Novel lineage depletion preserves autologous blood stem cells for gene therapy of Fanconi anemia complementation group A

A hallmark of Fanconi anemia is accelerated decline in hematopoietic stem and progenitor cells (CD34 +) leading to bone marrow failure. Long-term treatment requires hematopoietic cell transplantation from an unaffected donor but is associated with potentially severe side-effects. Gene therapy to correct the genetic defect in the patient's own CD34 cells has been limited by low CD34 cell numbers and viability. Here we demonstrate an altered ratio of CD34 to CD34 cells in Fanconi patients relative to healthy donors, with exclusive repopulating ability in only CD34 cells, underscoring a need for novel strategies to preserve limited CD34 cells. To address this need, we developed a clinical protocol to deplete lineage (CD3 , CD14 , CD16 and CD19 ) cells from blood and marrow products. This process depletes >90% of lineage cells while retaining ≥60% of the initial CD34 cell fraction, reduces total nucleated cells by 1-2 logs, and maintains transduction efficiency and cell viability following gene transfer. Importantly, transduced lineage cell products engrafted equivalently to that of purified CD34 cells from the same donor when xenotransplanted at matched CD34 cell doses. This novel selection strategy has been approved by the regulatory agencies in a gene therapy study for Fanconi anemia patients ( ).