Gene-Correction Rescues Reprogramming of Fanconi Anemia Fibroblasts and Enables Hematopoietic Differentiation of FA Induced Pluripotent Stem Cells in Vitro and In Vivo
Authors: Mueller Lars , Milsom Michael D. , Harris Chad , Vyas Rutesh , Brumme Kristina , Parmar Kalindi , Schambach Axel , Grassman Elke , Park In-Hyun , Wendy London , Strait Kelly , Schlaeger Thorsten , Devine Alexander L. , D'Andrea Alan D. , Daley George Q. , Williams David A. 
Published: October 2019

Abstract
Abstract 672
Fanconi anemia (FA) is a recessive syndrome characterized by progressive fatal bone marrow failure and chromosomal instability. FA cells have inactivating mutations in a signaling pathway that is critical for maintaining genomic integrity and repairing DNA damage caused by cross-linking agents. Transgenic expression of the implicated genes corrects the phenotype of hematopoietic cells but previous attempts at gene therapy failed largely due to inadequate numbers of hematopoietic stem cells available for gene correction and autologous engraftment. Induced pluripotent stem cells (iPSC) constitute an alternate source of autologous cells, which are amenable to ex vivo expansion and genetic correction. While fibroblasts from a limited number of FA patients have been reported to fail to undergo reprogramming (Raya et al., Nature, 2009), reproducible observations and mechanistic studies ascertained in an extended panel of patient cells and murine knock-out models are lacking to date. We undertook direct reprogramming of ten unique human FA primary fibroblast samples of the FA-A, FA-C, FA-G, and FA-D2 complementation groups. Using standard four-factor reprogramming, no human FA iPSC colonies were obtained in cells defective in the FA pathway. By contrast, reprogramming of gene-corrected patient samples, augmented by hypoxia (5%O2), yielded multiple pluripotent iPSC lines, confirming a critical cell-intrinsic role of the FA pathway in reprogramming. To determine if gene-corrected FA iPSC could be therapeutically useful, we performed karyotype analyses and evaluated in vitro hematopoietic differentiation in three FA-A iPSC lines. These FA patient iPSC lines were karyotypically normal and showed a robust multilineage hematopoietic differentiation potential, resulting in erythroid and myeloid hematopoietic colony forming units to a similar degree as compared to normal donor iPSC controls. We hypothesized that the reprogramming resistance of FA cells is due to defective DNA repair and genomic instability. To explore the mechanisms of the reprogramming defect, we transduced wild type (wt) tail-tip fibroblasts (TTF) with the reprogramming vectors. We observed significantly increased FANCD2 foci formation during reprogramming (median percentage of FANCD2 foci: mock-transduced TTF 2.5%, reprogrammed TTF 20.5%, n=8, p<0.01) indicating activation of the FA pathway. Next, we examined reprogramming in FA-deficient mouse cells. We observed a significantly higher incidence of reprogramming-induced double-strand DNA breaks and senescence in Fanca−/− TTF as compared to wt controls (γH2AX foci: wt 13%, Fanca−/− 19%; senescence: wt 47%, Fanca−/− 62%, median percentage, p<0.01). To evaluate whether these changes contribute to the reprogramming resistance of FA cells, we quantified the reprogramming efficiency of Fanca−/−, Fancc−/− and littermate wt TTF. The efficiency was 0.06% for Fanca−/− (n=8) and 0.38% for Fancc−/− (n=12) as compared to 0.55% for wt controls (n=13; p<0.01 and <0.05, respectively). To directly test the role of the FA pathway in reprogramming, TTF were transduced with retroviral vectors co-expressing FANCA and enhanced green fluorescent protein (eGFP) or encoding only eGFP as a control. Under hypoxic conditions, gene-correction of the Fanca−/− TTF with FANCA resulted in a significant reduction of senescence and rescued the reprogramming efficiency of Fanca−/− TTF to wt levels. While significant chromosomal aberrations were observed in uncorrected Fanca−/− iPCS, gene-corrected Fanca−/− iPSC did not show any significant chromosomal imbalances when analyzed by comparative genomic hybridization. To evaluate the capacity of FA iPSC to form blood cells in vivo, we injected wt, control transduced or gene-corrected Fanca−/− iPCS (CD45.1+) into wt blastocysts (CD45.2+) and analyzed the contribution of iPSC-derived hematopoietic cells in embryonic day 14.5 fetal livers. We observed 1.8–4% wt iPSC chimerism (n=15), 0.4–0.9% Fanca−/− iPSC chimerism (n=3) and 1.5 to 2.5% chimerism in gene-corrected Fanca−/− iPSC (n=11). Our data demonstrate that reprogramming activates the FA pathway. Gene-correction rescues the reprogramming block of FA cells and protects FA iPSC from genomic instability, thus yielding an expandable source of autologous stem cells with hematopoietic differentiation capacity that may be explored for future use in regenerative medicine.

Disclosures:
Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.