Recapture Lysosomal Enzyme Deficiency via Targeted Gene Disruption in the Human Near-Haploid Cell Line HAP1.

BACKGROUND: Advancement in genome engineering enables rapid and targeted disruption of any coding sequences to study gene functions or establish human disease models. We explored whether this approach can be used to study Gaucher disease, one of the most common types of lysosomal storage diseases (LSDs) in a near-haploid human cell line (HAP1). RESULTS: CRISPR-Cas9 targeting to coding sequences of ß-glucocerebrosidase (GBA), the causative gene of Gaucher disease, resulted in an insertional mutation and premature termination of GBA. We confirmed the GBA knockout at both the gene and enzyme levels by genotyping and GBA enzymatic assay. Characterization of the knockout line showed no significant changes in cell morphology and growth. Lysosomal staining revealed more granular lysosomes in the cytosol of the GBA-knockout line compared to its parental control. Flow cytometry analysis further confirmed that more lysosomes accumulated in the cytosol of the knockout line, recapturing the disease phenotype. Finally, we showed that this knockout cell line could be used to evaluate a replacement therapy by recombinant human GBA. CONCLUSIONS: Targeted gene disruption in human HAP1 cells enables rapid establishment of the Gaucher model to capture the key pathology and to test replacement therapy. We expect that this streamlined method can be used to generate human disease models of other LSDs, most of which are still lacking both appropriate human disease models and specific treatments to date.

Genetic labeling of extracellular vesicles for studying biogenesis and uptake in living mammalian cells.

Molecular imaging methods are powerful tools for gaining insight into the cellular organization of living cells. To understand the biogenesis and uptake of extracellular vesicles (EVs) as well as to engineer cell-derived vesicles for targeted drug delivery and therapy, genetic labeling with fluorescent proteins has increasingly been used to determine the structures, locations, and dynamics of EVs in vitro and in vivo. Here, we report a genetic method for the stable labeling of EVs to study their biogenesis and uptake in living human cells. Fusing a green fluorescent protein (GFP) with either the endogenous CD63 (CD63-GFP) or a vesicular stomatitis virus envelope glycoprotein, VSVG (VSVG-GFP), we successfully obtained distinct fluorescence signals in the cytoplasm, revealing the biogenesis of EVs in post-transfected cells. We describe experimental procedures in detail for EV isolation, imaging, and cellular uptake using both confocal microscopy and flow cytometry. We also provide a perspective on how genetic labeling methods can be used to study EV biology, characterization of engineered EVs, and development of EV-based nano-medicine.