Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs

Abstract

Fanconi Anemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration-free induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA-patient bone marrow cells.

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].…”

“…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]. Genome editing using CRISPR/Cas9-engineered nucleases corrects FANCA mutations in human FA fibroblasts [102].…”

“…Therefore, editing fibroblasts and IPS-cell genomes may soon translate directly to HSCs from FA patients (Figure 4a). FA-IPS cells offer a novel tool to model FA physiology and pathogenesis and provide a cell platform for drug screening [96]. Finally, therapies designed to enhance the correct mRNA processing at a mutant TT splice donor in FANCC using suppressor U1 snRNAs, suggests that correcting pathological mRNA processing at specific mutant splice sites might apply to FA complementation groups in a mutation-specific fashion [103].…”