Targeted gene therapy and cell reprogramming in Fanconi anemia

Abstract

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.

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].…”

“…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].…”

“…Genome editing using CRISPR/Cas9-engineered nucleases corrects FANCA mutations in human FA fibroblasts [102]. Although clinical translation of gene-corrected IPS cells and genome editing with engineered nucleases is difficult in the short term, successful engraftment of iPSC-derived and gene-corrected blood progenitor cells may cure FA and other blood disorders characterized by low numbers of bone marrow HSCs [97,101]. Therefore, editing fibroblasts and IPS-cell genomes may soon translate directly to HSCs from FA patients (Figure 4a).…”

Savior siblings and Fanconi anemia: analysis of success rates from the family’s perspective

“…9 Finally, disease-free blood progenitors from skin fibroblasts of patients with FA were recently generated via induced pluripotent stem cells, setting the path for a future cure through regenerative medicine for patients with FA and other blood disorders. [10][11][12] We hope that these therapeutic advances will make the generation of savior babies to cure FA and other genetic blood disorders unnecessary in the years to come.…”

Genetic Diagnosis of Fanconi Anemia

“…Methods of in vitro gene therapy might complement these approaches, mainly for FA patients with molecular defects in the commonly mutated FANC genes [88,89]. Gene therapy in this form will principally address hematological problems associated with FA [90].…”