Wolfram Syndrome is an uncommon, autosomal recessive disorder caused by a single mutation in the gene WFS1 which causes chronic endoplasmic reticulum stress and stimulation of unfolded protein response that ultimately lead to the death of insulin-producing pancreatic Beta (β) cells. Insulin-producing pancreatic β cells derived from induced pluripotent stem cells (iPSCs) of diabetic patients has previously been used as a possibility for cell replacement therapy. Researchers at the Washington University School of Medicine demonstrate how CRISPR-Cas9 was used to correct a pathogenic variant of Wolfram syndrome in induced pluripotent stem cells (iPSCs) derived from a patient with the disease and later transplanted into diabetic mice.
Diabetes mellitus is characterized by the death or dysfunction of β cells which reside in the pancreas and produce insulin. Pancreatic β cells derived from stem cells (SC- β cells) have shown to provide an autologous source of replacement cells, but some studies have reported generation of pancreatic or endocrine progenitors lacking B cell identity. To generate highly functional β Cells across cell lines, a differentiation protocol was developed that leveraged a previously unknown role of the cytoskeleton in pancreatic fate choice. The differentiation strategy yielded autologous, gene corrected stem cell-derived pancreatic SC- β cells. Efficacy of the differentiation strategy was further examined by glucose-stimulated insulin secretion and comparing undifferentiated cells to SC- β Cells in diabetic mouse models in vitro and in vivo.
Previous gene therapy for a Wolfram Syndrome patient involved generating pancreatic progenitors and polyhormonal endocrine cells that lack β cell features including high insulin per cell, dynamic function, or diabetes reversal in mice. Recent studies have generated mature SC- β cells from patients with diabetes, but have lacked robust function. Data is also lacking in comparison of differentiation from nuclear transfer stem cells and iPSCs in relation to SC- β cells. Studies which have used this cell therapy have only reported prevention and not reversal of diabetes. This study successfully demonstrated reversal of pre-existing diabetes in all mice with no observed teratomas or cystic structure formations.
Combining both iPSCs derived from a patient with Wolfram Syndrome along with CRISPR-Cas9 correction of the disease-causing pathogenic gene variants, the SC- β cells generated in the study were able to quickly aid mice with severe pre-existing diabetes. Editing of the WFS1 gene yield SC-B cell differentiation efficacy as well as functionality, and demonstrated 1st and 2nd phase insulin secretion. Endoplasmic reticulum and mitochondrial stress were also shown to be reduced as well as a reduction in apoptotic markers after CRISPR-Cas9 correction when assessed by scRNA-seq and direct assessment. Since cells were derived from a patient and gene corrected, immunosuppressant drugs could also be avoided for several forms of diabetes. Robust dynamic functionality in vitro, and first-and second- phase insulin stages were the major features corrected in the WS SC- β cells.
Further studies warrant diversity of patients with Wolfram Syndrome as iPSCs were only derived from one patient as well as to other monogenetic forms of diabetes. The mice in this study also had mild diabetes, while patients with WS typically have insulin-dependent diabetes. Limited understanding of human WS progression is also difficult for the development of effective therapies. Overall, this study validated the presence of elevated stress within the endoplasmic reticulum, activation of an unfolded protein response, and defects in insulin processing in Wolfram Syndrome.
This differentiation approach enables the possibility of study for disease modeling and drug screening for different forms of diabetes, and the use of the CRISPR-Cas9 gene therapy system as a means of modifying specific genes which cause them.
