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Childhood trauma (CT) can lead to long-term psychiatric disturbances. The current study investigated the prevalence of CT and its associated risk factors among Chinese adolescents with psychiatric disorders. Adolescents were recruited from a large study on mental health in the Hunan province of China in 2014. The study had a two-phase cross-sectional design. Patients with mental disorders (n = 907) and healthy subjects (n = 2,240) completed the Childhood Trauma Questionnaire–Short Form. Of all types of CT, emotional neglect (EN; 48.44%-68.82%) and physical neglect (PN; 60.0%-72.1%) were most common among adolescents. The experience of emotional abuse (EA), physical abuse (PA), sexual abuse (SA), EN, and multiple types of trauma (≥3) was higher among adolescents with psychiatric disorders than the healthy comparison group. Moreover, the results of logistic regression analysis showed that living in semi-urban areas and villages, having a sibling, and having bad-to-average academic performance were risk factors for CT among adolescents with psychiatric disorders ( p < .05). In additional, the results of logistic regression analysis revealed that being a left-behind child and bad academic performance were risk factors for experiencing multiple types (≥3) of CT ( p < .05), while being female and having siblings were found to be protective factors against (≥3) CT ( p < .05). In summary, the prevalence rate of CT (especially multiple types of trauma) is high among adolescents with psychiatric disorders. These results indicate the importance of reducing CT to limit the likelihood of psychiatric disorders, especially among adolescents.

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.

BackgroundTo date, around 4 per 100, 000 adolescent committed suicide within the 29 OECD countries. The suicidal behavior is related to psychological factors, genetics, neurobiology, and other biomarkers. We aimed to investigate the risk factors for the suicidal ideation, especially the association between suicide ideation and different levels of testosterone in the adolescent female with depression. The goal of this study is to aid the development of strategies to intervene the suicidal behavior for the depressed female adolescent. MethodIn this single center, prospective cohort study, we enrolled adolescent female with depression. We collected baseline demographic data, age adjusted level of testosterone, Symptom Self-rating Scale, and information about the suicidal ideation, non-suicidal self-injury (NSSI), and suicide attempt. We used multivariate logistic regression to determine the risk factors for the suicidal ideation. ResultsTotal 113 hospitalized adolescent female enrolled with a mean age of 13.5 (1.20). Among these patients, there were 86 (76.1%) subjects suffered from the suicidal ideation, while 59 (52.2%) of them had NSSI and 23 (20.4%) had suicide attempt behavior. In the final model, higher level of testosterone (p = 0.04) and higher age (p = 0.02) were associated with the higher odds of having suicidal ideation.ConclusionIn this exploratory cohort study, suicidal ideation was prevalent. It is important to assess the level of testosterone in the adolescent female with depression. This study is consistent with the other studies. It shows the age is a potential predictor for the suicidal ideation in the hospitalized adolescent female with depression.

Background. To date, around 4 per 100,000 adolescents committed suicide within the 29 OECD countries. The suicidal behavior is related to psychological factors, genetics, neurobiology, and other biomarkers. The aim of this study was to examine risk factors for the development of suicidal ideation in adolescent females with depression, focusing on the relationship between different testosterone levels and suicidal ideation, in order to help develop strategies to intervene in suicidal behavior in female adolescents with depression. Method. In this single-center prospective cohort study, we enrolled adolescent females with depression. We collected information on their baseline data, testosterone levels, symptom self-rating scale scores, suicidal ideation, non-suicidal self-injurious (NSSI) behaviours, and suicide attempts. We used multivariate logistic regression to identify risk factors for the development of suicidal ideation in adolescent females with depression. Results. A total of 113 hospitalized adolescent females were enrolled with a mean age of 13.5 (1.20). Among these patients, there were 86 (76.11%) subjects who suffered from suicidal ideation, 59 (52.21%) had NSSI and 23 (20.35%) had suicide attempt behavior. In the final model, higher level of testosterone ( p = 0.04 ) and higher age ( p = 0.02 ) were associated with the higher odds of having suicidal ideation. Conclusion. In this exploratory cohort study, the emergence of suicidal ideation was common among adolescent females with depression. This study is consistent with the other studies. It shows that the age is a potential predictor for suicidal ideation in hospitalized adolescent females with depression.

