Characterization and Evaluation of Cell Therapy Efficacy in Novel Cystinotic Mouse Lines

Abstract

Purpose : Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in the CTNS gene, which encodes the lysosomal cystine transporter, cystinosin. The purpose of the studies is to prepare novel mutant cystinotic mouse models with point and/or small insertion nonsense mutations for studying pathophysiology and developing therapeutic regimens for cystinosis in lieu of the large (57kb) deletion of CTNS allele observed in human.

Methods : Two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation in exon 7 of Ctns allele) and Ctnsis2 (pW138X, a common nonsense mutation in patients besides the 57 kb deletion of CTNS allele) were created by CRISPR gene editing techniques and identified by PCR, RT-PCR and DNA sequencing. The mouse lines were subjected to examinations by in vivo confocal HRTII microscope, histology, immunofluorescence staining, western blot analysis and renal function analysis. The experimental mice were then subjected to transplantation with human umbilical mesenchymal stem cells (hUMSC) via fibrin gel. The treatment efficacy was assessed by HRTII.

Results : Both Ctnsis1 and Ctnsis 2 encode truncated Ctns proteins and exhibit the expected cystinotic corneal crystal phenotype at 4 weeks of age. Therapeutic efficacies of different dosages (2x10⁴ cells and 10⁵ cells/cornea) of hUMSC for cystinotic cornea were examined. The results indicated that 10⁵ cells yield better therapeutic effects than that of 2X10^4 cells. However, the procedure of UMSC delivery to cornea caused inflammation that likely accounts for scar tissue formation. The kidney phenotype was observed in 20 months-old homozygous mutant mice, which exhibited an increase in urine volume, a reduction in urine osmolality, and impaired urinary concentrating defect associated with the loss of response to exogenous vasopressin treatment. Immunofluorescence staining and western blot analysis revealed a significant reduction in aquaporin 2 protein abundance in the inner medulla.

Conclusions : Further studies are needed to develop strategies that improve treatment efficacy of UMSC transplantation for treating cornea phenotypes. It would be also necessary to use strategies such as a combination of autologous transplantation of patient’s hematopoietic stem cells corrected by CRISPR gene editing to systematically ameliorate the pathogenesis caused by the loss of functional CTNS.

This is a 2020 ARVO Annual Meeting abstract.