novel gene therapy approach to treat Q188R mutation in a cellular model of Classic Galactosemia

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Authors
Chalhoub, Tina C.
Keyword
Base editing , Galactosemia , Classic Galactosemia , Adenoviral Vectors , Q188R Classic Galactosemia
Abstract
Classic Galactosemia (CG) is a rare genetic disorder represented by the inability to convert galactose to glucose. A mutation at the galactose-1-phosphate uridylyltransferase (GALT) enzyme coding gene halts galactose metabolism which leads to the accumulation of galactose-1-phosphate and galactitol. The Q188R mutation is caused by a base change from adenine to guanine in base pair 563. This mutation causes an amino acid change in position 188 from glutamine (Gln) to arginine (Arg) and is responsible for 60-70% of CG cases in homozygous state. Consequently, affected infants present with neurological, developmental, reproductive, and systemic impairments. Treatments are currently limited to lactose and galactose-free diets that only provide limited symptom management but do not provide any curative measures. In this study, we tested a CRISPR cytosine base editing strategy to restore the original GALT sequence in GALT KO patient cells. We observed base editing, but at a very low level of <2% from transducing homozygous Q188R patient fibroblasts with an adenovirus construct carrying the base editor. Further, we optimized a GALT activity assay that can detect GALT activity in murine liver lysates. This study demonstrates the need for further investigation of Q188R base editing to understand the low efficiencies and tackle possible causes.
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