Efficient and sustained locus editing in hematopoietic stem cells as a therapeutic approach for IPEX syndrome.
Swati Singh, Cole M Pugliano, Yuchi Honaker, Aidan Laird, M Quinn DeGottardi, Ezra Lopez, Stefan Lachkar, Claire Stoffers, Karen Sommer, Iram F Khan, David J Rawlings
Author Information
Swati Singh: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Cole M Pugliano: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Yuchi Honaker: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Aidan Laird: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
M Quinn DeGottardi: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Ezra Lopez: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Stefan Lachkar: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Claire Stoffers: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Karen Sommer: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Iram F Khan: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
David J Rawlings: Center for Immunity and Immunotherapies and the Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA 98101, USA.
Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic disorder caused by mutations in the gene, required for generation of regulatory T (T) cells. Loss of T cells leads to immune dysregulation characterized by multi-organ autoimmunity and early mortality. Hematopoietic stem cell (HSC) transplantation can be curative, but success is limited by autoimmune complications, donor availability and/or graft-vs.-host disease. Correction of FOXP3 in autologous HSC utilizing a homology-directed repair (HDR)-based platform may provide a safer alternative therapy. Here, we demonstrate efficient editing of utilizing co-delivery of Cas9 ribonucleoprotein complexes and adeno-associated viral vectors to achieve HDR rates of >40% using mobilized CD34 cells from multiple donors. Using this approach to deliver either a GFP or a FOXP3 cDNA donor cassette, we demonstrate sustained bone marrow engraftment of approximately 10% of HDR-edited cells in immune-deficient recipient mice at 16 weeks post-transplant. Further, we show targeted integration of FOXP3 cDNA in CD34 cells from an IPEX patient and expression of the introduced FOXP3 transcript in gene-edited primary T cells from both healthy individuals and IPEX patients. Our combined findings suggest that refinement of this approach is likely to provide future clinical benefit in IPEX.
