Overcoming reprogramming resistance of Fanconi anemia cells

Abstract

IntroductionFanconi anemia (FA) is a recessive syndrome characterized by BM failure, congenital anomalies, and a predisposition to malignancy. 1 In vitro myeloid and erythroid colony growth of BM and peripheral blood cells from FA patients is decreased, suggesting the contribution of an intrinsic cellular defect to the BM failure. 2,3 FA cells have a defect in DNA repair that leads to spontaneous chromosomal breakage and increased sensitivity to DNA bifunctional crosslinking agents such as mitomycin C and diepoxybutane. 4 Whereas the precise biochemical function of most FA proteins and the link between defective DNA repair and BM failure remain incompletely understood, human and murine knockout FA cells display G 2 phase arrest, increased sensitivity to oxidative damage, defective p53 induction, and increased apoptosis. 1,5 FA can be classified into 14 complementation groups. A loss of function in any one of these 14 genes, including FANCA, FANCB, FANCC, FANCD1 (BRCA2), FANCD2, FANCE, FANCF, FANCG, FANCL, FANCI, FANCJ, FANCM, FANCN, and FANCP (SLX4), causes the disease phenotype. 6,7 Expression of these cDNAs in cells from patients with FA in vitro corrects all cell-intrinsic defects, including BM progenitor growth, in vitro. 8,9 Gene-transfer studies have shown that correction of defects in mice with gene-targeted deficiency of FA proteins is feasible and corrects a significant engraftment defect-and in some mouse models provides a selective advantage in vivo. 10,11 Two clinical gene-therapy trials involving a total of 6 FA patients have been reported. 12,13 In both studies, the harvest of CD34 ϩ hematopoietic stem and progenitor cells (HSCs) from the BM or mobilized peripheral blood yielded lower than expected cell numbers and compromised in vitro expansion, resulting in a reduced number of cells available for gene transfer and autologous reinfusion. Therefore, whereas gene transfer per se is no longer a limitation to the therapeutic effectiveness of this approach, there are significant deficiencies in the number of autologous FA HSCs that can be collected and used in somatic gene-therapy trials.The unlimited proliferative capacity of iPSCs is particularly attractive with regard to regenerative therapies and disease models in FA. Successful differentiation of corrected iPSCs into transplantable HSCs would allow the generation of unlimited numbers of these cells and pretransplantation molecular characterization of gene-corrected cells. 14 Several recent studies have highlighted the utility of patient-specific iPSCs for in vitro disease modeling. 15,16 With regard to FA, knockdown of FANCA and FANCD2 in embryonic stem cells (ESCs) leads to reduced hemogenic potential after differentiation, suggesting that FA-deficient human pluripotent stem cells may be amenable to in vitro disease modeling. 17 Raya et al recently reported a failure of 4 FA-A and 2 FA-D2 patient samples to undergo direct reprogramming, concluding that restoration of the FA pathway is a prerequisite for iPSC generation from somatic cells of FA ...

Fanconi anemia: a model disease for studies on human genetics and advanced therapeutics

“…Because FA fibroblasts are difficult to reprogram, this study uncovered a novel role of the FA pathway in cell reprogramming. Consequently, correcting FA genes restores the reprogramming efficiency of FA fibroblasts to IPS cells [93][94][95][96][97][98]. Reprogramming induces the DDR [99] and activates the FA pathway [94], leading to P53-mediated apoptosis and low reprogramming efficiency [96].…”

“…Consequently, correcting FA genes restores the reprogramming efficiency of FA fibroblasts to IPS cells [93][94][95][96][97][98]. Reprogramming induces the DDR [99] and activates the FA pathway [94], leading to P53-mediated apoptosis and low reprogramming efficiency [96]. This is partially circumvented by preventing reactive oxygen species (ROS)-mediated DNA damage [94] or by suppressing P53 during reprogramming using RNA interference [96] or human papillomavirus P53-repressing E6 protein [100].…”

“…Reprogramming induces the DDR [99] and activates the FA pathway [94], leading to P53-mediated apoptosis and low reprogramming efficiency [96]. This is partially circumvented by preventing reactive oxygen species (ROS)-mediated DNA damage [94] or by suppressing P53 during reprogramming using RNA interference [96] or human papillomavirus P53-repressing E6 protein [100]. Safe and controlled FANCA gene correction was achieved using integration-free genome editing by genetic recombination with helper-dependent adenoviral vectors [96] or by targeting FANCA insertion into the safe locus AAVS1 [97] using engineered nucleases ("safe harbor" strategy) [101].…”

Brief Report: Impaired Cell Reprogramming in Nonhomologous End Joining Deficient Cells

“…Aiming to discern the biochemical pathways required for the efficient and safe reprogramming of adult somatic cells into iPSCs, recent studies have shown the relevance of telomere homeostasis [2][3][4], signal transducers, and DNA repair proteins [4][5][6][7] in cell reprogramming. These results have shown that a significant DNA stress-reflected by an increased number of DNA double strand breaks (DSBs)-is produced during the reprogramming process.…”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…Despite these potential advantages of iPSC-based modeling, there have only been very limited numbers of iPSCbased blood disease models that have shown either the disease features or the feasibility of gene therapy (Table 2) [14,[26][27][28][29][30]. The first comprehensive proof-of-principle study to demonstrate the potential of iPSCs in any disease modeling and treatment was conducted in the mouse model N/D not determined of sickle cell disease (SCD) [31].…”