Correction of IVS I-110(G>A) β-thalassemia by CRISPR/Cas-and TALEN-mediated disruption of aberrant regulatory elements in human hematopoietic stem and progenitor cells β-Hemoglobinopathies result from mutations in the β-globin (HBB) gene. 1 Whereas causative mutations may be corrected by precise gene correction based on homology-directed repair, imprecise disruption of genome elements by non-homologous end joining is inherently more efficient and more suitable for long-term repopulating cells. 2 This has already prompted the pursuit of disruption-based reactivation of the HBB paralog g-globin as a potentially universal genome-editing strategy to treat patients with β-hemoglobinopathies, 3 which is as yet unproven in the clinic. The common β-thalassemia allele IVSI-110 (HBB ) has an aberrant splice acceptor site that leads to abnormal splicing. 4 Here we investigated the use of a mutation-specific and disruption-based approach to correct HBB . Based on both transcription activator-like effector nucleases (TALEN) and CRISPR/Cas9 RNA-guided HBB -targeting nucleases we analyzed non-homologous end joining-based indel events at on-and off-target sites, and the efficiency of functional correction in patient-derived CD34 + -derived HBB IVS-110(G>A) -homozygous erythroblasts. Both platforms showed significant correction at the RNA, protein and morphological levels, with up to 95% on-target disruption, using a design that minimized d-globin (HBD) offtarget activity. The present study establishes suitable target sequences for effective restoration of normal splicing and validates gene disruption by virus-and DNA-free delivery of nucleases as potential therapy for HBB thalassemia.The HBB mutation resides 19 nucleotides upstream of the normal intron-1 splice acceptor site. We identified one CRISPR/Cas9 and two TALEN-pair target sites compatible with platform-specific sequence constraints, proximity of exon 2, and the need to discern HBB from HBD for therapy by disruption ( Figure 1, Online Supplementary Figure S1). Predicted double-stranded break sites were adjacent to the aberrant splice acceptor site for the RNA-guided nuclease (RGN) and upstream for TALEN pairs, TALEN R1/L1 (R1/L1) and TALEN

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Rare diseases pose a global challenge, in that their collective impact on health systems is considerable, whereas their individually rare occurrence impedes research and development of efficient therapies. In consequence, patients and their families are often unable to find an expert for their affliction, let alone a cure. The tide is turning as pharmaceutical companies embrace gene therapy development and as serviceable tools for the repair of primary mutations separate the ability to create cures from underlying disease expertise. Whereas gene therapy by gene addition took decades to reach the clinic by incremental diseasespecific refinements of vectors and methods, gene therapy by genome editing in its basic form merely requires certainty about the causative mutation. Suddenly we move from concept to trial in 3 years instead of 30: therapy development in the fast lane, with all the positive and negative implications of the phrase. Since their first application to eukaryotic cells in 2013, the proliferation and refinement in particular of tools based on clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein (Cas) prokaryotic RNA-guided nucleases has prompted a landslide of therapy-development studies for rare diseases. An estimated thousands of orphan diseases are up for adoption, and legislative, entrepreneurial, and research initiatives may finally conspire to find many of them a good home. Here we summarize the most significant recent achievements and remaining hurdles in the application of CRISPR/Cas technology to rare diseases and take a glimpse at the exciting road ahead. Marina Kleanthous and Carsten W. Lederer contributed equally to this review.

The β-hemoglobinopathies sickle cell anemia and β-thalassemia are the focus of many gene-therapy studies. A key disease parameter is the abundance of globin chains because it indicates the level of anemia, likely toxicity of excess or aberrant globins, and therapeutic potential of induced or exogenous β-like globins. Reversed-phase high-performance liquid chromatography (HPLC) allows versatile and inexpensive globin quantification, but commonly applied protocols suffer from long run times, high sample requirements, or inability to separate murine from human β-globin chains. The latter point is problematic for in vivo studies with gene-addition vectors in murine disease models and mouse/human chimeras. This study demonstrates HPLC-based measurements of globin expression (1) after differentiation of the commonly applied human umbilical cord blood–derived erythroid progenitor-2 cell line, (2) in erythroid progeny of CD34+ cells for the analysis of clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of the globin regulator BCL11A, and (3) of transgenic mice holding the human β-globin locus. At run times of 8 min for separation of murine and human β-globin chains as well as of human γ-globin chains, and with routine measurement of globin-chain ratios for 12 nL of blood (tested for down to 0.75 nL) or of 300,000 in vitro differentiated cells, the methods presented here and any variant-specific adaptations thereof will greatly facilitate evaluation of novel therapy applications for β-hemoglobinopathies.

MicroRNAs (miRNAs) are small non-coding RNAs crucial for post-transcriptional and translational regulation of cellular and developmental pathways. The study of miRNAs in erythropoiesis elucidates underlying regulatory mechanisms and facilitates related diagnostic and therapy development. Here, we used DNA Nanoball (DNB) small RNA sequencing to comprehensively characterize miRNAs in human erythroid cell cultures. Based on primary human peripheral-blood-derived CD34+ (hCD34+) cells and two influential erythroid cell lines with adult and fetal hemoglobin expression patterns, HUDEP-2 and HUDEP-1, respectively, our study links differential miRNA expression to erythroid differentiation, cell type, and hemoglobin expression profile. Sequencing results validated by reverse-transcription quantitative PCR (RT-qPCR) of selected miRNAs indicate shared differentiation signatures in primary and immortalized cells, characterized by reduced overall miRNA expression and reciprocal expression increases for individual lineage-specific miRNAs in late-stage erythropoiesis. Despite the high similarity of same-stage hCD34+ and HUDEP-2 cells, differential expression of several miRNAs highlighted informative discrepancies between both cell types. Moreover, a comparison between HUDEP-2 and HUDEP-1 cells displayed changes in miRNAs, transcription factors (TFs), target genes, and pathways associated with globin switching. In resulting TF-miRNA co-regulatory networks, major therapeutically relevant regulators of globin expression were targeted by many co-expressed miRNAs, outlining intricate combinatorial miRNA regulation of globin expression in erythroid cells.