First steps towards a kidney directed gene therapeutic approach for the lysosomal storage disease cystinosis
Presented by Dries David, KU Leuven, Belgium
Authors: D. David* L. Medaer* A. Jamalpoor** M. Janssen** E. Levtchenko* R. Gijsbers*
Defects in the lysosomal cystine transporter, cystinosin (CTNS), result in cystine accumulation in all organs, which in turn lead to the autosomal recessive lysosomal storage disease cystinosis. CTNS is ubiquitously expressed but the kidneys are most affected. The first manifestation of the disease is characterized by the renal Fanconi syndrome ending in kidney failure, due to defective reabsorption of nutrients by proximal tubule epithelial cells (PTECs). Currently, cysteamine is the only drug available to treat cystinosis. Cysteamine clears lysosomal cystine and delays kidney failure but cannot correct the renal Fanconi syndrome. In this work, isogenic PTECs were generated using CRISPR/Cas9 to knock out both alleles of CTNS (CTNSCRISPR/CRISPR) in an effort to unravel the effects of loss of CTNS and gene addition in the same genetic background. These CTNSCRISPR/CRISPR cells were complemented with lentiviral vectors (LV) expressing CTNS-WT driven by different promoters (EF1a, AQP1, CTNS, Podocin and EFS) ensuring on average 1 integrated LV copy. All promoters resulted in CTNS mRNA and protein. Strikingly, also promoters with a 10-fold lower CTNS expression compared to the EF1a promoter rescued cystine content and normalized GSH/GSSG ratio. This implies a low level of CTNS protein is sufficient for functional rescue. Transducing the cells with LVs expressing eGFP or a dead lysosomal ATPase (dATP13A2) did not rescue these phenotypes, indicating a specific CTNS effect. In all, these results show a proof-of-concept for a kidney directed gene therapy for cystinosis by targeting PTECs.
* KU Leuven, Belgium
** Utrecht University, Netherlands