SummarySickle cell anemia affects millions of people worldwide and is an emerging global health burden. As part of a large NIH-funded NextGen Consortium, we generated a diverse, comprehensive, and fully characterized library of sickle-cell-disease-specific induced pluripotent stem cells (iPSCs) from patients of different ethnicities, β-globin gene (HBB) haplotypes, and fetal hemoglobin (HbF) levels. iPSCs stand to revolutionize the way we study human development, model disease, and perhaps eventually, treat patients. Here, we describe this unique resource for the study of sickle cell disease, including novel haplotype-specific polymorphisms that affect disease severity, as well as for the development of patient-specific therapeutics for this phenotypically diverse disorder. As a complement to this library, and as proof of principle for future cell- and gene-based therapies, we also designed and employed CRISPR/Cas gene editing tools to correct the sickle hemoglobin (HbS) mutation.

Mutations in the gene Adenomatous Polyposis Coli or APC appear in most sporadic cases of colorectal cancer and it is the most frequent mutation causing hereditary Familial Adenomatous Polyposis. The detailed molecular mechanism by which APC mutations predispose to the development of colorectal cancer is not completely understood. This is in part due to the lack of accessibility to appropriate models that recapitulate the early events associated with APC mediated intestinal transformation. We have established a novel platform utilizing human induced Pluripotent Stem cells or iPSC from normal or FAP-specific APC mutant individuals and evaluated the effect of the mutation in the cells before and after differentiation into intestinal organoids. In order to minimize genetic background effects, we also established an isogenic platform using TALEN-mediated gene editing. Comparison of normal and APC mutant iPSC revealed a significant defect in cell identity and polarity due to the presence of APC in heterozygosity as well as chromosomal aberrations including abnormal anaphases and centrosome numbers. Importantly, upon specification into intestinal progeny, APC heterozygosity was responsible for a major change in the transcriptional identity of the cells with dysregulation of key signaling pathways, including metabolic reprogramming, abnormal lipid metabolism and intestinal-specific cadherin expression. In conclusion, we have developed a novel iPSC/intestinal model of APC mutagenesis and provide strong evidence that APC in heterozygosity imparts a clear phenotypic and molecular defect, affecting basic cellular functions and integrity, providing novel insights in the earlier events of APC-mediated tumorigenesis.

Fanconi anemia (FA) is a complex genetic disease associated with a defective DNA repair pathway known as the FA pathway. In contrast to many other FA proteins, BRCA 2 participates downstream in this pathway and has a critical role in homology-directed recombination (HDR). In our current studies, we have observed an extremely low reprogramming efficiency in cells with a hypomorphic mutation in Brca2 (Brca2), that was associated with increased apoptosis and defective generation of nuclear RAD51 foci during the reprogramming process. Gene complementation facilitated the generation of Brca2 D27/D27 induced pluripotent stem cells (iPSCs) with a disease-free FA phenotype. Karyotype analyses and comparative genome hybridization arrays of complemented Brca2 D27/D27 iPSCs showed, however, the presence of different genetic alterations in these cells, most of which were not evident in their parental Brca2 D27/D27 mouse embryonic fibroblasts. Gene-corrected Brca2 D27/D27 iPSCs could be differentiated in vitro toward the hematopoietic lineage, although with a more limited efficacy than WT iPSCs or mouse embryonic stem cells, and did not engraft in irradiated Brca2 D27/D27 recipients. Our results are consistent with previous studies proposing that HDR is critical for cell reprogramming and demonstrate that reprogramming defects characteristic of Brca2 mutant cells can be efficiently overcome by gene complementation. Finally, based on analysis of the phenotype, genetic stability, and hematopoietic differentiation potential of gene-corrected Brca2 D27/D27 iPSCs, achievements and limitations in the application of current reprogramming approaches in hematopoietic stem cell therapy are also discussed. STEM CELLS 2014;32:436-446

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Although there is an increasing interest in defining the role of DNA damage response mechanisms in cell reprogramming, the relevance of proteins participating in nonhomologous end joining (NHEJ), a major mechanism of DNA double-strand breaks repair, in this process remains to be investigated. Herein, we present data related to the reprogramming of primary mouse embryonic fibroblasts (MEF) from severe combined immunodeficient (Scid) mice defective in DNA-PKcs, a key protein for NHEJ. Reduced numbers of induced pluripotent stem cell (iPSC) colonies were generated from Scid cells using reprogramming lentiviral vectors (LV), being the reprogramming efficiency fourfold to sevenfold lower than that observed in wt cells. Moreover, these Scid iPSC-like clones were prematurely lost or differentiated spontaneously. While the Scid mutation neither reduce the proliferation rate nor the transduction efficacy of fibroblasts transduced with reprogramming LV, both the expression of SA-b-Gal and of P16/INK 4a senescence markers were highly increased in Scid versus wt MEFs during the reprogramming process, accounting for the reduced reprogramming efficacy of Scid MEFs. The use of improved Sleeping Beauty transposon/transposase systems allowed us, however, to isolate DNA-PKcs-deficient iPSCs which preserved their parental genotype and hypersensitivity to ionizing radiation. This new disease-specific iPSC model would be useful to understand the physiological consequences of the DNA-PKcs mutation during development and would help to improve current cell and gene therapy strategies for the disease.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

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