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Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

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. Keywords: hemoglobinopathies, BCL11a, genome editing, thalassemia, HbF reactivation, zinc finger nucleases

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system.

To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of the BCL11A 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 BCL11A resulting from bi-allelic frameshift mutations in BCL11A exon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer of BCL11A did not negatively impact enucleation. Furthermore, BCL11A 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 BCL11A 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 BCL11A erythroid-specific enhancer editing in BM-CD34+ HSPCs as an autologous stem cell therapy in SCD patients.

Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

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 co-delivering 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/IL2rγ(null) 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.

SummaryDistal 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.

SummaryChromatin 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.PaperFlick

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 antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR beta- and alpha-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

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 αβ 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 Beauty system to stably introduce the CD19-specific CAR with subsequent permanent deletion of α or β TCR chains with designer zinc finger nucleases. We show that these engineered T cells display the expected property of having redirected specificity for CD19 without responding to TCR stimulation. CAR+TCRneg T cells of this type may potentially have efficacy as an off-the-shelf therapy for investigational treatment of B-lineage malignancies.

Increases in fetal hemoglobin (HbF) are associated with improved clinical outcomes in the inherited hemoglobinopathies. We are developing a novel gene-edited cell therapy to treat patients with sickle cell disease (SCD) and beta-thalassemia (BT) using autologous hematopoietic stem and progenitor cells (HSPCs) genetically modified with zinc finger nucleases (ZFNs) to restore high levels of HbF expression. The ZFNs have been designed to specifically target the GATA motif within an intronic erythroid-specific enhancer (ESE) of BCL11A, the gene encoding a transcriptional regulator of the fetal-to-adult hemoglobin switch. Previously, we reported successful ZFN-mediated, ex vivoBCL11A gene editing in dual mobilized HSPCs, i.e. peripheral stem cells mobilized with a combination of granulocyte colony-stimulating factor (G-CSF) and plerixafor1. The editing procedure was optimized for high on-target/low off-target modification levels and increases in HbF in erythroid progeny. This drug product, ST-400, passed extensive safety testing and is currently in a phase 1/2a clinical trial for transfusion-dependent beta-thalassemia (ClinicalTrials.gov number NCT03432364). An analogous drug product, BIVV003, is being developed as a therapeutic for SCD. In patients with SCD, the use of G-CSF for stem cell mobilization is not recommended due to the risk of clinical complications. Therefore, peripheral HSPCs are obtained from SCD patients via single agent, plerixafor mobilization and apheresis2-5. Understanding the effect of this change in mobilization strategy on ZFN editing efficiency and specificity is a key element in preparing for SCD gene-edited cell therapy. In the present studies, we demonstrate comparability of ZFN editing outcomes in single and dual mobilized HSPCs obtained from healthy donors. In plerixafor mobilized HSPCs from five healthy donors at research scale, ZFN-mediated gene editing induced an efficient modification at the BCL11A ESE target site (>75% of alleles modified, as measured by MiSeq deep sequencing) with high post-editing viability (77%). Similar gene editing efficiencies (>70%) were obtained in HSPCs at clinical manufacturing scale (n=2). Further, in vitro HbF protein levels and HbF+ cell frequencies within erythroid progeny of edited cells were increased by >4 and 3-fold respectively - compared to non-edited cells in the same culture conditions, using reverse phase high-performance liquid chromatography and flow cytometry (n=4 healthy donors at research scale). Single cell clone analysis revealed that ZFN-mediated gene editing targeted both alleles of BCL11A at high frequency (91-94% of edited cells within erythroid progeny) with high levels of replicable GATA-disrupting indel patterns. On average, each edited allele contributed an additional 17.6% increase in HbF production in vitro, with a statistically-significant increase in HbF level for biallelic edited vs. unedited controls (3.4 fold). Critical to BIVV003 use in clinical trials, ZFN-mediated gene editing did not impair single agent mobilized HSPC function in vitro based on measurements of colony forming unit (CFU) production and frequencies of long-term HSC (LT-HSC) and common myeloid progenitor (CMP) cells by flow cytometry. In agreement with this data, injection of BIVV003 into immune-deficient NBSGW mice resulted in robust long-term engraftment (21 weeks) without any impact on the number of HSPCs and their differentiated progeny. Overall, these data demonstrate potential efficacy of ZFN-edited HSPCs (BIVV003) as a novel cell therapy for SCD patients.Holmes et al., 2017 (ASH abstract)Fitzhugh et al., 2009Lagresle-Peyrou et al., 2018Hsieh and Tisdale, 2018Yannaki et al., 2012 Disclosures Moran: Bioverativ, a Sanofi Company: Employment. Ling:Bioverativ, a Sanofi Company: Employment. Lessard:Bioverativ, a Sanofi Company: Employment. Vieira:Bioverativ a Sanofi Company: Employment. Hong:Bioverativ, a Sanofi Company: Employment. Holmes:Sangamo Therapeutics: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Gray:Sangamo Therapeutics: Employment. Levasseur:Bioverativ, a Sanofi Company: Employment. Rimmele:Bioverativ, a Sanofi Company: Employment.

Background: Beta-thalassemia and sickle cell disease are genetic disorders caused by mutations in the beta-globin gene which lead to significant anemia and serious medical complications. Increases in fetal hemoglobin (HbF) have been linked to improved clinical outcomes in patients with beta-thalassemia (BT) and sickle cell disease (SCD). Methods: We have developed engineered zinc finger nucleases (ZFNs) that precisely cleave and disrupt the erythroid enhancer of the BCL11A gene, which substantially boosts HbF production in erythroid progeny of genome-edited CD34 + hematopoietic stem/progenitor cells (HSPCs). The autologous modified HSPC drug product are named ST-400 (BT) and BIVV003 (SCD). Results: We demonstrate that ST-400 / BIVV003 can be manufactured by reproducible, high-level, ZFN-driven modification in peripheral blood mobilized HSPCs at clinical production scale (>10 8 cells). The drug product is made in a GMP-compliant setting using a clinical-grade electroporation device to deliver the ZFN mRNAs ex vivo . Unbiased specificity studies of ST-400 / BIVV003 demonstrated an exquisite amount of specificity, with high levels of on-target modification (~80%). Erythroid colony genotyping in enhancer targeted cells, showed bi-allelic modification of the BCL11A erythroid enhancer in >50% of HSPCs, resulting in >4-fold higher levels of gamma globin mRNA and protein compared to controls. Similarly, we observed high levels of modification in research-scale preparations of HSPCs from patients with beta-thalassemia. Injection of ST-400 / BIVV003 into immune-deficient mice resulted in robust long-term (19 week) engraftment. Targeted gene modification was maintained through multi-lineage differentiation in the bone marrow and peripheral blood. Conclusions: These results support further clinical development of ST-400 / BIVV003 as a potential therapy for beta-thalassemia and sickle cell disease. Disclosures No relevant conflicts of interest to declare.

Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to ∼500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells. Biotechnol. Bioeng. 2007; 97: 1180–1189. © 2006 Wiley Periodicals, Inc.

Mismatch of human leukocyte antigens (HLA) adversely impacts the outcome of patients after allogeneic hematopoietic stem-cell transplantation (alloHSCT). This translates into the clinical requirement to timely identify suitable HLA-matched donors which in turn curtails the chances of recipients, especially those from a racial minority, to successfully undergo alloHSCT. We thus sought to broaden the existing pool of registered unrelated donors based on analysis that eliminating the expression of the HLA-A increases the chance for finding a donor matched at HLA-B, -C and -DRB1 regardless of a patient’s race. Elimination of HLA-A expression in HSC was achieved using artificial zinc finger nucleases designed to target HLA-A alleles. Significantly, these engineered HSCs maintain their ability to engraft and reconstitute hematopoiesis in immunocompromised mice. This introduced loss of HLA-A expression decreases the need to recruit large number of donors to match with potential recipients and has particular importance for patients whose HLA repertoire is under-represented in the current donor pool. Furthermore, the genetic engineering of stem cells provides a translational approach to HLA-match a limited number of third-party donors with a wide number of recipients.

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather 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 HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs 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 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 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.

Regulatory regions harbor multiple transcription factor recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe a facile approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

Drug discovery requires high-quality, high-throughput bioassays for lead identification and optimization. These assays are usually based on immortalized cell lines, which express the selected drug target either naturally or as a consequence of transfection with the cDNA encoding the target. Natural untransfected cell lines often fail to achieve the levels of expression required to provide assays of sufficient quality with a high enough signal-to-noise ratio. Unfortunately, the use of cDNA is increasingly restricted, as the sequences for more and more genes become subject to patent restrictions. To overcome these limitations, the authors demonstrate that engineered transcription factors with Cys2-His2 zinc finger DNA-binding domains can be used to effectively activate an endogenous gene of interest without the use of isolated cDNA of the target gene. Using this approach, the authors have generated a cell line that provides a high-quality and pharmacologically validated G-protein-coupled receptor bioassay. In principle, this technology is applicable to any gene of pharmaceutical importance in any cell type.

We have investigated the mechanism, structural correlates, andcis-acting elements involved in chromatin opening and gene activation, using the human β-globin locus as a model. Full transcriptional activity of the human β-globin locus requires the locus control region (LCR), composed of a series of nuclease hypersensitive sites located upstream of this globin gene cluster. Our previous analysis of naturally occurring and targeted LCR deletions revealed that chromatin opening and transcriptional activity in the endogenous β-globin locus are dissociable and dependent on distinctcis-acting elements. We now report that general histone H3/H4 acetylation and relocation of the locus away from centromeric heterochromatin in the interphase nucleus are correlated and do not require the LCR. In contrast, LCR-dependent promoter activation is associated with localized histone H3 hyperacetylation at the LCR and the transcribed β-globin-promoter and gene. On the basis of these results, we suggest a multistep model for gene activation; localization away from centromeric heterochromatin is required to achieve general hyperacetylation and an open chromatin structure of the locus, whereas a mechanism involving LCR/promoter histone H3 hyperacetylation is required for high-level transcription of the β-globin genes.

We describe here a fundamentally novel way to approach the development of a therapeutic: use of data from genome-wide association studies (GWAS) as a guide to create, in a targeted fashion, a disease-ameliorating genotype in the patient's own cells. We exemplify this by focusing on the hemoglobinopathies, β-thalassemia and sickle cell disease (SCD): in both cases elevated levels of fetal hemoglobin (HbF) have been shown to lessen or eliminate disease symptoms. Thus, reversing HbF silencing in patients is an attractive strategy for the development of therapies. To this end, GWAS data pointed to loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, as causative for HbF elevation. While such regulatory elements are challenging to affect in a clinical setting via a conventional small molecule approach, the development of genome editing with engineered nucleases allows, in principle, a targeted intervention at the DNA level to disable enhancer function. Here, we reduce this notion to practice.The BCL11A enhancer consists of three separate cis-regulatory elements spanning a total of~1,500 bp that together drive erythroid-specific expression of BCL11A and lead to a silencing of fetal hemoglobin post-birth. Guided in part by fine-scale in vivo protein-DNA interaction data, we performed a reverse-genetic analysis of the enhancer at its endogenous location in the physiologically relevant cell type, primary human erythroid cells, using zinc finger nucleases (ZFNs). We developed highly optimized ZFNs that yield 60-80% target locus editing in human mobilized peripheral blood CD34 cells. Remarkably, we find that the ZFN-driven ablation of a single 5 bp element, GATAA, resident in the enhancer reproducibly elevates fetal globin in erythroid progeny of these CD34 cells to levels indistinguishable from those resulting from an essentially complete ZFN-driven coding knockout of BCL11A itself. We demonstrate high-efficiency ZFN-driven marking at the enhancer in peripheral blood mobilized CD34 cells at clinical scale production in a GMP-compliant setting, observe the successful engraftment and differentiation of genome-edited cells in an immunodeficient mouse model, and use an unbiased deep-sequencing based assay to comprehensively characterize the nucleus-wide specificity profile of ZFN action.Together these data illustrate the feasibility of using findings from genome-wide association studies to pursue novel therapeutic approaches in monogenic disease. In particular, our work supports the further development of fetal globin elevation via the targeted genome editing of the BCL11A erythroid-specific enhancer in both peripheral blood- and bone marrow-derived CD34 cells as a potential treatment for the hemoglobinopathies.

Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. Allogeneic hematopoietic cell transplantation (HCT) is the only curative treatment, but its application is limited by: (i) a paucity of HLA-matched donors and (ii) a fixed risk of graft vs host disease (GvHD) and transplant-related mortality. Sustained elevation of fetal hemoglobin (HbF) has a very strong clinical effect in both thalassemia and SCD with curative potential. HbF levels are under genetic control, and alleles of the transcription factor BCL11A that lower its expression result in elevation of HbF. BCL11A is thus an attractive target for gene editing of autologous hematopoietic stem cell (HSC) for HCT in β-hemoglobinopathies.We have designed zinc finger nucleases (ZFNs) that when transiently introduced into stem cells ex vivo can efficiently disrupt the BCL11A gene in hematopoietic stem cells (HSCs) by introduction of mutations following target site cleavage and non-homologous end joining. We have developed a clinical scale GMP process for mRNA electroporation of BC11A ZFNs into HSCs, and show that erythrocytes generated ex vivo from BCL11A-modified HSC have elevated fetal globin mRNA, and over 40% of all Hb is represented by HbF. A similar increase in HbF was seen in modified HSCs from subjects with β-thalassemia. Engraftment and differentiation potential of BCL11a modified and control HSCs in NSG mice were comparable. Furthermore, following engraftment into NSG mice, HSCs placed into erythropoiesis cultures maintained increased HbF levels. Preclinical safety studies included soft agar transformation, karyotyping, and double-strand break quantification following ZFN modification. Both bioinformatics guided and a novel, unbiased, oligonucleotide capture method were used to confirm low levels of off-target activity in BCL11A modified HSC as gauged by deep sequencing. In vivo carcinogenicity was evaluated by engraftment of HSC modified by BCL11A ZFNs in NSG mice. Engraftment and multilineage differentiation were comparable between groups and no evidence for carcinogenicity was found.The primary objective of the Phase I clinical study is to evaluate the safety of autologous BCL11A gene edited HSPC infusion after myeloablative (full dose busulfan) conditioning in subjects with transfusion dependent β-thalassemia major. The secondary objectives are to assess hematologic and immunological reconstitution, and to measure the treatment effects on anemia, RBC transfusion frequency, and iron burden.

Multiple inhibitory pathways are exploited by a number of cancers to block the body's immune response which may limit the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Importantly, its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor targeted T cells prior to ACT. Here we describe the elimination of PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PDCD1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 specific ZFNs, the clinical scale TIL production process yielded efficient modification of the PDCD1 gene locus (mean = 74.8% of alleles, n=3, range = 69.9 – 84.1%), which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 modified TIL products displayed an effector memory phenotype and expanded approximately 500 – 2000 fold during a rapid cell expansion. In addition, PD-1 modified and unmodified TILs showed equivalent levels of engraftment in a NSG-mouse model. PD-1 modified TILs demonstrated improved in vitro T cell effector functions relative to controls (statistically significant greater TNFα, GM-CSF and IFNγ cytokine secretion when co-cultured with MART-1+ tumor cells). Preliminary safety evaluations showed that PD-1 modified TILs maintained IL-2 dependent growth in vitro, and no tumors were observed in an NSG-mouse tumorigenicity study. These data support the further study of the efficacy and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma.

The most upstream hypersensitive site (HS) of the β-globin locus control region (LCR) in humans (5′ HS 5) and chickens (5′ HS 4) can act as an insulating element in some gain of function assays and may demarcate a β-globin domain. We have mapped the most upstream HSs of the mouse β-globin LCR and sequenced this region. We find that mice have a region homologous to human 5′ HS 5 that is associated with a minor HS. In addition we map a unique HS upstream of 5′ HS 5 and refer to this novel site as mouse 5′ HS 6. We have also generated mice containing a targeted deletion of the region containing 5′ HS 5 and 6. We find that after excision of the selectable marker in vivo, deletion of 5′ HS 5 and 6 has a minimal effect on transcription and does not prevent formation of the remaining LCR HSs. Taken together these findings suggest that the most upstream HSs of the mouse β-globin LCR are not necessary for maintaining the β-globin locus in an active configuration or to protect it from a surrounding repressive chromatin environment.

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-A(neg) 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.

Abstract 280 Distal enhancers physically contact target promoters to confer high level transcription. At the mammalian β-globin loci long-range chromosomal interactions between a distal enhancer, called the locus control region (LCR), and the globin genes are developmentally dynamic such that the LCR contacts the embryonic, fetal and adult globin genes in a stage-appropriate fashion. LCR-globin gene interactions require the nuclear factor Ldb1. Recently, we employed artificial zinc finger (ZF) proteins to target Ldb1 to the endogenous β-globin locus to force an LCR-promoter interaction. This led to substantial activation of β-globin transcription and suggested that forced chromatin looping could be employed as a powerful tool to manipulate gene expression in vivo (Deng et al., Cell 2012). Reactivation of the fetal globin genes in adult erythroid cells has been a long-standing goal in the treatment of patients with sickle cell anemia. Therefore, building on our findings, we investigated whether the developmentally silenced embryonic globin gene βh1 can be re-activated in adult murine erythroblasts by re-directing the LCR away from the adult type globin gene and towards its embryonic counterpart. To this end, Ldb1 was fused to artificial ZF proteins (ZF-Ldb1) designed to bind to the βh1 promoter. ZF-Ldb1 was introduced into definitive erythroid cells in which only the adult but not the embryonic β-like globin gene is expressed. In vivo binding of the ZF-Ldb1 to its intended target was verified by chromatin immunoprecipitation assay. Strikingly, expression of ZF-Ldb1 re-activated βh1 transcription up to approximately ∼15% of total cellular β-globin production. This suggests that forced tethering of a looping factor to a select promoter can be employed to override a pre-existing developmental long-range chromatin interaction to reprogram a developmentally controlled gene locus. We are now in the process of testing whether our approach might be suitable to reactivate the silent fetal globin genes in adult human erythroid cells. These studies are underway and the results will be discussed at the meeting. Disclosures: Reik: Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment.