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

Objective There is no systematic review or meta-analysis to evaluate the efficacy of adjuvant psychotherapy in early-stage bipolar disorder. Therefore, the goal of this meta-analysis is to examine the evidence supporting psychotherapy as an efficacious approach to treating bipolar disorder. Methods Seven electronic databases including Web of Science, Embase, PubMed, Wanfang Data, Scopus, Science Direct, Cochrane Library were searched in March 2021 by two independent reviewers. Data extraction was performed independently, and any conflict was resolved before final analysis. Only randomized clinical trials were included in this study. The trial entails 1 primary outcome measure (relapse) and several secondary outcome measures: time to relapse, relapse rate, days missed at work/school (record, interview), and social functioning level. The risk of bias assessment of the included studies was performed by 2 authors independently using the tool recommended in the Cochrane Handbook for Systematic Reviews of Interventions. Results We hypothesized that combined psychotherapy and pharmacological interventions would be superior to pharmacological interventions alone regarding the time to relapse into a manic or depressive episode. Conclusion This study expects to provide credible and scientific clinical evidence for the efficacy and safety of combined psychotherapy and pharmacological interventions in the treatment of bipolar disorder. Osf registration number 10.17605/OSF.IO/ZGS6W.

Background: Though several epidemiological surveys of psychiatric disorders have been carried out in China, only a few of them are concerned about the prevalence of psychiatric disorders in central Hunan and reveal the distribution of common psychiatric disorders and their comorbidities. Methods: Achenbach’s Child Behavior Checklist (CBCL), the Mini International Neuropsychiatric Interview for Children and Adolescents (MINI-KID), and Diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM-IV) were administered to a stratified sample of 17,071 participants aged 6 to 16 years old from two cities in the central part of Hunan province. Twelve-month prevalence rates were calculated. Results: Twelve-month prevalence of the population was 9.74%. The most common psychiatric disorders were attention deficit hyperactivity disorder (ADHD) (4.96%), oppositional defiant disorder (ODD) (2.98%) and generalized anxiety disorder (GAD) (1.77%). Of those with a 12-month prevalence diagnosis, 34.6% had one or more comorbid psychiatric disorders. Most notably, ADHD had comorbidity rates of 25.15% with ODD, 18.18% with CD, 6.38% with GAD, and 3.66% with MDD. Conclusions: Psychiatric disorders are common in Chinese children and adolescents. Being the most prevalent mental disorder, ADHD requires continued focus and support in awareness and education.

Although evidence is accumulating that autism spectrum disorder (ASD) is associated with disruption of functional connections between and within brain networks, it remains largely unknown whether these abnormalities are related to specific frequency bands. To address this question, network contingency analysis was performed on brain functional connectomes obtained from 213 adolescent participants across nine sites in the Autism Brain Imaging Data Exchange (ABIDE) multisite sample, to determine the disrupted connections between and within seven major cortical networks in adolescents with ASD at Slow-5, Slow-4 and Slow-3 frequency bands and further assess whether the aberrant intra- and inter-network connectivity varied as a function of ASD symptoms. Overall under-connectivity within and between large-scale intrinsic networks in ASD was revealed across the three frequency bands. Specifically, decreased connectivity strength within the default mode network (DMN), between DMN and visual network (VN), ventral attention network (VAN), and between dorsal attention network (DAN) and VAN was observed in the lower frequency band (slow-5, slow-4), while decreased connectivity between limbic network (LN) and frontal-parietal network (FPN) was observed in the higher frequency band (slow-3). Furthermore, weaker connectivity within and between specific networks correlated with poorer communication and social interaction skills in the slow-5 band, uniquely. These results demonstrate intrinsic under-connectivity within and between multiple brain networks within predefined frequency bands in ASD, suggesting that frequency-related properties underlie abnormal brain network organization in the disorder.

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.

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.

