Imaging flow cytometry of cells may be useful to help diagnose Wolman disease (WD), a severe form of lysosomal acid lipase deficiency (LAL-D), and to study the efficacy of gene correction strategies, according to an abstract presented at the American Society of Gene & Cell Therapy 25th Annual Meeting.

Higher fluorescence intensity and granularity were observed in both the fibroblasts of patients with WD and peripheral blood mononuclear cells (PBMCs) edited to become lysosomal acid lipase KO cells compared to healthy cells using imaging flow cytometry. The cells also had higher numbers of spots stained with Nile Red, a marker for neutral lipid, and Lysogreen, a marker for lysosomes.

Confocal microscopy showed that the increase in fluorescent intensity was due to both an increase in the number of lysosomes as well as more intense staining.


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“For the first time, we demonstrated that this spot counting could differentiate WD and healthy fibroblasts better than currently used imaging techniques,” the authors said.

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“Moreover, lentiviral vector mediated integration of a functional LIPA gene fully corrects the lipids accumulation in WD fibroblasts making this pipeline interesting to assess [gene therapy] efficacy,” they added.

Since fibroblasts are harder to collect than PBMCs, which are already collected for LAL-D assessment, the study’s authors wanted to test the quantification method on these cells. Due to the rarity of WD, however, the authors were not able to get access to patient blood so they created lysosomal acid lipase KO cells from T lymphocytes and monocytes using CRISPR-Cas9 editing to delete exon 4 of the LIPA gene.

The KO cells showed similar results as the WD fibroblasts with increased intensity and granularity due to increases in lipid and lysosome vesicles. These results showed that the findings could be generalized to more cell types and that PBMCs could potentially be used for WD diagnosis in the future.

“Overall, these analyses can be applied to diagnose the disease and to monitor over time the phenotypic progression and correction upon treatment,” the authors summarized. “We expect that this sensitive analysis pipeline could be used to evaluate the success of gene correction strategies and implemented to study and diagnose other lysosomal storage disorders.”

Reference

Laurent M, Pavani G, Bayol S, Stockholm D, Cosette J, Amendola M. 785. Novel cytometry based characterization of lysosomal storage disease affected and gene corrected patient’s cells. Mol Ther. 2022;30(4S1):367-368.