br Results br Discussion We generated a

Results
Discussion We generated a library of iPSCs from patients with sickle cell anemia of diverse ethnicities and HBB haplotypes to study the biology of these ras pathway and the feasibility of their generation from blood samples collected from patients in distant locations and shipped frozen to our laboratory. These fully characterized lines, along with accompanying genetic and hematologic data, are now freely available. Drug development is an expensive and time-consuming process that requires stringent specificity, potency, and toxicity validations of potential novel therapeutics. Traditionally, drug discovery proceeds from testing in in vitro cell-based assays in the laboratory to in vivo animal models, followed by three phases of clinical testing. Unfortunately, potential therapeutics usually are not extensively tested in humans until phase II clinical trials, which can occur many years after initial drug discovery. If in vitro testing is performed on human cells before clinical trials, these cells are typically immortalized cell lines, which have undergone genetic alterations to ensure their immortalization, possibly altering the fidelity of the drug screens. Use of immortalized cell lines is a common cause of high attrition rates for drug development, as what works in vitro and subsequently in animal models may not always translate to the clinic (Kola and Landis, 2004). Pluripotent stem cells, and in particular iPSCs, have the opportunity to revolutionize preclinical drug screening. iPSC technology offers the prospect of an unlimited supply of material and is ideal for screening drugs against the genetic variations found in a patient population, such as those suffering from sickle cell disease for which there is currently only a single FDA-approved drug. Sickle cell disease is phenotypically diverse, a quality that arises primarily from the known and unknown quantitative trait loci that regulate HbF expression and are polymorphic in diverse patient populations. This variance has led to many discoveries regarding transcriptional regulation of HbF and further elucidated the complexities of hemoglobin switching. Since there are still many unknown regulators of HbF expression, finding drugs that will be efficacious in patients with a variety of genetic backgrounds would be ideal, and the creation of the described iPSC bank may contribute to this effort. Cell-based treatments for sickle cell disease include blood transfusion, hematopoietic stem cell transplantation, and nascent trials of gene therapy. It is hoped that the gene editing tools described in this work, coupled with corrected sickle-cell-disease-specific iPSCs could one day provide a functional cure for the disorder. Erythroid-progenitor-derived iPSCs also hold promise for development as a potential, autologous, cellular therapeutic due to their constitutive HbF expression without progression to an adult globin phenotype (Smith et al., 2013). An autologously derived erythroid progenitor that makes high concentrations of HbF should render any remaining HbS incapable of damaging the sickle erythrocyte (Ngo et al., 2012).
Experimental Procedures
Author Contributions
Acknowledgments This work was funded by the NextGen Consortium U01HL107443 from the NIH/NHLBI, the University of Dammam, SP 11/2011, Office of Collaboration and Knowledge Exchange, University of Dammam, Training grant for Biostatistics5T32GM074905, and Training Grant for Hematology5T32 HL007501. Anthony Akimbami, MD, MPH, assisted with sample collection.
Introduction Due to their ability to differentiate into a variety of cell types, induced pluripotent stem cells (iPSCs) are a potentially powerful model system to study mechanisms underlying non-coding genetic variants associated with human traits, many of which lie in cell-type-specific regulatory regions (Maurano et al., 2012). However, because non-coding regulatory variants can have relatively small effect sizes, hundreds of lines from diverse individuals may be needed to measure genetic associations as opposed to the tens of different lines typically used to study disease-associated coding variants with strong effects (Avior et al., 2016). To enable the study of genetic variants associated with complex diseases and cell-type-specific molecular phenotypes, we and others are establishing large systematically generated collections of iPSCs toward the goal of generating large genomic datasets that will be openly available to researchers (Avior et al., 2016; Kilpinen et al., 2016; McKernan and Watt, 2013; Streeter et al., 2017). Ongoing collections, including large disease-focused iPSC repositories (www.cirm.ca.gov), however, are currently limited in sample diversity and in related individuals (e.g., pedigrees or twins), which would allow for the interrogation of population-associated genetic variation, rare variation, and family-based genetic study designs. Thus, the generation of a resource consisting of hundreds of systematically derived iPSCs with available genomic data including SNP arrays, RNA sequencing (RNA-seq), and whole-genome sequencing, and that includes a variety of familial architectures and individuals of multiple ethnicities, would further enable a wide variety of study designs to interrogate the genetic basis of phenotype and disease.