Background Parents of children with autism have higher rates of broad autism phenotype (BAP) features than parents of typically developing children (TDC) in Western countries. This study was designed to examine the rate of BAP features in parents of children with autism and the relationship between parental BAP and the social impairment of their children in a Chinese sample. Methods A total of 299 families with autistic children and 274 families with TDC participated in this study. Parents were assessed using the Broad Autism Phenotype Questionnaire (BAPQ), which includes self-report, informant-report, and best-estimate versions. Children were assessed using the Chinese version of the Social Responsiveness Scale (SRS). Results Parents of children with autism were significantly more likely to have BAP features than were parents of TDC; mothers and fathers in families with autistic children had various BAP features. The total scores of the informant and best-estimate BAPQ versions for fathers were significantly associated with their children’s SRS total scores in the autism group, whereas the total scores of the three BAPQ versions for mothers were significantly associated with their children’s SRS total scores in the TDC group. In the autism group, the total SRS scores of children with “BAP present” parents (informant and best-estimate) were higher than the total SRS scores of children with“BAP absent” parents. In the TDC group, the total SRS scores of children with “BAP present” parents were higher than the total SRS scores of children with“BAP absent” parents (best-estimate). Conclusions Parents of autistic children were found to have higher rates of BAP than parents of TDC in a sample of Chinese parents. The BAP features of parents are associated with their children’s social functioning in both autism families and TDC families, but the patterns of the associations are different.

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.

Eye-tracking studies in young children with autism spectrum disorder (ASD) have shown a visual attention preference for geometric patterns when viewing paired dynamic social images (DSIs) and dynamic geometric images (DGIs). In the present study, eye-tracking of two different paired presentations of DSIs and DGIs was monitored in a group of 13 children aged 4 to 6 years with ASD and 20 chronologically age-matched typically developing children (TDC). The results indicated that compared with the control group, children with ASD attended significantly less to DSIs showing two or more children playing than to similar DSIs showing a single child. Visual attention preference in 4- to 6-year-old children with ASDs, therefore, appears to be modulated by the type of visual stimuli.

Unregulated expression of vascular endothelial growth factor-A (VEGF-A) plays an important role in tumor growth. We have identified a cell type-specific enhancer, HS-1100, that contributes to VEGF-A transcriptional activation in tumorigenic glioblastoma cell lines. This enhancer exhibits increased accessibility to DNase I in glioblastoma cell lines that express high levels of VEGF-A but not in several other cell lines that express much lower levels of VEGF-A. HS-1100 contains a number of sequence elements that are highly conserved among human, mouse, and rat, including the hypoxia-response element (HRE). We show that the HRE contributes significantly to the cell type-specific enhancer activity of HS-1100 in U87MG glioblastoma cells. We use chromatin immunoprecipitation assays to show that endothelial PAS domain protein 1 (EPAS1) can efficiently bind to the endogenous HRE in U87MG cells but not in HEK293 cells in which the chromosomal HS-1100 is not accessible to DNase I. A dominant negative EPAS1 significantly reduces HS-1100 enhancer activity and VEGF-A levels in U87MG cells. Our results provide insight into the molecular mechanisms of VEGF-A up-regulation during cancer development.

The Silencing-Mediator for Retinoid/Thyroid hormone receptors (SMRT) interacts with, and mediates transcriptional repression by, a variety of eukaryotic transcription factors, including the nuclear hormone receptors. The ability of SMRT to function as a transcriptional ‘corepressor’ is regulated by a variety of signal transduction pathways. We report here that SMRT is a phosphoprotein in vivo, and is also phosphorylated in vitro by unfractionated cell extracts. A major site of phosphorylation of SMRT is a protein kinase CK2 motif centered on serine 1492, and located within a C-terminal SMRT domain that mediates interaction of the corepressor with the nuclear hormone receptors. Phosphorylation of SMRT by CK2 stabilizes the ability of the SMRT protein to interact with nuclear hormone receptors. Our results indicate that SMRT is a member of an expanding family of transcriptional regulators that are modified, and potentially regulated, in response to proteinkinase CK2.

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.

