Organ-on-a-Chip System for the pre-clinical evaluation of novel gene therapeutic approaches for the treatment of autosomal recessive osteopetrosis (ARO)
Sequential seeding of primary human cells on decellularized human cancellous bone allows engineering clinically relevant bone-on-a-chip systems to analyse genetic bone disorders like autosomal recessive osteopetrosis (ARO). ARO is a sclerosing bone dysplasia caused by defects in the bone-resorbing osteoclasts and is lethal if not timely treated by hematopoietic stem cell (HSC) transplantation. This treatment harbors still high risks since it relies on allografts. A solution will be autologous HSCs with corrected gene defect by somatic gene therapy. We have established induced pluripotent stem cell (iPSC) line from an ARO patient harboring CLCN7 gene mutations. We are able to efficiently differentiate iPSCs into osteoclasts. Osteoclasts derived from ARO iPSCs do not resorb bone in 2D cultures, but resorption activity is restored after gene correction. Corrected iPSCs are the positive controls for two gene therapy approaches under scrutiny: 1. CRISPR/Cas9-mediated integration of a rescue construct into a save harbor locus, 2. Random integration of a transposon-based rescue construct. Both will be first tested in iPSC-derived osteoclasts in short-term 2D cultures. Subsequently, the exact behavior and fate of the gene corrected cells and their interaction with other bone (marrow) cells will be tested in long-term 3D bone-on-a-chip system. This includes: (1.) iPSC-derived monocytes will be cultured alone in the 3D scaffold, (2.) monocytes will be combined with primary mesenchymal stromal cells (MSCs) and bone marrow cells, and (3.) iPSC-derived osteoblasts and monocytes will be combined. Our preliminary data also suggests efficient integration of the therapeutic constructs in HSCs. Thus, in a last step genetically modified iPSC-derived HSCs will be investigated in long-term cultures. Our stepwise approach allows for an extensive preclinical testing of the novel gene therapy strategies and thus replace mouse experiments, which only imperfectly mirror the human situation.