Gaucher disease is a genetic disorder caused by point mutations in the GBA gene encoding for glucocerebrosidase, an enzyme that breaks down the fat glucocerebroside. This work proposes the development of a Cas9-cytidine deaminase fusion protein to treat forms of Gaucher’s disease type 1 associated with an A1226G substitution on the GBA gene. This fusion protein would consist of the rAPOBEC1 cytidine deaminase, the SpCas9 nuclease, and two uracil glycosylase inhibitor (UGI) molecules connected with flexible serine/glycine linkers. This fusion protein, once complexed with an engineered guide RNA, would specifically bind to the mutated site and enzymatically convert the disease-associated cytosine base to thymine, reversing the disease mutation. The proposed system is unlikely to exhibit unintended edits; the designed guide RNA does not align with any other known locus on the human genome. Ultimately, we imagine expressing the fusion protein/guide RNA complex in vitro, encapsulating it in lipid nanoparticles, and injecting it into the body; to reach this goal, we propose three specific experimental aims: (1) expressing and purifying the fusion protein and guide RNA; (2) demonstrating consistent and on-target editing activity in human liver cell cultures; and (3) demonstrating successful delivery and on-target editing activity in a model organism. The completion of this project would put forth a vastly more accessible treatment for Gaucher’s disease type 1 and advance the precision of Cas9-based gene editing.

Engineering Cas9 fusion protein for the treatment of Gaucher’s disease type 1

ACS Fall 2021

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