ESPN 54th Annual Meeting

ESPN 2022


 
The potential of CTNS-mRNA based gene replacement therapy to treat nephropathic cystinosis
TJESSA BONDUE 1 LAMBERTUS VAN DEN HEUVEL 1 RIK GIJSBERS 2 ROLAND BROCK 3 ELENA LEVTCHENKO 1

1- DEPARTMENT OF PEDIATRIC NEPHROLOGY & GROWTH AND REGENERATION, UNIVERSITY HOSPITALS LEUVEN & KU LEUVEN, UZ HERESTRAAT 49-3000, LEUVEN, BELGIUM.
2- DEPARTMENT OF PHARMACEUTICAL AND PHARMACOLOGICAL SCIENCES, KU LEUVEN, HERESTRAAT 49 - 3000 LEUVEN, BELGIUM.
3- DEPARTMENT OF BIOCHEMISTRY, RADBOUD INSTITUTE FOR MOLECULAR LIFE SCIENCES, RADBOUD UNIVERSITY MEDICAL CENTER, GEERT GROOTEPLEIN 28, 6525 GA NIJMEGEN, THE NETHERLANDS
 
Introduction:

Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in CTNS, encoding the cystine transporter, cystinosin. Mutations result in lysosomal cystine accumulation in all cells of the body and the kidneys are the most affected organs. The current standard therapy, cysteamine, reduces cellular cystine levels, but does not cure the disease or improves the renal Fanconi syndrome (a generalized proximal tubular dysfunction).

mRNA has revolutionized the world of molecular therapy and mRNA-based therapeutics have started to emerge in the kidney field. Our aim is to investigate mRNA-based gene replacement to treat cystinosis.

Material and methods:

Patient derived proximal tubular epithelial cells (CYS PTECs) were transfected with synthetic CTNS-mRNA. A HA-tag was included in the sequence, allowing for immunostaining to assess the stability of the newly expressed cystinosin protein. Co-staining with the lysosomal associated membrane protein 1 (LAMP1) was used to assess the lysosomal localisation and cystine measurement was performed at 24 hours post transfection (hpt).

Results:

After transfection, PTECs were evaluated for the cystinosin protein from 12h to 10 days after transfection. Transfection efficiency was 72% (±11%) for the first 48hpt and protein half-life was estimated at 3-4 days, with no protein detectable at 7 days post-transfection. Co-staining with LAMP1 confirmed the lysosomal localisation of the protein at 24hpt. The functionality of the CTNS-mRNA was evaluated by cystine measurement at 24hpt and showed a significant decrease in cystine content after treatment.

Conclusions:

Our results shows that mRNA-based gene replacement results in detectable lysosomal cystinosin expression in CYS PTECs for up to 7 days and leads to cystine reduction.