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

Abstract

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.

AMPK downregulates ALK2 via increasing the interaction between Smurf1 and Smad6, leading to inhibition of osteogenic differentiation

“…In fulfilling this task, there are several available conditional knock-in or transgenic mouse models in which constitutively active ALK2 is expressed [9, 11]. In addition, induction of iPS cells derived from FOP to osteoblast differentiation and transplantation of the differentiated cells into nude mice has emerged as another type of model that is useful in studying the mechanism of HO, and for testing efficacy of drugs in the treatment of FOP [37, 38]. As such, it will be intriguing to apply clinically available AMPK activators to these FOP models.…”

Fibrodysplasia ossificans progressiva: Basic understanding and experimental models

“…The induction of iPSc in vitro rapidly and effectively indicates the phenotype of disease in an individual specific background. A variety of somatic cells can be (54) reprogrammed into stem cells, including skin fibroblasts (32) and kidney epithelial cells (48), and Sendai virus and non-integration vector can be used as programming tools (49). (50).…”

Urine-derived induced pluripotent stem cells as a modeling tool to study rare human diseases

“…A recent study suggested that a one-step procedure can generate gene-corrected ALK2-iPSCs, and the advantages of this approach are that it saves time, labor, and money (83). Activated BMP signaling by an FOP R206H mutation adversely affects the generation of FOP iPSCs since BMPs can induce differentiation of human ESCs (84).…”