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.

The anterior cingulate cortex (ACC) is frequently reported to have functionally distinct sub-regions that play key roles in different intrinsic networks. However, the contribution of the ACC, which is connected to several cortical areas and the limbic system, to autism is not clearly understood, although it may be involved in dysfunctions across several distinct but related functional domains. By comparing resting-state fMRI data from persons with autism and healthy controls, we sought to identify the abnormalities in the functional connectivity (FC) of ACC sub-regions in autism. The analyses found autism-related reductions in FC between the left caudal ACC and the right rolandic operculum, insula, postcentral gyrus, superior temporal gyrus, and the middle temporal gyrus. The FC (z-scores) between the left caudal ACC and the right insula was negatively correlated with the Stereotyped Behaviors and Restricted Interests scores of the autism group. These findings suggest that the caudal ACC is recruited selectively in the pathomechanism of autism.

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.

Background Allogeneic bone marrow transplant (BMT) is the only curative method for a number of monogenic blood disorders, including various forms of hemoglobinopathies and severe combined immunodeficiencies. Aside from the significant hurdle of finding an identical HLA-matched related donor, allogeneic BMT recipients require chronic immunosuppression to mitigate the significant risk of GVHD and are at greater risk for graft failure. Autologous gene modified HSPC potentially provide a much safer alternative to allogeneic BMT and abrogate the need for finding HLA-matched donors. Here, we report the development of a highly efficient process for generating gene modified human HSPC at clinical-scale (>150 x 10^6 cells for 2x10^6/kg) using clinical-grade equipment. Methods Healthy donors were administered Neupogen® (10mg/kg/day) for 4-5 consecutive days and then apheresed. Enrichment of CD34+ cells was performed with the Miltenyi CliniMACS® system. Genome editing was achieved via the introduction of mRNA encoding two engineered zinc finger nucleases (ZFN) using a scalable electroporation device. Cells were harvested and cryo-preserved with a controlled-rate freezer. Cell recovery and viability, gene modification efficiency, stem cell pluripotency and engraftment potential were evaluated by in vitro assays and in a humanized NSG mouse model. Results Enrichment of CD34+ cells from mobilized leukopak products was highly efficient with the Miltenyi CliniMACS® system (median recovery = 327 million CD34+ cells per 10L mobilized leukopak). The positively selected fractions were >98% CD34+ by FACS analysis. Two large scale electroporation devices (BTX AgilePulse Max® and MaxCyte GT®) were evaluated. Each device is capable of electroporating up to 300 million cells. The optimal transfection conditions for both devices were first identified by using a GFP mRNA to evaluate transfection efficiency by flow cytometry, which resulted in the identification of conditions (voltage and duration) that yielded gene transfer efficiencies of >90%. Compatibility of this protocol with driving endogenous gene modification was evaluated using mRNA encoding ZFNs that target various endogenous gene loci. Highly efficient levels of genome editing were observed in CD34+ HSPC each transfected with a different pair of ZFNs (median = 53%, 43%, 45% and 42% modified alleles at four distinct disease-relevant loci). At the optimal mRNA dose for each ZFN pair, cell viability post electroporation was >80%, comparable to untransfected controls. Process suitability was evaluated by in vitro colony forming cell assay. No significant differences in colony formation were observed between gene modified and untransfected control samples. The capacity of electroporated HSPC to engraft and support multi-lineage development of human hematopoietic cells was evaluated in NSG mice, and no differences were observed between the ZFN-treated and untransfected control cells. In addition, high levels of gene modification (19-28%) were detected in bulk human cells from the blood and tissues of engrafted mice, and in various sorted cell types (bone marrow CD34 and differentiated B and T cells). Conclusion We have developed a scalable process capable of deriving >300 million gene-modified CD34+ HSPC. This process supports high levels of ZFN-driven genome editing, is well tolerated, and causes no discernable defect in the hematopoietic potential of these cells to develop into multiple cell lineages, with high gene editing levels maintained in the differentiated progeny of the HSPC. These results support the use of gene modified autologus HSPC for the treatment of monogenic blood disorders. Disclosures: Lee: Sangamo BioSciences: Employment. Truong:Sangamo BioSciences: Employment. Wood:Sangamo BioSciences: Employment. Ya-Li:Sangamo BioSciences: Employment. Kim:Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Wang:Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment. Urnov:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Ando:Sangamo BioSciences: Employment. Giedlin:Sangamo BioSciences: Employment.

