Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.

Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for ß-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here we compared combined CRISPR-Cas9 endonuclease editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. We found that combined targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two sgRNAs resulted in superior HbF induction, including in engrafting erythroid cells from sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. We corroborated prior observations that double strand breaks (DSBs) could produce unintended on- target outcomes in hematopoietic stem and progenitor cells (HSPCs) such as long deletions and centromere-distal chromosome fragment loss. We show these unintended outcomes are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing HSPCs without cytokine culture bypassed long deletion and micronuclei formation while preserving efficient on-target editing and engraftment function. These results indicate that nuclease editing of quiescent hematopoietic stem cells (HSCs) limits DSB genotoxicity while maintaining therapeutic potency and encourages efforts for in vivo delivery of nucleases to HSCs.

Oral Cladribine Impairs Intermediate, but Not Conventional, Monocyte Transmigration in Multiple Sclerosis Patients across a Model Blood-Brain Barrier.

Multiple sclerosis (MS) is a disease in which the immune system damages components of the central nervous system (CNS), leading to the destruction of myelin and the formation of demyelinating plaques. This often occurs in episodic "attacks" precipitated by the transmigration of leukocytes across the blood-brain barrier (BBB), and repeated episodes of demyelination lead to substantial losses of axons within and removed from plaques, ultimately leading to progressive neurological dysfunction. Within leukocyte populations, macrophages and T and B lymphocytes are the predominant effectors. Among current immunotherapies, oral cladribine's impact on lymphocytes is well characterised, but little is known about its impact on other leukocytes such as monocytes and dendritic cells (DCs). The aim of this study was to determine the transmigratory ability of monocyte and DC subsets in healthy subjects and untreated and cladribine-treated relapse-remitting MS (RRMS) patients using a well-characterised model of the BBB. Peripheral blood mononuclear cells from subjects were added to an in vitro transmigration assay to assess cell migration. Our findings show that while prior treatment with oral cladribine inhibits the migration of intermediate monocytes, it has no impact on the transmigration of DC subsets. Overall, our data indicate a previously unrecognised role of cladribine on intermediate monocytes, known to accumulate in the brain active MS lesions.

Crystal Structure of an i-Motif from the HRAS Oncogene Promoter.

An i-motif is a non-canonical DNA structure implicated in gene regulation and linked to cancers. The C-rich strand of the HRAS oncogene, 5'-CGCCCGTGCCCTGCGCCCGCAACCCGA-3' (herein referred to as iHRAS), forms an i-motif in vitro but its exact structure was unknown. HRAS is a member of the RAS proto-oncogene family. About 19 % of US cancer patients carry mutations in RAS genes. We solved the structure of iHRAS at 1.77 Šresolution. The structure reveals that iHRAS folds into a double hairpin. The two double hairpins associate in an antiparallel fashion, forming an i-motif dimer capped by two loops on each end and linked by a connecting region. Six C-C+ base pairs form each i-motif core, and the core regions are extended by a G-G base pair and a cytosine stacking. Extensive canonical and non-canonical base pairing and stacking stabilizes the connecting region and loops. The iHRAS structure is the first atomic resolution structure of an i-motif from a human oncogene. This structure sheds light on i-motifs folding and function in the cell.