Macrophage tunneling nanotube-mediated HSPC transplantation therapy for the lysosomal storage disorder cystinosis remains effective in preclinical mouse study following Shpk elimination

Presented by Ms Meisha Khan, Department of Pediatrics, Division of Genetics, University of California, San Diego, La Jolla, California, USA

Authors*: Spencer Goodman, Meisha Khan, Jay Sharma, Jose Cano, Zijie Li, Ilya Gertsman, Charlie Castellanos, Valeria Estrada, and Stephanie Cherqui

Cystinosis is a lysosomal storage disorder caused by loss-of-function mutations in the CTNS gene. CTNS encodes the ubiquitously expressed lysosomal transporter cystinosin, whose functional absence leads to cystine accumulation and crystallization. Symptoms progressively manifest due to damage of the kidneys, thyroid, cornea, muscle and neurological systems and ultimately cystinosis leads to multi-organ failure and lethality. Current treatment can slow but not halt disease progression, so our group has developed a new investigational therapy based on transplantation of hematopoietic stem and progenitor cells (HSPCs). In mice, a single transplantation of heathy HSPCs into irradiated Ctns-/- recipients prevents tissue degeneration and restores biochemical function via delivery of functional cystinosinbearing lysosomes from HSPC-derived macrophages to diseased cells through tunneling nanotubes (TNTs). A Phase I/II clinical trial investigating the use of autologous transplantation of patients’ HSPCs ex vivo transduced with functional CTNS is currently being conducted at UC San Diego.

The most common mutation in cystinosis is a large 57-kb deletion eliminating not only the CTNS gene but also the neighboring SHPK (a.k.a. CARKL) locus. SHPK influences macrophage polarization and differentiation through regulation of glucose metabolism. Due to the central role of macrophages in HSPC therapy for cystinosis, and the fact that roughly 40% of all human cystinosis patients carry the 57-kb deletion at the homozygote state, we investigated if Shpk expression was necessary for effective transplantation therapy. Such studies will indicate if patients without SHPK are likely to benefit from ex vivo replacement of CTNS alone.

We generated the first two Shpk knockout (KO) models by introducing genomic deletions using CRISPR-Cas9. We confirmed elimination of Shpk expression at the mRNA and protein levels across multiple tissues, and also observed perturbations in the Pentose Phosphate Pathway, the metabolic shunt regulated by Shpk.

Shpk-/- HSPCs along with WT and Ctns-/- controls were then transplanted into lethally irradiated 2-month-old Ctns-/- mice, and analyzed 6 months post-transplantation. Shpk-/- HSPC recipients showed significant reduction in tissue cystine content, restoration of Ctns expression and improvements in renal function comparable to WT HSPC recipients, demonstrating that in vivo macrophage-mediated transplantation therapy remains effective despite elimination of Shpk in donor HSPCs. Furthermore, we investigated the phenotype of in vitro bone marrow derived macrophages (BMDMs) isolated Shpk-/- and note that despite loss of Shpk mRNA expression, they normally form TNTs in a co-culture system with cystinotic fibroblasts.

These data strongly suggest that cystinosis patients lacking Shpk will benefit from ex vivo gene therapy that restores only CTNS expression and can be included in the ongoing clinical trial.

* Author Afiliation

Department of Pediatrics, Division of Genetics, University of California, San Diego, La Jolla, California, USA