CTNSmRNA as a potential treatment for nephropathic cystinosis

Abstract

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to correct the genetic defect in nephropathic cystinosis using synthetic mRNA. Cystinosis is a prototype disorder of proximal tubular dysfunction caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and most severely affected organs, presenting glomerular and proximal tubular dysfunction. Cysteamine is the current therapeutic standard that reduces cellular cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA is safe and effective to reintroduce functional cystinosin using lipofection in CTNS-/- kidney cells and following direct injection in ctns-/- zebrafish larvae. CTNS mRNA therapy results in prompt lysosomal expression of the functional protein and decreases cellular cystine accumulation for up to 14 days. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. We propose that mRNA-based therapy, if sufficient kidney targeting can be achieved, may be a new approach to treat cystinosis.

The Zebrafish Embryo as a Model Organism for Testing mRNA-Based Therapeutics

“…In zebrafish, IVT mRNA has been used for ectopic expression or overexpression experiments to study gene function after injection at the one-cell stage and modified IVT mRNA has been applied to manipulate the expression levels [ 10 ]. It is worth noting that zebrafish can tolerate mRNA sequences from other species, which can increase the cost-effectiveness of a project [ 11 , 12 , 13 ].…”

“…Several modifications in the coding sequence, such as the use of pseudo-uridine instead of uridine, have shown promise in reducing immunogenic potential by preventing recognition of the mRNA by the mammalian innate immune system, which happens via toll-like receptor 7 (TLR7) and retinoic-acid-inducible gene I (RIG-I) [ 14 ]. The expression of TLR7 and RIG-I orthologues has been demonstrated in zebrafish, but studies using human mRNA in the zebrafish embryo have not provoked any toxicity or any signs of immune response after injection, as will be illustrated below [ 11 , 13 , 15 ]. Next, the use of artificial UTRs has been shown to increase protein expression when compared to the endogenous UTRs from the zebrafish genome.…”

“…Additionally, a zebrafish model of classic galactosemia (CG) ( galt KO ) , a hereditary disease of galactose metabolism caused by deficiency in galactose-1-phosphate:uridylyltransferase (GALT) activity, has been treated with human GALT mRNA by one-cell stage injection, resulting in enzyme activity and a decrease in the accumulated metabolites [ 11 ]. Finally, our group treated a zebrafish model for cystinosis ( ctns −/− ), an autosomal recessive lysosomal storage disorder caused by a defective or absent lysosomal cystine transporter (cystinosin), and resulting in cystine accumulation, with human CTNS mRNA at the one-cell stage and showed restoration of the kidney phenotype (proteinuria and proximal tubular reabsorption) along with reduction of whole body cystine levels [ 13 ].…”

“…Also, while qRT-PCR can be used to assess mRNA half-life, this is not always relevant and resources are mostly focused on mapping out protein half-life. Nevertheless, our study of CTNS mRNA in cystinotic zebrafish ( ctns −/− ) has detected mRNA for at least 120 h post-injection [ 13 ].…”

“…When using the one-cell stage injection method, the protein is expected to be ubiquitously expressed. This can be studied by classic methods like Western blotting and immunostaining [ 11 , 13 ]. When suitable antibodies are available, it is recommended to start by assessing the expression in the whole embryo by Western blot, providing amplification of the signal by combining multiple embryos in a single lysate.…”