While adoptive transfer of T-cells modified to express a chimeric antigen receptor or tumor antigen specific T-cell receptor (TCR) has shown great promise for the treatment of malignant cancers, most current clinical applications are limited by the use of autologous T-cell products. Targeting of the TCR and HLA Class I genes in primary T-cells thus represent attractive targets for genome editing in order to produce universal T cells from allogeneic donors. Elimination of the native TCR and HLA class I proteins on T-cells would, respectively, reduce the risk of graft-versus-host disease and host-versus-graft clearance mediated by the adaptive immune system. We have developed clinical grade zinc finger nuclease (ZFN) reagents that can efficiently target the T-cell receptor alpha constant (TRAC) and beta-2-microglobulin (B2M) loci. ZFN encoding mRNAs were introduced into purified T-cells by electroporation. Without selection, T-cells treated with TRAC specific ZFNs showed modification of up to 89% of alleles as gauged by deep sequencing. Flow analysis showed 91% of treated cells were negative for CD3 expression. Results from ZFN mRNA dose titration studies showed that the level of TRAC gene modification by deep sequencing was highly correlated with the percentage of cells that stain negative for CD3 expression by FACS (Spearman rho = 0.96; p < 0.0001). B2M is a subunit in all HLA class I molecules, and represents a conserved target for eliminating HLA class I presentation in cells from different donors. Analysis of T-cells transfected with mRNAs encoding B2M specific ZFNs showed up to 94% of alleles were modified, as determined by deep sequencing. FACS analysis showed that 89% of treated cells were negative for expression of HLA-A, B, C. Similar to TRAC modified cells, a marked correlation was observed between the percentage of B2M alleles modified and percentage of cells lacking surface expression of HLA-A,B, and C (Spearman rho = 0.57; p < 0.0001). ZFN mediated genome editing was well tolerated. T-cells treated with TRAC or B2M specific ZFNs showed similar viability and growth characteristics as mock transfected cells. Together, these highly efficient ZFN reagents permit the highly efficient double knockout of TCR and HLA class I surface expression in primary human T-cells for potential use in the development of allogeneic cell therapies.

Transcription of the rat tyrosine aminotransferase gene (TAT) is stimulated in liver by glucocorticoid hormones or by cAMP-increased protein kinase A activity via enhancers located 2.5 kilobases (kb) and 3.6 kb upstream of the start site of transcription. The proteins mediating induction have been characterized, and protein binding in the two enhancer regions has been analyzed in vivo and in vitro. The TAT gene is therefore a useful model system with which to study cross-talk between different signal transduction pathways. We find that activation of the second messenger pathway leading from protein kinase C to the transcription factor AP-1 by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) impairs induction of the TAT gene both by glucocorticoid hormones and cAMP. The effects of TPA treatment on chromatin structure of the TAT gene and protein-DNA interactions in vivo were assayed. Under conditions in which TPA impairs glucocorticoid induction of TAT mRNA, the glucocorticoid receptor and other proteins binding within the glucocorticoid-inducible enhancer occupy their binding sites, indicating that inhibition occurs at a later step necessary for transcriptional stimulation. On the other hand, inhibition of cAMP induction correlates with reduced occupancy of the cAMP response element in vivo.

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.

Sickle cell disease (SCD) is one of the most common inherited blood disorders and is caused by a mutation at the adult beta globin gene resulting in substitution of valine for glutamic acid at position 6 in the encoded protein. While SCD can be cured by hematopoietic stem cell transplant (HSCT), complete donor chimerism is not required to achieve clinical benefits. Stable mixed chimerism of 10-15% in bone marrow or peripheral blood nucleated cells with >70% donor-derived RBCs has been reported to achieve transfusion independence and a symptom-free state in a SCD patient. It has also been proposed that SCD can be treated by reactivating developmentally silenced fetal gamma globin to form fetal hemoglobin (alpha2gamma2, HbF), which inhibits polymerization of HbS. The effect of HbF is predicted to be maximal when HbF content per cell exceeds 10 pg (~30% of total Hb). Furthermore, pathology is prevented when protective F cells (>30% HbF per cell) constitute >70% of total RBCs. We hypothesize that in a gene therapy setting, if >15% of SCD patients' autologous HSCs are programmed to produce protective F cells during erythropoiesis, it will translate into >70% protective F cells in circulation and provide significant alleviation of clinical symptoms. Genome wide association studies have identified BCL11A as a major modifier of HbF levels. Subsequent studies have shown that BCL11A plays a critical role in the fetal to adult globin developmental switch and in repressing fetal globin expression in adult erythroid cells. Conditional inactivation of BCL11A in adult erythroid cells leads to high levels of pan-cellular fetal globin expression and correction of hematologic and pathologic defects in a humanized SCD mouse model. Previously, we have reported that zinc finger nucleases (ZFNs) targeting BCL11A either in the coding region or the GATAA motif in the erythroid-specific enhancer efficiently disrupt the BCL11A locus in human primary CD34+ cells following electroporation of ZFN-encoding mRNA. Elevated fetal globin expression in bulk erythroid cultures was observed following disruption. To determine what percentage of HSPCs have been modified and whether the HbF/F cell content has reached the hypothesized therapeutic level, we analyzed erythroid cells clonally derived from ZFN-transfected CD34+ cells. Genotype of each clonal culture was determined by deep sequencing and globin production was analyzed by a highly sensitive UPLC method. We found that up to 80% of the BFU-Es had both BCL11A alleles edited, half of which had KO/KO alleles (either out of frame mutations for coding region or elimination of the GATAA motif in the enhancer). BCL11A coding KO/KO cells expressed on average 79.1% ± 12.2% fetal globin (Mean ± SD) whereas GATAA motif enhancer region KO/KO cells expressed approximately 48.4% ± 14.1% fetal globin, in comparison with 14.5% ± 9.6% in WT/WT cells . These levels of fetal globin should be sufficiently high to confer protection against HbS polymerization in sickle cells. WT/KO cells in both coding and enhancer editing experiments showed an intermediate phenotype with fetal globin averaging 26.9%± 9.9% and 25.79% ± 12.6%, respectively. Interestingly, when background (WT/WT) fetal globin level was subtracted, the fetal globin levels in WT/KO cells are comparable to those observed in patients with BCL11A haploinsufficiency, which average 14.6%± 10.3%. Together, our data demonstrate that genome editing of BCL11A using highly efficient ZFNs can lead to clinically relevant levels of fetal globin expression in KO/KO erythroid cells. If the frequency of KO/KO BFU-Es we observed in vitro reflects the frequency of KO/KO HSCs in bone marrow after autologous transplantation, genome editing of BCL11A has the potential to provide significant clinical benefit for patients with SCD. Disclosures Chang: Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Yang:Biogen: Employment, Equity Ownership. Sun:Biogen: Employment, Equity Ownership. Vieira:Biogen: Employment, Equity Ownership. Zhang:Biogen: Employment. Hong:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

