Abstract: SA-OR12
Gene Surgery: A Potential CRISPR/Cas-Based Treatment Option for Nephropathic Cystinosis In Vitro
Session Information
- Genetics, Development, and Therapies
November 05, 2022 | Location: W308, Orange County Convention Center‚ West Building
Abstract Time: 04:39 PM - 04:48 PM
Category: Genetic Diseases of the Kidneys
- 1102 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Sendino Garví, Elena, Universiteit Utrecht Utrechts Instituut voor Farmaceutische Wetenschappen, Utrecht, Utrecht, Netherlands
- Öktem, Mert, Universiteit Utrecht Utrechts Instituut voor Farmaceutische Wetenschappen, Utrecht, Utrecht, Netherlands
- Masereeuw, Rosalinde, Universiteit Utrecht Utrechts Instituut voor Farmaceutische Wetenschappen, Utrecht, Utrecht, Netherlands
- Harrison, Patrick T., University College Cork, Cork, Cork, Ireland
- Mastrobattista, Enrico, Universiteit Utrecht Utrechts Instituut voor Farmaceutische Wetenschappen, Utrecht, Utrecht, Netherlands
- Janssen, Manoe J., Universiteit Utrecht Utrechts Instituut voor Farmaceutische Wetenschappen, Utrecht, Utrecht, Netherlands
Group or Team Name
- Pharmacology Group
Background
Nephropathic cystinosis is a rare monogenetic kidney disease caused by mutations in the CTNS gene, which encodes for the lysosomal cystine transporter, cystinosin. Mutations in the CTNS gene lead to the loss of the cystinosin transporter, resulting in intralysosomal cystine accumulation. There is no curative treatment for cystinosis to date, therefore, we aim to develop a novel gene repair strategy for the most predominant 57 kb deletion of CTNS using CRISPR-Cas9 technology, which allows for the delivery of a CTNS repair template into a specific location in the genome.
Methods
For this study, we used two conditionally immortalized proximal tubule epithelial cells (ciPTEC): a cystinotic patient-derived and a CTNS -/- knock-out. For the delivery of the repair complex, we used a novel non-viral peptide-mediated delivery system. The repair construct for CTNS (3.2 kb) contains the CTNS promoter and the first 10 exons of the CTNS gene, as well as a fluorescent reporter gene (mCherry). Additionally, a second repair construct was designed (1.7 Kb), excluding the mCherry gene, to study a more therapeutic-like construct.
Results
After transfection of the repair construct, the large and small repair templates achieved 5% and 20% insertion efficiency (N=3), respectively, indicating that the cells had successfully inserted the repair template into their DNA. Further analysis of individual clonal cells showed restoration of lysosomal cystine levels in 60% of the clones transfected with the big template (3.2 Kb), showing cystine values between 1-2nmol/mg protein (N=12; p<0.001). Strikingly, more than 70% of the clones transfected with the therapeutic-like template (1.7 kb), which were blind sorted, show cystine values between 0.5-2nmol/mg protein (N=15; p<0.001) thus, indicating that in most of the cells the repair template was inserted, consequently restoring the CTNS function.
Conclusion
In conclusion, these data show that the CTNS repair template can be precisely inserted into the genome, leading to the translation of a functional cystinosin transporter, which consequently restores the lysosomal cystine accumulation. Eventually, this gene repair system may offer a potential curative therapy for cystinosis, as well as a system for the in vitro restoration of several other genes involved in monogenic diseases.
Funding
- Government Support – Non-U.S.