The Design and In-Vitro Validation of Therapeutic CRISPR Base Editing and AAV-9 Gene Therapy for the Treatment of Classic Galactosemia

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Rahal, Mo
CRISPR, Base Editor, Classic Galactosemia, Galactosemia, AAV, Gene therapy, Gal-1P, galactose, gene editing, therapy, CG
Classic Galactosemia (CG) is a potentially fatal genetic metabolic illness characterized by the inability to effectively metabolize galactose. This disease manifests as various neurological, reproductive, and systemic symptoms and is currently incurable with limited treatment options. There are currently 240 genetic variants of CG with the most predominant being Q188R a point mutation resulting in a glutamine to arginine substitution. In this study, we tested in vitro two methods for the treatment of Q188R CG, an AAV-9 gene replacement therapy, and a CRISPR cytosine base editor therapy. We observed that our AAV-9 expression plasmid for human GALT enzyme successfully restored enzyme levels in the substitution in Q188R homozygous patient fibroblast cells as shown through western blotting. However, biochemical correction still needs to be confirmed. Further, we showed successful cytosine base editing at the mutation target site resulting in reversal of the amino acid transition in the same fibroblast cell line. Together these results demonstrate the potential of both AAV-9 gene therapy and cytosine base editing to treat Q188R CG.
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