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Mechanisms of Cystinosis Regenerative Therapies and Pathogenesis


Cystinosis is a multisystem genetic lysosomal storage disorder caused by mutations in the CTNS gene leading to cystine crystal accumulation within lysosomes. Current treatments merely delay disease progression so we have pioneered hematopoietic stem and progenitor cell (HSPC) transplantation in mice. Our group previously discovered that HSPC-derived macrophages are important effectors of this therapy by facilitating the delivery of functional lysosomes to diseased tissue through membranous protrusions called tunneling nanotubes (TNTs). This transplantation approach is being translated into human cystinosis patients in an ongoing Phase I/II clinical trial.

To understand the mechanistic underpinnings of this therapy, we investigated macrophage polarization and TNT-mediated intercellular trafficking. Employing an in vitro polarization co-culture system and automated image analysis workflows, we found that pro-inflammatory stimulation reduced TNT-like protrusion formation and lysosomal transport. However, we found that co-culture of macrophages with diseased cystinotic fibroblasts increased TNT formation and trafficking, signaling we further explored via metabolomic analysis of culture media.

Next, we addressed if SHPK, a metabolic gene involved in macrophage polarization that is frequently eliminated in cystinosis, is required for optimal HSPC therapy. We generated novel knockouts using CRISPR-Cas9, confirmed that Shpk expression was ablated and described a hepatic and urinary metabolic phenotype. We then transplanted Shpk-/- HSPCs into cystinotic mice and determined that they are equally efficacious as wildtype (WT) donors. Based on these findings, SHPK-/- patients will be enrolled as future subjects in the clinical trial.

Finally, we developed a novel clinical endpoint for the clinical trial by quantitating cystine crystals from intradermal confocal images of patients. We created an automated image analysis methodology to measure crystal density in both 2D and 3D. We found that patients have significantly higher normalized confocal crystal volume (nCCV) than healthy controls, and that this new metric correlates with patient age and several clinical outcomes. This methodology presents the potential to become a biomarker to monitor long-term disease trend, compliance with treatment, and anticipatory guidance for potential complications.

Overall, this work serves to highlight the potential of HSPC-based regenerative therapies to treat previously incurable genetic diseases. This work highlights how an understanding of basic molecular mechanisms can be directly translated into developing novel regenerative therapies for humans.

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