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.

Abstract 3766 Cell therapy by infusion of T cells can reconstitute immunity to combat pathogens and malignancies. However, the time required to manufacture T cells with the desired properties and in sufficient numbers ex vivo is often incompatible with the treatment window for patients. Furthermore, autologous T cells from patients with advanced disease may have compromised function and be tolerant to desired antigens. A potential solution would be an approach to infuse allogeneic T cells that avoids immune-mediated rejection caused by host T cells recognizing disparate major or minor histocompatibility antigens on the infused cells. To broaden the application of T cell therapy, we investigated whether HLA gene expression can be disrupted by designer zinc-finger nucleases (ZFNs). ZFNs comprise a zinc finger DNA binding domain designed to bind a specific DNA sequence fused to the cleavage domain of Fok I endonuclease. Since FokI dimerization is required to introduce a double strand break (DSB), we generated ZFN pairs that flank the intended DNA target sequences in the required spatial conformation. Cellular repair of the DSB by error-prone non-homologous end joining allows disruption of HLA gene expression. As an initial proof of concept experiment, transfection of ZFN pairs designed to target exon 3 of the HLA-A locus into the human kidney cell line HEK293 resulted in 10% genetic modification of the HLA-A loci. We generated clones of HEK293 cells that showed deletion or insertion mutations within the ZFN binding site of one or both HLA-A alleles leading to early termination of translation. These HLA-Anull HEK293 clones evaded HLA-A-restricted lysis by T cell clones, even after interferon-γ and TNF-α treatment was used to upregulate HLA expression. Since only transient expression of ZFNs is needed to disrupt a target gene, we tested the ability to disrupt HLA-A gene expression by electro-transfer of in vitro-transcribed ZFN mRNA into primary T cells. We show that a single administration of the mRNA encoding the ZFNs targeting HLA-A could render over 40% of primary T cells HLA-A negative. We enriched the HLA-Anull population by paramagnetic bead separation to obtain a pool of T cells >90% of which lack HLA-A expression. An attractive potential clinical application for HLAnull allogeneic T cells is to redirect their specificity independent of HLA via expression of a chimeric antigen receptor (CAR) targeting CD19. Thus, we eliminated HLA-A expression from CD19-specific CAR+ T cells and demonstrated that they (i) evade HLA-A-restricted lysis by T cell clones, and (ii) specifically lysed CD19+ tumor targets. Finally, to further improve this T cell product and eliminate potential deleterious immune mediated recognition by the endogenous T cell receptor (TCR) on allogeneic CAR+ T cells, we used ZFN pairs targeting the TCR α or the TCR β locus. Transient expression of these ZFNs resulted in permanent disruption of endogenous TCR expression and a highly enriched αβ TCRnull cell population could be generated by paramagnetic bead selection. These data support our plans to develop allogeneic T cells as “off-the-shelf” biologics that can be infused on demand as “drugs”. Disclosures: Reik: Sangamo BioSciences: Employment. Zhou:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment.

Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.

Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer. We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action. To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner. These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.

Achieving sufficient transgene expression poses a critical challenge for many gene therapies, particularly those designed to produce secreted factors for systemic or remote effects. Although expression may be boosted via increased transgene delivery, this approach is ultimately limited by vector cost or toxicity, often at expression levels which are still therapeutically insufficient. Expression may also be increased via the development of specially designed hybrid or synthetic promoters bearing target sites for potent natural transcriptional activators. Given the reliance of such approaches on the availability of appropriate endogenous factors, however, the resultant constructs often represent cell type-specific solutions to enhancing expression. Moreover, such promoters may also introduce the risk of deranging natural cellular regulatory pathways by squelching the expression of other endogenous genes.