Concurrent progress of reprogramming and gene correction to overcome therapeutic limitation of mutant ALK2-iPSC

Fibrodysplasia ossificans progressiva (FOP) syndrome is caused by mutation of the gene ACVR1 , encoding a constitutive active bone morphogenetic protein type I receptor (also called ALK2) to induce heterotopic ossification in the patient. To genetically correct it, we attempted to generate the mutant ALK2-iPSCs (mALK2-iPSCs) from FOP-human dermal fibroblasts. However, the mALK2 leads to inhibitory pluripotency maintenance, or impaired clonogenic potential after single-cell dissociation as an inevitable step, which applies gene-correction tools to induced pluripotent stem cells (iPSCs). Thus, current iPSC-based gene therapy approach reveals a limitation that is not readily applicable to iPSCs with ALK2 mutation. Here we developed a simplified one-step procedure by simultaneously introducing reprogramming and gene-editing components into human fibroblasts derived from patient with FOP syndrome, and genetically treated it. The mixtures of reprogramming and gene-editing components are composed of reprogramming episomal vectors, CRISPR/Cas9-expressing vectors and single-stranded oligodeoxynucleotide harboring normal base to correct ALK2 c.617G>A. The one-step-mediated ALK2 gene-corrected iPSCs restored global gene expression pattern, as well as mineralization to the extent of normal iPSCs. This procedure not only helps save time, labor and costs but also opens up a new paradigm that is beyond the current application of gene-editing methodologies, which is hampered by inhibitory pluripotency-maintenance requirements, or vulnerability of single-cell-dissociated iPSCs. Gene therapy: Reprogramming and editing stem cells in one step Researchers have found a way to reprogram skin cells into stem cells, and fix their genetic flaws at the same time. The standard way of generating induced pluripotent stem cells (iPSCs) that can be used to treat genetic conditions is to reprogram an adult cell, then fix the genetic problem. However, a team led by Sun-ku Chung at the Korea Institute of Oriental Medicine found that didn't work when dealing with fibrodysplasia ossificans progressiva, a condition in which muscle and connective tissue is gradually replaced by bone, because the mutation responsible also disables the iPSCs. Their solution was to combine the reprogramming and gene-editing steps. The researchers suggest this can save time, effort and money not only when dealing with rare diseases but also more generally in gene therapy and disease modeling.