Ablation of Bcl11A could be a viable approach for the treatment of β-hemoglobinopathies such as β-thalassemia and sickle cell disease (SCD), since patients with Bcl11A haploinsufficiency have persistently high levels of fetal hemoglobin (HbF) (up to 30%), which are associated with development of minimal to no disease symptoms. Genome editing by engineered zinc-finger nucleases that target either the exon 2 (exon ZFN) or the GATA motif of the erythroid specific enhancer (enhancer ZFN) of Bcl11A has been shown to increase HbF level in erythroid progeny from mobilized peripheral hematopoietic stem and progenitor cells (PB-CD34+ HSPCs). However, peripheral mobilization of CD34+ cells is associated with high risk and currently is not an option for SCD patients. Therefore, we investigated the efficacy of genome editing of Bcl11A in bone marrow derived CD34+ cells (BM-CD34+ HSPCs). We first established a clinically compatible large-scale process to isolate CD34+ HSPCs from human bone marrow aspirates and to transiently express the ZFN protein by mRNA electroporation. The CD34+ isolation process resulted in ~ 95% pure CD34+ cells with greater than 90% viability. Both the exon and the enhancer ZFN drove 50-60% Bcl11A gene editing, resulting in a robust elevation of HbF in the erythroid progeny. Notably, the BM-CD34+ HSPCs were found to contain a small population (10 to 25%) of CD34+CD19+ pro-B cells that were refractory to ZFN transfection under our current electroporation condition. Since CD34+CD19+ pro-B cells are not expected to contribute to reconstituting the hematopoietic system other than B-cell lineage, the Bcl11A editing efficiency in the multipotent BM-CD34+ HSPC could be even higher. The engraftment abilities of Bcl11A edited BM-CD34+ cells were then investigated in an immunodeficient NOD/scid/gamma (NSG) mouse model. At a dose of 1 million cells per mouse, treatment with either the exon ZFN or the enhancer ZFN did not detectably impact engraftment or multi-lineage reconstitution compared with untreated cells. However, Bcl11A marking in engrafted human cells was found to be markedly higher in the mice treated by the enhancer ZFN than that by the exon ZFN. The exon ZFN resulted in a strong bias towards in-frame mutations across multi-lineages with the strongest effect observed in the B-cell lineage, suggesting that a threshold level of Bcl11A is required for efficient hematopoietic reconstitution and that cells fully lacking it due to disruption of the coding sequence are at a disadvantage. In contrast, the enhancer ZFN resulted in comparable Bcl11A marking across all lineages with no apparent selection for cells with a functional GATA sequence. Collectively, these data indicate that genome editing of the erythroid specific enhancer of Bcl11A in BM-CD34+ promotes HbF reactivation in the erythroid progeny while maintaining the engraftment and multi-lineage repopulating activities of edited BM-CD34+ HSPCs, which supports further clinical development of this approach for the treatment of SCD. Disclosures Tan: Biogen: Employment, Equity Ownership. Chang:Biogen: Employment, Equity Ownership. Smith:Biogen: Employment, Equity Ownership. Chen:Biogen: Employment, Equity Ownership. Sullivan:Biogen: Employment, Equity Ownership. Zhou:Biogen: Employment, Equity Ownership. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Urnov:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Rebar:Sangamo BioSciences: Employment. Danos:Biogen: Employment, Equity Ownership. Jiang:Biogen: Employment, Equity Ownership.

We describe here a fundamentally novel way to develop a disease therapeutic: combining genome-wide association studies (GWAS) with targeted genome editing to create, in a clinically compliant setting, a disease-ameliorating genotype in the patient's own cells. In β-thalassemia, elevated levels of fetal hemoglobin (HbF) lessen or eliminate disease symptoms, thus making a reversal of HbF silencing in patients an appealing therapeutic strategy. Loss-of-function variants in the erythroid-specific enhancer of the fetal globin repressor, BCL11A, elevate HbF; rare individuals carrying a monoallelic knockout of BCL11A exhibit no known hematologic abnormality and up to 30% circulating HbF. We previously reported de novo knockout of BCL11A using targeted genome editing with engineered zinc finger nucleases (ZFNs) yielding up to 40% HbF in erythroid progeny of edited human CD34 cells in vitro. We now find that the targeted ablation of a single, specific GATAA motif in the BCL11A intronic enhancer does not affect in vitro erythroid differentiation, but reproducibly (n=6) activates fetal globin transcription in erythroid progeny of modified CD34 cells; importantly, at similar levels of on-target marking in CD34+ cells, these effects on fetal globin mRNA are comparable to those resulting from ZFN-driven coding knockout of BCL11A itself. We demonstrate reproducible (n=8), high-efficiency (up to 82%; average, 69%) ZFN-driven marking at the enhancer in peripheral blood mobilized human CD34 cells at clinical production scale (>1e8 cells) in a GMP-compliant setting for which we use a clinical-grade electroporation device to deliver nuclease-encoding transcribed mRNA ex vivo. Using erythroid colony assay genotyping we find that up to 70% of the cells in the resulting population are biallelically modified at the target locus, while ~10% remain wild-type, and find comparably high levels of marking in research-scale preparations of CD34 cells from patients with β-thalassemia. We observe robust long-term (18-24 week) engraftment and multilineage differentiation of genome-edited cells in immunodeficient mice, similar to control cells, and equivalent modification at the targeted enhancer locus at all timepoints in both differentiated (CD19+, CD3+, CD33+) and more primitive progenitor (CD34+CD38low) cells of human origin purified from bone marrow of long-term-engrafted animals. Our findings support clinical development of enhancer editing as a treatment of the β hemoglobinopathies with autologous hematopoietic stem cell transplant. Disclosures Urnov: Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Reik:Sangamo BioSciences: Employment, Equity Ownership, Patents & Royalties: Patent applications have been filed based on this work. Vierstra:University of Washington: Patents & Royalties: Patent applications have been filed based on this work. Chang:Biogen: Employment, Equity Ownership. Zhou:Sangamo BioSciences: Employment, Equity Ownership. Mich:Sangamo BioSciences: Employment, Equity Ownership. Adrian:Cellerant Therapeutics: Equity Ownership; Sangamo BioSciences: Employment, Equity Ownership. Fox:Sangamo BioSciences: Employment, Equity Ownership. Tan:Biogen: Employment, Equity Ownership. Craig:Sangamo BioSciences: Employment, Equity Ownership. Rebar:Sangamo BioSciences: Employment. Stamatoyannopoulos:University of Washington: Patents & Royalties: Patent applications have been filed based on this work.. Jiang:Biogen: Employment, Equity Ownership.

