Date of Award
Santa Clara : Santa Clara University, 2020.
Master of Science (MS)
Gaucher’s disease is a rare genetic lysosomal storage disorder. People suffering from Gaucher’s disease do not have functional beta-glucocerebrosidase (GBA), which results in toxic build-up of undegraded substrates within the cell. Currently, patients living with this disease rely on biologics to reduce their symptoms, however, they require frequent transfusions and are incapable of crossing the blood brain barrier (BBB) to address neurologic symptoms of the disease. To evaluate the efficacy of novel biologic therapeutics, a human Gaucher’s disease model is required. Here, we have utilized near-haploid human cells (Hap1) modified via CRISPR-Cas9 to model Gaucher’s disease in vitro. These cells contain a 479 bp insertion in the 6th exon of the GBA gene, resulting in non-functional GBA. PCR, enzyme activity assays, and flow cytometry have been employed to confirm the diseased genotype and phenotype. Characterization of GBA knock-out cells shows a total loss of GBA enzyme activity. Further characterization demonstrates a normal growth rate but an increased number of lysosomes, indicating a diseased phenotype. After confirmation of the diseased genotype and phenotype in this Hap1 GBA knock-out (KO) cell line, the diseased cells were treated with recombinant human GBA to determine their response. Evaluating the Hap1 GBA KO cells’ response to existing recombinant GBA enzyme shows the promise of utilizing this cell line to evaluate novel therapeutics for the disease.
Brown, Annie, "Characterizing a Gaucher’s Disease Model for the Evaluation of Novel Exosome-Based Enzyme Replacement Therapy" (2020). Bioengineering Master's Theses. 7.