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 triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases 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.
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•Tethering Ldb1 to embryonic or fetal globin genes activates them in adult erythroblasts•Activation of embryonic or fetal globin genes reduces adult type globin expression•Tethered Ldb1 reconfigures enhancer-promoter contacts commensurate with gene expression•Forced chromatin looping presents a novel therapeutic strategy for sickle cell anemia
In adult erythroblasts, forced juxtaposition of the distal enhancer of the β-globin locus with embryonic or fetal globin gene promoters leads to their reactivation, suggesting a potential new strategy for the treatment of β-hemoglobinopathies, including sickle cell anemia.
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 a critical effector of GATA1-mediated loop formation and indicate that chromatin looping causally underlies gene regulation.
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► Chromatin loops can be generated by targeted tethering of select looping factors ► Forced juxtaposition of a distant enhancer and promoter can trigger transcription ► Ldb1 functions as a looping factor ► Chromatin looping could be used to regulate genes for therapeutic intervention
Zinc-finger-mediated tethering of the nuclear factor Ldb1 to the native β-globin promoter is sufficient to juxtapose the promoter with the enhancer and activate transcription. Forced chromatin looping by engineered proteins offers a potential therapeutic strategy to modulate gene expression from a given promoter.
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the β-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would ...allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the β-globin locus with minimal off-target modification. By codelivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34+ hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγnull mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34+ cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.
•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.
Long-term engraftment of allogeneic cells necessitates eluding immune-mediated rejection, which is currently achieved by matching for human leukocyte antigen (HLA) expression, immunosuppression, ...and/or delivery of donor-derived cells to sanctuary sites. Genetic engineering provides an alternative approach to avoid clearance of cells that are recognized as “non-self” by the recipient. To this end, we developed designer zinc finger nucleases and employed a “hit-and-run” approach to genetic editing for selective elimination of HLA expression. Electro-transfer of mRNA species coding for these engineered nucleases completely disrupted expression of HLA-A on human T cells, including CD19-specific T cells. The HLA-Aneg T-cell pools can be enriched and evade lysis by HLA-restricted cytotoxic T-cell clones. Recognition by natural killer cells of cells that had lost HLA expression was circumvented by enforced expression of nonclassical HLA molecules. Furthermore, we demonstrate that zinc finger nucleases can eliminate HLA-A expression from embryonic stem cells, which broadens the applicability of this strategy beyond infusing HLA-disparate immune cells. These findings establish that clinically appealing cell types derived from donors with disparate HLA expression can be genetically edited to evade an immune response and provide a foundation whereby cells from a single donor can be administered to multiple recipients.
•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.
In the present report, we carried out clinical-scale editing in adult mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs) using zinc-finger nuclease-mediated disruption of BCL11a to ...upregulate the expression of γ-globin (fetal hemoglobin). In these cells, disruption of the erythroid-specific enhancer of the BCL11A gene increased endogenous γ-globin expression to levels that reached or exceeded those observed following knockout of the BCL11A coding region without negatively affecting survival or in vivo long-term proliferation of edited HSPCs and other lineages. In addition, BCL11A enhancer modification in mobilized CD34+ cells from patients with β-thalassemia major resulted in a readily detectable γ-globin increase with a preferential increase in G-gamma, leading to an improved phenotype and, likely, a survival advantage for maturing erythroid cells after editing. Furthermore, we documented that both normal and β-thalassemia HSPCs not only can be efficiently expanded ex vivo after editing but can also be successfully edited post-expansion, resulting in enhanced early in vivo engraftment compared with unexpanded cells. Overall, this work highlights a novel and effective treatment strategy for correcting the β-thalassemia phenotype by genome editing.
Nucleobase editors represent an emerging technology that enables precise single-base edits to the genomes of eukaryotic cells. Most nucleobase editors use deaminase domains that act upon ...single-stranded DNA and require RNA-guided proteins such as Cas9 to unwind the DNA prior to editing. However, the most recent class of base editors utilizes a deaminase domain, DddA
, that can act upon double-stranded DNA. Here, we target DddA
fragments and a FokI-based nickase to the human CIITA gene by fusing these domains to arrays of engineered zinc fingers (ZFs). We also identify a broad variety of Toxin-Derived Deaminases (TDDs) orthologous to DddA
that allow us to fine-tune properties such as targeting density and specificity. TDD-derived ZF base editors enable up to 73% base editing in T cells with good cell viability and favorable specificity.
To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of the
gene, either within exon 2 or at the GATAA motif in ...the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM) CD34
hematopoietic stem and progenitor cells (HSPCs). Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation of
resulting from bi-allelic frameshift mutations in
exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of
did not negatively impact enucleation. Furthermore,
exon 2-edited BM-CD34
cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed upon
erythroid-enhancer GATAA motif editing, because enhancer-edited CD34
cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of the
erythroid-specific enhancer editing in BM-CD34
HSPCs as an autologous stem cell therapy in SCD patients.
Mammalian cells with multi-gene knockouts could be of considerable utility in research, drug discovery, and cell-based therapeutics. However, existing methods for targeted gene deletion require ...sequential rounds of homologous recombination and drug selection to isolate rare desired events--a process sufficiently laborious to limit application to individual loci. Here we present a solution to this problem. Firstly, we report the development of zinc-finger nucleases (ZFNs) targeted to cleave three independent genes with known null phenotypes. Mammalian cells exposed to each ZFN pair in turn resulted in the generation of cell lines harboring single, double, and triple gene knockouts, that is, the successful disruption of two, four, and six alleles. All three biallelic knockout events were obtained at frequencies of >1% without the use of selection, displayed the expected knockout phenotype(s), and harbored DNA mutations centered at the ZFN binding sites. These data demonstrate the utility of ZFNs in multi-locus genome engineering. Biotechnol. Bioeng. 2010; 106: 97-105.
Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc ...finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.
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David Fenard and colleagues developed a lentiviral backbone for the multiplexing of up to three ZF-R sequences, allowing an efficient, stable, and specific epigenetic control of multiple genes in T cells or Tregs after a single lentiviral transduction event.
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