leukocyte antigens (HLA) displayed on cancer cells, but absent on normal neuroglia in the CNS. AlloCTL migrate through tumors, and at high effectorto-target (E:T) cell ratios achieve selective alloreactive killing, mediating a localized immunorejection effect. Adoptive alloCTL immunotherapy for glioma is also currently being evaluated in a Phase I study (www.clinicaltrials.gov, NCT01144247). Mixed lymphocyte-target cell assays demonstrated that alloCTL-based RRV producer cells (alloCTL/RRV) maintain migratory and effector function, and efficiently transmitted RRV to co-cultured cancer cells even at E:T ratios as low as 1:100. Combining alloCTL adoptive immunotherapy and RRV prodrug activator gene therapy in vivo showed both early tumor growth inhibition aswell as continued regression upon prodrug administration, achieving significantly improved therapeutic efficacy compared to either treatment alone in orthotopic brain tumor models. Thus, alloCTL/RRV can act both as immunotherapeutic effector cells that directly kill targets at high E:T ratios, and as motile cellular ‘carriers’ that release RRV and facilitate virus spread to unkilled targets at low E:T ratios.

Many monogenic recessive diseases of blood can, in principle, be cured by transfer of functional therapeutic transgene to the genome of the hematopoietic stem cell (HSC) – a strategy proven successful for multiple rare diseases using current integrating vector gene therapy. With a focus on X-linked chronic granulomatous disease (X-CGD), we report a directed approach orthogonal to randomly integrating retrovector gene therapy: the highly specific targeted placement of the curative transgene into a validated safe harbor locus in human HSCs via human genome editing with zinc finger nucleases (ZFNs) and donor insert delivery using an AAV6 vector.We describe here an integrated targeted delivery platform customized for targeted addition to human HSCs using a cGMP-compliant electroporation system compatible with clinical scale production. Using next-generation, highly optimized ZFNs against the AAVS1/PPP1R12C gene locus, we optimized conditions for addition of the fluorescent Venus cDNA into human peripheral blood G-CSF mobilized CD34+ HSCs. Venus expression in manipulated human HSCs in vitro reached >50% efficiency, while earlier experiments demonstrated persistence of gene-modified cells in NSG mice with 12-15% Venus+ human CD45+ cells retrieved from transplanted mouse bone marrow and overall human HSC engraftment levels of >15%. Targeted integration (TI) rates achieved in human CD45+ cells from mouse bone marrow were 28-57%. Similar levels of Venus+ (~10%) are observed in spleen and peripheral blood CD45+ cells, indicating differentiation of gene-modified CD34 HSCs into circulating blood cells.X-CGD patients suffer from severe bacterial and fungal infections with excessive inflammation due to a defect in the gp91phox subunit of phagocyte oxidase. To extend the results above to CGD, we therefore used the same approach to target addition of the relevant curative transgene, gp91phox, into the AAVS1 safe harbor locus of HSCs from patients with X-linked CGD. In vitro levels of gp91phox expression in gene-modified patient CD34+ HSCs population achieve 12-16% gp91phox expression by flow cytometric analysis, with an NSG xenograft study demonstrating 3-5% of the engrafted human CD45+ cells expressing gp91phox. Of note, the MND-driven gp91phox expression from the safe harbor locus in human neutrophils differentiating from CD34+ cells transplanted into the NSG mouse model parallels wildtype gp91phox levels produced at the native locus.Our studies demonstrate the feasibility of targeted addition of different genes at the AAVS1 safe harbor site of the genome in human HSCs at an unprecedented efficiency and specificity; we demonstrate the efficient correction of the enzymatic defect in neutrophils arising from patient-derived HSCs in vivo. In sum with the advances in GMP-scale cell processing for genome editing, and the charted regulatory path for ZFNs to the clinic provided by ongoing trials in HIV, our studies represent the foundation for a rapid translation of ZFN-driven targeted addition as a clinical modality for X-linked CGD.

Isogenic cell lines differing only in the expression of the protein of interest provide the ideal platform for cell-based screening. However, related natural lines differentially expressing the therapeutic target of choice are rare. Here the authors report a strategy for drug screening employing isogenic human cell lines in which the expression of the target protein is regulated by a gene-specific engineered zinc-finger protein (ZFP) transcription factor (TF). To demonstrate this approach, a ZFP TF activator of the human parathyroid hormone receptor 1 (PTHR1) gene was identified and introduced into HEK293 cells (negative for PTHR1). Following induction of ZFP TF expression, this cell line produced functional PTHR1 protein, resulting in a robust and ligand-specific cyclic adenosine monophosphate (cAMP) response. Reciprocally, the natural expression of PTHR1 observed in SAOS2 cells was dramatically reduced by the introduction of the appropriate PTHR1-specific ZFP TF repressor. Moreover, this ZFP-driven PTHR1 repression selectively eliminated the functional cAMP response invoked by known ligands of PTHR1. These data establish ZFP TF-generated isogenic lines as a general approach for the identification of therapeutic agents specific for the target gene of interest.

Designed zinc finger proteins (ZFPs) regulate expression of target genes when coupled to activator or repressor domains. Transfection of ZFPs into cell lines can create expression systems where the targeted endogenous gene is transcribed and the protein of interest can be investigated in its own cellular context. Here we describe the pharmacological investigation of an expression system generated using CCK2 receptor-selective ZFPs transfected into human embryonic kidney cells (HEKZFP system). The receptors expressed in this system, in response to ZFP expression, were functional in calcium mobilization studies and the potency of the agonists investigated was consistent with their action at CCK2 receptors (CCK-8S pA 50 = 9.05 ± 0.11, pentagastrin pA 50 = 9.11 ± 0.13). In addition, binding studies were conducted using [ 125 I]-BH-CCK-8S as radioligand. The saturation binding analysis of this radioligand was consistent with a single population of high affinity CCK receptors (pK D = 10.24). Competition studies were also conducted using a number of previously well-characterized CCK-receptor selective ligands; JB93182, YF476, PD-134,308, SR27897, dexloxiglumide, L-365,260 and L-364,718. Overall, the estimated affinity values for these ligands were consistent with their interaction at CCK2 receptors. Therefore, CCK2 receptors up-regulated using zinc finger protein technology can provide an alternative to standard transfection techniques for the pharmacological analysis of compounds.

