Summary Chromatin loops juxtapose distal enhancers with active promoters but their molecular architecture and relationship with transcription remain unclear. In erythroid cells, the locus control region (LCR) and β-globin promoter form a chromatin loop that requires transcription factor GATA1 and the associated molecule Ldb1. We employed artificial zinc fingers (ZF) to tether Ldb1 to the β-globin promoter in GATA1 null erythroblasts in which the β-globin locus is relaxed and inactive. Remarkably, targeting Ldb1 or only its self-association domain to the β-globin promoter substantially activated β-globin transcription in the absence of GATA1. Promoter-tethered Ldb1 interacted with endogenous Ldb1 complexes at the LCR to form a chromatin loop, causing recruitment and phosphorylation of RNA polymerase II. ZF-Ldb1 proteins were inactive at alleles lacking the LCR, demonstrating that their activities depend on long-range interactions. Our findings establish Ldb1 as critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.

Key Points Delivery of ZFNs and donor templates results in high levels of gene correction in human CD34+ cells from multiple sources, including SCD BM. Modified CD34+ cells are capable of engrafting immunocompromised NSG mice and produce cells from multiple lineages.

The transfer of high-avidity T-cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted specificities. We designed zinc-finger nucleases (ZFNs) promoting the disruption of endogenous TCR β and α chain genes. ZFN-treated lymphocytes lacked CD3/TCR surface expression and expanded with IL-7 and IL-15. Upon lentiviral transfer of a TCR for the WT1 tumor antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near-purity, and proved superior in specific antigen recognition to matched TCR-transferred cells. In contrast to TCR-transferred cells, TCR edited lymphocytes did not mediate off-target reactivity while maintaining anti-tumor activity in vivo, thus demonstrating that complete editing of T-cell specificity generate tumor-specific lymphocytes with improved biosafety profile.

Clinical-grade T cells are genetically modified ex vivo to express a chimeric antigen receptor (CAR) to redirect specificity to a tumor associated antigen (TAA) thereby conferring antitumor activity in vivo. T cells expressing a CD19-specific CAR recognize B-cell malignancies in multiple recipients independent of major histocompatibility complex (MHC) because the specificity domains are cloned from the variable chains of a CD19 monoclonal antibody. We now report a major step toward eliminating the need to generate patient-specific T cells by generating universal allogeneic TAA-specific T cells from one donor that might be administered to multiple recipients. This was achieved by genetically editing CD19-specific CAR ؉ T cells to eliminate expression of the endogenous ␣␤ T-cell receptor (TCR) to prevent a graft-versus-host response without compromising CAR-dependent effector functions. Genetically modified T cells were generated using the Sleeping

Summary Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts β-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the β-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggered its transcriptional reactivation. This activity depended on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting SA to the fetal γ-globin promoter in primary adult human erythroblasts increased γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total β-globin synthesis with a reciprocal reduction in adult β-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

Key Points Allogeneic-donor–derived cells can be genetically modified to eliminate expression of HLA-A. HLA-A disruption from donor cells is a step toward generating allogeneic cells as an off-the-shelf therapeutic.

Induction of the rat tyrosine aminotransferase gene (TAT) with glucocorticoid hormones leads to formation of a nuclease hypersensitive site at the hormone‐dependent enhancer located 2.5 kb upstream of the start site of transcription. This enhancer comprises binding sites for the glucocorticoid receptor and additional transcription factors which are only recognized after hormone administration, as demonstrated by genomic footprinting. We show here that the alteration in chromatin structure occurs very rapidly and is also rapidly reversible after withdrawal of the hormone. At all stages nuclease hypersensitivity at this enhancer parallels the transcriptional activity of the TAT gene indicating that transcriptional stimulation requires ongoing enhancer activity. The enhancer region is nucleosomal before induction and after removal of the hormone but the nucleosomal pattern is disturbed in the presence of the hormone. The rapidity of chromatin changes implies that neither disruption nor reassembly of the nucleosomes is dependent on DNA replication. Addition of the glucocorticoid antagonist RU486 to induced cells has effects similar to washing out the glucocorticoid hormone, showing that maintenance of the hypersensitive site requires the ongoing binding of agonist by glucocorticoid receptor. These results describe an inducible enhancer which is regulated by a dynamic balance between two types of protein‐DNA complexes, one of which is nucleosomal.

Gene therapy with genetically modified human CD34+ hematopoietic stem cells (HSCs) may be safer using targeted integration (TI) of transgenes into a genomic ‘safe harbor’ site than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno associated virus (AAV) 6 delivery of donor constructs in human HSCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus-positive HSCs with 6–16% human cell marking were observed following engraftment into mice. In HSCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 in resulted in ~15% gp91phox expression and increased NADPH oxidase activity in ex vivo–derived neutrophils. In mice transplanted with corrected HSCs, 4–11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.