As a monogenic blood disorder with potential survival disadvantage, Fanconi anemia has long been considered an attractive target for conventional gene therapy but success has been elusive. Consequently, there has been increasing interest in developing techniques to catalyze correction of the disease-causing mutation at the cognate site by homologous recombination (HR). By achieving gene correction rather than gene addition, the deleterious effects of random integration can be avoided.

Towards this end, engineered zinc finger nucleases (ZFNs), CRISPR/Cas9, and other targeted nucleases have shown promise and some have entered clinical trials. Recent developments have put CRISPR/Cas9 technology in the forefront because of its ease of use and facile reagent design. However, studies have shown that such nucleases exhibit frequent off-target effects because the nuclease activity of the molecules cannot be completely restricted to the intended sites. Although substantial effort is being directed toward reducing off-target nuclease effects, this is one area in which triplex-forming PNAs (TFPs), which have no intrinsic nuclease activity and simply trigger the cells’ own repair activity when they bind tightly to their target site, may offer an advantage.

We have developed active TFPs for targeted beta-globin gene editing. Recent results demonstrate that PNAs and donor DNAs delivered intravenously (IV) via PLGA nanoparticles (NPs) can mediate gene editing in vivo at a level sufficient to ameliorate the disease phenotype in thalassemic mice. We observed sustained reversal of the anemia, with normalization of blood hemoglobin concentrations and suppression of the reticulocytosis. We also noticed a morphologic improvement in RBC cytology, indicative of improved RBC stability, along with reduced extramedullary hematopoiesis and reduction in splenomegaly. This constellation of findings suggests that our therapeutic approach has the potential to deliver a significant clinical response that would relieve the morbidity and mortality associated with hematologic disorders like β-thalassemia and SCD. It is the goal of this proposal to apply this approach to Fanconi anemia.