Patient-Specific Induced Pluripotent Stem Cells in Hurler Syndrome
Authors: Tolar Jakub , Park In-Hyun , Xia Lily , Osborn Mark , McElmurry Ron T. , Orchard Paul J. , Daley George Q. , Blazar Bruce R 
Published: April 2020

Hurler syndrome (HS; mucopolysaccharidosis type I) is caused by severe mutations in the iduronidase (IDUA) gene, leading to multi-organ system dysfunction due to the toxic accumulation of glycosaminoglycans. Although allogeneic hematopoietic cell transplantation (HCT) has been shown to provide the IDUA protein and to reverse many of the manifestations of HS, allogeneic HCT is associated with significant morbidity and mortality. We hypothesized that an advantageous alternative strategy may be to induce gene-corrected autologous pluripotent cells to become hematopoietic stem cells, which then provide the missing IDUA enzyme. Because patient-specific embryonic stem cell isolation is not practical, recent strategies have been developed that reprogram adult cells to acquire pluripotency. Such induced pluripotent stem (iPS) cells can be created from fibroblasts or mesenchymal stromal cells (MSCs). As a first step in testing of iPS cells for gene-corrected HS treatment, we isolated host MSCs from the bone chips of a 9-year-old boy with HS who had undergone spinal surgery 8 years after successful allogeneic HCT. HS-MSCs expressed no IDUA, confirming a lack of contamination from either donor-derived hematopoietic cells or MSCs. To create HS-iPS cells, HS-MSCs were transduced with viral vectors carrying reprogramming transcription factors (OCT4, SOX2, KLF4, and c-MYC) that are typically associated with pluripotency and expressed at high levels in embryonic but not adult stem cells. Transduced cells were cultured on supportive stroma of irradiated mouse embryo fibroblasts. Within several weeks, colonies of iPS cells emerged from the two-dimensional culture. When compared to MSCs, the HS-iPS cells showed persistent mRNA expression of OCT3/4 and SOX2 and transient mRNA expression of c-MYC and KLF4, which is expected to occur in the wild-type iPS cells. HS-iPS cells expressed protein markers characteristic of reprogrammed immature cells: OCT3/4, NANOG, stage-specific embryonic antigens (SSEA) 3 and 4, tumor rejection antigens (TRA) 1–60 and 1–81, and alkaline phosphatase. HS-iPS cells had normal male karyotype as determined by chromosomal G-banding. As a second step in creating gene-corrected HS-iPS cells, we employed the non-viral Sleeping Beauty (SB) transposon system (because of the less random pattern of genome integration when compared to viral vectors). Human HS-iPS cells were co-nucleofected with an SB transposon that harbored the human IDUA gene and an expression cassette of the green fluorescent protein (GFP) along with an SB transposase plasmid that provides the enzymatic machinery necessary for integration into TA dinucleotide sites within the genome. Two weeks after nucleofection 10%-15% of HS-iPS cells expressed GFP. Total glycosaminoglycans (a hallmark of the biochemical defect in HS) in unsorted cultures were decreased to wild-type levels. IDUA expression in unsorted cultures was approximately 10% of wild-type IDUA levels, which is within the range sufficient for phenotypic rescue in HS patients after allogeneic HCT. Experiments are ongoing, and data will be presented in regards to: a) map transposon insertions in the genome to prove stable transgenesis by transposition; b) characterization of the differentiation potential of the corrected HS-iPS cells into various mesodermal lineages relevant to rescue of the clinical phenotype associated with HS (hematopoietic, chondrogenic, and osteogenic); c) assessment of development and consequences of cellular pathology in numerous tissue types affected by IDUA deficiency. To our knowledge these are the first data to report that autologous iPS cells can be obtained from HS patients. In summary, HS-iPS cells present an opportunity to use the hematopoietic progeny of gene-corrected autologous cells clinically in a manner that may preclude the immunologic complications of allogeneic transplantation.