Despite its central importance in gene regulation, chromatin in mammalian cells remains relatively poorly understood—a predicament due to the paucity of robust genetic tools in mammals, the complexity of the chromatin remodeling machinery, and the dynamic properties of chromatin in vivo. Here we review recent developments in understanding endogenous mammalian gene regulation via the use of designed transcription factors (TFs). These include mutated forms of naturally occurring TFs that exhibit dominant-negative activity, and designed proteins with novel, predetermined DNA-binding specificities. Systematic targeting of designed TFs to particular promoters is helping to illuminate the complex rules that chromatin imposes on TF access and action in vivo. We evaluate the potential applications of these proteins as probes of mammalian chromatin-based regulatory pathways and their potential for the therapy of human disease, highlighting leukemia in particular.

Multiple inhibitory pathways exist to block the immune response to cancer potentially limiting the effectiveness of adoptive cell transfer (ACT). Programmed cell death-1 (PD-1) is a member of the CD28 superfamily and is expressed on activated T cells. Its ligands, PDL-1 and PDL-2 are expressed on a variety of tumor cells, including melanoma. The binding of PD-1 to PDL-1 inhibits T cell effector function, and represents an important mechanism for PDL-1 expressing tumors to evade the host immune response to cancer. PD-1 thus represents an attractive target for gene-editing of tumor-targeted T cells prior to ACT. To this end, our aim was to eliminate PD-1 expression in tumor infiltrating lymphocytes (TIL) by genome-editing using zinc finger nucleases (ZFNs) directed against the PD-1 gene at a scale sufficient for patient treatment. Using the MaxCyte GT Flow Transfection System to deliver mRNA encoding the PD-1 ZFNs, we show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9 - 84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Importantly, the PD-1 gene-edited TIL product displayed an effector memory phenotype and expanded approximately 500 - 2000 fold during a rapid cell expansion in vitro while retaining T cell effector function. Thus further study to determine the safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.

DNA replication in the human beta-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the beta-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5'HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5'HS2-5 in the normal chromosomal context of the human beta-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5'HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5'HS2-5 in the classically defined LCR do not control replication in the human beta-globin locus. Recent studies also show that targeted deletion of 5'HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

The human β-globin locus provides a model system to study the interplay between chromatin structure and transcriptional regulation. The locus is located on chromosome 11 (11p15.5) and contains five developmentally regulated erythroid cell-specific genes arranged in the order in which they are expressed during development (5′-ɛ-Gγ-Aγ-δ-β-3′) and an upstream regulatory region characterized by five DNase I-hypersensitive sites (HSs; see Fig. ​Fig.1).1). By convention, these 5′HSs are denoted 5′HS1, 5′HS2, etc., with 5′HS1 being the most 3′ and located closest to the ɛ-globin gene. The importance of the upstream regulatory region was established by an analysis of the effects of a naturally occurring deletion which removes 5′HS2 to 5′HS5 and 25 kb of additional sequences 5′ of the HSs (Hispanic thalassemia). The chromosome carrying the deletion was transferred from lymphocytes of the thalassemic patient into murine erythroleukemia (MEL) cells to create the thalassemia line T-MEL. It was found that the mutation abolishes transcription of the adult human β-globin gene, prevents formation of 5′HS1 and other HSs throughout the locus, and renders the chromatin of the locus resistant to DNase I, indicative of a “closed” chromatin structure (19). In addition, the replication timing of the locus is changed from early to late in S phase (19) and a different origin of replication is used, even though the normal origin lies more than 50 kb from the site of the deletion (1, 36). The region containing the five HSs was termed the locus control region (LCR), because of its global effects on the locus. In transgenic mice, the LCR permits the expression of linked genes in all lines independent of the integration site (26), and regions with LCR properties have now been found in numerous other genes (reviewed in reference 35). FIG. 1 Homologous recombination in DT40 cells on a modified human chromosome 11 with the human β-globin locus. The genes of the β-globin locus and the upstream HSs are indicated, as well as the modification introduced into the locus in an earlier ... The properties of the β-globin LCR make it an interesting paradigm to study the global regulation of gene expression over large distances and the role that modulation of chromatin structure plays in transcriptional regulation. A variety of functional assays, including transient- and stable-transfection assays in cell lines and transgenic analyses, have been used in attempts to elucidate the function of the β-globin LCR and its component HSs. In general, these studies have demonstrated that the LCR and its component HSs demonstrate much higher activity in erythroid cells than in other cell types (5, 18, 44, 47, 63) and that for any given activity, the full LCR is more active than any individual component, suggesting interaction or additive effects among the individual sites (11, 18, 46, 55, 60). In addition, 5′HS2 and the full LCR are qualitatively equivalent: both function as classical enhancers in transient-transfection assays, increase the number of expressing clones when stably integrated in cell lines, and confer position independence and high-level expression in transgenic mice (5, 18, 41, 45, 58, 60, 63). In contrast to 5′HS2, both 5′HS3 and 5′HS4 have weak or no enhancer activity in transient-transfection assays but are active in colony assays when stably integrated and in transgenic mice (20, 30, 52, 63). Neither 5′HS1 nor 5′HS5 has appreciable activity in these transfection/transgenic-mouse assays (9, 21, 32). The failure of 5′HS1 to demonstrate activity in these assays is consistent with the lack of phenotypes associated with naturally occurring deletions of 5′HS1 in humans (37) and mice (3a). Although 5′HS5 does not demonstrate enhancer activity in transfection assays, it may play a functional role in the β-globin locus. Hypersensitivity at 5′HS5 is found in many nonerythroid cell lines, whereas 5′HS1 to 5′HS4 are predominantly erythroid cell specific (12, 61), and 5′HS5 is contained on a 2.5-kb restriction fragment which was characterized as one of the potential matrix attachment regions within the β-globin locus (33). In addition 5′HS5 increases the tendency for position-independent transcription of a linked reporter (32, 68) and it has also been reported to block the activation of a promoter when placed between the promoter and an enhancer (39). It has therefore been suggested that 5′HS5 serves as an insulator which shields the locus from neighboring chromatin. This role has also been postulated for HS4 of the chicken β-globin locus, which resembles human 5′HS5 in its broad tissue distribution and its relative position within the locus (8). While transfection and transgenic-mouse experiments have provided important information about the possible functions of the LCR, endogenous genes are regulated in defined chromosomal locations. Thus, analysis of unintegrated or randomly integrated constructs may overestimate, underestimate, or miss aspects of the activities of cis-regulatory elements, particularly those which influence chromatin, replication, and transcription over large distances. To examine how the LCR components interact to regulate the endogenous β-globin locus, we have developed homologous-recombination (HR) strategies for the mutational analysis of the endogenous human β-globin LCR in cell lines. Specifically, we studied the consequences of deleting those HSs of the LCR which are deleted in the Hispanic thalassemia deletion, as well as the consequences of the deletion of 5′HS5 on the chromatin structure and transcription of the β-globin locus. The results of these experiments show that in an erythroid background, the LCR is not required to maintain the open DNase I-sensitive chromatin structure of the locus but is essential for expression of all β-globin genes.

The understanding of the molecular mechanisms by which hormones regulate nuclear responses has been greatly extended by the cloning of the transcription factors that mediate hormone signals. Acquisition of these factors as cDNAs has permitted a range of residues that deliver information such as the modular structure of proteins, that are important residues for phosphorylation by kinases and dephos-phorylation by phosphatases, and the nature of the interaction between factors and pathways by protein-protein or post-translational means. Many of these new insights rely, however, on transient transfection or in vitro experiments in which the proteins are present at levelsl that are much higher than physiological concentrations. There is a need, therefore, to examine conclusions drawn from transfection and in vitro experiments in a physiological context, and the technique of genomic footprinting offers a powerful means of assessing hormonal responses in chromatin. In addition, genomic footprinting can ascertain mechanistic details of protein-DNA interactions during a hormone response that are unobtainable by other techniques. In this review, we will discuss the application of genomic footprinting to aspects of hormone signaling, concentrating on results obtained in our laboratory on the regulation of the tyrosine aminotransferase (TAT) gene, and on complementary examples from other systems. The TAT gene offers a good model for hormone studies, since it responds transcriptionally to representatives of both major classes of hormone signaling, namely glucocorticoids, which represent signaling by nuclear receptors, and cAMP, which represents signaling from cell-surface receptors via second messengers. Our intention is to present some of the insights that genomic footprinting has revealed as examples of how the technique can be applied and to emphasize the importance of information gained by observing hormone signaling under physiological conditions.

Beta-thalassemia (β-thal) and sickle cell disease (SCD) are monogenic diseases caused by mutations in the adult β-globin gene. A bone marrow transplant (BMT) is the only curative treatment, but its application is limited since (i) HLA-matched donors can be found for Recent insights into the regulation of γ-globin transcription by a network of transcription factors and regulatory elements both inside and outside the β-globin locus have revealed a set of new molecular targets, the modulation of which is expected to elevate γ-globin levels for potential therapeutic intervention. To this end, we and others have established that designed zinc finger nucleases (ZFNs) transiently introduced into stem cells ex vivo provide a safe and efficient way to permanently ablate the expression of a specific target gene in hematopoietic stem cells (HSC) by introduction of mutations following target site cleavage and error-prone DNA repair. Here we report the development and comparison of different ZFNs that target various regulators of γ-globin gene transcription in human HSCs: Bcl11a, Klf1, and specific positions in the γ-globin promoters that result in hereditary persistence of fetal hemoglobin (HPFH). In all cases these target sites / transcription factors have previously been identified as crucial repressors of γ-globin expression in humans, as well as by in vitro and in vivo experiments using human erythroid cells and mouse models. ZFN pairs with very high genome editing activity in CD34+ HSCs were identified for all targeted sites (>75% of alleles modified). In vitro differentiation of these ZFN-treated CD34+ HSCs into erythroid cells resulted in potent elevation of γ-globin mRNA and protein levels without significant effects on erythroid development. Importantly, a similar and specific elevation of γ-globin levels was observed with RBC progeny of genome-edited CD34+ cells obtained from SCD and β-thal patients. Notably, in the latter case a normalization of the β-like to α-globin ratio to ∼1.0 was observed in RBCs obtained from genome-edited CD34s from two individuals with β-thalassemia major. To deploy this strategy in a clinical setting, we developed protocols that yielded comparably high levels of target gene editing in mobilized adult CD34+ cells at large scale (>108 cells) using a clinical-grade electroporation device to deliver mRNA encoding the ZFN pair. Analysis of modification at the most likely off-target sites based on ZFN binding properties, combined with the maintenance of target genome editing observed throughout erythroid differentiation (and in isolated erythroid colonies) demonstrated that the ZFNs were both highly specific and well-tolerated when deployed at clinical scale. Finally, to assess the stemness of the genome-edited CD34+ HSCs we performed transplantation experiments in immunodeficient mice which revealed long term engraftment of the modified cells (>16 weeks, ∼25% human chimerism in mouse bone marrow) with maintenance of differentiation in vivo. Moreover, ex vivo erythroid differentiation of human precursor cells isolated from the bone marrow of transplanted animals confirmed the expected elevation of γ-globin. Taken together, these data suggest that a therapeutic level of γ-globin elevation can be obtained by the selective disruption, at the genome level, of specific regulators of the fetal to adult globin developmental switch. The ability to perform this modification at scale, with full retention of HSC engraftment and differentiation in vivo, provides a foundation for advancing this approach to a clinical trial for the hemoglobinopathies. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Lee:Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Luong:Sangamo BioSciences: Employment. Chan:Sangamo BioSciences: Employment. Liu:Sangamo BioSciences: Employment. Miller:Sangamo BioSciences: Employment. Paschon:Sangamo BioSciences: Employment. Guschin:Sangamo BioSciences: Employment. Zhang:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment.